Sample records for a53t-alpha-synuclein overexpression impairs

  1. Controlling aggregation propensity in A53T mutant of alpha-synuclein causing Parkinson's disease

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kumar, Sonu; Sarkar, Anita; Sundar, Durai, E-mail: sundar@dbeb.iitd.ac.in

    2009-09-18

    Understanding {alpha}-synuclein in terms of fibrillization, aggregation, solubility and stability is fundamental in Parkinson's disease (PD). The three familial mutations, namely, A30P, E46K and A53T cause PD because the hydrophobic regions in {alpha}-synuclein acquire {beta}-sheet configuration, and have a propensity to fibrillize and form amyloids that cause cytotoxicity and neurodegeneration. On simulating the native form and mutants (A30P, E46K and A53T) of {alpha}-synuclein in water solvent, clear deviations are observed in comparison to the all-helical 1XQ8 PDB structure. We have identified two crucial residues, {sup 40}Val and {sup 74}Val, which play key roles in {beta}-sheet aggregation in the hydrophobic regionsmore » 36-41 and 68-78, respectively, leading to fibrillization and amyloidosis in familial (A53T) PD. We have also identified V40D{sub V}74D, a double mutant of A53T (the most amyloidogenic mutant). The simultaneous introduction of these two mutations in A53T nearly ends its aggregation propensity, increases its solubility and positively enhances its thermodynamic stability.« less

  2. Early and progressive sensorimotor anomalies in mice overexpressing wild-type human alpha-synuclein.

    PubMed

    Fleming, Sheila M; Salcedo, Jonathan; Fernagut, Pierre-Olivier; Rockenstein, Edward; Masliah, Eliezer; Levine, Michael S; Chesselet, Marie-Françoise

    2004-10-20

    Accumulation of alpha-synuclein in brain is a hallmark of synucleinopathies, neurodegenerative diseases that include Parkinson's disease. Mice overexpressing alpha-synuclein under the Thy-1 promoter (ASO) show abnormal accumulation of alpha-synuclein in cortical and subcortical regions of the brain, including the substantia nigra. We examined the motor deficits in ASO mice with a battery of sensorimotor tests that are sensitive to alterations in the nigrostriatal dopaminergic system. Male wild-type and ASO mice were tested every 2 months for 8 months for motor performance and coordination on a challenging beam, inverted grid, and pole, sensorimotor deficits in an adhesive removal test, spontaneous activity in a cylinder, and gait. Fine motor skills were assessed by the ability to grasp cotton from a bin. ASO mice displayed significant impairments in motor performance and coordination and a reduction in spontaneous activity as early as 2 months of age. Motor performance and coordination impairments became progressively worse with age and sensorimotor deficits appeared at 6 months. Fine motor skills were altered at 4 months and worsened at 8 months. These data indicate that overexpression of alpha-synuclein induced an early and progressive behavioral phenotype that can be detected in multiple tests of sensorimotor function. These behavioral deficits provide a useful way to assess novel drug therapy in genetic models of synucleinopathies.

  3. AAV1/2-induced overexpression of A53T-α-synuclein in the substantia nigra results in degeneration of the nigrostriatal system with Lewy-like pathology and motor impairment: a new mouse model for Parkinson's disease.

    PubMed

    Ip, Chi Wang; Klaus, Laura-Christin; Karikari, Akua A; Visanji, Naomi P; Brotchie, Jonathan M; Lang, Anthony E; Volkmann, Jens; Koprich, James B

    2017-02-01

    α-Synuclein is a protein implicated in the etiopathogenesis of Parkinson's disease (PD). AAV1/2-driven overexpression of human mutated A53T-α-synuclein in rat and monkey substantia nigra (SN) induces degeneration of nigral dopaminergic neurons and decreases striatal dopamine and tyrosine hydroxylase (TH). Given certain advantages of the mouse, especially it being amendable to genetic manipulation, translating the AAV1/2-A53T α-synuclein model to mice would be of significant value. AAV1/2-A53T α-synuclein or AAV1/2 empty vector (EV) at a concentration of 5.16 x 10 12 gp/ml were unilaterally injected into the right SN of male adult C57BL/6 mice. Post-mortem examinations included immunohistochemistry to analyze nigral α-synuclein, Ser129 phosphorylated α-synuclein and TH expression, striatal dopamine transporter (DAT) levels by autoradiography and dopamine levels by high performance liquid chromatography. At 10 weeks, in AAV1/2-A53T α-synuclein mice there was a 33% reduction in TH+ dopaminergic nigral neurons (P < 0.001), 29% deficit in striatal DAT binding (P < 0.05), 38% and 33% reductions in dopamine (P < 0.001) and DOPAC (P < 0.01) levels and a 60% increase in dopamine turnover (homovanilic acid/dopamine ratio; P < 0.001). Immunofluorescence showed that the AAV1/2-A53T α-synuclein injected mice had widespread nigral and striatal expression of vector-delivered A53T-α-synuclein. Concurrent staining with human PD SN samples using gold standard histological methodology for Lewy pathology detection by proteinase K digestion and application of specific antibody raised against human Lewy body α-synuclein (LB509) and Ser129 phosphorylated α-synuclein (81A) revealed insoluble α-synuclein aggregates in AAV1/2-A53T α-synuclein mice resembling Lewy-like neurites and bodies. In the cylinder test, we observed significant paw use asymmetry in the AAV1/2-A53T α-synuclein group when compared to EV controls at 5 and 9 weeks post injection (P

  4. Transgenic mice overexpressing tyrosine-to-cysteine mutant human alpha-synuclein: a progressive neurodegenerative model of diffuse Lewy body disease.

    PubMed

    Zhou, Wenbo; Milder, Julie B; Freed, Curt R

    2008-04-11

    Abnormal aggregation of human alpha-synuclein in Lewy bodies and Lewy neurites is a pathological hallmark of Parkinson disease and dementia with Lewy bodies. Studies have shown that oxidation and nitration of alpha-synuclein lead to the formation of stable dimers and oligomers through dityrosine cross-linking. Previously we have reported that tyrosine-to-cysteine mutations, particularly at the tyrosine 39 residue (Y39C), significantly enhanced alpha-synuclein fibril formation and neurotoxicity. In the current study, we have generated transgenic mice expressing the Y39C mutant human alpha-synuclein gene controlled by the mouse Thy1 promoter. Mutant human alpha-synuclein was widely expressed in transgenic mouse brain, resulting in 150% overexpression relative to endogenous mouse alpha-synuclein. At age 9-12 months, transgenic mice began to display motor dysfunction in rotarod testing. Older animals aged 15-18 months showed progressive accumulation of human alpha-synuclein oligomers, associated with worse motor function and cognitive impairment in the Morris water maze. By age 21-24 months, alpha-synuclein aggregates were further increased, accompanied by severe behavioral deficits. At this age, transgenic mice developed neuropathology, such as Lewy body-like alpha-synuclein and ubiquitin-positive inclusions, phosphorylation at Ser(129) of human alpha-synuclein, and increased apoptotic cell death. In summary, Y39C human alpha-synuclein transgenic mice show age-dependent, progressive neuronal degeneration with motor and cognitive deficits similar to diffuse Lewy body disease. The time course of alpha-synuclein oligomer accumulation coincided with behavioral and pathological changes, indicating that these oligomers may initiate protein aggregation, disrupt cellular function, and eventually lead to neuronal death.

  5. A53T-α-synuclein overexpression in murine locus coeruleus induces Parkinson's disease-like pathology in neurons and glia.

    PubMed

    Henrich, Martin Timo; Geibl, Fanni Fruzsina; Lee, Bolam; Chiu, Wei-Hua; Koprich, James Benjamin; Brotchie, Jonathan Michael; Timmermann, Lars; Decher, Niels; Matschke, Lina Anita; Oertel, Wolfgang Hermann

    2018-05-10

    Degeneration of noradrenergic locus coeruleus neurons occurs during the prodromal phase of Parkinson's disease and contributes to a variety of non-motor symptoms, e.g. depression, anxiety and REM sleep behavior disorder. This study was designed to establish the first locus coeruleus α-synucleinopathy mouse model, which should provide sufficient information about the time-course of noradrenergic neurodegeneration, replicate cardinal histopathological features of the human Parkinson's disease neuropathology and finally lead to robust histological markers, which are sufficient to assess the pathological changes in a quantitative and qualitative way. We show that targeted viral vector-mediated overexpression of human mutant A53T-α-synuclein in vivo in locus coeruleus neurons of wild-type mice resulted in progressive noradrenergic neurodegeneration over a time frame of 9 weeks. Observed neuronal cell loss was accompanied by progressive α-synuclein phosphorylation, formation of proteinase K-resistant α-synuclein-aggregates, accumulation of Ubi-1- and p62-positive inclusions in microglia and induction of progressive micro- and astrogliosis. Apart from this local pathology, abundant α-synuclein-positive axons were found in locus coeruleus output regions, indicating rapid anterograde axonal transport of A53T-α-synuclein. Taken together, we present the first model of α-synucleinopathy in the murine locus coeruleus, replicating essential morphological features of human Parkinson's disease pathology. This new model may contribute to the research on prodromal Parkinson's disease, in respect to pathophysiology and the development of disease-modifying therapy.

  6. Sleep Dysfunction and EEG Alterations in Mice Overexpressing Alpha-Synuclein

    PubMed Central

    McDowell, Kimberly A.; Shin, David; Roos, Kenneth P.; Chesselet, Marie-Françoise

    2018-01-01

    Background: Sleep disruptions occur early and frequently in Parkinson’s disease (PD). PD patients also show a slowing of resting state activity. Alpha-synuclein is causally linked to PD and accumulates in sleep-related brain regions. While sleep problems occur in over 75% of PD patients and severely impact the quality of life of patients and caregivers, their study is limited by a paucity of adequate animal models. Objective: The objective of this study was to determine whether overexpression of wildtype alpha-synuclein could lead to alterations in sleep patterns reminiscent of those observed in PD by measuring sleep/wake activity with rigorous quantitative methods in a well-characterized genetic mouse model. Methods: At 10 months of age, mice expressing human wildtype alpha-synuclein under the Thy-1 promoter (Thy1-aSyn) and wildtype littermates underwent the subcutaneous implantation of a telemetry device (Data Sciences International) for the recording of electromyograms (EMG) and electroencephalograms (EEG) in freely moving animals. Surgeries and data collection were performed without knowledge of mouse genotype. Results: Thy1-aSyn mice showed increased non-rapid eye movement sleep during their quiescent phase, increased active wake during their active phase, and decreased rapid eye movement sleep over a 24-h period, as well as a shift in the density of their EEG power spectra toward lower frequencies with a significant decrease in gamma power during wakefulness. Conclusions: Alpha-synuclein overexpression in mice produces sleep disruptions and altered oscillatory EEG activity reminiscent of PD, and this model provides a novel platform to assess mechanisms and therapeutic strategies for sleep dysfunction in PD. PMID:24867919

  7. Pesticide exposure exacerbates alpha-synucleinopathy in an A53T transgenic mouse model.

    PubMed

    Norris, Erin H; Uryu, Kunihiro; Leight, Susan; Giasson, Benoit I; Trojanowski, John Q; Lee, Virginia M-Y

    2007-02-01

    The factors initiating or contributing to the pathogenesis of Parkinson's disease and related neurodegenerative synucleinopathies are still largely unclear, but environmental factors such as pesticides have been implicated. In this study, A53T mutant human alpha-synuclein transgenic mice (M83), which develop alpha-synuclein neuropathology, were treated with the pesticides paraquat and maneb (either singly or together), and their effects were analyzed. Immunohistochemical and biochemical analyses showed that chronic treatment of M83 transgenic mice with both pesticides (but not with either pesticide alone) drastically increased neuronal alpha-synuclein pathology throughout the central nervous system including the hippocampus, cerebellum, and sensory and auditory cortices. alpha-Synuclein-associated mitochondrial degeneration was observed in M83 but not in wild-type alpha-synuclein transgenic mice. Because alpha-synuclein inclusions accumulated in pesticide-exposed M83 transgenic mice without a motor phenotype, we conclude that alpha-synuclein aggregate formation precedes disease onset. These studies support the notion that environmental factors causing nitrative damage are closely linked to mechanisms underlying the formation of alpha-synuclein pathologies and the onset of Parkinson's-like neurodegeneration.

  8. Quantitative proteomics in A30P*A53T α-synuclein transgenic mice reveals upregulation of Sel1l.

    PubMed

    Yan, Jianguo; Zhang, Pei; Jiao, Fengjuan; Wang, Qingzhi; He, Feng; Zhang, Qian; Zhang, Zheng; Lv, Zexi; Peng, Xiang; Cai, Hongwei; Tian, Bo

    2017-01-01

    α-Synuclein is an abundantly expressed neuronal protein that is at the center of focus in understanding a group of neurodegenerative disorders called synucleinopathies, which are characterized by the intracellular presence of aggregated α-synuclein. However, the mechanism of α-synuclein biology in synucleinopathies pathogenesis is not fully understood. In this study, mice overexpressing human A30P*A53T α-synuclein were evaluated by a motor behavior test and count of TH-positive neurons, and then two-dimensional liquid chromatography-tandem mass spectrometry coupled with tandem mass tags (TMTs) labeling was employed to quantitatively identify the differentially expressed proteins of substantia nigra pars compacta (SNpc) tissue samples that were obtained from the α-synuclein transgenic mice and wild type controls. The number of SNpc dopaminergic neurons and the motor behavior were unchanged in A30P*A53T transgenic mice at the age of 6 months. Of the 4,715 proteins identified by proteomic techniques, 271 were differentially expressed, including 249 upregulated and 22 downregulated proteins. These alterations were primarily associated with mitochondrial dysfunction, oxidative stress, ubiquitin-proteasome system impairment, and endoplasmic reticulum (ER) stress. Some obviously changed proteins, which were validated by western blotting and immunofluorescence staining, including Sel1l and Sdhc, may be involved in the α-synuclein pathologies of synucleinopathies. A biological pathway analysis of common related proteins showed that the proteins were linked to a total of 31 KEGG pathways. Our findings suggest that these identified proteins may serve as novel therapeutic targets for synucleinopathies.

  9. PA700, the regulatory complex of the 26S proteasome, interferes with alpha-synuclein assembly.

    PubMed

    Ghee, Medeva; Melki, Ronald; Michot, Nadine; Mallet, Jacques

    2005-08-01

    Parkinson's disease is characterized by the loss of dopaminergic neurons in the nigrostriatal pathway accompanied by the presence of intracellular cytoplasmic inclusions, termed Lewy bodies. Fibrillized alpha-synuclein forms the major component of Lewy bodies. We reported a specific interaction between rat alpha-synuclein and tat binding protein 1, a subunit of PA700, the regulatory complex of the 26S proteasome. It has been demonstrated that PA700 prevents the aggregation of misfolded, nonubiquinated substrates. In this study, we examine the effect of PA700 on the aggregation of wild-type and A53T mutant alpha-synuclein. PA700 inhibits both wild-type and A53T alpha-synuclein fibril formation as measured by Thioflavin T fluorescence. Using size exclusion chromatography, we present evidence for a stable PA700-alpha-synuclein complex. Sedimentation analyses reveal that PA700 sequesters alpha-synuclein in an assembly incompetent form. Analysis of the morphology of wild-type and A53T alpha-synuclein aggregates during the course of fibrillization by electron microscopy demonstrate the formation of amyloid-like fibrils. Secondary structure analyses of wild-type and A53T alpha-synuclein assembled in the presence of PA700 revealed a decrease in the overall amount of assembled alpha-synuclein with no significant change in protein conformation. Thus, PA700 acts on alpha-synuclein assembly and not on the structure of fibrils. We hypothesize that PA700 sequesters alpha-synuclein oligomeric species that are the precursors of the fibrillar form of the protein, thus preventing its assembly into fibrils.

  10. Rasagiline protects against alpha-synuclein induced sensitivity to oxidative stress in dopaminergic cells

    PubMed Central

    Chau, K.Y.; Cooper, J.M.; Schapira, A.H.V.

    2010-01-01

    Rasagiline is a propargylamine and irreversible monoamine oxidase (MAO) B inhibitor used for the treatment of Parkinson's disease (PD). It has demonstrated neuroprotective properties in laboratory studies. Current concepts of PD aetiopathogenesis include the role of alpha-synuclein, protein aggregation, free radical metabolism and mitochondrial dysfunction in contributing to cell death. We have used a combination of alpha-synuclein and free radical mediated toxicity in a dopaminergic cell line to provide a model of nigral toxicity in order to investigate the potential molecular mechanisms that mediate rasagiline protection. We demonstrate that rasagiline protects against cell death induced by the combination of free radicals generated by paraquat and either wild-type or A53T mutant alpha-synuclein over-expression. This protection was associated with a reduction in caspase 3 activation, a reduction in superoxide generation and a trend to ameliorate the fall in mitochondrial membrane potential. Rasagiline induced an increase in cellular glutathione levels. The results support a role for rasagiline in protecting dopaminergic cells against free radical mediated damage and apoptosis in the presence of alpha-synuclein over-expression. The data are of relevance to the interpretation of the potential mechanisms of action of rasagiline in explaining the results of disease modification trials in PD. PMID:20624440

  11. Mutant alpha-synuclein overexpression mediates early proinflammatory activity.

    PubMed

    Su, Xiaomin; Federoff, Howard J; Maguire-Zeiss, Kathleen A

    2009-10-01

    Microglia provide immune surveillance for the brain through both the removal of cellular debris and protection against infection by microorganisms and "foreign" molecules. Upon activation, microglia display an altered morphology and increased expression of proinflammatory molecules. Increased numbers of activated microglia have been identified in a number of neurodegenerative diseases including Parkinson's disease (PD). What remains to be determined is whether activated microglia result from ongoing cell death or are involved in disease initiation and progression. To address this question we utilized a transgenic mouse model that expresses a mutated form of a key protein involved in Parkinson's disease, alpha-synuclein. Herein, we report an increase in activated microglia and proinflammatory molecules in 1-month-old transgenic mice well before cell death occurs in this model. Frank microglial activation is resolved by 6 months of age while a subset of proinflammatory molecules remain elevated for 12 months. Both tyrosine hydroxylase mRNA expression and alpha-synuclein protein are decreased in the striatum of older animals evidence of dystrophic neuritic projections. To determine whether mutated alpha-synuclein could directly activate microglia primary microglia-enriched cell cultures were treated with exogenous mutated alpha-synuclein. The data reveal an increase in activated microglia and proinflammatory molecules due to direct interaction with mutated alpha-synuclein. Together, these data demonstrate that mutated alpha-synuclein mediates a proinflammatory response in microglia and this activity may participate in PD pathogenesis.

  12. Non-motor parkinsonian pathology in aging A53T α-synuclein mice is associated with progressive synucleinopathy and altered enzymatic function.

    PubMed

    Farrell, Kaitlin F; Krishnamachari, Sesha; Villanueva, Ernesto; Lou, Haiyan; Alerte, Tshianda N M; Peet, Eloise; Drolet, Robert E; Perez, Ruth G

    2014-02-01

    Aging, the main risk factor for Parkinson's disease (PD), is associated with increased α-synuclein levels in substantia nigra pars compacta (SNc). Excess α-synuclein spurs Lewy-like pathology and dysregulates the activity of protein phosphatase 2A (PP2A). PP2A dephosphorylates many neuroproteins, including the catecholamine rate-limiting enzyme, tyrosine hydroxylase (TH). A loss of nigral dopaminergic neurons induces PD movement problems, but before those abnormalities occur, behaviors such as olfactory loss, anxiety, and constipation often manifest. Identifying mouse models with early PD behavioral changes could provide a model in which to test emerging therapeutic compounds. To this end, we evaluated mice expressing A53T mutant human (A53T) α-synuclein for behavior and α-synuclein pathology in olfactory bulb, adrenal gland, and gut. Aging A53T mice exhibited olfactory loss and anxiety that paralleled olfactory and adrenal α-synuclein aggregation. PP2A activity was also diminished in olfactory and adrenal tissues harboring insoluble α-synuclein. Low adrenal PP2A activity co-occurred with TH hyperactivity, making this the first study to link adrenal synucleinopathy to anxiety and catecholamine dysregulation. Aggregated A53T α-synuclein recombinant protein also had impaired stimulatory effects on soluble recombinant PP2A. Collectively, the data identify an excellent model in which to screen compounds for their ability to block the spread of α-synuclein pathology associated with pre-motor stages of PD. © 2013 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of The International Society for Neurochemistry.

  13. The different faces of the p. A53T alpha-synuclein mutation: A screening of Greek patients with parkinsonism and/or dementia.

    PubMed

    Breza, Marianthi; Koutsis, Georgios; Karadima, Georgia; Potagas, Constantin; Kartanou, Chrisoula; Papageorgiou, Sokratis G; Paraskevas, George P; Kapaki, Elisabeth; Stefanis, Leonidas; Panas, Marios

    2018-04-13

    The p. A53T mutation in the alpha-synuclein (SNCA) gene is a rare cause of autosomal dominant Parkinson's disease (PD). Although generally rare, it is particularly common in the Greek population due to a founder effect. A53T-positive PD patients often develop dementia during disease course and may very rarely present with dementia. We screened for the p. A53T SNCA mutation a total of 347 cases of Greek origin with parkinsonism and/or dementia, collected over 15 years at the Neurogenetics Unit, Eginition Hospital, University of Athens. Cases were classified into: "pure parkinsonism", "pure dementia" and "parkinsonism plus dementia". In total, 4 p. A53T SNCA mutation carriers were identified. All had autosomal dominant family history and early onset. Screening of the "pure parkinsonism" category revealed 2 cases with typical PD. The other two mutation carriers were identified in the "parkinsonism plus dementia" category. One had a diagnosis of PD dementia and the other of behavioral variant frontotemporal dementia. Screening of patients with "pure dementia" failed to identify any further A53T-positive cases. Our results confirm that the p. A53T SNCA mutation is relatively common in Greek patients with PD or PD plus dementia, particularly in cases with early onset and/or autosomal dominant family history. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Alpha-synuclein levels in blood plasma decline with healthy aging.

    PubMed

    Koehler, Niklas K U; Stransky, Elke; Meyer, Mirjam; Gaertner, Susanne; Shing, Mona; Schnaidt, Martina; Celej, Maria S; Jovin, Thomas M; Leyhe, Thomas; Laske, Christoph; Batra, Anil; Buchkremer, Gerhard; Fallgatter, Andreas J; Wernet, Dorothee; Richartz-Salzburger, Elke

    2015-01-01

    There is unequivocal evidence that alpha-synuclein plays a pivotal pathophysiological role in neurodegenerative diseases, and in particular in synucleinopathies. These disorders present with a variable extent of cognitive impairment and alpha-synuclein is being explored as a biomarker in CSF, blood serum and plasma. Considering key events of aging that include proteostasis, alpha-synuclein may not only be useful as a marker for differential diagnosis but also for aging per se. To explore this hypothesis, we developed a highly specific ELISA to measure alpha-synuclein. In healthy males plasma alpha-synuclein levels correlated strongly with age, revealing much lower concentrations in older (avg. 58.1 years) compared to younger (avg. 27.6 years) individuals. This difference between the age groups was enhanced after acidification of the plasmas (p<0.0001), possibly reflecting a decrease of alpha-synuclein-antibody complexes or chaperone activity in older individuals. Our results support the concept that alpha-synuclein homeostasis may be impaired early on, possibly due to disturbance of the proteostasis network, a key component of healthy aging. Thus, alpha-synuclein may be a novel biomarker of aging, a factor that should be considered when analyzing its presence in biological specimens.

  15. Aggresomes formed by alpha-synuclein and synphilin-1 are cytoprotective.

    PubMed

    Tanaka, Mikiei; Kim, Yong Man; Lee, Gwang; Junn, Eunsung; Iwatsubo, Takeshi; Mouradian, M Maral

    2004-02-06

    Lewy bodies (LBs), which are the hallmark pathologic features of Parkinson's disease and of dementia with LBs, have several morphologic and molecular similarities to aggresomes. Whether such cytoplasmic inclusions contribute to neuronal death or protect cells from the toxic effects of misfolded proteins remains controversial. In this report, the role of aggresomes in cell viability was addressed in the context of over-expressing alpha-synuclein and its interacting partner synphilin-1 using engineered 293T cells. Inhibition of proteasome activity elicited the formation of juxtanuclear aggregates with characteristics of aggresomes including immunoreactivity for vimentin, gamma-tubulin, ubiquitin, proteasome subunit, and hsp70. As expected from the properties of aggresomes, the microtubule disrupting agents, vinblastin and nocodazole, markedly prevented the formation of these inclusions. Similar to LBs, the phosphorylated form of alpha-synuclein co-localized in these synphilin-1-containing aggresomes. Although the caspase inhibitor z-VAD-fmk significantly reduced the number of apoptotic cells, it had no impact on the percentage of aggresome-positive cells. Finally, quantitative analysis revealed aggresomes in 60% of nonapoptotic cells but only in 10% of apoptotic cells. Additionally, alpha-synuclein-induced apoptosis was not coupled with increased prevalence of aggresome-bearing cells. Taken together, these observations indicate a disconnection between aggresome formation and apoptosis, and support a protective role for these inclusions from the toxicity associated with the combined over-expression of alpha-synuclein and synphilin-1.

  16. Mutant Alpha-Synuclein Causes Age-Dependent Neuropathology in Monkey Brain

    PubMed Central

    Yang, Weili; Wang, Guohao; Wang, Chuan-En; Guo, Xiangyu; Yin, Peng; Gao, Jinquan; Tu, Zhuchi; Wang, Zhengbo; Wu, Jing; Hu, Xintian; Li, Shihua

    2015-01-01

    Parkinson's disease (PD) is an age-dependent neurodegenerative disease that often occurs in those over age 60. Although rodents and small animals have been used widely to model PD and investigate its pathology, their short life span makes it difficult to assess the aging-related pathology that is likely to occur in PD patient brains. Here, we used brain tissues from rhesus monkeys at 2–3, 7–8, and >15 years of age to examine the expression of Parkin, PINK1, and α-synuclein, which are known to cause PD via loss- or gain-of-function mechanisms. We found that α-synuclein is increased in the older monkey brains, whereas Parkin and PINK1 are decreased or remain unchanged. Because of the gain of toxicity of α-synuclein, we performed stereotaxic injection of lentiviral vectors expressing mutant α-synuclein (A53T) into the substantia nigra of monkeys and found that aging also increases the accumulation of A53T in neurites and its associated neuropathology. A53T also causes more extensive reactive astrocytes and axonal degeneration in monkey brain than in mouse brain. Using monkey brain tissues, we found that A53T interacts with neurofascin, an adhesion molecule involved in axon subcellular targeting and neurite outgrowth. Aged monkey brain tissues show an increased interaction of neurofascin with A53T. Overexpression of A53T causes neuritic toxicity in cultured neuronal cells, which can be attenuated by transfected neurofascin. These findings from nonhuman primate brains reveal age-dependent pathological and molecular changes that could contribute to the age-dependent neuropathology in PD. PMID:26019347

  17. Mutant alpha-synuclein causes age-dependent neuropathology in monkey brain.

    PubMed

    Yang, Weili; Wang, Guohao; Wang, Chuan-En; Guo, Xiangyu; Yin, Peng; Gao, Jinquan; Tu, Zhuchi; Wang, Zhengbo; Wu, Jing; Hu, Xintian; Li, Shihua; Li, Xiao-Jiang

    2015-05-27

    Parkinson's disease (PD) is an age-dependent neurodegenerative disease that often occurs in those over age 60. Although rodents and small animals have been used widely to model PD and investigate its pathology, their short life span makes it difficult to assess the aging-related pathology that is likely to occur in PD patient brains. Here, we used brain tissues from rhesus monkeys at 2-3, 7-8, and >15 years of age to examine the expression of Parkin, PINK1, and α-synuclein, which are known to cause PD via loss- or gain-of-function mechanisms. We found that α-synuclein is increased in the older monkey brains, whereas Parkin and PINK1 are decreased or remain unchanged. Because of the gain of toxicity of α-synuclein, we performed stereotaxic injection of lentiviral vectors expressing mutant α-synuclein (A53T) into the substantia nigra of monkeys and found that aging also increases the accumulation of A53T in neurites and its associated neuropathology. A53T also causes more extensive reactive astrocytes and axonal degeneration in monkey brain than in mouse brain. Using monkey brain tissues, we found that A53T interacts with neurofascin, an adhesion molecule involved in axon subcellular targeting and neurite outgrowth. Aged monkey brain tissues show an increased interaction of neurofascin with A53T. Overexpression of A53T causes neuritic toxicity in cultured neuronal cells, which can be attenuated by transfected neurofascin. These findings from nonhuman primate brains reveal age-dependent pathological and molecular changes that could contribute to the age-dependent neuropathology in PD. Copyright © 2015 the authors 0270-6474/15/358345-14$15.00/0.

  18. Wild-Type Monomeric α-Synuclein Can Impair Vesicle Endocytosis and Synaptic Fidelity via Tubulin Polymerization at the Calyx of Held.

    PubMed

    Eguchi, Kohgaku; Taoufiq, Zacharie; Thorn-Seshold, Oliver; Trauner, Dirk; Hasegawa, Masato; Takahashi, Tomoyuki

    2017-06-21

    α-Synuclein is a presynaptic protein the function of which has yet to be identified, but its neuronal content increases in patients of synucleinopathies including Parkinson's disease. Chronic overexpression of α-synuclein reportedly expresses various phenotypes of synaptic dysfunction, but the primary target of its toxicity has not been determined. To investigate this, we acutely loaded human recombinant α-synuclein or its pathological mutants in their monomeric forms into the calyces of Held presynaptic terminals in slices from auditorily mature and immature rats of either sex. Membrane capacitance measurements revealed significant and specific inhibitory effects of WT monomeric α-synuclein on vesicle endocytosis throughout development. However, the α-synuclein A53T mutant affected vesicle endocytosis only at immature calyces, whereas the A30P mutant had no effect throughout. The endocytic impairment by WT α-synuclein was rescued by intraterminal coloading of the microtubule (MT) polymerization blocker nocodazole. Furthermore, it was reversibly rescued by presynaptically loaded photostatin-1, a photoswitcheable inhibitor of MT polymerization, in a light-wavelength-dependent manner. In contrast, endocytic inhibition by the A53T mutant at immature calyces was not rescued by nocodazole. Functionally, presynaptically loaded WT α-synuclein had no effect on basal synaptic transmission evoked at a low frequency, but significantly attenuated exocytosis and impaired the fidelity of neurotransmission during prolonged high-frequency stimulation. We conclude that monomeric WT α-synuclein primarily inhibits vesicle endocytosis via MT overassembly, thereby impairing high-frequency neurotransmission. SIGNIFICANCE STATEMENT Abnormal α-synuclein abundance is associated with synucleinopathies including Parkinson's disease, but neither the primary target of α-synuclein toxicity nor its mechanism is identified. Here, we loaded monomeric α-synuclein directly into mammalian

  19. D409H GBA1 mutation accelerates the progression of pathology in A53T α-synuclein transgenic mouse model.

    PubMed

    Kim, Donghoon; Hwang, Heehong; Choi, Seulah; Kwon, Sang Ho; Lee, Suhyun; Park, Jae Hong; Kim, SangMin; Ko, Han Seok

    2018-04-27

    Heterozygous mutations in glucocerebrosidase 1 (GBA1) are a major genetic risk factor for Parkinson's disease and Dementia with Lewy bodies. Mutations in GBA1 leads to GBA1 enzyme deficiency, and GBA1-associated parkinsonism has an earlier age of onset and more progressive parkinsonism. To investigate a potential influence of GBA1 deficiency caused by mutations in GBA1 on the disease progression of PD, GBA1 mice carrying D409H knock-in mutation were crossbred with the human A53T (hA53T) α-synuclein transgenic mice. Here, we show that GBA1 enzyme activity plays a significant role in the hA53T α-synuclein induced α-synucleinopathy. The expression of D409H GBA1 markedly shortens the lifespan of hA53T α-synuclein transgenic mice. Moreover, D409H GBA1 expression exacerbates the formation of insoluble aggregates of α-synuclein, glial activation, neuronal degeneration, and motor abnormalities in the hA53T α-synuclein transgenic mice. Interestingly, the expression of D409H GBA1 results in the loss of dopaminergic neurons in the substantia nigra pars compacta of hA53T transgenic mice. Taken together, these results indicate that GBA1 deficiency due to D409H mutation affects the disease onset and course in hA53T α-synuclein transgenic mice. Therefore, strategies aimed to maintain GBA1 enzyme activity could be employed to develop an effective novel therapy for GBA1 linked-PD and related α-synucleinopathies.

  20. Severely impaired hippocampal neurogenesis associates with an early serotonergic deficit in a BAC α-synuclein transgenic rat model of Parkinson's disease

    PubMed Central

    Kohl, Zacharias; Abdallah, Nada Ben; Vogelgsang, Jonathan; Tischer, Lucas; Deusser, Janina; Amato, Davide; Anderson, Scott; Müller, Christian P.; Riess, Olaf; Masliah, Eliezer; Nuber, Silke; Winkler, Jürgen

    2016-01-01

    Parkinson's disease (PD) is a multisystem disorder, involving several monoaminergic neurotransmitter systems resulting in a broad range of motor and non-motor symptoms. Pathological hallmarks of PD are the loss of dopaminergic neurons and the accumulation of alpha-synuclein, however also being present in the serotonergic raphe nuclei early in the disease course. The dysfunction of the serotonergic system projecting to the hippocampus might contribute to early non-motor symptoms such as anxiety and depression. The adult hippocampal dentate gyrus (DG), a unique niche of the forebrain continuously generating new neurons, may particularly present enhanced susceptibility towards accumulating alpha-synuclein levels. The underlying molecular mechanisms in the context of neuronal maturation and survival of new-born neurons are yet not well understood. To characterize the effects of overexpression of human full-length alpha-synuclein on hippocampal cellular and synaptic plasticity, we used a recently generated BAC alpha-synuclein transgenic rat model showing important features of PD such as widespread and progressive alpha-synuclein aggregation pathology, dopamine loss and age-dependent motor decline. At the age of four months, thus prior to the occurrence of the motor phenotype, we observed a profoundly impaired dendritogenesis of neuroblasts in the hippocampal DG resulting in severely reduced survival of adult new-born neurons. Diminished neurogenesis concurred with a serotonergic deficit in the hippocampus as defined by reduced levels of serotonin (5-HT) 1B receptor, decreased 5-HT neurotransmitter levels, and a loss of serotonergic nerve terminals innervating the DG/CA3 subfield, while the number of serotonergic neurons in the raphe nuclei remained unchanged. Moreover, alpha-synuclein overexpression reduced proteins involved in vesicle release, in particular synapsin-1 and Rab3 interacting molecule (RIM3), in conjunction with an altered ultrastructural architecture of

  1. Single-molecule FRET studies on alpha-synuclein oligomerization of Parkinson’s disease genetically related mutants

    NASA Astrophysics Data System (ADS)

    Tosatto, Laura; Horrocks, Mathew H.; Dear, Alexander J.; Knowles, Tuomas P. J.; Dalla Serra, Mauro; Cremades, Nunilo; Dobson, Christopher M.; Klenerman, David

    2015-11-01

    Oligomers of alpha-synuclein are toxic to cells and have been proposed to play a key role in the etiopathogenesis of Parkinson’s disease. As certain missense mutations in the gene encoding for alpha-synuclein induce early-onset forms of the disease, it has been suggested that these variants might have an inherent tendency to produce high concentrations of oligomers during aggregation, although a direct experimental evidence for this is still missing. We used single-molecule Förster Resonance Energy Transfer to visualize directly the protein self-assembly process by wild-type alpha-synuclein and A53T, A30P and E46K mutants and to compare the structural properties of the ensemble of oligomers generated. We found that the kinetics of oligomer formation correlates with the natural tendency of each variant to acquire beta-sheet structure. Moreover, A53T and A30P showed significant differences in the averaged FRET efficiency of one of the two types of oligomers formed compared to the wild-type oligomers, indicating possible structural variety among the ensemble of species generated. Importantly, we found similar concentrations of oligomers during the lag-phase of the aggregation of wild-type and mutated alpha-synuclein, suggesting that the properties of the ensemble of oligomers generated during self-assembly might be more relevant than their absolute concentration for triggering neurodegeneration.

  2. Alpha-synuclein Toxicity in the Early Secretory Pathway: How It Drives Neurodegeneration in Parkinsons Disease

    PubMed Central

    Wang, Ting; Hay, Jesse C.

    2015-01-01

    Alpha-synuclein is a predominant player in the pathogenesis of Parkinson's Disease. However, despite extensive study for two decades, its physiological and pathological mechanisms remain poorly understood. Alpha-synuclein forms a perplexing web of interactions with lipids, trafficking machinery, and other regulatory factors. One emerging consensus is that synaptic vesicles are likely the functional site for alpha-synuclein, where it appears to facilitate vesicle docking and fusion. On the other hand, the dysfunctions of alpha-synuclein are more dispersed and numerous; when mutated or over-expressed, alpha-synuclein affects several membrane trafficking and stress pathways, including exocytosis, ER-to-Golgi transport, ER stress, Golgi homeostasis, endocytosis, autophagy, oxidative stress, and others. Here we examine recent developments in alpha-synuclein's toxicity in the early secretory pathway placed in the context of emerging themes from other affected pathways to help illuminate its underlying pathogenic mechanisms in neurodegeneration. PMID:26617485

  3. Autophagy and Alpha-Synuclein: Relevance to Parkinson's Disease and Related Synucleopathies.

    PubMed

    Xilouri, Maria; Brekk, Oeystein Roed; Stefanis, Leonidas

    2016-02-01

    Evidence from human postmortem material, transgenic mice, and cellular/animal models of PD link alpha-synuclein accumulation to alterations in the autophagy lysosomal pathway. Conversely, alpha-synuclein mutations related to PD pathogenesis, as well as post-translational modifications of the wild-type protein, result in the generation of aberrant species that may impair further the function of the autophagy lysosomal pathway, thus generating a vicious cycle leading to neuronal death. Moreover, PD-linked mutations in lysosomal-related genes, such as glucocerebrosidase, have been also shown to contribute to alpha-synuclein accumulation and related toxicity, indicating that lysosomal dysfunction may, in part, account for the neurodegeneration observed in synucleinopathies. In the current review, we summarize findings related to the inter-relationship between alpha-synuclein and lysosomal proteolytic pathways, focusing especially on recent experimental strategies based on the manipulation of the autophagy lysosomal pathway to counteract alpha-synuclein-mediated neurotoxicity in vivo. Pinpointing the factors that regulate alpha-synuclein association to the lysosome may represent potential targets for therapeutic interventions in PD and related synucleinopathies. © 2016 International Parkinson and Movement Disorder Society.

  4. A Novel Hsp90 Inhibitor Activates Compensatory Heat Shock Protein Responses and Autophagy and Alleviates Mutant A53T α-Synuclein Toxicity

    PubMed Central

    Xiong, Rui; Zhou, Wenbo; Siegel, David; Kitson, Russell R. A.; Freed, Curt R.; Moody, Christopher J.

    2015-01-01

    A potential cause of neurodegenerative diseases, including Parkinson’s disease (PD), is protein misfolding and aggregation that in turn leads to neurotoxicity. Targeting Hsp90 is an attractive strategy to halt neurodegenerative diseases, and benzoquinone ansamycin (BQA) Hsp90 inhibitors such as geldanamycin (GA) and 17-(allylamino)-17-demethoxygeldanamycin have been shown to be beneficial in mutant A53T α-synuclein PD models. However, current BQA inhibitors result in off-target toxicities via redox cycling and/or arylation of nucleophiles at the C19 position. We developed novel 19-substituted BQA (19BQA) as a means to prevent arylation. In this study, our data demonstrated that 19-phenyl-GA, a lead 19BQA in the GA series, was redox stable and exhibited little toxicity relative to its parent quinone GA in human dopaminergic SH-SY5Y cells as examined by oxygen consumption, trypan blue, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and apoptosis assays. Meanwhile, 19-phenyl-GA retained the ability to induce autophagy and potentially protective heat shock proteins (HSPs) such as Hsp70 and Hsp27. We found that transduction of A53T, but not wild type (WT) α-synuclein, induced toxicity in SH-SY5Y cells. 19-Phenyl-GA decreased oligomer formation and toxicity of A53T α-synuclein in transduced cells. Mechanistic studies indicated that mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase signaling was activated by A53T but not WT α-synuclein, and 19-phenyl-GA decreased mTOR activation that may be associated with A53T α-synuclein toxicity. In summary, our results indicate that 19BQAs such as 19-phenyl-GA may provide a means to modulate protein-handling systems including HSPs and autophagy, thereby reducing the aggregation and toxicity of proteins such as mutant A53T α-synuclein. PMID:26405178

  5. Phthalocyanine tetrasulfonates affect the amyloid formation and cytotoxicity of alpha-synuclein.

    PubMed

    Lee, Eui-Nam; Cho, Hyun-Ju; Lee, Choong-Hwan; Lee, Daekyun; Chung, Kwang Chul; Paik, Seung R

    2004-03-30

    Alpha-synuclein is a pathological component of Parkinson's disease by constituting the filamentous component of Lewy bodies. Phthalocyanine (Pc) effects on the amyloidosis of alpha-synuclein have been examined. The copper complex of phthalocyanine tetrasulfonate (PcTS-Cu(2+)) caused the self-oligomerization of alpha-synuclein while Pc-Cu(2+) did not affect the protein, indicating that introduction of the sulfonate groups was critical for the selective protein interaction. The PcTS-Cu(2+) interaction with alpha-synuclein has occurred predominantly at the N-terminal region of the protein with a K(d) of 0.83 microM apart from the hydrophobic NAC (non-Abeta component of Alzheimer's disease amyloid) segment. Phthalocyanine tetrasulfonate (PcTS) lacking the intercalated copper ion also showed a considerable affinity toward alpha-synuclein with a K(d) of 3.12 microM, and its binding site, on the other hand, was located at the acidic C-terminus. These mutually exclusive interactions between PcTS and PcTS-Cu(2+) toward alpha-synuclein resulted in distinctive features on the kinetics of protein aggregation, morphologies of the final aggregates, and their in vitro cytotoxicities. The PcTS actually suppressed the fibrous amyloid formation of alpha-synuclein, but it produced the chopped-wood-looking protein aggregates. The aggregates showed rather low toxicity (9.5%) on human neuroblastoma cells (SH-SY5Y). In fact, the PcTS was shown to effectively rescue the cell death of alpha-synuclein overexpressing cells caused by the lactacystin treatment as a proteasome inhibitor. The anti-aggregative and anti-amyloidogenic properties of PcTS were also demonstrated with alcohol dehydrogenase, glutathione S-transferase, and amyloid beta/A4 protein under their aggregative conditions. The PcTS-Cu(2+), on the other hand, promoted the protein aggregation of alpha-synuclein, which gave rise to the fibrillar protein aggregates whose cytotoxicity became significant to 35.8%. Taken together, the

  6. Lrrk2 and alpha-synuclein are co-regulated in rodent striatum.

    PubMed

    Westerlund, Marie; Ran, Caroline; Borgkvist, Anders; Sterky, Fredrik H; Lindqvist, Eva; Lundströmer, Karin; Pernold, Karin; Brené, Stefan; Kallunki, Pekka; Fisone, Gilberto; Olson, Lars; Galter, Dagmar

    2008-12-01

    LRRK2, alpha-synuclein, UCH-L1 and DJ-1 are implicated in the etiology of Parkinson's disease. We show for the first time that increase in striatal alpha-synuclein levels induce increased Lrrk2 mRNA levels while Dj-1 and Uch-L1 are unchanged. We also demonstrate that a mouse strain lacking the dopamine signaling molecule DARPP-32 has significantly reduced levels of both Lrrk2 and alpha-synuclein, while mice carrying a disabling mutation of the DARPP-32 phosphorylation site T34A or lack alpha-synuclein do not show any changes. To test if striatal dopamine depletion influences Lrrk2 or alpha-synuclein expression, we used the neurotoxin 6-hydroxydopamine in rats and MitoPark mice in which there is progressive degeneration of dopamine neurons. Because striatal Lrrk2 and alpha-synuclein levels were not changed by dopamine depletion, we conclude that Lrrk2 and alpha-synuclein mRNA levels are possibly co-regulated, but they are not influenced by striatal dopamine levels.

  7. Drift diffusion model of reward and punishment learning in rare alpha-synuclein gene carriers.

    PubMed

    Moustafa, Ahmed A; Kéri, Szabolcs; Polner, Bertalan; White, Corey

    To understand the cognitive effects of alpha-synuclein polymorphism, we employed a drift diffusion model (DDM) to analyze reward- and punishment-guided probabilistic learning task data of participants with the rare alpha-synuclein gene duplication and age- and education-matched controls. Overall, the DDM analysis showed that, relative to controls, asymptomatic alpha-synuclein gene duplication carriers had significantly increased learning from negative feedback, while they tended to show impaired learning from positive feedback. No significant differences were found in response caution, response bias, or motor/encoding time. We here discuss the implications of these computational findings to the understanding of the neural mechanism of alpha-synuclein gene duplication.

  8. DNA damage preceding dopamine neuron degeneration in A53T human α-synuclein transgenic mice.

    PubMed

    Wang, Degui; Yu, Tianyu; Liu, Yongqiang; Yan, Jun; Guo, Yingli; Jing, Yuhong; Yang, Xuguang; Song, Yanfeng; Tian, Yingxia

    2016-12-02

    Defective DNA repair has been linked with age-associated neurodegenerative disorders. Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by genetic and environmental factors. Whether damages to nuclear DNA contribute to neurodegeneration of PD still remain obscure. in this study we aim to explore whether nuclear DNA damage induce dopamine neuron degeneration in A53T human α-Synuclein over expressed mouse model. We investigated the effects of X-ray irradiation on A53T-α-Syn MEFs and A53T-α-Syn transgene mice. Our results indicate that A53T-α-Syn MEFs show a prolonged DNA damage repair process and senescense phenotype. DNA damage preceded onset of motor phenotype in A53T-α-Syn transgenic mice and decrease the number of nigrostriatal dopaminergic neurons. Neurons of A53T-α-Syn transgenic mice are more fragile to DNA damages. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Asparagine endopeptidase cleaves α-synuclein and mediates pathologic activities in Parkinson's disease.

    PubMed

    Zhang, Zhentao; Kang, Seong Su; Liu, Xia; Ahn, Eun Hee; Zhang, Zhaohui; He, Li; Iuvone, P Michael; Duong, Duc M; Seyfried, Nicholas T; Benskey, Matthew J; Manfredsson, Fredric P; Jin, Lingjing; Sun, Yi E; Wang, Jian-Zhi; Ye, Keqiang

    2017-08-01

    Aggregated forms of α-synuclein play a crucial role in the pathogenesis of synucleinopathies such as Parkinson's disease (PD). However, the molecular mechanisms underlying the pathogenic effects of α-synuclein are not completely understood. Here we show that asparagine endopeptidase (AEP) cleaves human α-synuclein, triggers its aggregation and escalates its neurotoxicity, thus leading to dopaminergic neuronal loss and motor impairments in a mouse model. AEP is activated and cleaves human α-synuclein at N103 in an age-dependent manner. AEP is highly activated in human brains with PD, and it fragments α-synuclein, which is found aggregated in Lewy bodies. Overexpression of the AEP-cleaved α-synuclein 1-103 fragment in the substantia nigra induces both dopaminergic neuronal loss and movement defects in mice. In contrast, inhibition of AEP-mediated cleavage of α-synuclein (wild type and A53T mutant) diminishes α-synuclein's pathologic effects. Together, these findings support AEP's role as a key mediator of α-synuclein-related etiopathological effects in PD.

  10. Generation of gene-corrected iPSC line from Parkinson's disease patient iPSC line with alpha-SNCA A53T mutation.

    PubMed

    Lee, Seo-Young; Jeong, SangKyun; Kim, Janghwan; Chung, Sun-Ku

    2018-06-09

    Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder. PD can result from a mutation of alpha-synuclein (α-SNCA), such as α-SNCA A53T. Using episomal vectors, induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts with the α-SNCA A53T mutation. A huge bacterial artificial chromosome (BAC) harboring the normal α-SNCA gene successfully corrected the α-SNCA A53T-mutant iPSCs. Melting curve analysis for allelic composition indicated that the BAC DNA was precisely targeted to the α-SNCA A53T mutation allele, without random integration. The corrected PD-iPSCs displayed the normal karyotype and pluripotency, with the capability to differentiate to any cell type. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

  11. Deletion of alpha-synuclein decreases impulsivity in mice.

    PubMed

    Peña-Oliver, Y; Buchman, V L; Dalley, J W; Robbins, T W; Schumann, G; Ripley, T L; King, S L; Stephens, D N

    2012-03-01

    The presynaptic protein alpha-synuclein, associated with Parkinson's Disease (PD), plays a role in dopaminergic neurotransmission and is implicated in impulse control disorders (ICDs) such as drug addiction. In this study we investigated a potential causal relationship between alpha-synuclein and impulsivity, by evaluating differences in motor impulsivity in the 5-choice serial reaction time task (5-CSRTT) in strains of mice that differ in the expression of the alpha-synuclein gene. C57BL/6JOlaHsd mice differ from their C57BL/6J ancestors in possessing a chromosomal deletion resulting in the loss of two genes, snca, encoding alpha-synuclein, and mmrn1, encoding multimerin-1. C57BL/6J mice displayed higher impulsivity (more premature responding) than C57BL/6JOlaHsd mice when the pre-stimulus waiting interval was increased in the 5-CSRTT. In order to ensure that the reduced impulsivity was indeed related to snca, and not adjacent gene deletion, wild type (WT) and mice with targeted deletion of alpha-synuclein (KO) were tested in the 5-CSRTT. Similarly, WT mice were more impulsive than mice with targeted deletion of alpha-synuclein. Interrogation of our ongoing analysis of impulsivity in BXD recombinant inbred mouse lines revealed an association of impulsive responding with levels of alpha-synuclein expression in hippocampus. Expression of beta- and gamma-synuclein, members of the synuclein family that may substitute for alpha-synuclein following its deletion, revealed no differential compensations among the mouse strains. These findings suggest that alpha-synuclein may contribute to impulsivity and potentially, to ICDs which arise in some PD patients treated with dopaminergic medication. © 2011 The Authors. Genes, Brain and Behavior © 2011 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.

  12. DNA damage preceding dopamine neuron degeneration in A53T human α-synuclein transgenic mice

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Degui; Yu, Tianyu; Liu, Yongqiang

    Defective DNA repair has been linked with age-associated neurodegenerative disorders. Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by genetic and environmental factors. Whether damages to nuclear DNA contribute to neurodegeneration of PD still remain obscure. in this study we aim to explore whether nuclear DNA damage induce dopamine neuron degeneration in A53T human α-Synuclein over expressed mouse model. We investigated the effects of X-ray irradiation on A53T-α-Syn MEFs and A53T-α-Syn transgene mice. Our results indicate that A53T-α-Syn MEFs show a prolonged DNA damage repair process and senescense phenotype. DNA damage preceded onset of motor phenotype in A53T-α-Syn transgenicmore » mice and decrease the number of nigrostriatal dopaminergic neurons. Neurons of A53T-α-Syn transgenic mice are more fragile to DNA damages. - Highlights: • This study explore contribution of DNA damage to neurodegeneration in Parkinson's disease mice. • A53T-α-Syn MEF cells show a prolonged DNA damage repair process and senescense phenotype. • DNA damage preceded onset of motor phenotype in A53T-α-Syn transgenic mice. • DNA damage decrease the number of nigrostriatal dopaminergic neurons. • Neurons of A53T-α-Syn transgenic mice are more fragile to DNA damages.« less

  13. 5-HT2A Receptor Binding in the Frontal Cortex of Parkinson's Disease Patients and Alpha-Synuclein Overexpressing Mice: A Postmortem Study

    PubMed Central

    Rasmussen, Nadja Bredo; Olesen, Mikkel Vestergaard; Plenge, Per; Klein, Anders Bue; Westin, Jenny E.; Fog, Karina

    2016-01-01

    The 5-HT2A receptor is highly involved in aspects of cognition and executive function and seen to be affected in neurodegenerative diseases like Alzheimer's disease and related to the disease pathology. Even though Parkinson's disease (PD) is primarily a motor disorder, reports of impaired executive function are also steadily being associated with this disease. Not much is known about the pathophysiology behind this. The aim of this study was thereby twofold: (1) to investigate 5-HT2A receptor binding levels in Parkinson's brains and (2) to investigate whether PD associated pathology, alpha-synuclein (AS) overexpression, could be associated with 5-HT2A alterations. Binding density for the 5-HT2A-specific radioligand [3H]-MDL 100.907 was measured in membrane suspensions of frontal cortex tissue from PD patients. Protein levels of AS were further measured using western blotting. Results showed higher AS levels accompanied by increased 5-HT2A receptor binding in PD brains. In a separate study, we looked for changes in 5-HT2A receptors in the prefrontal cortex in 52-week-old transgenic mice overexpressing human AS. We performed region-specific 5-HT2A receptor binding measurements followed by gene expression analysis. The transgenic mice showed lower 5-HT2A binding in the frontal association cortex that was not accompanied by changes in gene expression levels. This study is one of the first to look at differences in serotonin receptor levels in PD and in relation to AS overexpression. PMID:27579212

  14. α-Synuclein transgenic mice reveal compensatory increases in Parkinson's disease-associated proteins DJ-1 and parkin and have enhanced α-synuclein and PINK1 levels after rotenone treatment.

    PubMed

    George, Sonia; Mok, Su San; Nurjono, Milawaty; Ayton, Scott; Finkelstein, David I; Masters, Colin L; Li, Qiao-Xin; Culvenor, Janetta G

    2010-10-01

    Parkinson's disease (PD) is a severe neurodegenerative disorder characterised by loss of dopaminergic neurons of the substantia nigra. The pathological hallmarks are cytoplasmic inclusions termed Lewy bodies consisting primarily of aggregated alpha-synuclein (alphaSN). Different lines of transgenic mice have been developed to model PD but have failed to recapitulate the hallmarks of this disease. Since treatment of rodents with the pesticide rotenone can reproduce nigrostriatal cell loss and other features of PD, we aimed to test chronic oral administration of rotenone to transgenic mice over-expressing human alphaSN with the A53T mutation. Initial assessment of this transgenic line for compensatory molecular changes indicated decreased brain beta-synuclein expression and significantly increased levels of the PD-associated oxidative stress response protein, DJ-1, and the E3 ubiquitin ligase enzyme, Parkin. Rotenone treatment of 30 mg/kg for 25 doses over a 35-day period was tolerated in the transgenic mice and resulted in decreased spontaneous locomotor movement and increased cytoplasmic alphaSN expression. The mitochondrial Parkinson's-associated PTEN-induced kinase 1 protein levels were also increased in transgenic mouse brain after rotenone treatment; there was no change in brain dopamine levels or nigrostriatal cell loss. These hA53T alphaSN transgenic mice provide a useful model for presymptomatic Parkinson's features and are valuable for study of associated compensatory changes in early Parkinson's disease stages.

  15. The heterozygous A53T mutation in the alpha-synuclein gene in a Chinese Han patient with Parkinson disease: case report and literature review.

    PubMed

    Xiong, Wei-Xi; Sun, Yi-Min; Guan, Rong-Yuan; Luo, Su-Shan; Chen, Chen; An, Yu; Wang, Jian; Wu, Jian-Jun

    2016-10-01

    The missense mutation A53T of alpha-synuclein gene (SNCA) was reported to be a rare but definite cause of sporadic and familial Parkinson disease (PD). It seemed to be restricted geographically in Greece and Italy. We aimed to identify the SNCA mutations in a Chinese PD cohort. Ninety-one early onset PD patients or familial PD probands were collected consecutively for the screening of PD-related genes. The genetic analysis was carried out by target sequencing of the exons and the corresponding flanking regions of the PD-related genes using Illumina HiSeq 2000 sequencer and further confirmed by Sanger sequencing or restriction fragment length polymorphism. Dosage mutations of exons in these genes were carried out by multiple ligation-dependent probe amplification. Among the 91 patients, we found only one heterozygous mutation of SNCA A53T, in a 23-year-old male patient with negative family history. The [(11)C]-2β-carbomethoxy-3β-(4-fluorophenyl) tropan (CFT) PET and PD-related spatial covariance pattern (PDRP) via [(18)F]-fluorodeoxyglucos (FDG) PET confirmed a typical pattern of PD. After examining his parents, we found his mother was an asymptomatic carrier, with declined hand dexterity detected by quantitative motor tests. Reduced dopamine transporter uptake of his mother was identified by CFT PET, and abnormal PDRP pattern was found by FDG PET. Our investigation expanded the clinical and genetic spectrum of Chinese PD patients, and we suggested SNCA mutations to be screened in familial and early onset Chinese PD patients.

  16. Alpha-synuclein aggregation induced by brief ischemia negatively impacts neuronal survival in vivo: a study in [A30P]alpha-synuclein transgenic mouse

    PubMed Central

    Unal-Cevik, Isin; Gursoy-Ozdemir, Yasemin; Yemisci, Muge; Lule, Sevda; Gurer, Gunfer; Can, Alp; Müller, Veronica; Kahle, Philip J; Dalkara, Turgay

    2011-01-01

    Alpha-synuclein oligomerization and aggregation are considered to have a role in the pathogenesis of neurodegenerative diseases. However, despite numerous in vitro studies, the impact of aggregates in the intact brain is unclear. In vitro, oxidative/nitrative stress and acidity induce α-synuclein oligomerization. These conditions favoring α-synuclein fibrillization are present in the ischemic brain, which may serve as an in vivo model to study α-synuclein aggregation. In this study, we show that 30-minute proximal middle cerebral artery (MCA) occlusion and 72 hours reperfusion induce oligomerization of wild-type α-synuclein in the ischemic mouse brain. The nonamyloidogenic isoform β-synuclein did not form oligomers. Alpha-synuclein aggregates were confined to neurons and colocalized with ubiquitin immunoreactivity. We also found that 30 minutes proximal MCA occlusion and 24 hours reperfusion induced larger infarcts in C57BL/6(Thy1)-h[A30P]alphaSYN transgenic mice, which have an increased tendency to form synuclein fibrils. Trangenics also developed more selective neuronal necrosis when subjected to 20 minutes distal MCA occlusion and 72 hours reperfusion. Enhanced 3-nitrotyrosine immunoreactivity in transgenic mice suggests that oxidative/nitrative stress may be one of the mechanisms mediating aggregate toxicity. Thus, the increased vulnerability of transgenic mice to ischemia suggests that α-synuclein aggregates not only form during ischemia but also negatively impact neuronal survival, supporting the idea that α-synuclein misfolding may be neurotoxic. PMID:20877387

  17. Drp-1 dependent mitochondrial fragmentation and protective autophagy in dopaminergic SH-SY5Y cells overexpressing alpha-synuclein.

    PubMed

    Martinez, Jimena Hebe; Alaimo, Agustina; Gorojod, Roxana Mayra; Porte Alcon, Soledad; Fuentes, Federico; Coluccio Leskow, Federico; Kotler, Mónica Lidia

    2018-04-01

    Parkinson's disease is a neurodegenerative movement disorder caused by the loss of dopaminergic neurons from substantia nigra. It is characterized by the accumulation of aggregated α-synuclein as the major component of the Lewy bodies. Additional common features of this disease are the mitochondrial dysfunction and the activation/inhibition of autophagy both events associated to the intracellular accumulation of α-synuclein. The mechanism by which these events contribute to neural degeneration remains unknown. In the present work we investigated the effect of α-synuclein on mitochondrial dynamics and autophagy/mitophagy in SH-SY5Y cells, an in vitro model of Parkinson disease. We demonstrated that overexpression of wild type α-synuclein causes moderated toxicity, ROS generation and mitochondrial dysfunction. In addition, α-synuclein induces the mitochondrial fragmentation on a Drp-1-dependent fashion. Overexpression of the fusion protein Opa-1 prevented both mitochondrial fragmentation and cytotoxicity. On the other hand, cells expressing α-synuclein showed activated autophagy and particularly mitophagy. Employing a genetic strategy we demonstrated that autophagy is triggered in order to protect cells from α-synuclein-induced cell death. Our results clarify the role of Opa-1 and Drp-1 in mitochondrial dynamics and cell survival, a controversial α-synuclein research issue. The findings presented point to the relevance of mitochondrial homeostasis and autophagy in the pathogenesis of PD. Better understanding of the molecular interaction between these processes could give rise to novel therapeutic methods for PD prevention and amelioration. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Curcumin inhibits aggregation of alpha-synuclein.

    PubMed

    Pandey, Neeraj; Strider, Jeffrey; Nolan, William C; Yan, Sherry X; Galvin, James E

    2008-04-01

    Aggregation of amyloid-beta protein (Abeta) is a key pathogenic event in Alzheimer's disease (AD). Curcumin, a constituent of the Indian spice Turmeric is structurally similar to Congo Red and has been demonstrated to bind Abeta amyloid and prevent further oligomerization of Abeta monomers onto growing amyloid beta-sheets. Reasoning that oligomerization kinetics and mechanism of amyloid formation are similar in Parkinson's disease (PD) and AD, we investigated the effect of curcumin on alpha-synuclein (AS) protein aggregation. In vitro model of AS aggregation was developed by treatment of purified AS protein (wild-type) with 1 mM Fe3+ (Fenton reaction). It was observed that the addition of curcumin inhibited aggregation in a dose-dependent manner and increased AS solubility. The aggregation-inhibiting effect of curcumin was next investigated in cell culture utilizing catecholaminergic SH-SY5Y cell line. A model system was developed in which the red fluorescent protein (DsRed2) was fused with A53T mutant of AS and its aggregation examined under different concentrations of curcumin. To estimate aggregation in an unbiased manner, a protocol was developed in which the images were captured automatically through a high-throughput cell-based screening microscope. The obtained images were processed automatically for aggregates within a defined dimension of 1-6 microm. Greater than 32% decrease in mutant alpha-synuclein aggregation was observed within 48 h subsequent to curcumin addition. Our data suggest that curcumin inhibits AS oligomerization into higher molecular weight aggregates and therefore should be further explored as a potential therapeutic compound for PD and related disorders.

  19. Triptolide Promotes the Clearance of α-Synuclein by Enhancing Autophagy in Neuronal Cells.

    PubMed

    Hu, Guanzheng; Gong, Xiaoli; Wang, Le; Liu, Mengru; Liu, Yang; Fu, Xia; Wang, Wei; Zhang, Ting; Wang, Xiaomin

    2017-04-01

    Parkinson's disease (PD) is an aging-associated neurodegenerative disease with a characteristic feature of α-synuclein accumulation. Point mutations (A53T, A30P) that increase the aggregation propensity of α-synuclein result in familial early onset PD. The abnormal metabolism of α-synuclein results in aberrant level changes of α-synuclein in PD. In pathological conditions, α-synuclein is degraded mainly by the autophagy-lysosome pathway. Triptolide (T10) is a monomeric compound isolated from a traditional Chinese herb. Our group demonstrated for the first time that T10 possesses potent neuroprotective properties both in vitro and in vivo PD models. In the present study, we reported T10 as a potent autophagy inducer in neuronal cells, which helped to promote the clearance of various forms of α-synuclein in neuronal cells. We transfected neuronal cells with A53T mutant (A53T) or wild-type (WT) α-synuclein plasmids and found T10 attenuated the cytotoxicity induced by pathogenic A53T α-synuclein overexpression. We observed that T10 significantly reduced both A53T and WT α-synuclein level in neuronal cell line, as well as in primary cultured cortical neurons. Excluding the changes of syntheses, secretion, and aggregation of α-synuclein, we further added autophagy inhibitor or proteasome inhibitor with T10, and we noticed that T10 promoted the clearance of α-synuclein mainly by the autophagic pathway. Lastly, we observed increased autophagy marker LC3-II expression and autophagosomes by GFP-LC3-II accumulation and ultrastructural characterization. However, the lysosome activity and cell viability were not modulated by T10. Our study revealed that T10 could induce autophagy and promote the clearance of both WT and A53T α-synuclein in neurons. These results provide evidence of T10 as a promising mean to treat PD and other neurodegenerative diseases by reducing pathogenic proteins in neurons.

  20. Preserved functional autonomic phenotype in adult mice overexpressing moderate levels of human alpha-synuclein in oligodendrocytes.

    PubMed

    Tank, Jens; da Costa-Goncalves, Andrey C; Kamer, Ilona; Qadri, Fatimunnisa; Ubhi, Kiren; Rockenstein, Edward; Diedrich, André; Masliah, Eliezer; Gross, Volkmar; Jordan, Jens

    2014-11-01

    Mice overexpressing human alpha-synuclein in oligodendrocytes (MBP1-α-syn) recapitulate some key functional and neuropathological features of multiple system atrophy (MSA). Whether or not these mice develop severe autonomic failure, which is a key feature of human MSA, remains unknown. We explored cardiovascular autonomic regulation using long-term blood pressure (BP) radiotelemetry and pharmacological testing. We instrumented 12 MBP1-α-syn mice and 11 wild-type mice aged 9 months for radiotelemetry. Animals were tested with atropine, metoprolol, clonidine, and trimethaphan at 9 and 12 months age. We applied spectral and cross-spectral analysis to assess heart rate (HR) and BP variability. At 9 months of age daytime BP (transgenic: 101 ± 2 vs. wild type: 99 ± 2 mmHg) and HR (497 ± 11 vs. 505 ± 16 beats/min) were similar. Circadian BP and HR rhythms were maintained. Nighttime BP (109 ± 2 vs. 108 ± 2 mmHg) and HR (575 ± 15 vs. 569 ± 14 beats/min), mean arterial BP responses to trimethaphan (-21 ± 8 vs. -10 ± 5 mmHg, P = 0.240) and to clonidine (-8 ± 3 vs. -5 ± 2 mmHg, P = 0.314) were similar. HR responses to atropine (+159 ± 24 vs. +146 ± 22 beats/min), and to clonidine (-188 ± 21 vs. -163 ± 33 beats/min) did not differ between strains. Baroreflex sensitivity (4 ± 1 vs. 4 ± 1 msec/mmHg) and HR variability (total power, 84 ± 17 vs. 65 ± 21 msec²) were similar under resting conditions and during pharmacological testing. Repeated measurements at 12 months of age provided similar results. In mice, moderate overexpression of human alpha-synuclein in oligodendrocytes is not sufficient to induce overt autonomic failure. Additional mechanisms may be required to express the autonomic failure phenotype including higher levels of expression or more advanced age. © 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  1. Genetic association between Alzheimer disease and the alpha-synuclein gene.

    PubMed

    Matsubara, M; Yamagata, H; Kamino, K; Nomura, T; Kohara, K; Kondo, I; Miki, T

    2001-01-01

    alpha-Synuclein has been isolated as a component of amyloid in addition to the major A beta peptide in Alzheimer disease (AD). However, there are conflicting reports regarding the association of alpha-synuclein gene polymorphism with AD. Using a novel and common polymorphism in intron 3, we examined the relationship between AD and alpha-synuclein and apolipoprotein E (ApoE) genes in 183 Japanese AD patients and 210 controls. Carriers of the alpha-synuclein deletion (D) allele had a 2.2-fold increased risk of developing AD than noncarriers in women. The odds ratio for the ApoE epsilon 4 and the alpha-synuclein D allele was 11.4 in women. The results showed that the alpha-synuclein gene is associated with sporadic AD in women, independent of ApoE epsilon 4 status. Copyright 2001 S. Karger AG, Basel

  2. A Focus on the Beneficial Effects of Alpha Synuclein and a Re-Appraisal of Synucleinopathies

    PubMed Central

    Ryskalin, Larisa; Busceti, Carla L.; Limanaqi, Fiona; Biagioni, Francesca; Gambardella, Stefano; Fornai, Francesco

    2018-01-01

    Alpha synuclein (α-syn) belongs to a class of proteins which are commonly considered to play a detrimental role in neuronal survival. This assumption is based on the occurrence of a severe neuronal degeneration in patients carrying a multiplication of the α-syn gene (SNCA) and in a variety of experi-mental models, where overexpression of α-syn leads to cell death and neurological impairment. In these conditions, a higher amount of normally structured α-syn produces a damage, which is even worse com-pared with that produced by α-syn owning an abnormal structure (as occurring following point gene muta-tions). In line with this, knocking out the expression of α-syn is reported to protect from specific neurotox-ins such as 1-methyl, 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the present review we briefly dis-cuss these well-known detrimental effects but we focus on findings showing that, in specific conditions α-syn is beneficial for cell survival. This occurs during methamphetamine intoxication which is counteracted by endogenous α-syn. Similarly, the dysfunction of the chaperone cysteine-string protein-alpha leads to cell pathology which is counteracted by over-expressing α-syn. In line with this, an increased expression of α-syn protects against oxidative damage produced by dopamine. Remarkably, when the lack of α-syn is combined with a depletion of β- and γ- synucleins, alterations in brain structure and function occur. This review tries to balance the evidence showing a beneficial effect with the bulk of data reporting a detri-mental effect of endogenous α-syn. The specific role of α-syn as a chaperone protein is discussed to ex-plain such a dual effect. PMID:29150919

  3. A Focus on the Beneficial Effects of Alpha Synuclein and a Re-Appraisal of Synucleinopathies.

    PubMed

    Ryskalin, Larisa; Busceti, Carla L; Limanaqi, Fiona; Biagioni, Francesca; Gambardella, Stefano; Fornai, Francesco

    2018-01-01

    Alpha synuclein (α-syn) belongs to a class of proteins which are commonly considered to play a detrimental role in neuronal survival. This assumption is based on the occurrence of a severe neuronal degeneration in patients carrying a multiplication of the α-syn gene (SNCA) and in a variety of experimental models, where overexpression of α-syn leads to cell death and neurological impairment. In these conditions, a higher amount of normally structured α-syn produces a damage, which is even worse compared with that produced by α-syn owning an abnormal structure (as occurring following point gene mutations). In line with this, knocking out the expression of α-syn is reported to protect from specific neurotoxins such as 1-methyl, 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the present review we briefly discuss these well-known detrimental effects but we focus on findings showing that, in specific conditions α-syn is beneficial for cell survival. This occurs during methamphetamine intoxication which is counteracted by endogenous α-syn. Similarly, the dysfunction of the chaperone cysteine-string protein- alpha leads to cell pathology which is counteracted by over-expressing α-syn. In line with this, an increased expression of α-syn protects against oxidative damage produced by dopamine. Remarkably, when the lack of α-syn is combined with a depletion of β- and γ- synucleins, alterations in brain structure and function occur. This review tries to balance the evidence showing a beneficial effect with the bulk of data reporting a detrimental effect of endogenous α-syn. The specific role of α-syn as a chaperone protein is discussed to explain such a dual effect. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  4. Structures and Free Energy Landscapes of the A53T Mutant-Type α-Synuclein Protein and Impact of A53T Mutation on the Structures of the Wild-Type α-Synuclein Protein with Dynamics

    PubMed Central

    2013-01-01

    The A53T genetic missense mutation of the wild-type α-synuclein (αS) protein was initially identified in Greek and Italian families with familial Parkinson’s disease. Detailed understanding of the structures and the changes induced in the wild-type αS structure by the A53T mutation, as well as establishing the direct relationships between the rapid conformational changes and free energy landscapes of these intrinsically disordered fibrillogenic proteins, helps to enhance our fundamental knowledge and to gain insights into the pathogenic mechanism of Parkinson’s disease. We employed extensive parallel tempering molecular dynamics simulations along with thermodynamic calculations to determine the secondary and tertiary structural properties as well as the conformational free energy surfaces of the wild-type and A53T mutant-type αS proteins in an aqueous solution medium using both implicit and explicit water models. The confined aqueous volume effect in the simulations of disordered proteins using an explicit model for water is addressed for a model disordered protein. We also assessed the stabilities of the residual secondary structure component interconversions in αS based on free energy calculations at the atomic level with dynamics using our recently developed theoretical strategy. To the best of our knowledge, this study presents the first detailed comparison of the structural properties linked directly to the conformational free energy landscapes of the monomeric wild-type and A53T mutant-type α-synuclein proteins in an aqueous solution environment. Results demonstrate that the β-sheet structure is significantly more altered than the helical structure upon A53T mutation of the monomeric wild-type αS protein in aqueous solution. The β-sheet content close to the mutation site in the N-terminal region is more abundant while the non-amyloid-β component (NAC) and C-terminal regions show a decrease in β-sheet abundance upon A53T mutation. Obtained

  5. Structures and free energy landscapes of the A53T mutant-type α-synuclein protein and impact of A53T mutation on the structures of the wild-type α-synuclein protein with dynamics.

    PubMed

    Coskuner, Orkid; Wise-Scira, Olivia

    2013-07-17

    The A53T genetic missense mutation of the wild-type α-synuclein (αS) protein was initially identified in Greek and Italian families with familial Parkinson's disease. Detailed understanding of the structures and the changes induced in the wild-type αS structure by the A53T mutation, as well as establishing the direct relationships between the rapid conformational changes and free energy landscapes of these intrinsically disordered fibrillogenic proteins, helps to enhance our fundamental knowledge and to gain insights into the pathogenic mechanism of Parkinson's disease. We employed extensive parallel tempering molecular dynamics simulations along with thermodynamic calculations to determine the secondary and tertiary structural properties as well as the conformational free energy surfaces of the wild-type and A53T mutant-type αS proteins in an aqueous solution medium using both implicit and explicit water models. The confined aqueous volume effect in the simulations of disordered proteins using an explicit model for water is addressed for a model disordered protein. We also assessed the stabilities of the residual secondary structure component interconversions in αS based on free energy calculations at the atomic level with dynamics using our recently developed theoretical strategy. To the best of our knowledge, this study presents the first detailed comparison of the structural properties linked directly to the conformational free energy landscapes of the monomeric wild-type and A53T mutant-type α-synuclein proteins in an aqueous solution environment. Results demonstrate that the β-sheet structure is significantly more altered than the helical structure upon A53T mutation of the monomeric wild-type αS protein in aqueous solution. The β-sheet content close to the mutation site in the N-terminal region is more abundant while the non-amyloid-β component (NAC) and C-terminal regions show a decrease in β-sheet abundance upon A53T mutation. Obtained results

  6. Characterization of antibodies that selectively detect alpha-synuclein in pathological inclusions.

    PubMed

    Waxman, Elisa A; Duda, John E; Giasson, Benoit I

    2008-07-01

    Sensitive detection of alpha-synuclein (alpha-syn) pathology is important in the diagnosis of disorders like Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy and in providing better insights into the etiology of these diseases. Several monoclonal antibodies that selectively react with aggregated alpha-syn in pathological inclusions and reveal extensive and underappreciated alpha-syn pathology in the brains of diseased patients were previously reported by Duda et al. (Ann Neurol 52:205-210, 2002). We sought to characterize the specificity of some of these antibodies (Syn 505, Syn 506 and Syn 514); using C-terminal and N-terminal truncations of alpha-syn, all three antibodies were determined to require N-terminal epitopes that minimally comprise amino acids 2-4, but possibly extend to amino acid 12 of alpha-syn. The selectivity of these antibodies was further assessed using biochemical analysis of human brains and reactivity to altered recombinant alpha-syn proteins with duplication variants of amino acids 1-12. In addition, by expressing wild-type or a double mutant (E46K/A53T) of alpha-syn in cultured cells and by comparing their immunoreactivities to another antibody (SNL-4), which has a similar primary epitope, it was determined that Syn 505, Syn 506 and Syn 514 recognize conformational variants of alpha-syn that is enhanced by the presence of the double mutations. These studies indicate that antibodies Syn 505, Syn 506 and Syn 514 preferentially recognize N-terminal epitopes in complex conformations, consistent with the dramatic conformational change associated with the polymerization of alpha-synuclein into amyloid fibrils that form pathological inclusions.

  7. Characterization of cognitive deficits in rats overexpressing human alpha-synuclein in the ventral tegmental area and medial septum using recombinant adeno-associated viral vectors.

    PubMed

    Hall, Hélène; Jewett, Michael; Landeck, Natalie; Nilsson, Nathalie; Schagerlöf, Ulrika; Leanza, Giampiero; Kirik, Deniz

    2013-01-01

    Intraneuronal inclusions containing alpha-synuclein (a-syn) constitute one of the pathological hallmarks of Parkinson's disease (PD) and are accompanied by severe neurodegeneration of A9 dopaminergic neurons located in the substantia nigra. Although to a lesser extent, A10 dopaminergic neurons are also affected. Neurodegeneration of other neuronal populations, such as the cholinergic, serotonergic and noradrenergic cell groups, has also been documented in PD patients. Studies in human post-mortem PD brains and in rodent models suggest that deficits in cholinergic and dopaminergic systems may be associated with the cognitive impairment seen in this disease. Here, we investigated the consequences of targeted overexpression of a-syn in the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways. Rats were injected with recombinant adeno-associated viral vectors encoding for either human wild-type a-syn or green fluorescent protein (GFP) in the ventral tegmental area and the medial septum/vertical limb of the diagonal band of Broca, two regions rich in dopaminergic and cholinergic neurons, respectively. Histopathological analysis showed widespread insoluble a-syn positive inclusions in all major projections areas of the targeted nuclei, including the hippocampus, neocortex, nucleus accumbens and anteromedial striatum. In addition, the rats overexpressing human a-syn displayed an abnormal locomotor response to apomorphine injection and exhibited spatial learning and memory deficits in the Morris water maze task, in the absence of obvious spontaneous locomotor impairment. As losses in dopaminergic and cholinergic immunoreactivity in both the GFP and a-syn expressing animals were mild-to-moderate and did not differ from each other, the behavioral impairments seen in the a-syn overexpressing animals appear to be determined by the long term persisting neuropathology in the surviving neurons rather than by neurodegeneration.

  8. FYCO1 mediates clearance of α-synuclein aggregates through a Rab7-dependent mechanism.

    PubMed

    Saridaki, Theodora; Nippold, Markus; Dinter, Elisabeth; Roos, Andreas; Diederichs, Leonie; Fensky, Luisa; Schulz, Jörg B; Falkenburger, Björn H

    2018-05-10

    Parkinson disease can be caused by mutations in the α-synuclein gene and is characterized by aggregates of α-synuclein protein. We have previously shown that overexpression of the small GTPase Rab7 can induce clearance of α-synuclein aggregates. In this study, we investigate which Rab7 effectors mediate this effect. To model Parkinson disease we expressed the pathogenic A53T mutant of α-synuclein in HEK293T cells and Drosophila melanogaster. We tested the Rab7 effectors FYVE and coiled-coil domain-containing protein 1 (FYCO1) and Rab-interacting lysosomal protein (RILP). FYCO1-EGFP decorated vesicles containing α-synuclein. RILP-EGFP also decorated vesicular structures, but they did not contain α-synuclein. FYCO1 overexpression reduced the number of cells with α-synuclein aggregates, defined as visible particles of EGFP-tagged α-synuclein, whereas RILP did not. FYCO1 but not RILP reduced the amount of α-synuclein protein as assayed by western blot, increased the disappearance of α-synuclein aggregates in time-lapse microscopy, and decreased α-synuclein-induced toxicity assayed by the Trypan blue assay. siRNA-mediated knockdown of FYCO1 but not RILP reduced Rab7 induced aggregate clearance. Collectively, these findings indicate that FYCO1 and not RILP mediates Rab7 induced aggregate clearance. The effect of FYCO1 on aggregate clearance was blocked by the dominant negative Rab7 indicating that FYCO1 requires active Rab7 to function. Electron microscopic analysis and insertion of lysosomal membranes into the plasma membrane indicate that FYCO1 could lead to secretion of α-synuclein aggregates. Extracellular α-synuclein as assayed by ELISA was, however, not increased with FYCO1. Coexpression of FYCO1 in the fly model decreased α-synuclein aggregates as shown by the filter trap assay and rescued the locomotor deficit resulting from neuronal A53T-α-synuclein expression. This latter finding confirms that a pathway involving Rab7 and FYCO1 stimulates

  9. Structure, function and toxicity of alpha-synuclein: the Bermuda triangle in synucleinopathies.

    PubMed

    Villar-Piqué, Anna; Lopes da Fonseca, Tomás; Outeiro, Tiago Fleming

    2016-10-01

    Parkinson's disease belongs to a group of currently incurable neurodegenerative disorders characterized by the misfolding and accumulation of alpha-synuclein aggregates that are commonly known as synucleinopathies. Clinically, synucleinopathies are heterogeneous, reflecting the somewhat selective neuronal vulnerability characteristic of each disease. The precise molecular underpinnings of synucleinopathies remain unclear, but the process of aggregation of alpha-synuclein appears as a central event. However, there is still no consensus with respect to the toxic forms of alpha-synuclein, hampering our ability to use the protein as a target for therapeutic intervention. To decipher the molecular bases of synucleinopathies, it is essential to understand the complex triangle formed between the structure, function and toxicity of alpha-synuclein. Recently, important steps have been undertaken to elucidate the role of the protein in both physiological and pathological conditions. Here, we provide an overview of recent findings in the field of alpha-synuclein research, and put forward a new perspective over paradigms that persist in the field. Establishing whether alpha-synuclein has a causative role in all synucleinopathies will enable the identification of targets for the development of novel therapeutic strategies for this devastating group of disorders. Alpha-synuclein is the speculated cornerstone of several neurodegenerative disorders known as Synucleinopathies. Nevertheless, the mechanisms underlying the pathogenic effects of this protein remain unknown. Here, we review the recent findings in the three corners of alpha-synuclein biology - structure, function and toxicity - and discuss the enigmatic roads that have accompanied alpha-synuclein from the beginning. This article is part of a special issue on Parkinson disease. © 2015 International Society for Neurochemistry.

  10. Assessment of brain metabolite correlates of adeno-associated virus-mediated over-expression of human alpha-synuclein in cortical neurons by in vivo (1) H-MR spectroscopy at 9.4 T.

    PubMed

    Cuellar-Baena, Sandra; Landeck, Natalie; Sonnay, Sarah; Buck, Kerstin; Mlynarik, Vladimir; In 't Zandt, René; Kirik, Deniz

    2016-06-01

    In this study, we used proton-localized spectroscopy ((1) H-MRS) for the acquisition of the neurochemical profile longitudinally in a novel rat model of human wild-type alpha-synuclein (α-syn) over-expression. Our goal was to find out if the increased α-syn load in this model could be linked to changes in metabolites in the frontal cortex. Animals injected with AAV vectors encoding for human α-syn formed the experimental group, whereas green fluorescent protein expressing animals were used as the vector-treated control group and a third group of uninjected animals were used as naïve controls. Data were acquired at 2, 4, and 8 month time points. Nineteen metabolites were quantified in the MR spectra using LCModel software. On the basis of 92 spectra, we evaluated any potential gender effect and found that lactate (Lac) levels were lower in males compared to females, while the opposite was observed for ascorbate (Asc). Next, we assessed the effect of age and found increased levels of GABA, Tau, and GPC+PCho. Finally, we analyzed the effect of treatment and found that Lac levels (p = 0.005) were specifically lower in the α-syn group compared to the green fluorescent protein and control groups. In addition, Asc levels (p = 0.05) were increased in the vector-injected groups, whereas glucose levels remained unchanged. This study indicates that the metabolic switch between glucose-lactate could be detectable in vivo and might be modulated by Asc. No concomitant changes were found in markers of neuronal integrity (e.g., N-acetylaspartate) consistent with the fact that α-syn over-expression in cortical neurons did not result in neurodegeneration in this model. We acquired the neurochemical profile longitudinally in a rat model of human wild-type alpha-synuclein (α-syn) over-expression in cortical neurons. We found that Lactate levels were reduced in the α-syn group compared to the control groups and Ascorbate levels were increased in the vector-injected groups

  11. Alpha-synuclein: relating metals to structure, function and inhibition.

    PubMed

    McDowall, J S; Brown, D R

    2016-04-01

    Alpha-synuclein has long been studied due to its involvement in the progression of Parkinson's disease (PD), a common neurodegenerative disorder, although a consensus on the exact function of this protein is elusive. This protein shows remarkable structural plasticity and this property is important for both correct cellular function and pathological progression of PD. Formation of intracellular oligomeric species within the substantia nigra correlates with disease progression and it has been proposed that formation of a partially folded intermediate is key to the initiation of the fibrillisation process. Many factors can influence changes in the structure of alpha-synuclein such as disease mutations and interaction with metals and neurotransmitters. High concentrations of both dopamine and metals are present in the substantia nigra making this an ideal location for both the structural alteration of alpha-synuclein and the production of toxic oxygen species. The recent proposal that alpha-synuclein is a ferrireductase is important as it can possibly catalyse the formation of such reactive species and as a result exacerbate neurodegeneration.

  12. HMGB1 promotes the starvation-induced autophagic degradation of α-synuclein in SH-SY5Y cells Atg 5-dependently.

    PubMed

    Guan, Yi; Li, Yiping; Zhao, Gang; Li, Yunqian

    2018-06-01

    Impaired autophagic clearance of aggregated α-synuclein is considered as one of key mechanisms underlining Parkinson disease (PD). High-mobility group protein B1 (HMGB1) has recently been demonstrated to mediate persistent neuroinflammation and consequent progressive neurodegeneration by promoting multiple inflammatory and neurotoxic factors. In this study, we examined the influence of the overexpression of wild-type (WT) and mutant-type (MT, A53T and A30P) α-synuclein on the autophagy in neuroblastoma SH-SY5Y cells under starvation, and then investigated the regulation of endogenous HMGB1 on the α-synuclein degradation and on the starvation-induced autophagy in the α-synuclein-overexpressed SH-SY5Y cells. It was demonstrated that the overexpression of WT or MT α-synuclein significantly downregulated the starvation-induced conversion of LC3I to LC3II and autophagy protein (Atg) 5 expression, whereas markedly inhibited the starvation-downregulated mTOR in SH-SY5Y cells. On the other side, the lentivirus-mediated upregulation of endogenous HMGB1 promoted the degradation of WT or MT α-synuclein in SH-SY5Y cells autophagy-dependently via promoting Atg 5, but not mTOR, the Atg 5 knockdown downregulated the HMGB1-mediated promotion to α-synuclein degeneration. Thus, we concluded that α-synuclein inhibited the starvation-induced autophagy in neuroblastoma SH-SY5Y cells via inhibiting the mTOR/Atg 5 signaling. However, the endogenous HMGB1 promoted the autophagic degradation of α-synuclein via the Atg 5-dependent autophagy-initiation pathway, implying the protective role of endogenous HMGB1 in the neuroblastoma cells against the α-synuclein accumulation. Copyright © 2018. Published by Elsevier Inc.

  13. Clearing Extracellular Alpha-Synuclein from Cerebrospinal Fluid: A New Therapeutic Strategy in Parkinson's Disease.

    PubMed

    Menéndez-González, Manuel; Padilla-Zambrano, Huber S; Tomás-Zapico, Cristina; García, Benjamin Fernández

    2018-03-23

    This concept article aims to show the rationale of targeting extracellular α-Synuclein (α-Syn) from cerebrospinal fluid (CSF) as a new strategy to remove this protein from the brain in Parkinson's disease (PD). Misfolding and intracellular aggregation of α-synuclein into Lewy bodies are thought to be crucial in the pathogenesis of PD. Recent research has shown that small amounts of monomeric and oligomeric α-synuclein are released from neuronal cells by exocytosis and that this extracellular alpha-synuclein contributes to neurodegeneration, progressive spreading of alpha-synuclein pathology, and neuroinflammation. In PD, extracellular oligomeric-α-synuclein moves in constant equilibrium between the interstitial fluid (ISF) and the CSF. Thus, we expect that continuous depletion of oligomeric-α-synuclein in the CSF will produce a steady clearance of the protein in the ISF, preventing transmission and deposition in the brain.

  14. Bridging the gap between high-throughput genetic and transcriptional data reveals cellular pathways responding to alpha-synuclein toxicity

    PubMed Central

    Yeger-Lotem, Esti; Riva, Laura; Su, Linhui Julie; Gitler, Aaron D.; Cashikar, Anil; King, Oliver D.; Auluck, Pavan K.; Geddie, Melissa L.; Valastyan, Julie S.; Karger, David R.; Lindquist, Susan; Fraenkel, Ernest

    2009-01-01

    Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression. Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens, yet the functional roles of the genes identified by these assays often remain enigmatic. By comparing the results of these two assays across various cellular responses, we found that they are consistently distinct. Moreover, genetic screens tend to identify response regulators, while mRNA profiling frequently detects metabolic responses. We developed an integrative approach that bridges the gap between these data using known molecular interactions, thus highlighting major response pathways. We harnessed this approach to reveal cellular pathways related to alpha-synuclein, a small lipid-binding protein implicated in several neurodegenerative disorders including Parkinson disease. For this we screened an established yeast model for alpha-synuclein toxicity to identify genes that when overexpressed alter cellular survival. Application of our algorithm to these data and data from mRNA profiling provided functional explanations for many of these genes and revealed novel relations between alpha-synuclein toxicity and basic cellular pathways. PMID:19234470

  15. Advancing Stem Cell Models of Alpha-Synuclein Gene Regulation in Neurodegenerative Disease.

    PubMed

    Piper, Desiree A; Sastre, Danuta; Schüle, Birgitt

    2018-01-01

    Alpha-synuclein ( non A4 component of amyloid precursor, SNCA, NM_000345.3 ) plays a central role in the pathogenesis of Parkinson's disease (PD) and related Lewy body disorders such as Parkinson's disease dementia, Lewy body dementia, and multiple system atrophy. Since its discovery as a disease-causing gene in 1997, alpha-synuclein has been a central point of scientific interest both at the protein and gene level. Mutations, including copy number variants, missense mutations, short structural variants, and single nucleotide polymorphisms, can be causative for PD and affect conformational changes of the protein, can contribute to changes in expression of alpha-synuclein and its isoforms, and can influence regulation of temporal as well as spatial levels of alpha-synuclein in different tissues and cell types. A lot of progress has been made to understand both the physiological transcriptional and epigenetic regulation of the alpha-synuclein gene and whether changes in transcriptional regulation could lead to disease and neurodegeneration in PD and related alpha-synucleinopathies. Although the histopathological changes in these neurodegenerative disorders are similar, the temporal and spatial presentation and progression distinguishes them which could be in part due to changes or disruption of transcriptional regulation of alpha-synuclein. In this review, we describe different genetic alterations that contribute to PD and neurodegenerative conditions and review aspects of transcriptional regulation of the alpha-synuclein gene in the context of the development of PD. New technologies, advanced gene engineering and stem cell modeling, are on the horizon to shed further light on a better understanding of gene regulatory processes and exploit them for therapeutic developments.

  16. Overexpression of IL-7R alpha provides a competitive advantage during early T-cell development.

    PubMed

    Laouar, Yasmina; Crispe, I Nicholas; Flavell, Richard A

    2004-03-15

    Critical checkpoints controlling early thymic T-cell development and homeostasis are set by the proper signaling function of the interleukin 7 receptor (IL-7R) and the pre-T-cell antigen receptor. Although alpha beta T-cell development is observed in IL-7- and IL-7R alpha-deficient mice, the number of thymocytes is significantly reduced, implying a role for the IL-7R in controlling the size of the thymic T-cell compartment. Here, we report the overexpression of IL-7R alpha that occurs in the early T-cell compartment from AKR/J mice, animals that are highly susceptible to the spontaneous development of thymoma. Increased IL-7R alpha was revealed by surface staining, and increased IL-7R alpha mRNA was documented by using reverse transcriptase-polymerase chain reaction (RT-PCR). This resulted in increased survival of AKR/J early thymocytes, shown by the decreased frequency of TUNEL(+) (terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate [dUTP]-fluorescein nick end labeling) cells. In an in vivo thymocyte repopulation model, AKR/J thymocytes had a selective advantage over healthy thymocytes. This advantage occurred at early stages of T-cell development. Our findings support the model that overexpression of growth factor receptors can contribute to proliferation and malignancy.

  17. Alpha-Synuclein Expression Restricts RNA Viral Infections in the Brain.

    PubMed

    Beatman, Erica L; Massey, Aaron; Shives, Katherine D; Burrack, Kristina S; Chamanian, Mastooreh; Morrison, Thomas E; Beckham, J David

    2015-12-30

    We have discovered that native, neuronal expression of alpha-synuclein (Asyn) inhibits viral infection, injury, and disease in the central nervous system (CNS). Enveloped RNA viruses, such as West Nile virus (WNV), invade the CNS and cause encephalitis, yet little is known about the innate neuron-specific inhibitors of viral infections in the CNS. Following WNV infection of primary neurons, we found that Asyn protein expression is increased. The infectious titer of WNV and Venezuelan equine encephalitis virus (VEEV) TC83 in the brains of Asyn-knockout mice exhibited a mean increase of 10(4.5) infectious viral particles compared to the titers in wild-type and heterozygote littermates. Asyn-knockout mice also exhibited significantly increased virus-induced mortality compared to Asyn heterozygote or homozygote control mice. Virus-induced Asyn localized to perinuclear, neuronal regions expressing viral envelope protein and the endoplasmic reticulum (ER)-associated trafficking protein Rab1. In Asyn-knockout primary neuronal cultures, the levels of expression of ER signaling pathways, known to support WNV replication, were significantly elevated before and during viral infection compared to those in Asyn-expressing primary neuronal cultures. We propose a model in which virus-induced Asyn localizes to ER-derived membranes, modulates virus-induced ER stress signaling, and inhibits viral replication, growth, and injury in the CNS. These data provide a novel and important functional role for the expression of native alpha-synuclein, a protein that is closely associated with the development of Parkinson's disease. Neuroinvasive viruses such as West Nile virus are able to infect neurons and cause severe disease, such as encephalitis, or infection of brain tissue. Following viral infection in the central nervous system, only select neurons are infected, implying that neurons exhibit innate resistance to viral infections. We discovered that native neuronal expression of alpha-synuclein

  18. Clearing Extracellular Alpha-Synuclein from Cerebrospinal Fluid: A New Therapeutic Strategy in Parkinson’s Disease

    PubMed Central

    Padilla-Zambrano, Huber S.; Tomás-Zapico, Cristina; García, Benjamin Fernández

    2018-01-01

    This concept article aims to show the rationale of targeting extracellular α-Synuclein (α-Syn) from cerebrospinal fluid (CSF) as a new strategy to remove this protein from the brain in Parkinson’s disease (PD). Misfolding and intracellular aggregation of α-synuclein into Lewy bodies are thought to be crucial in the pathogenesis of PD. Recent research has shown that small amounts of monomeric and oligomeric α-synuclein are released from neuronal cells by exocytosis and that this extracellular alpha-synuclein contributes to neurodegeneration, progressive spreading of alpha-synuclein pathology, and neuroinflammation. In PD, extracellular oligomeric-α-synuclein moves in constant equilibrium between the interstitial fluid (ISF) and the CSF. Thus, we expect that continuous depletion of oligomeric-α-synuclein in the CSF will produce a steady clearance of the protein in the ISF, preventing transmission and deposition in the brain. PMID:29570693

  19. Chronic administration of cholesterol oximes in mice increases transcription of cytoprotective genes and improves transcriptome alterations induced by alpha-synuclein overexpression in nigrostriatal dopaminergic neurons

    PubMed Central

    Richter, Franziska; Gao, Fuying; Medvedeva, Vera; Lee, Patrick; Bove, Nicholas; Fleming, Sheila M.; Michaud, Magali; Lemesre, Vincent; Patassini, Stefano; De La Rosa, Krystal; Mulligan, Caitlin K.; Sioshansi, Pedrom; Zhu, Chunni; Coppola, Giovanni; Bordet, Thierry; Pruss, Rebecca; Chesselet, Marie-Françoise

    2014-01-01

    Cholesterol-oximes TRO19622 and TRO40303 target outer mitochondrial membrane proteins and have beneficial effects in preclinical models of neurodegenerative diseases leading to their advancement to clinical trials. Dopaminergic neurons degenerate in Parkinson’s disease (PD) and are prone to oxidative stress and mitochondrial dysfunction. In order to provide insights into the neuroprotective potential of TRO19622 and TRO40303 for dopaminergic neurons in vivo, we assessed their effects on gene expression in laser captured nigrostriatal dopaminergic neurons of wildtype mice and of mice that over-express alpha-synuclein, a protein involved in both familial and sporadic forms of PD (Thy1-aSyn mice). Young mice were fed the drugs in food pellets or a control diet from 1 to 4 months of age, approximately 10 months before the appearance of striatal dopamine loss in this model. Unbiased weighted gene co-expression network analysis (WGCNA) of transcriptional changes revealed effects of cholesterol oximes on transcripts related to mitochondria, cytoprotection and anti-oxidant response in wild-type and transgenic mice, including increased transcription of stress defense (e.g. Prdx1, Prdx2, Glrx2, Hspa9, Pink1, Drp1, Trak1) and dopamine-related (Th, Ddc, Gch1, Dat, Vmat2, Drd2, Chnr6a) genes. Even at this young age transgenic mice showed alterations in transcripts implicated in mitochondrial function and oxidative stress (e.g. Bcl-2, Bax, Casp3, Nos2), and both drugs normalized about 20% of these alterations. Young Thy1-aSyn mice exhibit motor deficits that differ from parkinsonism and are established before the onset of treatment; these deficits were not improved by cholesterol oximes. However, high doses of TRO40303 improved olfaction and produced the same effects as dopamine agonists on a challenging beam test, specifically an increase in footslips, an observation congruent with its effects on transcripts involved in dopamine synthesis. High doses of TRO19622 increased

  20. Oral Exposure to Paraquat Triggers Earlier Expression of Phosphorylated α-Synuclein in the Enteric Nervous System of A53T Mutant Human α-Synuclein Transgenic Mice

    PubMed Central

    Naudet, Nicolas; Antier, Emilie; Gaillard, Damien; Morignat, Eric; Lakhdar, Latifa; Baron, Thierry; Bencsik, Anna

    2017-01-01

    Abstract The misfolded α-synuclein protein, phosphorylated at serine 129 (pSer129 α-syn), is the hallmark of Parkinson disease (PD). Detected also in the enteric nervous system (ENS), it supports the recent theory that PD could start in the gut, rather than the brain. In a previous study, using a transgenic mouse model of human synucleinopathies expressing the A53T mutant α-synuclein (TgM83), in which a neurodegenerative process associated with α-synuclein occurs spontaneously in the brain, we have shown earlier onset of pSer129 α-syn in the ENS. Here, we used this model to study the impact of paraquat (PQ) a neurotoxic herbicide incriminated in PD in agricultural workers) on the enteric pSer129 α-syn expression in young mice. Orally delivered in the drinking water at 10 mg/kg/day for 6–8 weeks, the impact of PQ was measured in a time-dependent manner on weight, locomotor abilities, pSer129 α-syn, and glial fibrillary acidic protein (GFAP) expression levels in the ENS. Remarkably, pSer129 α-syn was detected in ENS earlier under PQ oral exposure and enteric GFAP expression was also increased. These findings bring additional support to the theory that neurotoxic agents such as PQ initiate idiopathic PD after oral delivery. PMID:29040593

  1. Oral Exposure to Paraquat Triggers Earlier Expression of Phosphorylated α-Synuclein in the Enteric Nervous System of A53T Mutant Human α-Synuclein Transgenic Mice.

    PubMed

    Naudet, Nicolas; Antier, Emilie; Gaillard, Damien; Morignat, Eric; Lakhdar, Latifa; Baron, Thierry; Bencsik, Anna

    2017-12-01

    The misfolded α-synuclein protein, phosphorylated at serine 129 (pSer129 α-syn), is the hallmark of Parkinson disease (PD). Detected also in the enteric nervous system (ENS), it supports the recent theory that PD could start in the gut, rather than the brain. In a previous study, using a transgenic mouse model of human synucleinopathies expressing the A53T mutant α-synuclein (TgM83), in which a neurodegenerative process associated with α-synuclein occurs spontaneously in the brain, we have shown earlier onset of pSer129 α-syn in the ENS. Here, we used this model to study the impact of paraquat (PQ) a neurotoxic herbicide incriminated in PD in agricultural workers) on the enteric pSer129 α-syn expression in young mice. Orally delivered in the drinking water at 10 mg/kg/day for 6-8 weeks, the impact of PQ was measured in a time-dependent manner on weight, locomotor abilities, pSer129 α-syn, and glial fibrillary acidic protein (GFAP) expression levels in the ENS. Remarkably, pSer129 α-syn was detected in ENS earlier under PQ oral exposure and enteric GFAP expression was also increased. These findings bring additional support to the theory that neurotoxic agents such as PQ initiate idiopathic PD after oral delivery. © 2017 American Association of Neuropathologists, Inc.

  2. Role of alpha-synuclein in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism in mice.

    PubMed

    Schlüter, O M; Fornai, F; Alessandrí, M G; Takamori, S; Geppert, M; Jahn, R; Südhof, T C

    2003-01-01

    In humans, mutations in the alpha-synuclein gene or exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produce Parkinson's disease with loss of dopaminergic neurons and depletion of nigrostriatal dopamine. alpha-Synuclein is a vertebrate-specific component of presynaptic nerve terminals that may function in modulating synaptic transmission. To test whether MPTP toxicity involves alpha-synuclein, we generated alpha-synuclein-deficient mice by homologous recombination, and analyzed the effect of deleting alpha-synuclein on MPTP toxicity using these knockout mice. In addition, we examined commercially available mice that contain a spontaneous loss of the alpha-synuclein gene. As described previously, deletion of alpha-synuclein had no significant effects on brain structure or composition. In particular, the levels of synaptic proteins were not altered, and the concentrations of dopamine, dopamine metabolites, and dopaminergic proteins were unchanged. Upon acute MPTP challenge, alpha-synuclein knockout mice were partly protected from chronic depletion of nigrostriatal dopamine when compared with littermates of the same genetic background, whereas mice carrying the spontaneous deletion of the alpha-synuclein gene exhibited no protection. Furthermore, alpha-synuclein knockout mice but not the mice with the alpha-synuclein gene deletion were slightly more sensitive to methamphetamine than littermate control mice. These results demonstrate that alpha-synuclein is not obligatorily coupled to MPTP sensitivity, but can influence MPTP toxicity on some genetic backgrounds, and illustrate the need for extensive controls in studies aimed at describing the effects of mouse knockouts on MPTP sensitivity.

  3. Structural Characteristics of the Alpha-Synuclein Oligomers Stabilized By the Flavonoid Baicalein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hong, D.-P.; Fink, A.L.; Uversky, V.N.

    The flavonoid baicalein inhibits fibrillation of alpha-synuclein, which is a major component of Lewy bodies in Parkinson's disease. It has been known that baicalein induces the formation of alpha-synuclein oligomers and consequently prevents their fibrillation. In order to evaluate the structural properties of baicalein-stabilized oligomers, we purified oligomer species by HPLC and examined their stability and structure by CD, Fourier transform infrared spectroscopy, size exclusion chromatography HPLC, small-angle X-ray scattering, and atomic force microscopy. Baicalein-stabilized oligomers are beta-sheet-enriched according to CD and Fourier transform infrared spectroscopy analyses. They did not form fibrils even after very prolonged incubation. From small-angle X-raymore » scattering data and atomic force microscopy images, the oligomers were characterized as quite compact globular species. Oligomers were extremely stable, with a GdmCl C(m)=3.3 M. This high stability explains the previously observed inhibition properties of baicalein against alpha-synuclein fibrillation. These baicalein-stabilized oligomers, added to the solution of aggregating alpha-synuclein, were able to noticeably inhibit its fibrillation. After prolonged coincubation, short fibrils were formed, suggesting an effective interaction of oligomers with monomeric alpha-synuclein. Membrane permeability tests suggested that the baicalein-stabilized oligomers had a mild effect on the integrity of the membrane surface. This effect was rather similar to that of the monomeric protein, suggesting that targeted stabilization of certain alpha-synuclein oligomers might offer a potential strategy for the development of novel Parkinson's disease therapies.« less

  4. α-Synuclein Mutation Inhibits Endocytosis at Mammalian Central Nerve Terminals.

    PubMed

    Xu, Jianhua; Wu, Xin-Sheng; Sheng, Jiansong; Zhang, Zhen; Yue, Hai-Yuan; Sun, Lixin; Sgobio, Carmelo; Lin, Xian; Peng, Shiyong; Jin, Yinghui; Gan, Lin; Cai, Huaibin; Wu, Ling-Gang

    2016-04-20

    α-Synuclein (α-syn) missense and multiplication mutations have been suggested to cause neurodegenerative diseases, including Parkinson's disease (PD) and dementia with Lewy bodies. Before causing the progressive neuronal loss, α-syn mutations impair exocytosis, which may contribute to eventual neurodegeneration. To understand how α-syn mutations impair exocytosis, we developed a mouse model that selectively expressed PD-related human α-syn A53T (h-α-synA53T) mutation at the calyx of Held terminals, where release mechanisms can be dissected with a patch-clamping technique. With capacitance measurement of endocytosis, we reported that h-α-synA53T, either expressed transgenically or dialyzed in the short term in calyces, inhibited two of the most common forms of endocytosis, the slow and rapid vesicle endocytosis at mammalian central synapses. The expression of h-α-synA53Tin calyces also inhibited vesicle replenishment to the readily releasable pool. These findings may help to understand how α-syn mutations impair neurotransmission before neurodegeneration. α-Synuclein (α-syn) missense or multiplication mutations may cause neurodegenerative diseases, such as Parkinson's disease and dementia with Lewy bodies. The initial impact of α-syn mutations before neuronal loss is impairment of exocytosis, which may contribute to eventual neurodegeneration. The mechanism underlying impairment of exocytosis is poorly understood. Here we report that an α-syn mutant, the human α-syn A53T, inhibited two of the most commonly observed forms of endocytosis, slow and rapid endocytosis, at a mammalian central synapse. We also found that α-syn A53T inhibited vesicle replenishment to the readily releasable pool. These results may contribute to accounting for the widely observed early synaptic impairment caused by α-syn mutations in the progression toward neurodegeneration. Copyright © 2016 the authors 0270-6474/16/364408-07$15.00/0.

  5. α-Synuclein induced toxicity in brain stem serotonin neurons mediated by an AAV vector driven by the tryptophan hydroxylase promoter.

    PubMed

    Wan, Oi Wan; Shin, Eunju; Mattsson, Bengt; Caudal, Dorian; Svenningsson, Per; Björklund, Anders

    2016-05-23

    We studied the impact of α-synuclein overexpression in brainstem serotonin neurons using a novel vector construct where the expression of human wildtype α-synuclein is driven by the tryptophan hydroxylase promoter, allowing expression of α-synuclein at elevated levels, and with high selectivity, in serotonergic neurons. α-Synuclein induced degenerative changes in axons and dendrites, displaying a distorted appearance, suggesting accumulation and aggregation of α-synuclein as a result of impaired axonal transport, accompanied by a 40% loss of terminals, as assessed in the hippocampus. Tissue levels of serotonin and its major metabolite 5-HIAA remained largely unaltered, and the performance of the α-synuclein overexpressing rats in tests of spatial learning (water maze), anxiety related behavior (elevated plus maze) and depressive-like behavior (forced swim test) was not different from control, suggesting that the impact of the developing axonal pathology on serotonin neurotransmission was relatively mild. Overexpression of α-synuclein in the raphe nuclei, combined with overexpression in basal forebrain cholinergic neurons, resulted in more pronounced axonal pathology and significant impairment in the elevated plus maze. We conclude that α-synuclein pathology in serotonergic or cholinergic neurons alone is not sufficient to impair non-motor behaviors, but that it is their simultaneous involvement that determines severity of such symptoms.

  6. Alpha-synuclein mitochondrial interaction leads to irreversible translocation and complex I impairment.

    PubMed

    Martínez, Jimena H; Fuentes, Federico; Vanasco, Virginia; Alvarez, Silvia; Alaimo, Agustina; Cassina, Adriana; Coluccio Leskow, Federico; Velazquez, Francisco

    2018-08-01

    α-synuclein is involved in both familial and sporadic Parkinson's disease. Although its interaction with mitochondria has been well documented, several aspects remains unknown or under debate such as the specific sub-mitochondrial localization or the dynamics of the interaction. It has been suggested that α-synuclein could only interact with ER-associated mitochondria. The vast use of model systems and experimental conditions makes difficult to compare results and extract definitive conclusions. Here we tackle this by analyzing, in a simplified system, the interaction between purified α-synuclein and isolated rat brain mitochondria. This work shows that wild type α-synuclein interacts with isolated mitochondria and translocates into the mitochondrial matrix. This interaction and the irreversibility of α-synuclein translocation depend on incubation time and α-synuclein concentration. FRET experiments show that α-synuclein localizes close to components of the TOM complex suggesting a passive transport of α-synuclein through the outer membrane. In addition, α-synuclein binding alters mitochondrial function at the level of Complex I leading to a decrease in ATP synthesis and an increase of ROS production. Copyright © 2018. Published by Elsevier Inc.

  7. Behavioral Characterization of A53T Mice Reveals Early and Late Stage Deficits Related to Parkinson’s Disease

    PubMed Central

    Paumier, Katrina L.; Sukoff Rizzo, Stacey J.; Berger, Zdenek; Chen, Yi; Gonzales, Cathleen; Kaftan, Edward; Li, Li; Lotarski, Susan; Monaghan, Michael; Shen, Wei; Stolyar, Polina; Vasilyev, Dmytro; Zaleska, Margaret; D. Hirst, Warren; Dunlop, John

    2013-01-01

    Parkinson's disease (PD) pathology is characterized by the formation of intra-neuronal inclusions called Lewy bodies, which are comprised of alpha-synuclein (α-syn). Duplication, triplication or genetic mutations in α-syn (A53T, A30P and E46K) are linked to autosomal dominant PD; thus implicating its role in the pathogenesis of PD. In both PD patients and mouse models, there is increasing evidence that neuronal dysfunction occurs before the accumulation of protein aggregates (i.e., α-syn) and neurodegeneration. Characterization of the timing and nature of symptomatic dysfunction is important for understanding the impact of α-syn on disease progression. Furthermore, this knowledge is essential for identifying pathways and molecular targets for therapeutic intervention. To this end, we examined various functional and morphological endpoints in the transgenic mouse model expressing the human A53T α-syn variant directed by the mouse prion promoter at specific ages relating to disease progression (2, 6 and 12 months of age). Our findings indicate A53T mice develop fine, sensorimotor, and synaptic deficits before the onset of age-related gross motor and cognitive dysfunction. Results from open field and rotarod tests show A53T mice develop age-dependent changes in locomotor activity and reduced anxiety-like behavior. Additionally, digigait analysis shows these mice develop an abnormal gait by 12 months of age. A53T mice also exhibit spatial memory deficits at 6 and 12 months, as demonstrated by Y-maze performance. In contrast to gross motor and cognitive changes, A53T mice display significant impairments in fine- and sensorimotor tasks such as grooming, nest building and acoustic startle as early as 1–2 months of age. These mice also show significant abnormalities in basal synaptic transmission, paired-pulse facilitation and long-term depression (LTD). Combined, these data indicate the A53T model exhibits early- and late-onset behavioral and synaptic impairments

  8. Antiapoptotic property of human alpha-synuclein in neuronal cell lines is associated with the inhibition of caspase-3 but not caspase-9 activity.

    PubMed

    Li, Wenxue; Lee, Michael K

    2005-06-01

    Abnormalities of alpha-synuclein (alpha-Syn) are mechanistically linked to Parkinson's disease (PD) and other alpha-synucleinopathies. To gain additional insights into the relationships between alpha-Syn expression and cell death, we examined the effects of expressing human alpha-Syn (Hualpha-Syn) variants on the cellular vulnerability to apoptotic stimuli. We show that the expression of wild-type (WT) and A30P mutant, but not A53T mutant, Hualpha-Syn leads to the protection of neuronal cell lines from apoptosis but not necrosis. Significantly, Hualpha-Syn did not protect non-neuronal cell lines from apoptosis. We also show that A53T mutant is a loss of function in regards to the antiapoptotic property since the expression of WT Hualpha-Syn with an excess of A53T mutant Hualpha-Syn leads to protection of the cells from apoptosis. The antiapoptotic property is specific to human alpha-Syn as neither beta-Syn nor mouse alpha-Syn protected cells from apoptosis, and the carboxy-terminal 20 amino acids are required for the antiapoptotic property. Analyses of capase-3 and caspase-9 activation reveal that the antiapoptotic property of Hualpha-Syn in neuronal cell lines is associated with the attenuation of caspase-3 activity without affecting the caspase-9 activity or the levels of cleaved, active caspase-3. We conclude that Hualpha-Syn modulates the activity of cleaved caspase-3 product in neuronal cell lines.

  9. Treatment with Trehalose Prevents Behavioral and Neurochemical Deficits Produced in an AAV α-Synuclein Rat Model of Parkinson's Disease.

    PubMed

    He, Qing; Koprich, James B; Wang, Ying; Yu, Wen-bo; Xiao, Bao-guo; Brotchie, Jonathan M; Wang, Jian

    2016-05-01

    The accumulation of misfolded α-synuclein in dopamine (DA) neurons is believed to be of major importance in the pathogenesis of Parkinson's disease (PD). Animal models of PD, based on viral-vector-mediated over-expression of α-synuclein, have been developed and show evidence of dopaminergic toxicity, providing us a good tool to investigate potential therapies to interfere with α-synuclein-mediated pathology. An efficient disease-modifying therapeutic molecule should be able to interfere with the neurotoxicity of α-synuclein aggregation. Our study highlighted the ability of an autophagy enhancer, trehalose (at concentrations of 5 and 2% in drinking water), to protect against A53T α-synuclein-mediated DA degeneration in an adeno-associated virus serotype 1/2 (AAV1/2)-based rat model of PD. Behavioral tests and neurochemical analysis demonstrated a significant attenuation in α-synuclein-mediated deficits in motor asymmetry and DA neurodegeneration including impaired DA neuronal survival and DA turnover, as well as α-synuclein accumulation and aggregation in the nigrostriatal system by commencing 5 and 2% trehalose at the same time as delivery of AAV. Trehalose (0.5%) was ineffective on the above behavioral and neurochemical deficits. Further investigation showed that trehalose enhanced autophagy in the striatum by increasing formation of LC3-II. This study supports the concept of using trehalose as a novel therapeutic strategy that might prevent/reverse α-synuclein aggregation for the treatment of PD.

  10. The Identification of Alpha-Synuclein as the First Parkinson Disease Gene.

    PubMed

    Nussbaum, Robert L

    2017-01-01

    In this Commentary, I describe the events that led from an NINDS-sponsored Workshop on Parkinson Disease Research in 1995, where I was asked to speak about the genetics of Parkinson disease, to the identification a mere two years later of a mutation in alpha-synuclein as the cause of autosomal dominant Parkinson disease in the Contursi kindred. I review the steps we took to first map and then find the mutation in the alpha-synuclein locus and describe the obstacles and the role of serendipity in facilitating the work. Although alpha-synuclein mutations are a rare cause of hereditary PD, the importance of this finding goes far beyond the rare families with hereditary disease because it pinpointed alpha-synuclein as a key contributor to the far more common sporadic form of Parkinson disease. This work confirms William Harvey's observation from 350 years ago that studying rarer forms of a disease is an excellent way to understand the more common forms of that disease. The identification of synuclein's role in hereditary Parkinson disease has opened new avenues of research into the pathogenesis and potential treatments of the common form of Parkinson disease that affects many millions of Americans and tens of millions of human beings worldwide.

  11. Aggregates assembled from overexpression of wild-type alpha-synuclein are not toxic to human neuronal cells.

    PubMed

    Ko, Li-Wen; Ko, Hwai-Hwa C; Lin, Wen-Lang; Kulathingal, Jayanranyan G; Yen, Shu-Hui C

    2008-11-01

    Filamentous alpha-synuclein (alpha-syn) aggregates form Lewy bodies (LBs), the neuropathologic hallmarks of Parkinson disease and related alpha-synucleinopathies. To model Lewy body-associated neurodegeneration, we generated transfectant 3D5 of human neuronal-type in which expression of human wild-type alpha-syn is regulated by the tetracycline off (TetOff)-inducible mechanism. Retinoic acid-elicited differentiation promoted assembly of alpha-syn aggregates after TetOff induction in 3D5 cells. The aggregates accumulated 14 days after TetOff induction were primarily soluble and showed augmented thioflavin affinity with concomitant phosphorylation and nitration of alpha-syn. Extension of the induction led to the formation of sarkosyl-insoluble aggregates that appeared concurrently with thioflavin-positive inclusions. Immunoelectron microscopy revealed that the inclusions consist of dense bundles of 8- to 12-nm alpha-syn fibrils that congregate in the perikarya and resemble Lewy bodies. Most importantly, accumulation of soluble and insoluble aggregates after TetOff induction for 14 and 28 days was reversible and did not compromise the viability of the cells or their subsequent survival. Thus, this chemically defined culture paradigm provides a useful means to elucidate how oxidative injuries and other insults that are associated with aging promote alpha-syn to self-assemble or interact with other molecules leading to neuronal degeneration in alpha-synucleinopathies.

  12. Expression of alpha-synuclein during eye development of mice (Mus musculus), chick (Gallus gallus domisticus) and fish (Ctenopharyngodon idella) in a comparison study.

    PubMed

    Seleem, Amin A

    2015-08-01

    Synucleins are small proteins associated with neurodegenerative diseases, alpha-synuclein is a Parkinson's disease-linked protein of ubiquitous expression in the central nervous system. This study aimed at the localization of alpha-synuclein during eye development of mice (Mus musculus), chick (Gallus gallus domisticus) and fish (Ctenopharyngodon idella) by immunohistochemical staining in a comparison study. The results showed that alpha-synuclein expression increased gradually with the development of ciliary body, iris, retina and cornea of mice at E17, P1, P3, P7 and chick at E5, E10, E15 with unequal appearance of alpha-synuclein expression. Also, it was not detected in iridocorneal angle during eye development of mice and chick. Alpha-synuclein expression during fish eye development at P10, P15, P20 was not detected either in the ciliray body or Iris regions and it was pronounced with sharp signals in the highly specialized tissue of the iridocorneal angle at P20. Also, the expression was gradually increased from P15 to P20 in fish retina and cornea. The pattern of expression and distribution of alpha-synuclein during the development of ciliary body and iris of mice, chick and fish has not been previously characterized, The data concluded that alpha-synuclein has important cellular function during eye development of studied animals. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. The chaperonin CCT inhibits assembly of α-synuclein amyloid fibrils by a specific, conformation-dependent interaction

    PubMed Central

    Sot, Begoña; Rubio-Muñoz, Alejandra; Leal-Quintero, Ahudrey; Martínez-Sabando, Javier; Marcilla, Miguel; Roodveldt, Cintia; Valpuesta, José M.

    2017-01-01

    The eukaryotic chaperonin CCT (chaperonin containing TCP-1) uses cavities built into its double-ring structure to encapsulate and to assist folding of a large subset of proteins. CCT can inhibit amyloid fibre assembly and toxicity of the polyQ extended mutant of huntingtin, the protein responsible for Huntington’s disease. This raises the possibility that CCT modulates other amyloidopathies, a still-unaddressed question. We show here that CCT inhibits amyloid fibre assembly of α-synuclein A53T, one of the mutants responsible for Parkinson’s disease. We evaluated fibrillation blockade in α-synuclein A53T deletion mutants and CCT interactions of full-length A53T in distinct oligomeric states to define an inhibition mechanism specific for α-synuclein. CCT interferes with fibre assembly by interaction of its CCTζ and CCTγ subunits with the A53T central hydrophobic region (NAC). This interaction is specific to NAC conformation, as it is produced once soluble α-synuclein A53T oligomers form and blocks the reaction before fibres begin to grow. Finally, we show that this association inhibits α-synuclein A53T oligomer toxicity in neuroblastoma cells. In summary, our results and those for huntingtin suggest that CCT is a general modulator of amyloidogenesis via a specific mechanism. PMID:28102321

  14. The Identification of Alpha-Synuclein as the First Parkinson Disease Gene

    PubMed Central

    Nussbaum, Robert L.

    2017-01-01

    In this Commentary, I describe the events that led from an NINDS-sponsored Workshop on Parkinson Disease Research in 1995, where I was asked to speak about the genetics of Parkinson disease, to the identification a mere two years later of a mutation in alpha-synuclein as the cause of autosomal dominant Parkinson disease in the Contursi kindred. I review the steps we took to first map and then find the mutation in the alpha-synuclein locus and describe the obstacles and the role of serendipity in facilitating the work. Although alpha-synuclein mutations are a rare cause of hereditary PD, the importance of this finding goes far beyond the rare families with hereditary disease because it pinpointed alpha-synuclein as a key contributor to the far more common sporadic form of Parkinson disease. This work confirms William Harvey’s observation from 350 years ago that studying rarer forms of a disease is an excellent way to understand the more common forms of that disease. The identification of synuclein’s role in hereditary Parkinson disease has opened new avenues of research into the pathogenesis and potential treatments of the common form of Parkinson disease that affects many millions of Americans and tens of millions of human beings worldwide. PMID:28282812

  15. Rotenone Upregulates Alpha-Synuclein and Myocyte Enhancer Factor 2D Independently from Lysosomal Degradation Inhibition

    PubMed Central

    Stefanoni, Giovanni; Melchionda, Laura; Riva, Chiara; Brighina, Laura

    2013-01-01

    Dysfunctions of chaperone-mediated autophagy (CMA), the main catabolic pathway for alpha-synuclein, have been linked to the pathogenesis of Parkinson's disease (PD). Since till now there is limited information on how PD-related toxins may affect CMA, in this study we explored the effect of mitochondrial complex I inhibitor rotenone on CMA substrates, alpha-synuclein and MEF2D, and effectors, lamp2A and hsc70, in a human dopaminergic neuroblastoma SH-SY5Y cell line. Rotenone induced an upregulation of alpha-synuclein and MEF2D protein levels through the stimulation of their de novo synthesis rather than through a reduction of their CMA-mediated degradation. Moreover, increased MEF2D transcription resulted in higher nuclear protein levels that exert a protective role against mitochondrial dysfunction and oxidative stress. These results were compared with those obtained after lysosome inhibition with ammonium chloride. As expected, this toxin induced the cytosolic accumulation of both alpha-synuclein and MEF2D proteins, as the result of the inhibition of their lysosome-mediated degradation, while, differently from rotenone, ammonium chloride decreased MEF2D nuclear levels through the downregulation of its transcription, thus reducing its protective function. These results highlight that rotenone affects alpha-synuclein and MEF2D protein levels through a mechanism independent from lysosomal degradation inhibition. PMID:23984410

  16. Small heat shock proteins protect against {alpha}-synuclein-induced toxicity and aggregation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Outeiro, Tiago Fleming; Klucken, Jochen; Strathearn, Katherine E.

    Protein misfolding and inclusion formation are common events in neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD) or Huntington's disease (HD). {alpha}-Synuclein (aSyn) is the main protein component of inclusions called Lewy bodies (LB) which are pathognomic of PD, Dementia with Lewy bodies (DLB), and other diseases collectively known as LB diseases. Heat shock proteins (HSPs) are one class of the cellular quality control system that mediate protein folding, remodeling, and even disaggregation. Here, we investigated the role of the small heat shock proteins Hsp27 and {alpha}B-crystallin, in LB diseases. We demonstrate, via quantitative PCR, that Hsp27 messengermore » RNA levels are {approx}2-3-fold higher in DLB cases compared to control. We also show a corresponding increase in Hsp27 protein levels. Furthermore, we found that Hsp27 reduces aSyn-induced toxicity by {approx}80% in a culture model while {alpha}B-crystallin reduces toxicity by {approx}20%. In addition, intracellular inclusions were immunopositive for endogenous Hsp27, and overexpression of this protein reduced aSyn aggregation in a cell culture model.« less

  17. The Interplay between Alpha-Synuclein Clearance and Spreading

    PubMed Central

    Lopes da Fonseca, Tomás; Villar-Piqué, Anna; Outeiro, Tiago Fleming

    2015-01-01

    Parkinson’s Disease (PD) is a complex neurodegenerative disorder classically characterized by movement impairment. Pathologically, the most striking features of PD are the loss of dopaminergic neurons and the presence of intraneuronal protein inclusions primarily composed of alpha-synuclein (α-syn) that are known as Lewy bodies and Lewy neurites in surviving neurons. Though the mechanisms underlying the progression of PD pathology are unclear, accumulating evidence suggests a prion-like spreading of α-syn pathology. The intracellular homeostasis of α-syn requires the proper degradation of the protein by three mechanisms: chaperone-mediated autophagy, macroautophagy and ubiquitin-proteasome. Impairment of these pathways might drive the system towards an alternative clearance mechanism that could involve its release from the cell. This increased release to the extracellular space could be the basis for α-syn propagation to different brain areas and, ultimately, for the spreading of pathology and disease progression. Here, we review the interplay between α-syn degradation pathways and its intercellular spreading. The understanding of this interplay is indispensable for obtaining a better knowledge of the molecular basis of PD and, consequently, for the design of novel avenues for therapeutic intervention. PMID:25874605

  18. Dopamine induces soluble α-synuclein oligomers and nigrostriatal degeneration

    PubMed Central

    Mor, Danielle E.; Tsika, Elpida; Mazzulli, Joseph R.; Gould, Neal S.; Kim, Hanna; Daniels, Malcolm J.; Doshi, Shachee; Gupta, Preetika; Grossman, Jennifer L.; Tan, Victor X.; Kalb, Robert G.; Caldwell, Kim A.; Caldwell, Guy A.; Wolfe, John H.; Ischiropoulos, Harry

    2018-01-01

    Parkinson’s disease is defined by the loss of dopaminergic neurons in the substantia nigra and formation of Lewy body inclusions containing aggregated α-synuclein. Efforts to explain dopamine neuron vulnerability are hindered by the lack of dopaminergic cell death in α-synuclein transgenic mice. To address this, we manipulated dopamine levels in addition to α-synuclein expression. Nigra-targeted expression of mutant tyrosine hydroxylase with enhanced catalytic activity increased dopamine without damaging neurons in non-transgenic mice. In contrast, raising dopamine in mice expressing human A53T mutant α-synuclein induced progressive nigrostriatal degeneration and reduced locomotion. Dopamine elevation in A53T mice increased levels of potentially toxic α-synuclein oligomers, resulting in conformationally and functionally modified species. Moreover, in genetically tractable C. elegans models expression of α-synuclein mutated at the site of interaction with dopamine prevented dopamine-induced toxicity. The data suggest a unique mechanism linking two cardinal features of Parkinson’s disease, dopaminergic cell death and α-synuclein aggregation. PMID:28920936

  19. Dopamine induces soluble α-synuclein oligomers and nigrostriatal degeneration.

    PubMed

    Mor, Danielle E; Tsika, Elpida; Mazzulli, Joseph R; Gould, Neal S; Kim, Hanna; Daniels, Malcolm J; Doshi, Shachee; Gupta, Preetika; Grossman, Jennifer L; Tan, Victor X; Kalb, Robert G; Caldwell, Kim A; Caldwell, Guy A; Wolfe, John H; Ischiropoulos, Harry

    2017-11-01

    Parkinson's disease (PD) is defined by the loss of dopaminergic neurons in the substantia nigra and the formation of Lewy body inclusions containing aggregated α-synuclein. Efforts to explain dopamine neuron vulnerability are hindered by the lack of dopaminergic cell death in α-synuclein transgenic mice. To address this, we manipulated both dopamine levels and α-synuclein expression. Nigrally targeted expression of mutant tyrosine hydroxylase with enhanced catalytic activity increased dopamine levels without damaging neurons in non-transgenic mice. In contrast, raising dopamine levels in mice expressing human A53T mutant α-synuclein induced progressive nigrostriatal degeneration and reduced locomotion. Dopamine elevation in A53T mice increased levels of potentially toxic α-synuclein oligomers, resulting in conformationally and functionally modified species. Moreover, in genetically tractable Caenorhabditis elegans models, expression of α-synuclein mutated at the site of interaction with dopamine prevented dopamine-induced toxicity. These data suggest that a unique mechanism links two cardinal features of PD: dopaminergic cell death and α-synuclein aggregation.

  20. Overexpression of {alpha}-catenin increases osteoblastic differentiation in mouse mesenchymal C3H10T1/2 cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kim, Dohee; Yang, Jae-Yeon; Shin, Chan Soo, E-mail: csshin@snu.ac.kr

    2009-05-15

    {alpha}- and {beta}-Catenin link cadherins to the actin-based cytoskeleton at adherens junctions and regulate cell-cell adhesion. Although roles of cadherins and canonical Wnt-/{beta}-catenin-signaling in osteoblastic differentiation have been extensively studied, the role of {alpha}-catenin is not known. Murine embryonic mesenchymal stem cells, C3H10T1/2 cells, were transduced with retrovirus encoding {alpha}-catenin (MSCV-{alpha}-catenin-HA-GFP). In the presence of Wnt-3A conditioned medium or osteogenic medium ({beta}-glycerol phosphate and ascorbic acid), cells overexpressing {alpha}-catenin showed enhanced osteoblastic differentiation as measured by alkaline phosphatase (ALP) staining and ALP activity assay compared to cells transduced with empty virus (MSCV-GFP). In addition, mRNA expression of osteocalcin and Runx2more » was significantly increased compared to control. Cell aggregation assay revealed that {alpha}-catenin overexpression has significantly increased cell-cell aggregation. However, cellular {beta}-catenin levels (total, cytoplasmic-nuclear ratio) and {beta}-catenin-TCF/LEF transcriptional activity did not change by overexpression of {alpha}-catenin. Knock-down of {alpha}-catenin using siRNA decreased osteoblastic differentiation as measured by ALP assay. These results suggest that {alpha}-catenin overexpression increases osteoblastic differentiation by increasing cell-cell adhesion rather than Wnt-/{beta}-catenin-signaling.« less

  1. Formation and implications of alpha-synuclein radical in Maneb- and paraquat-induced models of Parkinson’s disease

    PubMed Central

    Kumar, Ashutosh; Leinisch, Fabian; Kadiiska, Maria B.; Corbett, Jean; Mason, Ronald P.

    2015-01-01

    Parkinson’s disease (PD) is a debilitating, progressive, neurodegenerative disorder characterized by progressive loss of dopaminergic neurons and motor deficits. Alpha-synuclein-containing aggregates represent a feature of a variety of neurodegenerative disorders, including PD; however, the mechanism that initiates and promotes intraneuronal alpha-synuclein aggregation remains unknown. We hypothesized protein radical formation as an initiating mechanism for alpha-synuclein aggregation. Therefore, we used the highly sensitive immuno-spin trapping technique to investigate protein radical formation as a possible mechanism of alpha-synuclein aggregation as well as to investigate the source of protein radical formation in the midbrains of Maneb and paraquat coexposed mice. Coexposure to Maneb and paraquat for 6 weeks resulted in active microgliosis, NADPH oxidase activation, and inducible nitric oxide synthase (iNOS) induction, which culminated in protein radical formation in the midbrains of mice. Results obtained with immuno-spin trapping and immunoprecipitation experiments confirmed formation of alpha-synuclein radicals in dopaminergic neurons of exposed mice. Free radical formation requires NADPH oxidase and iNOS, as indicated by decreased protein radical formation in knockout mice (P47phox−/− and iNOS−/−) and in mice treated with inhibitors such as FeTPPS (a peroxynitrite decomposition catalyst), 1400W (an iNOS inhibitor), or apocynin (a NADPH oxidase inhibitor). Concurrence of protein radical formation with dopaminergic neuronal death indicated a link between protein radicals and disease progression. Taken together, these results show for the first time the formation and detection of the alpha-synuclein radical and suggest that NADPH oxidase and iNOS play roles in peroxynitrite-mediated protein radical formation and subsequent neuronal death in the midbrains of Maneb and paraquat coexposed mice. PMID:25952542

  2. Pesticide Exposure Exacerbates α-Synucleinopathy in an A53T Transgenic Mouse Model

    PubMed Central

    Norris, Erin H.; Uryu, Kunihiro; Leight, Susan; Giasson, Benoit I.; Trojanowski, John Q.; Lee, Virginia M.-Y.

    2007-01-01

    The factors initiating or contributing to the pathogenesis of Parkinson’s disease and related neurodegenerative synucleinopathies are still largely unclear, but environmental factors such as pesticides have been implicated. In this study, A53T mutant human α-synuclein transgenic mice (M83), which develop α-synuclein neuropathology, were treated with the pesticides paraquat and maneb (either singly or together), and their effects were analyzed. Immunohistochemical and biochemical analyses showed that chronic treatment of M83 transgenic mice with both pesticides (but not with either pesticide alone) drastically increased neuronal α-synuclein pathology throughout the central nervous system including the hippocampus, cerebellum, and sensory and auditory cortices. α-Synuclein-associated mitochondrial degeneration was observed in M83 but not in wild-type α-synuclein transgenic mice. Because α-synuclein inclusions accumulated in pesticide-exposed M83 transgenic mice without a motor phenotype, we conclude that α-synuclein aggregate formation precedes disease onset. These studies support the notion that environmental factors causing nitrative damage are closely linked to mechanisms underlying the formation of α-synuclein pathologies and the onset of Parkinson’s-like neurodegeneration. PMID:17255333

  3. Over-expression of C/EBP-{alpha} induces apoptosis in cultured rat hepatic stellate cells depending on p53 and peroxisome proliferator-activated receptor-{gamma}

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang Xueqing; Huang Guangcun; Mei Shuang

    2009-03-06

    Hepatic stellate cells (HSCs) play a key role in the pathogenesis of hepatic fibrosis. In our previous studies, CCAAT enhancer binding protein-{alpha} (C/EBP-{alpha}) has been shown to be involved in the activation of HSCs and to have a repression effect on hepatic fibrosis in vivo. However, the mechanisms are largely unknown. In this study, we show that the infection of adenovirus vector expressing C/EBP-{alpha} gene (Ad-C/EBP-{alpha}) could induce HSCs apoptosis in a dose- and time-dependent manner by Annexin V/PI staining, caspase-3 activation assay, and flow cytometry. Also, over-expression of C/EBP-{alpha} resulted in the up-regulation of peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) andmore » P53, while P53 expression was regulated by PPAR-{gamma}. In addition, Fas, FasL, DR4, DR5, and TRAIL were studied. The results indicated that the death receptor pathway was mainly involved and regulated by PPAR-{gamma} and p53 in the process of apoptosis triggered by C/EBP-{alpha} in HSCs.« less

  4. A microRNA embedded AAV alpha-synuclein gene silencing vector for dopaminergic neurons

    PubMed Central

    Han, Ye; Khodr, Christina E.; Sapru, Mohan K.; Pedapati, Jyothi; Bohn, Martha C.

    2011-01-01

    Alpha-synuclein (SNCA), an abundantly expressed presynaptic protein, is implicated in Parkinson disease (PD). Since over-expression of human SNCA (hSNCA) leads to death of dopaminergic (DA) neurons in human, rodent and fly brain, hSNCA gene silencing may reduce levels of toxic forms of SNCA and ameliorate degeneration of DA neurons in PD. To begin to develop a gene therapy for PD based on hSNCA gene silencing, two AAV gene silencing vectors were designed, and tested for efficiency and specificity of silencing, as well as toxicity in vitro. The same hSNCA silencing sequence (shRNA) was used in both vectors, but in one vector, the shRNA was embedded in a microRNA backbone and driven by a pol II promoter, and in the other the shRNA was not embedded in a microRNA and was driven by a pol III promoter. Both vectors silenced hSNCA to the same extent in 293T cells transfected with hSNCA. In DA PC12 cells, neither vector decreased expression of rat SNCA, tyrosine hydroxylase (TH), dopamine transporter (DAT) or the vesicular monoamine transporter (VMAT). However, the mir30 embedded vector was significantly less toxic to both PC12 and SH-SY5Y cells. Our in vitro data suggest that this miRNA-embedded silencing vector may be ideal for chronic in vivo SNCA gene silencing in DA neurons. PMID:21338582

  5. Quantitative measurement of intact alpha-synuclein proteoforms from post-mortem control and Parkinson's disease brain tissue by intact protein mass spectrometry.

    PubMed

    Kellie, John F; Higgs, Richard E; Ryder, John W; Major, Anthony; Beach, Thomas G; Adler, Charles H; Merchant, Kalpana; Knierman, Michael D

    2014-07-23

    A robust top down proteomics method is presented for profiling alpha-synuclein species from autopsied human frontal cortex brain tissue from Parkinson's cases and controls. The method was used to test the hypothesis that pathology associated brain tissue will have a different profile of post-translationally modified alpha-synuclein than the control samples. Validation of the sample processing steps, mass spectrometry based measurements, and data processing steps were performed. The intact protein quantitation method features extraction and integration of m/z data from each charge state of a detected alpha-synuclein species and fitting of the data to a simple linear model which accounts for concentration and charge state variability. The quantitation method was validated with serial dilutions of intact protein standards. Using the method on the human brain samples, several previously unreported modifications in alpha-synuclein were identified. Low levels of phosphorylated alpha synuclein were detected in brain tissue fractions enriched for Lewy body pathology and were marginally significant between PD cases and controls (p = 0.03).

  6. Regional deficiencies in chaperone-mediated autophagy underlie α-synuclein aggregation and neurodegeneration

    PubMed Central

    Malkus, Kristen A.; Ischiropoulos, Harry

    2012-01-01

    In neurodegenerative diseases, it remains unclear why certain brain regions are selectively vulnerable to protein aggregation. In transgenic mice expressing human A53T α-synuclein, the brainstem and spinal cord develop the most prominent α-synuclein inclusions which correlate with age-dependent motor dysfunction. Herein we present the novel finding that this selective aggregation is in part dependent on the inability of chaperone-mediated autophagy (CMA) to effectively degrade α-synuclein in these brain regions. Lysosomal assays revealed that CMA activity was significantly decreased in aggregation-prone regions compared to the remainder of the brain. Previously, CMA activity has been shown to be proportional to levels of the CMA receptor Lamp-2a. Using antibodies, brain tissue from Lamp-2a null mice, enzymatic deglycosylation, and mass spectrometry, we identified Lamp2a as a novel 72 kDa glycoprotein in the mouse brain. Examination of Lamp-2a levels revealed differences in expression across brain regions. The brainstem and the spinal cord had a more than three-fold greater levels of Lamp-2a as compared to regions less vulnerable to aggregation and exhibited a selective upregulation of Lamp-2a during development of α-synuclein inclusions. Despite this dynamic response of Lamp-2a, the levels of substrates bound to the brain lysosomes as well as the rates of substrate uptake and degradation were not proportional to the levels of Lamp-2a. These regional differences in CMA activity and Lamp-2a expression were found in both non-transgenic mice as well as A53T α-syn mice. Therefore, these are inherent variations and not a transgene-specific effect. However, differences in CMA activity may render select brain regions vulnerable to homeostatic dysfunction in the presence of stressors such as overexpression of human A53T α-syn. Collectively, the data provide a potential mechanism to explain the dichotomy of vulnerability or resistance that underlies brain regions

  7. Diffusion Kurtosis Imaging Detects Microstructural Alterations in Brain of α-Synuclein Overexpressing Transgenic Mouse Model of Parkinson's Disease: A Pilot Study.

    PubMed

    Khairnar, Amit; Latta, Peter; Drazanova, Eva; Ruda-Kucerova, Jana; Szabó, Nikoletta; Arab, Anas; Hutter-Paier, Birgit; Havas, Daniel; Windisch, Manfred; Sulcova, Alexandra; Starcuk, Zenon; Rektorova, Irena

    2015-11-01

    Evidence suggests that accumulation and aggregation of α-synuclein contribute to the pathogenesis of Parkinson's disease (PD). The aim of this study was to evaluate whether diffusion kurtosis imaging (DKI) will provide a sensitive tool for differentiating between α-synuclein-overexpressing transgenic mouse model of PD (TNWT-61) and wild-type (WT) littermates. This experiment was designed as a proof-of-concept study and forms a part of a complex protocol and ongoing translational research. Nine-month-old TNWT-61 mice and age-matched WT littermates underwent behavioral tests to monitor motor impairment and MRI scanning using 9.4 Tesla system in vivo. Tract-based spatial statistics (TBSS) and the DKI protocol were used to compare the whole brain white matter of TNWT-61 and WT mice. In addition, region of interest (ROI) analysis was performed in gray matter regions such as substantia nigra, striatum, hippocampus, sensorimotor cortex, and thalamus known to show higher accumulation of α-synuclein. For the ROI analysis, both DKI (6 b-values) protocol and conventional (2 b-values) diffusion tensor imaging (cDTI) protocol were used. TNWT-61 mice showed significant impairment of motor coordination. With the DKI protocol, mean, axial, and radial kurtosis were found to be significantly elevated, whereas mean and radial diffusivity were decreased in the TNWT-61 group compared to that in the WT controls with both TBSS and ROI analysis. With the cDTI protocol, the ROI analysis showed decrease in all diffusivity parameters in TNWT-61 mice. The current study provides evidence that DKI by providing both kurtosis and diffusivity parameters gives unique information that is complementary to cDTI for in vivo detection of pathological changes that underlie PD-like symptomatology in TNWT-61 mouse model of PD. This result is a crucial step in search for a candidate diagnostic biomarker with translational potential and relevance for human studies.

  8. Plasma α-synuclein and cognitive impairment in the Parkinson's Associated Risk Syndrome: A pilot study.

    PubMed

    Wang, Hua; Atik, Anzari; Stewart, Tessandra; Ginghina, Carmen; Aro, Patrick; Kerr, Kathleen F; Seibyl, John; Jennings, Danna; Jensen, Poul Henning; Marek, Kenneth; Shi, Min; Zhang, Jing

    2018-04-27

    Plasma total and nervous system derived exosomal (NDE) α-synuclein have been determined as potential biomarkers of Parkinson's disease (PD). To explore the utility of plasma α-synuclein in the prodromal phase of PD, plasma total and NDE α-synuclein were evaluated in baseline and 2-year follow-up samples from 256 individuals recruited as part of the Parkinson's Associated Risk Syndrome (PARS) study. The results demonstrated that baseline and longitudinal increases in total α-synuclein predicted progression of cognitive decline in hyposmic individuals with dopamine transporter (DAT) binding reduction. On the other hand, a longitudinal decrease in NDE α-synuclein predicted worsening cognitive scores in hyposmic individuals with DAT binding reduction. Finally, in individuals with faster DAT progression, decreasing NDE/total α-synuclein ratio was associated with a larger reduction in DAT from baseline to follow-up. These results suggest that, though underlying mechanisms remain to be defined, alterations in plasma total and NDE α-synuclein concentrations are likely associated with PD progression, especially in the aspect of cognitive impairment, at early stages of the disease. Copyright © 2018. Published by Elsevier Inc.

  9. Overexpression of hypoxia-inducible factor 1 alpha improves immunomodulation by dental mesenchymal stem cells.

    PubMed

    Martinez, Victor G; Ontoria-Oviedo, Imelda; Ricardo, Carolina P; Harding, Sian E; Sacedon, Rosa; Varas, Alberto; Zapata, Agustin; Sepulveda, Pilar; Vicente, Angeles

    2017-09-29

    Human dental mesenchymal stem cells (MSCs) are considered as highly accessible and attractive MSCs for use in regenerative medicine, yet some of their features are not as well characterized as other MSCs. Hypoxia-preconditioning and hypoxia-inducible factor 1 (HIF-1) alpha overexpression significantly improves MSC therapeutics, but the mechanisms involved are not fully understood. In the present study, we characterize immunomodulatory properties of dental MSCs and determine changes in their ability to modulate adaptive and innate immune populations after HIF-1 alpha overexpression. Human dental MSCs were stably transduced with green fluorescent protein (GFP-MSCs) or GFP-HIF-1 alpha lentivirus vectors (HIF-MSCs). A hypoxic-like metabolic profile was confirmed by mitochondrial and glycolysis stress test. Capacity of HIF-MSCs to modulate T-cell activation, dendritic cell differentiation, monocyte migration, and polarizations towards macrophages and natural killer (NK) cell lytic activity was assessed by a number of functional assays in co-cultures. The expression of relevant factors were determined by polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA). While HIF-1 alpha overexpression did not modify the inhibition of T-cell activation by MSCs, HIF-MSCs impaired dendritic cell differentiation more efficiently. In addition, HIF-MSCs showed a tendency to induce higher attraction of monocytes, which differentiate into suppressor macrophages, and exhibited enhanced resistance to NK cell-mediated lysis, which supports the improved therapeutic capacity of HIF-MSCs. HIF-MSCs also displayed a pro-angiogenic profile characterized by increased expression of CXCL12/SDF1 and CCL5/RANTES and complete loss of CXCL10/IP10 transcription. Immunomodulation and expression of trophic factors by dental MSCs make them perfect candidates for cell therapy. Overexpression of HIF-1 alpha enhances these features and increases their resistance to allogenic NK

  10. Comparison of kinetics, toxicity, oligomers formation and membrane binding capacity of α-synuclein familial mutations at A53 site including newly discovered A53V mutation.

    PubMed

    Mohite, Ganesh M; Kumar, Rakesh; Panigrahi, Rajlaxmi; Navalkar, Ambuja; Singh, Nitu; Datta, Debalina; Mehra, Surabhi; Ray, Soumik; Gadhe, Laxmikant G; Das, Subhadeep; Singh, Namrata; Chatterjee, Debdeep; Kumar, Ashutosh; Maji, Samir K

    2018-05-17

    The involvement of α-synuclein (α-Syn) amyloid formation in Parkinson's disease (PD) pathogenesis is supported by the discovery of α-Syn gene (SNCA) mutations linked with familial PD, which are known to modulate the oligomerization and aggregation of α-Syn. Recently, the A53V mutation has been discovered, which leads to the late-onset PD. In the present study, we characterized for the first time the biophysical properties including the aggregation propensities, toxicity of aggregated species and membrane binding capability of A53V along with all familial mutations at A53 position. Present data suggest that A53V accelerate fibrillation of α-Syn without affecting the overall morphology and cytotoxicity of fibrils compared to wild-type protein. The aggregation propensity for A53 mutants is found to be; A53T>A53V>WT>A53E. Further, time course aggregation study reveals that A53V mutant promotes early oligomerization similar to A53T mutation. It promotes the highest amount of oligomer formation immediate after dissolution, which are cytotoxic. Although in the presence of membrane-mimicking environments, A53V mutation showed similar extent of helix-induction capacity as of WT protein, however, it exhibited lesser binding to lipid vesicle. The NMR study revealed unique chemical shift perturbation by A53V mutation com-pared to other mutations at A53 site. The present study might help to establish the disease-causing mechanism of A53V in PD pathology.

  11. A role of BAG3 in regulating SNCA/α-synuclein clearance via selective macroautophagy.

    PubMed

    Cao, Yu-Lan; Yang, Ya-Ping; Mao, Cheng-Jie; Zhang, Xiao-Qi; Wang, Chen-Tao; Yang, Jing; Lv, Dong-Jun; Wang, Fen; Hu, Li-Fang; Liu, Chun-Feng

    2017-12-01

    Many studies reveal that BAG3 plays a critical role in the regulation of protein degradation via macroautophagy. However, it remains unknown whether BAG3 affects the quality control of α-synuclein (SNCA), a Parkinson's disease-related protein. In this study, we demonstrated the increases of BAG3 expression in the ventral midbrain of SNCA A53T transgenic mice and also in MG132-treated PC12 cells overexpressing wild-type SNCA (SNCA WT -PC12). Moreover, we showed that BAG3 overexpression was sufficient to enhance the autophagy activity while knockdown of Bag3 reduced it in SNCA WT -PC12 cells. Immunoprecipitation revealed that BAG3 interacted with heat shock protein 70 and sequestosome 1. The immunostaining also showed the perinuclear accumulation and colocalization of BAG3 with these 2 proteins, as well as with LC3 dots in tyrosine hydroxylase-positive neurons in the midbrain of SNCA A53T mice. BAG3 overexpression was able to modulate SNCA degradation via macroautophagy which was prevented by Atg5 knockdown. Taken together, these results indicate that BAG3 plays a relevant role in regulating SNCA clearance via macroautophagy, and the heat shock protein 70-BAG3-sequestosome 1 complex may be involved in this process. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. A prolyl oligopeptidase inhibitor, KYP-2047, reduces α-synuclein protein levels and aggregates in cellular and animal models of Parkinson's disease

    PubMed Central

    Myöhänen, TT; Hannula, MJ; Van Elzen, R; Gerard, M; Van Der Veken, P; García-Horsman, JA; Baekelandt, V; Männistö, PT; Lambeir, AM

    2012-01-01

    BACKGROUND AND PURPOSE The aggregation of α-synuclein is connected to the pathology of Parkinson's disease and prolyl oligopeptidase (PREP) accelerates the aggregation of α-synuclein in vitro. The aim of this study was to investigate the effects of a PREP inhibitor, KYP-2047, on α-synuclein aggregation in cell lines overexpressing wild-type or A30P/A53T mutant human α-syn and in the brains of two A30P α-synuclein transgenic mouse strains. EXPERIMENTAL APPROACH Cells were exposed to oxidative stress and then incubated with the PREP inhibitor during or after the stress. Wild-type or transgenic mice were treated for 5 days with KYP-2047 (2 × 3 mg·kg−1 a day). Besides immunohistochemistry and thioflavin S staining, soluble and insoluble α-synuclein protein levels were measured by Western blot. α-synuclein mRNA levels were quantified by PCR. The colocalization of PREP and α-synuclein,and the effect of KYP-2047 on cell viability were also investigated. KEY RESULTS In cell lines, oxidative stress induced a robust aggregation of α-synuclein,and low concentrations of KYP-2047 significantly reduced the number of cells with α-synuclein inclusions while abolishing the colocalization of α-synuclein and PREP. KYP-2047 significantly reduced the amount of aggregated α-synuclein,and it had beneficial effects on cell viability. In the transgenic mice, a 5-day treatment with the PREP inhibitor reduced the amount of α-synuclein immunoreactivity and soluble α-synuclein protein in the brain. CONCLUSIONS AND IMPLICATIONS The results suggest that the PREP may play a role in brain accumulation and aggregation of α-synuclein, while KYP-2047 seems to effectively prevent these processes. PMID:22233220

  13. Ageing enhances alpha-synuclein, ubiquitin and endoplasmic reticular stress protein expression in the nigral neurons of Asian Indians.

    PubMed

    Alladi, Phalguni Anand; Mahadevan, Anita; Vijayalakshmi, K; Muthane, Uday; Shankar, S K; Raju, T R

    2010-11-01

    Accumulating evidences suggest that dopaminergic neuronal loss in the substantia nigra pars compacta (SNpc) during ageing and in Parkinson's disease (PD) is linked to neurodegenerative changes like exponential increase in alpha-synuclein expression and protein misfolding. Lewy body formation is also a quintessential observation in neurodegeneration and PD. In experimental models of PD, GRP78 a neuroprotective endoplasmic reticulum (ER) chaperone protein targets misfolded proteins for degradation and prevents release of caspase12 from the ER. Release of active caspase12 and its translocation to the nucleus induces ER mediated apoptosis. The effect of ageing on these proteins in human nigra is not known. We evaluated alpha-synuclein, caspase12, GRP78 and ubiquitin expression in the SNpc of Asian Indians, using immunohistochemistry and stereology. The number of alpha-synuclein and caspase12 immunoreactive neurons increased gradually with age whereas the number of GRP78-labeled neurons remained stable. In contrast, GRP78 protein expression was significantly upregulated with age, while alpha-synuclein and caspase12 increased slightly. An increase in the size and numbers of marinesco bodies was prominent after the sixth decade. The mild increase in alpha-synuclein expression and occurrence of marinesco bodies suggests ageing induced protein misfolding and GRP78 upregulation indicates presence of ER stress. The logarithmic upregulation of GRP78 could even be an indicator of neuroprotective or neuromodulatory response of ER to protein misfolding and initiation of unfolded protein response pathway. Since dopaminergic neurons are preserved in ageing Asian Indians, our study possibly signifies better proteasomal or ER response and partially explains the lower prevalence of PD in them. Copyright 2010 Elsevier Ltd. All rights reserved.

  14. Alpha-synuclein, epigenetics, mitochondria, metabolism, calcium traffic, & circadian dysfunction in Parkinson's disease. An integrated strategy for management.

    PubMed

    Phillipson, Oliver T

    2017-11-01

    The motor deficits which characterise the sporadic form of Parkinson's disease arise from age-related loss of a subset of dopamine neurons in the substantia nigra. Although motor symptoms respond to dopamine replacement therapies, the underlying disease process remains. This review details some features of the progressive molecular pathology and proposes deployment of a combination of nutrients: R-lipoic acid, acetyl-l-carnitine, ubiquinol, melatonin (or receptor agonists) and vitamin D3, with the collective potential to slow progression of these features. The main nutrient targets include impaired mitochondria and the associated oxidative/nitrosative stress, calcium stress and impaired gene transcription induced by pathogenic forms of alpha- synuclein. Benefits may be achieved via nutrient influence on epigenetic signaling pathways governing transcription factors for mitochondrial biogenesis, antioxidant defences and the autophagy-lysosomal pathway, via regulation of the metabolic energy sensor AMP activated protein kinase (AMPK) and the mammalian target of rapamycin mTOR. Nutrients also benefit expression of the transcription factor for neuronal survival (NR4A2), trophic factors GDNF and BDNF, and age-related calcium signals. In addition a number of non-motor related dysfunctions in circadian control, clock genes and associated metabolic, endocrine and sleep-wake activity are briefly addressed, as are late-stage complications in respect of cognitive decline and osteoporosis. Analysis of the network of nutrient effects reveals how beneficial synergies may counter the accumulation and promote clearance of pathogenic alpha-synuclein. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  15. Monomeric Alpha-Synuclein Exerts a Physiological Role on Brain ATP Synthase

    PubMed Central

    Ludtmann, Marthe H.R.; Angelova, Plamena R.; Ninkina, Natalia N.; Gandhi, Sonia

    2016-01-01

    Misfolded α-synuclein is a key factor in the pathogenesis of Parkinson's disease (PD). However, knowledge about a physiological role for the native, unfolded α-synuclein is limited. Using brains of mice lacking α-, β-, and γ-synuclein, we report that extracellular monomeric α-synuclein enters neurons and localizes to mitochondria, interacts with ATP synthase subunit α, and modulates ATP synthase function. Using a combination of biochemical, live-cell imaging and mitochondrial respiration analysis, we found that brain mitochondria of α-, β-, and γ-synuclein knock-out mice are uncoupled, as characterized by increased mitochondrial respiration and reduced mitochondrial membrane potential. Furthermore, synuclein deficiency results in reduced ATP synthase efficiency and lower ATP levels. Exogenous application of low unfolded α-synuclein concentrations is able to increase the ATP synthase activity that rescues the mitochondrial phenotypes observed in synuclein deficiency. Overall, the data suggest that α-synuclein is a previously unrecognized physiological regulator of mitochondrial bioenergetics through its ability to interact with ATP synthase and increase its efficiency. This may be of particular importance in times of stress or PD mutations leading to energy depletion and neuronal cell toxicity. SIGNIFICANCE STATEMENT Misfolded α-synuclein aggregations in the form of Lewy bodies have been shown to be a pathological hallmark in histological staining of Parkinson's disease (PD) patient brains. It is known that misfolded α-synuclein is a key driver in PD pathogenesis, but the physiological role of unfolded monomeric α-synuclein remains unclear. Using neuronal cocultures and isolated brain mitochondria of α-, β-, and γ-synuclein knock-out mice and monomeric α-synuclein, this current study shows that α-synuclein in its unfolded monomeric form improves ATP synthase efficiency and mitochondrial function. The ability of monomeric α-synuclein to enhance

  16. Dementia with Lewy bodies in an elderly Greek male due to alpha-synuclein gene mutation.

    PubMed

    Morfis, Litsa; Cordato, Dennis John

    2006-11-01

    We report the case of an elderly man of Greek background who presented with progressive cognitive decline and motor parkinsonism on a background of a strong family history of Parkinson's disease. Associated symptoms included visual hallucinations, excessive daytime drowsiness, recurrent falls, orthostatic hypotension and urinary incontinence. His major clinical symptoms and signs fulfilled consensus criteria for a clinical diagnosis of dementia with Lewy bodies. An alpha-synuclein gene mutation analysis for the G209A substitution was positive. We conclude that the alpha-synuclein (G209A) gene mutation genotype should be considered in the differential diagnosis of dementia with Lewy bodies, particularly in patients with European ancestry and a family history of Parkinson's disease.

  17. Curcumin Treatment Improves Motor Behavior in α-Synuclein Transgenic Mice

    PubMed Central

    Spinelli, Kateri J.; Osterberg, Valerie R.; Meshul, Charles K.; Soumyanath, Amala; Unni, Vivek K.

    2015-01-01

    The curry spice curcumin plays a protective role in mouse models of neurodegenerative diseases, and can also directly modulate aggregation of α-synuclein protein in vitro, yet no studies have described the interaction of curcumin and α-synuclein in genetic synucleinopathy mouse models. Here we examined the effect of chronic and acute curcumin treatment in the Syn-GFP mouse line, which overexpresses wild-type human α-synuclein protein. We discovered that curcumin diet intervention significantly improved gait impairments and resulted in an increase in phosphorylated forms of α-synuclein at cortical presynaptic terminals. Acute curcumin treatment also caused an increase in phosphorylated α-synuclein in terminals, but had no direct effect on α-synuclein aggregation, as measured by in vivo multiphoton imaging and Proteinase-K digestion. Using LC-MS/MS, we detected ~5 ng/mL and ~12 ng/mL free curcumin in the plasma of chronic or acutely treated mice, with a glucuronidation rate of 94% and 97%, respectively. Despite the low plasma levels and extensive metabolism of curcumin, these results show that dietary curcumin intervention correlates with significant behavioral and molecular changes in a genetic synucleinopathy mouse model that mimics human disease. PMID:26035833

  18. Folate receptor {alpha} regulates cell proliferation in mouse gonadotroph {alpha}T3-1 cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yao, Congjun; Evans, Chheng-Orn; Stevens, Victoria L.

    We have previously found that the mRNA and protein levels of the folate receptor alpha (FR{alpha}) are uniquely over-expressed in clinically human nonfunctional (NF) pituitary adenomas, but the mechanistic role of FR{alpha} has not fully been determined. We investigated the effect of FR{alpha} over-expression in the mouse gonadotroph {alpha}T3-1 cell line as a model for NF pituitary adenomas. We found that the expression and function of FR{alpha} were strongly up-regulated, by Western blotting and folic acid binding assay. Furthermore, we found a higher cell growth rate, an enhanced percentage of cells in S-phase by BrdU assay, and a higher PCNAmore » staining. These observations indicate that over-expression of FR{alpha} promotes cell proliferation. These effects were abrogated in the same {alpha}T3-1 cells when transfected with a mutant FR{alpha} cDNA that confers a dominant-negative phenotype by inhibiting folic acid binding. Finally, by real-time quantitative PCR, we found that mRNA expression of NOTCH3 was up-regulated in FR{alpha} over-expressing cells. In summary, our data suggests that FR{alpha} regulates pituitary tumor cell proliferation and mechanistically may involve the NOTCH pathway. Potentially, this finding could be exploited to develop new, innovative molecular targeted treatment for human NF pituitary adenomas.« less

  19. Alpha-Synuclein: From Early Synaptic Dysfunction to Neurodegeneration.

    PubMed

    Ghiglieri, Veronica; Calabrese, Valeria; Calabresi, Paolo

    2018-01-01

    Over the last two decades, many experimental and clinical studies have provided solid evidence that alpha-synuclein (α-syn), a small, natively unfolded protein, is closely related to Parkinson's disease (PD) pathology. To provide an overview on the different roles of this protein, here we propose a synopsis of seminal and recent studies that explored the many aspects of α-syn. Ranging from the physiological functions to its neurodegenerative potential, the relationship with the possible pathogenesis of PD will be discussed. Close attention will be paid on early cellular and molecular alterations associated with the presence of α-syn aggregates.

  20. Role of Different Alpha-Synuclein Strains in Synucleinopathies, Similarities with other Neurodegenerative Diseases

    PubMed Central

    Melki, Ronald

    2015-01-01

    Abstract Misfolded protein aggregates are the hallmark of several neurodegenerative diseases in humans. The main protein constituent of these aggregates and the regions within the brain that are affected differ from one neurodegenerative disorder to another. A plethora of reports suggest that distinct diseases have in common the ability of protein aggregates to spread and amplify within the central nervous system. This review summarizes briefly what is known about the nature of the protein aggregates that are infectious and the reason they are toxic to cells. The chameleon property of polypeptides which aggregation into distinct high-molecular weight assemblies is associated to different diseases, in particular, that of alpha-synuclein which aggregation is the hallmark of distinct synucleinopathies, is discussed. Finally, strategies targeting the formation and propagation of structurally distinct alpha-synuclein assemblies associated to different synucleinopathies are presented and their therapeutic and diagnostic potential is discussed. PMID:25757830

  1. CPT1{alpha} over-expression increases long-chain fatty acid oxidation and reduces cell viability with incremental palmitic acid concentration in 293T cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jambor de Sousa, Ulrike L.; Koss, Michael D.; Fillies, Marion

    2005-12-16

    To test the cellular response to an increased fatty acid oxidation, we generated a vector for an inducible expression of the rate-limiting enzyme carnitine palmitoyl-transferase 1{alpha} (CPT1{alpha}). Human embryonic 293T kidney cells were transiently transfected and expression of the CPT1{alpha} transgene in the tet-on vector was activated with doxycycline. Fatty acid oxidation was measured by determining the conversion of supplemented, synthetic cis-10-heptadecenoic acid (C17:1n-7) to C15:ln-7. CPT1{alpha} over-expression increased mitochondrial long-chain fatty acid oxidation about 6-fold. Addition of palmitic acid (PA) decreased viability of CPT1{alpha} over-expressing cells in a concentration-dependent manner. Both, PA and CPT1{alpha} over-expression increased cell death. Interestingly,more » PA reduced total cell number only in cells over-expressing CPT1{alpha}, suggesting an effect on cell proliferation that requires PA translocation across the mitochondrial inner membrane. This inducible expression system should be well suited to study the roles of CPT1 and fatty acid oxidation in lipotoxicity and metabolism in vivo.« less

  2. In vivo silencing of alpha-synuclein using naked siRNA

    PubMed Central

    Lewis, Jada; Melrose, Heather; Bumcrot, David; Hope, Andrew; Zehr, Cynthia; Lincoln, Sarah; Braithwaite, Adam; He, Zhen; Ogholikhan, Sina; Hinkle, Kelly; Kent, Caroline; Toudjarska, Ivanka; Charisse, Klaus; Braich, Ravi; Pandey, Rajendra K; Heckman, Michael; Maraganore, Demetrius M; Crook, Julia; Farrer, Matthew J

    2008-01-01

    Background Overexpression of α-synuclein (SNCA) in families with multiplication mutations causes parkinsonism and subsequent dementia, characterized by diffuse Lewy Body disease post-mortem. Genetic variability in SNCA contributes to risk of idiopathic Parkinson's disease (PD), possibly as a result of overexpression. SNCA downregulation is therefore a valid therapeutic target for PD. Results We have identified human and murine-specific siRNA molecules which reduce SNCA in vitro. As a proof of concept, we demonstrate that direct infusion of chemically modified (naked), murine-specific siRNA into the hippocampus significantly reduces SNCA levels. Reduction of SNCA in the hippocampus and cortex persists for a minimum of 1 week post-infusion with recovery nearing control levels by 3 weeks post-infusion. Conclusion We have developed naked gene-specific siRNAs that silence expression of SNCA in vivo. This approach may prove beneficial toward our understanding of the endogenous functional equilibrium of SNCA, its role in disease, and eventually as a therapeutic strategy for α-synucleinopathies resulting from SNCA overexpression. PMID:18976489

  3. α-Synuclein staging in the amygdala of a Parkinson's disease model: cell types involved.

    PubMed

    Flores-Cuadrado, Alicia; Ubeda-Bañon, Isabel; Saiz-Sanchez, Daniel; de la Rosa-Prieto, Carlos; Martinez-Marcos, Alino

    2015-01-01

    Lewy bodies (ubiquitin and α-synuclein aggregates) can be detected in brain areas in a predictable sequence of six neuropathological stages in Parkinson's disease. Brainstem and olfactory structures are involved in stage 1, whereas the substantia nigra and amygdala are involved in stage 3, prior to cortical spreading. Amygdaloid pathology has been suggested to contribute to non-motor symptoms such as olfactory dysfunction and emotional impairment. This work analysed the distribution of α-synuclein at 16, 30, 43 and 56 weeks in the basolateral, central and cortical amygdaloid complexes of A53T transgenic mice. The expression of calbindin, calretinin and somatostatin was compared in control and transgenic animals. Co-localisation of these markers with α-synuclein was performed. Triple labeling of calbindin, somatostatin and α-synuclein was also investigated. Quantification was carried out using an optical dissector, ImageJ software and confocal microscopy. α-Synuclein-positive cells were mainly concentrated in the basolateral and cortical amygdaloid complexes with a non-significant increase over time from 16 to 30-43 weeks and a significant decrease thereafter. The expression of interneuron markers showed a significant decrease with aging in control animals. When comparing these markers between control and transgenic mice, calretinin was moderately decreased, but calbindin and somatostatin were highly reduced, particularly in the cortical amygdaloid complex. α-Synuclein mostly co-localised with calbindin and a number of these cells also co-expressed somatostatin. These data on α-synucleinopathy staging in the amygdala could help to explain non-motor symptoms as well as to understand the progression of Parkinson's disease in the brain. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  4. The association of metal ion exposure with alpha-synuclein-like immunoreactivity in the central nervous system of fish, Catostomus commersoni.

    PubMed

    Boudreau, Heather S; Krol, Karmen M; Eibl, Joseph K; Williams, Linda D; Rossiter, John P; Palace, Vincent P; Ross, Gregory M

    2009-05-17

    Alpha-synuclein protein aggregates are a major component of Lewy bodies, the intracytoplasmic inclusions found in dopaminergic neurons that are a defining characteristic of Parkinson's disease. Other "synucleopathies" include dementia with Lewy bodies and multisystem atrophy. In vitro, the formation of these deposits can be induced by a number of substances, including metal ions. Fish provide a useful model to study the long-term biological effects of metal ion exposure, but to date no studies have been reported concerning such exposures with respect to alpha-synuclein aggregation. Mature white sucker fish (Catostomus commersoni; aged 5-8 years) were sampled from two sites within the Red Lake area of Northwestern Ontario, a region highly contaminated by metal ions due to mining activity. Individual fish were characterized with respect to liver metal ion uptake and metallothionein levels. Central nervous system (CNS) tissues of fish from test sites representing high and low metal ion contamination were examined immunohistochemically using a polyclonal antibody recognising alpha-synuclein protein. We demonstrate here that the CNS of fish exposed to elevated metal ion environments had increased alpha-synuclein-like immunoreactive aggregates, potentially reflecting metal ion exposure leading to CNS toxicity. These findings demonstrate that fish may be an important new model for studying environmental risk factors and the pathology associated with Parkinson's disease.

  5. Brain region-dependent differential expression of alpha-synuclein.

    PubMed

    Taguchi, Katsutoshi; Watanabe, Yoshihisa; Tsujimura, Atsushi; Tanaka, Masaki

    2016-04-15

    α-Synuclein, the major constituent of Lewy bodies (LBs), is normally expressed in presynapses and is involved in synaptic function. Abnormal intracellular aggregation of α-synuclein is observed as LBs and Lewy neurites in neurodegenerative disorders, such as Parkinson's disease (PD) or dementia with Lewy bodies. Accumulated evidence suggests that abundant intracellular expression of α-synuclein is one of the risk factors for pathological aggregation. Recently, we reported differential expression patterns of α-synuclein between excitatory and inhibitory hippocampal neurons. Here we further investigated the precise expression profile in the adult mouse brain with special reference to vulnerable regions along the progression of idiopathic PD. The results show that α-synuclein was highly expressed in the neuronal cell bodies of some early PD-affected brain regions, such as the olfactory bulb, dorsal motor nucleus of the vagus, and substantia nigra pars compacta. Synaptic expression of α-synuclein was mostly accompanied by expression of vesicular glutamate transporter-1, an excitatory presynaptic marker. In contrast, expression of α-synuclein in the GABAergic inhibitory synapses was different among brain regions. α-Synuclein was clearly expressed in inhibitory synapses in the external plexiform layer of the olfactory bulb, globus pallidus, and substantia nigra pars reticulata, but not in the cerebral cortex, subthalamic nucleus, or thalamus. These results suggest that some neurons in early PD-affected human brain regions express high levels of perikaryal α-synuclein, as happens in the mouse brain. Additionally, synaptic profiles expressing α-synuclein are different in various brain regions. © 2015 Wiley Periodicals, Inc.

  6. Extracellular Alpha-Synuclein Oligomers Induce Parkin S-Nitrosylation: Relevance to Sporadic Parkinson's Disease Etiopathology.

    PubMed

    Wilkaniec, Anna; Lenkiewicz, Anna M; Czapski, Grzegorz A; Jęśko, Henryk M; Hilgier, Wojciech; Brodzik, Robert; Gąssowska-Dobrowolska, Magdalena; Culmsee, Carsten; Adamczyk, Agata

    2018-04-21

    α-Synuclein (ASN) and parkin, a multifunctional E3 ubiquitin ligase, are two proteins that are associated with the pathophysiology of Parkinson's disease (PD). Excessive release of ASN, its oligomerization, aggregation, and deposition in the cytoplasm contribute to neuronal injury and cell death through oxidative-nitrosative stress induction, mitochondrial impairment, and synaptic dysfunction. In contrast, overexpression of parkin provides protection against cellular stresses and prevents dopaminergic neural cell loss in several animal models of PD. However, the influence of ASN on the function of parkin is largely unknown. Therefore, the aim of this study was to investigate the effect of extracellular ASN oligomers on parkin expression, S-nitrosylation, as well as its activity. For these investigations, we used rat pheochromocytoma (PC12) cell line treated with exogenous oligomeric ASN as well as PC12 cells with parkin overexpression and parkin knock-down. The experiments were performed using spectrophotometric, spectrofluorometric, and immunochemical methods. We found that exogenous ASN oligomers induce oxidative/nitrosative stress leading to parkin S-nitrosylation. Moreover, this posttranslational modification induced the elevation of parkin autoubiquitination and degradation of the protein. The decreased parkin levels resulted in significant cell death, whereas parkin overexpression protected against toxicity induced by extracellular ASN oligomers. We conclude that lowering parkin levels by extracellular ASN may significantly contribute to the propagation of neurodegeneration in PD pathology through accumulation of defective proteins as a consequence of parkin degradation.

  7. Arachidonic acid mediates the formation of abundant alpha-helical multimers of alpha-synuclein

    NASA Astrophysics Data System (ADS)

    Iljina, Marija; Tosatto, Laura; Choi, Minee L.; Sang, Jason C.; Ye, Yu; Hughes, Craig D.; Bryant, Clare E.; Gandhi, Sonia; Klenerman, David

    2016-09-01

    The protein alpha-synuclein (αS) self-assembles into toxic beta-sheet aggregates in Parkinson’s disease, while it is proposed that αS forms soluble alpha-helical multimers in healthy neurons. Here, we have made αS multimers in vitro using arachidonic acid (ARA), one of the most abundant fatty acids in the brain, and characterized them by a combination of bulk experiments and single-molecule Fӧrster resonance energy transfer (sm-FRET) measurements. The data suggest that ARA-induced oligomers are alpha-helical, resistant to fibril formation, more prone to disaggregation, enzymatic digestion and degradation by the 26S proteasome, and lead to lower neuronal damage and reduced activation of microglia compared to the oligomers formed in the absence of ARA. These multimers can be formed at physiologically-relevant concentrations, and pathological mutants of αS form less multimers than wild-type αS. Our work provides strong biophysical evidence for the formation of alpha-helical multimers of αS in the presence of a biologically relevant fatty acid, which may have a protective role with respect to the generation of beta-sheet toxic structures during αS fibrillation.

  8. Accumulation of phosphorylated alpha-synuclein (p129S) and retinal pathology in a mouse model of Parkinson's disease

    USDA-ARS?s Scientific Manuscript database

    Aims: Parkinson's disease (PD) is a neurodegenerative disorder characterized by accumulation of misfolded alpha-synuclein within the CNS. Although non-motor clinical phenotypes of PD such as visual dysfunction have become increasingly apparent, retinal pathology associated with PD is not well under...

  9. Parkinson's disease: acid-glucocerebrosidase activity and alpha-synuclein clearance.

    PubMed

    Blanz, Judith; Saftig, Paul

    2016-10-01

    The role of mutations in the gene GBA1 encoding the lysosomal hydrolase β-glucocerebrosidase for the development of synucleinopathies, such as Parkinson's disease and dementia with Lewy bodies, was only very recently uncovered. The knowledge obtained from the study of carriers or patients suffering from Gaucher disease (a common lysosomal storage disorder because of GBA1 mutations) is of particular importance for understanding the role of the enzyme and its catabolic pathway in the development of synucleinopathies. Decreased activity of β-glucocerebrosidase leads to lysosomal dysfunction and the accumulation of its substrate glucosylceramide and related lipid derivatives. Glucosylceramide is suggested to stabilize toxic oligomeric forms of α-synuclein that negatively influence the activity of β-glucocerebrosidase and to partially block export of newly synthesized β-glucocerebrosidase from the endoplasmic reticulum to late endocytic compartments, amplifying the pathological effects of α-synuclein and ultimately resulting in neuronal cell death. This pathogenic molecular feedback loop and most likely other factors (such as impaired endoplasmic reticulum-associated degradation, activation of the unfolded protein response and dysregulation of calcium homeostasis induced by misfolded GC mutants) are involved in shifting the cellular homeostasis from monomeric α-synuclein towards oligomeric neurotoxic and aggregated forms, which contribute to Parkinson's disease progression. From a therapeutic point of view, strategies aiming to increase either the expression, stability or delivery of the β-glucocerebrosidase to lysosomes are likely to decrease the α-synuclein burden and may be useful for an in depth evaluation at the organismal level. Lysosomes are critical for protein and lipid homeostasis. Recent research revealed that dysfunction of this organelle contributes to the development of neurodegenerative diseases such as Parkinson's disease (PD). Mutations in the

  10. Preclinical development of a vaccine against oligomeric alpha-synuclein based on virus-like particles.

    PubMed

    Doucet, Marika; El-Turabi, Aadil; Zabel, Franziska; Hunn, Benjamin H M; Bengoa-Vergniory, Nora; Cioroch, Milena; Ramm, Mauricio; Smith, Amy M; Gomes, Ariane Cruz; Cabral de Miranda, Gustavo; Wade-Martins, Richard; Bachmann, Martin F

    2017-01-01

    Parkinson's disease (PD) is a progressive and currently incurable neurological disorder characterised by the loss of midbrain dopaminergic neurons and the accumulation of aggregated alpha-synuclein (a-syn). Oligomeric a-syn is proposed to play a central role in spreading protein aggregation in the brain with associated cellular toxicity contributing to a progressive neurological decline. For this reason, a-syn oligomers have attracted interest as therapeutic targets for neurodegenerative conditions such as PD and other alpha-synucleinopathies. In addition to strategies using small molecules, neutralisation of the toxic oligomers by antibodies represents an attractive and highly specific strategy for reducing disease progression. Emerging active immunisation approaches using vaccines are already being trialled to induce such antibodies. Here we propose a novel vaccine based on the RNA bacteriophage (Qbeta) virus-like particle conjugated with short peptides of human a-syn. High titres of antibodies were successfully and safely generated in wild-type and human a-syn over-expressing (SNCA-OVX) transgenic mice following vaccination. Antibodies from vaccine candidates targeting the C-terminal regions of a-syn were able to recognise Lewy bodies, the hallmark aggregates in human PD brains. Furthermore, antibodies specifically targeted oligomeric and aggregated a-syn as they exhibited 100 times greater affinity for oligomeric species over monomer a-syn proteins in solution. In the SNCA-OVX transgenic mice used, vaccination was, however, unable to confer significant changes to oligomeric a-syn bioburden. Similarly, there was no discernible effect of vaccine treatment on behavioural phenotype as compared to control groups. Thus, antibodies specific for oligomeric a-syn induced by vaccination were unable to treat symptoms of PD in this particular mouse model.

  11. Preclinical development of a vaccine against oligomeric alpha-synuclein based on virus-like particles

    PubMed Central

    Zabel, Franziska; Hunn, Benjamin H.M.; Bengoa-Vergniory, Nora; Cioroch, Milena; Ramm, Mauricio; Smith, Amy M.; Gomes, Ariane Cruz; Cabral de Miranda, Gustavo; Wade-Martins, Richard; Bachmann, Martin F.

    2017-01-01

    Parkinson's disease (PD) is a progressive and currently incurable neurological disorder characterised by the loss of midbrain dopaminergic neurons and the accumulation of aggregated alpha-synuclein (a-syn). Oligomeric a-syn is proposed to play a central role in spreading protein aggregation in the brain with associated cellular toxicity contributing to a progressive neurological decline. For this reason, a-syn oligomers have attracted interest as therapeutic targets for neurodegenerative conditions such as PD and other alpha-synucleinopathies. In addition to strategies using small molecules, neutralisation of the toxic oligomers by antibodies represents an attractive and highly specific strategy for reducing disease progression. Emerging active immunisation approaches using vaccines are already being trialled to induce such antibodies. Here we propose a novel vaccine based on the RNA bacteriophage (Qbeta) virus-like particle conjugated with short peptides of human a-syn. High titres of antibodies were successfully and safely generated in wild-type and human a-syn over-expressing (SNCA-OVX) transgenic mice following vaccination. Antibodies from vaccine candidates targeting the C-terminal regions of a-syn were able to recognise Lewy bodies, the hallmark aggregates in human PD brains. Furthermore, antibodies specifically targeted oligomeric and aggregated a-syn as they exhibited 100 times greater affinity for oligomeric species over monomer a-syn proteins in solution. In the SNCA-OVX transgenic mice used, vaccination was, however, unable to confer significant changes to oligomeric a-syn bioburden. Similarly, there was no discernible effect of vaccine treatment on behavioural phenotype as compared to control groups. Thus, antibodies specific for oligomeric a-syn induced by vaccination were unable to treat symptoms of PD in this particular mouse model. PMID:28797124

  12. Overexpression of p53 mRNA in colorectal cancer and its relationship to p53 gene mutation.

    PubMed Central

    el-Mahdani, N.; Vaillant, J. C.; Guiguet, M.; Prévot, S.; Bertrand, V.; Bernard, C.; Parc, R.; Béréziat, G.; Hermelin, B.

    1997-01-01

    We analysed the frequency of p53 mRNA overexpression in a series of 109 primary colorectal carcinomas and its association with p53 gene mutation, which has been correlated with short survival. Sixty-nine of the 109 cases (63%) demonstrated p53 mRNA overexpression, without any correlation with stage or site of disease. Comparison with p53 gene mutation indicated that, besides cases in which p53 gene mutation and p53 mRNA overexpression were either both present (40 cases) or both absent (36 cases), there were also cases in which p53 mRNA was overexpressed in the absence of any mutation (29 cases) and those with a mutant gene in which the mRNA was not overexpressed (four cases). Moreover, the mutant p53 tumours exhibited an increase of p53 mRNA expression, which was significantly higher in tumours expressing the mutated allele alone than in tumours expressing both wild- and mutated-type alleles. These data (1) show that p53 mRNA overexpression is a frequent event in colorectal tumours and is not predictive of the status of the gene, i.e. whether or not a mutation is present; (2) provide further evidence that p53 protein overexpression does not only result from an increase in the half-life of mutated p53 and suggest that inactivation of the p53 function in colorectal cancers involves at least two distinct mechanisms, including p53 overexpression and/or mutation; and (3) suggest that p53 mRNA overexpression is an early event, since it is not correlated with Dukes stage. PMID:9052405

  13. Solid surface dependent layering of self-arranged structures with fibril-like assemblies of alpha-synuclein

    NASA Astrophysics Data System (ADS)

    Bukauskas, V.; Šetkus, A.; Šimkienė, I.; Tumėnas, S.; Kašalynas, I.; Rėza, A.; Babonas, J.; Časaitė, V.; Povilonienė, S.; Meškys, R.

    2012-03-01

    In present work the formation of hybrid constructions composed of alpha-synuclein-based colloidal solutions on various solid surfaces (silica coated Si, mica, CaF2 and KBr) is investigated by scanning probe microscopy, spectrocopic ellipsometry, Fourier transformed infrared spectroscopy and vibrational circular dichroism. Prior to the modification of the solids, the proteins were intentionally fibrilled under special conditions. It is proved that the multi-component coatings are self-arranged on the solid substrates. Depending on the substrate material, the interface films consisting of individual biomolecules can be detected on the solid surfaces. The coatings with fibril-like alpha-synuclein objects can be obtained on solid surfaces with negligible or comparatively thick interface films. The results are interpreted in terms of the charged surface-controlled electrostatic interaction between the substrate and the biomolecules. Solubility of solids is considered in this interpretation.

  14. Extracellular alpha-synuclein oligomers modulate synaptic transmission and impair LTP via NMDA-receptor activation.

    PubMed

    Diógenes, Maria José; Dias, Raquel B; Rombo, Diogo M; Vicente Miranda, Hugo; Maiolino, Francesca; Guerreiro, Patrícia; Näsström, Thomas; Franquelim, Henri G; Oliveira, Luís M A; Castanho, Miguel A R B; Lannfelt, Lars; Bergström, Joakim; Ingelsson, Martin; Quintas, Alexandre; Sebastião, Ana M; Lopes, Luísa V; Outeiro, Tiago Fleming

    2012-08-22

    Parkinson's disease (PD) is the most common representative of a group of disorders known as synucleinopathies, in which misfolding and aggregation of α-synuclein (a-syn) in various brain regions is the major pathological hallmark. Indeed, the motor symptoms in PD are caused by a heterogeneous degeneration of brain neurons not only in substantia nigra pars compacta but also in other extrastriatal areas of the brain. In addition to the well known motor dysfunction in PD patients, cognitive deficits and memory impairment are also an important part of the disorder, probably due to disruption of synaptic transmission and plasticity in extrastriatal areas, including the hippocampus. Here, we investigated the impact of a-syn aggregation on AMPA and NMDA receptor-mediated rat hippocampal (CA3-CA1) synaptic transmission and long-term potentiation (LTP), the neurophysiological basis for learning and memory. Our data show that prolonged exposure to a-syn oligomers, but not monomers or fibrils, increases basal synaptic transmission through NMDA receptor activation, triggering enhanced contribution of calcium-permeable AMPA receptors. Slices treated with a-syn oligomers were unable to respond with further potentiation to theta-burst stimulation, leading to impaired LTP. Prior delivery of a low-frequency train reinstated the ability to express LTP, implying that exposure to a-syn oligomers drives the increase of glutamatergic synaptic transmission, preventing further potentiation by physiological stimuli. Our novel findings provide mechanistic insight on how a-syn oligomers may trigger neuronal dysfunction and toxicity in PD and other synucleinopathies.

  15. Immunotherapy targeting α-synuclein protofibrils reduced pathology in (Thy-1)-h[A30P] α-synuclein mice.

    PubMed

    Lindström, Veronica; Fagerqvist, Therese; Nordström, Eva; Eriksson, Fredrik; Lord, Anna; Tucker, Stina; Andersson, Jessica; Johannesson, Malin; Schell, Heinrich; Kahle, Philipp J; Möller, Christer; Gellerfors, Pär; Bergström, Joakim; Lannfelt, Lars; Ingelsson, Martin

    2014-09-01

    Several lines of evidence suggest that accumulation of aggregated alpha-synuclein (α-synuclein) in the central nervous system (CNS) is an early pathogenic event in Parkinson's disease and other Lewy body disorders. In recent years, animal studies have indicated immunotherapy with antibodies directed against α-synuclein as a promising novel treatment strategy. Since large α-synuclein oligomers, or protofibrils, have been demonstrated to possess pronounced cytotoxic properties, such species should be particularly attractive as therapeutic targets. In support of this, (Thy-1)-h[A30P] α-synuclein transgenic mice with motor dysfunction symptoms were found to display increased levels of α-synuclein protofibrils in the CNS. An α-synuclein protofibril-selective monoclonal antibody (mAb47) was evaluated in this α-synuclein transgenic mouse model. As measured by ELISA, 14month old mice treated for 14weeks with weekly intraperitoneal injections of mAb47 displayed significantly lower levels of both soluble and membrane-associated protofibrils in the spinal cord. Besides the lower levels of pathogenic α-synuclein demonstrated, a reduction of motor dysfunction in transgenic mice upon peripheral administration of mAb47 was indicated. Thus, immunotherapy with antibodies targeting toxic α-synuclein species holds promise as a future disease-modifying treatment in Parkinson's disease and related disorders. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Silencing Alpha Synuclein in Mature Nigral Neurons Results in Rapid Neuroinflammation and Subsequent Toxicity

    PubMed Central

    Benskey, Matthew J.; Sellnow, Rhyomi C.; Sandoval, Ivette M.; Sortwell, Caryl E.; Lipton, Jack W.; Manfredsson, Fredric P.

    2018-01-01

    Human studies and preclinical models of Parkinson’s disease implicate the involvement of both the innate and adaptive immune systems in disease progression. Further, pro-inflammatory markers are highly enriched near neurons containing pathological forms of alpha synuclein (α-syn), and α-syn overexpression recapitulates neuroinflammatory changes in models of Parkinson’s disease. These data suggest that α-syn may initiate a pathological inflammatory response, however the mechanism by which α-syn initiates neuroinflammation is poorly understood. Silencing endogenous α-syn results in a similar pattern of nigral degeneration observed following α-syn overexpression. Here we aimed to test the hypothesis that loss of α-syn function within nigrostriatal neurons results in neuronal dysfunction, which subsequently stimulates neuroinflammation. Adeno-associated virus (AAV) expressing an short hairpin RNA (shRNA) targeting endogenous α-syn was unilaterally injected into the substantia nigra pars compacta (SNc) of adult rats, after which nigrostriatal pathology and indices of neuroinflammation were examined at 7, 10, 14 and 21 days post-surgery. Removing endogenous α-syn from nigrostriatal neurons resulted in a rapid up-regulation of the major histocompatibility complex class 1 (MHC-1) within transduced nigral neurons. Nigral MHC-1 expression occurred prior to any overt cell death and coincided with the recruitment of reactive microglia and T-cells to affected neurons. Following the induction of neuroinflammation, α-syn knockdown resulted in a 50% loss of nigrostriatal neurons in the SNc and a corresponding loss of nigrostriatal terminals and dopamine (DA) concentrations within the striatum. Expression of a control shRNA did not elicit any pathological changes. Silencing α-syn within glutamatergic neurons of the cerebellum did not elicit inflammation or cell death, suggesting that toxicity initiated by α-syn silencing is specific to DA neurons. These data provide

  17. Elevated α-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells

    PubMed Central

    Oliveira, L M A; Falomir-Lockhart, L J; Botelho, M G; Lin, K-H; Wales, P; Koch, J C; Gerhardt, E; Taschenberger, H; Outeiro, T F; Lingor, P; Schüle, B; Arndt-Jovin, D J; Jovin, T M

    2015-01-01

    We have assessed the impact of α-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson's disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of α-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson's disease progression, particularly in the context of bioenergetic dysfunction. PMID:26610207

  18. Drug Targets from Genetics: Alpha-Synuclein

    PubMed Central

    Danzer, Karin M.; McLean, Pamela J.

    2012-01-01

    One of the critical issues in Parkinson disease (PD) research is the identity of the specific toxic, pathogenic moiety. In PD, mutations in alpha-synuclein (αsyn) or multiplication of the SNCA gene encoding αsyn, result in a phenotype of cellular inclusions, cell death, and brain dysfunction. While the historical point of view has been that the macroscopic aggregates containing αsyn are the toxic species, in the last several years evidence has emerged that suggests instead that smaller soluble species - likely oligomers containing misfolded αsyn - are actually the toxic moiety and that the fibrillar inclusions may even be a cellular detoxification pathway and less harmful. If soluble misfolded species of αsyn are the toxic moieties, then cellular mechanisms that degrade misfolded αsyn would be neuroprotective and a rational target for drug development. In this review we will discuss the fundamental mechanisms underlying αsyn toxicity including oligomer formation, oxidative stress, and degradation pathways and consider rational therapeutic strategies that may have the potential to prevent or halt αsyn induced pathogenesis in PD. PMID:21838671

  19. Synuclein impairs trafficking and signaling of BDNF in a mouse model of Parkinson's disease.

    PubMed

    Fang, Fang; Yang, Wanlin; Florio, Jazmin B; Rockenstein, Edward; Spencer, Brian; Orain, Xavier M; Dong, Stephanie X; Li, Huayan; Chen, Xuqiao; Sung, Kijung; Rissman, Robert A; Masliah, Eliezer; Ding, Jianqing; Wu, Chengbiao

    2017-06-20

    Recent studies have demonstrated that hyperphosphorylation of tau protein plays a role in neuronal toxicities of α-synuclein (ASYN) in neurodegenerative disease such as familial Alzheimer's disease (AD), dementia with Lewy bodies (DLB) and Parkinson's disease. Using a transgenic mouse model of Parkinson's disease (PD) that expresses GFP-ASYN driven by the PDGF-β promoter, we investigated how accumulation of ASYN impacted axonal function. We found that retrograde axonal trafficking of brain-derived neurotrophic factor (BDNF) in DIV7 cultures of E18 cortical neurons was markedly impaired at the embryonic stage, even though hyperphosphorylation of tau was not detectable in these neurons at this stage. Interestingly, we found that overexpressed ASYN interacted with dynein and induced a significant increase in the activated levels of small Rab GTPases such as Rab5 and Rab7, both key regulators of endocytic processes. Furthermore, expression of ASYN resulted in neuronal atrophy in DIV7 cortical cultures of either from E18 transgenic mouse model or from rat E18 embryos that were transiently transfected with ASYN-GFP for 72 hrs. Our studies suggest that excessive ASYN likely alters endocytic pathways leading to axonal dysfunction in embryonic cortical neurons in PD mouse models.

  20. Aggregation of alpha-synuclein by a coarse-grained Monte Carlo simulation

    NASA Astrophysics Data System (ADS)

    Farmer, Barry; Pandey, Ras

    Alpha-synuclein, an intrinsic protein abundant in neurons, is believed to be a major cause of neurodegenerative diseases (e.g. Alzheimer, Parkinson's disease). Abnormal aggregation of ASN leads to Lewy bodies with specific morphologies. We investigate the self-organizing structures in a crowded environment of ASN proteins by a coarse-grained Monte Carlo simulation. ASN is a chain of 140 residues. Structure detail of residues is neglected but its specificity is captured via unique knowledge-based residue-residue interactions. Large-scale simulations are performed to analyze a number local and global physical quantities (e.g. mobility profile, contact map, radius of gyration, structure factor) as a function of temperature and protein concentration. Trend in multi-scale structural variations of the protein in a crowded environment is compared with that of a free protein chain.

  1. Interaction of Synuclein and Inflammation in Dopaminergic Neurodegeneration

    DTIC Science & Technology

    2010-07-01

    synuclein in cell culture. Specific Aim II In vivo studies: we are now optimizing immunostaining for human -synuclein in order to distinguish...responsible for this response. Recent cell culture experiments seem to point us in the right direction for the answer as it has been shown that human BE-M17...synuclein. Furthermore, Zhou et al (2002) showed that, in primary cell cultures derived from the embryonic human mesencephalon overexpressing either

  2. Differences in extinction of conditioned fear in C57BL/6 substrains are unrelated to expression of alpha-synuclein.

    PubMed

    Siegmund, Anja; Langnaese, Kristina; Wotjak, Carsten T

    2005-02-28

    C57BL/6 mice are commonly used as background strains for genetically modified mice, and little attention is usually paid to the notification of the specific substrain. However, it is known that C57BL/6NCrl (B6N) and C57BL/6JOlaHsd (B6JOla) mice differ in the course of extinction of conditioned fear (Stiedl O, Radulovic J, Lohmann R, Birkenfeld K, Palve M, Kammermeier J, et al. Strain and substrain differences in context- and tone-dependent fear conditioning of inbred mice. Behav Brain Res 1999;104:1-12), as well as in the expression of alpha-synuclein (Specht CG, Schoepfer R. Deletion of the alpha-synuclein locus in a subpopulation of C57BL/6J inbred mice. BMC Neurosci 2001;2:11). We tested for a causal relationship between the two findings by employing B6N (expressing alpha-synuclein), B6JOla (not expressing alpha-syn) and the third strain C57BL/6JCrl (B6Jax, expressing alpha-syn). We show that alpha-syn does not account for differences in extinction in a fear conditioning task, as its expression did not covary with the decrease of freezing on repeated non-reinforced tone and context exposure in the three strains: B6Jax exhibited fastest extinction followed by B6JOla. In contrast, B6N showed persistent fear over the course of extinction training. The differences in extinction between B6JOla and B6N were unrelated to sensorimotor processing (pain threshold and basal tone reaction) and innate fear (light-dark test). However, B6Jax displayed less innate fear than B6JOla and B6N. Our results of marked differences in innate and conditioned fear in three B6 substrains illustrate the necessity of a strict adherence to an exact mouse strain nomenclature.

  3. Transient β-hairpin formation in α-synuclein monomer revealed by coarse-grained molecular dynamics simulation

    NASA Astrophysics Data System (ADS)

    Yu, Hang; Han, Wei; Ma, Wen; Schulten, Klaus

    2015-12-01

    Parkinson's disease, originating from the intrinsically disordered peptide α-synuclein, is a common neurodegenerative disorder that affects more than 5% of the population above age 85. It remains unclear how α-synuclein monomers undergo conformational changes leading to aggregation and formation of fibrils characteristic for the disease. In the present study, we perform molecular dynamics simulations (over 180 μs in aggregated time) using a hybrid-resolution model, Proteins with Atomic details in Coarse-grained Environment (PACE), to characterize in atomic detail structural ensembles of wild type and mutant monomeric α-synuclein in aqueous solution. The simulations reproduce structural properties of α-synuclein characterized in experiments, such as secondary structure content, long-range contacts, chemical shifts, and 3J(HNHCα)-coupling constants. Most notably, the simulations reveal that a short fragment encompassing region 38-53, adjacent to the non-amyloid-β component region, exhibits a high probability of forming a β-hairpin; this fragment, when isolated from the remainder of α-synuclein, fluctuates frequently into its β-hairpin conformation. Two disease-prone mutations, namely, A30P and A53T, significantly accelerate the formation of a β-hairpin in the stated fragment. We conclude that the formation of a β-hairpin in region 38-53 is a key event during α-synuclein aggregation. We predict further that the G47V mutation impedes the formation of a turn in the β-hairpin and slows down β-hairpin formation, thereby retarding α-synuclein aggregation.

  4. Alpha-synuclein levels in patients with multiple system atrophy: a meta-analysis.

    PubMed

    Yang, Fei; Li, Wan-Jun; Huang, Xu-Sheng

    2018-05-01

    This study evaluates the relationship between multiple system atrophy and α-synuclein levels in the cerebrospinal fluid, plasma and neural tissue. Literature search for relevant research articles was undertaken in electronic databases and study selection was based on a priori eligibility criteria. Random-effects meta-analyses of standardized mean differences in α-synuclein levels between multiple system atrophy patients and normal controls were conducted to obtain the overall and subgroup effect sizes. Meta-regression analyses were performed to evaluate the effect of age, gender and disease severity on standardized mean differences. Data were obtained from 11 studies involving 378 multiple system atrophy patients and 637 healthy controls (age: multiple system atrophy patients 64.14 [95% confidence interval 62.05, 66.23] years; controls 64.16 [60.06, 68.25] years; disease duration: 44.41 [26.44, 62.38] months). Cerebrospinal fluid α-synuclein levels were significantly lower in multiple system atrophy patients than in controls but in plasma and neural tissue, α-synuclein levels were significantly higher in multiple system atrophy patients (standardized mean difference: -0.99 [-1.65, -0.32]; p = 0.001). Percentage of male multiple system atrophy patients was significantly positively associated with the standardized mean differences of cerebrospinal fluid α-synuclein levels (p = 0.029) whereas the percentage of healthy males was not associated with the standardized mean differences of cerebrospinal fluid α-synuclein levels (p = 0.920). In multiple system atrophy patients, α-synuclein levels were significantly lower in the cerebrospinal fluid and were positively associated with the male gender.

  5. Inflammation kinase PKR phosphorylates α-synuclein and causes α-synuclein-dependent cell death.

    PubMed

    Reimer, Lasse; Vesterager, Louise Buur; Betzer, Cristine; Zheng, Jin; Nielsen, Lærke Dalsgaard; Kofoed, Rikke Hahn; Lassen, Louise Berkhoudt; Bølcho, Ulrik; Paludan, Søren Riis; Fog, Karina; Jensen, Poul Henning

    2018-07-01

    Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy comprise a group of neurodegenerative diseases termed synucleinopathies. Synucleinopathie are, characterized by presence of inclusion bodies in degenerating brain cells which contain aggregated α-synuclein phosphorylated on Ser129. Although the inflammation-associated serine-threonine kinase, PKR (EIF2AK2), promotes cellular protection against infection, we demonstrate a pro-degenerative role of activated PKR in an α-synuclein-dependent cell model of multiple system atrophy, where inhibition and silencing of PKR decrease cellular degeneration. In vitro phosphorylation demonstrates that PKR can directly bind and phosphorylate monomeric and filamenteous α-synuclein on Ser129. Inhibition and knockdown of PKR reduce Ser129 phosphorylation in different models (SH-SY5Y ASYN cells, OLN-AS7 cells, primary mouse hippocampal neurons, and acute brain slices), while overexpression of constitutively active PKR increases Ser129 α-syn phosphorylation. Treatment with pre-formed α-synuclein fibrils, proteostatic stress-promoting MG-132 and known PKR activators, herpes simplex virus-1-∆ICP34.5 and LPS, as well as PKR inducer, IFN-β-1b, lead to increased levels of phosphorylated Ser129 α-synuclein that is completely blocked by simultaneous PKR inhibition. These results reveal a direct link between PKR and the phosphorylation and toxicity of α-synuclein, and they support that neuroinflammatory processes play a role in modulating the pathogenicity of α-synuclein. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. FTY720/Fingolimod Reduces Synucleinopathy and Improves Gut Motility in A53T Mice

    PubMed Central

    Vidal-Martínez, Guadalupe; Vargas-Medrano, Javier; Gil-Tommee, Carolina; Medina, David; Garza, Nathan T.; Yang, Barbara; Segura-Ulate, Ismael; Dominguez, Samantha J.; Perez, Ruth G.

    2016-01-01

    Patients with Parkinson's disease (PD) often have aggregated α-synuclein (aSyn) in enteric nervous system (ENS) neurons, which may be associated with the development of constipation. This occurs well before the onset of classic PD motor symptoms. We previously found that aging A53T transgenic (Tg) mice closely model PD-like ENS aSyn pathology, making them appropriate for testing potential PD therapies. Here we show that Tg mice overexpressing mutant human aSyn develop ENS pathology by 4 months. We then evaluated the responses of Tg mice and their WT littermates to the Food and Drug Administration-approved drug FTY720 (fingolimod, Gilenya) or vehicle control solution from 5 months of age. Long term oral FTY720 in Tg mice reduced ENS aSyn aggregation and constipation, enhanced gut motility, and increased levels of brain-derived neurotrophic factor (BDNF) but produced no significant change in WT littermates. A role for BDNF was directly assessed in a cohort of young A53T mice given vehicle, FTY720, the Trk-B receptor inhibitor ANA-12, or FTY720 + ANA-12 from 1 to 4 months of age. ANA-12-treated Tg mice developed more gut aSyn aggregation as well as constipation, whereas FTY720-treated Tg mice had reduced aSyn aggregation and less constipation, occurring in part by increasing both pro-BDNF and mature BDNF levels. The data from young and old Tg mice revealed FTY720-associated neuroprotection and reduced aSyn pathology, suggesting that FTY720 may also benefit PD patients and others with synucleinopathy. Another finding was a loss of tyrosine hydroxylase immunoreactivity in gut neurons with aggregated aSyn, comparable with our prior findings in the CNS. PMID:27528608

  7. Involvement of Cellular Prion Protein in α-Synuclein Transport in Neurons.

    PubMed

    Urrea, Laura; Segura-Feliu, Miriam; Masuda-Suzukake, Masami; Hervera, Arnau; Pedraz, Lucas; García Aznar, José Manuel; Vila, Miquel; Samitier, Josep; Torrents, Eduard; Ferrer, Isidro; Gavín, Rosalina; Hagesawa, Masato; Del Río, José Antonio

    2018-03-01

    The cellular prion protein, encoded by the gene Prnp, has been reported to be a receptor of β-amyloid. Their interaction is mandatory for neurotoxic effects of β-amyloid oligomers. In this study, we aimed to explore whether the cellular prion protein participates in the spreading of α-synuclein. Results demonstrate that Prnp expression is not mandatory for α-synuclein spreading. However, although the pathological spreading of α-synuclein can take place in the absence of Prnp, α-synuclein expanded faster in PrP C -overexpressing mice. In addition, α-synuclein binds strongly on PrP C -expressing cells, suggesting a role in modulating the effect of α-synuclein fibrils.

  8. (Poly)phenol-digested metabolites modulate alpha-synuclein toxicity by regulating proteostasis.

    PubMed

    Macedo, Diana; Jardim, Carolina; Figueira, Inês; Almeida, A Filipa; McDougall, Gordon J; Stewart, Derek; Yuste, Jose E; Tomás-Barberán, Francisco A; Tenreiro, Sandra; Outeiro, Tiago F; Santos, Cláudia N

    2018-05-03

    Parkinson's disease (PD) is an age-related neurodegenerative disease associated with the misfolding and aggregation of alpha-synuclein (aSyn). The molecular underpinnings of PD are still obscure, but nutrition may play an important role in the prevention, onset, and disease progression. Dietary (poly)phenols revert and prevent age-related cognitive decline and neurodegeneration in model systems. However, only limited attempts were made to evaluate the impact of digestion on the bioactivities of (poly)phenols and determine their mechanisms of action. This constitutes a challenge for the development of (poly)phenol-based nutritional therapies. Here, we subjected (poly)phenols from Arbutus unedo to in vitro digestion and tested the products in cell models of PD based on the cytotoxicity of aSyn. The (poly)phenol-digested metabolites from A. unedo leaves (LPDMs) effectively counteracted aSyn and H 2 O 2 toxicity in yeast and human cells, improving viability by reducing aSyn aggregation and inducing its clearance. In addition, LPDMs modulated pathways associated with aSyn toxicity, such as oxidative stress, endoplasmic reticulum (ER) stress, mitochondrial impairment, and SIR2 expression. Overall, LPDMs reduced aSyn toxicity, enhanced the efficiency of ER-associated protein degradation by the proteasome and autophagy, and reduced oxidative stress. In total, our study opens novel avenues for the exploitation of (poly)phenols in nutrition and health.

  9. The contribution of alpha synuclein to neuronal survival and function - Implications for Parkinson's disease.

    PubMed

    Benskey, Matthew J; Perez, Ruth G; Manfredsson, Fredric P

    2016-05-01

    The aggregation of alpha synuclein (α-syn) is a neuropathological feature that defines a spectrum of disorders collectively termed synucleinopathies, and of these, Parkinson's disease (PD) is arguably the best characterized. Aggregated α-syn is the primary component of Lewy bodies, the defining pathological feature of PD, while mutations or multiplications in the α-syn gene result in familial PD. The high correlation between α-syn burden and PD has led to the hypothesis that α-syn aggregation produces toxicity through a gain-of-function mechanism. However, α-syn has been implicated to function in a diverse range of essential cellular processes such as the regulation of neurotransmission and response to cellular stress. As such, an alternative hypothesis with equal explanatory power is that the aggregation of α-syn results in toxicity because of a toxic loss of necessary α-syn function, following sequestration of functional forms α-syn into insoluble protein aggregates. Within this review, we will provide an overview of the literature linking α-syn to PD and the knowledge gained from current α-syn-based animal models of PD. We will then interpret these data from the viewpoint of the α-syn loss-of-function hypothesis and provide a potential mechanistic model by which loss of α-syn function could result in at least some of the neurodegeneration observed in PD. By providing an alternative perspective on the etiopathogenesis of PD and synucleinopathies, this may reveal alternative avenues of research in order to identify potential novel therapeutic targets for disease modifying strategies. The correlation between α-synuclein burden and Parkinson's disease pathology has led to the hypothesis that α-synuclein aggregation produces toxicity through a gain-of-function mechanism. However, in this review, we discuss data supporting the alternative hypothesis that the aggregation of α-synuclein results in toxicity because of loss of necessary α-synuclein

  10. Alpha-synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation.

    PubMed

    Betzer, Cristine; Lassen, Louise Berkhoudt; Olsen, Anders; Kofoed, Rikke Hahn; Reimer, Lasse; Gregersen, Emil; Zheng, Jin; Calì, Tito; Gai, Wei-Ping; Chen, Tong; Moeller, Arne; Brini, Marisa; Fu, Yuhong; Halliday, Glenda; Brudek, Tomasz; Aznar, Susana; Pakkenberg, Bente; Andersen, Jens Peter; Jensen, Poul Henning

    2018-05-01

    Aggregation of α-synuclein is a hallmark of Parkinson's disease and dementia with Lewy bodies. We here investigate the relationship between cytosolic Ca 2+ and α-synuclein aggregation. Analyses of cell lines and primary culture models of α-synuclein cytopathology reveal an early phase with reduced cytosolic Ca 2+ levels followed by a later Ca 2+ increase. Aggregated but not monomeric α-synuclein binds to and activates SERCA in vitro , and proximity ligation assays confirm this interaction in cells. The SERCA inhibitor cyclopiazonic acid (CPA) normalises both the initial reduction and the later increase in cytosolic Ca 2+ CPA protects the cells against α-synuclein-aggregate stress and improves viability in cell models and in Caenorhabditis elegans in vivo Proximity ligation assays also reveal an increased interaction between α-synuclein aggregates and SERCA in human brains affected by dementia with Lewy bodies. We conclude that α-synuclein aggregates bind SERCA and stimulate its activity. Reducing SERCA activity is neuroprotective, indicating that SERCA and down-stream processes may be therapeutic targets for treating α-synucleinopathies. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  11. Seeking a Mechanism for the Toxicity of Oligomeric α-Synuclein

    PubMed Central

    Roberts, Hazel L.; Brown, David R.

    2015-01-01

    In a number of neurological diseases including Parkinson’s disease (PD), α‑synuclein is aberrantly folded, forming abnormal oligomers, and amyloid fibrils within nerve cells. Strong evidence exists for the toxicity of increased production and aggregation of α-synuclein in vivo. The toxicity of α-synuclein is popularly attributed to the formation of “toxic oligomers”: a heterogenous and poorly characterized group of conformers that may share common molecular features. This review presents the available evidence on the properties of α-synuclein oligomers and the potential molecular mechanisms of their cellular disruption. Toxic α-synuclein oligomers may impact cells in a number of ways, including the disruption of membranes, mitochondrial depolarization, cytoskeleton changes, impairment of protein clearance pathways, and enhanced oxidative stress. We also examine the relationship between α-synuclein toxic oligomers and amyloid fibrils, in the light of recent studies that paint a more complex picture of α-synuclein toxicity. Finally, methods of studying and manipulating oligomers within cells are described. PMID:25816357

  12. Network Analysis Implicates Alpha-Synuclein (Snca) in the Regulation of Ovariectomy-Induced Bone Loss

    PubMed Central

    Calabrese, Gina; Mesner, Larry D.; Foley, Patricia L.; Rosen, Clifford J.; Farber, Charles R.

    2016-01-01

    The postmenopausal period in women is associated with decreased circulating estrogen levels, which accelerate bone loss and increase the risk of fracture. Here, we gained novel insight into the molecular mechanisms mediating bone loss in ovariectomized (OVX) mice, a model of human menopause, using co-expression network analysis. Specifically, we generated a co-expression network consisting of 53 gene modules using expression profiles from intact and OVX mice from a panel of inbred strains. The expression of four modules was altered by OVX, including module 23 whose expression was decreased by OVX across all strains. Module 23 was enriched for genes involved in the response to oxidative stress, a process known to be involved in OVX-induced bone loss. Additionally, module 23 homologs were co-expressed in human bone marrow. Alpha synuclein (Snca) was one of the most highly connected “hub” genes in module 23. We characterized mice deficient in Snca and observed a 40% reduction in OVX-induced bone loss. Furthermore, protection was associated with the altered expression of specific network modules, including module 23. In summary, the results of this study suggest that Snca regulates bone network homeostasis and ovariectomy-induced bone loss. PMID:27378017

  13. Dynamic Changes in Striatal mGluR1 But Not mGluR5 during Pathological Progression of Parkinson's Disease in Human Alpha-Synuclein A53T Transgenic Rats: A Multi-PET Imaging Study.

    PubMed

    Yamasaki, Tomoteru; Fujinaga, Masayuki; Kawamura, Kazunori; Furutsuka, Kenji; Nengaki, Nobuki; Shimoda, Yoko; Shiomi, Satoshi; Takei, Makoto; Hashimoto, Hiroki; Yui, Joji; Wakizaka, Hidekatsu; Hatori, Akiko; Xie, Lin; Kumata, Katsushi; Zhang, Ming-Rong

    2016-01-13

    Parkinson's disease (PD) is a prevalent degenerative disorder affecting the CNS that is primarily characterized by resting tremor and movement deficits. Group I metabotropic glutamate receptor subtypes 1 and 5 (mGluR1 and mGluR5, respectively) are important targets for investigation in several CNS disorders. In the present study, we investigated the in vivo roles of mGluR1 and mGluR5 in chronic PD pathology by performing longitudinal positron emission tomography (PET) imaging in A53T transgenic (A53T-Tg) rats expressing an abnormal human α-synuclein (ASN) gene. A53T-Tg rats showed a dramatic decline in general motor activities with age, along with abnormal ASN aggregation and striatal neuron degeneration. In longitudinal PET imaging, striatal nondisplaceable binding potential (BPND) values for [(11)C]ITDM (N-[4-[6-(isopropylamino) pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methyl-4-[(11)C]methylbenzamide), a selective PET ligand for mGluR1, temporarily increased before PD symptom onset and dramatically decreased afterward with age. However, striatal BPND values for (E)-[(11)C]ABP688 [3-(6-methylpyridin-2-ylethynyl)-cyclohex-2-enone-(E)-O-[(11)C]methyloxime], a specific PET ligand for mGluR5, remained constant during experimental terms. The dynamic changes in striatal mGluR1 BPND values also showed a high correlation in pathological decreases in general motor activities. Furthermore, declines in mGluR1 BPND values were correlated with decreases in BPND values for [(18)F]FE-PE2I [(E)-N-(3-iodoprop-2E-enyl)-2β-carbo-[(18)F]fluoroethoxy-3β-(4-methylphenyl) nortropane], a specific PET ligand for the dopamine transporter, a biomarker for dopaminergic neurons. In conclusion, our results have demonstrated for the first time that dynamic changes occur in mGluR1, but not mGluR5, that accompany pathological progression in a PD animal model. Synaptic signaling by glutamate, the principal excitatory neurotransmitter in the brain, is modulated by group I metabotropic glutamate

  14. α-Synuclein expression in the mouse cerebellum is restricted to VGluT1 excitatory terminals and is enriched in unipolar brush cells.

    PubMed

    Lee, Sun Kyong; Sillitoe, Roy V; Silva, Coralie; Martina, Marco; Sekerkova, Gabriella

    2015-10-01

    α-Synuclein has a crucial role in synaptic vesicle release and synaptic membrane recycling. Although its general expression pattern has been described in the cerebellum, the precise cerebellar structures where α-synuclein is localized are poorly understood. To address this question, we used α-synuclein immunohistochemistry in adult mice cerebellar sections. We found that α-synuclein labels glutamatergic but not glycinergic and GABAergic synaptic terminals in the molecular and granule cell layers. α-Synuclein was preferentially expressed in parallel and mossy fiber synaptic terminals that also express vesicular glutamate transporter 1 (VGluT1), while it was not detected in VGluT2-positive climbing fibers. α-Synuclein was particularly enriched in lobules IX and X, a region known to contain a high density of unipolar brush cells (UBCs). To elucidate whether the α-synuclein-positive mossy fibers belong to UBCs, we double-labeled cerebellar sections with antibodies to α-synuclein and UBC-type-specific markers (calretinin for type I and metabotropic glutamate receptor 1α (mGluR1α) for type II UBCs) and took advantage of organotypic cerebellar cultures (in which all mossy fibers are UBC axons) and moonwalker mice (in which almost all UBCs are ablated) and found that both type I and type II UBCs express α-synuclein. In moonwalker mutant cerebella, the α-synuclein/VGluT1 immunolabeling showed a dramatic decrease in the vestibulocerebellum that correlated with the absence of UBC. α-Synuclein appears to be an excellent marker for intrinsic mossy fibers of the VGluT1 subset in conjunction with UBCs of both subtypes.

  15. Structural and functional characterization of two alpha-synuclein strains

    NASA Astrophysics Data System (ADS)

    Bousset, Luc; Pieri, Laura; Ruiz-Arlandis, Gemma; Gath, Julia; Jensen, Poul Henning; Habenstein, Birgit; Madiona, Karine; Olieric, Vincent; Böckmann, Anja; Meier, Beat H.; Melki, Ronald

    2013-10-01

    α-synuclein aggregation is implicated in a variety of diseases including Parkinson’s disease, dementia with Lewy bodies, pure autonomic failure and multiple system atrophy. The association of protein aggregates made of a single protein with a variety of clinical phenotypes has been explained for prion diseases by the existence of different strains that propagate through the infection pathway. Here we structurally and functionally characterize two polymorphs of α-synuclein. We present evidence that the two forms indeed fulfil the molecular criteria to be identified as two strains of α-synuclein. Specifically, we show that the two strains have different structures, levels of toxicity, and in vitro and in vivo seeding and propagation properties. Such strain differences may account for differences in disease progression in different individuals/cell types and/or types of synucleinopathies.

  16. α-synuclein expression in the mouse cerebellum is restricted to VGluT1 excitatory terminals and is enriched in unipolar brush cells

    PubMed Central

    Lee, Sun Kyong; Sillitoe, Roy V.; Silva, Coralie; Martina, Marco; Sekerkova, Gabriella

    2015-01-01

    α-synuclein has a crucial role in synaptic vesicle release and synaptic membrane recycling. Although its general expression pattern has been described in the cerebellum, the precise cerebellar structures where α-synuclein is localized are poorly understood. To address this question, we used α-synuclein immunohistochemistry in adult mice cerebellar sections. We found that α-synuclein labels glutamatergic but not glycinergic and GABAergic synaptic terminals in the molecular and granule cell layers. α-synuclein was preferentially expressed in parallel and mossy fiber synaptic terminals that also express vesicular glutamate transporter 1 (VGluT1) while it was not detected in VGluT2-positive climbing fibers. α-synuclein was particularly enriched in lobules IX and X, a region known to contain high density of unipolar brush cells (UBCs). To elucidate whether the α-synuclein-positive mossy fibers belong to UBCs, we double labeled cerebellar sections with antibodies to α-synuclein and UBCs type specific markers(calretinin for type I and metabotropic glutamate receptor 1α (mGluR1α) for type II UBCs), and took advantage of organotypic cerebellar cultures (in which all mossy fibers are UBC axons) and moonwalker mice (in which almost all UBCs are ablated) and found that both type I and type II UBCs express α-synuclein. In moonwalker mutant cerebella, the α-synuclein/VGluT1 immunolabeling showed dramatic decrease in the vestibulocerebellum that correlated with the absence of UBC. α-synuclein appears to be an excellent marker for intrinsic mossy fibers of the VGluT1 subset in conjunction with UBCs of both subtypes. PMID:25917213

  17. Redistribution of DAT/α-Synuclein Complexes Visualized by “In Situ” Proximity Ligation Assay in Transgenic Mice Modelling Early Parkinson's Disease

    PubMed Central

    Bellucci, Arianna; Navarria, Laura; Falarti, Elisa; Zaltieri, Michela; Bono, Federica; Collo, Ginetta; Grazia, Maria; Missale, Cristina; Spano, PierFranco

    2011-01-01

    Alpha-synuclein, the major component of Lewy bodies, is thought to play a central role in the onset of synaptic dysfunctions in Parkinson's disease (PD). In particular, α-synuclein may affect dopaminergic neuron function as it interacts with a key protein modulating dopamine (DA) content at the synapse: the DA transporter (DAT). Indeed, recent evidence from our “in vitro” studies showed that α-synuclein aggregation decreases the expression and membrane trafficking of the DAT as the DAT is retained into α-synuclein-immunopositive inclusions. This notwithstanding, “in vivo” studies on PD animal models investigating whether DAT distribution is altered by the pathological overexpression and aggregation of α-synuclein are missing. By using the proximity ligation assay, a technique which allows the “in situ” visualization of protein-protein interactions, we studied the occurrence of alterations in the distribution of DAT/α-synuclein complexes in the SYN120 transgenic mouse model, showing insoluble α-synuclein aggregates into dopaminergic neurons of the nigrostriatal system, reduced striatal DA levels and an altered distribution of synaptic proteins in the striatum. We found that DAT/α-synuclein complexes were markedly redistributed in the striatum and substantia nigra of SYN120 mice. These alterations were accompanied by a significant increase of DAT striatal levels in transgenic animals when compared to wild type littermates. Our data indicate that, in the early pathogenesis of PD, α-synuclein acts as a fine modulator of the dopaminergic synapse by regulating the subcellular distribution of key proteins such as the DAT. PMID:22163275

  18. Structural and functional characterization of two alpha-synuclein strains

    PubMed Central

    Bousset, Luc; Pieri, Laura; Ruiz-Arlandis, Gemma; Gath, Julia; Jensen, Poul Henning; Habenstein, Birgit; Madiona, Karine; Olieric, Vincent; Böckmann, Anja; Meier, Beat H.; Melki, Ronald

    2013-01-01

    α-synuclein aggregation is implicated in a variety of diseases including Parkinson’s disease, dementia with Lewy bodies, pure autonomic failure and multiple system atrophy. The association of protein aggregates made of a single protein with a variety of clinical phenotypes has been explained for prion diseases by the existence of different strains that propagate through the infection pathway. Here we structurally and functionally characterize two polymorphs of α-synuclein. We present evidence that the two forms indeed fulfil the molecular criteria to be identified as two strains of α-synuclein. Specifically, we show that the two strains have different structures, levels of toxicity, and in vitro and in vivo seeding and propagation properties. Such strain differences may account for differences in disease progression in different individuals/cell types and/or types of synucleinopathies. PMID:24108358

  19. Glycation potentiates α-synuclein-associated neurodegeneration in synucleinopathies.

    PubMed

    Vicente Miranda, Hugo; Szego, Éva M; Oliveira, Luís M A; Breda, Carlo; Darendelioglu, Ekrem; de Oliveira, Rita M; Ferreira, Diana G; Gomes, Marcos A; Rott, Ruth; Oliveira, Márcia; Munari, Francesca; Enguita, Francisco J; Simões, Tânia; Rodrigues, Eva F; Heinrich, Michael; Martins, Ivo C; Zamolo, Irina; Riess, Olaf; Cordeiro, Carlos; Ponces-Freire, Ana; Lashuel, Hilal A; Santos, Nuno C; Lopes, Luisa V; Xiang, Wei; Jovin, Thomas M; Penque, Deborah; Engelender, Simone; Zweckstetter, Markus; Klucken, Jochen; Giorgini, Flaviano; Quintas, Alexandre; Outeiro, Tiago F

    2017-05-01

    α-Synuclein misfolding and aggregation is a hallmark in Parkinson's disease and in several other neurodegenerative diseases known as synucleinopathies. The toxic properties of α-synuclein are conserved from yeast to man, but the precise underpinnings of the cellular pathologies associated are still elusive, complicating the development of effective therapeutic strategies. Combining molecular genetics with target-based approaches, we established that glycation, an unavoidable age-associated post-translational modification, enhanced α-synuclein toxicity in vitro and in vivo, in Drosophila and in mice. Glycation affected primarily the N-terminal region of α-synuclein, reducing membrane binding, impaired the clearance of α-synuclein, and promoted the accumulation of toxic oligomers that impaired neuronal synaptic transmission. Strikingly, using glycation inhibitors, we demonstrated that normal clearance of α-synuclein was re-established, aggregation was reduced, and motor phenotypes in Drosophila were alleviated. Altogether, our study demonstrates glycation constitutes a novel drug target that can be explored in synucleinopathies as well as in other neurodegenerative conditions. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. The novel compound PBT434 prevents iron mediated neurodegeneration and alpha-synuclein toxicity in multiple models of Parkinson's disease.

    PubMed

    Finkelstein, David I; Billings, Jessica L; Adlard, Paul A; Ayton, Scott; Sedjahtera, Amelia; Masters, Colin L; Wilkins, Simon; Shackleford, David M; Charman, Susan A; Bal, Wojciech; Zawisza, Izabela A; Kurowska, Ewa; Gundlach, Andrew L; Ma, Sheri; Bush, Ashley I; Hare, Dominic J; Doble, Philip A; Crawford, Simon; Gautier, Elisabeth Cl; Parsons, Jack; Huggins, Penny; Barnham, Kevin J; Cherny, Robert A

    2017-06-28

    Elevated iron in the SNpc may play a key role in Parkinson's disease (PD) neurodegeneration since drug candidates with high iron affinity rescue PD animal models, and one candidate, deferirpone, has shown efficacy recently in a phase two clinical trial. However, strong iron chelators may perturb essential iron metabolism, and it is not yet known whether the damage associated with iron is mediated by a tightly bound (eg ferritin) or lower-affinity, labile, iron pool. Here we report the preclinical characterization of PBT434, a novel quinazolinone compound bearing a moderate affinity metal-binding motif, which is in development for Parkinsonian conditions. In vitro, PBT434 was far less potent than deferiprone or deferoxamine at lowering cellular iron levels, yet was found to inhibit iron-mediated redox activity and iron-mediated aggregation of α-synuclein, a protein that aggregates in the neuropathology. In vivo, PBT434 did not deplete tissue iron stores in normal rodents, yet prevented loss of substantia nigra pars compacta neurons (SNpc), lowered nigral α-synuclein accumulation, and rescued motor performance in mice exposed to the Parkinsonian toxins 6-OHDA and MPTP, and in a transgenic animal model (hA53T α-synuclein) of PD. These improvements were associated with reduced markers of oxidative damage, and increased levels of ferroportin (an iron exporter) and DJ-1. We conclude that compounds designed to target a pool of pathological iron that is not held in high-affinity complexes in the tissue can maintain the survival of SNpc neurons and could be disease-modifying in PD.

  1. An Efficient Procedure for Removal and Inactivation of Alpha-Synuclein Assemblies from Laboratory Materials.

    PubMed

    Bousset, Luc; Brundin, Patrik; Böckmann, Anja; Meier, Beat; Melki, Ronald

    2016-01-01

    Preformed α-synuclein fibrils seed the aggregation of soluble α-synuclein in cultured cells and in vivo. This, and other findings, has kindled the idea that α-synuclein fibrils possess prion-like properties. As α-synuclein fibrils should not be considered as innocuous, there is a need for decontamination and inactivation procedures for laboratory benches and non-disposable laboratory material. We assessed the effectiveness of different procedures designed to disassemble α-synuclein fibrils and reduce their infectivity. We examined different commercially available detergents to remove α-synuclein assemblies adsorbed on materials that are not disposable and that are most found in laboratories (e.g. plastic, glass, aluminum or stainless steel surfaces). We show that methods designed to decrease PrP prion infectivity neither effectively remove α-synuclein assemblies adsorbed to different materials commonly used in the laboratory nor disassemble the fibrillar form of the protein with efficiency. In contrast, both commercial detergents and SDS detached α-synuclein assemblies from contaminated surfaces and disassembled the fibrils. We describe three cleaning procedures that effectively remove and disassemble α-synuclein seeds. The methods rely on the use of detergents that are compatible with most non-disposable tools in a laboratory. The procedures are easy to implement and significantly decrease any potential risks associated to handling α-synuclein assemblies.

  2. α-Synuclein Sequesters Dnmt1 from the Nucleus

    PubMed Central

    Desplats, Paula; Spencer, Brian; Coffee, Elizabeth; Patel, Pruthul; Michael, Sarah; Patrick, Christina; Adame, Anthony; Rockenstein, Edward; Masliah, Eliezer

    2011-01-01

    DNA methylation is a major epigenetic modification that regulates gene expression. Dnmt1, the maintenance DNA methylation enzyme, is abundantly expressed in the adult brain and is mainly located in the nuclear compartment, where it has access to chromatin. Hypomethylation of CpG islands at intron 1 of the SNCA gene has recently been reported to result in overexpression of α-synuclein in Parkinson disease (PD) and related disorders. We therefore investigated the mechanisms underlying altered DNA methylation in PD and dementia with Lewy bodies (DLB). We present evidence of reduction of nuclear Dnmt1 levels in human postmortem brain samples from PD and DLB patients as well as in the brains of α-synuclein transgenic mice models. Furthermore, sequestration of Dnmt1 in the cytoplasm results in global DNA hypomethylation in human and mouse brains, involving CpG islands upstream of SNCA, SEPW1, and PRKAR2A genes. We report that association of Dnmt1 and α-synuclein might mediate aberrant subcellular localization of Dnmt1. Nuclear Dnmt1 levels were partially rescued by overexpression of Dnmt1 in neuronal cell cultures and in α-synuclein transgenic mice brains. Our results underscore a novel mechanism for epigenetic dysregulation in Lewy body diseases, which might underlie the decrease in DNA methylation reported for PD and DLB. PMID:21296890

  3. An Efficient Procedure for Removal and Inactivation of Alpha-Synuclein Assemblies from Laboratory Materials

    PubMed Central

    Bousset, Luc; Brundin, Patrik; Böckmann, Anja; Meier, Beat; Melki, Ronald

    2015-01-01

    Background: Preformed α-synuclein fibrils seed the aggregation of soluble α-synuclein in cultured cells and in vivo. This, and other findings, has kindled the idea that α-synuclein fibrils possess prion-like properties. Objective: As α-synuclein fibrils should not be considered as innocuous, there is a need for decontamination and inactivation procedures for laboratory benches and non-disposable laboratory material. Methods: We assessed the effectiveness of different procedures designed to disassemble α-synuclein fibrils and reduce their infectivity. We examined different commercially available detergents to remove α-synuclein assemblies adsorbed on materials that are not disposable and that are most found in laboratories (e.g. plastic, glass, aluminum or stainless steel surfaces). Results: We show that methods designed to decrease PrP prion infectivity neither effectively remove α-synuclein assemblies adsorbed to different materials commonly used in the laboratory nor disassemble the fibrillar form of the protein with efficiency. In contrast, both commercial detergents and SDS detached α-synuclein assemblies from contaminated surfaces and disassembled the fibrils. Conclusions: We describe three cleaning procedures that effectively remove and disassemble α-synuclein seeds. The methods rely on the use of detergents that are compatible with most non-disposable tools in a laboratory. The procedures are easy to implement and significantly decrease any potential risks associated to handling α-synuclein assemblies. PMID:26639448

  4. Neuroprotective effects of voluntary running on cognitive dysfunction in an α-synuclein rat model of Parkinson's disease.

    PubMed

    Crowley, Erin K; Nolan, Yvonne M; Sullivan, Aideen M

    2018-05-01

    Parkinson's disease (PD) is no longer primarily classified as a motor disorder due to increasing recognition of the impact on patients of several nonmotor PD symptoms, including cognitive dysfunction. These nonmotor symptoms are highly prevalent and greatly affect the quality of life of patients with PD, and so, therapeutic interventions to alleviate these symptoms are urgently needed. The aim of this study was to investigate the potential neuroprotective effects of voluntary running on cognitive dysfunction in an adeno-associated virus-α-synuclein rat model of PD. Bilateral intranigral administration of adeno-associated virus-α-synuclein was found to induce motor dysfunction and a significant loss of nigral dopaminergic neurons, neither of which were rescued by voluntary running. Overexpression of α-synuclein also resulted in significant impairment on hippocampal neurogenesis-dependent pattern separation, a cognitive task; this was rescued by voluntary running. This was substantiated by an effect of running on neurogenesis levels in the dorsal dentate gyrus, suggesting that the functional effects of running on pattern separation were mediated via increased neurogenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. p53 functional impairment and high p21waf1/cip1 expression in human T-cell lymphotropic/leukemia virus type I-transformed T cells.

    PubMed

    Cereseto, A; Diella, F; Mulloy, J C; Cara, A; Michieli, P; Grassmann, R; Franchini, G; Klotman, M E

    1996-09-01

    Human T-cell lymphotropic/leukemia virus type I (HTLV-I) is associated with T-cell transformation both in vivo and in vitro. Although some of the mechanisms responsible for transformation remain unknown, increasing evidence supports a direct role of viral as well as dysregulated cellular proteins in transformation. We investigated the potential role of the tumor suppressor gene p53 and of the p53-regulated gene, p21waf1/cip1 (wild-type p53 activated fragment 1/cycling dependent kinases [cdks] interacting protein 1), in HTLV-I-infected T cells. We have found that the majority of HTLV-I-infected T cells have the wild-type p53 gene. However, its function in HTLV-I-transformed cells appears to be impaired, as shown by the lack of appropriate p53-mediated responses to ionizing radiation (IR). Interestingly, the expression of the p53 inducible gene, p21waf1/cip1, is elevated at the messenger ribonucleic acid and protein levels in all HTLV-I-infected T-cell lines examined as well as in Taxl-1, a human T-cell line stably expressing Tax. Additionally, Tax induces upregulation of a p21waf1/cip1 promoter-driven luciferase gene in p53 null cells, and increases p21waf1/cip1 expression in Jurkat T cells. These findings suggest that the Tax protein is at least partially responsible for the p53-independent expression of p21waf1/cip1 in HTLV-I-infected cells. Dysregulation of p53 and p21waf1/cip1 proteins regulating cell-cycle progression, may represent an important step in HTLV-I-induced T-cell transformation.

  6. Human T-cell lymphotropic virus type 1-infected T lymphocytes impair catabolism and uptake of glutamate by astrocytes via Tax-1 and tumor necrosis factor alpha.

    PubMed

    Szymocha, R; Akaoka, H; Dutuit, M; Malcus, C; Didier-Bazes, M; Belin, M F; Giraudon, P

    2000-07-01

    Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of a chronic progressive myelopathy called tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). In this disease, lesions of the central nervous system (CNS) are associated with perivascular infiltration by lymphocytes. We and others have hypothesized that these T lymphocytes infiltrating the CNS may play a prominent role in TSP/HAM. Here, we show that transient contact of human or rat astrocytes with T lymphocytes chronically infected by HTLV-1 impairs some of the major functions of brain astrocytes. Uptake of extracellular glutamate by astrocytes was significantly decreased after transient contact with infected T cells, while the expression of the glial transporters GLAST and GLT-1 was decreased. In two-compartment cultures avoiding direct cell-to-cell contact, similar results were obtained, suggesting possible involvement of soluble factors, such as cytokines and the viral protein Tax-1. Recombinant Tax-1 and tumor necrosis factor alpha (TNF-alpha) decreased glutamate uptake by astrocytes. Tax-1 probably acts by inducing TNF-alpha, as the effect of Tax-1 was abolished by anti-TNF-alpha antibody. The expression of glutamate-catabolizing enzymes in astrocytes was increased for glutamine synthetase and decreased for glutamate dehydrogenase, the magnitudes of these effects being correlated with the level of Tax-1 transcripts. In conclusion, Tax-1 and cytokines produced by HTLV-1-infected T cells impair the ability of astrocytes to manage the steady-state level of glutamate, which in turn may affect neuronal and oligodendrocytic functions and survival.

  7. Alpha-Synuclein Produces Early Behavioral Alterations via Striatal Cholinergic Synaptic Dysfunction by Interacting With GluN2D N-Methyl-D-Aspartate Receptor Subunit.

    PubMed

    Tozzi, Alessandro; de Iure, Antonio; Bagetta, Vincenza; Tantucci, Michela; Durante, Valentina; Quiroga-Varela, Ana; Costa, Cinzia; Di Filippo, Massimiliano; Ghiglieri, Veronica; Latagliata, Emanuele Claudio; Wegrzynowicz, Michal; Decressac, Mickael; Giampà, Carmela; Dalley, Jeffrey W; Xia, Jing; Gardoni, Fabrizio; Mellone, Manuela; El-Agnaf, Omar Mukhtar; Ardah, Mustafa Taleb; Puglisi-Allegra, Stefano; Björklund, Anders; Spillantini, Maria Grazia; Picconi, Barbara; Calabresi, Paolo

    2016-03-01

    Advanced Parkinson's disease (PD) is characterized by massive degeneration of nigral dopaminergic neurons, dramatic motor and cognitive alterations, and presence of nigral Lewy bodies, whose main constituent is α-synuclein (α-syn). However, the synaptic mechanisms underlying behavioral and motor effects induced by early selective overexpression of nigral α-syn are still a matter of debate. We performed behavioral, molecular, and immunohistochemical analyses in two transgenic models of PD, mice transgenic for truncated human α-synuclein 1-120 and rats injected with the adeno-associated viral vector carrying wild-type human α-synuclein. We also investigated striatal synaptic plasticity by electrophysiological recordings from spiny projection neurons and cholinergic interneurons. We found that overexpression of truncated or wild-type human α-syn causes partial reduction of striatal dopamine levels and selectively blocks the induction of long-term potentiation in striatal cholinergic interneurons, producing early memory and motor alterations. These effects were dependent on α-syn modulation of the GluN2D-expressing N-methyl-D-aspartate receptors in cholinergic interneurons. Acute in vitro application of human α-syn oligomers mimicked the synaptic effects observed ex vivo in PD models. We suggest that striatal cholinergic dysfunction, induced by a direct interaction between α-syn and GluN2D-expressing N-methyl-D-aspartate receptors, represents a precocious biological marker of the disease. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  8. Preserved functional autonomic phenotype in adult mice overexpressing moderate levels of human alpha‐synuclein in oligodendrocytes

    PubMed Central

    Tank, Jens; da Costa‐Goncalves, Andrey C.; Kamer, Ilona; Qadri, Fatimunnisa; Ubhi, Kiren; Rockenstein, Edward; Diedrich, André; Masliah, Eliezer; Gross, Volkmar; Jordan, Jens

    2014-01-01

    Abstract Mice overexpressing human alpha‐synuclein in oligodendrocytes (MBP1‐α‐syn) recapitulate some key functional and neuropathological features of multiple system atrophy (MSA). Whether or not these mice develop severe autonomic failure, which is a key feature of human MSA, remains unknown. We explored cardiovascular autonomic regulation using long‐term blood pressure (BP) radiotelemetry and pharmacological testing. We instrumented 12 MBP1‐α‐syn mice and 11 wild‐type mice aged 9 months for radiotelemetry. Animals were tested with atropine, metoprolol, clonidine, and trimethaphan at 9 and 12 months age. We applied spectral and cross‐spectral analysis to assess heart rate (HR) and BP variability. At 9 months of age daytime BP (transgenic: 101 ± 2 vs. wild type: 99 ± 2 mmHg) and HR (497 ± 11 vs. 505 ± 16 beats/min) were similar. Circadian BP and HR rhythms were maintained. Nighttime BP (109 ± 2 vs. 108 ± 2 mmHg) and HR (575 ± 15 vs. 569 ± 14 beats/min), mean arterial BP responses to trimethaphan (−21 ± 8 vs. −10 ± 5 mmHg, P = 0.240) and to clonidine (−8 ± 3 vs. −5 ± 2 mmHg, P = 0.314) were similar. HR responses to atropine (+159 ± 24 vs. +146 ± 22 beats/min), and to clonidine (−188 ± 21 vs. −163 ± 33 beats/min) did not differ between strains. Baroreflex sensitivity (4 ± 1 vs. 4 ± 1 msec/mmHg) and HR variability (total power, 84 ± 17 vs. 65 ± 21 msec²) were similar under resting conditions and during pharmacological testing. Repeated measurements at 12 months of age provided similar results. In mice, moderate overexpression of human alpha‐synuclein in oligodendrocytes is not sufficient to induce overt autonomic failure. Additional mechanisms may be required to express the autonomic failure phenotype including higher levels of expression or more advanced age. PMID:25428949

  9. Studies of protein aggregation in A53T α-synuclein transgenic, Tg2576 transgenic, and P246L presenilin-1 knock-in cross bred mice.

    PubMed

    Emmer, Kristel L; Covy, Jason P; Giasson, Benoit I

    2012-01-24

    Synucleinopathies are a group of neurodegenerative disorders, including Parkinson disease, associated with neuronal amyloid inclusions comprised of the presynaptic protein α-synuclein (α-syn); however the biological events that initiate and lead to the formation of these inclusions are still poorly understood. There is mounting evidence that intracellular α-syn aggregation may proceed via a seeding mechanism and could spread between neurons through a prion-like mechanism that may involve other amyloidogenic proteins. Several lines of evidence suggest that Aβ peptides and/or extracellular Aβ deposits may directly or indirectly promote intracellular α-syn aggregation. To assess the effects of Aβ peptides and extracellular Aβ deposits on α-syn aggregate formation, transgenic mice (line M83) expressing A53T human α-syn that are sensitive to developing α-syn pathological inclusions were cross bred to Tg2576 transgenic mice that generated elevated levels of Aβ peptides and develop abundant Aβ plaques. In addition these mice were bred to mice with the P264L presenilin-1 knock-in mutation that further promotes Aβ plaque formation. These mice demonstrated the expected formation of Aβ plaques; however despite the accumulation of hyperphosphorylated α-syn dystrophic neurites within or surrounding Aβ plaques, no additional α-syn pathologies were observed. These studies show that Aβ amyloid deposits can cause the local aggregation of α-syn, but these did not lead to more extensive α-syn pathology. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  10. Epigallocatechin Gallate (EGCG) Inhibits Alpha-Synuclein Aggregation: A Potential Agent for Parkinson's Disease.

    PubMed

    Xu, Yan; Zhang, Yanyan; Quan, Zhenzhen; Wong, Winnie; Guo, Jianping; Zhang, Rongkai; Yang, Qinghu; Dai, Rongji; McGeer, Patrick L; Qing, Hong

    2016-10-01

    Protein aggregation is a prominent feature of many neurodegenerative disorders including Parkinson's disease (PD). Aggregation of alpha-synuclein (SNCA) may underlie the pathology of PD. They are the main components of Lewy bodies and dystrophic neurites that are the intraneuronal inclusions characteristic of the disease. We have demonstrated that the polyphenol (-)-epi-gallocatechine gallate (EGCG) inhibited SNCA aggregation, which made it a candidate for therapeutic intervention in PD. Three methods were used: SNCA fibril formation inhibition by EGCG in incubates; inhibition of the SNCA fluorophore A-Syn-HiLyte488 binding to plated SNCA in microwells; and inhibition of the A-Syn-HiLyte488 probe binding to aggregated SNCA in postmortem PD tissue. Recombinant human SNCA was incubated under conditions that result in fibril formation. The aggregation was blocked by 100 nM EGCG in a concentration-dependent manner, as shown by an absence of thioflavin T binding. In the microplate assay system, the ED 50 of EGCG inhibition of A-Syn-HiLyte488 binding to coated SNCA was 250 nM. In the PD tissue based assay, SNCA aggregates were recognized by incubation with 7 nM of A-Syn-HiLyte488. This binding was blocked by EGCG in a concentration dependent manner. The SNCA amino acid sites, which potentially interacted with EGCG, were detected on peptide membranes. It was implicated that EGCG binds to SNCA by instable hydrophobic interactions. In this study, we suggested that EGCG could be a potent remodeling agent of SNCA aggregates and a potential disease modifying drug for the treatment of PD and other α-synucleinopathies.

  11. KLK6 proteolysis is implicated in the turnover and uptake of extracellular alpha-synuclein species.

    PubMed

    Pampalakis, Georgios; Sykioti, Vasia-Samantha; Ximerakis, Methodios; Stefanakou-Kalakou, Ioanna; Melki, Ronald; Vekrellis, Kostas; Sotiropoulou, Georgia

    2017-02-28

    KLK6 is a serine protease highly expressed in the nervous system. In synucleinopathies, including Parkinson disease, the levels of KLK6 inversely correlate with α-synuclein in CSF. Recently, we suggested that recombinant KLK6 mediates the degradation of extracellular α-synuclein directly and via a proteolytic cascade that involves unidentified metalloproteinase(s). Here, we show that recombinant and naturally secreted KLK6 can readily cleave α-synuclein fibrils that have the potential for cell-to-cell propagation in "a prion-like mechanism". Importantly, KLK6-deficient primary cortical neurons have increased ability for α-synuclein fibril uptake. We also demonstrate that KLK6 activates proMMP2, which in turn can cleave α-synuclein. The repertoire of proteases activated by KLK6 in a neuronal environment was analyzed by degradomic profiling, which also identified ADAMTS19 and showed that KLK6 has a limited number of substrates indicating specific biological functions such as the regulation of α-synuclein turnover. We generated adenoviral vectors for KLK6 delivery and demonstrated that the levels of extracellular α-synuclein can be reduced by neuronally secreted KLK6. Our findings open the possibility to exploit KLK6 as a novel therapeutic target for Parkinson disease and other synucleinopathies.

  12. KLK6 proteolysis is implicated in the turnover and uptake of extracellular alpha-synuclein species

    PubMed Central

    Pampalakis, Georgios; Sykioti, Vasia-Samantha; Ximerakis, Methodios; Stefanakou-Kalakou, Ioanna; Melki, Ronald; Vekrellis, Kostas; Sotiropoulou, Georgia

    2017-01-01

    KLK6 is a serine protease highly expressed in the nervous system. In synucleinopathies, including Parkinson disease, the levels of KLK6 inversely correlate with α-synuclein in CSF. Recently, we suggested that recombinant KLK6 mediates the degradation of extracellular α-synuclein directly and via a proteolytic cascade that involves unidentified metalloproteinase(s). Here, we show that recombinant and naturally secreted KLK6 can readily cleave α-synuclein fibrils that have the potential for cell-to-cell propagation in “a prion-like mechanism”. Importantly, KLK6-deficient primary cortical neurons have increased ability for α-synuclein fibril uptake. We also demonstrate that KLK6 activates proMMP2, which in turn can cleave α-synuclein. The repertoire of proteases activated by KLK6 in a neuronal environment was analyzed by degradomic profiling, which also identified ADAMTS19 and showed that KLK6 has a limited number of substrates indicating specific biological functions such as the regulation of α-synuclein turnover. We generated adenoviral vectors for KLK6 delivery and demonstrated that the levels of extracellular α-synuclein can be reduced by neuronally secreted KLK6. Our findings open the possibility to exploit KLK6 as a novel therapeutic target for Parkinson disease and other synucleinopathies. PMID:27845893

  13. Nanotized PPARα Overexpression Targeted to Hypertrophied Myocardium Improves Cardiac Function by Attenuating the p53-GSK3β-Mediated Mitochondrial Death Pathway.

    PubMed

    Rana, Santanu; Datta, Ritwik; Chaudhuri, Ratul Datta; Chatterjee, Emeli; Chawla-Sarkar, Mamta; Sarkar, Sagartirtha

    2018-05-09

    Metabolic remodeling of cardiac muscles during pathological hypertrophy is characterized by downregulation of fatty acid oxidation (FAO) regulator, peroxisome proliferator-activated receptor alpha (PPARα). Thereby, we hypothesized that a cardiac-specific induction of PPARα might restore the FAO-related protein expression and resultant energy deficit. In the present study, consequences of PPARα augmentation were evaluated for amelioration of chronic oxidative stress, myocyte apoptosis, and cardiac function during pathological cardiac hypertrophy. Nanotized PPARα overexpression targeted to myocardium was done by a stearic acid-modified carboxymethyl-chitosan (CMC) conjugated to a 20-mer myocyte-targeted peptide (CMCP). Overexpression of PPARα ameliorated pathological hypertrophy and improved cardiac function. Augmented PPARα in hypertrophied myocytes revealed downregulated p53 acetylation (lys 382), leading to reduced apoptosis. Such cells showed increased binding of PPARα with p53 that in turn reduced interaction of p53 with glycogen synthase kinase-3β (GSK3β), which upregulated inactive phospho-GSK3β (serine [Ser]9) expression within mitochondrial protein fraction. Altogether, the altered molecular milieu in PPARα-overexpressed hypertrophy groups restored mitochondrial structure and function both in vitro and in vivo. Cardiomyocyte-targeted overexpression of a protein of interest (PPARα) by nanotized plasmid has been described for the first time in this study. Our data provide a novel insight towards regression of pathological hypertrophy by ameliorating mitochondrial oxidative stress in targeted PPARα-overexpressed myocardium. PPARα-overexpression during pathological hypertrophy showed substantial betterment of mitochondrial structure and function, along with downregulated apoptosis. Myocardium-targeted overexpression of PPARα during pathological cardiac hypertrophy led to an overall improvement of cardiac energy deficit and subsequent cardiac

  14. α-Synuclein Fibrils Exhibit Gain of Toxic Function, Promoting Tau Aggregation and Inhibiting Microtubule Assembly*

    PubMed Central

    Oikawa, Takayuki; Nonaka, Takashi; Terada, Makoto; Tamaoka, Akira; Hisanaga, Shin-ichi; Hasegawa, Masato

    2016-01-01

    α-Synuclein is the major component of Lewy bodies and Lewy neurites in Parkinson disease and dementia with Lewy bodies and of glial cytoplasmic inclusions in multiple system atrophy. It has been suggested that α-synuclein fibrils or intermediate protofibrils in the process of fibril formation may have a toxic effect on neuronal cells. In this study, we investigated the ability of soluble monomeric α-synuclein to promote microtubule assembly and the effects of conformational changes of α-synuclein on Tau-promoted microtubule assembly. In marked contrast to previous findings, monomeric α-synuclein had no effect on microtubule polymerization. However, both α-synuclein fibrils and protofibrils inhibited Tau-promoted microtubule assembly. The inhibitory effect of α-synuclein fibrils was greater than that of the protofibrils. Dot blot overlay assay and spin-down techniques revealed that α-synuclein fibrils bind to Tau and inhibit microtubule assembly by depleting the Tau available for microtubule polymerization. Using various deletion mutants of α-synuclein and Tau, the acidic C-terminal region of α-synuclein and the basic central region of Tau were identified as regions involved in the binding. Furthermore, introduction of α-synuclein fibrils into cultured cells overexpressing Tau protein induced Tau aggregation. These results raise the possibility that α-synuclein fibrils interact with Tau, inhibit its function to stabilize microtubules, and also promote Tau aggregation, leading to dysfunction of neuronal cells. PMID:27226637

  15. p27Kip1 regulates alpha-synuclein expression

    PubMed Central

    Gallastegui, Edurne; Domuro, Carla; Serratosa, Joan; Larrieux, Alejandra; Sin, Laura; Martinez, Jonatan; Besson, Arnaud; Morante-Redolat, José Manuel; Orlando, Serena; Aligue, Rosa; Fariñas, Isabel; Pujol, María Jesús; Bachs, Oriol

    2018-01-01

    Alpha-synuclein (α-SYN) is the main component of anomalous protein aggregates (Lewy bodies) that play a crucial role in several neurodegenerative diseases (synucleinopathies) like Parkinson’s disease and multiple system atrophy. However, the mechanisms involved in its transcriptional regulation are poorly understood. We investigated here the role of the cyclin-dependent kinase (Cdk) inhibitor and transcriptional regulator p27Kip1 (p27) in the regulation of α-SYN expression. We observed that selective deletion of p27 by CRISPR/Cas9 technology in neural cells resulted in increased levels of α-SYN. Knock-down of the member of the same family p21Cip1 (p21) also led to increased α-SYN levels, indicating that p27 and p21 collaborate in the repression of α-SYN transcription. We demonstrated that this repression is mediated by the transcription factor E2F4 and the member of the retinoblastoma protein family p130 and that it is dependent of Cdk activity. Chromatin immunoprecipitation analysis revealed specific binding sites for p27, p21 and E2F4 in the proximal α-SYN gene promoter. Finally, luciferase assays revealed a direct action of p27, p21 and E2F4 in α-SYN gene expression. Our findings reveal for the first time a negative regulatory mechanism of α-SYN expression, suggesting a putative role for cell cycle regulators in the etiology of synucleinopathies. PMID:29662651

  16. MECHANISM OF ALPHA-SYNUCLEIN OLIGOMERIZATION AND MEMBRANE INTERACTION: THEORETICAL APPROACH TO UNSTRUCTURED PROTEINS STUDIES

    PubMed Central

    Tsigelny, Igor F.; Sharikov, Yuriy; Miller, Mark A.; Masliah, Eliezer

    2008-01-01

    Misfolding and oligomerization of unstructured proteins is involved in the pathogenesis of Parkinson’s (PD), Alzheimer’s (AD), Huntington’s, and other neurodegenerative disorders. Elucidation of possible conformations of these proteins and their interactions with the membrane is necessary to understand the molecular mechanisms of neurodegeneration. We developed a strategy that makes it possible to elucidate the molecular mechanisms of of alpha-synuclein aggregation- a key molecular event in the pathogenesis of PD. This strategy can be also useful for the study of other unstructured proteins involved in neurodegeneration. The results of these theoretical studies have been confirmed with biochemical and electrophysiological studies. Our studies provide insights into the molecular mechanism for PD initiation and progression, and provide a useful paradigm for identifying possible therapeutic interventions through computational modeling. PMID:18640077

  17. Direct Correlation Between Ligand-Induced α-Synuclein Oligomers and Amyloid-like Fibril Growth

    PubMed Central

    Nors Perdersen, Martin; Foderà, Vito; Horvath, Istvan; van Maarschalkerweerd, Andreas; Nørgaard Toft, Katrine; Weise, Christoph; Almqvist, Fredrik; Wolf-Watz, Magnus; Wittung-Stafshede, Pernilla; Vestergaard, Bente

    2015-01-01

    Aggregation of proteins into amyloid deposits is the hallmark of several neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. The suggestion that intermediate oligomeric species may be cytotoxic has led to intensified investigations of pre-fibrillar oligomers, which are complicated by their transient nature and low population. Here we investigate alpha-synuclein oligomers, enriched by a 2-pyridone molecule (FN075), and the conversion of oligomers into fibrils. As probed by leakage assays, the FN075 induced oligomers potently disrupt vesicles in vitro, suggesting a potential link to disease related degenerative activity. Fibrils formed in the presence and absence of FN075 are indistinguishable on microscopic and macroscopic levels. Using small angle X-ray scattering, we reveal that FN075 induced oligomers are similar, but not identical, to oligomers previously observed during alpha-synuclein fibrillation. Since the levels of FN075 induced oligomers correlate with the amounts of fibrils among different FN075:protein ratios, the oligomers appear to be on-pathway and modeling supports an ‘oligomer stacking model’ for alpha-synuclein fibril elongation. PMID:26020724

  18. CDIP, a novel pro-apoptotic gene, regulates TNFalpha-mediated apoptosis in a p53-dependent manner.

    PubMed

    Brown, Lauren; Ongusaha, Pat P; Kim, Hyung-Gu; Nuti, Shanthy; Mandinova, Anna; Lee, Ji Won; Khosravi-Far, Roya; Aaronson, Stuart A; Lee, Sam W

    2007-07-25

    We have identified a novel pro-apoptotic p53 target gene named CDIP (Cell Death Involved p53-target). Inhibition of CDIP abrogates p53-mediated apoptotic responses, demonstrating that CDIP is an important p53 apoptotic effector. CDIP itself potently induces apoptosis that is associated with caspase-8 cleavage, implicating the extrinsic cell death pathway in apoptosis mediated by CDIP. siRNA-directed knockdown of caspase-8 results in a severe impairment of CDIP-dependent cell death. In investigating the potential involvement of extrinsic cell death pathway in CDIP-mediated apoptosis, we found that TNF-alpha expression tightly correlates with CDIP expression, and that inhibition of TNF-alpha signaling attenuates CDIP-dependent apoptosis. We also demonstrate that TNF-alpha is upregulated in response to p53 and p53 inducing genotoxic stress, in a CDIP-dependent manner. Consistently, knockdown of TNF-alpha impairs p53-mediated stress-induced apoptosis. Together, these findings support a novel p53 --> CDIP --> TNF-alpha apoptotic pathway that directs apoptosis after exposure of cells to genotoxic stress. Thus, CDIP provides a new link between p53-mediated intrinsic and death receptor-mediated extrinsic apoptotic signaling, providing a novel target for cancer therapeutics aimed at maximizing the p53 apoptotic response of cancer cells to drug therapy.

  19. Is trehalose an autophagic inducer? Unraveling the roles of non-reducing disaccharides on autophagic flux and alpha-synuclein aggregation.

    PubMed

    Yoon, Ye-Seul; Cho, Eun-Duk; Jung Ahn, Woo; Won Lee, Kyung; Lee, Seung-Jae; Lee, He-Jin

    2017-10-05

    Autophagy is a pivotal intracellular process by which cellular macromolecules are degraded upon various stimuli. A failure in the degradation of autophagic substrates such as impaired organelles and protein aggregates leads to their accumulations, which are characteristics of many neurodegenerative diseases. Pharmacological activation of autophagy has thus been considered a prospective therapeutic approach for treating neurodegenerative diseases. Among a number of autophagy-inducing agents, trehalose has received attention for its beneficial effects in different disease models of neurodegeneration. However, how trehalose promotes autophagy has not been fully revealed. We investigated the influence of trehalose and other disaccharides upon autophagic flux and aggregation of α-synuclein, a protein linked to Parkinson's disease. In differentiated human neuroblastoma and primary rat cortical neuron culture models, treatment with trehalose and other disaccharides resulted in accumulation of lipidated LC3 (LC3-II), p62, and autophagosomes, whereas it decreased autolysosomes. On the other hand, addition of Bafilomycin A1 to trehalose treatments had relatively marginal effect, an indicative of autophagic flux blockage. In concordance with these results, the cells treated with trehalose exhibited an incremental tendency in α-synuclein aggregation. Secretion of α-synuclein was also elevated in the culture medium upon trehalose treatment, thereby significantly increasing intercellular transmission of this protein. Despite the substantial increase in α-synuclein aggregation, which normally leads to cell death, cell viability was not affected upon treatment with trehalose, suggesting an autophagy-independent protective function of trehalose against protein aggregates. This study demonstrates that, although trehalose has been widely considered an autophagic inducer, it may be actually a potent blocker of the autophagic flux.

  20. Overexpression of protein kinase FA/GSK-3 alpha (a proline-directed protein kinase) correlates with human hepatoma dedifferentiation/progression.

    PubMed

    Yang, S D; Yu, J S; Yang, C C; Lee, S C; Lee, T T; Ni, M H; Kuan, C Y; Chen, H C

    1996-05-01

    Computer analysis of protein phosphorylation sites sequence revealed that transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of the proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3 alpha (kinase F(A)/GSK-3 alpha) (a member of PDPK family) has been optimized for human hepatoma and used to demonstrate for the first time significantly increased (P < 0.01) activity in poorly differentiated SK-Hep-1 hepatoma (24.2 +/- 2.8 units/mg) and moderately differentiated Mahlavu hepatoma (14.5 +/- 2.2 units/mg) when compared to well differentiated Hep 3B hepatoma (8.0 +/- 2.4 units/mg). Immunoblotting analysis revealed that increased activity of kinase FA/GSK-3 alpha is due to overexpression of the protein. Elevated kinase FA/GSK-3 alpha expression in human hepatoma biopsies relative to normal liver tissue was found to be even more profound. This kinase appeared to be fivefold overexpressed in well differentiated hepatoma and 13-fold overexpressed in poorly differentiated hepatoma when compared to normal liver tissue. Taken together, the results provide initial evidence that overexpression of kinase FA/GSK-3 alpha is involved in human hepatoma dedifferentiation/progression. Since kinase FA/GSK-3 alpha is a PDPK, the results further support a potential role of this kinase in human liver tumorigenesis, especially in its dedifferentiation/progression.

  1. Sodium butyrate rescues dopaminergic cells from alpha-synuclein-induced transcriptional deregulation and DNA damage.

    PubMed

    Paiva, Isabel; Pinho, Raquel; Pavlou, Maria Angeliki; Hennion, Magali; Wales, Pauline; Schütz, Anna-Lena; Rajput, Ashish; Szego, Éva M; Kerimoglu, Cemil; Gerhardt, Ellen; Rego, Ana Cristina; Fischer, André; Bonn, Stefan; Outeiro, Tiago F

    2017-06-15

    Alpha-synuclein (aSyn) is considered a major culprit in Parkinson's disease (PD) pathophysiology. However, the precise molecular function of the protein remains elusive. Recent evidence suggests that aSyn may play a role on transcription regulation, possibly by modulating the acetylation status of histones. Our study aimed at evaluating the impact of wild-type (WT) and mutant A30P aSyn on gene expression, in a dopaminergic neuronal cell model, and decipher potential mechanisms underlying aSyn-mediated transcriptional deregulation. We performed gene expression analysis using RNA-sequencing in Lund Human Mesencephalic (LUHMES) cells expressing endogenous (control) or increased levels of WT or A30P aSyn. Compared to control cells, cells expressing both aSyn variants exhibited robust changes in the expression of several genes, including downregulation of major genes involved in DNA repair. WT aSyn, unlike A30P aSyn, promoted DNA damage and increased levels of phosphorylated p53. In dopaminergic neuronal cells, increased aSyn expression led to reduced levels of acetylated histone 3. Importantly, treatment with sodium butyrate, a histone deacetylase inhibitor (HDACi), rescued WT aSyn-induced DNA damage, possibly via upregulation of genes involved in DNA repair. Overall, our findings provide novel and compelling insight into the mechanisms associated with aSyn neurotoxicity in dopaminergic cells, which could be ameliorated with an HDACi. Future studies will be crucial to further validate these findings and to define novel possible targets for intervention in PD. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. THE EFFECT OF GESTATIONAL MERCURY VAPOR EXPOSURE ON RAT BRAIN A-SYNUCLEIN EXPRESSION.

    EPA Science Inventory

    Alpha-synuclein is a highly conserved protein that localizes to pre-synaptic terminals and is thought to play a role in neuronal plasticity. It is upregulated developmentally and continues to be expressed at high levels in the adult brain. Its presence in a number of neuronal (A...

  3. Critical radius in the organisation of synuclein-alpha interacting protein in living cells

    NASA Astrophysics Data System (ADS)

    Narayanan, Arjun; Meriin, Anatoli; Sherman, Michael; Cisse, Ibrahim

    We report a super-resolution imaging study of protein aggregation in the living cell. Focusing on the aggregation of the Parkinsons's disease linked Synuclein-alpha interacting protein, we found and characterized sub-diffraction aggregates in healthy cells and studied the progression of these aggregates in stressed cells. Our results allowed us to establish the aggregation process as amenable to a simple physical description - the well-established thermodynamics of condensation phenomena. This description turned out to be both robust and useful. Not only did the distribution of aggregate sizes fit exceedingly well to the thermodynamic predictions in all tested conditions, but its evolving shape under pharmacological and genetic perturbations correlated intuitively with predictions from cell biology. The picture emerging from measurements in different genetic and pharmacological states is a view of protein aggregate size distribution as resulting from a non-equilibrium steady state maintained - even in healthy cells - with continuous and concurrent aggregate production and clearance.

  4. Geniposide reduces α-synuclein by blocking microRNA-21/lysosome-associated membrane protein 2A interaction in Parkinson disease models.

    PubMed

    Su, Chunhe; Yang, Xiaopeng; Lou, Jiyu

    2016-08-01

    This study aimed to explore whether the regulatory effect of miR-21 on α-synuclein expression in neurons is a potential mechanism by which geniopside (GP) protects the central nervous system from Parkinson disease (PD). The human neuroblastoma cell line SH-SY5Y was induced to differentiate in vitro and treated with dimethyl sulfoxide (DMSO), N-methyl-4-phenylpyridinium iodide (MPP(+)), and MPP(+) together with GP. To identify the role of miR-21 in the regulation of lysosome-associated membrane protein 2 (LAMP2A) and α-synuclein, SH-SY5Y cells pretreated with MPP(+) were transfected with miR-21 mimic and miR-21 inhibitor. To identify whether GP could reduce the level of α-synuclein through miR-21/LAMP2A, SHSY5Y cells pretreated with GP were treated with miR-21 mimic or miR-21 inhibitor; meanwhile, a luciferase reporter assay was performed to confirm the direct target of miR-21. LAMP2A was overexpressed using a pCMV6-XL5-LAMP2A vector to confirm the role of LAMP2A in the regulation of α-synuclein by miR-21. In these in vitro experiments, the RNA and/or protein expressions of miR-21, LAMP2A, and α-synuclein in SH-SY5Y cells were determined by quantitative real-time polymerase chain reaction and/or western blotting, respectively. An in vivo PD mouse model was established through intraperitoneal injection with N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP). The mice were treated with saline, MPTP, MPTP+GP, and MPTP+GP+miR-21 agomir. The numbers of TH(+) cells in the substantia nigra in different groups of mice were compared. The RNA and/or protein expressions of miR-21, LAMP2A, and α-synuclein were also determined. The level of miR-21 in the cells or mice models was significantly higher than that in normal cells or normal mice, respectively, and GP significantly downregulated miR-21. GP also raised the protein and mRNA expressions of LAMP2A and reduced the protein level of α-synuclein in PD models. MiR-21 upregulated the expression of α-synuclein by

  5. A rationally designed six-residue swap generates comparability in the aggregation behavior of α-synuclein and β-synuclein.

    PubMed

    Roodveldt, Cintia; Andersson, August; De Genst, Erwin J; Labrador-Garrido, Adahir; Buell, Alexander K; Dobson, Christopher M; Tartaglia, Gian Gaetano; Vendruscolo, Michele

    2012-11-06

    The aggregation process of α-synuclein, a protein closely associated with Parkinson's disease, is highly sensitive to sequence variations. It is therefore of great importance to understand the factors that define the aggregation propensity of specific mutational variants as well as their toxic behavior in the cellular environment. In this context, we investigated the extent to which the aggregation behavior of α-synuclein can be altered to resemble that of β-synuclein, an aggregation-resistant homologue of α-synuclein not associated with disease, by swapping residues between the two proteins. Because of the vast number of possible swaps, we have applied a rational design procedure to single out a mutational variant, called α2β, in which two short stretches of the sequence in the NAC region have been replaced in α-synuclein from β-synuclein. We find not only that the aggregation rate of α2β is close to that of β-synuclein, being much lower than that of α-synuclein, but also that α2β effectively changes the cellular toxicity of α-synuclein to a value similar to that of β-synuclein upon exposure of SH-SY5Y cells to preformed oligomers. Remarkably, control experiments on the corresponding mutational variant of β-synuclein, called β2α, confirmed that the mutations that we have identified also shift the aggregation behavior of this protein toward that of α-synuclein. These results demonstrate that it is becoming possible to control in quantitative detail the sequence code that defines the aggregation behavior and toxicity of α-synuclein.

  6. Selective Impairment of TH17-Differentiation and Protection against Autoimmune Arthritis after Overexpression of BCL2A1 in T Lymphocytes.

    PubMed

    Iglesias, Marcos; Augustin, Juan Jesús; Alvarez, Pilar; Santiuste, Inés; Postigo, Jorge; Merino, Jesús; Merino, Ramón

    2016-01-01

    The inhibition of apoptotic cell death in T cells through the dysregulated expression of BCL2 family members has been associated with the protection against the development of different autoimmune diseases. However, multiple mechanisms were proposed to be responsible for such protective effect. The purpose of this study was to explore the effect of the T-cell overexpression of BCL2A1, an anti-apoptotic BCL2 family member without an effect on cell cycle progression, in the development of collagen-induced arthritis. Our results demonstrated an attenuated development of arthritis in these transgenic mice. The protective effect was unrelated to the suppressive activity of regulatory T cells but it was associated with a defective activation of p38 mitogen-activated protein kinase in CD4+ cells after in vitro TCR stimulation. In addition, the in vitro and in vivo TH17 differentiation were impaired in BCL2A1 transgenic mice. Taken together, we demonstrated here a previously unknown role for BCL2A1 controlling the activation of CD4+ cells and their differentiation into pathogenic proinflammatory TH17 cells and identified BCL2A1 as a potential target in the control of autoimmune/inflammatory diseases.

  7. Inactivation of p53 by Human T-Cell Lymphotropic Virus Type 1 Tax Requires Activation of the NF-κB Pathway and Is Dependent on p53 Phosphorylation

    PubMed Central

    Pise-Masison, Cynthia A.; Mahieux, Renaud; Jiang, Hua; Ashcroft, Margaret; Radonovich, Michael; Duvall, Janet; Guillerm, Claire; Brady, John N.

    2000-01-01

    p53 plays a key role in guarding cells against DNA damage and transformation. We previously demonstrated that the human T-cell lymphotropic virus type 1 (HTLV-1) Tax can inactivate p53 transactivation function in lymphocytes. The present study demonstrates that in T cells, Tax-induced p53 inactivation is dependent upon NF-κB activation. Analysis of Tax mutants demonstrated that Tax inactivation of p53 function correlates with the ability of Tax to induce NF-κB but not p300 binding or CREB transactivation. The Tax-induced p53 inactivation can be overcome by overexpression of a dominant IκB mutant. Tax-NF-κB-induced p53 inactivation is not due to p300 squelching, since overexpression of p300 does not recover p53 activity in the presence of Tax. Further, using wild-type and p65 knockout mouse embryo fibroblasts (MEFs), we demonstrate that the p65 subunit of NF-κB is critical for Tax-induced p53 inactivation. While Tax can inactivate endogenous p53 function in wild-type MEFs, it fails to inactivate p53 function in p65 knockout MEFs. Importantly, Tax-induced p53 inactivation can be restored by expression of p65 in the knockout MEFs. Finally, we present evidence that phosphorylation of serines 15 and 392 correlates with inactivation of p53 by Tax in T cells. This study provides evidence that the divergent NF-κB proliferative and p53 cell cycle arrest pathways may be cross-regulated at several levels, including posttranslational modification of p53. PMID:10779327

  8. Selective sensitization to DNA-damaging agents in a human rhabdomyosarcoma cell line with inducible wild-type p53 overexpression.

    PubMed

    Gibson, A A; Harwood, F G; Tillman, D M; Houghton, J A

    1998-01-01

    Drug-induced cytotoxicity or apoptosis may be influenced by the expression of the p53 tumor suppressor gene and by the specific oncogene expressed, which may dictate the threshold at which a cytotoxic response may by induced. The objective of the study was to elucidate how DNA-damaging agents with different mechanisms of action were sensitized in the context of expression of the Pax3/FKHR fusion protein, a transformation event unique to alveolar rhabdomyosarcomas (ARMSs), and wild-type p53 (wtp53). A wtp53 cDNA was subcloned into the pGRE5-2/EBV vector with dexamethasone-inducible overexpression and transfected into Rh30 ARMS cells that express Pax3/FKHR and a mutant p53 phenotype. Following dexamethasone induction of wtp53 overexpression in a derived clone (Cl.#27), growth was slowed, and cells accumulated in G1. Functional wtp53 activity was demonstrated by selective transactivation of p50-2, a wtp53 chloramphenicol acetyltransferase reporter construct, and by up-regulated expression of endogenous p21Waf1. Data demonstrated p53-dependent sensitization (> or = 4-fold) to bleomycin, actinomycin D, and 5-fluorouracil and considerably less p53-dependence (< or = 2-fold) for doxorubicin, topotecan, etoposide, and cisplatin in Cl.#27 compared to an equivalent clone containing the pGRE5-EBV vector alone (VC#3). Data demonstrate that ARMS cells show a selective sensitization to DNA-damaging agents when wtp53 is overexpressed. The cytotoxic activity of agents that are not potentiated substantially must, therefore, depend upon p53-independent factors that relate to the mechanism of drug action.

  9. Validation of a quantitative cerebrospinal fluid alpha-synuclein assay in a European-wide interlaboratory study.

    PubMed

    Kruse, Niels; Persson, Staffan; Alcolea, Daniel; Bahl, Justyna M C; Baldeiras, Ines; Capello, Elisabetta; Chiasserini, Davide; Bocchio Chiavetto, Luisella; Emersic, Andreja; Engelborghs, Sebastiaan; Eren, Erden; Fladby, Tormod; Frisoni, Giovanni; García-Ayllón, María-Salud; Genc, Sermin; Gkatzima, Olymbia; Heegaard, Niels H H; Janeiro, André M; Kováčech, Branislav; Kuiperij, H Bea; Leitão, Maria J; Lleó, Alberto; Martins, Madalena; Matos, Mafalda; Mollergard, Hanne M; Nobili, Flavio; Öhrfelt, Annika; Parnetti, Lucilla; de Oliveira, Catarina Resende; Rot, Uros; Sáez-Valero, Javier; Struyfs, Hanne; Tanassi, Julia T; Taylor, Peggy; Tsolaki, Magda; Vanmechelen, Eugeen; Verbeek, Marcel M; Zilka, Norbert; Blennow, Kaj; Zetterberg, Henrik; Mollenhauer, Brit

    2015-09-01

    Decreased levels of alpha-synuclein (aSyn) in cerebrospinal fluid (CSF) in Parkinson's disease and related synucleinopathies have been reported, however, not consistently in all cross-sectional studies. To test the performance of one recently released human-specific enzyme-linked immunosorbent assay (ELISA) for the quantification of aSyn in CSF, we carried out a round robin trial with 18 participating laboratories trained in CSF ELISA analyses within the BIOMARKAPD project in the EU Joint Program - Neurodegenerative Disease Research. CSF samples (homogeneous aliquots from pools) and ELISA kits (one lot) were provided centrally and data reported back to one laboratory for data analysis. Our study showed that although factors such as preanalytical sample handling and lot-to-lot variability were minimized by our study design, we identified high variation in absolute values of CSF aSyn even when the same samples and same lots of assays were applied. We further demonstrate that although absolute concentrations differ between laboratories the quantitative results are comparable. With further standardization this assay may become an attractive tool for comparing aSyn measurements in diverse settings. Recommendations for further validation experiments and improvement of the interlaboratory results obtained are given. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. One single method to produce native and Tat-fused recombinant human α-synuclein in Escherichia coli.

    PubMed

    Caldinelli, Laura; Albani, Diego; Pollegioni, Loredano

    2013-04-04

    Human α-synuclein is a small-sized, natively unfolded protein that in fibrillar form is the primary component of Lewy bodies, the pathological hallmark of Parkinson's disease. Experimental evidence suggests that α-synuclein aggregation is the key event that triggers neurotoxicity although additional findings have proposed a protective role of α-synuclein against oxidative stress. One way to address the mechanism of this protective action is to evaluate α-synuclein-mediated protection by delivering this protein inside cells using a chimeric protein fused with the Tat-transduction domain of HIV Tat, named TAT-α-synuclein. A reliable protocol was designed to efficiently express and purify two different forms of human α-synuclein. The synthetic cDNAs encoding for the native α-synuclein and the fusion protein with the transduction domain of Tat protein from HIV were overexpressed in a BL21(DE3) E. coli strain as His-tagged proteins. The recombinant proteins largely localized (≥ 85%) to the periplasmic space. By using a quick purification protocol, based on recovery of periplasmic space content and metal-chelating chromatography, the recombinant α-synuclein protein forms could be purified in a single step to ≥ 95% purity. Both α-synuclein recombinant proteins form fibrils and the TAT-α-synuclein is also cytotoxic in the micromolar concentration range. To further characterize the molecular mechanisms of α-synuclein neurotoxicity both in vitro and in vivo and to evaluate the relevance of extracellular α-synuclein for the pathogenesis and progression of Parkinson's disease, a suitable method to produce different high-quality forms of this pathological protein is required. Our optimized expression and purification procedure offers an easier and faster means of producing different forms (i.e., both the native and the TAT-fusion form) of soluble recombinant α-synuclein than previously described procedures.

  11. RNF38 encodes a nuclear ubiquitin protein ligase that modifies p53

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sheren, Jamie E.; Kassenbrock, C. Kenneth, E-mail: ken.kassenbrock@ucdenver.edu; Department of Biology, Colorado State University, Fort Collins, CO 80523-1878

    2013-11-01

    Highlights: •RNF38 is shown to be a nuclear protein with a bipartite nuclear localization signal. •RNF38 protein is purified and shown to have ubiquitin protein ligase (E3) activity. •We show that RNF38 binds p53 and can ubiquitinate p53 in vitro. •Overexpression of RNF38 increases p53 ubiquitination in HEK293T cells. •Overexpression of RNF38 in HEK293T cells alters p53 localization. -- Abstract: The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequencemore » (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.« less

  12. The impairment of learning and memory and synaptic loss in mouse after chronic nitrite exposure.

    PubMed

    Chen, Yongfang; Cui, Zhanjun; Wang, Lai; Liu, Hongliang; Fan, Wenjuan; Deng, Jinbo; Deng, Jiexin

    2016-12-01

    The objective of this study is to understand the impairment of learning and memory in mouse after chronic nitrite exposure. The animal model of nitrite exposure in mouse was created with the daily intubation of nitrite in adult healthy male mice for 3 months. Furthermore, the mouse's learning and memory abilities were tested with Morris water maze, and the expression of Synaptophysin and γ-Synuclein was visualized with immunocytochemistry and Western blot. Our results showed that nitrite exposure significantly prolonged the escape latency period (ELP) and decreased the values of the frequency across platform (FAP) as well as the accumulative time in target quadrant (ATITQ) compared to control, in dose-dependent manner. In addition, after nitrite exposure, synaptophysin (SYN) positive buttons in the visual cortex was reduced, in contrast the increase of γ-synuclein positive cells. The results above were supported by Western blot as well. We conclude that nitrite exposure could lead to a decline in mice's learning and memory. The overexpression of γ-synuclein contributed to the synaptic loss, which is most likely the cause of learning and memory impairment. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1720-1730, 2016. © 2015 Wiley Periodicals, Inc.

  13. FTY720/Fingolimod Reduces Synucleinopathy and Improves Gut Motility in A53T Mice: CONTRIBUTIONS OF PRO-BRAIN-DERIVED NEUROTROPHIC FACTOR (PRO-BDNF) AND MATURE BDNF.

    PubMed

    Vidal-Martínez, Guadalupe; Vargas-Medrano, Javier; Gil-Tommee, Carolina; Medina, David; Garza, Nathan T; Yang, Barbara; Segura-Ulate, Ismael; Dominguez, Samantha J; Perez, Ruth G

    2016-09-23

    Patients with Parkinson's disease (PD) often have aggregated α-synuclein (aSyn) in enteric nervous system (ENS) neurons, which may be associated with the development of constipation. This occurs well before the onset of classic PD motor symptoms. We previously found that aging A53T transgenic (Tg) mice closely model PD-like ENS aSyn pathology, making them appropriate for testing potential PD therapies. Here we show that Tg mice overexpressing mutant human aSyn develop ENS pathology by 4 months. We then evaluated the responses of Tg mice and their WT littermates to the Food and Drug Administration-approved drug FTY720 (fingolimod, Gilenya) or vehicle control solution from 5 months of age. Long term oral FTY720 in Tg mice reduced ENS aSyn aggregation and constipation, enhanced gut motility, and increased levels of brain-derived neurotrophic factor (BDNF) but produced no significant change in WT littermates. A role for BDNF was directly assessed in a cohort of young A53T mice given vehicle, FTY720, the Trk-B receptor inhibitor ANA-12, or FTY720 + ANA-12 from 1 to 4 months of age. ANA-12-treated Tg mice developed more gut aSyn aggregation as well as constipation, whereas FTY720-treated Tg mice had reduced aSyn aggregation and less constipation, occurring in part by increasing both pro-BDNF and mature BDNF levels. The data from young and old Tg mice revealed FTY720-associated neuroprotection and reduced aSyn pathology, suggesting that FTY720 may also benefit PD patients and others with synucleinopathy. Another finding was a loss of tyrosine hydroxylase immunoreactivity in gut neurons with aggregated aSyn, comparable with our prior findings in the CNS. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Novel immunolocalization of alpha-synuclein in human muscle of inclusion-body myositis, regenerating and necrotic muscle fibers, and at neuromuscular junctions.

    PubMed

    Askanas, V; Engel, W K; Alvarez, R B; McFerrin, J; Broccolini, A

    2000-07-01

    Alpha-synuclein (alpha-syn) is an important component of neuronal and glial inclusions in brains of patients with several neurodegenerative disorders. Sporadic inclusion-body myositis (s-IBM) is the most common progressive muscle disease of older patients. Its muscle phenotype shows several similarities with Alzheimer disease brain. A distinct feature of s-IBM pathology is specific vacuolar degeneration of muscle fibers characterized by intracellular amyloid inclusions formed by both amyloid-beta (Abeta) and paired-helical filaments composed of phosphorylated tau. We immunostained alpha-syn in muscle biopsies of s-IBM, disease-control, and normal patients. Approximately 60% of Abeta-positive vacuolated muscle fibers (VMF) contained well-defined inclusions immunoreactive with antibodies against alpha-syn. In those fibers. alpha-syn co-localized with Abeta, both by light microscopy, and ultrastructurally. Paired-helical filaments did not contain alpha-syn immunoreactivity. In all muscle biopsies, alpha-syn was strongly immunoreactive at the postsynaptic region of the neuromuscular junctions. alpha-syn immunoreactivity also occurred diffusely in regenerating and necrotic muscle fibers. In cultured human muscle fibers, alpha-syn and its mRNA were expressed by immunocytochemistry, immunoblots, and Northern blots. Our study provides the first demonstration that alpha-syn participates in normal and pathologic processes of human muscle. Therefore. its function is not exclusive to the brain and neurodegenerative diseases.

  15. cFLIP overexpression in T cells in thymoma-associated myasthenia gravis

    PubMed Central

    Belharazem, Djeda; Schalke, Berthold; Gold, Ralf; Nix, Wilfred; Vitacolonna, Mario; Hohenberger, Peter; Roessner, Eric; Schulze, Torsten J; Saruhan-Direskeneli, Güher; Yilmaz, Vuslat; Ott, German; Ströbel, Philipp; Marx, Alexander

    2015-01-01

    Objective The capacity of thymomas to generate mature CD4+ effector T cells from immature precursors inside the tumor and export them to the blood is associated with thymoma-associated myasthenia gravis (TAMG). Why TAMG(+) thymomas generate and export more mature CD4+ T cells than MG(−) thymomas is unknown. Methods Unfixed thymoma tissue, thymocytes derived thereof, peripheral blood mononuclear cells (PBMCs), T-cell subsets and B cells were analysed using qRT-PCR and western blotting. Survival of PBMCs was measured by MTT assay. FAS-mediated apoptosis in PBMCs was quantified by flow cytometry. NF-κB in PBMCs was inhibited by the NF-κB-Inhibitor, EF24 prior to FAS-Ligand (FASLG) treatment for apoptosis induction. Results Expression levels of the apoptosis inhibitor cellular FLICE-like inhibitory protein (c-FLIP) in blood T cells and intratumorous thymocytes were higher in TAMG(+) than in MG(−) thymomas and non-neoplastic thymic remnants. Thymocytes and PBMCs of TAMG patients showed nuclear NF-κB accumulation and apoptosis resistance to FASLG stimulation that was sensitive to NF-κB blockade. Thymoma removal reduced cFLIP expression in PBMCs. Interpretation We conclude that thymomas induce cFLIP overexpression in thymocytes and their progeny, blood T cells. We suggest that the stronger cFLIP overexpression in TAMG(+) compared to MG(−) thymomas allows for the more efficient generation of mature CD4+ T cells in TAMG(+) thymomas. cFLIP overexpression in thymocytes and exported CD4+ T cells of patients with TAMG might contribute to the pathogenesis of TAMG by impairing central and peripheral T-cell tolerance. PMID:26401511

  16. MicroRNA-7 facilitates the degradation of alpha-synuclein and its aggregates by promoting autophagy.

    PubMed

    Choi, Doo Chul; Yoo, Myungsik; Kabaria, Savan; Junn, Eunsung

    2018-05-05

    Alpha-Synuclein (α-Syn) is an important protein in the pathogenesis of Parkinson disease (PD) as it accumulates as fibrillar inclusions in affected brain regions including dopaminergic neurons in the substantia nigra. Elevated levels of α-Syn seem to be crucial in mediating its toxicity. Thus, detailed information regarding the regulatory mechanism of α-Syn expression in several layers such as transcription, post-transcription and post-translation is needed in order to devise therapeutic interventions for PD. Previously, we reported that expression of α-Syn is repressed by microRNA-7 (miR-7) through its effect on the 3'-untranslated region (UTR) of α-Syn mRNA. Here, we show that miR-7 also accelerates the clearance of α-Syn and its aggregates by promoting autophagy in differentiated ReNcell VM cells. Further, miR-7 facilitates the degradation of pre-formed fibrils of α-Syn transported from outside the cells. This additional mechanism for reducing α-Syn levels show miR-7 to be an important molecular target for PD and other alpha-synucleinopathies. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Glucose Metabolism and AMPK Signaling Regulate Dopaminergic Cell Death Induced by Gene (α-Synuclein)-Environment (Paraquat) Interactions.

    PubMed

    Anandhan, Annadurai; Lei, Shulei; Levytskyy, Roman; Pappa, Aglaia; Panayiotidis, Mihalis I; Cerny, Ronald L; Khalimonchuk, Oleh; Powers, Robert; Franco, Rodrigo

    2017-07-01

    While environmental exposures are not the single cause of Parkinson's disease (PD), their interaction with genetic alterations is thought to contribute to neuronal dopaminergic degeneration. However, the mechanisms involved in dopaminergic cell death induced by gene-environment interactions remain unclear. In this work, we have revealed for the first time the role of central carbon metabolism and metabolic dysfunction in dopaminergic cell death induced by the paraquat (PQ)-α-synuclein interaction. The toxicity of PQ in dopaminergic N27 cells was significantly reduced by glucose deprivation, inhibition of hexokinase with 2-deoxy-D-glucose (2-DG), or equimolar substitution of glucose with galactose, which evidenced the contribution of glucose metabolism to PQ-induced cell death. PQ also stimulated an increase in glucose uptake, and in the levels of glucose transporter type 4 (GLUT4) and Na + -glucose transporters isoform 1 (SGLT1) proteins, but only inhibition of GLUT-like transport with STF-31 or ascorbic acid reduced PQ-induced cell death. Importantly, while autophagy protein 5 (ATG5)/unc-51 like autophagy activating kinase 1 (ULK1)-dependent autophagy protected against PQ toxicity, the inhibitory effect of glucose deprivation on cell death progression was largely independent of autophagy or mammalian target of rapamycin (mTOR) signaling. PQ selectively induced metabolomic alterations and adenosine monophosphate-activated protein kinase (AMPK) activation in the midbrain and striatum of mice chronically treated with PQ. Inhibition of AMPK signaling led to metabolic dysfunction and an enhanced sensitivity of dopaminergic cells to PQ. In addition, activation of AMPK by PQ was prevented by inhibition of the inducible nitric oxide syntase (iNOS) with 1400W, but PQ had no effect on iNOS levels. Overexpression of wild type or A53T mutant α-synuclein stimulated glucose accumulation and PQ toxicity, and this toxic synergism was reduced by inhibition of glucose metabolism

  18. Validation of a commercially available enzyme-linked immunoabsorbent assay for the quantification of human α-Synuclein in cerebrospinal fluid.

    PubMed

    Kruse, Niels; Mollenhauer, Brit

    2015-11-01

    The quantification of α-Synuclein in cerebrospinal fluid (CSF) as a biomarker has gained tremendous interest in the last years. Several commercially available immunoassays are emerging. We here describe the full validation of one commercially available ELISA assay for the quantification of α-Synuclein in human CSF (Covance alpha-Synuclein ELISA kit). The study was conducted within the BIOMARKAPD project in the European initiative Joint Program for Neurodegenerative Diseases (JPND). We investigated the effect of several pre-analytical and analytical confounders: i.e. (1) need for centrifugation of freshly drawn CSF, (2) sample stability, (3) delay of freezing, (4) volume of storage aliquots, (5) freeze/thaw cycles, (6) thawing conditions, (7) dilution linearity, (8) parallelism, (9) spike recovery, and (10) precision. None of these confounders influenced the levels of α-Synuclein in CSF significantly. We found a very high intra-assay precision. The inter-assay precision was lower than expected due to different performances of kit lots used. Overall the validated immunoassay is useful for the quantification of α-Synuclein in human CSF. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. The p53 inhibitor, pifithrin-{alpha}, suppresses self-renewal of embryonic stem cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Abdelalim, Essam Mohamed, E-mail: essam_abdelalim@yahoo.com; Department of Cytology and Histology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522; Tooyama, Ikuo

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer We determine the role of p53 in ES cells under unstressful conditions. Black-Right-Pointing-Pointer PFT-{alpha} suppresses ES cell proliferation. Black-Right-Pointing-Pointer PFT-{alpha} induces ES cell cycle arrest. Black-Right-Pointing-Pointer PFT-{alpha} downregulates Nanog and cyclin D1. -- Abstract: Recent studies have reported the role of p53 in suppressing the pluripotency of embryonic stem (ES) cells after DNA damage and blocking the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. However, to date no evidence has been presented to support the function of p53 in unstressed ES cells. In this study, we investigated the effect of pifithrin (PFT)-{alpha}, an inhibitor ofmore » p53-dependent transcriptional activation, on self-renewal of ES cells. Our results revealed that treatment of ES cells with PFT-{alpha} resulted in the inhibition of ES cell propagation in a dose-dependent manner, as indicated by a marked reduction in the cell number and colony size. Also, PFT-{alpha} caused a cell cycle arrest and significant reduction in DNA synthesis. In addition, inhibition of p53 activity reduced the expression levels of cyclin D1 and Nanog. These findings indicate that p53 pathway in ES cells rather than acting as an inactive gene, is required for ES cell proliferation and self-renewal under unstressful conditions.« less

  20. Overexpression of cellular repressor of E1A-stimulated genes inhibits TNF-{alpha}-induced apoptosis via NF-{kappa}B in mesenchymal stem cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Peng, Cheng-Fei; Cardiovascular Research Institute and Department of Cardiology, Shenyang Northern Hospital, Shenyang; Han, Ya-Ling, E-mail: hanyaling53@gmail.com

    2011-03-25

    Research highlights: {yields} CREG protected MSCs from tumor necrosis factor-{alpha} (TNF-{alpha}) induced apoptosis. {yields} CREG inhibits the phosphorylation of I{kappa}B{alpha} and prevents the activation of NF-{kappa}B. {yields} CREG inhibits NF-{kappa}B nuclear translocation and pro-apoptosis protein transcription. {yields} CREG anti-apoptotic effect involves inhibition of the death receptor pathway. {yields} p53 is downregulated by CREG via NF-{kappa}B pathway under TNF-{alpha} stimulation. -- Abstract: Bone marrow-derived mesenchymal stem cells (MSCs) show great potential for therapeutic repair after myocardial infarction. However, poor viability of transplanted MSCs in the ischemic heart has limited their use. Cellular repressor of E1A-stimulated genes (CREG) has been identified asmore » a potent inhibitor of apoptosis. This study therefore aimed to determine if rat bone marrow MSCs transfected with CREG-were able to effectively resist apoptosis induced by inflammatory mediators, and to demonstrate the mechanism of CREG action. Apoptosis was determined by flow cytometric and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays. The pathways mediating these apoptotic effects were investigated by Western blotting. Overexpression of CREG markedly protected MSCs from tumor necrosis factor-{alpha} (TNF-{alpha}) induced apoptosis by 50% after 10 h, through inhibition of the death-receptor-mediated apoptotic pathway, leading to attenuation of caspase-8 and caspase-3. Moreover, CREG resisted the serine phosphorylation of I{kappa}B{alpha} and prevented the nuclear translocation of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) under TNF-{alpha} stimulation. Treatment of cells with the NF-{kappa}B inhibitor pyrrolidine dithiocarbamate (PDTC) significantly increased the transcription of pro-apoptosis proteins (p53 and Fas) by NF-{kappa}B, and attenuated the anti-apoptotic effects of CREG on MSCs. The results of this

  1. Specific pesticide-dependent increases in α-synuclein levels in human neuroblastoma (SH-SY5Y) and melanoma (SK-MEL-2) cell lines.

    PubMed

    Chorfa, Areski; Bétemps, Dominique; Morignat, Eric; Lazizzera, Corinne; Hogeveen, Kevin; Andrieu, Thibault; Baron, Thierry

    2013-06-01

    Epidemiological studies indicate a role of genetic and environmental factors in Parkinson's disease involving alterations of the neuronal α-synuclein (α-syn) protein. In particular, a relationship between Parkinson's disease and occupational exposure to pesticides has been repeatedly suggested. Our objective was to precisely assess changes in α-syn levels in human neuroblastoma (SH-SY5Y) and melanoma (SK-MEL-2) cell lines following acute exposure to pesticides (rotenone, paraquat, maneb, and glyphosate) using Western blot and flow cytometry. These human cell lines express α-syn endogenously, and overexpression of α-syn (wild type or mutated A53T) can be obtained following recombinant adenoviral transduction. We found that endogenous α-syn levels in the SH-SY5Y neuroblastoma cell line were markedly increased by paraquat, and to a lesser extent by rotenone and maneb, but not by glyphosate. Rotenone also clearly increased endogenous α-syn levels in the SK-MEL-2 melanoma cell line. In the SH-SY5Y cell line, similar differences were observed in the α-syn adenovirus-transduced cells, with a higher increase of the A53T mutated protein. Paraquat markedly increased α-syn in the SK-MEL-2 adenovirus-transduced cell line, similarly for the wild-type or A53T proteins. The observed differences in the propensities of pesticides to increase α-syn levels are in agreement with numerous reports that indicate a potential role of exposure to certain pesticides in the development of Parkinson's disease. Our data support the hypothesis that pesticides can trigger some molecular events involved in this disease and also in malignant melanoma that consistently shows a significant but still unexplained association with Parkinson's disease.

  2. The Contribution of α-Synuclein Spreading to Parkinson's Disease Synaptopathy

    PubMed Central

    Faustini, Gaia; Missale, Cristina; Pizzi, Marina; Spano, PierFranco

    2017-01-01

    Synaptopathies are diseases with synapse defects as shared pathogenic features, encompassing neurodegenerative disorders such as Parkinson's disease (PD). In sporadic PD, the most common age-related neurodegenerative movement disorder, nigrostriatal dopaminergic deficits are responsible for the onset of motor symptoms that have been related to α-synuclein deposition at synaptic sites. Indeed, α-synuclein accumulation can impair synaptic dopamine release and induces the death of nigrostriatal neurons. While in physiological conditions the protein can interact with and modulate synaptic vesicle proteins and membranes, numerous experimental evidences have confirmed that its pathological aggregation can compromise correct neuronal functioning. In addition, recent findings indicate that α-synuclein pathology spreads into the brain and can affect the peripheral autonomic and somatic nervous system. Indeed, monomeric, oligomeric, and fibrillary α-synuclein can move from cell to cell and can trigger the aggregation of the endogenous protein in recipient neurons. This novel “prion-like” behavior could further contribute to synaptic failure in PD and other synucleinopathies. This review describes the major findings supporting the occurrence of α-synuclein pathology propagation in PD and discusses how this phenomenon could induce or contribute to synaptic injury and degeneration. PMID:28133550

  3. Intercellular transfer of pathogenic α-synuclein by extracellular vesicles is induced by the lipid peroxidation product 4-hydroxynonenal.

    PubMed

    Zhang, Shi; Eitan, Erez; Wu, Tsung-Yu; Mattson, Mark P

    2018-01-01

    Parkinson's disease (PD) is characterized by accumulations of toxic α-synuclein aggregates in vulnerable neuronal populations in the brainstem, midbrain, and cerebral cortex. Recent findings suggest that α-synuclein pathology can be propagated transneuronally, but the underlying molecular mechanisms are unknown. Advances in the genetics of rare early-onset familial PD indicate that increased production and/or reduced autophagic clearance of α-synuclein can cause PD. The cause of the most common late-onset PD is unclear, but may involve metabolic compromise and oxidative stress upstream of α-synuclein accumulation. As evidence, the lipid peroxidation product 4-hydroxynonenal (HNE) is elevated in the brain during normal aging and moreso in brain regions afflicted with α-synuclein pathology. Here, we report that HNE increases aggregation of endogenous α-synuclein in primary neurons and triggers the secretion of extracellular vesicles (EVs) containing cytotoxic oligomeric α-synuclein species. EVs released from HNE-treated neurons are internalized by healthy neurons which as a consequence degenerate. Levels of endogenously generated HNE are elevated in cultured cells overexpressing human α-synuclein, and EVs released from those cells are toxic to neurons. The EV-associated α-synuclein is located both inside the vesicles and on their surface, where it plays a role in EV internalization by neurons. On internalization, EVs harboring pathogenic α-synuclein are transported both anterogradely and retrogradely within axons. Focal injection of EVs containing α-synuclein into the striatum of wild-type mice results in spread of synuclein pathology to anatomically connected brain regions. Our findings suggest a scenario for late-onset PD in which lipid peroxidation promotes intracellular accumulation and then extrusion of EVs containing toxic α-synuclein species; the EVs are then internalized by adjacent neurons, so propagating the neurodegenerative process. Published

  4. Association of Cerebrospinal Fluid β-Amyloid 1-42, T-tau, P-tau181, and α-Synuclein Levels With Clinical Features of Drug-Naive Patients With Early Parkinson Disease

    PubMed Central

    Kang, Ju-Hee; Irwin, David J.; Chen-Plotkin, Alice S.; Siderowf, Andrew; Caspell, Chelsea; Coffey, Christopher S.; Waligórska, Teresa; Taylor, Peggy; Pan, Sarah; Frasier, Mark; Marek, Kenneth; Kieburtz, Karl; Jennings, Danna; Simuni, Tanya; Tanner, Caroline M.; Singleton, Andrew; Toga, Arthur W.; Chowdhury, Sohini; Mollenhauer, Brit; Trojanowski, John Q.; Shaw, Leslie M.

    2014-01-01

    Importance We observed a significant correlation between cerebrospinal fluid (CSF) levels of tau proteins and α-synuclein, but not β-amyloid 1–42 (Aβ1–42), and lower concentration of CSF biomarkers, as compared with healthy controls, in a cohort of entirely untreated patients with Parkinson disease (PD) at the earliest stage of the disease studied so far. Objective To evaluate the baseline characteristics and relationship to clinical features of CSF biomarkers (Aβ1–42, total tau [T-tau], tau phosphorylated at threonine 181 [P-tau181], and α-synuclein) in drug-naive patients with early PD and demographically matched healthy controls enrolled in the Parkinson’s Progression Markers Initiative (PPMI) study. Design, Setting, and Participants Cross-sectional study of the initial 102 research volunteers (63 patients with PD and 39 healthy controls) of the PPMI cohort. Main Outcomes and Measures The CSF biomarkers were measured by INNO-BIA AlzBio3 immunoassay (Aβ1–42, T-tau, and P-tau181; Innogenetics Inc) or by enzyme-linked immunosorbent assay (α-synuclein). Clinical features including diagnosis, demographic characteristics, motor, neuropsychiatric, and cognitive assessments, and DaTscan were systematically assessed according to the PPMI study protocol. Results Slightly, but significantly, lower levels of Aβ1–42, T-tau, P-tau181, α-synuclein, and T-tau/Aβ1–42 were seen in subjects with PD compared with healthy controls but with a marked overlap between groups. Using multivariate regression analysis, we found that lower Aβ1–42 and P-tau181 levels were associated with PD diagnosis and that decreased CSF T-tau and α-synuclein were associated with increased motor severity. Notably, when we classified patients with PD by their motor phenotypes, lower CSF Aβ1–42 and P-tau181 concentrations were associated with the postural instability–gait disturbance–dominant phenotype but not with the tremor-dominant or intermediate phenotype. Finally, we

  5. Slower EEG alpha generation, synchronization and "flow"-possible biomarkers of cognitive impairment and neuropathology of minor stroke.

    PubMed

    Petrovic, Jelena; Milosevic, Vuk; Zivkovic, Miroslava; Stojanov, Dragan; Milojkovic, Olga; Kalauzi, Aleksandar; Saponjic, Jasna

    2017-01-01

    We investigated EEG rhythms, particularly alpha activity, and their relationship to post-stroke neuropathology and cognitive functions in the subacute and chronic stages of minor strokes. We included 10 patients with right middle cerebral artery (MCA) ischemic strokes and 11 healthy controls. All the assessments of stroke patients were done both in the subacute and chronic stages. Neurological impairment was measured using the National Institute of Health Stroke Scale (NIHSS), whereas cognitive functions were assessed using the Montreal Cognitive Assessment (MoCA) and MoCA memory index (MoCA-MIS). The EEG was recorded using a 19 channel EEG system with standard EEG electrode placement. In particular, we analyzed the EEGs derived from the four lateral frontal (F3, F7, F4, F8), and corresponding lateral posterior (P3, P4, T5, T6) electrodes. Quantitative EEG analysis included: the group FFT spectra, the weighted average of alpha frequency (αAVG), the group probability density distributions of all conventional EEG frequency band relative amplitudes (EEG microstructure), the inter- and intra-hemispheric coherences, and the topographic distribution of alpha carrier frequency phase potentials (PPs). Statistical analysis was done using a Kruskal-Wallis ANOVA with a post-hoc Mann-Whitney U two-tailed test, and Spearman's correlation. We demonstrated transient cognitive impairment alongside a slower alpha frequency ( α AVG) in the subacute right MCA stroke patients vs. the controls. This slower alpha frequency showed no amplitude change, but was highly synchronized intra-hemispherically, overlying the ipsi-lesional hemisphere, and inter-hemispherically, overlying the frontal cortex. In addition, the disturbances in EEG alpha activity in subacute stroke patients were expressed as a decrease in alpha PPs over the frontal cortex and an altered "alpha flow", indicating the sustained augmentation of inter-hemispheric interactions. Although the stroke induced slower alpha was a

  6. DCAF1 controls T-cell function via p53-dependent and -independent mechanisms.

    PubMed

    Guo, Zengli; Kong, Qing; Liu, Cui; Zhang, Song; Zou, Liyun; Yan, Feng; Whitmire, Jason K; Xiong, Yue; Chen, Xian; Wan, Yisong Y

    2016-01-05

    On activation, naive T cells grow in size and enter cell cycle to mount immune response. How the fundamental processes of T-cell growth and cell cycle entry are regulated is poorly understood. Here we report that DCAF1 (Ddb1-cullin4-associated-factor 1) is essential for these processes. The deletion of DCAF1 in T cells impairs their peripheral homeostasis. DCAF1 is upregulated on T-cell receptor activation and critical for activation-induced T-cell growth, cell cycle entry and proliferation. In addition, DCAF1 is required for T-cell expansion and function during anti-viral and autoimmune responses in vivo. DCAF1 deletion leads to a drastic stabilization of p53 protein, which can be attributed to a requirement of DCAF1 for MDM2-mediated p53 poly-ubiquitination. Importantly, p53 deletion rescues the cell cycle entry defect but not the growth defect of DCAF1-deficient cells. Therefore, DCAF1 is vital for T-cell function through p53-dependent and -independent mechanisms.

  7. DCAF1 controls T-cell function via p53-dependent and -independent mechanisms

    PubMed Central

    Guo, Zengli; Kong, Qing; Liu, Cui; Zhang, Song; Zou, Liyun; Yan, Feng; Whitmire, Jason K.; Xiong, Yue; Chen, Xian; Wan, Yisong Y.

    2016-01-01

    On activation, naive T cells grow in size and enter cell cycle to mount immune response. How the fundamental processes of T-cell growth and cell cycle entry are regulated is poorly understood. Here we report that DCAF1 (Ddb1–cullin4-associated-factor 1) is essential for these processes. The deletion of DCAF1 in T cells impairs their peripheral homeostasis. DCAF1 is upregulated on T-cell receptor activation and critical for activation-induced T-cell growth, cell cycle entry and proliferation. In addition, DCAF1 is required for T-cell expansion and function during anti-viral and autoimmune responses in vivo. DCAF1 deletion leads to a drastic stabilization of p53 protein, which can be attributed to a requirement of DCAF1 for MDM2-mediated p53 poly-ubiquitination. Importantly, p53 deletion rescues the cell cycle entry defect but not the growth defect of DCAF1-deficient cells. Therefore, DCAF1 is vital for T-cell function through p53-dependent and -independent mechanisms. PMID:26728942

  8. Neurogranin binds α-synuclein in the human superior temporal cortex and interaction is decreased in Parkinson's disease.

    PubMed

    Koob, Andrew O; Shaked, Gideon M; Bender, Andreas; Bisquertt, Alejandro; Rockenstein, Edward; Masliah, Eliezer

    2014-12-03

    Neurogranin is a calmodulin binding protein that has been implicated in learning and memory, long-term potentiation and synaptic plasticity. Neurons expressing neurogranin in the cortex degenerate in late stages of Parkinson's disease with widespread α-synuclein pathology. While analyzing neurogranin gene expression levels through rtPCR in brains of mouse models overexpressing human α-synuclein, we found levels were elevated 2.5 times when compared to nontransgenic animals. Immunohistochemistry in the cortex revealed colocalization between α-synuclein and neurogranin in mouse transgenics when compared to control mice. Coimmunoprecipitation studies in the superior temporal cortex in humans confirmed interaction between α-synuclein and neurogranin, and decreased interaction between α-synuclein and neurogranin was noticed in patients diagnosed with Parkinson's disease when compared to normal control brains. Additionally, phosphorylated neurogranin levels were also decreased in the human superior temporal cortex in patients diagnosed with Parkinson's disease and patients diagnosed with dementia with Lewy bodies. Here, we show for the first time that neurogranin binds to α-synuclein in the human cortex, and this interaction decreases in Parkinson's disease along with the phosphorylation of neurogranin, a molecular process thought to be involved in learning and memory. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Sustained expression of PGC-1α in the rat nigrostriatal system selectively impairs dopaminergic function

    PubMed Central

    Ciron, C.; Lengacher, S.; Dusonchet, J.; Aebischer, P.; Schneider, B.L.

    2012-01-01

    Mitochondrial dysfunction and oxidative stress have been implicated in the etiology of Parkinson's disease. Therefore, pathways controlling mitochondrial activity rapidly emerge as potential therapeutic targets. Here, we explore the neuronal response to prolonged overexpression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), a transcriptional regulator of mitochondrial function, both in vitro and in vivo. In neuronal primary cultures from the ventral midbrain, PGC-1α induces mitochondrial biogenesis and increases basal respiration. Over time, we observe an increasing proportion of the oxygen consumed by neurons which are dedicated to adenosine triphosphate production. In parallel to enhanced oxidative phosphorylation, PGC-1α progressively leads to a decrease in mitochondrial polarization. In the adult rat nigrostriatal system, adeno-associated virus (AAV)-mediated overexpression of PGC-1α induces the selective loss of dopaminergic markers and increases dopamine (DA) catabolism, leading to a reduction in striatal DA content. In addition, PGC-1α prevents the labeling of nigral neurons following striatal injection of the fluorogold retrograde tracer. When PGC-1α is expressed at higher levels following intranigral AAV injection, it leads to overt degeneration of dopaminergic neurons. Finally, PGC-1α overexpression does not prevent nigrostriatal degeneration in pathologic conditions induced by α-synuclein overexpression. Overall, we find that lasting overexpression of PGC-1α leads to major alterations in the metabolic activity of neuronal cells which dramatically impair dopaminergic function in vivo. These results highlight the central role of PGC-1α in the function and survival of dopaminergic neurons and the critical need for maintaining physiological levels of PGC-1α activity. PMID:22246294

  10. Synucleins: are they two-edged swords?

    PubMed

    Surguchov, Andrei

    2013-02-01

    The synuclein family consists of three distinct highly homologous genes, α-synuclein, β-synuclein, and γ-synuclein, which have so far been found only in vertebrates. Proteins encoded by these genes are characterized by an acidic C-terminal region and five or six imperfect repeat motifs (KTKEGV) distributed throughout the highly conserved N-terminal region. Numerous data demonstrate that synucleins are implicated in two groups of the most devastating human disorders, i.e., neurodegenerative diseases (NDDs) and cancer. Mutations in the α-synuclein gene are associated with familial forms of Parkinson's disease (PD), and accumulation of α-synuclein inclusions is a hallmark of this disorder. In breast cancer, increased expression of γ-synuclein correlates with disease progression. Conversely, some results indicate that the members of the synuclein family may have a protective effect. How might these small proteins combine such controversial properties? We present evidence that synuclein's features are basically regulated by two mechanisms, i.e., posttranslational modifications (PTMs) and the level of their expression. We also discuss a new, emerging area of investigation of synucleins, namely, their role in the cell-to-cell propagation of pathology. Copyright © 2012 Wiley Periodicals, Inc.

  11. In vitro study of α-synuclein protofibrils by cryo-EM suggests a Cu(2+)-dependent aggregation pathway.

    PubMed

    Zhang, Hangyu; Griggs, Amy; Rochet, Jean-Christophe; Stanciu, Lia A

    2013-06-18

    The aggregation of α-synuclein is thought to play a role in the death of dopamine neurons in Parkinson's disease (PD). Alpha-synuclein transitions itself through an aggregation pathway consisting of pathogenic species referred to as protofibrils (or oligomer), which ultimately convert to mature fibrils. The structural heterogeneity and instability of protofibrils has significantly impeded advance related to the understanding of their structural characteristics and the amyloid aggregation mystery. Here, we report, to our knowledge for the first time, on α-synuclein protofibril structural characteristics with cryo-electron microscopy. Statistical analysis of annular protofibrils revealed a constant wall thickness as a common feature. The visualization of the assembly steps enabled us to propose a novel, to our knowledge, mechanisms for α-synuclein aggregation involving ring-opening and protofibril-protofibril interaction events. The ion channel-like protofibrils and their membrane permeability have also been found in other amyloid diseases, suggesting a common molecular mechanism of pathological aggregation. Our direct visualization of the aggregation pathway of α-synuclein opens up fresh opportunities to advance the understanding of protein aggregation mechanisms relevant to many amyloid diseases. In turn, this information would enable the development of additional therapeutic strategies aimed at suppressing toxic protofibrils of amyloid proteins involved in neurological disorders. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  12. Tyrosinase overexpression promotes ATM-dependent p53 phosphorylation by quercetin and sensitizes melanoma cells to dacarbazine.

    PubMed

    Thangasamy, Thilakavathy; Sittadjody, Sivanandane; Limesand, Kirsten H; Burd, Randy

    2008-01-01

    Dacarbazine (DTIC) has been used for the treatment of melanoma for decades. However, monotherapy with this chemotherapeutic agent results only in moderate response rates. To improve tumor response to DTIC current clinical trials in melanoma focus on combining a novel targeted agent with chemotherapy. Here, we demonstrate that tyrosinase which is commonly overexpressed in melanoma activates the bioflavonoid quercetin (Qct) and promotes an ataxia telangiectasia mutated (ATM)-dependent DNA damage response. This response sensitizes melanoma cells that overexpress tyrosinase to DTIC. In DB-1 melanoma cells that overexpress tyrosinase (Tyr(+) cells), the threshold for phosphorylation of ATM and p53 at serine 15 was observed at a low dose of Qct (25 microM) when compared to the mock transfected pcDNA3 cells, which required a higher dose (75 microM). Both pcDNA3 and Tyr(+) DB-1 cells demonstrated similar increases in phosphorylation of p53 at other serine sites, but in the Tyr(+) cells, DNApk expression was found to be reduced compared to control cells, indicating a shift towards an ATM-mediated response. The DB-1 control cells were resistant to DTIC, but were sensitized to apoptosis with high dose Qct, while Tyr(+) cells were sensitized to DTIC with low or high dose Qct. Qct also sensitized SK Mel 5 (p53 wildtype) and 28 (p53 mutant) cells to DTIC. However, when SK Mel 5 cells were transiently transfected with tyrosinase and treated with Qct plus DTIC, SK Mel 5 cells demonstrated a more than additive induction of apoptosis. Therefore, this study demonstrates that tyrosinase overexpression promotes an ATM-dependent p53 phosphorylation by Qct treatment and sensitizes melanoma cells to dacarbazine. In conclusion, these results suggest that Qct or Qct analogues may significantly improve DTIC response rates in tumors that express tyrosinase.

  13. Tyrosinase Overexpression Promotes ATM-Dependent p53 Phosphorylation by Quercetin and Sensitizes Melanoma Cells to Dacarbazine

    PubMed Central

    Thangasamy, Thilakavathy; Sittadjody, Sivanandane; H. Limesand, Kirsten; Burd, Randy

    2008-01-01

    Dacarbazine (DTIC) has been used for the treatment of melanoma for decades. However, monotherapy with this chemotherapeutic agent results only in moderate response rates. To improve tumor response to DTIC current clinical trials in melanoma focus on combining a novel targeted agent with chemotherapy. Here, we demonstrate that tyrosinase which is commonly overexpressed in melanoma activates the bioflavonoid quercetin (Qct) and promotes an ataxia telangiectasia mutated (ATM)-dependent DNA damage response. This response sensitizes melanoma cells that overexpress tyrosinase to DTIC. In DB-1 melanoma cells that overexpress tyrosinase (Tyr cells), the threshold for phosphorylation of ATM and p53 at serine 15 was observed at a low dose of Qct (25 μM) when compared to the mock transfected pcDNA3 cells, which required a higher dose (75 μM). Both pcDNA3 and Tyr DB-1 cells demonstrated similar increases in phosphorylation of p53 at other serine sites, but in the Tyr cells, DNApk expression was found to be reduced compared to control cells, indicating a shift towards an ATM-mediated response. The DB-1 control cells were resistant to DTIC, but were sensitized to apoptosis with high dose Qct, while Tyr cells were sensitized to DTIC with low or high dose Qct. Qct also sensitized SK Mel 5 (p53 wildtype) and 28 (p53 mutant) cells to DTIC. However, when SK Mel 5 cells were transiently transfected with tyrosinase and treated with Qct plus DTIC, SK Mel 5 cells demonstrated a more than additive induction of apoptosis. Therefore, this study demonstrates that tyrosinase overexpression promotes an ATM-dependent p53 phosphorylation by Qct treatment and sensitizes melanoma cells to dacarbazine. In conclusion, these results suggest that Qct or Qct analogues may significantly improve DTIC response rates in tumors that express tyrosinase. PMID:18791269

  14. Alpha-synuclein functions in the nucleus to protect against hydroxyurea-induced replication stress in yeast

    PubMed Central

    Liu, Xianpeng; Lee, Yong Joo; Liou, Liang-Chun; Ren, Qun; Zhang, Zhaojie; Wang, Shaoxiao; Witt, Stephan N.

    2011-01-01

    Hydroxyurea (HU) inhibits ribonucleotide reductase (RNR), which catalyzes the rate-limiting synthesis of deoxyribonucleotides for DNA replication. HU is used to treat HIV, sickle-cell anemia and some cancers. We found that, compared with vector control cells, low levels of alpha-synuclein (α-syn) protect S. cerevisiae cells from the growth inhibition and reactive oxygen species (ROS) accumulation induced by HU. Analysis of this effect using different α-syn mutants revealed that the α-syn protein functions in the nucleus and not the cytoplasm to modulate S-phase checkpoint responses: α-syn up-regulates histone acetylation and RNR levels, maintains helicase minichromosome maintenance protein complexes (Mcm2–7) on chromatin and inhibits HU-induced ROS accumulation. Strikingly, when residues 2–10 or 96–140 are deleted, this protective function of α-syn in the nucleus is abolished. Understanding the mechanism by which α-syn protects against HU could expand our knowledge of the normal function of this neuronal protein. PMID:21642386

  15. SM22{alpha}-induced activation of p16{sup INK4a}/retinoblastoma pathway promotes cellular senescence caused by a subclinical dose of {gamma}-radiation and doxorubicin in HepG2 cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kim, Tae Rim; Lee, Hee Min; Lee, So Yong

    Research highlights: {yields} SM22{alpha} overexpression in HepG2 cells leads cells to a growth arrest state, and the treatment of a subclinical dose of {gamma}-radiation or doxorubicin promotes cellular senescence. {yields} SM22{alpha} overexpression elevates p16{sup INK4a} followed by pRB activation, but there are no effects on p53/p21{sup WAF1/Cip1} pathway. {yields} SM22{alpha}-induced MT-1G activates p16{sup INK4a}/pRB pathway, which promotes cellular senescence by damaging agents. -- Abstract: Smooth muscle protein 22-alpha (SM22{alpha}) is known as a transformation- and shape change-sensitive actin cross-linking protein found in smooth muscle tissue and fibroblasts; however, its functional role remains uncertain. We reported previously that SM22{alpha} overexpression confersmore » resistance against anti-cancer drugs or radiation via induction of metallothionein (MT) isozymes in HepG2 cells. In this study, we demonstrate that SM22{alpha} overexpression leads cells to a growth arrest state and promotes cellular senescence caused by treatment with a subclinical dose of {gamma}-radiation (0.05 and 0.1 Gy) or doxorubicin (0.01 and 0.05 {mu}g/ml), compared to control cells. Senescence growth arrest is known to be controlled by p53 phosphorylation/p21{sup WAF1/Cip1} induction or p16{sup INK4a}/retinoblastoma protein (pRB) activation. SM22{alpha} overexpression in HepG2 cells elevated p16{sup INK4a} followed by pRB activation, but did not activate the p53/p21{sup WAF1/Cip1} pathway. Moreover, MT-1G, which is induced by SM22{alpha} overexpression, was involved in the activation of the p16{sup INK4a}/pRB pathway, which led to a growth arrest state and promoted cellular senescence caused by damaging agents. Our findings provide the first demonstration that SM22{alpha} modulates cellular senescence caused by damaging agents via regulation of the p16{sup INK4a}/pRB pathway in HepG2 cells and that these effects of SM22{alpha} are partially mediated by MT-1G.« less

  16. Lewy Body-like α-Synuclein Aggregates Resist Degradation and Impair Macroautophagy*♦

    PubMed Central

    Tanik, Selcuk A.; Schultheiss, Christine E.; Volpicelli-Daley, Laura A.; Brunden, Kurt R.; Lee, Virginia M. Y.

    2013-01-01

    Cytoplasmic α-synuclein (α-syn) aggregates, referred to as Lewy bodies, are pathological hallmarks of a number of neurodegenerative diseases, most notably Parkinson disease. Activation of macroautophagy is suggested to facilitate degradation of certain proteinaceous inclusions, but it is unclear if this pathway is capable of degrading α-syn aggregates. Here, we examined this issue by utilizing cellular models in which intracellular Lewy body-like α-syn inclusions accumulate after internalization of pre-formed α-syn fibrils into α-syn-expressing HEK293 cells or cultured primary neurons. We demonstrate that α-syn inclusions cannot be effectively degraded, even though they co-localize with essential components of both the autophagic and proteasomal protein degradation pathways. The α-syn aggregates persist even after soluble α-syn levels have been substantially reduced, suggesting that once formed, the α-syn inclusions are refractory to clearance. Importantly, we also find that α-syn aggregates impair overall macroautophagy by reducing autophagosome clearance, which may contribute to the increased cell death that is observed in aggregate-bearing cells. PMID:23532841

  17. Neurogranin binds α-synuclein in the human superior temporal cortex and interaction is decreased in Parkinson’s disease

    PubMed Central

    Koob, Andrew O.; Shaked, Gideon M.; Bender, Andreas; Bisquertt, Alejandro; Rockenstein, Edward; Masliah, Eliezer

    2016-01-01

    Neurogranin is a calmodulin binding protein that has been implicated in learning and memory, long-term potentiation and synaptic plasticity. Neurons expressing neurogranin in the cortex degenerate in late stages of Parkinson’s disease with widespread α-synuclein pathology. While analyzing neurogranin gene expression levels through rtPCR in brains of mouse models overexpressing human α-synuclein, we found levels were elevated 2.5 times when compared to nontransgenic animals. Immunohistochemistry in the cortex revealed colocalization between α-synuclein and neurogranin in mouse transgenics when compared to control mice. Coimmunoprecipitation studies in the superior temporal cortex in humans confirmed interaction between α-synuclein and neurogranin, and decreased interaction between α-synuclein and neurogranin was noticed in patients diagnosed with Parkinson’s disease when compared to normal control brains. Additionally, phosphorylated neurogranin levels were also decreased in the human superior temporal cortex in patients diagnosed with Parkinson’s disease and patients diagnosed with dementia with Lewy bodies. Here, we show for the first time that neurogranin binds to α-synuclein in the human cortex, and this interaction decreases in Parkinson’s disease along with the phosphorylation of neurogranin, a molecular process thought to be involved in learning and memory. PMID:25446004

  18. Comparison of alpha-synuclein immunoreactivity in the spinal cord between the adult and aged beagle dog

    PubMed Central

    Ahn, Ji-Hyeon; Choi, Jung-Hoon; Park, Joon-Ha; Yan, Bing-Chun; Kim, In-Hye; Lee, Jae-Chul; Lee, Dae-Hwan; Kim, Jin-Sang

    2012-01-01

    Alpha-synuclein (α-syn) is a presynaptic protein that is richly expressed in the central and peripheral nervous systems of mammals, and it is related to the pathogenesis of Parkinson's disease and other neurodegenerative disorders. In the present study, we compared the distribution of the immunoreactivity of α-syn and its related gliosis in the spinal cord of young adult (2-3 years) and aged (10-12 years) beagle dogs. We discovered that α-syn immunoreactivity was present in many neurons in the thoracic level of the aged spinal cord, however, its protein level was not distinct inform that of the adult spinal cord. In addition, ionized calcium-binding adapter molecule-1 (a marker for microglia) immunoreactivity, and not glial fibrillary acidic protein (a marker for astrocytes) immunoreactivity, was somewhat increased in the aged group compared to the adult group. These results indicate that α-syn immunoreactivity was not dramatically changed in the dog spinal cord during aging. PMID:23091516

  19. SIRT1 inhibition restores apoptotic sensitivity in p53-mutated human keratinocytes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Herbert, Katharine J.; Cook, Anthony L., E-mail: Anthony.Cook@utas.edu.au; Snow, Elizabeth T., E-mail: elizabeth.snow@utas.edu.au

    2014-06-15

    Mutations to the p53 gene are common in UV-exposed keratinocytes and contribute to apoptotic resistance in skin cancer. P53-dependent activity is modulated, in part, by a complex, self-limiting feedback loop imposed by miR-34a-mediated regulation of the lysine deacetylase, SIRT1. Expression of numerous microRNAs is dysregulated in squamous and basal cell carcinomas; however the contribution of specific microRNAs to the pathogenesis of skin cancer remains untested. Through use of RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors, this study explored the influence of p53 mutational status, SIRT1 activity and miR-34a levels on apoptotic sensitivity in primary (NHEK) and p53-mutatedmore » (HaCaT) keratinocyte cell lines. SIRT1 and p53 are overexpressed in p53-mutated keratinocytes, whilst miR-34a levels are 90% less in HaCaT cells. HaCaTs have impaired responses to p53/SIRT1/miR-34a axis manipulation which enhanced survival during exposure to the chemotherapeutic agent, camptothecin. Inhibition of SIRT1 activity in this cell line increased p53 acetylation and doubled camptothecin-induced cell death. Our results demonstrate that p53 mutations increase apoptotic resistance in keratinocytes by interfering with miR-34a-mediated regulation of SIRT1 expression. Thus, SIRT1 inhibitors may have a therapeutic potential for overcoming apoptotic resistance during skin cancer treatment. - Highlights: • Impaired microRNA biogenesis promotes apoptotic resistance in HaCaT keratinocytes. • TP53 mutations suppress miR-34a-mediated regulation of SIRT1 expression. • SIRT1 inhibition increases p53 acetylation in HaCaTs, restoring apoptosis.« less

  20. ER Stress Induced by Tunicamycin Triggers α-Synuclein Oligomerization, Dopaminergic Neurons Death and Locomotor Impairment: a New Model of Parkinson's Disease.

    PubMed

    Cóppola-Segovia, Valentín; Cavarsan, Clarissa; Maia, Flavia G; Ferraz, Anete C; Nakao, Lia S; Lima, Marcelo Ms; Zanata, Silvio M

    2017-10-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive death of dopaminergic neurons of the substantia nigra pars compacta (SNpc), leading to the major clinical abnormalities that characterize this disease. Although PD's etiology is unknown, α-synuclein aggregation plays a pivotal role in PD pathogenesis, which could be associated to some pathological processes such as oxidative stress, endoplasmic reticulum (ER) stress, impaired protein degradation, and mitochondrial dysfunction. Increasing experimental evidence indicates that ER stress is involved in PD, however most of the described results employed cultured cell lines and genetically modified animal models. In this study, we developed a new ER stress rat model employing the well-known ER stressor tunicamycin (Tm). To evaluate if ER stress was able to induce PD features, we performed an intranigral injection of Tm (0.1 μg/cerebral hemisphere) and animals (male Wistar rats) were analyzed 7 days post injection. The classical 6-OHDA neurotoxin model (1 μg/cerebral hemisphere) was used as an established positive control for PD. We show that Tm injection induced locomotor impairment, dopaminergic neurons death, and activation of astroglia. In addition, we observed an extensive α-synuclein oligomerization in SNpc of Tm-injected animals when compared with DMSO-injected controls. Finally, both Tm and 6-OHDA treated animals presented increased levels of ER stress markers. Taken together, these findings show for the first time that the ER stressor Tm recapitulates some of the phenotypic characteristics observed in rodent models of PD, reinforcing the concept that ER stress could be an important contributor to the pathophysiology of PD. Therefore, we propose the intranigral Tm injection as a new ER stress-based model for the study of PD in vivo.

  1. Mitochondria and α-Synuclein: Friends or Foes in the Pathogenesis of Parkinson's Disease?

    PubMed

    Faustini, Gaia; Bono, Federica; Valerio, Alessandra; Pizzi, Marina; Spano, PierFranco; Bellucci, Arianna

    2017-12-08

    Parkinson's disease (PD) is a movement disorder characterized by dopaminergic nigrostriatal neuron degeneration and the formation of Lewy bodies (LB), pathological inclusions containing fibrils that are mainly composed of α-synuclein. Dopaminergic neurons, for their intrinsic characteristics, have a high energy demand that relies on the efficiency of the mitochondria respiratory chain. Dysregulations of mitochondria, deriving from alterations of complex I protein or oxidative DNA damage, change the trafficking, size and morphology of these organelles. Of note, these mitochondrial bioenergetics defects have been related to PD. A series of experimental evidence supports that α-synuclein physiological action is relevant for mitochondrial homeostasis, while its pathological aggregation can negatively impinge on mitochondrial function. It thus appears that imbalances in the equilibrium between the reciprocal modulatory action of mitochondria and α-synuclein can contribute to PD onset by inducing neuronal impairment. This review will try to highlight the role of physiological and pathological α-synuclein in the modulation of mitochondrial functions.

  2. Overexpression of TGF-alpha increases lung tissue hysteresivity in transgenic mice.

    PubMed

    Pillow, J J; Korfhagen, T R; Ikegami, M; Sly, P D

    2001-12-01

    Increased transforming growth factor (TGF)-alpha has been observed in neonatal chronic lung disease. Lungs of transgenic mice that overexpress TGF-alpha develop enlarged air spaces and pulmonary fibrosis compared with wild-type mice. We hypothesized that these pathological changes may alter the mechanical coupling of viscous and elastic forces within lung parenchyma. Respiratory impedance was measured in open-chested, tracheostomized adult wild-type and TGF-alpha mice by using the forced oscillation technique (0.25-19.63 Hz) delivered by flexiVent (Scireq, Montreal, PQ). Estimates of airway resistance (Raw), inertance (I), and the coefficients of tissue damping (G(L)) and tissue elastance (H(L)) were obtained by fitting a model to each impedance spectrum. Hysteresivity (eta) was calculated as G(L)/H(L). There was a significant increase in eta (P < 0.01) and a trend to a decrease in H(L) (P = 0.07) of TGF-alpha mice compared with the wild-type group. There was no significant change in Raw, I, or G(L). Structural abnormality present in the lungs of adult TGF-alpha mice alters viscoelastic coupling of the tissues, as evidenced by a change in eta.

  3. The G209A mutation in the alpha-synuclein gene is not detected in familial cases of Parkinson disease in non-Greek and/or Italian populations.

    PubMed

    Wang, W W; Khajavi, M; Patel, B J; Beach, J; Jankovic, J; Ashizawa, T

    1998-12-01

    To determine whether the G-to-A substitution at nucleotide 209 (G209A) mutation in the alpha-synuclein gene is responsible for familial Parkinson disease (PD) in the US population. Polymerase chain reaction-based DNA analysis of consecutive patients with PD and family history of PD. A university-affiliated movement disorder clinic and a Veterans Affairs clinical research laboratory. Forty-four patients with PD and family history of PD and 29 patients with sporadic PD, all with no known Greek and/or Italian background. None of the DNA samples showed the G209A mutation. The G209A mutation is rare in US patients with familial PD.

  4. Transient overexpression of striatal D2 receptors impairs operant motivation and interval timing.

    PubMed

    Drew, Michael R; Simpson, Eleanor H; Kellendonk, Christoph; Herzberg, William G; Lipatova, Olga; Fairhurst, Stephen; Kandel, Eric R; Malapani, Chara; Balsam, Peter D

    2007-07-18

    The striatum receives prominent dopaminergic innervation that is integral to appetitive learning, performance, and motivation. Signaling through the dopamine D2 receptor is critical for all of these processes. For instance, drugs with high affinity for the D2 receptor potently alter timing of operant responses and modulate motivation. Recently, in an attempt to model a genetic abnormality encountered in schizophrenia, mice were generated that reversibly overexpress D2 receptors specifically in the striatum (Kellendonk et al., 2006). These mice have impairments in working memory and behavioral flexibility, components of the cognitive symptoms of schizophrenia, that are not rescued when D2 overexpression is reversed in the adult. Here we report that overexpression of striatal D2 receptors also profoundly affects operant performance, a potential index of negative symptoms. Mice overexpressing D2 exhibited impairments in the ability to time food rewards in an operant interval timing task and reduced motivation to lever press for food reward in both the operant timing task and a progressive ratio schedule of reinforcement. The motivational deficit, but not the timing deficit, was rescued in adult mice by reversing D2 overexpression with doxycycline. These results suggest that early D2 overexpression alters the organization of interval timing circuits and confirms that striatal D2 signaling in the adult regulates motivational process. Moreover, overexpression of D2 under pathological conditions such as schizophrenia and Parkinson's disease could give rise to motivational and timing deficits.

  5. Parkinson-causing α-synuclein missense mutations shift native tetramers to monomers as a mechanism for disease initiation

    PubMed Central

    Dettmer, Ulf; Newman, Andrew J.; Soldner, Frank; Luth, Eric S.; Kim, Nora C.; von Saucken, Victoria E.; Sanderson, John B.; Jaenisch, Rudolf; Bartels, Tim; Selkoe, Dennis

    2015-01-01

    β-Sheet-rich α-synuclein (αS) aggregates characterize Parkinson's disease (PD). αS was long believed to be a natively unfolded monomer, but recent work suggests it also occurs in α-helix-rich tetramers. Crosslinking traps principally tetrameric αS in intact normal neurons, but not after cell lysis, suggesting a dynamic equilibrium. Here we show that freshly biopsied normal human brain contains abundant αS tetramers. The PD-causing mutation A53T decreases tetramers in mouse brain. Neurons derived from an A53T patient have decreased tetramers. Neurons expressing E46K do also, and adding 1-2 E46K-like mutations into the canonical αS repeat motifs (KTKEGV) further reduces tetramers, decreases αS solubility and induces neurotoxicity and round inclusions. The other three fPD missense mutations likewise decrease tetramer:monomer ratios. The destabilization of physiological tetramers by PD-causing missense mutations and the neurotoxicity and inclusions induced by markedly decreasing tetramers suggest that decreased α-helical tetramers and increased unfolded monomers initiate pathogenesis. Tetramer-stabilizing compounds should prevent this. PMID:26076669

  6. β-Synuclein suppresses both the initiation and amplification steps of α-synuclein aggregation via competitive binding to surfaces

    NASA Astrophysics Data System (ADS)

    Brown, James W. P.; Buell, Alexander K.; Michaels, Thomas C. T.; Meisl, Georg; Carozza, Jacqueline; Flagmeier, Patrick; Vendruscolo, Michele; Knowles, Tuomas P. J.; Dobson, Christopher M.; Galvagnion, Céline

    2016-11-01

    α-Synuclein is an intrinsically disordered protein that is associated with the pathogenesis of Parkinson’s disease through the processes involved in the formation of amyloid fibrils. α and β-synuclein are homologous proteins found at comparable levels in presynaptic terminals but β-synuclein has a greatly reduced propensity to aggregate and indeed has been found to inhibit α-synuclein aggregation. In this paper, we describe how sequence differences between α- and β-synuclein affect individual microscopic processes in amyloid formation. In particular, we show that β-synuclein strongly suppresses both lipid-induced aggregation and secondary nucleation of α-synuclein by competing for binding sites at the surfaces of lipid vesicles and fibrils, respectively. These results suggest that β-synuclein can act as a natural inhibitor of α-synuclein aggregation by reducing both the initiation of its self-assembly and the proliferation of its aggregates.

  7. Convergence of pathology in dementia with Lewy bodies and Alzheimer's disease: a role for the novel interaction of alpha-synuclein and presenilin 1 in disease.

    PubMed

    Winslow, Ashley R; Moussaud, Simon; Zhu, Liya; Post, Kathryn L; Post, Katherine L; Dickson, Dennis W; Berezovska, Oksana; McLean, Pamela J

    2014-07-01

    A growing number of PSEN1 mutations have been associated with dementia with Lewy bodies and familial Alzheimer's disease with concomitant α-synuclein pathology. The objective of this study was to determine if PSEN1 plays a direct role in the development of α-synuclein pathology in these diseases. Using mass spectrometry, immunoelectron microscopy and fluorescence lifetime image microscopy based on Forster resonance energy transfer (FLIM-FRET) we identified α-synuclein as a novel interactor of PSEN1 in wild-type mouse brain tissue. The interaction of α-synuclein with PSEN1 was detected in post-mortem brain tissue from cognitively normal cases and was significantly increased in tissue from cases with dementia with Lewy bodies and familial Alzheimer's disease associated with known PSEN1 mutations. We confirmed an increased interaction of PSEN1 and α-synuclein in cell lines expressing well characterized familial Alzheimer's disease PSEN1 mutations, L166P and delta exon 9, and demonstrated that PSEN1 mutations associate with increased membrane association and accumulation of α-synuclein. Our data provides evidence of a molecular interaction of PSEN1 and α-synuclein that may explain the clinical and pathophysiological overlap seen in synucleinopathies, including Parkinson's disease, dementia with Lewy bodies, and some forms of Alzheimer's disease. © The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  8. Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers.

    PubMed

    Fusco, Giuliana; Chen, Serene W; Williamson, Philip T F; Cascella, Roberta; Perni, Michele; Jarvis, James A; Cecchi, Cristina; Vendruscolo, Michele; Chiti, Fabrizio; Cremades, Nunilo; Ying, Liming; Dobson, Christopher M; De Simone, Alfonso

    2017-12-15

    Oligomeric species populated during the aggregation process of α-synuclein have been linked to neuronal impairment in Parkinson's disease and related neurodegenerative disorders. By using solution and solid-state nuclear magnetic resonance techniques in conjunction with other structural methods, we identified the fundamental characteristics that enable toxic α-synuclein oligomers to perturb biological membranes and disrupt cellular function; these include a highly lipophilic element that promotes strong membrane interactions and a structured region that inserts into lipid bilayers and disrupts their integrity. In support of these conclusions, mutations that target the region that promotes strong membrane interactions by α-synuclein oligomers suppressed their toxicity in neuroblastoma cells and primary cortical neurons. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  9. α-Synuclein Regulates Neuronal Cholesterol Efflux.

    PubMed

    Hsiao, Jen-Hsiang T; Halliday, Glenda M; Kim, Woojin Scott

    2017-10-19

    α-Synuclein is a neuronal protein that is at the center of focus in understanding the etiology of a group of neurodegenerative diseases called α-synucleinopathies, which includes Parkinson's disease (PD). Despite much research, the exact physiological function of α-synuclein is still unclear. α-Synuclein has similar biophysical properties as apolipoproteins and other lipid-binding proteins and has a high affinity for cholesterol. These properties suggest a possible role for α-synuclein as a lipid acceptor mediating cholesterol efflux (the process of removing cholesterol out of cells). To test this concept, we "loaded" SK-N-SH neuronal cells with fluorescently-labelled cholesterol, applied exogenous α-synuclein, and measured the amount of cholesterol removed from the cells using a classic cholesterol efflux assay. We found that α-synuclein potently stimulated cholesterol efflux. We found that the process was dose and time dependent, and was saturable at 1.0 µg/mL of α-synuclein. It was also dependent on the transporter protein ABCA1 located on the plasma membrane. We reveal for the first time a novel role of α-synuclein that underscores its importance in neuronal cholesterol regulation, and identify novel therapeutic targets for controlling cellular cholesterol levels.

  10. Convergence of pathology in dementia with Lewy bodies and Alzheimer’s disease: a role for the novel interaction of alpha-synuclein and presenilin 1 in disease

    PubMed Central

    Winslow, Ashley R.; Moussaud, Simon; Zhu, Liya; Post, Katherine L.; Dickson, Dennis W.

    2014-01-01

    A growing number of PSEN1 mutations have been associated with dementia with Lewy bodies and familial Alzheimer’s disease with concomitant α-synuclein pathology. The objective of this study was to determine if PSEN1 plays a direct role in the development of α-synuclein pathology in these diseases. Using mass spectrometry, immunoelectron microscopy and fluorescence lifetime image microscopy based on Forster resonance energy transfer (FLIM-FRET) we identified α-synuclein as a novel interactor of PSEN1 in wild-type mouse brain tissue. The interaction of α-synuclein with PSEN1 was detected in post-mortem brain tissue from cognitively normal cases and was significantly increased in tissue from cases with dementia with Lewy bodies and familial Alzheimer’s disease associated with known PSEN1 mutations. We confirmed an increased interaction of PSEN1 and α-synuclein in cell lines expressing well characterized familial Alzheimer’s disease PSEN1 mutations, L166P and delta exon 9, and demonstrated that PSEN1 mutations associate with increased membrane association and accumulation of α-synuclein. Our data provides evidence of a molecular interaction of PSEN1 and α-synuclein that may explain the clinical and pathophysiological overlap seen in synucleinopathies, including Parkinson’s disease, dementia with Lewy bodies, and some forms of Alzheimer’s disease. PMID:24860142

  11. Brain propagation of transduced α-synuclein involves non-fibrillar protein species and is enhanced in α-synuclein null mice.

    PubMed

    Helwig, Michael; Klinkenberg, Michael; Rusconi, Raffaella; Musgrove, Ruth E; Majbour, Nour K; El-Agnaf, Omar M A; Ulusoy, Ayse; Di Monte, Donato A

    2016-03-01

    Aggregation and neuron-to-neuron transmission are attributes of α-synuclein relevant to its pathogenetic role in human synucleinopathies such as Parkinson's disease. Intraparenchymal injections of fibrillar α-synuclein trigger widespread propagation of amyloidogenic protein species via mechanisms that require expression of endogenous α-synuclein and, possibly, its structural corruption by misfolded conformers acting as pathological seeds. Here we describe another paradigm of long-distance brain diffusion of α-synuclein that involves inter-neuronal transfer of monomeric and/or oligomeric species and is independent of recruitment of the endogenous protein. Targeted expression of human α-synuclein was induced in the mouse medulla oblongata through an injection of viral vectors into the vagus nerve. Enhanced levels of intra-neuronal α-synuclein were sufficient to initiate its caudo-rostral diffusion that likely involved at least one synaptic transfer and progressively reached specific brain regions such as the locus coeruleus, dorsal raphae and amygdala in the pons, midbrain and forebrain. Transfer of human α-synuclein was compared in two separate lines of α-synuclein-deficient mice versus their respective wild-type controls and, interestingly, lack of endogenous α-synuclein expression did not counteract diffusion but actually resulted in a more pronounced and advanced propagation of exogenous α-synuclein. Self-interaction of adjacent molecules of human α-synuclein was detected in both wild-type and mutant mice. In the former, interaction of human α-synuclein with mouse α-synuclein was also observed and might have contributed to differences in protein transmission. In wild-type and α-synuclein-deficient mice, accumulation of human α-synuclein within recipient axons in the pons, midbrain and forebrain caused morphological evidence of neuritic pathology. Tissue sections from the medulla oblongata and pons were stained with different antibodies recognizing

  12. Ca2+ is a key factor in α-synuclein-induced neurotoxicity

    PubMed Central

    Angelova, Plamena R.; Ludtmann, Marthe H. R.; Horrocks, Mathew H.; Negoda, Alexander; Cremades, Nunilo; Klenerman, David; Dobson, Christopher M.; Wood, Nicholas W.; Pavlov, Evgeny V.; Gandhi, Sonia

    2016-01-01

    ABSTRACT Aggregation of α-synuclein leads to the formation of oligomeric intermediates that can interact with membranes to form pores. However, it is unknown how this leads to cell toxicity in Parkinson's disease. We investigated the species-specific effects of α-synuclein on Ca2+ signalling in primary neurons and astrocytes using live neuronal imaging and electrophysiology on artificial membranes. We demonstrate that α-synuclein induces an increase in basal intracellular Ca2+ in its unfolded monomeric state as well as in its oligomeric state. Electrophysiology of artificial membranes demonstrated that α-synuclein monomers induce irregular ionic currents, whereas α-synuclein oligomers induce rare discrete channel formation events. Despite the ability of monomeric α-synuclein to affect Ca2+ signalling, it is only the oligomeric form of α-synuclein that induces cell death. Oligomer-induced cell death was abolished by the exclusion of extracellular Ca2+, which prevented the α-synuclein-induced Ca2+ dysregulation. The findings of this study confirm that α-synuclein interacts with membranes to affect Ca2+ signalling in a structure-specific manner and the oligomeric β-sheet-rich α-synuclein species ultimately leads to Ca2+ dysregulation and Ca2+-dependent cell death. PMID:26989132

  13. Curcumin Pyrazole and its derivative (N-(3-Nitrophenylpyrazole) Curcumin inhibit aggregation, disrupt fibrils and modulate toxicity of Wild type and Mutant α-Synuclein

    PubMed Central

    Ahsan, Nuzhat; Mishra, Satyendra; Jain, Manish Kumar; Surolia, Avadhesha; Gupta, Sarika

    2015-01-01

    Accumulating evidence suggests that deposition of neurotoxic α-synuclein aggregates in the brain during the development of neurodegenerative diseases like Parkinson’s disease can be curbed by anti-aggregation strategies that either disrupt or eliminate toxic aggregates. Curcumin, a dietary polyphenol exhibits anti-amyloid activity but the use of this polyphenol is limited owing to its instability. As chemical modifications in curcumin confiscate this limitation, such efforts are intensively performed to discover molecules with similar but enhanced stability and superior properties. This study focuses on the inhibitory effect of two stable analogs of curcumin viz. curcumin pyrazole and curcumin isoxazole and their derivatives against α-synuclein aggregation, fibrillization and toxicity. Employing biochemical, biophysical and cell based assays we discovered that curcumin pyrazole (3) and its derivative N-(3-Nitrophenylpyrazole) curcumin (15) exhibit remarkable potency in not only arresting fibrillization and disrupting preformed fibrils but also preventing formation of A11 conformation in the protein that imparts toxic effects. Compounds 3 and 15 also decreased neurotoxicity associated with fast aggregating A53T mutant form of α-synuclein. These two analogues of curcumin described here may therefore be useful therapeutic inhibitors for the treatment of α-synuclein amyloidosis and toxicity in Parkinson’s disease and other synucleinopathies. PMID:25985292

  14. Passive immunization reduces behavioral and neuropathological deficits in an alpha-synuclein transgenic model of Lewy body disease.

    PubMed

    Masliah, Eliezer; Rockenstein, Edward; Mante, Michael; Crews, Leslie; Spencer, Brian; Adame, Anthony; Patrick, Christina; Trejo, Margarita; Ubhi, Kiren; Rohn, Troy T; Mueller-Steiner, Sarah; Seubert, Peter; Barbour, Robin; McConlogue, Lisa; Buttini, Manuel; Games, Dora; Schenk, Dale

    2011-04-29

    Dementia with Lewy bodies (DLB) and Parkinson's Disease (PD) are common causes of motor and cognitive deficits and are associated with the abnormal accumulation of alpha-synuclein (α-syn). This study investigated whether passive immunization with a novel monoclonal α-syn antibody (9E4) against the C-terminus (CT) of α-syn was able to cross into the CNS and ameliorate the deficits associated with α-syn accumulation. In this study we demonstrate that 9E4 was effective at reducing behavioral deficits in the water maze, moreover, immunization with 9E4 reduced the accumulation of calpain-cleaved α-syn in axons and synapses and the associated neurodegenerative deficits. In vivo studies demonstrated that 9E4 traffics into the CNS, binds to cells that display α-syn accumulation and promotes α-syn clearance via the lysosomal pathway. These results suggest that passive immunization with monoclonal antibodies against the CT of α-syn may be of therapeutic relevance in patients with PD and DLB.

  15. Cell Biology and Pathophysiology of α-Synuclein

    PubMed Central

    Burré, Jacqueline; Sharma, Manu; Südhof, Thomas C.

    2017-01-01

    α-Synuclein is an abundant neuronal protein that is highly enriched in presynaptic nerve terminals. Genetics and neuropathology studies link α-synuclein to Parkinson’s disease (PD) and other neurodegenerative disorders. Accumulation of misfolded oligomers and larger aggregates of α-synuclein defines multiple neurodegenerative diseases called synucleino-pathies, but the mechanisms by which α-synuclein acts in neurodegeneration are unknown. Moreover, the normal cellular function of α-synuclein remains debated. In this perspective, we review the structural characteristics of α-synuclein, its developmental expression pattern, its cellular and subcellular localization, and its function in neurons. We also discuss recent progress on secretion of α-synuclein, which may contribute to its interneuronal spread in a prion-like fashion, and describe the neurotoxic effects of α-synuclein that are thought to be responsible for its role in neurodegeneration. PMID:28108534

  16. Mechanisms of α-Synuclein Induced Synaptopathy in Parkinson's Disease

    PubMed Central

    Bridi, Jessika C.; Hirth, Frank

    2018-01-01

    Parkinson's disease (PD) is characterized by intracellular inclusions of aggregated and misfolded α-Synuclein (α-Syn), and the loss of dopaminergic (DA) neurons in the brain. The resulting motor abnormalities mark the progression of PD, while non-motor symptoms can already be identified during early, prodromal stages of disease. Recent studies provide evidence that during this early prodromal phase, synaptic and axonal abnormalities occur before the degenerative loss of neuronal cell bodies. These early phenotypes can be attributed to synaptic accumulation of toxic α-Syn. Under physiological conditions, α-Syn functions in its native conformation as a soluble monomer. However, PD patient brains are characterized by intracellular inclusions of insoluble fibrils. Yet, oligomers and protofibrils of α-Syn have been identified to be the most toxic species, with their accumulation at presynaptic terminals affecting several steps of neurotransmitter release. First, high levels of α-Syn alter the size of synaptic vesicle pools and impair their trafficking. Second, α-Syn overexpression can either misregulate or redistribute proteins of the presynaptic SNARE complex. This leads to deficient tethering, docking, priming and fusion of synaptic vesicles at the active zone (AZ). Third, α-Syn inclusions are found within the presynaptic AZ, accompanied by a decrease in AZ protein levels. Furthermore, α-Syn overexpression reduces the endocytic retrieval of synaptic vesicle membranes during vesicle recycling. These presynaptic alterations mediated by accumulation of α-Syn, together impair neurotransmitter exocytosis and neuronal communication. Although α-Syn is expressed throughout the brain and enriched at presynaptic terminals, DA neurons are the most vulnerable in PD, likely because α-Syn directly regulates dopamine levels. Indeed, evidence suggests that α-Syn is a negative modulator of dopamine by inhibiting enzymes responsible for its synthesis. In addition,

  17. Introduction of an Aliphatic Ketone into Recombinant Proteins in a Bacterial Strain that Overexpresses an Editing-Impaired Leucyl-tRNA Synthetase

    PubMed Central

    Tang, Yi; Wang, Pin; Van Deventer, James A.; Link, A. James; Tirrell, David A.

    2011-01-01

    A leucine analog containing a ketone has been incorporated into proteins in E. coli. Only E. coli strains overexpressing an editing-deficient leucyl-tRNA synthetase were capable of synthesizing proteins with the aliphatic ketone amino acid. Modification of ketone-containing proteins under mild conditions has been demonstrated. PMID:19670197

  18. Disruption of alpha beta but not of gamma delta T cell development by overexpression of the helix-loop-helix protein Id3 in committed T cell progenitors.

    PubMed Central

    Blom, B; Heemskerk, M H; Verschuren, M C; van Dongen, J J; Stegmann, A P; Bakker, A Q; Couwenberg, F; Res, P C; Spits, H

    1999-01-01

    Enforced expression of Id3, which has the capacity to inhibit many basic helix-loop-helix (bHLH) transcription factors, in human CD34(+) hematopoietic progenitor cells that have not undergone T cell receptor (TCR) gene rearrangements inhibits development of the transduced cells into TCRalpha beta and gamma delta cells in a fetal thymic organ culture (FTOC). Here we document that overexpression of Id3, in progenitors that have initiated TCR gene rearrangements (pre-T cells), inhibits development into TCRalpha beta but not into TCRgamma delta T cells. Furthermore, Id3 impedes expression of recombination activating genes and downregulates pre-Talpha mRNA. These observations suggest possible mechanisms by which Id3 overexpression can differentially affect development of pre-T cells into TCRalpha beta and gamma delta cells. We also observed that cell surface CD4(-)CD8(-)CD3(-) cells with rearranged TCR genes developed from Id3-transduced but not from control-transduced pre-T cells in an FTOC. These cells had properties of both natural killer (NK) and pre-T cells. These findings suggest that bHLH factors are required to control T cell development after the T/NK developmental checkpoint. PMID:10329625

  19. Lysosomal impairment in Parkinson's disease.

    PubMed

    Dehay, Benjamin; Martinez-Vicente, Marta; Caldwell, Guy A; Caldwell, Kim A; Yue, Zhenyue; Cookson, Mark R; Klein, Christine; Vila, Miquel; Bezard, Erwan

    2013-06-01

    Impairment of autophagy-lysosomal pathways (ALPs) is increasingly regarded as a major pathogenic event in neurodegenerative diseases, including Parkinson's disease (PD). ALP alterations are observed in sporadic PD brains and in toxic and genetic rodent models of PD-related neurodegeneration. In addition, PD-linked mutations and post-translational modifications of α-synuclein impair its own lysosomal-mediated degradation, thereby contributing to its accumulation and aggregation. Furthermore, other PD-related genes, such as leucine-rich repeat kinase-2 (LRRK2), parkin, and phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), have been mechanistically linked to alterations in ALPs. Conversely, mutations in lysosomal-related genes, such as glucocerebrosidase (GBA) and lysosomal type 5 P-type ATPase (ATP13A2), have been linked to PD. New data offer mechanistic molecular evidence for such a connection, unraveling a causal link between lysosomal impairment, α-synuclein accumulation, and neurotoxicity. First, PD-related GBA deficiency/mutations initiate a positive feedback loop in which reduced lysosomal function leads to α-synuclein accumulation, which, in turn, further decreases lysosomal GBA activity by impairing the trafficking of GBA from the endoplasmic reticulum-Golgi to lysosomes, leading to neurodegeneration. Second, PD-related mutations/deficiency in the ATP13A2 gene lead to a general lysosomal impairment characterized by lysosomal membrane instability, impaired lysosomal acidification, decreased processing of lysosomal enzymes, reduced degradation of lysosomal substrates, and diminished clearance of autophagosomes, collectively contributing to α-synuclein accumulation and cell death. According to these new findings, primary lysosomal defects could potentially account for Lewy body formation and neurodegeneration in PD, laying the groundwork for the prospective development of new neuroprotective/disease-modifying therapeutic strategies

  20. Binding Interactions of Agents That Alter α-Synuclein Aggregation

    PubMed Central

    Sivanesam, K.; Byrne, A.; Bisaglia, M.; Bubacco, L.

    2015-01-01

    Further examination of peptides with well-folded antiparallel β strands as inhibitors of amyloid formation from α-synuclein has resulted in more potent inhibitors. Several of these had multiple Tyr residues and represent a new lead for inhibitor design by small peptides that do not divert α-synuclein to non-amyloid aggregate formation. The most potent inhibitor obtained in this study is a backbone cyclized version of a previously studied β hairpin, designated as WW2, with a cross-strand Trp/Trp cluster. The cyclization was accomplished by adding a d-Pro-l-Pro turn locus across strand termini. At a 2:1 peptide to α-synuclein ratio, cyclo-WW2 displays complete inhibition of β-structure formation. Trp-bearing antiparallel β-sheets held together by a disulphide bond are also potent inhibitors. 15N HSQC spectra of α-synuclein provided new mechanistic details. The time course of 15N HSQC spectral changes observed during β-oligomer formation has revealed which segments of the structure become part of the rigid core of an oligomer at early stages of amyloidogenesis and that the C-terminus remains fully flexible throughout the process. All of the effective peptide inhibitors display binding-associated titration shifts in 15N HSQC spectra of α-synuclein in the C-terminal Q109-E137 segment. Cyclo-WW2, the most potent inhibitor, also displays titration shifts in the G41-T54 span of α-synuclein, an additional binding site. The earliest aggregation event appears to be centered about H50 which is also a binding site for our most potent inhibitor. PMID:25705374

  1. Binding Interactions of Agents That Alter α-Synuclein Aggregation.

    PubMed

    Sivanesam, K; Byrne, A; Bisaglia, M; Bubacco, L; Andersen, N

    Further examination of peptides with well-folded antiparallel β strands as inhibitors of amyloid formation from α-synuclein has resulted in more potent inhibitors. Several of these had multiple Tyr residues and represent a new lead for inhibitor design by small peptides that do not divert α-synuclein to non-amyloid aggregate formation. The most potent inhibitor obtained in this study is a backbone cyclized version of a previously studied β hairpin, designated as WW2, with a cross-strand Trp/Trp cluster. The cyclization was accomplished by adding a d-Pro-l-Pro turn locus across strand termini. At a 2:1 peptide to α-synuclein ratio, cyclo-WW2 displays complete inhibition of β-structure formation. Trp-bearing antiparallel β-sheets held together by a disulphide bond are also potent inhibitors. 15 N HSQC spectra of α-synuclein provided new mechanistic details. The time course of 15 N HSQC spectral changes observed during β-oligomer formation has revealed which segments of the structure become part of the rigid core of an oligomer at early stages of amyloidogenesis and that the C-terminus remains fully flexible throughout the process. All of the effective peptide inhibitors display binding-associated titration shifts in 15 N HSQC spectra of α-synuclein in the C-terminal Q109-E137 segment. Cyclo-WW2, the most potent inhibitor, also displays titration shifts in the G41-T54 span of α-synuclein, an additional binding site. The earliest aggregation event appears to be centered about H50 which is also a binding site for our most potent inhibitor.

  2. Novel One-step Immunoassays to Quantify α-Synuclein

    PubMed Central

    Bidinosti, Michael; Shimshek, Derya R.; Mollenhauer, Brit; Marcellin, David; Schweizer, Tatjana; Lotz, Gregor P.; Schlossmacher, Michael G.; Weiss, Andreas

    2012-01-01

    Familial Parkinson disease (PD) can result from α-synuclein gene multiplication, implicating the reduction of neuronal α-synuclein as a therapeutic target. Moreover, α-synuclein content in human cerebrospinal fluid (CSF) represents a PD biomarker candidate. However, capture-based assays for α-synuclein quantification in CSF (such as by ELISA) have shown discrepancies and have limited suitability for high-throughput screening. Here, we describe two sensitive, in-solution, time-resolved Förster's resonance energy transfer (TR-FRET)-based immunoassays for total and oligomeric α-synuclein quantification. CSF analysis showed strong concordance for total α-synuclein content between two TR-FRET assays and, in agreement with a previously characterized 36 h protocol-based ELISA, demonstrated lower α-synuclein levels in PD donors. Critically, the assay suitability for high-throughput screening of siRNA constructs and small molecules aimed at reducing endogenous α-synuclein levels was established and validated. In a small-scale proof of concept compound screen using 384 well plates, signals ranged from <30 to >120% of the mean of vehicle-treated cells for molecules known to lower and increase cellular α-synuclein, respectively. Furthermore, a reverse genetic screen of a kinase-directed siRNA library identified seven genes that modulated α-synuclein protein levels (five whose knockdown increased and two that decreased cellular α-synuclein protein). This provides critical new biological insight into cellular pathways regulating α-synuclein steady-state expression that may help guide further drug discovery efforts. Moreover, we describe an inherent limitation in current α-synuclein oligomer detection methodology, a finding that will direct improvement of future assay design. Our one-step TR-FRET-based platform for α-synuclein quantification provides a novel platform with superior performance parameters for the rapid screening of large biomarker cohorts and of

  3. α-Synuclein binds to the ER-mitochondria tethering protein VAPB to disrupt Ca2+ homeostasis and mitochondrial ATP production.

    PubMed

    Paillusson, Sébastien; Gomez-Suaga, Patricia; Stoica, Radu; Little, Daniel; Gissen, Paul; Devine, Michael J; Noble, Wendy; Hanger, Diane P; Miller, Christopher C J

    2017-07-01

    α-Synuclein is strongly linked to Parkinson's disease but the molecular targets for its toxicity are not fully clear. However, many neuronal functions damaged in Parkinson's disease are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. This signalling involves close physical associations between the two organelles that are mediated by binding of the integral ER protein vesicle-associated membrane protein-associated protein B (VAPB) to the outer mitochondrial membrane protein, protein tyrosine phosphatase-interacting protein 51 (PTPIP51). VAPB and PTPIP51 thus act as a scaffold to tether the two organelles. Here we show that α-synuclein binds to VAPB and that overexpression of wild-type and familial Parkinson's disease mutant α-synuclein disrupt the VAPB-PTPIP51 tethers to loosen ER-mitochondria associations. This disruption to the VAPB-PTPIP51 tethers is also seen in neurons derived from induced pluripotent stem cells from familial Parkinson's disease patients harbouring pathogenic triplication of the α-synuclein gene. We also show that the α-synuclein induced loosening of ER-mitochondria contacts is accompanied by disruption to Ca 2+ exchange between the two organelles and mitochondrial ATP production. Such disruptions are likely to be particularly damaging to neurons that are heavily dependent on correct Ca 2+ signaling and ATP.

  4. Selective lowering of synapsins induced by oligomeric α-synuclein exacerbates memory deficits.

    PubMed

    Larson, Megan E; Greimel, Susan J; Amar, Fatou; LaCroix, Michael; Boyle, Gabriel; Sherman, Mathew A; Schley, Hallie; Miel, Camille; Schneider, Julie A; Kayed, Rakez; Benfenati, Fabio; Lee, Michael K; Bennett, David A; Lesné, Sylvain E

    2017-06-06

    Mounting evidence indicates that soluble oligomeric forms of amyloid proteins linked to neurodegenerative disorders, such as amyloid-β (Aβ), tau, or α-synuclein (αSyn) might be the major deleterious species for neuronal function in these diseases. Here, we found an abnormal accumulation of oligomeric αSyn species in AD brains by custom ELISA, size-exclusion chromatography, and nondenaturing/denaturing immunoblotting techniques. Importantly, the abundance of αSyn oligomers in human brain tissue correlated with cognitive impairment and reductions in synapsin expression. By overexpressing WT human αSyn in an AD mouse model, we artificially enhanced αSyn oligomerization. These bigenic mice displayed exacerbated Aβ-induced cognitive deficits and a selective decrease in synapsins. Following isolation of various soluble αSyn assemblies from transgenic mice, we found that in vitro delivery of exogenous oligomeric αSyn but not monomeric αSyn was causing a lowering in synapsin-I/II protein abundance. For a particular αSyn oligomer, these changes were either dependent or independent on endogenous αSyn expression. Finally, at a molecular level, the expression of synapsin genes SYN1 and SYN2 was down-regulated in vivo and in vitro by αSyn oligomers, which decreased two transcription factors, cAMP response element binding and Nurr1, controlling synapsin gene promoter activity. Overall, our results demonstrate that endogenous αSyn oligomers can impair memory by selectively lowering synapsin expression.

  5. t-Darpp overexpression in HER2-positive breast cancer confers a survival advantage in lapatinib.

    PubMed

    Christenson, Jessica L; Denny, Erin C; Kane, Susan E

    2015-10-20

    Drug resistance is a major barrier to successful cancer treatment. For patients with HER2-positive breast cancer who initially respond to therapy, the majority develop resistance within one year of treatment. Patient outcomes could improve significantly if we can find and exploit common mechanisms of acquired resistance to different targeted therapies. Overexpression of t-Darpp, a truncated form of the dual kinase/phosphatase inhibitor Darpp-32, has been linked to acquired resistance to trastuzumab, a front-line therapy for HER2-positive breast cancer. Darpp-32 reverses t-Darpp's effect on trastuzumab resistance. In this study, we examined whether t-Darpp could be involved in resistance to lapatinib, another HER2-targeted therapeutic. Lapatinib-resistant SKBR3 cells (SK/LapR) showed a marked change in the Darpp-32:t-Darpp ratio toward a predominance of t-Darpp. Overexpression of t-Darpp alone was not sufficient to confer lapatinib resistance, but cells that overexpress t-Darpp partially mimicked the molecular resistance phenotype observed in SK/LapR cells exposed to lapatinib. SK/LapR cells failed to down-regulate Survivin and failed to induce BIM accumulation in response to lapatinib; cells overexpressing t-Darpp exhibited only the failed BIM accumulation. t-Darpp knock-down reversed this phenotype. Using a fluorescence-based co-culture system, we found that cells overexpressing t-Darpp formed colonies in lapatinib within 3-4 weeks, whereas parental cells in the same co-culture did not. Overall, t-Darpp appears to mediate a survival advantage in lapatinib, possibly linked to failed lapatinib-induced BIM accumulation. t-Darpp might also be relevant to acquired resistance to other cancer drugs that rely on BIM accumulation to induce apoptosis.

  6. The Parkinson disease-related protein DJ-1 counteracts mitochondrial impairment induced by the tumour suppressor protein p53 by enhancing endoplasmic reticulum-mitochondria tethering.

    PubMed

    Ottolini, Denis; Calì, Tito; Negro, Alessandro; Brini, Marisa

    2013-06-01

    DJ-1 was first identified as an oncogene. More recently, mutations in its gene have been found causative for autosomal recessive familial Parkinson disease. Numerous studies support the DJ-1 role in the protection against oxidative stress and maintenance of mitochondria structure; however, the mechanism of its protective function remains largely unknown. We investigated whether mitochondrial Ca(2+) homeostasis, a key parameter in cell physiology, could be a target for DJ-1 action. Here, we show that DJ-1 modulates mitochondrial Ca(2+) transients induced upon cell stimulation with an 1,4,5-inositol-tris-phosphate agonist by favouring the endoplasmic reticulum (ER)-mitochondria tethering. A reduction of DJ-1 levels results in mitochondria fragmentation and decreased mitochondrial Ca(2+) uptake in stimulated cells. To functionally couple these effects with the well-recognized cytoprotective role of DJ-1, we investigated its action in respect to the tumour suppressor p53. p53 overexpression in HeLa cells impairs their ability to accumulate Ca(2+) in the mitochondrial matrix, causes alteration of the mitochondrial morphology and reduces ER-mitochondria contact sites. Mitochondrial impairments are independent from Drp1 activation, since the co-expression of the dominant negative mutant of Drp1 failed to abolish them. DJ-1 overexpression prevents these alterations by re-establishing the ER-mitochondria tethering. Similarly, the co-expression of the pro-fusion protein Mitofusin 2 blocks the effects induced by p53 on mitochondria, confirming that the modulation of the ER-mitochondria contact sites is critical to mitochondria integrity. Thus, the impairment of ER-mitochondria communication, as a consequence of DJ-1 loss-of-function, may be detrimental for mitochondria-related processes and be at the basis of mitochondrial dysfunction observed in Parkinson disease.

  7. Calpastatin overexpression impairs postinfarct scar healing in mice by compromising reparative immune cell recruitment and activation.

    PubMed

    Wan, Feng; Letavernier, Emmanuel; Le Saux, Claude Jourdan; Houssaini, Amal; Abid, Shariq; Czibik, Gabor; Sawaki, Daigo; Marcos, Elisabeth; Dubois-Rande, Jean-Luc; Baud, Laurent; Adnot, Serge; Derumeaux, Geneviève; Gellen, Barnabas

    2015-12-01

    The activation of the calpain system is involved in the repair process following myocardial infarction (MI). However, the impact of the inhibition of calpain by calpastatin, its natural inhibitor, on scar healing and left ventricular (LV) remodeling is elusive. Male mice ubiquitously overexpressing calpastatin (TG) and wild-type (WT) controls were subjected to an anterior coronary artery ligation. Mortality at 6 wk was higher in TG mice (24% in WT vs. 44% in TG, P < 0.05) driven by a significantly higher incidence of cardiac rupture during the first week post-MI, despite comparable infarct size and LV dysfunction and dilatation. Calpain activation post-MI was blunted in TG myocardium. In TG mice, inflammatory cell infiltration and activation were reduced in the infarct zone (IZ), particularly affecting M2 macrophages and CD4(+) T cells, which are crucial for scar healing. To elucidate the role of calpastatin overexpression in macrophages, we stimulated peritoneal macrophages obtained from TG and WT mice in vitro with IL-4, yielding an abrogated M2 polarization in TG but not in WT cells. Lymphopenic Rag1(-/-) mice receiving TG splenocytes before MI demonstrated decreased T-cell recruitment and M2 macrophage activation in the IZ day 5 after MI compared with those receiving WT splenocytes. Calpastatin overexpression prevented the activation of the calpain system after MI. It also impaired scar healing, promoted LV rupture, and increased mortality. Defective scar formation was associated with blunted CD4(+) T-cell and M2-macrophage recruitment. Copyright © 2015 the American Physiological Society.

  8. Exploring the structural diversity in inhibitors of α-synuclein amyloidogenic folding, aggregation and neurotoxicity

    NASA Astrophysics Data System (ADS)

    Das, Sukanya; Pukala, Tara L.; Smid, Scott D.

    2018-05-01

    Aggregation of α-Synuclein (αS) protein to amyloid fibrils is a neuropathological hallmark of Parkinson’s disease (PD). Growing evidence suggests that extracellular αS aggregation plays a pivotal role in neurodegeneration found in PD in addition to the intracellular αS aggregates in Lewy bodies (LB). Here, we identified and compared a diverse set of molecules capable of mitigating protein aggregation and exogenous toxicity of αSA53T, a more aggregation-prone αS mutant found in familial PD. For the first time, we investigated the αS anti-amyloid activity of semi-synthetic flavonoid 2', 3', 4' trihydroxyflavone or 2-D08, which was compared with natural flavones myricetin and transilitin, as well as such structurally diverse polyphenols as honokiol and punicalagin. Additionally, two novel synthetic compounds with a dibenzyl imidazolidine scaffold, Compound 1 and Compound 2, were also investigated as they exhibited favourable binding with αSA53T. All seven compounds inhibited αSA53T aggregation as demonstrated by Thioflavin T fluorescence assays, with modified fibril morphology observed by transmission electron microscopy. Ion mobility-mass spectrometry (IM-MS) was used to monitor the structural conversion of native αSA53T into amyloidogenic conformations and all seven compounds preserved the native unfolded conformations of αSA53T following 48 hrs incubation. The presence of each test compound in a 1:2 molar ratio was also shown to inhibit the neurotoxicity of preincubated αSA53T using phaeochromocytoma (PC12) cell viability assays. Among the seven tested compounds 2-D08, honokiol and the synthetic Compound 2 demonstrated the highest inhibition of aggregation, coupled with neuroprotection from preincubated αSA53T in vitro. Molecular docking predicted that all compounds bound near the lysine-rich region of the N-terminus of αSA53T, where the flavonoids and honokiol predominantly interacted with Lys 23. Overall, these findings highlight that i

  9. Overexpression of SIRT1 in Mouse Forebrain Impairs Lipid/Glucose Metabolism and Motor Function

    PubMed Central

    Wu, Dongmei; Qiu, Yifu; Gao, Xiang; Yuan, Xiao-Bing; Zhai, Qiwei

    2011-01-01

    SIRT1 plays crucial roles in glucose and lipid metabolism, and has various functions in different tissues including brain. The brain-specific SIRT1 knockout mice display defects in somatotropic signaling, memory and synaptic plasticity. And the female mice without SIRT1 in POMC neuron are more sensitive to diet-induced obesity. Here we created transgenic mice overexpressing SIRT1 in striatum and hippocampus under the control of CaMKIIα promoter. These mice, especially females, exhibited increased fat accumulation accompanied by significant upregulation of adipogenic genes in white adipose tissue. Glucose tolerance of the mice was also impaired with decreased Glut4 mRNA levels in muscle. Moreover, the SIRT1 overexpressing mice showed decreased energy expenditure, and concomitantly mitochondria-related genes were decreased in muscle. In addition, these mice showed unusual spontaneous physical activity pattern, decreased activity in open field and rotarod performance. Further studies demonstrated that SIRT1 deacetylated IRS-2, and upregulated phosphorylation level of IRS-2 and ERK1/2 in striatum. Meanwhile, the neurotransmitter signaling in striatum and the expression of endocrine hormones in hypothalamus and serum T3, T4 levels were altered. Taken together, our findings demonstrate that SIRT1 in forebrain regulates lipid/glucose metabolism and motor function. PMID:21738790

  10. Mitochondrial dynamics in Parkinson's disease: a role for α-synuclein?

    PubMed Central

    Pozo Devoto, Victorio M.

    2017-01-01

    ABSTRACT The distinctive pathological hallmarks of Parkinson's disease are the progressive death of dopaminergic neurons and the intracellular accumulation of Lewy bodies enriched in α-synuclein protein. Several lines of evidence from the study of sporadic, familial and pharmacologically induced forms of human Parkinson's disease also suggest that mitochondrial dysfunction plays an important role in disease progression. Although many functions have been proposed for α-synuclein, emerging data from human and animal models of Parkinson's disease highlight a role for α-synuclein in the control of neuronal mitochondrial dynamics. Here, we review the α-synuclein structural, biophysical and biochemical properties that influence relevant mitochondrial dynamic processes such as fusion-fission, transport and clearance. Drawing on current evidence, we propose that α-synuclein contributes to the mitochondrial defects that are associated with the pathology of this common and progressive neurodegenerative disease. PMID:28883016

  11. Palmitic acid induces neurotoxicity and gliatoxicity in SH-SY5Y human neuroblastoma and T98G human glioblastoma cells.

    PubMed

    Ng, Yee-Wen; Say, Yee-How

    2018-01-01

    Obesity-related central nervous system (CNS) pathologies like neuroinflammation and reactive gliosis are associated with high-fat diet (HFD) related elevation of saturated fatty acids like palmitic acid (PA) in neurons and astrocytes of the brain. Human neuroblastoma cells SH-SY5Y (as a neuronal model) and human glioblastoma cells T98G (as an astrocytic model), were treated with 100-500 µM PA, oleic acid (OA) or lauric acid (LA) for 24 h or 48 h, and their cell viability was assessed by 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of stable overexpression of γ-synuclein (γ-syn), a neuronal protein recently recognized as a novel regulator of lipid handling in adipocytes, and transient overexpression of Parkinson's disease (PD) α-synuclein [α-syn; wild-type (wt) and its pathogenic mutants A53T, A30P and E46K] in SH-SY5Y and T98G cells, were also evaluated. The effects of co-treatment of PA with paraquat (PQ), a Parkinsonian pesticide, and leptin, a hormone involved in the brain-adipose axis, were also assessed. Cell death mode and cell cycle were analyzed by Annexin V/PI flow cytometry. Reactive oxygen species (ROS) level was determined using 2',7'-dichlorofluorescien diacetate (DCFH-DA) assay and lipid peroxidation level was determined using thiobarbituric acid reactive substances (TBARS) assay. MTT assay revealed dose- and time-dependent PA cytotoxicity on SH-SY5Y and T98G cells, but not OA and LA. The cytotoxicity was significantly lower in SH-SY5Y-γ-syn cells, while transient overexpression of wt α-syn or its PD mutants (A30P and E46K, but not A53T) modestly (but still significantly) rescued the cytotoxicity of PA in SH-SY5Y and T98G cells. Co-treatment of increasing concentrations of PQ exacerbated PA's neurotoxicity. Pre-treatment of leptin, an anti-apoptotic adipokine, did not successfully rescue SH-SY5Y cells from PA-induced cytotoxicity-suggesting a mechanism of PA-induced leptin resistance. Annexin V/PI flow

  12. The p16INK4alpha/p19ARF gene mutations are infrequent and are mutually exclusive to p53 mutations in Indian oral squamous cell carcinomas.

    PubMed

    Kannan, K; Munirajan, A K; Krishnamurthy, J; Bhuvarahamurthy, V; Mohanprasad, B K; Panishankar, K H; Tsuchida, N; Shanmugam, G

    2000-03-01

    Eighty-seven untreated primary oral squamous cell carcinomas (SCCs) associated with betel quid and tobacco chewing from Indian patients were analysed for the presence of mutations in the commonly shared exon 2 of p16INK4alpha/p19ARF genes. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing analysis were used to detect mutations. SSCP analysis indicated that only 9% (8/87) of the tumours had mutation in p16INK4alpha/p19ARF genes. Seventy-two tumours studied here were previously analysed for p53 mutations and 21% (15/72) of them were found to have mutations in p53 gene. Only one tumour was found to have mutation at both p53 and p16INK4alpha/p19ARF genes. Thus, the mutation rates observed were 21% for p53, 9% for p16INK4alpha/p19ARF, and 1% for both. Sequencing analysis revealed two types of mutations; i) G to C (GCAG to CCAG) transversion type mutation at intron 1-exon 2 splice junction and ii) another C to T transition type mutation resulting in CGA to TGA changing arginine to a termination codon at p16INK4alpha gene codon 80 and the same mutation will alter codon 94 of p19ARF gene from CCG to CTG (proline to leucine). These results suggest that p16INK4alpha/p19ARF mutations are less frequent than p53 mutations in Indian oral SCCs. The p53 and p16INK4alpha/p19ARF mutational events are independent and are mutually exclusive suggesting that mutational inactivation of either p53 or p16INK4alpha/p19ARF may alleviate the need for the inactivation of the other gene.

  13. Passive Immunization Reduces Behavioral and Neuropathological Deficits in an Alpha-Synuclein Transgenic Model of Lewy Body Disease

    PubMed Central

    Masliah, Eliezer; Rockenstein, Edward; Mante, Michael; Crews, Leslie; Spencer, Brian; Adame, Anthony; Patrick, Christina; Trejo, Margarita; Ubhi, Kiren; Rohn, Troy T.; Mueller-Steiner, Sarah; Seubert, Peter; Barbour, Robin; McConlogue, Lisa; Buttini, Manuel; Games, Dora; Schenk, Dale

    2011-01-01

    Dementia with Lewy bodies (DLB) and Parkinson's Disease (PD) are common causes of motor and cognitive deficits and are associated with the abnormal accumulation of alpha-synuclein (α-syn). This study investigated whether passive immunization with a novel monoclonal α-syn antibody (9E4) against the C-terminus (CT) of α-syn was able to cross into the CNS and ameliorate the deficits associated with α-syn accumulation. In this study we demonstrate that 9E4 was effective at reducing behavioral deficits in the water maze, moreover, immunization with 9E4 reduced the accumulation of calpain-cleaved α-syn in axons and synapses and the associated neurodegenerative deficits. In vivo studies demonstrated that 9E4 traffics into the CNS, binds to cells that display α-syn accumulation and promotes α-syn clearance via the lysosomal pathway. These results suggest that passive immunization with monoclonal antibodies against the CT of α-syn may be of therapeutic relevance in patients with PD and DLB. PMID:21559417

  14. α-Synuclein in Parkinson's Disease

    PubMed Central

    Stefanis, Leonidas

    2012-01-01

    α-Synuclein is a presynaptic neuronal protein that is linked genetically and neuropathologically to Parkinson's disease (PD). α-Synuclein may contribute to PD pathogenesis in a number of ways, but it is generally thought that its aberrant soluble oligomeric conformations, termed protofibrils, are the toxic species that mediate disruption of cellular homeostasis and neuronal death, through effects on various intracellular targets, including synaptic function. Furthermore, secreted α-synuclein may exert deleterious effects on neighboring cells, including seeding of aggregation, thus possibly contributing to disease propagation. Although the extent to which α-synuclein is involved in all cases of PD is not clear, targeting the toxic functions conferred by this protein when it is dysregulated may lead to novel therapeutic strategies not only in PD, but also in other neurodegenerative conditions, termed synucleinopathies. PMID:22355802

  15. Mitochondrial dynamics in Parkinson's disease: a role for α-synuclein?

    PubMed

    Pozo Devoto, Victorio M; Falzone, Tomas L

    2017-09-01

    The distinctive pathological hallmarks of Parkinson's disease are the progressive death of dopaminergic neurons and the intracellular accumulation of Lewy bodies enriched in α-synuclein protein. Several lines of evidence from the study of sporadic, familial and pharmacologically induced forms of human Parkinson's disease also suggest that mitochondrial dysfunction plays an important role in disease progression. Although many functions have been proposed for α-synuclein, emerging data from human and animal models of Parkinson's disease highlight a role for α-synuclein in the control of neuronal mitochondrial dynamics. Here, we review the α-synuclein structural, biophysical and biochemical properties that influence relevant mitochondrial dynamic processes such as fusion-fission, transport and clearance. Drawing on current evidence, we propose that α-synuclein contributes to the mitochondrial defects that are associated with the pathology of this common and progressive neurodegenerative disease. © 2017. Published by The Company of Biologists Ltd.

  16. Overexpression of PBK/TOPK relates to tumour malignant potential and poor outcome of gastric carcinoma

    PubMed Central

    Ohashi, Takuma; Komatsu, Shuhei; Ichikawa, Daisuke; Miyamae, Mahito; Okajima, Wataru; Imamura, Taisuke; Kiuchi, Jun; Kosuga, Toshiyuki; Konishi, Hirotaka; Shiozaki, Atsushi; Fujiwara, Hitoshi; Okamoto, Kazuma; Tsuda, Hitoshi; Otsuji, Eigo

    2017-01-01

    Background: PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK) is a serine–threonine kinase and overexpressed in various types of cancer by inhibiting the transactivation activities of p53 and PTEN. We tested whether PBK/TOPK acts as a cancer-promoting gene through its activation/overexpression in gastric cancer (GC). Methods: We analysed five GC cell lines and 144 primary tumours, which were curatively resected in our hospital between 2001 and 2003. Results: Overexpression of the PBK/TOPK protein was frequently detected in GC cell lines (4 out of 5 lines, 80.0%) was detected in primary tumour samples of GC (24 out of 144 cases, 16.6%) and was significantly correlated with venous invasion, tumour depth and recurrence rate. PDZ-binding kinase/T-LAK cell-originated protein kinase-overexpressing tumours had a worse survival rate than those with non-expressing tumours (P=0.0009, log-rank test). PDZ-binding kinase/T-LAK cell-originated protein kinase positivity was independently associated with a worse outcome in multivariate analysis (P<0.0001, hazard ratio 6.40 (2.71–14.49)). In PBK/TOPK-overexpressing GC cells, knockdown of PBK/TOPK inhibited the cell proliferation through the p53 activation in a TP53 mutation-dependent manner and inhibited the migration/invasion through the PTEN upregulation in a TP53 mutation-independent manner. Conclusions: These findings suggest PBK/TOPK plays a crucial role in tumour malignant potential through its overexpression and highlight its usefulness as a prognostic factor and potential therapeutic target in GC. PMID:27898655

  17. Relationship between human papillomavirus infection and overexpression of p53 protein in cervical carcinomas and lymph node metastases.

    PubMed

    Cavuslu, S; Goodlad, J; Hobbs, C; Connor, A M; Raju, K S; Best, J M; Cason, J

    1997-10-01

    Overexpression of p53 protein is common in cervical carcinoma. We investigated archival biopsies from 26 cervical cancer patients (24 with available lymph nodes) to determine the relationship between p53 overexpression and HPV infection at the cervix and lymph nodes. Twelve cervical carcinoma patients had p53 protein in cervical biopsies detectable by immunohistochemistry using monoclonal antibody DO-1, and 22 were positive for HPV DNA in polymerase chain reaction assays (16, contained HPV-16; 3, HPV-18; and, 3 HPV-X). Seven cervical cancer patients had one or more lymph nodes positive for p53 protein, and all but one of these were concordantly p53 positive at the cervix. However, detection of p53 protein in cervical biopsies was predictive neither of the expression of p53 at draining lymph nodes (P > 0.1) nor of the occurrence of metastases (P > 0.1). Fourteen patients were positive at one or more lymph nodes for HPV DNA. Cervical positivity for HPV DNA was associated significantly with concordant HPV positivity at the lymph nodes (P = 0.039) and was predictive of metastases (P = 0.019). There was no association between positivity for p53 and for HPV DNA at primary cervical carcinomas or at the lymph nodes (all P > 0.1). We conclude that, although detectable p53 protein is a common feature of cervical carcinomas, it is not predictive of metastases and is independent of HPV infection.

  18. Low-level overexpression of p53 promotes warfarin-induced calcification of porcine aortic valve interstitial cells by activating Slug gene transcription.

    PubMed

    Gao, Li; Ji, Yue; Lu, Yan; Qiu, Ming; Shen, Yejiao; Wang, Yaqing; Kong, Xiangqing; Shao, Yongfeng; Sheng, Yanhui; Sun, Wei

    2018-03-09

    The most frequently used oral anti-coagulant warfarin has been implicated in inducing calcification of aortic valve interstitial cells (AVICs), whereas the mechanism is not fully understood. The low-level activation of p53 is found to be involved in osteogenic transdifferentiation and calcification of AVICs. Whether p53 participates in warfarin-induced AVIC calcification remains unknown. In this study, we investigated the role of low-level p53 overexpression in warfarin-induced porcine AVIC (pAVIC) calcification. Immunostaining, quantitative PCR, and Western blotting revealed that p53 was expressed in human and pAVICs and that p53 expression was slightly increased in calcific human aortic valves compared with non-calcific valves. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining indicated that apoptosis slightly increased in calcific aortic valves than in non-calcific valves. Warfarin treatment led to a low-level increase of p53 mRNA and protein in both pAVICs and mouse aortic valves. Low-level overexpression of p53 in pAVICs via an adenovirus vector did not affect pAVIC apoptosis but promoted warfarin-induced calcium deposition and expression of osteogenic markers. shRNA-mediated p53 knockdown attenuated the pAVIC calcium deposition and osteogenic marker expression. Moreover, ChIP and luciferase assays showed that p53 was recruited to the slug promoter and activated slug expression in calcific pAVICs. Of note, overexpression of Slug increased osteogenic marker Runx2 expression, but not pAVIC calcium deposition, and Slug knockdown attenuated pAVIC calcification and p53-mediated pAVIC calcium deposition and expression of osteogenic markers. In conclusion, we found that p53 plays an important role in warfarin induced pAVIC calcification, and increased slug transcription by p53 is required for p53-mediated pAVIC calcification. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Expression of alpha-AR subtypes in T lymphocytes and role of the alpha-ARs in mediating modulation of T cell function.

    PubMed

    Bao, Jing-Yin; Huang, Yan; Wang, Feng; Peng, Yu-Ping; Qiu, Yi-Hua

    2007-01-01

    Previous work in our laboratory has shown that alpha-adrenoreceptors (alpha-ARs) and beta-ARs exist on lymphocytes from functional profile, and that the receptors mediate the regulation of lymphocyte function by catecholamines. In the present study, we directly examined the expression of alpha-AR subtypes, alpha(1)-AR and alpha(2)-AR mRNAs, in T lymphocytes and explored the roles of the alpha-AR subtypes and intracellular signal transduction mechanisms linked to the receptors in mediating the modulation of T lymphocyte function. T lymphocytes from mesenteric lymph nodes of rats were purified by using a nylon wool column. Reverse transcription polymerase chain reaction was used to detect the expression of alpha(1)-AR and alpha(2)-AR mRNAs in the freshly isolated T cells and the mitogen concanavalin A (Con A)-activated lymphocytes. Colorimetric methylthiazoletetrazolium assay was employed to measure lymphocyte proliferation induced by Con A. Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) levels in the Con A-stimulated lymphocyte culture supernatants were examined by enzyme-linked immunosorbent assay. T cells expressed both alpha(1)-AR and alpha(2)-AR mRNAs. The expression of both alpha(1)-AR and alpha(2)-AR mRNAs was significantly higher in the Con A-activated lymphocytes than in the resting lymphocytes. Phenylephrine, a selective alpha(1)-AR agonist, had no evident effect on lymphocyte proliferation nor on IFN-gamma and IL-4 production induced by Con A. However, the selective alpha(2)-AR agonist clonidine attenuated Con A-induced lymphocyte proliferation as well as IFN-gamma and IL-4 production. The inhibited lymphocyte proliferation and IFN-gamma and IL-4 production by clonidine were blocked by yohimbine, an alpha(2)-AR antagonist. Either phospholipase C inhibitor U-73122 or protein kinase C inhibitor chelerythrine partially prevented the suppressive effect of clonidine on Con A-stimulated lymphocyte proliferation and IL-4 production. T lymphocytes express

  20. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bae, Song Yi; Kim, Seulgi; Hwang, Heejin

    Research highlights: {yields} Formation of the {alpha}-synuclein amyloid fibrils by [BIMbF{sub 3}Im]. {yields} Disaggregation of amyloid fibrils by epigallocatechin gallate (EGCG) and baicalein. {yields} Amyloid formation of {alpha}-synuclein tandem repeat ({alpha}-TR). -- Abstract: The aggregation of {alpha}-synuclein is clearly related to the pathogenesis of Parkinson's disease. Therefore, detailed understanding of the mechanism of fibril formation is highly valuable for the development of clinical treatment and also of the diagnostic tools. Here, we have investigated the interaction of {alpha}-synuclein with ionic liquids by using several biochemical techniques including Thioflavin T assays and transmission electron microscopy (TEM). Our data shows a rapidmore » formation of {alpha}-synuclein amyloid fibrils was stimulated by 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [BIMbF{sub 3}Im], and these fibrils could be disaggregated by polyphenols such as epigallocatechin gallate (EGCG) and baicalein. Furthermore, the effect of [BIMbF{sub 3}Im] on the {alpha}-synuclein tandem repeat ({alpha}-TR) in the aggregation process was studied.« less

  1. Common key-signals in learning and neurodegeneration: focus on excito-amino acids, beta-amyloid peptides and alpha-synuclein.

    PubMed

    Agnati, L F; Leo, G; Genedani, S; Piron, L; Rivera, A; Guidolin, D; Fuxe, K

    2009-08-01

    In this paper a hypothesis that some special signals ("key-signals" excito-amino acids, beta-amyloid peptides and alpha-synuclein) are not only involved in information handling by the neuronal circuits, but also trigger out substantial structural and/or functional changes in the Central Nervous System (CNS) is introduced. This forces the neuronal circuits to move from one stable state towards a new state, but in doing so these signals became potentially dangerous. Several mechanisms are put in action to protect neurons and glial cells from these potentially harmful signals. However, in agreement with the Red Queen Theory of Ageing (Agnati et al. in Acta Physiol Scand 145:301-309, 1992), it is proposed that during ageing these neuroprotective processes become less effective while, in the meantime, a shortage of brain plasticity occurs together with an increased need of plasticity for repairing the wear and tear of the CNS. The paper presents findings supporting the concept that such key-signals in instances such as ageing may favour neurodegenerative processes in an attempt of maximizing neuronal plasticity.

  2. β-Cell-Specific Mafk Overexpression Impairs Pancreatic Endocrine Cell Development

    PubMed Central

    Abdellatif, Ahmed M.; Oishi, Hisashi; Itagaki, Takahiro; Jung, Yunshin; Shawki, Hossam H.; Okita, Yukari; Hasegawa, Yoshikazu; Suzuki, Hiroyuki; El-Morsy, Salah E.; El-Sayed, Mesbah A.; Shoaib, Mahmoud B.; Sugiyama, Fumihiro; Takahashi, Satoru

    2016-01-01

    The MAF family transcription factors are homologs of v-Maf, the oncogenic component of the avian retrovirus AS42. They are subdivided into 2 groups, small and large MAF proteins, according to their structure, function, and molecular size. MAFK is a member of the small MAF family and acts as a dominant negative form of large MAFs. In previous research we generated transgenic mice that overexpress MAFK in order to suppress the function of large MAF proteins in pancreatic β-cells. These mice developed hyperglycemia in adulthood due to impairment of glucose-stimulated insulin secretion. The aim of the current study is to examine the effects of β-cell-specific Mafk overexpression in endocrine cell development. The developing islets of Mafk-transgenic embryos appeared to be disorganized with an inversion of total numbers of insulin+ and glucagon+ cells due to reduced β-cell proliferation. Gene expression analysis by quantitative RT-PCR revealed decreased levels of β-cell-related genes whose expressions are known to be controlled by large MAF proteins. Additionally, these changes were accompanied with a significant increase in key β-cell transcription factors likely due to compensatory mechanisms that might have been activated in response to the β-cell loss. Finally, microarray comparison of gene expression profiles between wild-type and transgenic pancreata revealed alteration of some uncharacterized genes including Pcbd1, Fam132a, Cryba2, and Npy, which might play important roles during pancreatic endocrine development. Taken together, these results suggest that Mafk overexpression impairs endocrine development through a regulation of numerous β-cell-related genes. The microarray analysis provided a unique data set of differentially expressed genes that might contribute to a better understanding of the molecular basis that governs the development and function of endocrine pancreas. PMID:26901059

  3. TNF-alpha infusion impairs corpora cavernosa reactivity.

    PubMed

    Carneiro, Fernando S; Zemse, Saiprazad; Giachini, Fernanda R C; Carneiro, Zidonia N; Lima, Victor V; Webb, R Clinton; Tostes, Rita C

    2009-03-01

    Erectile dysfunction (ED), as well as cardiovascular diseases (CVDs), is associated with endothelial dysfunction and increased levels of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha). We hypothesized that increased TNF-alpha levels impair cavernosal function. In vitro organ bath studies were used to measure cavernosal reactivity in mice infused with vehicle or TNF-alpha (220 ng/kg/min) for 14 days. Gene expression of nitric oxide synthase isoforms was evaluated by real-time polymerase chain reaction. Corpora cavernosa from TNF-alpha-infused mice exhibited decreased nitric oxide (NO)-dependent relaxation, which was associated with decreased endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) cavernosal expression. Cavernosal strips from the TNF-alpha-infused mice displayed decreased nonadrenergic-noncholinergic (NANC)-induced relaxation (59.4 +/- 6.2 vs. control: 76.2 +/- 4.7; 16 Hz) compared with the control animals. These responses were associated with decreased gene expression of eNOS and nNOS (P < 0.05). Sympathetic-mediated, as well as phenylephrine (PE)-induced, contractile responses (PE-induced contraction; 1.32 +/- 0.06 vs. control: 0.9 +/- 0.09, mN) were increased in cavernosal strips from TNF-alpha-infused mice. Additionally, infusion of TNF-alpha increased cavernosal responses to endothelin-1 and endothelin receptor A subtype (ET(A)) receptor expression (P < 0.05) and slightly decreased tumor necrosis factor-alpha receptor 1 (TNFR1) expression (P = 0.063). Corpora cavernosa from TNF-alpha-infused mice display increased contractile responses and decreased NANC nerve-mediated relaxation associated with decreased eNOS and nNOS gene expression. These changes may trigger ED and indicate that TNF-alpha plays a detrimental role in erectile function. Blockade of TNF-alpha actions may represent an alternative therapeutic approach for ED, especially in pathologic conditions associated with increased levels

  4. The lysosomal enzyme alpha-Galactosidase A is deficient in Parkinson's disease brain in association with the pathologic accumulation of alpha-synuclein.

    PubMed

    Nelson, Michael P; Boutin, Michel; Tse, Tonia E; Lu, Hailin; Haley, Emily D; Ouyang, Xiaosen; Zhang, Jianhua; Auray-Blais, Christiane; Shacka, John J

    2018-02-01

    The aberrant accumulation of alpha-synuclein (α-syn) is believed to contribute to the onset and pathogenesis of Parkinson's disease (PD). The autophagy-lysosome pathway (ALP) is responsible for the high capacity clearance of α-syn. ALP dysfunction is documented in PD and pre-clinical evidence suggests that inhibiting the ALP promotes the pathological accumulation of α-syn. We previously identified the pathological accumulation of α-syn in the brains of mice deficient for the soluble lysosomal enzyme alpha-Galactosidase A (α-Gal A), a member of the glycosphingolipid metabolism pathway. In the present study, we quantified α-Gal A activity and levels of its glycosphingolipid metabolites in postmortem temporal cortex specimens from control individuals and in PD individuals staged with respect to α-syn containing Lewy body pathology. In late-state PD temporal cortex we observed significant decreases in α-Gal A activity and the 46kDa "active" species of α-Gal A as determined respectively by fluorometric activity assay and western blot analysis. These decreases in α-Gal A activity/levels correlated significantly with increased α-syn phosphorylated at serine 129 (p129S-α-syn) that was maximal in late-stage PD temporal cortex. Mass spectrometric analysis of 29 different isoforms of globotriaosylceramide (Gb 3 ), a substrate of α-Gal A indicated no significant differences with respect to different stages of PD temporal cortex. However, significant correlations were observed between increased levels of several Gb 3 isoforms and with decreased α-Gal A activity and/or increased p129S-α-syn. Deacylated Gb 3 (globotriaosylsphingosine or lyso-Gb 3 ) was also analyzed in PD brain tissue but was below the limit of detection of 20pmol/g. Analysis of other lysosomal enzymes revealed a significant decrease in activity for the lysosomal aspartic acid protease cathepsin D but not for glucocerebrosidase (GCase) or cathepsin B in late-stage PD temporal cortex. However, a

  5. Human β-Synuclein Rendered Fibrillogenic by Designed Mutations

    PubMed Central

    Zibaee, Shahin; Fraser, Graham; Jakes, Ross; Owen, David; Serpell, Louise C.; Crowther, R. Anthony; Goedert, Michel

    2010-01-01

    Filamentous inclusions made of α-synuclein are found in nerve cells and glial cells in a number of human neurodegenerative diseases, including Parkinson disease, dementia with Lewy bodies, and multiple system atrophy. The assembly and spreading of these inclusions are likely to play an important role in the etiology of common dementias and movement disorders. Both α-synuclein and the homologous β-synuclein are abundantly expressed in the central nervous system; however, β-synuclein is not present in the pathological inclusions. Previously, we observed a poor correlation between filament formation and the presence of residues 73–83 of α-synuclein, which are absent in β-synuclein. Instead, filament formation correlated with the mean β-sheet propensity, charge, and hydrophilicity of the protein (global physicochemical properties) and β-strand contiguity calculated by a simple algorithm of sliding averages (local physicochemical property). In the present study, we rendered β-synuclein fibrillogenic via one set of point mutations engineered to enhance global properties and a second set engineered to enhance predominantly β-strand contiguity. Our findings show that the intrinsic physicochemical properties of synucleins influence their fibrillogenic propensity via two distinct but overlapping modalities. The implications for filament formation and the pathogenesis of neurodegenerative diseases are discussed. PMID:20833719

  6. Exploring the Structural Diversity in Inhibitors of α-Synuclein Amyloidogenic Folding, Aggregation, and Neurotoxicity

    PubMed Central

    Das, Sukanya; Pukala, Tara L.; Smid, Scott D.

    2018-01-01

    Aggregation of α-Synuclein (αS) protein to amyloid fibrils is a neuropathological hallmark of Parkinson's disease (PD). Growing evidence suggests that extracellular αS aggregation plays a pivotal role in neurodegeneration found in PD in addition to the intracellular αS aggregates in Lewy bodies (LB). Here, we identified and compared a diverse set of molecules capable of mitigating protein aggregation and exogenous toxicity of αSA53T, a more aggregation-prone αS mutant found in familial PD. For the first time, we investigated the αS anti-amyloid activity of semi-synthetic flavonoid 2′, 3′, 4′ trihydroxyflavone or 2-D08, which was compared with natural flavones myricetin and transilitin, as well as such structurally diverse polyphenols as honokiol and punicalagin. Additionally, two novel synthetic compounds with a dibenzyl imidazolidine scaffold, Compound 1 and Compound 2, were also investigated as they exhibited favorable binding with αSA53T. All seven compounds inhibited αSA53T aggregation as demonstrated by Thioflavin T fluorescence assays, with modified fibril morphology observed by transmission electron microscopy. Ion mobility-mass spectrometry (IM-MS) was used to monitor the structural conversion of native αSA53T into amyloidogenic conformations and all seven compounds preserved the native unfolded conformations of αSA53T following 48 h incubation. The presence of each test compound in a 1:2 molar ratio was also shown to inhibit the neurotoxicity of preincubated αSA53T using phaeochromocytoma (PC12) cell viability assays. Among the seven tested compounds 2-D08, honokiol, and the synthetic Compound 2 demonstrated the highest inhibition of aggregation, coupled with neuroprotection from preincubated αSA53T in vitro. Molecular docking predicted that all compounds bound near the lysine-rich region of the N-terminus of αSA53T, where the flavonoids and honokiol predominantly interacted with Lys 23. Overall, these findings highlight that (i

  7. Overexpression and localization of heat shock proteins mRNA in pancreatic carcinoma.

    PubMed

    Ogata, M; Naito, Z; Tanaka, S; Moriyama, Y; Asano, G

    2000-06-01

    In the present study we examined the localization and overexpression of heat shock proteins (hsps), mainly hsp90, in pancreatic carcinoma tissue compared with control tissue (including chronic pancreatitis and normal pancreas tissue), with the aid of immunohistochemical staining, in situ hybridization and reverse transcriptase polymerase chain reaction. Hsp90 alpha mRNA was overexpressed more highly in pancreatic carcinoma than in the control tissue. The proliferating-cell-nuclear-antigen labeling index was also high in pancreatic carcinoma tissue compared with the other tissue. These findings suggest that the overexpression of hsp90 alpha mRNA in carcinomas may be correlated with cell proliferation. However, hsp90 beta was constitutively overexpressed almost equally in all groups of pancreatic tissue including pancreatic carcinoma, chronic pancreatitis and normal pancreas tissue. Immunohistochemical staining demonstrated a differentiation in the expression of hsp90 between histological types of pancreatic carcinoma. These findings suggest that hsp90 alpha is involved in carcinogenesis and that hsp90 beta is correlated to structural conformation. Hsp90 alpha and hsp90 beta seem to perform different functions in tissue containing malignant cells. P53, MDM2 and WAF1, that were cell-cycle-related oncogene product were more strongly expressed in the nuclei of the cancer cells of the cancer tissue. Especially, MDM2 was more strongly expressed in mucinous carcinoma and the mucin secreting tissues surrounding pancreatic carcinoma tissue. The expression of MDM2 protein might also be correlated to secretion systems during structural conformation and be correlated to hsp90 beta.

  8. Tristetraprolin inhibits mitochondrial function through suppression of α-Synuclein expression in cancer cells

    PubMed Central

    Vo, Mai-Tram; Choi, Seong Hee; Lee, Ji-Heon; Hong, Chung Hwan; Kim, Jong Soo; Lee, Unn Hwa; Chung, Hyung-Min; Lee, Byung Ju; Park, Jeong Woo; Cho, Wha Ja

    2017-01-01

    Mitochondrial dynamics play critical roles in maintaining mitochondrial functions. Here, we report a novel mechanism for regulation of mitochondrial dynamics mediated by tristetraprolin (TTP), an AU-rich element (ARE)-binding protein. Overexpression of TTP resulted in elongated mitochondria, down-regulation of mitochondrial oxidative phosphorylation, reduced membrane potential, cytochrome c release, and increased apoptotic cell death in cancer cells. TTP overexpression inhibited the expression of α-Synuclein (α-Syn). TTP bound to the ARE within the mRNA 3′-untranslated regions (3′-UTRs) of α-Syn and enhanced the decay of α-Syn mRNA. Overexpression of α-Syn without the 3′-UTR restored TTP-induced defects in mitochondrial morphology, mitochondrial oxidative phosphorylation, membrane potential, and apoptotic cell death. Taken together, our data demonstrate that TTP acts as a regulator of mitochondrial dynamics through enhancing degradation of α-Syn mRNA in cancer cells. This finding will increase understanding of the molecular basis of mitochondrial dynamics. PMID:28410208

  9. Intact blood-brain barrier transport of small molecular drugs in animal models of amyloid beta and alpha-synuclein pathology.

    PubMed

    Gustafsson, Sofia; Lindström, Veronica; Ingelsson, Martin; Hammarlund-Udenaes, Margareta; Syvänen, Stina

    2018-01-01

    Pathophysiological impairment of the neurovascular unit, including the integrity and dynamics of the blood-brain barrier (BBB), has been denoted both a cause and consequence of neurodegenerative diseases. Pathological impact on BBB drug delivery has also been debated. The aim of the present study was to investigate BBB drug transport, by determining the unbound brain-to-plasma concentration ratio (K p,uu,brain ), in aged AβPP-transgenic mice, α-synuclein transgenic mice, and wild type mice. Mice were dosed with a cassette of five compounds, including digoxin, levofloxacin (1 mg/kg, s.c.), paliperidone, oxycodone, and diazepam (0.25 mg/kg, s.c.). Brain and blood were collected at 0.5, 1, or 3 h after dosage. Drug concentrations were measured using LC-MS/MS. The total brain-to-plasma concentration ratio was calculated and equilibrium dialysis was used to determine the fraction of unbound drug in brain and plasma for all compounds. Together, these three measures were used to determine the K p,uu,brain value. Despite Aβ or α-synuclein pathology in the current animal models, no difference was observed in the extent of drug transport across the BBB compared to wild type animals for any of the compounds investigated. Hence, the present study shows that the concept of a leaking barrier within neurodegenerative conditions has to be interpreted with caution when estimating drug transport into the brain. The capability of the highly dynamic BBB to regulate brain drug exposure still seems to be intact despite the presence of pathology. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. An alpha-synuclein MRM assay with diagnostic potential for Parkinson's disease and monitoring disease progression.

    PubMed

    Yang, Li; Stewart, Tessandra; Shi, Min; Pottiez, Gwenael; Dator, Romel; Wu, Rui; Aro, Patrick; Schuster, Robert J; Ginghina, Carmen; Pan, Catherine; Gao, Yuqian; Qian, Weijun; Zabetian, Cyrus P; Hu, Shu-Ching; Quinn, Joseph F; Zhang, Jing

    2017-07-01

    The alpha-synuclein (α-syn) level in human cerebrospinal fluid (CSF), as measured by immunoassays, is promising as a Parkinson's disease (PD) biomarker. However, the levels of total α-syn are inconsistent among studies with large cohorts and different measurement platforms. Total α-syn level also does not correlate with disease severity or progression. Here, the authors developed a highly sensitive MRM method to measure absolute CSF α-syn peptide concentrations without prior enrichment or fractionation, aiming to discover new candidate biomarkers. Six peptides covering 73% of protein sequence were reliably identified, and two were consistently quantified in cross-sectional and longitudinal cohorts. Absolute concentration of α-syn in human CSF was determined to be 2.1 ng/mL. A unique α-syn peptide, TVEGAGSIAAATGFVK (81-96), displayed excellent correlation with previous immunoassay results in two independent PD cohorts (p < 0.001), correlated with disease severity, and its changes significantly tracked the disease progression longitudinally. An MRM assay to quantify human CSF α-syn was developed and optimized. Sixty clinical samples from cross-sectional and longitudinal PD cohorts were analyzed with this approach. Although further larger scale validation is needed, the results suggest that α-syn peptide could serve as a promising biomarker in PD diagnosis and progression. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. A de novo compound targeting α-synuclein improves deficits in models of Parkinson’s disease

    PubMed Central

    Wrasidlo, Wolfgang; Tsigelny, Igor F.; Price, Diana L.; Dutta, Garima; Rockenstein, Edward; Schwarz, Thomas C.; Ledolter, Karin; Bonhaus, Douglas; Paulino, Amy; Eleuteri, Simona; Skjevik, Åge A.; Kouznetsova, Valentina L.; Spencer, Brian; Desplats, Paula; Gonzalez-Ruelas, Tania; Trejo-Morales, Margarita; Overk, Cassia R.; Winter, Stefan; Zhu, Chunni; Chesselet, Marie-Francoise; Meier, Dieter; Moessler, Herbert; Konrat, Robert; Masliah, Eliezer

    2016-01-01

    Abnormal accumulation and propagation of the neuronal protein α-synuclein has been hypothesized to underlie the pathogenesis of Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy. Here we report a de novo-developed compound (NPT100-18A) that reduces α-synuclein toxicity through a novel mechanism that involves displacing α-synuclein from the membrane. This compound interacts with a domain in the C-terminus of α-synuclein. The E83R mutation reduces the compound interaction with the 80–90 amino acid region of α-synuclein and prevents the effects of NPT100-18A. In vitro studies showed that NPT100-18A reduced the formation of wild-type α-synuclein oligomers in membranes, reduced the neuronal accumulation of α-synuclein, and decreased markers of cell toxicity. In vivo studies were conducted in three different α-synuclein transgenic rodent models. Treatment with NPT100-18A ameliorated motor deficits in mThy1 wild-type α-synuclein transgenic mice in a dose-dependent manner at two independent institutions. Neuropathological examination showed that NPT100-18A decreased the accumulation of proteinase K-resistant α-synuclein aggregates in the CNS and was accompanied by the normalization of neuronal and inflammatory markers. These results were confirmed in a mutant line of α-synuclein transgenic mice that is prone to generate oligomers. In vivo imaging studies of α-synuclein-GFP transgenic mice using two-photon microscopy showed that NPT100-18A reduced the cortical synaptic accumulation of α-synuclein within 1 h post-administration. Taken together, these studies support the notion that altering the interaction of α-synuclein with the membrane might be a feasible therapeutic approach for developing new disease-modifying treatments of Parkinson’s disease and other synucleinopathies. PMID:27679481

  12. An alpha-synuclein MRM assay with diagnostic potential for Parkinson's disease and monitoring disease progression

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yang, Li; Stewart, Tessandra; Shi, Min

    Aim: The alpha-synuclein (α-syn) level in human cerebrospinal fluid (CSF), as measured by immunoassays, is promising as a Parkinson’s disease (PD) biomarker. However, the levels of total α-syn are inconsistent among studies with large cohorts and different measurement platforms. Total α-syn level also does not correlate with disease severity or progression. Here, we developed a highly sensitive Multiple Reaction Monitoring (MRM) method to measure absolute CSF α-syn peptide concentrations without prior enrichment or fractionation, aiming to discover new candidate biomarkers. Results: Six peptides covering 73% of protein sequence were reliably identified, and two were consistently quantified in cross-sectional and longitudinalmore » cohorts. Absolute concentration of α-syn in human CSF was determined to be 2.1ng/mL. A unique α-syn peptide, TVEGAGSIAAATGFVK (81-96), displayed excellent correlation with previous immunoassay results in two independent PD cohorts (p < 0.001), correlated with disease severity, and its changes significantly tracked the disease progression longitudinally. Conclusions: An MRM assay to quantify human CSF α-syn was developed and optimized. Sixty clinical samples from cross-sectional and longitudinal PD cohorts were analyzed with this approach. Although further larger-scale validation is needed, the results suggest that α-syn peptide could serve as a promising biomarker in PD diagnosis and progression.« less

  13. Inhibition of Protein Ubiquitination by Paraquat and 1-Methyl-4-Phenylpyridinium Impairs Ubiquitin-Dependent Protein Degradation Pathways.

    PubMed

    Navarro-Yepes, Juliana; Anandhan, Annadurai; Bradley, Erin; Bohovych, Iryna; Yarabe, Bo; de Jong, Annemieke; Ovaa, Huib; Zhou, You; Khalimonchuk, Oleh; Quintanilla-Vega, Betzabet; Franco, Rodrigo

    2016-10-01

    Intracytoplasmic inclusions of protein aggregates in dopaminergic cells (Lewy bodies) are the pathological hallmark of Parkinson's disease (PD). Ubiquitin (Ub), alpha (α)-synuclein, p62/sequestosome 1, and oxidized proteins are the major components of Lewy bodies. However, the mechanisms involved in the impairment of misfolded/oxidized protein degradation pathways in PD are still unclear. PD is linked to mitochondrial dysfunction and environmental pesticide exposure. In this work, we evaluated the effects of the pesticide paraquat (PQ) and the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP(+)) on Ub-dependent protein degradation pathways. No increase in the accumulation of Ub-bound proteins or aggregates was observed in dopaminergic cells (SK-N-SH) treated with PQ or MPP(+), or in mice chronically exposed to PQ. PQ decreased Ub protein content, but not its mRNA transcription. Protein synthesis inhibition with cycloheximide depleted Ub levels and potentiated PQ-induced cell death. The inhibition of proteasomal activity by PQ was found to be a late event in cell death progression and had neither effect on the toxicity of either MPP(+) or PQ, nor on the accumulation of oxidized sulfenylated, sulfonylated (DJ-1/PARK7 and peroxiredoxins), and carbonylated proteins induced by PQ. PQ- and MPP(+)-induced Ub protein depletion prompted the dimerization/inactivation of the Ub-binding protein p62 that regulates the clearance of ubiquitinated proteins by autophagy. We confirmed that PQ and MPP(+) impaired autophagy flux and that the blockage of autophagy by the overexpression of a dominant-negative form of the autophagy protein 5 (dnAtg5) stimulated their toxicity, but there was no additional effect upon inhibition of the proteasome. PQ induced an increase in the accumulation of α-synuclein in dopaminergic cells and membrane-associated foci in yeast cells. Our results demonstrate that the inhibition of protein ubiquitination by PQ and MPP(+) is involved in the

  14. Inhibition of protein ubiquitination by paraquat and 1-methyl-4-phenylpyridinium impairs ubiquitin-dependent protein degradation pathways

    PubMed Central

    Navarro-Yepes, Juliana; Anandhan, Annadurai; Bradley, Erin; Bohovych, Iryna; Yarabe, Bo; de Jong, Annemieke; Ovaa, Huib; Zhou, You; Khalimonchuk, Oleh; Quintanilla-Vega, Betzabet; Franco, Rodrigo

    2016-01-01

    Intracytoplasmic inclusions of protein aggregates in dopaminergic cells (Lewy bodies) are the pathological hallmark of Parkinson’s disease (PD). Ubiquitin (Ub), alpha [α]-synuclein, p62/sequestosome 1 and oxidized proteins are major components of Lewy bodies. However, the mechanisms involved in the impairment of misfolded/oxidized protein degradation pathways in PD are still unclear. PD is linked to mitochondrial dysfunction and environmental pesticide exposure. In this work, we evaluated the effect of the pesticide paraquat (PQ) and the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+) on Ub-dependent protein degradation pathways. No increase in the accumulation of Ub-bound proteins or aggregates was observed in dopaminergic cells (SK-N-SH) treated with PQ or MPP+, or in mice chronically exposed to PQ. PQ decreased Ub protein content, but not its mRNA transcription. Protein synthesis inhibition with cycloheximide depleted Ub levels and potentiated PQ–induced cell death. Inhibition of proteasomal activity by PQ was found to be a late event in cell death progression, and had no effect on either the toxicity of MPP+ or PQ, or the accumulation of oxidized sulfenylated, sulfonylated (DJ-1/PARK7 and peroxiredoxins) and carbonylated proteins induced by PQ. PQ- and MPP+-induced Ub protein depletion prompted the dimerization/inactivation of the Ub-binding protein p62 that regulates the clearance of ubiquitinated proteins by autophagic. We confirmed that PQ and MPP+ impaired autophagy flux, and that the blockage of autophagy by the overexpression of a dominant-negative form of the autophagy protein 5 (dnAtg5) stimulated their toxicity, but there was no additional effect upon inhibition of the proteasome. PQ induced an increase in the accumulation of α-synuclein in dopaminergic cells and membrane associated foci in yeast cells. Our results demonstrate that inhibition of protein ubiquitination by PQ and MPP+ is involved in the dysfunction of Ub-dependent protein

  15. Estrogen receptor alpha deletion enhances the metastatic phenotype of Ron overexpressing mammary tumors in mice

    PubMed Central

    2012-01-01

    Background The receptor tyrosine kinase family includes many transmembrane proteins with diverse physiological and pathophysiological functions. The involvement of tyrosine kinase signaling in promoting a more aggressive tumor phenotype within the context of chemotherapeutic evasion is gaining recognition. The Ron receptor is a tyrosine kinase receptor that has been implicated in the progression of breast cancer and evasion of tamoxifen therapy. Results Here, we report that Ron expression is correlated with in situ, estrogen receptor alpha (ERα)-positive tumors, and is higher in breast tumors following neoadjuvant tamoxifen therapy. We also demonstrate that the majority of mammary tumors isolated from transgenic mice with mammary specific-Ron overexpression (MMTV-Ron mice), exhibit appreciable ER expression. Moreover, genetic-ablation of ERα, in the context of Ron overexpression, leads to delayed mammary tumor initiation and growth, but also results in an increased metastasis. Conclusions Ron receptor overexpression is associated with ERα-positive human and murine breast tumors. In addition, loss of ERα on a Ron overexpressing background in mice leads to the development of breast tumors which grow slower but which exhibit more metastasis and suggests that targeting of ERα, as in the case of tamoxifen therapy, may reduce the growth of Ron overexpressing breast cancers but may cause these tumors to be more metastatic. PMID:22226043

  16. Over-expression of two different forms of the alpha-secretase ADAM10 affects learning and memory in mice.

    PubMed

    Schmitt, Ulrich; Hiemke, Christoph; Fahrenholz, Falk; Schroeder, Anja

    2006-12-15

    Members of the ADAM family (adisintegrin and metalloprotease) are the main candidates for physiologically relevant alpha-secretases. The alpha-secretase cleaves in the non-amyloidogenic pathway the amyloid precursor protein within the region of the Abeta peptides preventing their aggregation in the brain. The increase of alpha-secretase activity in the brain provides a plausible strategy to prevent Abeta formation. Concerning this possibility two transgenic mouse lines (FVB/N) have been created: mice over-expressing the bovine form of the alpha-secretase (ADAM10) and mice over-expressing an inactive form of the alpha-secretase (ADAM10-E348A-HA; ADAM10-dn). For behavioral examination a F1 generation of transgenic mice (C57Bl/6 x FVB/N (tg)) was generated and compared to wild type F1 generation (C57Bl/6 x FVB/N). Behavior was characterized in the following tasks: standard open field, enriched open field, elevated plus-maze, and the Morris water maze hidden platform task. Concerning basal activity, exploration, and anxiety, transgenic mice behaved similar to controls. With respect to learning and memory both transgenic lines showed a significant deficit compared to controls. ADAM10 mice however, showed thigmotaxis with passive floating behavior in the Morris water maze indicating differences in motivation, whereas, ADAM10-dn mice displayed an inconspicuous but limited goal-directed search pattern. Thus variation of the enzymatic activity of alpha-secretase ADAM10 alters learning and memory differentially. Nevertheless, it could be concluded that both, ADAM10 and ADAM10-dn mice are suitable control mice for the assessment of alpha-secretase-related effects in animal models of Alzheimer's disease.

  17. 14 CFR 382.53 - What information must carriers give individuals with a vision or hearing impairment at airports?

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false What information must carriers give individuals with a vision or hearing impairment at airports? 382.53 Section 382.53 Aeronautics and Space... information must carriers give individuals with a vision or hearing impairment at airports? (a)(1) As a U.S...

  18. 14 CFR 382.53 - What information must carriers give individuals with a vision or hearing impairment at airports?

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false What information must carriers give individuals with a vision or hearing impairment at airports? 382.53 Section 382.53 Aeronautics and Space... information must carriers give individuals with a vision or hearing impairment at airports? (a)(1) As a U.S...

  19. Apolipoprotein Eε4: A Biomarker for Executive Dysfunction among Parkinson's Disease Patients with Mild Cognitive Impairment.

    PubMed

    Samat, Nor A; Abdul Murad, Nor A; Mohamad, Khairiyah; Abdul Razak, Mohd R; Mohamed Ibrahim, Norlinah

    2017-01-01

    Background: Cognitive impairment is prevalent in Parkinson's disease (PD), affecting 15-20% of patients at diagnosis. α-synuclein expression and genetic polymorphisms of Apolipoprotein E ( ApoE ) have been associated with the presence of cognitive impairment in PD although data have been inconsistent. Objectives: To determine the prevalence of cognitive impairment in patients with PD using Montreal Cognitive Assessment (MoCA), Comprehensive Trail Making Test (CTMT) and Parkinson's disease-cognitive rating scale (PDCRS), and its association with plasma α-synuclein and ApoE genetic polymorphisms. Methods: This was across-sectional study involving 46 PD patients. Patients were evaluated using Montreal cognitive assessment test (MoCA), and detailed neuropsychological tests. The Parkinson's disease cognitive rating scale (PDCRS) was used for cognitive function and comprehensive trail making test (CTMT) for executive function. Blood was drawn for plasma α-synuclein measurements and ApoE genetic analysis. ApoE polymorphism was detected using MutaGEL APoE from ImmunDiagnostik. Plasma α-synuclein was detected using the ELISA Technique (USCN Life Science Inc.) according to the standard protocol. Results: Based on MoCA, 26 (56.5%) patients had mild cognitive impairment (PD-MCI) and 20 (43.5%) had normal cognition (PD-NC). Based on the PDCRS, 18 (39.1%) had normal cognition (PDCRS-NC), 17 (37%) had mild cognitive impairment (PDCRS-MCI), and 11 (23.9%) had dementia (PDCRS-PDD). In the PDCRS-MCI group, 5 (25%) patients were from PD-NC group and all PDCRS-PDD patients were from PD-MCI group. CTMT scores were significantly different between patients with MCI and normal cognition on MoCA ( p = 0.003). Twenty one patients (72.4%) with executive dysfunction were from the PD-MCI group; 17 (77.3%) with severe executive dysfunction and 4 (57.1%) had mild to moderate executive dysfunction. There were no differences in the plasma α-synuclein concentration between the presence or

  20. Potential Modes of Intercellular α-Synuclein Transmission

    PubMed Central

    Valdinocci, Dario; Radford, Rowan A. W.; Siow, Sue Maye; Chung, Roger S.; Pountney, Dean L.

    2017-01-01

    Intracellular aggregates of the α-synuclein protein result in cell loss and dysfunction in Parkinson’s disease and atypical Parkinsonism, such as multiple system atrophy and dementia with Lewy bodies. Each of these neurodegenerative conditions, known collectively as α-synucleinopathies, may be characterized by a different suite of molecular triggers that initiate pathogenesis. The mechanisms whereby α-synuclein aggregates mediate cytotoxicity also remain to be fully elucidated. However, recent studies have implicated the cell-to-cell spread of α-synuclein as the major mode of disease propagation between brain regions during disease progression. Here, we review the current evidence for different modes of α-synuclein cellular release, movement and uptake, including exocytosis, exosomes, tunneling nanotubes, glymphatic flow and endocytosis. A more detailed understanding of the major modes by which α-synuclein pathology spreads throughout the brain may provide new targets for therapies that halt the progression of disease. PMID:28241427

  1. Potential Modes of Intercellular α-Synuclein Transmission.

    PubMed

    Valdinocci, Dario; Radford, Rowan A W; Siow, Sue Maye; Chung, Roger S; Pountney, Dean L

    2017-02-22

    Intracellular aggregates of the α-synuclein protein result in cell loss and dysfunction in Parkinson's disease and atypical Parkinsonism, such as multiple system atrophy and dementia with Lewy bodies. Each of these neurodegenerative conditions, known collectively as α-synucleinopathies, may be characterized by a different suite of molecular triggers that initiate pathogenesis. The mechanisms whereby α-synuclein aggregates mediate cytotoxicity also remain to be fully elucidated. However, recent studies have implicated the cell-to-cell spread of α-synuclein as the major mode of disease propagation between brain regions during disease progression. Here, we review the current evidence for different modes of α-synuclein cellular release, movement and uptake, including exocytosis, exosomes, tunneling nanotubes, glymphatic flow and endocytosis. A more detailed understanding of the major modes by which α-synuclein pathology spreads throughout the brain may provide new targets for therapies that halt the progression of disease.

  2. Two major pathways of penile carcinogenesis: HPV-induced penile cancers overexpress p16ink4a, HPV-negative cancers associated with dermatoses express p53, but lack p16ink4a overexpression.

    PubMed

    Mannweiler, Sebastian; Sygulla, Stephan; Winter, Elke; Regauer, Sigrid

    2013-07-01

    Penile squamous cell carcinomas (SCC) arise either through transforming infections with human papillomavirus (HPV) or independent of HPV, often in the background of lichen sclerosus (LS) and lichen planus (LP). Despite impact on therapy and prognosis, etiologic stratifications are missing in most histological diagnoses and publications about penile cancers/precursors. Classification of penile lesions into HPV-induced or HPV-negative via immunohistochemical demonstration of p16(ink4a) overexpression, a surrogate marker for transforming HPV-high-risk infections, and p53 expression in the absence of p16(ink4a) overexpression. Archival formalin-fixed material of 123 invasive penile cancers and 43 pre-invasive lesions was evaluated for the presence of LS, LP, 28 HPV genotypes, and expression of p53 and p16(ink4a). Seventy-two of 123 SCCs and 33 of 43 pre-invasive lesions showed p16(ink4a) overexpression independent of HPV-HR genotypes involved; 66 of 72 SCCs and 29 of 43 precursor lesions revealed a single HPV-high-risk-genotype (HPV-HR16 in 76% followed by HPV33, HPV31, HPV45, HPV18, HPV56); 5 of 72 SCCs and 4 of 43 precursor lesions revealed multiple HPV-HR-genotypes. One SCC revealed HPV-LR and HR-DNA. Fifty-one of 123 SCCs and 10 precursor lesions were p16(ink4a) negative, but showed nuclear p53 expression in tumor cells and basal keratinocytes. Forty-nine of 51 SCCs and 10 of 10 precursor lesions lacked HPV DNA. Two of 51 SCCs contained HPV18 and HPV45 DNA, respectively, but p16(ink4a) negativity classified them as non-HPV-induced. Twenty-seven of 51 SCCs showed peritumoral LS, 13 of 51 SCCs showed peritumoral LP, and 11 SCCs revealed no peritumoral tissue. Histologically, HPV-negative precursors showed hyperkeratotic, verrucous, atrophic, and basaloid differentiation. This was a retrospective study. p16(ink4a) overexpression identifies HPV-HR-induced penile carcinogenesis independent of HPV-HR genotype. p53 expression along with p16(ink4a) negativity identifies

  3. VPS35 in Dopamine Neurons Is Required for Endosome-to-Golgi Retrieval of Lamp2a, a Receptor of Chaperone-Mediated Autophagy That Is Critical for α-Synuclein Degradation and Prevention of Pathogenesis of Parkinson's Disease

    PubMed Central

    Tang, Fu-Lei; Erion, Joanna R.; Tian, Yun; Liu, Wei; Yin, Dong-Min; Ye, Jian; Tang, Baisha; Mei, Lin

    2015-01-01

    Vacuolar protein sorting-35 (VPS35) is essential for endosome-to-Golgi retrieval of membrane proteins. Mutations in the VPS35 gene have been identified in patients with autosomal dominant PD. However, it remains poorly understood if and how VPS35 deficiency or mutation contributes to PD pathogenesis. Here we provide evidence that links VPS35 deficiency to PD-like neuropathology. VPS35 was expressed in mouse dopamine (DA) neurons in substantia nigra pars compacta (SNpc) and STR (striatum)—regions that are PD vulnerable. VPS35-deficient mice exhibited PD-relevant deficits including accumulation of α-synuclein in SNpc-DA neurons, loss of DA transmitter and DA neurons in SNpc and STR, and impairment of locomotor behavior. Further mechanical studies showed that VPS35-deficient DA neurons or DA neurons expressing PD-linked VPS35 mutant (D620N) had impaired endosome-to-Golgi retrieval of lysosome-associated membrane glycoprotein 2a (Lamp2a) and accelerated Lamp2a degradation. Expression of Lamp2a in VPS35-deficient DA neurons reduced α-synuclein, supporting the view for Lamp2a as a receptor of chaperone-mediated autophagy to be critical for α-synuclein degradation. These results suggest that VPS35 deficiency or mutation promotes PD pathogenesis and reveals a crucial pathway, VPS35-Lamp2a-α-synuclein, to prevent PD pathogenesis. SIGNIFICANCE STATEMENT VPS35 is a key component of the retromer complex that is essential for endosome-to-Golgi retrieval of membrane proteins. Mutations in the VPS35 gene have been identified in patients with PD. However, if and how VPS35 deficiency or mutation contributes to PD pathogenesis remains unclear. We demonstrated that VPS35 deficiency or mutation (D620N) in mice leads to α-synuclein accumulation and aggregation in the substantia nigra, accompanied with DA neurodegeneration. VPS35-deficient DA neurons exhibit impaired endosome-to-Golgi retrieval of Lamp2a, which may contribute to the reduced α-synuclein degradation through

  4. Agrochemicals, α-synuclein, and Parkinson's disease.

    PubMed

    Silva, Blanca A; Breydo, Leonid; Fink, Anthony L; Uversky, Vladimir N

    2013-04-01

    Epidemiological, population-based case-control, and experimental studies at the molecular, cellular, and organism levels revealed that exposure to various environmental agents, including a number of structurally different agrochemicals, may contribute to the pathogenesis of Parkinson's disease (PD) and several other neurodegenerative disorders. The role of genetic predisposition in PD has also been increasingly acknowledged, driven by the identification of a number of disease-related genes [e.g., α-synuclein, parkin, DJ-1, ubiquitin C-terminal hydrolase isozyme L1 (UCH-L1), and nuclear receptor-related factor 1]. Therefore, the etiology of this multifactorial disease is likely to involve both genetic and environmental factors. Various neurotoxicants, including agrochemicals, have been shown to elevate the levels of α-synuclein expression in neurons and to promote aggregation of this protein in vivo. Many agrochemicals physically interact with α-synuclein and accelerate the fibrillation and aggregation rates of this protein in vitro. This review analyzes some of the aspects linking α-synuclein to PD, provides brief structural and functional descriptions of this important protein, and represents some data connecting exposure to agrochemicals with α-synuclein aggregation and PD pathogenesis.

  5. Astrocyte-specific DJ-1 overexpression protects against rotenone-induced neurotoxicity in a rat model of Parkinson's disease.

    PubMed

    De Miranda, Briana R; Rocha, Emily M; Bai, Qing; El Ayadi, Amina; Hinkle, David; Burton, Edward A; Timothy Greenamyre, J

    2018-07-01

    DJ-1 is a redox-sensitive protein with several putative functions important in mitochondrial physiology, protein transcription, proteasome regulation, and chaperone activity. High levels of DJ-1 immunoreactivity are reported in astrocytes surrounding pathology associated with idiopathic Parkinson's disease, possibly reflecting the glial response to oxidative damage. Previous studies showed that astrocytic over-expression of DJ-1 in vitro prevented oxidative stress and mitochondrial dysfunction in primary neurons. Based on these observations, we developed a pseudotyped lentiviral gene transfer vector with specific tropism for CNS astrocytes in vivo to overexpress human DJ-1 protein in astroglial cells. Following vector delivery to the substantia nigra and striatum of adult Lewis rats, the DJ-1 transgene was expressed robustly and specifically within astrocytes. There was no observable transgene expression in neurons or other glial cell types. Three weeks after vector infusion, animals were exposed to rotenone to induce Parkinson's disease-like pathology, including loss of dopaminergic neurons, accumulation of endogenous α-synuclein, and neuroinflammation. Animals over-expressing hDJ-1 in astrocytes were protected from rotenone-induced neurodegeneration, and displayed a marked reduction in neuronal oxidative stress and microglial activation. In addition, α-synuclein accumulation and phosphorylation were decreased within substantia nigra dopaminergic neurons in DJ-1-transduced animals, and expression of LAMP-2A, a marker of chaperone mediated autophagy, was increased. Together, these data indicate that astrocyte-specific overexpression of hDJ-1 protects neighboring neurons against multiple pathologic features of Parkinson's disease and provides the first direct evidence in vivo of a cell non-autonomous neuroprotective function of astroglial DJ-1. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Interaction between subclinical doses of the Parkinson's disease associated gene, α-synuclein, and the pesticide, rotenone, precipitates motor dysfunction and nigrostriatal neurodegeneration in rats.

    PubMed

    Naughton, Carol; O'Toole, Daniel; Kirik, Deniz; Dowd, Eilís

    2017-01-01

    In most patients, Parkinson's disease is thought to emerge after a lifetime of exposure to, and interaction between, various genetic and environmental risk factors. One of the key genetic factors linked to this condition is α-synuclein, and the α-synuclein protein is pathologically associated with idiopathic cases. However, α-synuclein pathology is also present in presymptomatic, clinically "normal" individuals suggesting that environmental factors, such as Parkinson's disease-linked agricultural pesticides, may be required to precipitate Parkinson's disease in these individuals. In this context, the aim of this study was to assess the behavioural and neuropathological impact of exposing rats with a subclinical load of α-synuclein to subclinical doses of the organic pesticide, rotenone. Rats were randomly assigned to two groups for intra-nigral infusion of AAV 2/5- GFP or AAV 2/5 -α-synuclein. Post viral motor function was assessed at 8, 10 and 12 weeks in the Corridor, Stepping and Whisker tests of lateralised motor function. At week 12, animals were performance-matched to receive a subsequent intra-striatal challenge of the organic pesticide rotenone (or its vehicle) to yield four final groups (Control, Rotenone, AAV 2/5 -α-synuclein and Combined). Behavioural testing resumed one week after rotenone surgery and continued for 5 weeks. We found that, when administered alone, neither intra-nigral AAV-α-synuclein nor intra-striatal rotenone caused sufficient nigrostriatal neurodegeneration to induce a significant motor impairment in their own right. However, when these were administered sequentially to the same rats, the interaction between the two Parkinsonian challenges significantly exacerbated nigrostriatal neurodegeneration which precipitated a pronounced impairment in motor function. These results indicate that exposing rats with a subclinical α-synuclein-induced pathology to the pesticide, rotenone, profoundly exacerbates their Parkinsonian

  7. Adenovirally mediated p53 overexpression diversely influence the cell cycle of HEp-2 and CAL 27 cell lines upon cisplatin and methotrexate treatment.

    PubMed

    Kraljević Pavelić, Sandra; Marjanović, Marko; Poznić, Miroslav; Kralj, Marijeta

    2009-12-01

    p53 gene plays a crucial role in the response to therapy. Since it is inactivated in the majority of human cancers, it is strongly believed that the p53 mutations confer resistance to therapeutics. In this paper we analyzed the influence of two mechanistically diverse antitumor agents--cisplatin and methotrexate on the proliferation and cell cycle of two head and neck squamous cancer cell lines HEp-2 (wild type p53 gene, but HPV 18/E6-inactivated protein) and CAL 27 (mutated p53 gene), along with the influence of adenovirally mediated p53 overexpression in modulation of cisplatin and methoterexate effects, whereby subtoxic vector/compound concentrations were employed. p53 gene was introduced into tumor cells using adenoviral vector (AdCMV-p53). The cell cycle perturbations were measured by two parameter flow cytometry. The expression of p53, p21(WAF1/CIP1) and cyclin B1 proteins was examined using immunocytochemistry and western blot methods. In CAL 27 cells overexpression of p53 completely abrogated high S phase content observed in methotrexate-treated cells into a G1 and slight G2 arrest, while it sustained G2 arrest of the cells treated with cisplatin, along with the reduction of DNA synthesis and cyclin B1 expression. On the other hand, in HEp-2 cell line p53 overexpression slightly slowed down the progression through S phase in cells treated with methotrexate, decreased the cyclin B1 expression only after 24 h, and failed to sustain the G2 arrest after treatment with cisplatin alone. Instead, it increased the population of S phase cells that were not actively synthesizing DNA, sustained cyclin B1 expression and allowed the G2 cells to progress through mitosis. This study demonstrates that adenovirally mediated p53 overexpression at sub-cytotoxic levels enhanced the activity of low doses of cisplatin and methotrexate in HEp-2 and CAL 27 cells through changes in the cell cycle. However, the mechanisms of these effects differ depending on the genetic context and

  8. 46 CFR 32.53-10 - General-T/ALL.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false General-T/ALL. 32.53-10 Section 32.53-10 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS SPECIAL EQUIPMENT, MACHINERY, AND HULL REQUIREMENTS Inert Gas System § 32.53-10 General—T/ALL. (a) Each tankship to which this subpart applies must have an...

  9. 46 CFR 32.53-10 - General-T/ALL.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 1 2011-10-01 2011-10-01 false General-T/ALL. 32.53-10 Section 32.53-10 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS SPECIAL EQUIPMENT, MACHINERY, AND HULL REQUIREMENTS Inert Gas System § 32.53-10 General—T/ALL. (a) Each tankship to which this subpart applies must have an...

  10. Cytokine dysregulation in AIDS: in vivo overexpression of mRNA of tumor necrosis factor-alpha and its correlation with that of the inflammatory cytokine GRO.

    PubMed

    Dezube, B J; Pardee, A B; Beckett, L A; Ahlers, C M; Ecto, L; Allen-Ryan, J; Anisowicz, A; Sager, R; Crumpacker, C S

    1992-01-01

    The human immunodeficiency virus establishes an intimate interaction with the immune system. The virus can use cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (Il-1), to regulate its own expression by modifying the normal immunoregulatory network. We demonstrate that mRNA of the cytokine TNF-alpha from peripheral blood mononuclear cells is overexpressed in virtually all patients with AIDS who do not have active opportunistic infections compared with uninfected volunteers (p < 0.0001). This overexpression correlates with elevated mRNA levels of the recently discovered GRO (p < 0.05), a cytokine involved in the inflammatory response.

  11. Nigrostriatal neuronal death following chronic dichlorvos exposure: crosstalk between mitochondrial impairments, α synuclein aggregation, oxidative damage and behavioral changes

    PubMed Central

    2010-01-01

    Background In recent years, several lines of evidence have shown an increase in Parkinson's disease prevalence in rural environments where pesticides are heavily used. Although, the underlying mechanism for neuronal degeneration in sporadic PD remains unknown, mitochondrial dysfunction, oxidative stress and proteasomal dysfunction are proposed as contributing factors. In this study rats were chronically and continuously exposed to the pesticide, dichlorvos to identify the molecular mechanism of nigrostaital neuronal degeneration. Result Chronic dichlorvos exposure (2.50 mg/kg b.wt.s.c/daily for 12 weeks) caused nigrostriatal dopaminergic degeneration. The degenerative changes were accompanied by a loss of 60-80% of the nigral dopamine neurons and 60-70% reduction in striatal dopamine and tyrosine hydroxylase levels. Dichlorvos exposed animals also showed α -synuclein and ubiquitin positive inclusions along with swollen, dystrophic neurites and mitochondrial abnormalities like decreased complex I&IV activities, increased mitochondrial size, axonal degeneration and presence of electron dense perinuclear cytoplasmic inclusions in the substantia nigra of rats. These animals also showed evidence of oxidative stress, including increased mitochondrial ROS levels, decreased MnSOD activity and increased lipid peroxidation. Measurable impairments in neurobehavioral indices were also observed. Notable exacerbations in motor impairments, open field and catalepsy were also evident in dichlorvos exposed animals. Conclusion All these findings taken together indicate that chronic dichlorvos exposure may cause nigrostaital neurodegenaration and significant behavioral impairments. PMID:21073741

  12. Potential role of alpha-synuclein and metallothionein in lead-induced inclusion body formation.

    PubMed

    Zuo, Peijun; Qu, Wei; Cooper, Ryan N; Goyer, Robert A; Diwan, Bhalchandra A; Waalkes, Michael P

    2009-09-01

    Lead (Pb) produces aggresome-like inclusion bodies (IBs) in target cells as a toxic response. Our prior work shows metallothionein (MT) is required for this process. We used MT-I/II double knockout (MT-null) and parental wild-type (WT) cell lines to further explore the formation process of Pb-induced IBs. Unlike WT cells, MT-null cells did not form IBs after Pb exposure. Western blot of cytosol showed soluble MT protein in WT cells was lost during Pb exposure as IBs formed. Transfection of MT-I into MT-null cells allowed IBs formation after Pb exposure. Considering Pb-induced IBs may be like disease-related aggresomes, which often contain alpha-synuclein (Scna), we investigated Scna expression in cells capable (WT) and incapable (MT-null) of producing IBs after Pb exposure. Scna protein showed poor basal expression in MT-null cells. Pb exposure increased Scna expression only in WT cells. MT transfection increased Scna transcript to WT levels. In WT or MT-transfected MT-null cells, Pb-induced Scna expression rapidly increased and then decreased over 48 h as Pb-induced IBs were formed. A direct interaction between Scna and MT was confirmed ex vivo by antibody pulldown assay where the proteins coprecipitated with an antibody to MT. Pb exposure caused increased colocalization of MT and Scna proteins with time only in WT cells. In WT mice after chronic Pb exposure Scna was localized in renal cells containing forming IBs, whereas MT-null mice did not form IBs. Thus, Scna could be component of Pb-induced IBs and, with MT, may play a role in IBs formation.

  13. Enriching the Environment of [alpha]CaMKII[superscript T286A] Mutant Mice Reveals that LTD Occurs in Memory Processing but Must be Subsequently Reversed by LTP

    ERIC Educational Resources Information Center

    Soto, Florentina; Giese, K. Peter; Edwards, Frances A.; Parsley, Stephanie L.; Pilgram, Sara M.

    2007-01-01

    [alpha]CaMKII[superscript T286A] mutant mice lack long-term potentiation (LTP) in the hippocampal CA1 region and are impaired in spatial learning. In situ hybridization confirms that the mutant mice show the same developmental expression of [alpha]CaMKII as their wild-type littermates. A simple hypothesis would suggest that if LTP is a substrate…

  14. Study of the biologic behavior of odontogenic keratocyst and orthokeratinaized odontogenic cyst using TGF-alpha and P53 markers.

    PubMed

    Deyhimi, Parviz; Hashemzadeh, Zahra

    2014-04-01

    Odontogenic keratocyst (OKC) is an aggressive cyst, and its recurrence rate is higher than that of other odontogenic cysts. Orthokeratinized odontogenic cyst (OOC) is less aggressive than OKC, but bears the probability of carcinomatous changes. In this study, we evaluated the expression and intensity of P53 and TGF-alpha in order to compare the biologic behavior or probable carcinomatous changes of these two cysts. In this cross-sectional study, 15 OKC and 15 OOC were stained immunohistochemically for P53 and TGF-alpha using the Novolink polymer method. Then, all slides were examined by an optical microscope with 400× magnification, and the stained cells in the basal and parabasal layers were counted. Finally, the results were analyzed by the Mann-Whitney and Wilcoxon tests (P-value<0.05). The difference between the expression of P53 and TGF alpha in the basal layer of OKC and OOC was not statistically significant (P-value>0.05), but the expression of P53 and TGF-alpha in the parabasal layer in OKC was statistically higher compared to OOC (P<0.05). Considering the known role of P53 and TGF-alpha in malignant changes and the higher expression of P53 and TGF-alpha in OKC compared to those in OOC, the probability of carcinomatous changes was higher in OKC than in OOC. Copyright © 2013 Elsevier GmbH. All rights reserved.

  15. {alpha}-Lipoic acid prevents lipotoxic cardiomyopathy in acyl CoA-synthase transgenic mice

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Young; Department of Internal Medicine, University of Texas, Southwestern Medical Center, Dallas, TX 75390-8854; Naseem, R. Haris

    2006-05-26

    {alpha}-Lipoic acid ({alpha}-LA) mimics the hypothalamic actions of leptin on food intake, energy expenditure, and activation of AMP-activated protein kinase (AMPK). To determine if, like leptin, {alpha}-LA protects against cardiac lipotoxicity, {alpha}-LA was fed to transgenic mice with cardiomyocyte-specific overexpression of the acyl CoA synthase (ACS) gene. Untreated ACS-transgenic mice died prematurely with increased triacylglycerol content and dilated cardiomyopathy, impaired systolic function and myofiber disorganization, apoptosis, and interstitial fibrosis on microscopy. In {alpha}-LA-treated ACS-transgenic mice heart size, echocardiogram and TG content were normal. Plasma TG fell 50%, hepatic-activated phospho-AMPK rose 6-fold, sterol regulatory element-binding protein-1c declined 50%, and peroxisome proliferator-activatedmore » receptor-{gamma} cofactor-1{alpha} mRNA rose 4-fold. Since food restriction did not prevent lipotoxicity, we conclude that {alpha}-LA treatment, like hyperleptinemia, protects the heart of ACS-transgenic mice from lipotoxicity.« less

  16. Effect of alpha lipoic acid on intracerebroventricular streptozotocin model of cognitive impairment in rats.

    PubMed

    Sharma, Monisha; Gupta, Y K

    2003-08-01

    In the present study, the effect of alpha lipoic acid, a potent free radical scavenger, was investigated against the intracerebroventricular streptozotocin model of cognitive impairment in rats, which is characterized by a progressive deterioration of memory, cerebral glucose and energy metabolism, and oxidative stress. Wistar rats were injected with intracerebroventricular streptozotocin bilaterally. The rats were treated chronically with alpha lipoic acid (50, 100 and 200 mg/kg) orally for 21 days starting from day 1 of streptozotocin injection in separate groups. The learning and memory behavior was evaluated and the rats were sacrificed for estimation of oxidative stress. The intracerebroventricular streptozotocin rats treated with alpha lipoic acid (200 mg/kg, p.o.) showed significantly less cognitive impairment as compared to the vehicle treated rats. There was also an insignificant increase in oxidative stress in the alpha lipoic acid treated groups. The study demonstrated the effectiveness of alpha lipoic acid in preventing cognitive impairment and oxidative stress induced by intracerebroventricular streptozotocin and its potential in dementia associated with age and age related neurodegenerative disorders where oxidative stress is involved such as Alzheimer's disease.

  17. Hippocampal α-Synuclein in Dementia with Lewy Bodies Contributes to Memory Impairment and Is Consistent with Spread of Pathology

    PubMed Central

    Roy, Subhojit; Galasko, Douglas R.; Hansen, Lawrence A.; Masliah, Eliezer

    2017-01-01

    Despite considerable research to uncover them, the anatomic and neuropathologic correlates of memory impairment in dementia with Lewy bodies (DLB) remain unclear. While some studies have implicated Lewy bodies in the neocortex, others have pointed to α-synuclein pathology in the hippocampus. We systematically examined hippocampal Lewy pathology and its distribution in hippocampal subfields in 95 clinically and neuropathologically characterized human cases of DLB, finding that α-synuclein pathology was highest in two hippocampal-related subregions: the CA2 subfield and the entorhinal cortex (EC). While the EC had numerous classic somatic Lewy bodies, CA2 contained mainly Lewy neurites in presumed axon terminals, suggesting the involvement of the EC → CA2 circuitry in the pathogenesis of DLB symptoms. Clinicopathological correlations with measures of verbal and visual memory supported a role for EC Lewy pathology, but not CA2, in causing these memory deficits. Lewy pathology in CA1—the main output region for CA2—correlated best with results from memory testing despite a milder pathology. This result indicates that CA1 may be more functionally relevant than CA2 in the context of memory impairment in DLB. These correlations remained significant after controlling for several factors, including concurrent Alzheimer's pathology (neuritic plaques and neurofibrillary tangles) and the interval between time of testing and time of death. Our data suggest that although hippocampal Lewy pathology in DLB is predominant in CA2 and EC, memory performance correlates most strongly with CA1 burden. SIGNIFICANCE STATEMENT This study provides a detailed neuropathologic analysis of hippocampal Lewy pathology in human patients with autopsy-confirmed dementia with Lewy bodies. The approach—informed by regional molecular markers, concurrent Alzheimer's pathology analysis, and relevant clinical data—helps tease out the relative contribution of Lewy pathology to memory dysfunction

  18. Alterations of p53 in tumorigenic human bronchial epithelial cells correlate with metastatic potential

    NASA Technical Reports Server (NTRS)

    Piao, C. Q.; Willey, J. C.; Hei, T. K.; Hall, E. J. (Principal Investigator)

    1999-01-01

    The cellular and molecular mechanisms of radiation-induced lung cancer are not known. In the present study, alterations of p53 in tumorigenic human papillomavirus-immortalized human bronchial epithelial (BEP2D) cells induced by a single low dose of either alpha-particles or 1 GeV/nucleon (56)Fe were analyzed by PCR-single-stranded conformation polymorphism (SSCP) coupled with sequencing analysis and immunoprecipitation assay. A total of nine primary and four secondary tumor cell lines, three of which were metastatic, together with the parental BEP2D and primary human bronchial epithelial (NHBE) cells were studied. The immunoprecipitation assay showed overexpression of mutant p53 proteins in all the tumor lines but not in NHBE and BEP2D cells. PCR-SSCP and sequencing analysis found band shifts and gene mutations in all four of the secondary tumors. A G-->T transversion in codon 139 in exon 5 that replaced Lys with Asn was detected in two tumor lines. One mutation each, involving a G-->T transversion in codon 215 in exon 6 (Ser-->lle) and a G-->A transition in codon 373 in exon 8 (Arg-->His), was identified in the remaining two secondary tumors. These results suggest that p53 alterations correlate with tumorigenesis in the BEP2D cell model and that mutations in the p53 gene may be indicative of metastatic potential.

  19. Structure and properties of a complex of α-synuclein and a single-domain camelid antibody.

    PubMed

    De Genst, Erwin J; Guilliams, Tim; Wellens, Joke; O'Day, Elizabeth M; Waudby, Christopher A; Meehan, Sarah; Dumoulin, Mireille; Hsu, Shang-Te Danny; Cremades, Nunilo; Verschueren, Koen H G; Pardon, Els; Wyns, Lode; Steyaert, Jan; Christodoulou, John; Dobson, Christopher M

    2010-09-17

    The aggregation of the intrinsically disordered protein α-synuclein to form fibrillar amyloid structures is intimately associated with a variety of neurological disorders, most notably Parkinson's disease. The molecular mechanism of α-synuclein aggregation and toxicity is not yet understood in any detail, not least because of the paucity of structural probes through which to study the behavior of such a disordered system. Here, we describe an investigation involving a single-domain camelid antibody, NbSyn2, selected by phage display techniques to bind to α-synuclein, including the exploration of its effects on the in vitro aggregation of the protein under a variety of conditions. We show using isothermal calorimetric methods that NbSyn2 binds specifically to monomeric α-synuclein with nanomolar affinity and by means of NMR spectroscopy that it interacts with the four C-terminal residues of the protein. This latter finding is confirmed by the determination of a crystal structure of NbSyn2 bound to a peptide encompassing the nine C-terminal residues of α-synuclein. The NbSyn2:α-synuclein interaction is mediated mainly by side-chain interactions while water molecules cross-link the main-chain atoms of α-synuclein to atoms of NbSyn2, a feature we believe could be important in intrinsically disordered protein interactions more generally. The aggregation behavior of α-synuclein at physiological pH, including the morphology of the resulting fibrillar structures, is remarkably unaffected by the presence of NbSyn2 and indeed we show that NbSyn2 binds strongly to the aggregated as well as to the soluble forms of α-synuclein. These results give strong support to the conjecture that the C-terminal region of the protein is not directly involved in the mechanism of aggregation and suggest that binding of NbSyn2 could be a useful probe for the identification of α-synuclein aggregation in vitro and possibly in vivo. Copyright © 2010. Published by Elsevier Ltd.

  20. Progressive accumulation of ubiquitin and disappearance of alpha-synuclein epitope in multiple system atrophy-associated glial cytoplasmic inclusions: triple fluorescence study combined with Gallyas-Braak method.

    PubMed

    Sakamoto, Masaki; Uchihara, Toshiki; Nakamura, Ayako; Mizutani, Toshio; Mizusawa, Hidehiro

    2005-10-01

    Alpha-synuclein (alphaS) and ubiquitin (Ub) are shared constituents of glial cytoplasmic inclusions (GCIs) and Lewy bodies (LBs), both composed of fibrillary structures. Staining profiles of GCIs were investigated with triple immunofluorescence involving immunostaining for alphaS and Ub, both amplified with catalyzed reporter deposition, and a fluorochrome, thiazin red (TR) that has an affinity to fibrillary structures. After observation for the triple-fluorescent images, the sections were subsequently stained with the Gallyas-Braak method. Sections of putamen, cerebellar white matter and motor cortex from patients suffering from multiple system atrophy (MSA) with varying duration of the disease (4-15 years) were quantified for these staining profiles of Gallyas-positive GCIs. Although most of GCIs were positive for Ub and variably positive for alphaS, they were consistently negative for TR. The result was opposite in LBs in Lewy body disease with variable affinity to TR, suggesting that the construction of GCIs is different from that of LBs. These four staining features (alphaS, Ub, TR and Gallyas) alone failed to exhibit apparent correlation with disease duration, lesion site or severity of degeneration as reported previously. The fraction of alphaS-negative and Ub-positive GCIs, however, linearly increased along the disease progression, while that of alphaS-positive and Ub-negative GCIs decreased in contrast. This reciprocal change suggests that alphaS immunoreactivity in GCIs is being replaced by Ub immunoreactivity during the disease progression, which resulted in the ultimate predominance of alphaS-negative and Ub-positive GCIs in the most advanced case. Interestingly, this predominance of alphaS-negative and Ub-positive GCIs was a feature of motor cortex, where degeneration usually remains mild in spite of robust appearance of Gallyas-positive GCIs. Another fraction, alphaS-positive and Ub-positive GCIs were frequent in cerebellar white matter, suggesting

  1. Overexpression of Wnt5a in mouse epidermis causes no psoriasis phenotype but an impairment of hair follicle anagen development.

    PubMed

    Zhu, Xuming; Wu, Yumei; Huang, Sixia; Chen, Yingwei; Tao, Yixin; Wang, Yushu; He, Shigang; Shen, Sanbing; Wu, Ji; Guo, Xizhi; Li, Baojie; He, Lin; Ma, Gang

    2014-12-01

    Increased Wnt5a expression has been observed in psoriatic plaques. However, whether Wnt5a overexpression directly causes psoriasis is unknown. In this study, we generated transgenic (TG) mice with epidermal Wnt5a overexpression under the control of the human K14 promoter. The skin of Wnt5a TG mice was not psoriatic, but characterized with normal proliferation and homeostasis of epidermis. Instead, these TG mice displayed impaired hair follicle transition from telogen to anagen, most likely due to impaired canonical Wnt signalling. These results suggest that increased Wnt5a expression alone is inadequate to induce psoriasis in the skin and possible involvement of Wnt5a in hair follicle cycling. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo

    PubMed Central

    Rockenstein, Edward; Nuber, Silke; Overk, Cassia R.; Ubhi, Kiren; Mante, Michael; Patrick, Christina; Adame, Anthony; Trejo-Morales, Margarita; Gerez, Juan; Picotti, Paola; Jensen, Poul H.; Campioni, Silvia; Riek, Roland; Winkler, Jürgen; Gage, Fred H.; Winner, Beate

    2014-01-01

    In Parkinson’s disease and dementia with Lewy bodies, α-synuclein aggregates to form oligomers and fibrils; however, the precise nature of the toxic α-synuclein species remains unclear. A number of synthetic α-synuclein mutations were recently created (E57K and E35K) that produce species of α-synuclein that preferentially form oligomers and increase α-synuclein-mediated toxicity. We have shown that acute lentiviral expression of α-synuclein E57K leads to the degeneration of dopaminergic neurons; however, the effects of chronic expression of oligomer-prone α-synuclein in synapses throughout the brain have not been investigated. Such a study could provide insight into the possible mechanism(s) through which accumulation of α-synuclein oligomers in the synapse leads to neurodegeneration. For this purpose, we compared the patterns of neurodegeneration and synaptic damage between a newly generated mThy-1 α-synuclein E57K transgenic mouse model that is prone to forming oligomers and the mThy-1 α-synuclein wild-type mouse model (Line 61), which accumulates various forms of α-synuclein. Three lines of α-synuclein E57K (Lines 9, 16 and 54) were generated and compared with the wild-type. The α-synuclein E57K Lines 9 and 16 were higher expressings of α-synuclein, similar to α-synuclein wild-type Line 61, and Line 54 was a low expressing of α-synuclein compared to Line 61. By immunoblot analysis, the higher-expressing α-synuclein E57K transgenic mice showed abundant oligomeric, but not fibrillar, α-synuclein whereas lower-expressing mice accumulated monomeric α-synuclein. Monomers, oligomers, and fibrils were present in α-synuclein wild-type Line 61. Immunohistochemical and ultrastructural analyses demonstrated that α-synuclein accumulated in the synapses but not in the neuronal cells bodies, which was different from the α-synuclein wild-type Line 61, which accumulates α-synuclein in the soma. Compared to non-transgenic and lower-expressing mice, the

  3. Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo.

    PubMed

    Rockenstein, Edward; Nuber, Silke; Overk, Cassia R; Ubhi, Kiren; Mante, Michael; Patrick, Christina; Adame, Anthony; Trejo-Morales, Margarita; Gerez, Juan; Picotti, Paola; Jensen, Poul H; Campioni, Silvia; Riek, Roland; Winkler, Jürgen; Gage, Fred H; Winner, Beate; Masliah, Eliezer

    2014-05-01

    In Parkinson's disease and dementia with Lewy bodies, α-synuclein aggregates to form oligomers and fibrils; however, the precise nature of the toxic α-synuclein species remains unclear. A number of synthetic α-synuclein mutations were recently created (E57K and E35K) that produce species of α-synuclein that preferentially form oligomers and increase α-synuclein-mediated toxicity. We have shown that acute lentiviral expression of α-synuclein E57K leads to the degeneration of dopaminergic neurons; however, the effects of chronic expression of oligomer-prone α-synuclein in synapses throughout the brain have not been investigated. Such a study could provide insight into the possible mechanism(s) through which accumulation of α-synuclein oligomers in the synapse leads to neurodegeneration. For this purpose, we compared the patterns of neurodegeneration and synaptic damage between a newly generated mThy-1 α-synuclein E57K transgenic mouse model that is prone to forming oligomers and the mThy-1 α-synuclein wild-type mouse model (Line 61), which accumulates various forms of α-synuclein. Three lines of α-synuclein E57K (Lines 9, 16 and 54) were generated and compared with the wild-type. The α-synuclein E57K Lines 9 and 16 were higher expressings of α-synuclein, similar to α-synuclein wild-type Line 61, and Line 54 was a low expressing of α-synuclein compared to Line 61. By immunoblot analysis, the higher-expressing α-synuclein E57K transgenic mice showed abundant oligomeric, but not fibrillar, α-synuclein whereas lower-expressing mice accumulated monomeric α-synuclein. Monomers, oligomers, and fibrils were present in α-synuclein wild-type Line 61. Immunohistochemical and ultrastructural analyses demonstrated that α-synuclein accumulated in the synapses but not in the neuronal cells bodies, which was different from the α-synuclein wild-type Line 61, which accumulates α-synuclein in the soma. Compared to non-transgenic and lower-expressing mice, the

  4. Amyloid Precursor Protein and Alpha Synuclein Translation, Implications for Iron and Inflammation in Neurodegenerative diseases

    PubMed Central

    Cahill, Catherine M.; Lahiri, Debomoy K.; Huang, Xudong; Rogers, Jack T.

    2014-01-01

    Summary Recent studies that alleles in the hemochromatosis gene may accelerate the onset of Alzheimer's disease (AD) by five years have validated interest in the model in which metals (particularly iron) accelerate disease course. Biochemical and biophysical measurements demonstrated the presence of elevated levels of neurotoxic copper, zinc and iron in the brains of AD patients. Intracellular levels of amyloid precursor protein (APP) holoprotein were shown to be modulated via iron by a mechanism that is similar to the translation control of the ferritin L- and H mRNAs by Iron-responsive Element (IRE) RNA stem loops in their 5′untranslated regions (5′UTRs). Recently, we reported a putative IRE-like sequence to be present in the 5′UTR of the Parkinson's disease (PD) specific alpha synuclein (ASYN) transcript. ASYN encodes the non-Aβ component (NAC) of amyloid plaques. The demonstration of iron-dependent translation of APP mRNA, the involvement of metals in the plaque of AD patients and of increased iron in striatal neurons in the Substantia nigra (SN) of PD patients, have each encouraged the development of metal attenuating agents and iron chelators as a major new therapeutic strategy for the treatment of these neurodegenerative diseases. In the case of AD, metal based therapeutics may ultimately prove more cost effective than the use of an amyloid vaccine as the preferred anti-amyloid therapeutic strategy to ameliorate the cognitive decline of AD patients. PMID:19166904

  5. Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-{alpha}

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tsukasaki, Masayuki; Yamada, Atsushi, E-mail: yamadaa@dent.showa-u.ac.jp; Suzuki, Dai

    2011-07-15

    Highlights: {yields} TNF-{alpha} inhibits POEM gene expression. {yields} Inhibition of POEM gene expression is caused by NF-{kappa}B activation by TNF-{alpha}. {yields} Over-expression of POEM recovers inhibition of osteoblast differentiation by TNF-{alpha}. -- Abstract: POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-{alpha} (TNF-{alpha}), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-{alpha}-induced down-regulation of POEM gene expression occurred in both time- andmore » dose-dependent manners through the nuclear factor kappa B (NF-{kappa}B) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-{alpha} in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-{alpha}-induced inhibition of osteoblast differentiation. These results suggest that TNF-{alpha} inhibits POEM expression through the NF-{kappa}B signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-{alpha}.« less

  6. Applying chaperones to protein-misfolding disorders: molecular chaperones against α-synuclein in Parkinson's disease.

    PubMed

    Chaari, Ali; Hoarau-Véchot, Jessica; Ladjimi, Moncef

    2013-09-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the accumulation of a protein called α-synuclein (α-syn) into inclusions known as lewy bodies (LB) within neurons. This accumulation is also due to insufficient formation and activity of dopamine produced in certain neurons within the substantia nigra. Lewy bodies are the pathological hallmark of the idiopathic disorder and the cascade that allows α-synuclein to misfold, aggregate and form these inclusions has been the subject of intensive research. Targeting these early steps of oligomerization is one of the main therapeutic approaches in order to develop neurodegenerative-modifying agents. Because the folding and refolding of alpha synuclein is the key point of this cascade, we are interested in this review to summarize the role of some molecular chaperones proteins such as Hsp70, Hsp90 and small heat shock proteins (sHsp) and Hsp 104. Hsp70 and its co-chaperone, Hsp70 and small heat shock proteins can prevent neurodegeneration by preventing α-syn misfolding, oligomerization and aggregation in vitro and in Parkinson disease animal models. Hsp104 is able to resolve disordered protein aggregates and cross beta amyloid conformers. Together, these chaperones have a complementary effect and can be a target for therapeutic intervention in PD. Published by Elsevier B.V.

  7. Variation in the miRNA-433 Binding Site of FGF20 Confers Risk for Parkinson Disease by Overexpression of α-Synuclein

    PubMed Central

    Wang, Gaofeng; van der Walt, Joelle M.; Mayhew, Gregory; Li, Yi-Ju; Züchner, Stephan; Scott, William K.; Martin, Eden R.; Vance, Jeffery M.

    2008-01-01

    Parkinson disease (PD) is a common neurodegenerative disorder caused by environmental and genetic factors. We have previously shown linkage of PD to chromosome 8p. Subsequently, fibroblast growth factor 20 (FGF20) at 8p21.3–22 was identified as a risk factor in several association studies. To identify the risk-conferring polymorphism in FGF20, we performed genetic and functional analysis of single-nucleotide polymorphisms within the gene. In a sample of 729 nuclear families with 1089 affected and 1165 unaffected individuals, the strongest evidence of association came from rs12720208 in the 3′ untranslated region of FGF20. We show in several functional assays that the risk allele for rs12720208 disrupts a binding site for microRNA-433, increasing translation of FGF20 in vitro and in vivo. In a cell-based system and in PD brains, this increase in translation of FGF20 is correlated with increased α-synuclein expression, which has previously been shown to cause PD through both overexpression and point mutations. We suggest a novel mechanism of action for PD risk in which the modulation of the susceptibility gene's translation by common variations interfere with the regulation mechanisms of microRNA. We propose this is likely to be a common mechanism of genetic modulation of individual susceptibility to complex disease. PMID:18252210

  8. Activation of tyrosine kinase c-Abl contributes to α-synuclein–induced neurodegeneration

    PubMed Central

    Lee, Su Hyun; Kim, Donghoon; Karuppagounder, Senthilkumar S.; Kumar, Manoj; Mao, Xiaobo; Shin, Joo Ho; Lee, Yunjong; Pletnikova, Olga; Troncoso, Juan C.; Dawson, Valina L.; Dawson, Ted M.; Ko, Han Seok

    2016-01-01

    Aggregation of α-synuclein contributes to the formation of Lewy bodies and neurites, the pathologic hallmarks of Parkinson disease (PD) and α-synucleinopathies. Although a number of human mutations have been identified in familial PD, the mechanisms that promote α-synuclein accumulation and toxicity are poorly understood. Here, we report that hyperactivity of the nonreceptor tyrosine kinase c-Abl critically regulates α-synuclein–induced neuropathology. In mice expressing a human α-synucleinopathy–associated mutation (hA53Tα-syn mice), deletion of the gene encoding c-Abl reduced α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Conversely, overexpression of constitutively active c-Abl in hA53Tα-syn mice accelerated α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Moreover, c-Abl activation led to an age-dependent increase in phosphotyrosine 39 α-synuclein. In human postmortem samples, there was an accumulation of phosphotyrosine 39 α-synuclein in brain tissues and Lewy bodies of PD patients compared with age-matched controls. Furthermore, in vitro studies show that c-Abl phosphorylation of α-synuclein at tyrosine 39 enhances α-synuclein aggregation. Taken together, this work establishes a critical role for c-Abl in α-synuclein–induced neurodegeneration and demonstrates that selective inhibition of c-Abl may be neuroprotective. This study further indicates that phosphotyrosine 39 α-synuclein is a potential disease indicator for PD and related α-synucleinopathies. PMID:27348587

  9. α-Synuclein Activates Innate Immunity but Suppresses Interferon-γ Expression in Murine Astrocytes.

    PubMed

    Wang, Jintang; Chen, Zheng; Walston, Jeremy; Gao, Peisong; Gao, Maolong; Leng, Sean X

    2018-05-19

    Glial activation and neuroinflammation contribute to pathogenesis of neurodegenerative diseases, linked to neuron loss and dysfunction. α-Synuclein (α-syn), as a metabolite of neuron, can induce microglia activation to trigger innate immune response. However, whether α-syn, as well as its mutants (A53T, A30P and E46K), induces astrocyte activation and inflammatory response is not fully elucidated. In this study, we used A53T mutant and wildtype α-syns to stimulate primary astrocytes in dose- and time-dependent manners (0.5, 2, 8 and 20 μg/mL for 24 hour or 3, 12, 24 and 48 hour at 2 μg/mL), and evaluated activation of several canonical inflammatory pathway components. The results showed that A53T mutant or wildtype α-syn significantly upregulated mRNA expression of toll-like receptor (TLR)2, TLR3, nuclear factor-κB and interleukin (IL)-1β, displaying a pattern of positive dose-effect correlation or negative time-effect correlation. Such upregulation was confirmed at protein levels of TLR2 (at 20 μg/mL), TLR3 (at most doses) and IL-1β (at 3 hour) by western blotting. Blockage of TLR2 other than TLR4 inhibited TLR3 and IL-1β mRNA expressions. By contrast, interferon (IFN)-γ was significantly downregulated at mRNA, protein and protein release levels, especially at high concentrations of α-syns or early time-points. These findings indicate that α-syn was a TLRs-mediated immunogenic agent (A53T mutant stronger than wildtype α-syn). The stimulation patterns suggest that persistent release and accumulation of α-syn is required for maintenance of innate immunity activation, and IFN-γ expression inhibition by α-syn suggests a novel immune molecule interaction mechanism underlying pathogenesis of neurodegenerative diseases. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  10. 46 CFR 32.53-5 - Operation-T/ALL.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Operation-T/ALL. 32.53-5 Section 32.53-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS SPECIAL EQUIPMENT, MACHINERY, AND HULL REQUIREMENTS Inert Gas System § 32.53-5 Operation-T/ALL. Unless the cargo tanks are gas free, the master of each...

  11. 46 CFR 32.53-5 - Operation-T/ALL.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 1 2011-10-01 2011-10-01 false Operation-T/ALL. 32.53-5 Section 32.53-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS SPECIAL EQUIPMENT, MACHINERY, AND HULL REQUIREMENTS Inert Gas System § 32.53-5 Operation-T/ALL. Unless the cargo tanks are gas free, the master of each...

  12. In-silico analysis for RNA-interference mechanism of α-synuclein to treat Parkinson's disease.

    PubMed

    Seema, S; Seenivasagam, R; Hemavathi, K

    2013-01-01

    Parkinson's Disease (PD) causing mutations in α-synuclein gene are ALA30PRO, GLU46LYS and ALA53THR. The conformational changes in proteins with respect to all the three mutations were analysed. These were used to predict the structures of Short Interfering RNA (siRNA) antisense strand and siRNA region. The siRNA binds with the argonaute protein forming RNA Induced Silencing Complex (RISC). Then, siRNA antisense-strand was attached to RISC. The structure of dicer (RNase-III-enzyme) cleaves double-stranded RNA (dsRNA) into two siRNA-strands. Incorporation of single siRNA-strand into RISC guides to pair with the complementary α-synuclein target-messenger RNA (mRNA) thereby enabling it to cleave the target.

  13. Chorionic gonadotropin regulates the transcript level of VHL, p53, and HIF-2alpha in human granulosa lutein cells.

    PubMed

    Herr, D; Keck, C; Tempfer, C; Pietrowski, Detlef

    2004-12-01

    The ovarian corpus luteum plays a critical role in reproduction being the primary source of circulating progesterone. After ovulation the corpus luteum is build by avascular granulosa lutein cells through rapid vascularization regulated by gonadotropic hormones. The present study was performed to investigate whether this process might be influenced by the human chorionic gonadotropin (hCG)-dependent expression of different tumor suppressor genes and hypoxia dependent transcription factors. RNA was isolated from cultured granulosa lutein cells, transcribed into cDNA, and the transcript level of following genes were determined: RB-1, VHL, NF-1, NF-2, Wt-1, p53, APC, and hypoxia inducible factor-1 (HIF-1), -2, and -3alpha. Additionally, the influence of hCG on the expression of VHL, p53, and HIf2alpha were investigated. We demonstrate that in human granulosa lutein cells the tumor suppressor genes RB-1, VHL, NF-1, NF-2, Wt-1, p53, and APC and the hypoxia dependent transcription factors HIF-1alpha, -2alpha, and -3alpha are expressed. In addition, we showed that hCG regulates the expression of p53, VHL, and HIF-2alpha. Our results indicate that hCG may determine the growth and development of the corpus luteum by mediating hypoxic and apoptotic pathways in human granulosa lutein cells. Copyright 2004 Wiley-Liss, Inc.

  14. Electroacupuncture remediates glial dysfunction and ameliorates neurodegeneration in the astrocytic α-synuclein mutant mouse model.

    PubMed

    Deng, Jiahui; Lv, E; Yang, Jian; Gong, Xiaoli; Zhang, Wenzhong; Liang, Xibin; Wang, Jiazeng; Jia, Jun; Wang, Xiaomin

    2015-05-28

    The acupuncture or electroacupuncture (EA) shows the therapeutic effect on various neurodegenerative diseases. This effect was thought to be partially achieved by its ability to alleviate existing neuroinflammation and glial dysfunction. In this study, we systematically investigated the effect of EA on abnormal neurochemical changes and motor symptoms in a mouse neurodegenerative disease model. The transgenic mouse which expresses a mutant α-synuclein (α-syn) protein, A53T α-syn, in brain astrocytic cells was used. These mice exhibit extensive neuroinflammatory and motor phenotypes of neurodegenerative disorders. In this study, the effects of EA on these phenotypic changes were examined in these mice. EA improved the movement detected in multiple motor tests in A53T mutant mice. At the cellular level, EA significantly reduced the activation of microglia and prevented the loss of dopaminergic neurons in the midbrain and motor neurons in the spinal cord. At the molecular level, EA suppressed the abnormal elevation of proinflammatory factors (tumor necrosis factor-α and interleukin-1β) in the striatum and midbrain of A53T mice. In contrast, EA increased striatal and midbrain expression of a transcription factor, nuclear factor E2-related factor 2, and its downstream antioxidants (heme oxygenase-1 and glutamate-cysteine ligase modifier subunits). These results suggest that EA possesses the ability to ameliorate mutant α-syn-induced motor abnormalities. This ability may be due to that EA enhances both anti-inflammatory and antioxidant activities and suppresses aberrant glial activation in the diseased sites of brains.

  15. Adsorption and decontamination of α-synuclein from medically and environmentally-relevant surfaces.

    PubMed

    Phan, Hanh T M; Bartz, Jason C; Ayers, Jacob; Giasson, Benoit I; Schubert, Mathias; Rodenhausen, Keith B; Kananizadeh, Negin; Li, Yusong; Bartelt-Hunt, Shannon L

    2018-06-01

    The assembly and accumulation of α-synuclein fibrils are implicated in the development of several neurodegenerative disorders including multiple system atrophy and Parkinson's disease. Pre-existing α-synuclein fibrils can recruit and convert soluble non-fibrillar α-synuclein to the fibrillar form similar to what is observed in prion diseases. This raises concerns regarding attachment of fibrillary α-synuclein to medical instruments and subsequent exposure of patients to α-synuclein similar to what has been observed in iatrogenic transmission of prions. Here, we evaluated adsorption and desorption of α-synuclein to two surfaces: stainless steel and a gold surface coated with a 11-Amino-1-undecanethiol hydrochloride self-assembled-monolayer (SAM) using in-situ combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry. α-Synuclein was found to attach to both surfaces, however, increased α-synuclein adsorption was observed onto the positively charged SAM surface compared to the stainless steel surface. Dynamic light scattering data showed that larger α-synuclein fibrils were preferentially attached to the stainless steel surface when compared with the distributions in the original α-synuclein solution and on the SAM surface. We determined that after attachment, introduction of a 1N NaOH solution could completely remove α-synuclein adsorbed on the stainless steel surface while α-synuclein was retained on the SAM surface. Our results indicate α-synuclein can bind to multiple surface types and that decontamination is surface-dependent. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. [Rat cardiomyocyte remodeling after neonatal cryptosporidiosis. II. Elongation, excessive polyploidization and HIF-1alpha overexpression].

    PubMed

    Anatskaia, O V; Sidorenko, N V; Matveev, I V; Kropotov, A V; Vinogradov, A E

    2012-01-01

    Retrospective epidemyological studies evidence that infant diseases leave survivors with an increased susceptibility to cardiovascular diseases in later life. At the same time, the mechanisms of this link remain poorly understood. Based on medical statistics reporting that infectious gastroenteritis is the most common cause of maladies in babies, infants and children, we analysed the effects of moderate cryptosporidial gastroenteritis on the heart and ventricular cardiomyocyte remodelling in rats of the first month of life. The disease was challenged by a worldwide human protozoic pathogen Cryptosporidium parvum (Apicomplexa, Sporozoa). The main symptoms manifested in the growth retardation moderate diarrhea. Using real-time PCR, cytophotometry, confocal microscopy and image analysis, we indicated that cryptosporidiosis was associated, with the atrophy heart and the elongation, narrowing, protein content decrease and hyperpolyploidization of cardiomyocytes and the moderate overexpression of hypoxia inducible factor 1alpha (HIF-1alpha) mRNA. Cardiomyocyte shape remodeling and heart atrophy presented in all age groups. The severity of these changes, hovewer, declined gradually from younger to older groups. In contrast, hyperpolyploidization and HIF-1alpha mRNA overexpression were registered mainly among animals aged between 6 and 13 days, and were barely detected and non-significant in older age groups. In the rat the time period covering 6-13 days after birth is known to coincide with the intensive cardiomyocyte polyploidization and the switch from proliferation to hypertrophy. Thus, our data indicate that neonatal cryptosporidiosis may be potential cardiovascular diseases risk factor and that one of the critical time windows for the growing heart covers the time period when cardiomyocyte undergo polyploidization.

  17. Endonuclease G mediates α-synuclein cytotoxicity during Parkinson's disease.

    PubMed

    Büttner, Sabrina; Habernig, Lukas; Broeskamp, Filomena; Ruli, Doris; Vögtle, F Nora; Vlachos, Manolis; Macchi, Francesca; Küttner, Victoria; Carmona-Gutierrez, Didac; Eisenberg, Tobias; Ring, Julia; Markaki, Maria; Taskin, Asli Aras; Benke, Stefan; Ruckenstuhl, Christoph; Braun, Ralf; Van den Haute, Chris; Bammens, Tine; van der Perren, Anke; Fröhlich, Kai-Uwe; Winderickx, Joris; Kroemer, Guido; Baekelandt, Veerle; Tavernarakis, Nektarios; Kovacs, Gabor G; Dengjel, Jörn; Meisinger, Chris; Sigrist, Stephan J; Madeo, Frank

    2013-11-27

    Malfunctioning of the protein α-synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaining this pathogenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triggers DNA degradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminergic neurons of human Parkinson-diseased patients, while EndoG depletion largely reduces α-synuclein-induced cell death in human neuroblastoma cells. Xenogenic expression of human α-synuclein in yeast cells triggers mitochondria-nuclear translocation of EndoG and EndoG-mediated DNA degradation through a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α-synuclein-driven degeneration of dopaminergic neurons. Moreover, the locomotion and survival of α-synuclein-expressing flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phylogenetically conserved pathway that involves EndoG as a critical downstream executor of α-synuclein cytotoxicity.

  18. The Synaptic Function of α-Synuclein

    PubMed Central

    Burré, Jacqueline

    2015-01-01

    α-Synuclein is an abundant neuronal protein which localizes predominantly to presynaptic terminals, and is strongly linked genetically and pathologically to Parkinson’s disease and other neurodegenerative diseases. While the accumulation of α-synuclein in the form of misfolded oligomers and large aggregates defines multiple neurodegenerative diseases called “synucleinopathies”, its cellular function has remained largely unclear, and is the subject of intense investigation. In this review, I focus on the structural characteristics of α-synuclein, its cellular and subcellular localization, and discuss how this relates to its function in neurons, in particular at the neuronal synapse. PMID:26407041

  19. Identification of p53 unbound to T-antigen in human cells transformed by simian virus 40 T-antigen.

    PubMed

    O'Neill, F J; Hu, Y; Chen, T; Carney, H

    1997-02-27

    In several clones of SV40-transformed human cells, we investigated the relative amounts of large T-Antigen (T-Ag) and p53 proteins, both unbound and associated within complexes, with the goal of identifying changes associated with transformation and immortalization. Cells were transformed by wild type (wt) T-Ag, a functionally temperature sensitive T-Ag (tsA58) and other T-Ag variants. Western analysis showed that while most of the T-Ag was ultimately bound by p53, most of the p53 remained unbound to T-Ag. Unbound p53 remained in the supernatant after a T-Ag immunoprecipitation and p53 was present in two to fourfold excess of T-Ag. In one transformant there was five to tenfold more p53 than T-Ag. p53 was present in transformants in amounts at least 200-fold greater than in untransformed human cells. In wt and variant T-Ag transformants, including those generated with tsA58 T-Ag, large amounts of unbound p53 were present in both pre-crisis and immortal cells and when the cells were grown at permissive or non-permissive temperatures. We also found that in transformants produced by tsA58, an SV40/JCV chimeric T-Ag and other variants, T-Ag appeared to form a complex with p53 slowly perhaps because one or both proteins matured slowly. The presence in transformed human cells of large amounts of unbound p53 and in excess of T-Ag suggests that sequestration of p53 by T-Ag, resulting from complex formation, is required neither for morphological transformation nor immortalization of human cells. Rather, these results support the proposal that high levels of p53, the T-Ag/p53 complexes, or other biochemical event(s), lead to transformation and immortalization of human cells by T-Ag.

  20. Alpha-synuclein is present in dental calculus but not altered in Parkinson's disease patients in comparison to controls.

    PubMed

    Schmid, Sabrina; Goldberg-Bockhorn, Eva; Schwarz, Silke; Rotter, Nicole; Kassubek, Jan; Del Tredici, Kelly; Pinkhardt, Elmar; Otto, Markus; Ludolph, Albert C; Oeckl, Patrick

    2018-06-01

    In autopsy cases staged for sporadic Parkinson's disease (PD), the neuropathology is characterized by a preclinical phase that targets the enteric nervous system of the gastrointestinal tract (GIT). Therefore, the ENS might be a source of potential (presymptomatic) PD biomarkers. In this clinically based study, we examined the alpha-synuclein (αSyn) concentration in an easily accessible protein storage medium of the GIT, dental calculus, in 21/50 patients with PD and 28/50 age- and gender-matched controls using ELISA. αSyn was detectable in dental calculus and the median concentration in the control patients was 8.6 pg/mg calculus (interquartile range 2.6-13.1 pg/mg). αSyn concentrations were significantly influenced by blood contamination and samples with a hemoglobin concentration of > 4000 ng/mL were excluded. There was no significant difference of αSyn concentrations in the dental calculus of PD patients (5.76 pg/mg, interquartile range 2.91-9.74 pg/mg) compared to those in controls (p = 0.40). The total αSyn concentration in dental calculus is not a suitable biomarker for sporadic PD. Disease-related variants such as oligomeric or phosphorylated αSyn in calculus might prove to be more specific.

  1. Measurements of auto-antibodies to α-synuclein in the serum and cerebral spinal fluids of patients with Parkinson's disease.

    PubMed

    Akhtar, Rizwan S; Licata, Joseph P; Luk, Kelvin C; Shaw, Leslie M; Trojanowski, John Q; Lee, Virginia M-Y

    2018-03-03

    Biomarkers for α-synuclein are needed for diagnosis and prognosis in Parkinson's disease (PD). Endogenous auto-antibodies to α-synuclein could serve as biomarkers for underlying synucleinopathy, but previous assessments of auto-antibodies have shown variability and inconsistent clinical correlations. We hypothesized that auto-antibodies to α-synuclein could be diagnostic for PD and explain its clinical heterogeneity. To test this hypothesis, we developed an enzyme-linked immunosorbent assay for measuring α-synuclein auto-antibodies in human samples. We evaluated 69 serum samples (16 healthy controls (HC) and 53 PD patients) and 145 CSF samples (52 HC and 93 PD patients) from our Institution. Both serum and CSF were available for 24 participants. Males had higher auto-antibody levels than females in both fluids. CSF auto-antibody levels were significantly higher in PD patients as compared to HC, whereas serum levels were not significantly different. CSF auto-antibody levels did not associate with amyloid-β 1-42 , total tau, or phosphorylated tau. CSF auto-antibody levels correlated with performance on the Montreal Cognitive Assessment, even when controlled for CSF amyloidβ 1-42 . CSF hemoglobin levels, as a proxy for contamination of CSF by blood during lumbar puncture, did not influence these observations. Using recombinant α-synuclein with N- and C-terminal truncations, we found that CSF auto-antibodies target amino acids 100 through 120 of α-synuclein. We conclude that endogenous CSF auto-antibodies are significantly higher in PD patients as compared to HC, suggesting that they could indicate the presence of underlying synucleinopathy. These auto-antibodies associate with poor cognition, independently of CSF amyloidβ 1-42 ., and target a select C-terminal region of α-synuclein. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  2. Role of advanced glycation on aggregation and DNA binding properties of α-synuclein.

    PubMed

    Padmaraju, Vasudevaraju; Bhaskar, Jamuna J; Prasada Rao, Ummiti J S; Salimath, Paramahans V; Rao, K S

    2011-01-01

    Parkinson's disease (PD) is a neurodegenerative disease with multiple etiologies. Advanced glycation end products (AGEs) accumulate in the aging brain and could be one of the reasons for age-related diseases like PD. Oxidative stress also leads to the formation of AGEs and may be involved in neurodegeneration by altering the properties of proteins. α-Synuclein is involved in pathogenesis of PD and there are limited studies on the role of AGE-α-synuclein in neurodegeneration. We studied the aggregation and DNA binding ability of AGE-α-synuclein in vitro. α-Synuclein is glycated using methylglyoxal and formation of AGE-α-synuclein is characterized using fluorescence studies, intrinsic tyrosine fluorescence, and fructosamine estimation. The results indicated that AGE-α-synuclein aggregates into smaller globular-like aggregates compared to fibrils formed with native α-synuclein. Further, it is found that AGE-α-synuclein induced conformational changes in scDNA from B-form to B-C-A mixed conformation. Additionally, AGE-α-synuclein altered DNA integrity as evidenced by the melting temperature, ethidium bromide, and DNAse I sensitivity studies. AGE-α-synuclein converted biphasic Tm to higher monophasic Tm. The Tm of AGE-α-synuclein-scDNA complex is more than that of native α-synuclein-scDNA complex, indicating that AGE-α-synuclein stabilized the uncoiled scDNA. AGE-α-synuclein could stabilize the uncoiled scDNA, as shown by the decrease in the number of ethidium bromide binding molecules per base pair of DNA. DNAse I sensitive studies indicated that both AGE-α-synuclein-scDNA and α-synuclein-scDNA are resistant to DNAse I digestion. The relevance of these findings to neuronal cell death is discussed.

  3. A Blood-Brain Barrier (BBB) Disrupter Is Also a Potent α-Synuclein (α-syn) Aggregation Inhibitor

    PubMed Central

    Shaltiel-Karyo, Ronit; Frenkel-Pinter, Moran; Rockenstein, Edward; Patrick, Christina; Levy-Sakin, Michal; Schiller, Abigail; Egoz-Matia, Nirit; Masliah, Eliezer; Segal, Daniel; Gazit, Ehud

    2013-01-01

    The development of disease-modifying therapy for Parkinson disease has been a main drug development challenge, including the need to deliver the therapeutic agents to the brain. Here, we examined the ability of mannitol to interfere with the aggregation process of α-synuclein in vitro and in vivo in addition to its blood-brain barrier-disrupting properties. Using in vitro studies, we demonstrated the effect of mannitol on α-synuclein aggregation. Although low concentration of mannitol inhibited the formation of fibrils, high concentration significantly decreased the formation of tetramers and high molecular weight oligomers and shifted the secondary structure of α-synuclein from α-helical to a different structure, suggesting alternative potential pathways for aggregation. When administered to a Parkinson Drosophila model, mannitol dramatically corrected its behavioral defects and reduced the amount of α-synuclein aggregates in the brains of treated flies. In the mThy1-human α-synuclein transgenic mouse model, a decrease in α-synuclein accumulation was detected in several brain regions following treatment, suggesting that mannitol promotes α-synuclein clearance in the cell bodies. It appears that mannitol has a general neuroprotective effect in the transgenic treated mice, which includes the dopaminergic system. We therefore suggest mannitol as a basis for a dual mechanism therapeutic agent for the treatment of Parkinson disease. PMID:23637226

  4. Long-term air pollution exposure is associated with neuroinflammation, an altered innate immune response, disruption of the blood-brain barrier, ultrafine particulate deposition, and accumulation of amyloid beta-42 and alpha-synuclein in children and young adults.

    PubMed

    Calderón-Garcidueñas, Lilian; Solt, Anna C; Henríquez-Roldán, Carlos; Torres-Jardón, Ricardo; Nuse, Bryan; Herritt, Lou; Villarreal-Calderón, Rafael; Osnaya, Norma; Stone, Ida; García, Raquel; Brooks, Diane M; González-Maciel, Angelica; Reynoso-Robles, Rafael; Delgado-Chávez, Ricardo; Reed, William

    2008-02-01

    Air pollution is a serious environmental problem. We investigated whether residency in cities with high air pollution is associated with neuroinflammation/neurodegeneration in healthy children and young adults who died suddenly. We measured mRNA cyclooxygenase-2, interleukin-1beta, and CD14 in target brain regions from low (n = 12) or highly exposed residents (n = 35) aged 25.1 +/- 1.5 years. Upregulation of cyclooxygenase-2, interleukin-1beta, and CD14 in olfactory bulb, frontal cortex, substantia nigrae and vagus nerves; disruption of the blood-brain barrier; endothelial activation, oxidative stress, and inflammatory cell trafficking were seen in highly exposed subjects. Amyloid beta42 (Abeta42) immunoreactivity was observed in 58.8% of apolipoprotein E (APOE) 3/3 < 25 y, and 100% of the APOE 4 subjects, whereas alpha-synuclein was seen in 23.5% of < 25 y subjects. Particulate material (PM) was seen in olfactory bulb neurons, and PM < 100 nm were observed in intraluminal erythrocytes from lung, frontal, and trigeminal ganglia capillaries. Exposure to air pollution causes neuroinflammation, an altered brain innate immune response, and accumulation of Abeta42 and alpha-synuclein starting in childhood. Exposure to air pollution should be considered a risk factor for Alzheimer's and Parkinson's diseases, and carriers of the APOE 4 allele could have a higher risk of developing Alzheimer's disease if they reside in a polluted environment.

  5. Overexpression of alpha(1)-acid glycoprotein in transgenic mice leads to sensitisation to acute colitis.

    PubMed

    Hochepied, T; Wullaert, A; Berger, F G; Baumann, H; Brouckaert, P; Steidler, L; Libert, C

    2002-09-01

    alpha(1)-Acid glycoprotein (alpha(1)-AGP) is an acute phase protein in most mammalian species whose concentration rises 2-5-fold during an acute phase reaction. Its serum concentration has often been used as a marker of disease, including inflammatory bowel disease (IBD). High alpha(1)-AGP levels were found to have a prognostic value for an increased risk of relapse in IBD. To investigate a possible role for increased serum levels of alpha(1)-AGP in the development of IBD. Dextran sodium sulphate (DSS) 2% was added to the drinking water of transgenic mice, overexpressing the rat alpha(1)-AGP gene, to induce acute colitis, thus mimicking the conditions of relapse. Clinical parameters, inflammatory parameters, and histological analyses on colon sections were performed. Homozygous alpha(1)-AGP-transgenic mice started losing weight and showed rectal bleeding significantly earlier than heterozygous transgenic or wild-type mice. Survival time of homozygous transgenic mice was significantly shorter compared with heterozygous and wild-type mice. The higher susceptibility of homozygous alpha(1)-AGP-transgenic mice to DSS induced acute colitis was also reflected in higher local myeloperoxidase levels, higher inflammation scores of the colon, and higher systemic levels of interleukin 6 and serum amyloid P component. Local inflammatory parameters were also significantly different in heterozygous transgenic mice compared with wild-type mice, indicating a local dosage effect. In homozygous transgenic mice, significantly higher amounts of bacteria were found in organs but IgA levels were only slightly lower than those of control mice. Sufficiently high serum levels of alpha(1)-AGP result in a more aggressive development of acute colitis.

  6. Spectral Line Polarisation Atlases for 53 Cam (A4p) and alpha 2 CVn (A0p)

    NASA Astrophysics Data System (ADS)

    Wade, G. A.

    2002-08-01

    Wade, Donati & Landstreet (2000) presented a atlas of the R=35,000 Stokes IQUV spectrum of the cool magnetic Ap star beta CrB in the spectral range 450-660 nm. In this report we present analogous atlases for the well-studied magnetic Ap stars 53 Cam (HD 65339, A4p) and alpha 2 CVn (HD 112413, A0p).

  7. 14 CFR 382.53 - What information must carriers give individuals with a vision or hearing impairment at airports?

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... foreign airport, to the extent that this does not interfere with employees' safety and security duties as... individuals with a vision or hearing impairment at airports? 382.53 Section 382.53 Aeronautics and Space... NONDISCRIMINATION ON THE BASIS OF DISABILITY IN AIR TRAVEL Accessibility of Airport Facilities § 382.53 What...

  8. 14 CFR 382.53 - What information must carriers give individuals with a vision or hearing impairment at airports?

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... foreign airport, to the extent that this does not interfere with employees' safety and security duties as... individuals with a vision or hearing impairment at airports? 382.53 Section 382.53 Aeronautics and Space... NONDISCRIMINATION ON THE BASIS OF DISABILITY IN AIR TRAVEL Accessibility of Airport Facilities § 382.53 What...

  9. 14 CFR 382.53 - What information must carriers give individuals with a vision or hearing impairment at airports?

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... foreign airport, to the extent that this does not interfere with employees' safety and security duties as... individuals with a vision or hearing impairment at airports? 382.53 Section 382.53 Aeronautics and Space... NONDISCRIMINATION ON THE BASIS OF DISABILITY IN AIR TRAVEL Accessibility of Airport Facilities § 382.53 What...

  10. The human T-cell leukemia virus type-1 p30II protein activates p53 and induces the TIGAR and suppresses oncogene-induced oxidative stress during viral carcinogenesis.

    PubMed

    Romeo, Megan; Hutchison, Tetiana; Malu, Aditi; White, Averi; Kim, Janice; Gardner, Rachel; Smith, Katie; Nelson, Katherine; Bergeson, Rachel; McKee, Ryan; Harrod, Carolyn; Ratner, Lee; Lüscher, Bernhard; Martinez, Ernest; Harrod, Robert

    2018-05-01

    In normal cells, aberrant oncogene expression leads to the accumulation of cytotoxic metabolites, including reactive oxygen species (ROS), which can cause oxidative DNA-damage and apoptosis as an intrinsic barrier against neoplastic disease. The c-Myc oncoprotein is overexpressed in many lymphoid cancers due to c-myc gene amplification and/or 8q24 chromosomal translocations. Intriguingly, p53 is a downstream target of c-Myc and hematological malignancies, such as adult T-cell leukemia/lymphoma (ATL), frequently contain wildtype p53 and c-Myc overexpression. We therefore hypothesized that p53-regulated pro-survival signals may thwart the cell's metabolic anticancer defenses to support oncogene-activation in lymphoid cancers. Here we show that the Tp53-induced glycolysis and apoptosis regulator (TIGAR) promotes c-myc oncogene-activation by the human T-cell leukemia virus type-1 (HTLV-1) latency-maintenance factor p30 II , associated with c-Myc deregulation in ATL clinical isolates. TIGAR prevents the intracellular accumulation of c-Myc-induced ROS and inhibits oncogene-induced cellular senescence in ATL, acute lymphoblastic leukemia, and multiple myeloma cells with elevated c-Myc expression. Our results allude to a pivotal role for p53-regulated antioxidant signals as mediators of c-Myc oncogenic functions in viral and non-viral lymphoid tumors. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. Parkinson's disease and α-synuclein expression.

    PubMed

    Devine, Michael J; Gwinn, Katrina; Singleton, Andrew; Hardy, John

    2011-10-01

    Genetic studies of Parkinson's disease over the last decade or more have revolutionized our understanding of this condition. α-Synuclein was the first gene to be linked to Parkinson's disease, and is arguably the most important: the protein is the principal constituent of Lewy bodies, and variation at its locus is the major genetic risk factor for sporadic disease. Intriguingly, duplications and triplications of the locus, as well as point mutations, cause familial disease. Therefore, subtle alterations of α-synuclein expression can manifest with a dramatic phenotype. We outline the clinical impact of α-synuclein locus multiplications, and the implications that this has for Parkinson's disease pathogenesis. Finally, we discuss potential strategies for disease-modifying therapies for this currently incurable disorder. Copyright © 2011 Movement Disorder Society.

  12. A neuroprotective role for angiogenin in models of Parkinson’s Disease

    PubMed Central

    Steidinger, Trent U.; Standaert, David G.; Yacoubian, Talene A.

    2010-01-01

    We previously observed marked downregulation of the mRNA for angiogenin, a potent inducer of neovascularization, in a mouse model of Parkinson’s disease (PD) based on overexpression of alpha-synuclein. Angiogenin has also been recently implicated in the pathogenesis of amyotrophic lateral sclerosis. In this study, we confirmed that mouse angiogenin-1 protein is dramatically reduced in this transgenic alpha-synuclein mouse model of PD, and examined the effect of angiogenin in cellular models of PD. We found that endogenous angiogenin is present in two dopamine-producing neuroblastoma cell lines, SH-SY5Y and M17, and that exogenous angiogenin is taken up by these cells and leads to phosphorylation of Akt. Applied angiogenin protects against the cell death induced by the neurotoxins MPP+ and rotenone and reduces the activation of caspase-3. Together our data supports the importance of angiogenin in protecting against dopaminergic neuronal cell death and suggests its potential as a therapy for PD. PMID:21091473

  13. p53 targets chromatin structure alteration to repress alpha-fetoprotein gene expression.

    PubMed

    Ogden, S K; Lee, K C; Wernke-Dollries, K; Stratton, S A; Aronow, B; Barton, M C

    2001-11-09

    Many of the functions ascribed to p53 tumor suppressor protein are mediated through transcription regulation. We have shown that p53 represses hepatic-specific alpha-fetoprotein (AFP) gene expression by direct interaction with a composite HNF-3/p53 DNA binding element. Using solid-phase, chromatin-assembled AFP DNA templates and analysis of chromatin structure and transcription in vitro, we find that p53 binds DNA and alters chromatin structure at the AFP core promoter to regulate transcription. Chromatin assembled in the presence of hepatoma extracts is activated for AFP transcription with an open, accessible core promoter structure. Distal (-850) binding of p53 during chromatin assembly, but not post-assembly, reverses transcription activation concomitant with promoter inaccessibility to restriction enzyme digestion. Inhibition of histone deacetylase activity by trichostatin-A (TSA) addition, prior to and during chromatin assembly, activated chromatin transcription in parallel with increased core promoter accessibility. Chromatin immunoprecipitation analyses showed increased H3 and H4 acetylated histones at the core promoter in the presence of TSA, while histone acetylation remained unchanged at the site of distal p53 binding. Our data reveal that p53 targets chromatin structure alteration at the core promoter, independently of effects on histone acetylation, to establish repressed AFP gene expression.

  14. Minnelide/Triptolide Impairs Mitochondrial Function by Regulating SIRT3 in P53-Dependent Manner in Non-Small Cell Lung Cancer.

    PubMed

    Kumar, Ajay; Corey, Catherine; Scott, Iain; Shiva, Sruti; D'Cunha, Jonathan

    2016-01-01

    Minnelide/Triptolide (TL) has recently emerged as a potent anticancer drug in non-small cell lung cancer (NSCLC). However, the precise mechanism of its action remains ambiguous. In this study, we elucidated the molecular basis for TL-induced cell death in context to p53 status. Cell death was attributed to dysfunction of mitochondrial bioenergetics in p53-deficient cells, which was characterized by decreased mitochondrial respiration, steady-state ATP level and membrane potential, but augmented reactive oxygen species (ROS). Increased ROS production resulted in oxidative stress in TL-treated cells. This was exhibited by elevated nuclear levels of a redox-sensitive transcriptional factor, NF-E2-related factor-2 (NRF2), along with diminished cellular glutathione (GSH) content. We further demonstrated that in the absence of p53, TL blunted the expression of mitochondrial SIRT3 triggering increased acetylation of NDUAF9 and succinate dehydrogenase, components of complexes I and II of the electron transport chain (ETC). TL-mediated hyperacetylation of complexes I and II proteins and these complexes displayed decreased enzymatic activities. We also provide the evidence that P53 regulate steady-state level of SIRT3 through Proteasome-Pathway. Finally, forced overexpression of Sirt3, but not deacetylase-deficient mutant of Sirt3 (H243Y), restored the deleterious effect of TL on p53-deficient cells by rescuing mitochondrial bioenergetics. On contrary, Sirt3 deficiency in the background of wild-type p53 triggered TL-induced mitochondrial impairment that echoed TL effect in p53-deficeint cells. These findings illustrate a novel mechanism by which TL exerts its potent effects on mitochondrial function and ultimately the viability of NSCLC tumor.

  15. 46 CFR 32.53-30 - Positive pressure-T/ALL.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Positive pressure-T/ALL. 32.53-30 Section 32.53-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS SPECIAL EQUIPMENT, MACHINERY, AND HULL REQUIREMENTS Inert Gas System § 32.53-30 Positive pressure—T/ALL. Each inert gas system must be designed to...

  16. 46 CFR 32.53-30 - Positive pressure-T/ALL.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 1 2011-10-01 2011-10-01 false Positive pressure-T/ALL. 32.53-30 Section 32.53-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS SPECIAL EQUIPMENT, MACHINERY, AND HULL REQUIREMENTS Inert Gas System § 32.53-30 Positive pressure—T/ALL. Each inert gas system must be designed to...

  17. α-Synuclein aggregation, seeding and inhibition by scyllo-inositol

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ibrahim, Tarek; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, M4N 3M5, ON; McLaurin, JoAnne, E-mail: jmclaurin@sri.utoronto.ca

    2016-01-15

    Recent literature demonstrates the accelerated aggregation of α-synuclein, a protein implicated in the pathogenesis of Parkinson's disease (PD), by the presence of preformed fibrillar conformers in vitro. Furthermore, these preformed fibrillar seeds are suggested to accelerate pathological induction in vivo when injected into the brains of mice. Variation in the results of in vivo studies is proposed to be caused by α-synuclein conformational variants. To investigate the impact of amino acid sequence on seeding efficiency, human and mouse α-synuclein seeds, which vary at 7 amino acid residues, were generated and cross-seeding kinetics studied. Using transmission electron microscopy (TEM), we confirmed that mouse α-synucleinmore » aggregated more rapidly than human α-synuclein. Subsequently, we determined that seeding of human and mouse α-synuclein was more rapid in the presence of seeds generated from the same species. In addition, an established amyloid inhibitor, scyllo-inositol, was examined for potential inhibitory effects on α-synuclein aggregation. TEM analysis of protein:inhibitor assays demonstrated that scyllo-inositol inhibits the aggregation of α-synuclein, suggesting the therapeutic potential of the small molecule in PD. - Highlights: • Mouse α-syn fibrillizes in a significantly shorter timeframe than human α-syn. • Seeding of monomers is more efficient when seeds originate from the same species. • scyllo-Inositol has anti-aggregation effects on mouse and human α-syn.« less

  18. Impaired coactivator activity of the Gly{sub 482} variant of peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) on mitochondrial transcription factor A (Tfam) promoter

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Choi, Yon-Sik; Hong, Jung-Man; Lim, Sunny

    2006-06-09

    Mitochondrial dysfunction may cause diabetes or insulin resistance. Peroxisome proliferation-activated receptor-{gamma} (PPAR-{gamma}) coactivator-1 {alpha} (PGC-1{alpha}) increases mitochondrial transcription factor A (Tfam) resulting in mitochondrial DNA content increase. An association between a single nucleotide polymorphism (SNP), G1444A(Gly482Ser), of PGC-1{alpha} coding region and insulin resistance has been reported in some ethnic groups. In this study, we investigated whether a change of glycine to serine at codon 482 of PGC-1{alpha} affected the Tfam promoter activity. The cDNA of PGC-1{alpha} variant bearing either glycine or serine at 482 codon was transfected into Chang human hepatocyte cells. The PGC-1{alpha} protein bearing glycine had impaired coactivatormore » activity on Tfam promoter-mediated luciferase. We analyzed the PGC-1{alpha} genotype G1444A and mitochondrial DNA (mtDNA) copy number from 229 Korean leukocyte genomic DNAs. Subjects with Gly/Gly had a 20% lower amount of peripheral blood mtDNA than did subjects with Gly/Ser and Ser/Ser (p < 0.05). No correlation was observed between diabetic parameters and PGC-1{alpha} genotypes in Koreans. These results suggest that PGC-1{alpha} variants with Gly/Gly at 482nd amino acid may impair the Tfam transcription, a regulatory function of mitochondrial biogenesis, resulting in dysfunctional mtDNA replication.« less

  19. Mitochondrial translocation of α-synuclein is promoted by intracellular acidification

    PubMed Central

    Cole, Nelson B.; DiEuliis, Diane; Leo, Paul; Mitchell, Drake C.; Nussbaum, Robert L.

    2008-01-01

    Mitochondrial dysfunction plays a central role in the selective vulnerability of dopaminergic neurons in Parkinson’s disease (PD) and is influenced by both environmental and genetic factors. Expression of the PD protein α-synuclein or its familial mutants often sensitizes neurons to oxidative stress and to damage by mitochondrial toxins. This effect is thought to be indirect, since little evidence physically linking α-synuclein to mitochondria has been reported. Here, we show that the distribution of α-synuclein within neuronal and non-neuronal cells is dependent on intracellular pH. Cytosolic acidification induces translocation of α-synuclein from the cytosol onto the surface of mitochondria. Translocation occurs rapidly under artificially-induced low pH conditions and as a result of pH changes during oxidative or metabolic stress. Binding is likely facilitated by low pH-induced exposure of the mitochondria-specific lipid cardiolipin. These results imply a direct role for α-synuclein in mitochondrial physiology, especially under pathological conditions, and in principle, link α-synuclein to other PD genes in regulating mitochondrial homeostasis. PMID:18440504

  20. Trehalose does not improve neuronal survival on exposure to alpha-synuclein pre-formed fibrils.

    PubMed

    Redmann, Matthew; Wani, Willayat Y; Volpicelli-Daley, Laura; Darley-Usmar, Victor; Zhang, Jianhua

    2017-04-01

    Parkinson's disease is a debilitating neurodegenerative disorder that is pathologically characterized by intracellular inclusions comprised primarily of alpha-synuclein (αSyn) that can also be transmitted from neuron to neuron. Several lines of evidence suggest that these inclusions cause neurodegeneration. Thus exploring strategies to improve neuronal survival in neurons with αSyn aggregates is critical. Previously, exposure to αSyn pre-formed fibrils (PFFs) has been shown to induce aggregation of endogenous αSyn resulting in cell death that is exacerbated by either starvation or inhibition of mTOR by rapamycin, both of which are able to induce autophagy, an intracellular protein degradation pathway. Since mTOR inhibition may also inhibit protein synthesis and starvation itself can be detrimental to neuronal survival, we investigated the effects of autophagy induction on neurons with αSyn inclusions by a starvation and mTOR-independent autophagy induction mechanism. We exposed mouse primary cortical neurons to PFFs to induce inclusion formation in the presence and absence of the disaccharide trehalose, which has been proposed to induce autophagy and stimulate lysosomal biogenesis. As expected, we observed that on exposure to PFFs, there was increased abundance of pS129-αSyn aggregates and cell death. Trehalose alone increased LC3-II levels, consistent with increased autophagosome levels that remained elevated with PFF exposure. Interestingly, trehalose alone increased cell viability over a 14-d time course. Trehalose was also able to restore cell viability to control levels, but PFFs still exhibited toxic effects on the cells. These data provide essential information regarding effects of trehalose on αSyn accumulation and neuronal survival on exposure to PFF. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Late stages of hematopoiesis and B cell lymphopoiesis are regulated by α-synuclein, a key player in Parkinson's disease.

    PubMed

    Xiao, Wenbin; Shameli, Afshin; Harding, Clifford V; Meyerson, Howard J; Maitta, Robert W

    2014-11-01

    α-Synuclein plays a crucial role in Parkinson's disease and dementias defined as synucleinopathies. α-Synuclein is expressed in hematopoietic and immune cells, but its functions in hematopoiesis and immune responses are unknown. We utilized α-synuclein(-/-) (KO) mice to investigate its role in hematopoiesis and B cell lymphopoiesis. We demonstrated hematologic abnormalities including mild anemia, smaller platelets, lymphopenia but relatively normal early hematopoiesis in KO mice compared to wild-type (WT) as measured in hematopoietic stem cells and progenitors of the different cell lineages. However, the absolute number of B220(+)IgM(+) B cells in bone marrow was reduced by 4-fold in KO mice (WT: 104±23×10(5) vs. KO: 27±5×10(5)). B cells were also reduced in KO spleens associated with effacement of splenic and lymph node architecture. KO mice showed reduced total serum IgG but no abnormality in serum IgM was noted. When KO mice were challenged with a T cell-dependent antigen, production of antigen specific IgG1 and IgG2b was abolished, but antigen specific IgM was not different from WT mice. Our study shows hematologic abnormalities including anemia and smaller platelets, reduced B cell lymphopoiesis and defects in IgG production in the absence of α-synuclein. This is the first report to show an important role of α-synuclein late in hematopoiesis, B cell lymphopoiesis and adaptive immune response. Copyright © 2014 Elsevier GmbH. All rights reserved.

  2. Overexpression of Escherichia coli udk mimics the absence of T7 Gp2 function and thereby abrogates successful infection by T7 phage.

    PubMed

    Shadrin, Andrey; Sheppard, Carol; Savalia, Dhruti; Severinov, Konstantin; Wigneshweraraj, Sivaramesh

    2013-02-01

    Successful infection of Escherichia coli by bacteriophage T7 relies upon the transcription of the T7 genome by two different RNA polymerases (RNAps). The bacterial RNAp transcribes early T7 promoters, whereas middle and late T7 genes are transcribed by the T7 RNAp. Gp2, a T7-encoded transcription factor, is a 7 kDa product of an essential middle T7 gene 2, and is a potent inhibitor of the host RNAp. The essential biological role of Gp2 is to inhibit transcription of early T7 genes that fail to terminate efficiently in order to facilitate the coordinated usage of the T7 genome by both host and phage RNAps. Overexpression of the E. coli udk gene, which encodes a uridine/cytidine kinase, interferes with T7 infection. We demonstrate that overexpression of udk antagonizes Gp2 function in E. coli in the absence of T7 infection and thus independently of T7-encoded factors. It seems that overexpression of udk reduces Gp2 stability and functionality during T7 infection, which consequently results in inadequate inhibition of host RNAp and in the accumulation of early T7 transcripts. In other words, overexpression of udk mimics the absence of Gp2 during T7 infection. Our study suggests that the transcriptional regulation of the T7 genome is surprisingly complex and might potentially be affected at many levels by phage- and host-encoded factors.

  3. Effects of different isoforms of apoE on aggregation of the α-synuclein protein implicated in Parkinson's disease.

    PubMed

    Emamzadeh, Fatemeh Nouri; Aojula, Harmesh; McHugh, Patrick C; Allsop, David

    2016-04-08

    Parkinson's disease is a progressive brain disorder due to the degeneration of dopaminergic neurons in the substantia nigra. The accumulation of aggregated forms of α-synuclein protein into Lewy bodies is one of the characteristic features of this disease although the pathological role of any such protein deposits in causing neurodegeneration remains elusive. Here, the effects of different apolipoprotein E isoforms (apoE2, apoE3, apoE4) on the aggregation of α-synuclein in vitro were examined using thioflavin T assays and also an immunoassay to detect the formation of multimeric forms. Our results revealed that the aggregation of α-synuclein is influenced by apoE concentration. At low concentrations of apoE (<15nM), all of the isoforms were able to increase the aggregation of α-synuclein (50μM), with apoE4 showing the greatest stimulatory effect. This is in contrast to a higher concentration (>15nM) of these isoforms, where a decrease in the aggregation of α-synuclein was noted. The data show that exceptionally low levels of apoE may seed α-syn aggregation, which could potentially lead to the pathogenesis of α-synuclein-induced neurodegeneration. On the other hand, higher levels of apoE could potentially lower the degree of α-synuclein aggregation and confer protection. The differential effects noted with apoE4 could explain why this particular isoform results in an earlier age of onset for Parkinson's disease. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes.

    PubMed

    Abounit, Saïda; Bousset, Luc; Loria, Frida; Zhu, Seng; de Chaumont, Fabrice; Pieri, Laura; Olivo-Marin, Jean-Christophe; Melki, Ronald; Zurzolo, Chiara

    2016-10-04

    Synucleinopathies such as Parkinson's disease are characterized by the pathological deposition of misfolded α-synuclein aggregates into inclusions throughout the central and peripheral nervous system. Mounting evidence suggests that intercellular propagation of α-synuclein aggregates may contribute to the neuropathology; however, the mechanism by which spread occurs is not fully understood. By using quantitative fluorescence microscopy with co-cultured neurons, here we show that α-synuclein fibrils efficiently transfer from donor to acceptor cells through tunneling nanotubes (TNTs) inside lysosomal vesicles. Following transfer through TNTs, α-synuclein fibrils are able to seed soluble α-synuclein aggregation in the cytosol of acceptor cells. We propose that donor cells overloaded with α-synuclein aggregates in lysosomes dispose of this material by hijacking TNT-mediated intercellular trafficking. Our findings thus reveal a possible novel role of TNTs and lysosomes in the progression of synucleinopathies. © 2016 The Authors.

  5. Regulation of Id2 expression in EL4 T lymphoma cells overexpressing growth hormone.

    PubMed

    Weigent, Douglas A

    2009-01-01

    In previous studies, we have shown that overexpression of growth hormone (GH) in cells of the immune system upregulates proteins involved in cell growth and protects from apoptosis. Here, we report that overexpression of GH in EL4 T lymphoma cells (GHo) also significantly increased levels of the inhibitor of differentiation-2 (Id2). The increase in Id2 was suggested in both Id2 promoter luciferase assays and by Western analysis for Id2 protein. To identify the regulatory elements that mediate transcriptional activation by GH in the Id2 promoter, promoter deletion analysis was performed. Deletion analysis revealed that transactivation involved a 301-132bp region upstream to the Id2 transcriptional start site. The pattern in the human GHo Jurkat T lymphoma cell line paralleled that found in the mouse GHo EL4 T lymphoma cell line. Significantly less Id2 was detected in the nucleus of GHo EL4 T lymphoma cells compared to vector alone controls. Although serum increased the levels of Id2 in control vector alone cells, no difference was found in the total levels of Id2 in GHo EL4 T lymphoma cells treated with or without serum. The increase in Id2 expression in GHo EL4 T lymphoma cells measured by Id2 promoter luciferase expression and Western blot analysis was blocked by the overexpression of a dominant-negative mutant of STAT5. The results suggest that in EL4 T lymphoma cells overexpressing GH, there is an upregulation of Id2 protein that appears to involve STAT protein activity.

  6. SIRT1 deacetylase is overexpressed in human melanoma and its small molecule inhibition imparts anti-proliferative response via p53 activation.

    PubMed

    Wilking, Melissa J; Singh, Chandra; Nihal, Minakshi; Zhong, Weixiong; Ahmad, Nihal

    2014-12-01

    Melanoma causes more deaths than any other skin cancer, and its incidence in the US continues to rise. Current medical therapies are insufficient to control this deadly neoplasm, necessitating the development of new target-based approaches. The objective of this study was to determine the role and functional significance of the class III histone deacetylase SIRT1 in melanoma. We have found that SIRT1 is overexpressed in clinical human melanoma tissues and human melanoma cell lines (Sk-Mel-2, WM35, G361, A375, and Hs294T) compared to normal skin and normal melanocytes, respectively. In addition, treatment of melanoma cell lines A375, Hs294T, and G361 with Tenovin-1, a small molecule SIRT1 inhibitor, resulted in a significant decrease in cell growth and cell viability. Further, Tenovin-1 treatment also resulted in a marked decrease in the clonogenic survival of melanoma cells. Further experiments showed that the anti-proliferative response of Tenovin-1 was accompanied by an increase in the protein as well as activity of the tumor suppressor p53. This increase in p53 activity was substantiated by an increase in the protein level of its downstream target p21. Overall, these data suggest that small molecule inhibition of SIRT1 causes anti-proliferative effects in melanoma cells. SIRT1 appears to be acting through the activity of the tumor suppressor p53, which is not mutated in the majority of melanomas. However, future detailed studies are needed to further explore the role and mechanism of SIRT1 in melanoma development and progression and its usefulness in melanoma treatment.

  7. Calcium accelerates SNARE-mediated lipid mixing through modulating α-synuclein membrane interaction.

    PubMed

    Zhang, Zeting; Jiang, Xin; Xu, Danrui; Zheng, Wenwen; Liu, Maili; Li, Conggang

    2018-04-04

    α-Synuclein is involved in Parkinson's disease, and its interaction with cell membrane is vital to its pathological and physiological functions. We have shown that Ca 2+ can regulate α-synuclein membrane interaction, but the physiological role of Ca 2+ in modulating α-synuclein membrane interaction is still unexplored. Based on the previous findings that α-synuclein inhibits membrane fusion and its inhibitory effect is highly related to its membrane binding, here we employed solution state Nuclear Magnetic Resonance (NMR) spectroscopy and the ensemble fluorescence fusion assay to show that Ca 2+ can modulate the inhibitory effect of α-synuclein on SNARE-mediated membrane fusion through disrupting α-synuclein membrane interaction, resulting in acceleration of SNARE-mediated membrane fusion. These results suggest a modulatory effect of Ca 2+ on membrane mediated normal function of α-synuclein, which of importance for the study of the Parkinson's disease. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Overexpressed ubiquitin ligase Cullin7 in breast cancer promotes cell proliferation and invasion via down-regulating p53

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Guo, Hongsheng; Wu, Fenping; Wang, Yan

    2014-08-08

    Highlights: • Cullin7 is overexpressed in human breast cancer samples. • Cullin7 stimulated proliferation and invasion of breast cancer cells. • Inhibition of p53 contributes to Cullin7-induced proliferation and invasion. - Abstract: Ubiquitin ligase Cullin7 has been identified as an oncogene in some malignant diseases such as choriocarcinoma and neuroblastoma. However, the role of Cullin7 in breast cancer carcinogenesis remains unclear. In this study, we compared Cullin7 protein levels in breast cancer tissues with normal breast tissues and identified significantly higher expression of Cullin7 protein in breast cancer specimens. By overexpressing Cullin7 in breast cancer cells HCC1937, we found thatmore » Cullin7 could promote cell growth and invasion in vitro. In contrast, the cell growth and invasion was inhibited by silencing Cullin7 in breast cancer cell BT474. Moreover, we demonstrated that Cullin7 promoted breast cancer cell proliferation and invasion via down-regulating p53 expression. Thus, our study provided evidence that Cullin7 functions as a novel oncogene in breast cancer and may be a potential therapeutic target for breast cancer management.« less

  9. Plasma exosomal α-synuclein is likely CNS-derived and increased in Parkinson’s disease

    PubMed Central

    Cook, Travis J.; Bullock, Kristin M.; Zhao, Yanchun; Ginghina, Carmen; Li, Yanfei; Aro, Patrick; Dator, Romel; He, Chunmei; Hipp, Michael J.; Zabetian, Cyrus P.; Peskind, Elaine R.; Hu, Shu-Ching; Quinn, Joseph F.; Galasko, Douglas R.; Banks, William A.; Zhang, Jing

    2014-01-01

    Extracellular α-synuclein is important in the pathogenesis of Parkinson disease (PD) and also as a potential biomarker when tested in the cerebrospinal fluid (CSF). The performance of blood plasma or serum α-synuclein as a biomarker has been found to be inconsistent and generally ineffective, largely due to the contribution of peripherally derived α-synuclein. In this study, we discovered, via an intracerebroventricular injection of radiolabeled α-synuclein into mouse brain, that CSF α-synuclein was readily transported to blood, with a small portion being contained in exosomes that are relatively specific to the central nervous system (CNS). Consequently, we developed a technique to evaluate the levels of α-synuclein in these exosomes in individual plasma samples. When applied to a large cohort of clinical samples (267 PD, 215 controls), we found that in contrast to CSF α-synuclein concentrations, which are consistently reported to be lower in PD patients compared to controls, the levels of plasma exosomal α-synuclein were substantially higher in PD patients, suggesting an increased efflux of the protein to the peripheral blood of these patients. Furthermore, although no association was observed between plasma exosomal and CSF α-synuclein, a significant correlation between plasma exosomal α-synuclein and disease severity (r=0.176, p=0.004) was observed, and the diagnostic sensitivity and specificity achieved by plasma exosomal α-synuclein were comparable to those determined by CSF α-synuclein. Further studies are clearly needed to elucidate the mechanism involved in the transport of CNS α-synuclein to the periphery, which may lead to a more convenient and robust assessment of PD clinically. PMID:24997849

  10. Parkinson’s Disease and α-synuclein Expression

    PubMed Central

    Devine, Michael J.; Gwinn, Katrina; Singleton, Andrew; Hardy, John

    2015-01-01

    Genetic studies of Parkinson’s disease over the last decade or more have revolutionized our understanding of this condition. α-Synuclein was the first gene to be linked to Parkinson’s disease, and is arguably the most important: the protein is the principal constituent of Lewy bodies, and variation at its locus is the major genetic risk factor for sporadic disease. Intriguingly, duplications and triplications of the locus, as well as point mutations, cause familial disease. Therefore, subtle alterations of α-synuclein expression can manifest with a dramatic phenotype. We outline the clinical impact of α-synuclein locus multiplications, and the implications that this has for Parkinson’s disease pathogenesis. Finally, we discuss potential strategies for disease-modifying therapies for this currently incurable disorder. PMID:21887711

  11. Reduction of the tumorigenic potential of human retinoblastoma cell lines by TFF1 overexpression involves p53/caspase signaling and miR-18a regulation.

    PubMed

    Busch, Maike; Große-Kreul, Jan; Wirtz, Janina Jasmin; Beier, Manfred; Stephan, Harald; Royer-Pokora, Brigitte; Metz, Klaus; Dünker, Nicole

    2017-08-01

    Trefoil factor family (TFF) peptides have been shown to play a pivotal role in oncogenic transformation, tumorigenesis and metastasis by changing cell proliferation, apoptosis, migration and invasion behavior of various cancer cell lines. In the study presented, we investigated the effect of TFF1 overexpression on cell growth, viability, migration and tumorigenicity of different retinoblastoma (RB) cell lines. Transient TFF1 overexpression significantly increases RB cell apoptosis levels. Stable, lentiviral TFF1 overexpression likewise decreases RB cell viability, proliferation and growth and significantly increases apoptosis as revealed by WST-1 assays, BrdU and DAPI cell counts. TFF1-induced apoptosis is executed via cleaved caspase-3 activation as revealed by caspase blockage experiments and caspase-3 immunocytochemistry. Results from pG13-luciferase reporter assays and Western blot analyses indicate that TFF1-induced apoptosis is mediated through transcriptional activity of p53 with concurrently downregulated miR-18a expression. In ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF1 overexpression significantly decreases the size of tumors forming from Y79 and RB355 cells and reduces the migration potential of RB355 cells. Differentially expressed genes and pathways involved in cancer progression were identified after TFF1 overexpression in Y79 cells by gene expression array analysis, underlining the effects on reduced tumorigenicity. TFF1 knockdown in RBL30 cells revealed caspase-3/7-independent apoptosis induction, but no changes on cell proliferation level. In summary, the in vitro and in vivo data demonstrate for the first time a tumor suppressor function of TFF1 in RB cells which is at least partly mediated by p53 activation and miR-18a downregulation. © 2017 UICC.

  12. Broad neutralization of calcium-permeable amyloid pore channels with a chimeric Alzheimer/Parkinson peptide targeting brain gangliosides.

    PubMed

    Di Scala, Coralie; Yahi, Nouara; Flores, Alessandra; Boutemeur, Sonia; Kourdougli, Nazim; Chahinian, Henri; Fantini, Jacques

    2016-02-01

    Growing evidence supports a role for brain gangliosides in the pathogenesis of neurodegenerative diseases including Alzheimer's and Parkinson's. Recently we deciphered the ganglioside-recognition code controlling specific ganglioside binding to Alzheimer's β-amyloid (Aβ1-42) peptide and Parkinson's disease-associated protein α-synuclein. Cracking this code allowed us to engineer a short chimeric Aβ/α-synuclein peptide that recognizes all brain gangliosides. Here we show that ganglioside-deprived neural cells do no longer sustain the formation of zinc-sensitive amyloid pore channels induced by either Aβ1-42 or α-synuclein, as assessed by single-cell Ca(2+) fluorescence microscopy. Thus, amyloid channel formation, now considered a key step in neurodegeneration, is a ganglioside-dependent process. Nanomolar concentrations of chimeric peptide competitively inhibited amyloid pore formation induced by Aβ1-42 or α-synuclein in cultured neural cells. Moreover, this peptide abrogated the intracellular calcium increases induced by Parkinson's-associated mutant forms of α-synuclein (A30P, E46K and A53T). The chimeric peptide also prevented the deleterious effects of Aβ1-42 on synaptic vesicle trafficking and decreased the Aβ1-42-induced impairment of spontaneous activity in rat hippocampal slices. Taken together, these data show that the chimeric peptide has broad anti-amyloid pore activity, suggesting that a common therapeutic strategy based on the prevention of amyloid-ganglioside interactions is a reachable goal for both Alzheimer's and Parkinson's diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Neuron-to-neuron transmission of α-synuclein fibrils through axonal transport

    PubMed Central

    Freundt, Eric C.; Maynard, Nate; Clancy, Eileen K.; Roy, Shyamali; Bousset, Luc; Sourigues, Yannick; Covert, Markus; Melki, Ronald; Kirkegaard, Karla; Brahic, Michel

    2012-01-01

    Objective The lesions of Parkinson's disease spread through the brain in a characteristic pattern that corresponds to axonal projections. Previous observations suggest that misfolded α-synuclein could behave as a prion, moving from neuron to neuron and causing endogenous α-synuclein to misfold. Here, we characterized and quantified the axonal transport of α-synuclein fibrils and showed that fibrils could be transferred from axons to second-order neurons following anterograde transport. Methods We grew primary cortical mouse neurons in microfluidic devices to separate soma from axonal projections in fluidically isolated microenvironments. We used live-cell imaging and immunofluorescence to characterize the transport of fluorescent α-synuclein fibrils and their transfer to second-order neurons. Results Fibrillar α-synuclein was internalized by primary neurons and transported in axons with kinetics consistent with slow component-b of axonal transport (fast axonal transport with saltatory movement). Fibrillar α-synuclein was readily observed in the cell bodies of second-order neurons following anterograde axonal transport. Axon-to-soma transfer appeared not to require synaptic contacts. Interpretation These results support the hypothesis that the progression of Parkinson's disease can be caused by neuron-to-neuron spread of α-synuclein aggregates and that the anatomical pattern of progression of lesions between axonally connected areas results from the axonal transport of such aggregates. That the transfer did not appear to be transsynaptic gives hope that α-synuclein fibrils could be intercepted by drugs during the extra-cellular phase of their journey. PMID:23109146

  14. Geraniol attenuates α-synuclein expression and neuromuscular impairment through increase dopamine content in MPTP intoxicated mice by dose dependent manner.

    PubMed

    Rekha, Karamkolly R; Selvakumar, Govindasamy P; Santha, Karunanithi; Inmozhi Sivakamasundari, Ramu

    2013-11-01

    Parkinson's disease (PD) is characterized by progressive loss of dopamine (DA) neurons in the nigrostriatal system and by the presence of Lewy bodies (LB), proteinaceous inclusions mainly composed of filamentous α-synuclein (α-Syn) aggregates. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was adopted to generate PD models in C57BL/6 mice. In the present study, we investigated the effect of geraniol (GE) against α-Syn aggregation on MPTP induced mouse model of PD in dose dependant manner. When pretreatment of GE improved neuromuscular impairment, TH expressions and decreases α-Syn expressions in MPTP intoxicated PD mice by dose dependent manner. In addition, we confirmed that sub-chronic administration of MPTP in mice leads to permanent neuromuscular deficits and depletion of dopamine and its metabolites. Our results suggest that GE is beneficial for the treatment of PD associated with neuromuscular disability and LB aggregation. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

  15. α-Synuclein binds the KATP channel at insulin-secretory granules and inhibits insulin secretion

    PubMed Central

    Geng, Xuehui; Lou, Haiyan; Wang, Jian; Li, Lehong; Swanson, Alexandra L.; Sun, Ming; Beers-Stolz, Donna; Watkins, Simon; Perez, Ruth G.

    2011-01-01

    α-Synuclein has been studied in numerous cell types often associated with secretory processes. In pancreatic β-cells, α-synuclein might therefore play a similar role by interacting with organelles involved in insulin secretion. We tested for α-synuclein localizing to insulin-secretory granules and characterized its role in glucose-stimulated insulin secretion. Immunohistochemistry and fluorescent sulfonylureas were used to test for α-synuclein localization to insulin granules in β-cells, immunoprecipitation with Western blot analysis for interaction between α-synuclein and KATP channels, and ELISA assays for the effect of altering α-synuclein expression up or down on insulin secretion in INS1 cells or mouse islets, respectively. Differences in cellular phenotype between α-synuclein knockout and wild-type β-cells were found by using confocal microscopy to image the fluorescent insulin biosensor Ins-C-emGFP and by using transmission electron microscopy. The results show that anti-α-synuclein antibodies labeled secretory organelles within β-cells. Anti-α-synuclein antibodies colocalized with KATP channel, anti-insulin, and anti-C-peptide antibodies. α-Synuclein coimmunoprecipitated in complexes with KATP channels. Expression of α-synuclein downregulated insulin secretion at 2.8 mM glucose with little effect following 16.7 mM glucose stimulation. α-Synuclein knockout islets upregulated insulin secretion at 2.8 and 8.4 mM but not 16.7 mM glucose, consistent with the depleted insulin granule density at the β-cell surface membranes observed in these islets. These findings demonstrate that α-synuclein interacts with KATP channels and insulin-secretory granules and functionally acts as a brake on secretion that glucose stimulation can override. α-Synuclein might play similar roles in diabetes as it does in other degenerative diseases, including Alzheimer's and Parkinson's diseases. PMID:20858756

  16. [Targeted inactivation of gamma-synuclein gene affects anxiety and exploratory behaviour of mice].

    PubMed

    Kokhan, V S; Bolkunov, A V; Ustiugov, A A; Van'kin, G I; Shelkovnikova, T A; Redkozubova, O M; Strekalova, T V; Bukhman, V L; Ninkina, N N; Bachurin, S O

    2011-01-01

    Gamma(gamma)-synuclein is a member of synuclein family of cytoplasmic and predominantly neuronal proteins found only in vertebrates. Gamma-synuclein is abundant in axons and presynaptic terminals of neurons localized in brain regions involved in emotions, learning and memory. However, the role of gamma-synuclein in these brain functions was not previously assessed. We have demonstrated for the first time that the loss of gamma-synuclein results in a significant increase in the level of orientation response in novel environment and decrease in the level of state anxiety.

  17. A stapled p53 helix overcomes HDMX-mediated suppression of p53.

    PubMed

    Bernal, Federico; Wade, Mark; Godes, Marina; Davis, Tina N; Whitehead, David G; Kung, Andrew L; Wahl, Geoffrey M; Walensky, Loren D

    2010-11-16

    Cancer cells neutralize p53 by deletion, mutation, proteasomal degradation, or sequestration to achieve a pathologic survival advantage. Targeting the E3 ubiquitin ligase HDM2 can lead to a therapeutic surge in p53 levels. However, the efficacy of HDM2 inhibition can be compromised by overexpression of HDMX, an HDM2 homolog that binds and sequesters p53. Here, we report that a stapled p53 helix preferentially targets HDMX, blocks the formation of inhibitory p53-HDMX complexes, induces p53-dependent transcriptional upregulation, and thereby overcomes HDMX-mediated cancer resistance in vitro and in vivo. Importantly, our analysis of p53 interaction dynamics provides a blueprint for reactivating the p53 pathway in cancer by matching HDM2, HDMX, or dual inhibitors to the appropriate cellular context. Copyright © 2010 Elsevier Inc. All rights reserved.

  18. Targeting α-synuclein oligomers by protein-fragment complementation for drug discovery in synucleinopathies.

    PubMed

    Moussaud, Simon; Malany, Siobhan; Mehta, Alka; Vasile, Stefan; Smith, Layton H; McLean, Pamela J

    2015-05-01

    Reducing the burden of α-synuclein oligomeric species represents a promising approach for disease-modifying therapies against synucleinopathies such as Parkinson's disease and dementia with Lewy bodies. However, the lack of efficient drug discovery strategies that specifically target α-synuclein oligomers has been a limitation to drug discovery programs. Here we describe an innovative strategy that harnesses the power of bimolecular protein-fragment complementation to monitor synuclein-synuclein interactions. We have developed two robust models to monitor α-synuclein oligomerization by generating novel stable cell lines expressing α-synuclein fusion proteins for either fluorescent or bioluminescent protein-fragment complementation under the tetracycline-controlled transcriptional activation system. A pilot screen was performed resulting in the identification of two potential hits, a p38 MAPK inhibitor and a casein kinase 2 inhibitor, thereby demonstrating the suitability of our protein-fragment complementation assay for the measurement of α-synuclein oligomerization in living cells at high throughput. The application of the strategy described herein to monitor α-synuclein oligomer formation in living cells with high throughput will facilitate drug discovery efforts for disease-modifying therapies against synucleinopathies and other proteinopathies.

  19. TP53INP1 is a novel p73 target gene that induces cell cycle arrest and cell death by modulating p73 transcriptional activity.

    PubMed

    Tomasini, Richard; Seux, Mylène; Nowak, Jonathan; Bontemps, Caroline; Carrier, Alice; Dagorn, Jean-Charles; Pébusque, Marie-Josèphe; Iovanna, Juan L; Dusetti, Nelson J

    2005-12-08

    TP53INP1 is an alternatively spliced gene encoding two nuclear protein isoforms (TP53INP1alpha and TP53INP1beta), whose transcription is activated by p53. When overexpressed, both isoforms induce cell cycle arrest in G1 and enhance p53-mediated apoptosis. TP53INP1s also interact with the p53 gene and regulate p53 transcriptional activity. We report here that TP53INP1 expression is induced during experimental acute pancreatitis in p53-/- mice and in cisplatin-treated p53-/- mouse embryo fibroblasts (MEFs). We demonstrate that ectopic expression of p73, a p53 homologue, leads to TP53INP1 induction in p53-deficient cells. In turn, TP53INP1s alters the transactivation capacity of p73 on several p53-target genes, including TP53INP1 itself, demonstrating a functional association between p73 and TP53INP1s. Also, when overexpressed in p53-deficient cells, TP53INP1s inhibit cell growth and promote cell death as assessed by cell cycle analysis and colony formation assays. Finally, we show that TP53INP1s potentiate the capacity of p73 to inhibit cell growth, that effect being prevented when the p53 mutant R175H is expressed or when p73 expression is blocked by a siRNA. These results suggest that TP53INP1s are functionally associated with p73 to regulate cell cycle progression and apoptosis, independently from p53.

  20. Cardiac-specific overexpression of sarcolipin inhibits sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2a) activity and impairs cardiac function in mice

    PubMed Central

    Asahi, Michio; Otsu, Kinya; Nakayama, Hiroyuki; Hikoso, Shungo; Takeda, Toshihiro; Gramolini, Anthony O.; Trivieri, Maria G.; Oudit, Gavin Y.; Morita, Takashi; Kusakari, Yoichiro; Hirano, Shuta; Hongo, Kenichi; Hirotani, Shinichi; Yamaguchi, Osamu; Peterson, Alan; Backx, Peter H.; Kurihara, Satoshi; Hori, Masatsugu; MacLennan, David H.

    2004-01-01

    Sarcolipin (SLN) inhibits the cardiac sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2a) by direct binding and is superinhibitory if it binds through phospholamban (PLN). To determine whether overexpression of SLN in the heart might impair cardiac function, transgenic (TG) mice were generated with cardiac-specific overexpression of NF-SLN (SLN tagged at its N terminus with the FLAG epitope). The level of NF-SLN expression (the NF-SLN/PLN expression ratio) was equivalent to that which induces profound superinhibition when coexpressed with PLN and SERCA2a in HEK-293 cells. In TG hearts, the apparent affinity of SERCA2a for Ca2+ was decreased compared with non-TG littermate control hearts. Invasive hemodynamic and echocardiographic analyses revealed impaired cardiac contractility and ventricular hypertrophy in TG mice. Basal PLN phosphorylation was reduced. In isolated papillary muscle subjected to isometric tension, peak amplitudes of Ca2+ transients and peak tensions were reduced, whereas decay times of Ca2+ transients and relaxation times of tension were increased in TG mice. Isoproterenol largely restored contractility in papillary muscle and stimulated PLN phosphorylation to wild-type levels in intact hearts. No compensatory changes in expression of SERCA2a, PLN, ryanodine receptor, and calsequestrin were observed in TG hearts. Coimmunoprecipitation indicated that overexpressed NF-SLN was bound to both SERCA2a and PLN, forming a ternary complex. These data suggest that NF-SLN overexpression inhibits SERCA2a through stabilization of SERCA2a–PLN interaction in the absence of PLN phosphorylation and through the inhibition of PLN phosphorylation. Inhibition of SERCA2a impairs contractility and calcium cycling, but responsiveness to β-adrenergic agonists may prevent progression to heart failure. PMID:15201433

  1. Biophysical characterization of α-synuclein and its controversial structure

    PubMed Central

    Alderson, T Reid; Markley, John L

    2013-01-01

    α-synuclein, a presynaptic protein of poorly defined function, constitutes the main component of Parkinson disease-associated Lewy bodies. Extensive biophysical investigations have provided evidence that isolated α-synuclein is an intrinsically disordered protein (IDP) in vitro. Subsequently serving as a model IDP in numerous studies, α-synuclein has aided in the development of many technologies used to characterize IDPs and arguably represents the most thoroughly analyzed IDP to date. Recent reports, however, have challenged the disordered nature of α-synuclein inside cells and have instead proposed a physiologically relevant helical tetramer. Despite α-synuclein’s rich biophysical history, a single coherent picture has not yet emerged concerning its in vivo structure, dynamics, and physiological role(s). We present herein a review of the biophysical discoveries, developments, and models pertinent to the characterization of α-synuclein’s structure and analysis of the native tetramer controversy. PMID:24634806

  2. Evidence for Intramolecular Antiparallel Beta-Sheet Structure in Alpha-Synuclein Fibrils from a Combination of Two-Dimensional Infrared Spectroscopy and Atomic Force Microscopy

    NASA Astrophysics Data System (ADS)

    Roeters, Steven J.; Iyer, Aditya; Pletikapić, Galja; Kogan, Vladimir; Subramaniam, Vinod; Woutersen, Sander

    2017-01-01

    The aggregation of the intrinsically disordered protein alpha-synuclein (αS) into amyloid fibrils is thought to play a central role in the pathology of Parkinson’s disease. Using a combination of techniques (AFM, UV-CD, XRD, and amide-I 1D- and 2D-IR spectroscopy) we show that the structure of αS fibrils varies as a function of ionic strength: fibrils aggregated in low ionic-strength buffers ([NaCl] ≤ 25 mM) have a significantly different structure than fibrils grown in higher ionic-strength buffers. The observations for fibrils aggregated in low-salt buffers are consistent with an extended conformation of αS molecules, forming hydrogen-bonded intermolecular β-sheets that are loosely packed in a parallel fashion. For fibrils aggregated in high-salt buffers (including those prepared in buffers with a physiological salt concentration) the measurements are consistent with αS molecules in a more tightly-packed, antiparallel intramolecular conformation, and suggest a structure characterized by two twisting stacks of approximately five hydrogen-bonded intermolecular β-sheets each. We find evidence that the high-frequency peak in the amide-I spectrum of αS fibrils involves a normal mode that differs fundamentally from the canonical high-frequency antiparallel β-sheet mode. The high sensitivity of the fibril structure to the ionic strength might form the basis of differences in αS-related pathologies.

  3. Down-regulation of natural resistance-associated macrophage protein-1 (Nramp1) is associated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/1-methyl-4-phenylpyridinium (MPP+ )-induced α-synuclein accumulation and neurotoxicity.

    PubMed

    Wu, K-C; Liou, H-H; Lee, C-Y; Lin, C-J

    2018-04-21

    The accumulation of α-synuclein is a hallmark in the pathogenesis of Parkinson's disease (PD). Natural resistance-associated macrophage protein-1 (Nramp1) was previously shown to contribute to the degradation of extracellular α-synuclein in microglia under conditions of iron overload. This study was aimed at investigating the role of Nramp1 in α-synuclein pathology in the neurone under 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/1-methyl-4-phenylpyridinium (MPP + ) treatment. The expression of Nramp1 and pathological features (including iron and α-synuclein accumulation) were examined in the dopaminergic neurones of humans (with and without PD) and of mice [with and without receiving chronic MPTP intoxication]. The effects of Nramp1 expression on low-dose MPP + -induced α-synuclein expression and neurotoxicity were determined in human dopaminergic neuroblastoma SH-SY5Y cells. Similar to the findings in the substantia nigra of human PD, lower expression of Nramp1 but higher levels of iron and α-synuclein were identified in the dopaminergic neurones of mice receiving chronic MPTP intoxication, compared to controls. In parallel to the loss of dopaminergic neurones, the numbers of glial fibrillary acidic protein- and ionized calcium-binding adapter molecule-1-positive cells were significantly increased in the substantia nigra of MPTP-treated mice. Likewise, in human neuroblastoma SH-SY5Y cells exposed to low-dose MPP + , Nramp1 expression and cathepsin D activity were decreased, along with an increase in α-synuclein protein expression and aggregation. Overexpression of functional Nramp1 restored cathepsin D activity and attenuated α-synuclein up-regulation and neuronal cell death caused by MPP + treatment. These data suggest that the neuronal expression of Nramp1 is important for protecting against the development of MPTP/MPP + -induced α-synuclein pathology and neurotoxicity. © 2018 British Neuropathological Society.

  4. Recombinant α- β- and γ-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays

    PubMed Central

    Lek, Sovanarak; Vargas-Medrano, Javier; Villanueva, Ernesto; Marcus, Brian; Godfrey, Wesley; Perez, Ruth G.

    2017-01-01

    α-Synuclein (aSyn), β-Synuclein (bSyn), and γ-Synuclein (gSyn) are members of a conserved family of chaperone-like proteins that are highly expressed in vertebrate neuronal tissues. Of the three synucleins, only aSyn has been strongly implicated in neurodegenerative disorders such as Parkinson's disease, Dementia with Lewy Bodies, and Multiple System Atrophy. In studying normal aSyn function, data indicate that aSyn stimulates the activity of the catalytic subunit of an abundantly expressed dephosphorylating enzyme, PP2Ac in vitro and in vivo. Prior data show that aSyn aggregation in human brain reduces PP2Ac activity in regions with Lewy body pathology, where soluble aSyn has become insoluble. However, because all three synucleins have considerable homology in the amino acid sequences, experiments were designed to test if all can modulate PP2Ac activity. Using recombinant synucleins and recombinant PP2Ac protein, activity was assessed by malachite green colorimetric assay. Data revealed that all three recombinant synucleins stimulated PP2Ac activity in cell-free assays, raising the possibility that the conserved homology between synucleins may endow all three homologs with the ability to bind to and activate the PP2Ac. Co-immunoprecipitation data, however, suggest that PP2Ac modulation likely occurs through endogenous interactions between aSyn and PP2Ac in vivo. PMID:28829427

  5. In Situ Proximity Ligation Assay Reveals Co-Localization of Alpha-Synuclein and SNARE Proteins in Murine Primary Neurons.

    PubMed

    Almandoz-Gil, Leire; Persson, Emma; Lindström, Veronica; Ingelsson, Martin; Erlandsson, Anna; Bergström, Joakim

    2018-01-01

    The aggregation of alpha-synuclein (αSyn) is the pathological hallmark of Parkinson's disease, dementia with Lewy bodies and related neurological disorders. However, the physiological function of the protein and how this function relates to its pathological effects remain poorly understood. One of the proposed roles of αSyn is to promote the soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly by binding to VAMP-2. The objective of this study was to visualize the co-localization between αSyn and the SNARE proteins (VAMP-2, SNAP-25, and syntaxin-1) for the first time using in situ proximity ligation assay (PLA). Cortical primary neurons were cultured from either non-transgenic or transgenic mice expressing human αSyn with the A30P mutation under the Thy-1 promoter. With an antibody recognizing both mouse and human αSyn, a PLA signal indicating close proximity between αSyn and the three SNARE proteins was observed both in the soma and throughout the processes. No differences in the extent of PLA signals were seen between non-transgenic and transgenic neurons. With an antibody specific against human αSyn, the PLA signal was mostly located to the soma and was only present in a few cells. Taken together, in situ PLA is a method that can be used to investigate the co-localization of αSyn and the SNARE proteins in primary neuronal cultures.

  6. α-Synuclein interferes with the ESCRT-III complex contributing to the pathogenesis of Lewy body disease.

    PubMed

    Spencer, Brian; Kim, Changyoun; Gonzalez, Tania; Bisquertt, Alejandro; Patrick, Christina; Rockenstein, Edward; Adame, Anthony; Lee, Seung-Jae; Desplats, Paula; Masliah, Eliezer

    2016-03-15

    α-Synuclein (α-syn) has been implicated in neurological disorders with parkinsonism, including Parkinson's disease and Dementia with Lewy body. Recent studies have shown α-syn oligomers released from neurons can propagate from cell-to-cell in a prion-like fashion exacerbating neurodegeneration. In this study, we examined the role of the endosomal sorting complex required for transport (ESCRT) pathway on the propagation of α-syn. α-syn, which is transported via the ESCRT pathway through multivesicular bodies for degradation, can also target the degradation of the ESCRT protein-charged multivesicular body protein (CHMP2B), thus generating a roadblock of endocytosed α-syn. Disruption of the ESCRT transport system also resulted in increased exocytosis of α-syn thus potentially increasing cell-to-cell propagation of synuclein. Conversely, delivery of a lentiviral vector overexpressing CHMP2B rescued the neurodegeneration in α-syn transgenic mice. Better understanding of the mechanisms of intracellular trafficking of α-syn might be important for understanding the pathogenesis and developing new treatments for synucleinopathies. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. The 200-year journey of Parkinson disease: Reflecting on the past and looking towards the future.

    PubMed

    Fahn, Stanley

    2018-01-01

    It took almost 100 years before a meaningful advance occurred in any basic science understanding of Parkinson disease (PD) following James Parkinson's description in 1817. The Lewy body was described in 1912, and the substantia nigra was found to be depigmented with neuronal loss and gliosis in 1919. The link between dopamine and PD began in 1957, 140 years after Parkinson's Essay. Arvid Carlsson and Oleh Hornykiewicz were the major pioneers. The revolutionary therapeutic breakthrough was the introduction of high dosage levodopa therapy by George Cotzias in 1967. Following 40 years of the dopa/dopamine era, we have entered the era of alpha-synuclein, the protein present in Lewy bodies. Heiko Braak found that alpha-synuclein accumulates initially in the olfactory system and lower brainstem and then travels in an anatomic pattern to involve other regions of the brain and thereby cause progressive symptoms. Alpha-synuclein was somehow converted to a rogue protein. Where this originates and how it is propagated are under intense investigation. At the same time that the alpha-synuclein era was developing, clinical advances took place by recognizing PD as hosting a wide variety of nonmotor features with eventual cognitive impairment in many. Therapeutics has also evolved. Although the most effective therapy for the motor features remains levodopa, surgical approaches and drugs for nonmotor problems continue to expand our ability to treat people with PD. We can expect therapeutic advances in neuroprotection as the basic science discoveries uncovered in the alpha-synuclein era are translated into effective treatments. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Atomic force microscopy based nanoassay: a new method to study α-Synuclein-dopamine bioaffinity interactions

    NASA Astrophysics Data System (ADS)

    Corvaglia, Stefania; Sanavio, Barbara; Sorce, Barbara; Bosco, Alessandro; Sabella, Stefania; Pompa, Pierpaolo; Scoles, Giacinto; Casalis, Loredana

    2015-03-01

    Intrinsically Disordered Proteins (IDPs) are characterized by the lack of well-defined 3-D structure and show high conformational plasticity. For this reason, they are a strong challenge for the traditional characterization of structure, supramolecular assembly and biorecognition phenomena. We show here how the fine tuning of protein orientation on a surface turns useful in the reliable testing of biorecognition interactions of IDPs, in particular α-Synuclein. We exploited atomic force microscopy (AFM) for the selective, nanoscale confinement of α-Synuclein on gold to study the early stages of α-Synuclein aggregation and the effect of small molecules, like dopamine, on the aggregation process. Capitalizing on the high sensitivity of AFM topographic height measurements we determined, for the first time in the literature, the dissociation constant of dopamine- α-Synuclein adducts.

  9. Atomic force microscopy based nanoassay: a new method to study α-Synuclein-dopamine bioaffinity interactions

    NASA Astrophysics Data System (ADS)

    Corvaglia, Stefania; Sanavio, Barbara; Hong Enriquez, Rolando P.; Sorce, Barbara; Bosco, Alessandro; Scaini, Denis; Sabella, Stefania; Pompa, Pier Paolo; Scoles, Giacinto; Casalis, Loredana

    2014-06-01

    Intrinsically Disordered Proteins (IDPs) are characterized by the lack of well-defined 3-D structure and show high conformational plasticity. For this reason, they are a strong challenge for the traditional characterization of structure, supramolecular assembly and biorecognition phenomena. We show here how the fine tuning of protein orientation on a surface turns useful in the reliable testing of biorecognition interactions of IDPs, in particular α-Synuclein. We exploited atomic force microscopy (AFM) for the selective, nanoscale confinement of α-Synuclein on gold to study the early stages of α-Synuclein aggregation and the effect of small molecules, like dopamine, on the aggregation process. Capitalizing on the high sensitivity of AFM topographic height measurements we determined, for the first time in the literature, the dissociation constant of dopamine-α-Synuclein adducts.

  10. Onjisaponin B derived from Radix Polygalae enhances autophagy and accelerates the degradation of mutant α-synuclein and huntingtin in PC-12 cells.

    PubMed

    Wu, An-Guo; Wong, Vincent Kam-Wai; Xu, Su-Wei; Chan, Wai-Kit; Ng, Choi-In; Liu, Liang; Law, Betty Yuen-Kwan

    2013-11-15

    Emerging evidence indicates important protective roles being played by autophagy in neurodegenerative disorders through clearance of aggregate-prone or mutant proteins. In the current study, we aimed to identify autophagy inducers from Chinese medicinal herbs as a potential neuroprotective agent that enhances the clearance of mutant huntingtin and α-synuclein in PC-12 cells. Through intensive screening using the green fluorescent protein-light chain 3 (GFP-LC3) autophagy detection platform, we found that the ethanol extracts of Radix Polygalae (Yuan Zhi) were capable of inducing autophagy. Further investigation showed that among three single components derived from Radix Polygalae--i.e., polygalacic acid, senegenin and onjisaponin B--onjisaponin B was able to induce autophagy and accelerate both the removal of mutant huntingtin and A53T α-synuclein, which are highly associated with Huntington disease and Parkinson disease, respectively. Our study further demonstrated that onjisaponin B induces autophagy via the AMPK-mTOR signaling pathway. Therefore, findings in the current study provide detailed insights into the protective mechanism of a novel autophagy inducer, which is valuable for further investigation as a new candidate agent for modulating neurodegenerative disorders through the reduction of toxicity and clearance of mutant proteins in the cellular level.

  11. Onjisaponin B Derived from Radix Polygalae Enhances Autophagy and Accelerates the Degradation of Mutant α-Synuclein and Huntingtin in PC-12 Cells

    PubMed Central

    Wu, An-Guo; Wong, Vincent Kam-Wai; Xu, Su-Wei; Chan, Wai-Kit; Ng, Choi-In; Liu, Liang; Law, Betty Yuen-Kwan

    2013-01-01

    Emerging evidence indicates important protective roles being played by autophagy in neurodegenerative disorders through clearance of aggregate-prone or mutant proteins. In the current study, we aimed to identify autophagy inducers from Chinese medicinal herbs as a potential neuroprotective agent that enhances the clearance of mutant huntingtin and α-synuclein in PC-12 cells. Through intensive screening using the green fluorescent protein-light chain 3 (GFP-LC3) autophagy detection platform, we found that the ethanol extracts of Radix Polygalae (Yuan Zhi) were capable of inducing autophagy. Further investigation showed that among three single components derived from Radix Polygalae—i.e., polygalacic acid, senegenin and onjisaponin B—onjisaponin B was able to induce autophagy and accelerate both the removal of mutant huntingtin and A53T α-synuclein, which are highly associated with Huntington disease and Parkinson disease, respectively. Our study further demonstrated that onjisaponin B induces autophagy via the AMPK-mTOR signaling pathway. Therefore, findings in the current study provide detailed insights into the protective mechanism of a novel autophagy inducer, which is valuable for further investigation as a new candidate agent for modulating neurodegenerative disorders through the reduction of toxicity and clearance of mutant proteins in the cellular level. PMID:24248062

  12. Alpha1B-adrenoceptor signaling and cell motility: GTPase function of Gh/transglutaminase 2 inhibits cell migration through interaction with cytoplasmic tail of integrin alpha subunits.

    PubMed

    Kang, Sung Koo; Yi, Kye Sook; Kwon, Nyoun Soo; Park, Kwang-Hyun; Kim, Uh-Hyun; Baek, Kwang Jin; Im, Mie-Jae

    2004-08-27

    A multifunctional enzyme, G(h), is a GTP-binding protein that couples to the alpha(1B)-adrenoreceptor and stimulates phospholipase C-delta1 but also displays transglutaminase 2 (TG2) activity. G(h)/TG2 has been implicated to play a role in cell motility. In this study we have examined which function of G(h)/TG2 is involved in this cellular response and the molecular basis. Treatment of human aortic smooth muscle cell with epinephrine inhibits migration to fibronectin and vitronectin, and the inhibition is blocked by the alpha(1)-adrenoreceptor antagonist prazosin or chloroethylclonidine. Up-regulation or overexpression of G(h)/TG2 in human aortic smooth muscle cells, DDT1-MF2, or human embryonic kidney cells, HEK 293 cells, results in inhibition of the migratory activity, and stimulation of the alpha(1B)-adrenoreceptor with the alpha(1) agonist further augments the inhibition of migration of human aortic smooth muscle cells and DDT1-MF2. G(h)/TG2 is coimmunoprecipitated by an integrin alpha(5) antibody and binds to the cytoplasmic tail peptide of integrins alpha(5), alpha(v), and alpha(IIb) subunits in the presence of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). Mutation of Lys-Arg residues in the GFFKR motif, present in the alpha(5)-tail, significantly reduces the binding of GTPgammaS-G(h)/TG2. Moreover, the motif-containing integrin alpha(5)-tail peptides block G(h)/TG2 coimmunoprecipitation and reverse the inhibition of the migratory activity of HEK 293 cells caused by overexpression G(h)/TG2. These results provide evidence that G(h) function initiates the modulation of cell motility via association of GTP-bound G(h)/TG2 with the GFFKR motif located in integrin alpha subunits.

  13. Alpha oscillations and their impairment in affective and post-traumatic stress disorders.

    PubMed

    Eidelman-Rothman, Moranne; Levy, Jonathan; Feldman, Ruth

    2016-09-01

    Affective and anxiety disorders are debilitating conditions characterized by impairments in cognitive and social functioning. Elucidating their neural underpinnings may assist in improving diagnosis and developing targeted interventions. Neural oscillations are fundamental for brain functioning. Specifically, oscillations in the alpha frequency range (alpha rhythms) are prevalent in the awake, conscious brain and play an important role in supporting perceptual, cognitive, and social processes. We review studies utilizing various alpha power measurements to assess abnormalities in brain functioning in affective and anxiety disorders as well as obsessive compulsive and post-traumatic stress disorders. Despite some inconsistencies, studies demonstrate associations between aberrant alpha patterns and these disorders both in response to specific cognitive and emotional tasks and during a resting state. We conclude by discussing methodological considerations and future directions, and underscore the need for much further research on the role of alpha functionality in social contexts. As social dysfunction accompanies most psychiatric conditions, research on alpha's involvement in social processes may provide a unique window into the neural mechanisms underlying these disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Myeloid leukemia factor 1 regulates p53 by suppressing COP1 via COP9 signalosome subunit 3.

    PubMed

    Yoneda-Kato, Noriko; Tomoda, Kiichiro; Umehara, Mari; Arata, Yukinobu; Kato, Jun-ya

    2005-05-04

    Myeloid leukemia factor 1 (MLF1) was first identified as the leukemic fusion protein NPM-MLF1 generated by the t(3;5)(q25.1;q34) chromosomal translocation. Although MLF1 expresses normally in a variety of tissues including hematopoietic stem cells and the overexpression of MLF1 correlates with malignant transformation in human cancer, little is known about how MLF1 is involved in the regulation of cell growth. Here we show that MLF1 is a negative regulator of cell cycle progression functioning upstream of the tumor suppressor p53. MLF1 induces p53-dependent cell cycle arrest in murine embryonic fibroblasts. This action requires a novel binding partner, subunit 3 of the COP9 signalosome (CSN3). A reduction in the level of CSN3 protein with small interfering RNA abrogated MLF1-induced G1 arrest and impaired the activation of p53 by genotoxic stress. Furthermore, ectopic MLF1 expression and CSN3 knockdown inversely affect the endogenous level of COP1, a ubiquitin ligase for p53. Exogenous expression of COP1 overcomes MLF1-induced growth arrest. These results indicate that MLF1 is a critical regulator of p53 and suggest its involvement in leukemogenesis through a novel CSN3-COP1 pathway.

  15. TaGS5-3A, a grain size gene selected during wheat improvement for larger kernel and yield.

    PubMed

    Ma, Lin; Li, Tian; Hao, Chenyang; Wang, Yuquan; Chen, Xinhong; Zhang, Xueyong

    2016-05-01

    Grain size is a dominant component of grain weight in cereals. Earlier studies have shown that OsGS5 plays a major role in regulating both grain size and weight in rice via promotion of cell division. In this study, we isolated TaGS5 homoeologues in wheat and mapped them on chromosomes 3A, 3B and 3D. Temporal and spatial expression analysis showed that TaGS5 homoeologues were preferentially expressed in young spikes and developing grains. Two alleles of TaGS5-3A, TaGS5-3A-T and TaGS5-3A-G were identified in wheat accessions, and a functional marker was developed to discriminate them. Association analysis revealed that TaGS5-3A-T was significantly correlated with larger grain size and higher thousand kernel weight. Biochemical assays showed that TaGS5-3A-T possesses a higher enzymatic activity than TaGS5-3A-G. Transgenic rice lines overexpressing TaGS5-3A-T also exhibited larger grain size and higher thousand kernel weight than TaGS5-3A-G lines, and the transcript levels of cell cycle-related genes in TaGS5-3A-T lines were higher than those in TaGS5-3A-G lines. Furthermore, systematic evolution analysis in diploid, tetraploid and hexaploid wheat showed that TaGS5-3A underwent strong artificial selection during wheat polyploidization events and the frequency changes of two alleles demonstrated that TaGS5-3A-T was favoured in global modern wheat cultivars. These results suggest that TaGS5-3A is a positive regulator of grain size and its favoured allele TaGS5-3A-T exhibits a larger potential application in wheat high-yield breeding. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  16. Age-dependent α-synuclein aggregation in the Microcebus murinus lemur primate

    PubMed Central

    Canron, Marie-Hélène; Perret, Martine; Vital, Anne; Bézard, Erwan; Dehay, Benjamin

    2012-01-01

    Since age-dependent deposition of Aβ-amyloid has been reported in the Microcebus murinus, we posited that this animal could as well be a model of age-related synucleinopathy. We characterized the distribution of Aβ-amyloid, α-synuclein and two of its modified forms in the brain of Microcebus murinus aged from 1.5 to 10 years. Intracytoplasmic α-synuclein aggregates were observed only in aged animals in different brain regions, which were also phospho-Ser129 and nitrated α-synuclein immunoreactive. Age-dependent α-synuclein aggregation occurs spontaneously in mouse lemur primates. Microcebus murinus may provide a model to study age-associated α-synucleinopathy and for testing putative therapeutic interventions for both Alzheimer's and Parkinson's diseases. PMID:23205271

  17. Interaction of α-synuclein with Rhus typhina tannin - Implication for Parkinson's disease.

    PubMed

    Sekowski, Szymon; Ionov, Maksim; Abdulladjanova, Nodira; Makhmudov, Rustam; Mavlyanov, Saidmukhtar; Milowska, Katarzyna; Bryszewska, Maria; Zamaraeva, Maria

    2017-07-01

    The etiology of Parkinson's disease (PD) relates to α-synuclein, a small protein with the ability to aggregate and form Lewy bodies. One of its prevention strategies is inhibition of α-synuclein oligomerization. We have investigated the interaction of α-synuclein and human serum albumin with 3,6-bis-О-di-О-galloyl-1,2,4-tri-О-galloyl-β-d-glucose (a tannin isolated from the plant Rhus typhina). Using fluorescence spectroscopy method we found that this tannin interacts strongly with α-synuclein forming complexes. Circular dichroism analysis showed a time-dependent inhibition of α-synuclein aggregation in the presence of the tannin. On the other hand, 3,6-bis-О-di-О-galloyl-1,2,4-tri-О-galloyl-β-d-glucose had a much stronger interaction with human serum albumin than α-synuclein. The calculated binding constant for tannin-protein interaction was considerably higher for albumin than α-synuclein. This tannin interacted with albumin through a "sphere of action" mechanism. The results lead to the conclusion that 3,6-bis-О-di-О-galloyl-1,2,4-tri-О-galloyl-β-d-glucose is a potent preventive compound against Parkinson's disease. However, this tannin interacts very strongly with human serum albumin, significantly reducing the bioavailability of this compound. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Conditional expression of constitutively active estrogen receptor {alpha} in chondrocytes impairs longitudinal bone growth in mice

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ikeda, Kazuhiro; Tsukui, Tohru; Imazawa, Yukiko

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Conditional transgenic mice expressing constitutively active estrogen receptor {alpha} (caER{alpha}) in chondrocytes were developed. Black-Right-Pointing-Pointer Expression of caER{alpha} in chondrocytes impaired longitudinal bone growth in mice. Black-Right-Pointing-Pointer caER{alpha} affects chondrocyte proliferation and differentiation. Black-Right-Pointing-Pointer This mouse model is useful for understanding the physiological role of ER{alpha}in vivo. -- Abstract: Estrogen plays important roles in the regulation of chondrocyte proliferation and differentiation, which are essential steps for longitudinal bone growth; however, the mechanisms of estrogen action on chondrocytes have not been fully elucidated. In the present study, we generated conditional transgenic mice, designated as caER{alpha}{sup ColII}, expressing constitutively activemore » mutant estrogen receptor (ER) {alpha} in chondrocytes, using the chondrocyte-specific type II collagen promoter-driven Cre transgenic mice. caER{alpha}{sup ColII} mice showed retardation in longitudinal growth, with short bone lengths. BrdU labeling showed reduced proliferation of hypertrophic chondrocytes in the proliferating layer of the growth plate of tibia in caER{alpha}{sup ColII} mice. In situ hybridization analysis of type X collagen revealed that the maturation of hypertrophic chondrocytes was impaired in caER{alpha}{sup ColII} mice. These results suggest that ER{alpha} is a critical regulator of chondrocyte proliferation and maturation during skeletal development, mediating longitudinal bone growth in vivo.« less

  19. Sodium Butyrate Improves Locomotor Impairment and Early Mortality in a Rotenone-Induced Drosophila Model of Parkinson’s Disease

    PubMed Central

    St. Laurent, Robyn; O’Brien, Liam M.; Ahmad, S. Tariq

    2013-01-01

    Parkinson’s disease (PD) is a neurodegenerative disorder primarily affecting the dopaminergic neurons in the nigrastriatal pathway resulting in debilitating motor impairment in both familial and sporadic cases. Histone deacetylase (HDAC) inhibitors have been recently implicated as a therapeutic candidate because of their ability to correct the disrupted HDAC activity in PD and other neurodegenerative diseases. Sodium butyrate (SB), an HDAC inhibitor, reduces degeneration of dopaminergic neurons in a mutant alpha-synuclein Drosophila transgenic model of familial PD. Chronic exposure to the pesticide rotenone also causes selective degeneration of dopaminergic neurons and causes locomotor impairment and early mortality in a Drosophila model of chemically-induced PD. This study investigated the effects of sodium butyrate on locomotor impairment and early mortality in a rotenone-induced PD model. We show that treatment with 10 mM SB-supplemented food rescued the rotenone-induced locomotor impairment and early mortality in flies. Additionally, flies with the genetic knockdown of HDAC activity through Sin3A loss-of-function mutation (Sin3Alof) were resistant to rotenone-induced locomotor impairment and early mortality. Furthermore, SB-supplemented Sin3Alof flies had a modest additive effect for improving locomotor impairment. We also show SB-mediated improvement of rotenone-induced locomotor impairment was associated with elevated dopamine levels in the brain. However, the possibility of SB-mediated protective role through mechanisms independent from dopamine system is also discussed. These findings demonstrate that HDAC inhibitors like SB can ameliorate locomotor impairment in a rotenone-induced PD model. PMID:23623990

  20. Effects of different force fields on the structural character of α synuclein β-hairpin peptide (35-56) in aqueous environment.

    PubMed

    Kundu, Sangeeta

    2018-02-01

    The hallmark of Parkinson's disease (PD) is the intracellular protein aggregation forming Lewy Bodies (LB) and Lewy neuritis which comprise mostly of a protein, alpha synuclein (α-syn). Molecular dynamics (MD) simulation methods can augment experimental techniques to understand misfolding and aggregation pathways with atomistic resolution. The quality of MD simulations for proteins and peptides depends greatly on the accuracy of empirical force fields. The aim of this work is to investigate the effects of different force fields on the structural character of β hairpin fragment of α-syn (residues 35-56) peptide in aqueous solution. Six independent MD simulations are done in explicit solvent using, AMBER03, AMBER99SB, GROMOS96 43A1, GROMOS96 53A6, OPLS-AA, and CHARMM27 force fields with CMAP corrections. The performance of each force field is assessed from several structural parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), formation of β-turn, the stability of folded β-hairpin structure, and the favourable conformations obtained for different force fields. In this study, CMAP correction of CHARMM27 force field is found to overestimate the helical conformation, while GROMOS96 53A6 is found to most successfully capture the conformational dynamics of α-syn β-hairpin fragment as elicited from NMR.

  1. The role of p53 in cancer drug resistance and targeted chemotherapy.

    PubMed

    Hientz, Karin; Mohr, André; Bhakta-Guha, Dipita; Efferth, Thomas

    2017-01-31

    Cancer has long been a grievous disease complicated by innumerable players aggravating its cure. Many clinical studies demonstrated the prognostic relevance of the tumor suppressor protein p53 for many human tumor types. Overexpression of mutated p53 with reduced or abolished function is often connected to resistance to standard medications, including cisplatin, alkylating agents (temozolomide), anthracyclines, (doxorubicin), antimetabolites (gemcitabine), antiestrogenes (tamoxifen) and EGFR-inhibitors (cetuximab). Such mutations in the TP53 gene are often accompanied by changes in the conformation of the p53 protein. Small molecules that restore the wild-type conformation of p53 and, consequently, rebuild its proper function have been identified. These promising agents include PRIMA-1, MIRA-1, and several derivatives of the thiosemicarbazone family. In addition to mutations in p53 itself, p53 activity may be also be impaired due to alterations in p53's regulating proteins such as MDM2. MDM2 functions as primary cellular p53 inhibitor and deregulation of the MDM2/p53-balance has serious consequences. MDM2 alterations often result in its overexpression and therefore promote inhibition of p53 activity. To deal with this problem, a judicious approach is to employ MDM2 inhibitors. Several promising MDM2 inhibitors have been described such as nutlins, benzodiazepinediones or spiro-oxindoles as well as novel compound classes such as xanthone derivatives and trisubstituted aminothiophenes. Furthermore, even naturally derived inhibitor compounds such as α-mangostin, gambogic acid and siladenoserinols have been discovered. In this review, we discuss in detail such small molecules that play a pertinent role in affecting the p53-MDM2 signaling axis and analyze their potential as cancer chemotherapeutics.

  2. Immunohistochemical detection of p53 protein in ameloblastoma types.

    PubMed

    el-Sissy, N A

    1999-05-01

    Overexpression of p53 protein in unicystic ameloblastoma (uAB) is denser than in the conventional ameloblastoma (cAB) type, indicating increased wild type p53--suppressing the growth potential of uAB and denoting the early event of neoplastic transformation, probably of a previous odontogenic cyst. Overexpression of p53 in borderline cAB and malignant ameloblastoma (mAB) types might reflect a mutational p53 protein playing an oncogenic role, promoting tumour growth. Overexpression of p53 protein could be a valid screening method for predicting underlying malignant genetic changes in AB types, through increased frequency of immunoreactive cells or increased staining density.

  3. Retinoic acid receptor alpha drives cell cycle progression and is associated with increased sensitivity to retinoids in T-cell lymphoma.

    PubMed

    Wang, Xueju; Dasari, Surendra; Nowakowski, Grzegorz S; Lazaridis, Konstantinos N; Wieben, Eric D; Kadin, Marshall E; Feldman, Andrew L; Boddicker, Rebecca L

    2017-04-18

    Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with generally poor outcomes following standard therapy. Few candidate therapeutic targets have been identified to date. Retinoic acid receptor alpha (RARA) is a transcription factor that modulates cell growth and differentiation in response to retinoids. While retinoids have been used to treat some cutaneous T-cell lymphomas (CTCLs), their mechanism of action and the role of RARA in CTCL and other mature T-cell lymphomas remain poorly understood. After identifying a PTCL with a RARAR394Q mutation, we sought to characterize the role of RARA in T-cell lymphoma cells. Overexpressing wild-type RARA or RARAR394Q significantly increased cell growth in RARAlow cell lines, while RARA knockdown induced G1 arrest and decreased expression of cyclin-dependent kinases CDK2/4/6 in RARAhigh cells. The retinoids, AM80 (tamibarotene) and all-trans retinoic acid, caused dose-dependent growth inhibition, G1 arrest, and CDK2/4/6 down-regulation. Genes down-regulated in transcriptome data were enriched for cell cycle and G1-S transition. Finally, RARA overexpression augmented chemosensitivity to retinoids. In conclusion, RARA drives cyclin-dependent kinase expression, G1-S transition, and cell growth in T-cell lymphoma. Synthetic retinoids inhibit these functions in a dose-dependent fashion and are most effective in cells with high RARA expression, indicating RARA may represent a therapeutic target in some PTCLs.

  4. Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson’s Disease in Rodents

    PubMed Central

    Polinski, Nicole K.; Volpicelli-Daley, Laura A.; Sortwell, Caryl E.; Luk, Kelvin C.; Cremades, Nunilo; Gottler, Lindsey M.; Froula, Jessica; Duffy, Megan F.; Lee, Virginia M.Y.; Martinez, Terina N.; Dave, Kuldip D.

    2018-01-01

    Parkinson’s disease (PD) is the second most common neurodegenerative disease, affecting approximately one-percent of the population over the age of sixty. Although many animal models have been developed to study this disease, each model presents its own advantages and caveats. A unique model has arisen to study the role of alpha-synuclein (aSyn) in the pathogenesis of PD. This model involves the conversion of recombinant monomeric aSyn protein to a fibrillar form—the aSyn pre-formed fibril (aSyn PFF)—which is then injected into the brain or introduced to the media in culture. Although many groups have successfully adopted and replicated the aSyn PFF model, issues with generating consistent pathology have been reported by investigators. To improve the replicability of this model and diminish these issues, The Michael J. Fox Foundation for Parkinson’s Research (MJFF) has enlisted the help of field leaders who performed key experiments to establish the aSyn PFF model to provide the research community with guidelines and practical tips for improving the robustness and success of this model. Specifically, we identify key pitfalls and suggestions for avoiding these mistakes as they relate to generating the aSyn PFFs from monomeric protein, validating the formation of pathogenic aSyn PFFs, and using the aSyn PFFs in vivo or in vitro to model PD. With this additional information, adoption and use of the aSyn PFF model should present fewer challenges, resulting in a robust and widely available model of PD. PMID:29400668

  5. Targeting α-synuclein for treating Parkinson’s disease: mechanistic and therapeutic considerations

    PubMed Central

    Gorry, Philippe; Przedborski, Serge; Vila, Miquel; Hunot, Stephane; Singleton, Andrew; Olanow, C. Warren; Merchant, Kalpana M.; Bezard, Erwan; Petsko, Gregory A.; Meissner, Wassilios G.

    2016-01-01

    Summary Progressive neuronal cell loss in a small subset of brainstem and mesencephalic nuclei and widespread aggregation of the α-synuclein protein in the form of Lewy bodies and Lewy neurites are neuropathological hallmarks of Parkinson’s disease. Most cases occur sporadically, but mutations in several genes, including α-synuclein, are associated with disease development. The mechanisms driving neurodegeneration remain unknown, hence limiting therapeutic strategies aimed at blocking neuronal death. This review describes current evidence for a predominant role of α-synuclein in the pathogenesis of PD, as well as some of the most promising α-synuclein-based strategies currently in development for this incurable neurodegenerative disorder. PMID:26050140

  6. Immunohistochemical study of DNA topoisomerase I, DNA topoisomerase II alpha, p53, and Ki-67 in oral preneoplastic lesions and oral squamous cell carcinomas.

    PubMed

    Hafian, Hilal; Venteo, Lydie; Sukhanova, Alyona; Nabiev, Igor; Lefevre, Benoît; Pluot, Michel

    2004-06-01

    Human DNA topoisomerase I (topo I) is the molecular target of the camptothecin group of anticancer drugs. Laboratory studies have shown that the cellular response to topo I-targeted drugs depends on the topo I expression and DNA replication rate and the apoptotic pathway activity. In this study, we tested potential indicators of the sensitivity of topo I-targeted drugs in 36 cases of oral squamous cell carcinoma (OSCC). Formalin-fixed, paraffin-embedded tissue sections were immunostained with monoclonal antibodies against Ki-67, p53, and topo I, and with polyclonal antibodies against DNA topoisomerase II-alpha (topo II-alpha). These markers were also tested in 18 epithelial hyperplastic lesions and 18 mild dysplasias. Immunostaining was quantified by the percentage of stained nuclei in each sample (the labeling index); 200 immunoreactive epithelial nuclei were counted per case for each antibody. The results support the possibility of using topo II-alpha staining for assessing the proliferative activity. High expression of topo II-alpha and topo I in OSCCs suggests that they may serve as potential indicators of sensitivity to topo I inhibitors. However, the apoptotic pathway assessed by p53 immunostaining was found to be uninformative. Analysis of the relationship between immunohistochemical results and clinical and pathologic parameters (the T and N stages and differentiation) showed that only the differentiation parameter correlated with the topo I expression rate. Thus, significant increase in the topo I expression in the poorly differentiated OSCCs suggests their higher sensitivity to drug treatment.

  7. Evidence for Intramolecular Antiparallel Beta-Sheet Structure in Alpha-Synuclein Fibrils from a Combination of Two-Dimensional Infrared Spectroscopy and Atomic Force Microscopy

    PubMed Central

    Roeters, Steven J.; Iyer, Aditya; Pletikapić, Galja; Kogan, Vladimir; Subramaniam, Vinod; Woutersen, Sander

    2017-01-01

    The aggregation of the intrinsically disordered protein alpha-synuclein (αS) into amyloid fibrils is thought to play a central role in the pathology of Parkinson’s disease. Using a combination of techniques (AFM, UV-CD, XRD, and amide-I 1D- and 2D-IR spectroscopy) we show that the structure of αS fibrils varies as a function of ionic strength: fibrils aggregated in low ionic-strength buffers ([NaCl] ≤ 25 mM) have a significantly different structure than fibrils grown in higher ionic-strength buffers. The observations for fibrils aggregated in low-salt buffers are consistent with an extended conformation of αS molecules, forming hydrogen-bonded intermolecular β-sheets that are loosely packed in a parallel fashion. For fibrils aggregated in high-salt buffers (including those prepared in buffers with a physiological salt concentration) the measurements are consistent with αS molecules in a more tightly-packed, antiparallel intramolecular conformation, and suggest a structure characterized by two twisting stacks of approximately five hydrogen-bonded intermolecular β-sheets each. We find evidence that the high-frequency peak in the amide-I spectrum of αS fibrils involves a normal mode that differs fundamentally from the canonical high-frequency antiparallel β-sheet mode. The high sensitivity of the fibril structure to the ionic strength might form the basis of differences in αS-related pathologies. PMID:28112214

  8. α-Synuclein inclusions in the skin of Parkinson's disease and parkinsonism.

    PubMed

    Rodríguez-Leyva, Ildefonso; Calderón-Garcidueñas, Ana Laura; Jiménez-Capdeville, María E; Rentería-Palomo, Ana Arely; Hernandez-Rodriguez, Héctor Gerardo; Valdés-Rodríguez, Rodrigo; Fuentes-Ahumada, Cornelia; Torres-Álvarez, Bertha; Sepúlveda-Saavedra, Julio; Soto-Domínguez, Adolfo; Santoyo, Martha E; Rodriguez-Moreno, José Ildefonso; Castanedo-Cázares, Juan Pablo

    2014-07-01

    The presence in the brain of α-synuclein containing Lewy neurites, or bodies, is the histological hallmark of Parkinson's disease (PD). The discovery of α-synuclein aggregates in nerve endings of the heart, digestive tract, and skin has lent support to the concept of PD as a systemic disease. Our goals were, first, to demonstrate the presence of α-synuclein inclusions in the skin and, second, to detect quantitative differences between patients with PD and atypical parkinsonism (AP). Skin biopsies were taken from 67 patients and 20 controls. The biopsies underwent immunohistochemistry (IHC) and immunofluorescence (IF) testing for α-synuclein, whereupon its presence was quantified as the percentage of positive cells. Patients were divided into those with PD and those with AP. AP patients included AP with neurodegenerative disease (proteinopathies) and secondary AP. Sixty-seven patients (34 with PD) and 20 controls were recruited. In the PD group, α-synuclein was detected in 58% of the cells in the spinous cell layer (SCL), 62% in the pilosebaceous unit (PSU), and 58% in the eccrine glands (EG). The AP-proteinopathies group showed 7%, 7%, and 0% expression of α-synuclein, respectively. No expression was found in the skin of the control group. The expression of α-synuclein in the skin was relatively high in the PD group, scarce in AP, and null for the individuals in the control group. While these findings require further confirmation, this minimally invasive technique may aid in the improvement of the accuracy of PD diagnoses.

  9. Analysis of alpha hemoglobin stabilizing protein overexpression in murine β-thalassemia

    PubMed Central

    Nasimuzzaman, Md; Khandros, Eugene; Wang, Xiaomei; Kong, Yi; Zhao, Huifen; Weiss, David; Rivella, Stefano; Weiss, Mitchell J.; Persons, Derek A.

    2013-01-01

    Excess free α-globin is cytotoxic and contributes to the pathophysiology of β-thalassemia. Alpha hemoglobin stabilizing protein (AHSP) is a molecular chaperone that binds free α-globin to promote its folding and inhibit its ability to produce damaging reactive oxygen species. Reduced AHSP levels correlate with increased severity of β-thalassemia in some human cohorts, but causal mechanistic relationships are not established for these associations. We used transgenic and lentiviral gene transfer methods to investigate whether supraphysiologic AHSP levels could mitigate the severity of β-thalassemia intermedia by providing an increased sink for the excess pool of α-globin chains. We tested wild-type AHSP and two mutant versions with amino acid substitutions that confer 3- or 13-fold higher affinity for α-globin. Erythroid overexpression of these AHSP proteins up to 11-fold beyond endogenous levels had no major effects on hematologic parameters in β-thalassemic animals. Our results demonstrate that endogenous AHSP is not limiting for α-globin detoxification in a murine model of β-thalassemia. PMID:20815047

  10. Direct Membrane Association Drives Mitochondrial Fission by the Parkinson Disease-associated Protein α-Synuclein*♦

    PubMed Central

    Nakamura, Ken; Nemani, Venu M.; Azarbal, Farnaz; Skibinski, Gaia; Levy, Jon M.; Egami, Kiyoshi; Munishkina, Larissa; Zhang, Jue; Gardner, Brooke; Wakabayashi, Junko; Sesaki, Hiromi; Cheng, Yifan; Finkbeiner, Steven; Nussbaum, Robert L.; Masliah, Eliezer; Edwards, Robert H.

    2011-01-01

    The protein α-synuclein has a central role in Parkinson disease, but the mechanism by which it contributes to neural degeneration remains unknown. We now show that the expression of α-synuclein in mammalian cells, including neurons in vitro and in vivo, causes the fragmentation of mitochondria. The effect is specific for synuclein, with more fragmentation by α- than β- or γ-isoforms, and it is not accompanied by changes in the morphology of other organelles or in mitochondrial membrane potential. However, mitochondrial fragmentation is eventually followed by a decline in respiration and neuronal death. The fragmentation does not require the mitochondrial fission protein Drp1 and involves a direct interaction of synuclein with mitochondrial membranes. In vitro, synuclein fragments artificial membranes containing the mitochondrial lipid cardiolipin, and this effect is specific for the small oligomeric forms of synuclein. α-Synuclein thus exerts a primary and direct effect on the morphology of an organelle long implicated in the pathogenesis of Parkinson disease. PMID:21489994

  11. Comparison of independent screens on differentially vulnerable motor neurons reveals alpha-synuclein as a common modifier in motor neuron diseases.

    PubMed

    Kline, Rachel A; Kaifer, Kevin A; Osman, Erkan Y; Carella, Francesco; Tiberi, Ariana; Ross, Jolill; Pennetta, Giuseppa; Lorson, Christian L; Murray, Lyndsay M

    2017-03-01

    The term "motor neuron disease" encompasses a spectrum of disorders in which motor neurons are the primary pathological target. However, in both patients and animal models of these diseases, not all motor neurons are equally vulnerable, in that while some motor neurons are lost very early in disease, others remain comparatively intact, even at late stages. This creates a valuable system to investigate the factors that regulate motor neuron vulnerability. In this study, we aim to use this experimental paradigm to identify potential transcriptional modifiers. We have compared the transcriptome of motor neurons from healthy wild-type mice, which are differentially vulnerable in the childhood motor neuron disease Spinal Muscular Atrophy (SMA), and have identified 910 transcriptional changes. We have compared this data set with published microarray data sets on other differentially vulnerable motor neurons. These neurons were differentially vulnerable in the adult onset motor neuron disease Amyotrophic Lateral Sclerosis (ALS), but the screen was performed on the equivalent population of neurons from neurologically normal human, rat and mouse. This cross species comparison has generated a refined list of differentially expressed genes, including CELF5, Col5a2, PGEMN1, SNCA, Stmn1 and HOXa5, alongside a further enrichment for synaptic and axonal transcripts. As an in vivo validation, we demonstrate that the manipulation of a significant number of these transcripts can modify the neurodegenerative phenotype observed in a Drosophila line carrying an ALS causing mutation. Finally, we demonstrate that vector-mediated expression of alpha-synuclein (SNCA), a transcript decreased in selectively vulnerable motor neurons in all four screens, can extend life span, increase weight and decrease neuromuscular junction pathology in a mouse model of SMA. In summary, we have combined multiple data sets to identify transcripts, which are strong candidates for being phenotypic modifiers

  12. Altered S-nitrosylation of p53 is responsible for impaired antioxidant response in skeletal muscle during aging.

    PubMed

    Baldelli, Sara; Ciriolo, Maria Rosa

    2016-12-20

    p53 transcriptional activity has been proposed to regulate both homeostasis and sarcopenia of skeletal muscle during aging. However, the exact molecular function of p53 remains to be clearly defined. We demonstrated a requirement of nuclear p53 S-nitrosylation in inducing a nitric oxide/PGC-1α-mediated antioxidant pathway in skeletal muscle. Importantly, mutant form of p53-DNA binding domain (C124S) did not undergo nuclear S-nitrosylation and failed in inducing the expression of antioxidant genes (i.e. SOD2 and GCLC). Moreover, we found that during aging the nuclear S-nitrosylation of p53 significantly declines in gastrocnemius/soleus leading to an impairment of redox homeostasis of skeletal muscle. We suggested that decreased level of nuclear neuronal nitric oxide synthase (nNOS)/Syntrophin complex, which we observed during aging, could be responsible for impaired nuclear S-nitrosylation. Taken together, our data indicate that altered S-nitrosylation of p53 during aging could be a contributing factor of sarcopenia condition and of other skeletal muscle pathologies associated with oxidative/nitrosative stress.

  13. Altered S-nitrosylation of p53 is responsible for impaired antioxidant response in skeletal muscle during aging

    PubMed Central

    Baldelli, Sara; Ciriolo, Maria Rosa

    2016-01-01

    p53 transcriptional activity has been proposed to regulate both homeostasis and sarcopenia of skeletal muscle during aging. However, the exact molecular function of p53 remains to be clearly defined. We demonstrated a requirement of nuclear p53 S-nitrosylation in inducing a nitric oxide/PGC-1α-mediated antioxidant pathway in skeletal muscle. Importantly, mutant form of p53-DNA binding domain (C124S) did not undergo nuclear S-nitrosylation and failed in inducing the expression of antioxidant genes (i.e. SOD2 and GCLC). Moreover, we found that during aging the nuclear S-nitrosylation of p53 significantly declines in gastrocnemius/soleus leading to an impairment of redox homeostasis of skeletal muscle. We suggested that decreased level of nuclear neuronal nitric oxide synthase (nNOS)/Syntrophin complex, which we observed during aging, could be responsible for impaired nuclear S-nitrosylation. Taken together, our data indicate that altered S-nitrosylation of p53 during aging could be a contributing factor of sarcopenia condition and of other skeletal muscle pathologies associated with oxidative/nitrosative stress. PMID:28025407

  14. α-Synuclein-induced dopaminergic neurodegeneration in a rat model of Parkinson's disease occurs independent of ATP13A2 (PARK9).

    PubMed

    Daniel, Guillaume; Musso, Alessandra; Tsika, Elpida; Fiser, Aris; Glauser, Liliane; Pletnikova, Olga; Schneider, Bernard L; Moore, Darren J

    2015-01-01

    Mutations in the ATP13A2 (PARK9) gene cause early-onset, autosomal recessive Parkinson's disease (PD) and Kufor-Rakeb syndrome. ATP13A2 mRNA is spliced into three distinct isoforms encoding a P5-type ATPase involved in regulating heavy metal transport across vesicular membranes. Here, we demonstrate that three ATP13A2 mRNA isoforms are expressed in the normal human brain and are modestly increased in the cingulate cortex of PD cases. ATP13A2 can mediate protection toward a number of stressors in mammalian cells and can protect against α-synuclein-induced toxicity in cellular and invertebrate models of PD. Using a primary cortical neuronal model combined with lentiviral-mediated gene transfer, we demonstrate that human ATP13A2 isoforms 1 and 2 display selective neuroprotective effects toward toxicity induced by manganese and hydrogen peroxide exposure through an ATPase-independent mechanism. The familial PD mutations, F182L and G504R, abolish the neuroprotective effects of ATP13A2 consistent with a loss-of-function mechanism. We further demonstrate that the AAV-mediated overexpression of human ATP13A2 is not sufficient to attenuate dopaminergic neurodegeneration, neuropathology, and striatal dopamine and motoric deficits induced by human α-synuclein expression in a rat model of PD. Intriguingly, the delivery of an ATPase-deficient form of ATP13A2 (D513N) to the substantia nigra is sufficient to induce dopaminergic neuronal degeneration and motor deficits in rats, potentially suggesting a dominant-negative mechanism of action. Collectively, our data demonstrate a distinct lack of ATP13A2-mediated protection against α-synuclein-induced neurotoxicity in the rat nigrostriatal dopaminergic pathway, and limited neuroprotective capacity overall, and raise doubts about the potential of ATP13A2 as a therapeutic target for PD. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Reduced glucocerebrosidase is associated with increased α-synuclein in sporadic Parkinson's disease.

    PubMed

    Murphy, Karen E; Gysbers, Amanda M; Abbott, Sarah K; Tayebi, Nahid; Kim, Woojin S; Sidransky, Ellen; Cooper, Antony; Garner, Brett; Halliday, Glenda M

    2014-03-01

    Heterozygous mutations in GBA1, the gene encoding lysosomal glucocerebrosidase, are the most frequent known genetic risk factor for Parkinson's disease. Reduced glucocerebrosidase and α-synuclein accumulation are directly related in cell models of Parkinson's disease. We investigated relationships between Parkinson's disease-specific glucocerebrosidase deficits, glucocerebrosidase-related pathways, and α-synuclein levels in brain tissue from subjects with sporadic Parkinson's disease without GBA1 mutations. Brain regions with and without a Parkinson's disease-related increase in α-synuclein levels were assessed in autopsy samples from subjects with sporadic Parkinson's disease (n = 19) and age- and post-mortem delay-matched controls (n = 10). Levels of glucocerebrosidase, α-synuclein and related lysosomal and autophagic proteins were assessed by western blotting. Glucocerebrosidase enzyme activity was measured using a fluorimetric assay, and glucocerebrosidase and α-synuclein messenger RNA expression determined by quantitative polymerase chain reaction. Related sphingolipids were analysed by mass spectrometry. Multivariate statistical analyses were performed to identify differences between disease groups and regions, with non-parametric correlations used to identify relationships between variables. Glucocerebrosidase protein levels and enzyme activity were selectively reduced in the early stages of Parkinson's disease in regions with increased α-synuclein levels although limited inclusion formation, whereas GBA1 messenger RNA expression was non-selectively reduced in Parkinson's disease. The selective loss of lysosomal glucocerebrosidase was directly related to reduced lysosomal chaperone-mediated autophagy, increased α-synuclein and decreased ceramide. Glucocerebrosidase deficits in sporadic Parkinson's disease are related to the abnormal accumulation of α-synuclein and are associated with substantial alterations in lysosomal chaperone-mediated autophagy

  16. Reynosin protects against neuronal toxicity in dopamine-induced SH-SY5Y cells and 6-hydroxydopamine-lesioned rats as models of Parkinson's disease: Reciprocal up-regulation of E6-AP and down-regulation of α-synuclein.

    PubMed

    Ham, Ahrom; Kim, Dong-Woo; Kim, Kyeong Ho; Lee, Sung-Jin; Oh, Ki-Bong; Shin, Jongheon; Mar, Woongchon

    2013-08-02

    Aggregation of α-synuclein (ASYN) is considered a major determinant of neuronal loss in Parkinson's disease (PD). E6-associated protein (E6-AP), an E3 ubiquitin protein ligase, has been known to promote the degradation of α-synuclein. The aim of this study was to assess the effects of the sesquiterpene lactone reynosin on dopamine (DA)-induced neuronal toxicity and regulation of E6-associated protein and α-synuclein proteins in both in vitro and in vivo models of Parkinson's disease. Usi"ng flow cytometry and western blot analysis, we determined that reynosin significantly protected both against cell death from dopamine-induced toxicity in human neuroblastoma SH-SY5Y cells and against the loss of tyrosine hydroxylase (TH)-positive cells in 6-hydroxydopamine (6-OHDA)-lesioned rats (a rodent Parkinson's disease model system). In addition, reynosin made up-regulation of E6-associated protein expression and down-regulation of the over-expression of α-synuclein protein in both dopamine-treated SH-SY5Y cells and 6-hydroxydopamine-lesioned rats. These results suggest that the protective effect of reynosin against dopamine-induced neuronal cell death may be due to the reciprocal up-regulation of E6-associated protein and down-regulation of α-synuclein protein expression. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Overexpression of angiotensin II type 2 receptor promotes apoptosis and impairs insulin secretion in rat insulinoma cells.

    PubMed

    Liu, Min; Jing, Danqing; Wang, Yan; Liu, Yu; Yin, Shinan

    2015-02-01

    Angiotensin II (Ang II), the major effector hormone of renin-angiotensin system, acts as a promoter of insulin resistance and diabetes mellitus type 2 pathogenesis. Activation of Ang II type 2 receptor (AT2R) has been examined as a potential therapeutic strategy. However, there are conflicting findings regarding the role of AT2R. In the current study, we evaluated the effects of overexpressing AT2R by viral vector transduction on the apoptosis and function of pancreatic β-islet cells. The rat insulinoma cell line, INS-1, was transduced with a recombinant adenoviral vector expressing AT2R (Ad-G-AT2R-EGFP). AT2R overexpression resulted in significantly reduced cell viability and subsequently impaired glucose-stimulated insulin secretion (GSIS) function in INS-1 cells. Down-regulated expressions of GSIS pathway components, insulin, glucose transporter 2, and glucokinase were associated with AT2R overexpression. Further analysis determined that overexpression of AT2R induced G1-phase cell cycle arrest and Ang II-independent apoptotic cell death as indicated by increased Annexin V staining. To understand the apoptosis signaling triggered by AT2R overexpression, levels of caspase proteins were measured. Overexpression of AT2R significantly induced caspase-8, caspase-9, and caspase-3 cleavage, and decreased Bcl-2, pAkt, and pERK expression levels. AT2R-induced cell apoptosis was successfully blocked by the caspase inhibitor Z-VAD-FMK. Our findings suggested that AT2R overexpression triggers the apoptosis of INS-1 cells and dysfunction in insulin secretion. In conclusion, more careful design and consideration are required when applying AT2R-related therapies in treating diabetes.

  18. Overexpression of SIRT6 in the hippocampal CA1 impairs the formation of long-term contextual fear memory

    PubMed Central

    Yin, Xi; Gao, Yuan; Shi, Hai-Shui; Song, Li; Wang, Jie-Chao; Shao, Juan; Geng, Xu-Hong; Xue, Gai; Li, Jian-Li; Hou, Yan-Ning

    2016-01-01

    Histone modifications have been implicated in learning and memory. Our previous transcriptome data showed that expression of sirtuins 6 (SIRT6), a member of Histone deacetylases (HDACs) family in the hippocampal cornu ammonis 1 (CA1) was decreased after contextual fear conditioning. However, the role of SIRT6 in the formation of memory is still elusive. In the present study, we found that contextual fear conditioning inhibited translational expression of SIRT6 in the CA1. Microinfusion of lentiviral vector-expressing SIRT6 into theCA1 region selectively enhanced the expression of SIRT6 and impaired the formation of long-term contextual fear memory without affecting short-term fear memory. The overexpression of SIRT6 in the CA1 had no effect on anxiety-like behaviors or locomotor activity. Also, we also found that SIRT6 overexpression significantly inhibited the expression of insulin-like factor 2 (IGF2) and amounts of proteins and/or phosphoproteins (e.g. Akt, pAkt, mTOR and p-mTOR) related to the IGF2 signal pathway in the CA1. These results demonstrate that the overexpression of SIRT6 in the CA1 impaired the formation of long-term fear memory, and SIRT6 in the CA1 may negatively modulate the formation of contextual fear memory via inhibiting the IGF signaling pathway. PMID:26732053

  19. Transglutaminase 2 overexpression induces depressive-like behavior and impaired TrkB signaling in mice

    PubMed Central

    Pandya, Chirayu D; Hoda, Nasrul; Crider, Amanda; Peter, Diya; Kutiyanawalla, Ammar; Kumar, Sanjiv; Ahmed, Anthony O; Turecki, Gustavo; Hernandez, Caterina M; Terry, Alvin V

    2016-01-01

    Serotonin (5-HT) and brain derived neurotrophic factor (BDNF) are two signaling molecules that play important regulatory roles in the development and plasticity of neural circuits that are known to be altered in depression. However, the mechanism by which 5-HT regulates BDNF signaling is unknown. In the present study, we found that 5-HT treatment increases BDNF receptor, TrkB (tropomyosin related kinase B) levels in mouse primary cortical neurons via a Rac1 (RAS-related C3 botulinum toxin substrate 1)-dependent mechanism. Significant increases in the levels of transglutaminase 2 (TG2, which is implicated in transamidation of 5-HT to Rac1) are observed in the mouse prefrontal cortex (PFC) following chronic exposure to stress. We also found that TG2 levels are increased in the postmortem PFC of depressed suicide subjects relative to matched controls. Moreover, in mice, neuronal overexpression of TG2 resulted in the atrophy of neurons and reduced levels of TrkB in the PFC as well as a depressive-like phenotype. Overexpression of TG2 in mouse cortical neurons reduced TrkB levels as a result of impaired endocytosis of TrkB. TG2 inhibition by either a viral particle or pharmacological approach attenuated behavioral deficits caused by chronic unpredictable stress. Moreover, the overexpression of TrkB in the mouse PFC ameliorated the depressive-like phenotype of TG2 overexpressed mice. Taken together, these postmortem and preclinical findings identify TG2 as a critical mediator of the altered TrkB expression and depressive-like behaviors associated with chronic exposure to stress and suggest that TG2 may represent a novel therapeutic target in depression. PMID:27620841

  20. TNF-Alpha in Peripheral Neuropathy Patients with Impaired Glucose Regulation.

    PubMed

    Li, Xia; Zhu, Ju; Liu, Na; Liu, Jie; Zhang, Zhecheng

    2017-01-01

    Impaired glucose regulation (IGR) is the prestate of diabetes; about 1/3 of IGR patients will develop to diabetes finally. In this study, we investigated the serum tumor necrosis factor-alpha (TNF- α ) and interleukin-6 (IL-6) levels in peripheral neuropathy impaired patients with impaired glucose regulation (IGR). A total of 70 IGR patients received the conventional nerve conduction test, including 30 patients with peripheral neuropathy (PN) and 40 patients without peripheral neuropathy (NPN). The other 40 healthy individuals were recruited as controls. The serum TNF- α and IL-6 in IGR patients were higher than in control group, and serum TNF- α and IL-6 levels in IGR-PN group were higher than in IGR-NPN group (27.7 ± 17.8 versus 13.1 ± 6.7 pg/mL and 18.1 ± 17.7 versus 6.4 ± 3.7 pg/mL, resp., both p < 0.05). Multifactors logistic regression analysis showed that TNF- α (OR = 0.893; p = 0.009) was an independent factor affecting whether IGR could combine with peripheral neuropathy. TNF- α and IL-6 could aggregate peripheral neuropathy in impaired glucose regulation patients; TNF- α might be independent risk factor for peripheral neuropathy in glucose regulation impaired patients.

  1. Interaction of Synuclein and Inflammation in Dopaminergic Neurodegeneration

    DTIC Science & Technology

    2014-06-01

    are less responsive to the blocking effects of anti-CD36 antibodies. The CD36 inhibitor, ursolic acid , at concentrations of 10-5 and 10-6, reduced...N9 chemotaxis across native synuclein by almost 50%. Equally interesting is the fact that ursolic acid had a small effect on CD36-deficient N9 cell...chemotaxis across synuclein which suggests that ursolic acid may also inhibit other scavenger receptors such as SR1 and RAGE. Ursolic acid was

  2. Immunohistochemical detection of HIF-1alpha and CAIX in advanced head-and-neck cancer. Prognostic role and correlation with tumor markers and tumor oxygenation parameters.

    PubMed

    Kappler, Matthias; Taubert, Helge; Holzhausen, Hans-Jürgen; Reddemann, Rolf; Rot, Swetlann; Becker, Axel; Kuhnt, Thomas; Dellas, Kathrin; Dunst, Jürgen; Vordermark, Dirk; Hänsgen, Gabriele; Bache, Matthias

    2008-08-01

    Tumor hypoxia has an impact on the outcome of cancer patients treated with radiotherapy. The validity of endogenous markers such as hypoxia-inducible factor-1alpha (HIF-1alpha) and carbonic anhydrase isozyme IX (CAIX) to detect therapeutically relevant Levels of hypoxia within tumors is controversially discussed. Furthermore, the association of these hypoxia markers with tumor markers or tumor oxygenation parameters is of importance for understanding the relationship between the different factors. Tumortissue sections of 34 patients with advanced head-and-neck cancertreated with radio(chemo)therapy were assessed by immunohistochemistry for the expression of HIF-1alpha and CAIX. The relationships of both markers with tumor oxygenation parameters, molecular factors like P53, OPN, VEGF, VHL, survivin, and Ki67 levels, and clinical parameters were studied. Bivariate analysis showed a significant correlation of HIF-1alpha expression with high P53 and high OPN expression, high serum VEGF Levels, and low VHL and low Ki67 expression. The CAIX expression was inversely correlated with pH value and directly correlated with T-stage. However, no correlation was found between HIF-1alpha and CAIX expression. Neither in a univariate Cox proportional hazard regression nor in a Kaplan-Meier analysis did expression of HIF-1alpha or CAIX have a significant impact on clinical outcome. However, in a Kaplan-Meier analysis, the combination of both factors showed that patients with intratumoral overexpression of either HIF-1alpha or CAIX or both markers died on average 2 years earlier than patients whose tumors had low expression of both factors (p < 0.05). Expression of HIF-1alpha and CAIX was correlated with different tumor parameters. Only combined HIF-1alpha and CAIX expression was significantly predictive of patients' overall survival.

  3. Myeloid leukemia factor 1 regulates p53 by suppressing COP1 via COP9 signalosome subunit 3

    PubMed Central

    Yoneda-Kato, Noriko; Tomoda, Kiichiro; Umehara, Mari; Arata, Yukinobu; Kato, Jun-ya

    2005-01-01

    Myeloid leukemia factor 1 (MLF1) was first identified as the leukemic fusion protein NPM-MLF1 generated by the t(3;5)(q25.1;q34) chromosomal translocation. Although MLF1 expresses normally in a variety of tissues including hematopoietic stem cells and the overexpression of MLF1 correlates with malignant transformation in human cancer, little is known about how MLF1 is involved in the regulation of cell growth. Here we show that MLF1 is a negative regulator of cell cycle progression functioning upstream of the tumor suppressor p53. MLF1 induces p53-dependent cell cycle arrest in murine embryonic fibroblasts. This action requires a novel binding partner, subunit 3 of the COP9 signalosome (CSN3). A reduction in the level of CSN3 protein with small interfering RNA abrogated MLF1-induced G1 arrest and impaired the activation of p53 by genotoxic stress. Furthermore, ectopic MLF1 expression and CSN3 knockdown inversely affect the endogenous level of COP1, a ubiquitin ligase for p53. Exogenous expression of COP1 overcomes MLF1-induced growth arrest. These results indicate that MLF1 is a critical regulator of p53 and suggest its involvement in leukemogenesis through a novel CSN3–COP1 pathway. PMID:15861129

  4. Alpha-synuclein mRNA isoform formation and translation affected by polymorphism in the human SNCA 3'UTR.

    PubMed

    Barrie, Elizabeth S; Lee, Sung-Ha; Frater, John T; Kataki, Maria; Scharre, Douglas W; Sadee, Wolfgang

    2018-05-06

    Multiple variants in SNCA, encoding alpha-synuclein, a main component of Lewy bodies, are implicated in Parkinson's disease. We searched for cis-acting SNCA variants using allelic mRNA ratios in human brain tissues. In a SNCA 3'UTR (2,520 bp) luciferase reporter gene assay, translation in SH-SY5Y cells in the presence of the rs17016074 G/A alleles was measured. To assess clinical impact, we queried neurocognitive genome-wide association studies. Allelic ratios deviated up to twofold, measured at a marker SNP in the middle of a long 3' untranslated region (3'UTR), but not at a marker at its start, suggesting regulation of 3'UTR processing. 3'UTR SNP rs17016074 G/A, minor allele frequency (MAF) <1% in Caucasians, 13% in Africans, strongly associates with large allelic mRNA expression imbalance (AEI), resulting in reduced expression of long 3'UTR isoforms. A second 3'UTR SNP (rs356165) associates with moderate AEI and enhances SNCA mRNA expression. The rs17016074 A allele reduces overall 3'UTR expression in luciferase reporter gene assays but supports more efficient translation, resolving previous contradictory results. We failed to detect significant genome-wide associations for rs17016074, possibly a result of low MAF in Caucasians or its absence from most genotyping panels. In the "Genome Wide Association Study of Yoruba in Nigeria," rs356165 was associated with reduced memory performance. Here, we identify two cis-acting regulatory variants affecting SNCA mRNA expression, measured by allelic ratios in the 3'UTR. The rs17016074 minor A allele is associated with higher expression of luciferase protein activity. Resolving the genetic influence of SNCA polymorphisms requires study of the interactions between multiple regulatory variants with distinct frequencies among populations. © 2018 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

  5. Lysosomal response in relation to α-synuclein pathology differs between Parkinson's disease and multiple system atrophy.

    PubMed

    Puska, Gina; Lutz, Mirjam I; Molnar, Kinga; Regelsberger, Günther; Ricken, Gerda; Pirker, Walter; Laszlo, Lajos; Kovacs, Gabor G

    2018-06-01

    Intracellular deposition of pathologically altered α-synuclein mostly in neurons characterises Parkinson's disease (PD), while its accumulation predominantly in oligodendrocytes is a feature of multiple system atrophy (MSA). Recently a prion-like spreading of pathologic α-synuclein has been suggested to play a role in the pathogenesis of PD and MSA. This implicates a role of protein processing systems, including lysosomes, supported also by genetic studies in PD. However, particularly for MSA, the mechanism of cell-to-cell propagation of α-synuclein is yet not fully understood. To evaluate the significance of lysosomal response, we systematically compared differently affected neuronal populations in PD, MSA, and non-diseased brains using morphometric immunohistochemistry (cathepsin D), double immunolabelling (cathepsin D/α-synuclein) laser confocal microscopy, and immunogold electron microscopy for the disease associated α-synuclein. We found that i) irrespective of the presence of neuronal inclusions, the volume density of cathepsin D immunoreactivity significantly increases in affected neurons of the pontine base in MSA brains; ii) volume density of cathepsin D immunoreactivity increases in nigral neurons in PD without inclusions and with non-ubiquitinated pre-aggregates of α-synuclein, but not in neurons with Lewy bodies; iii) cathepsin D immunoreactivity frequently colocalises with α-synuclein pre-aggregates in nigral neurons in PD; iv) ultrastructural observations confirm disease-associated α-synuclein in neuronal and astrocytic lysosomes in PD; v) lysosome-associated α-synuclein is observed in astroglia and rarely in oligodendroglia and in neurons in MSA. Our observations support a crucial role for the neuronal endosomal-lysosomal system in the processing of α-synuclein in PD. We suggest a distinct contribution of lysosomes to the pathogenesis of MSA, including the possibility of oligodendroglial and eventually neuronal uptake of exogenous α-synuclein

  6. Suppression of α-synuclein toxicity and vesicle trafficking defects by phosphorylation at S129 in yeast depends on genetic context

    PubMed Central

    Sancenon, Vicente; Lee, Sue-Ann; Patrick, Christina; Griffith, Janice; Paulino, Amy; Outeiro, Tiago F.; Reggiori, Fulvio; Masliah, Eliezer; Muchowski, Paul J.

    2012-01-01

    The aggregation of α-synuclein (αSyn) is a neuropathologic hallmark of Parkinson's disease and other synucleinopathies. In Lewy bodies, αSyn is extensively phosphorylated, predominantly at serine 129 (S129). Recent studies in yeast have shown that, at toxic levels, αSyn disrupts Rab homeostasis, causing an initial endoplasmic reticulum-to-Golgi block that precedes a generalized trafficking collapse. However, whether αSyn phosphorylation modulates trafficking defects has not been evaluated. Here, we show that constitutive expression of αSyn in yeast impairs late-exocytic, early-endocytic and/or recycling trafficking. Although members of the casein kinase I (CKI) family phosphorylate αSyn at S129, they attenuate αSyn toxicity and trafficking defects by an S129 phosphorylation-independent mechanism. Surprisingly, phosphorylation of S129 modulates αSyn toxicity and trafficking defects in a manner strictly determined by genetic background. Abnormal endosome morphology, increased levels of the endosome marker Rab5 and co-localization of mammalian CKI with αSyn aggregates are observed in brain sections from αSyn-overexpressing mice and human synucleinopathies. Our results contribute to evidence that suggests αSyn-induced defects in endocytosis, exocytosis and/or recycling of vesicles involved in these cellular processes might contribute to the pathogenesis of synucleinopathies. PMID:22357655

  7. Acquisition of T regulatory function in cathepsin L-inhibited T cells by eye-derived CTLA-2alpha during inflammatory conditions.

    PubMed

    Sugita, Sunao; Horie, Shintaro; Nakamura, Orie; Maruyama, Kazuichi; Takase, Hiroshi; Usui, Yoshihiko; Takeuchi, Masaru; Ishidoh, Kazumi; Koike, Masato; Uchiyama, Yasuo; Peters, Christoph; Yamamoto, Yoshimi; Mochizuki, Manabu

    2009-10-15

    Pigment epithelium isolated from the eye possesses immunosuppressive properties such as regulatory T (Treg) cell induction; e.g., cultured retinal pigment epithelium (RPE) converts CD4(+) T cells into Treg cells in vitro. RPE constitutively expresses a novel immunosuppressive factor, CTLA-2alpha, which is a cathepsin L (CathL) inhibitor, and this molecule acts via RPE to induce Treg cells. To clarify CTLA-2alpha's role in the T cell response to RPE in ocular inflammation, we used the experimental autoimmune uveitis (EAU) animal model to examine this new immunosuppressive property of RPE. In EAU models, TGF-beta, but not IFN-gamma inflammatory cytokines, promotes the up-regulation of the expression of CTLA-2alpha in RPE. Similarly, CTLA-2alpha via RPE was able to promote TGF-beta production by the CD4(+) T cells. The RPE-exposed T cells (RPE-induced Treg cells) greatly produced TGF-beta and suppressed bystander effector T cells. There was less expression of CathL by the RPE-exposed T cells, and CathL-inhibited T cells were able to acquire the Treg phenotype. Moreover, CathL-deficient mice spontaneously produced Treg cells, with the increase in T cells potentially providing protection against ocular inflammation. More importantly, CD4(+) T cells from EAU in CathL knockout mice or rCTLA-2alpha from EAU animals were found to contain a high population of forkhead box p3(+) T cells. In both EAU models, there was significant suppression of the ocular inflammation. These results indicate that RPE secretes CTLA-2alpha, thereby enabling the bystander T cells to be converted into Treg cells via TGF-beta promotion.

  8. Gamma Synuclein Promotes a Metastatic Phenotype in Breast and Ovarian Tumor Cells by Modulating the Rho Signal Transduction Activity

    DTIC Science & Technology

    2002-05-01

    peripheral nervous system, such as dorsal root ganglia and trigeminal ganglia . Synoretin, the newest member of the synuclein family is expressed at high...involved in neuron development and function (43). The involvement of y-synuclein in human neoplastic diseases came to light when y-synuclein was...wed) are a famnily of small, highly conserved proteins exprssed predominanly in neurons . While a- synfucein is impicatad i neurodegenerative diseases

  9. Neuronal overexpression of Ube3a isoform 2 causes behavioral impairments and neuroanatomical pathology relevant to 15q11.2-q13.3 duplication syndrome.

    PubMed

    Copping, Nycole A; Christian, Sarah G B; Ritter, Dylan J; Islam, M Saharul; Buscher, Nathalie; Zolkowska, Dorota; Pride, Michael C; Berg, Elizabeth L; LaSalle, Janine M; Ellegood, Jacob; Lerch, Jason P; Reiter, Lawrence T; Silverman, Jill L; Dindot, Scott V

    2017-10-15

    Maternally derived copy number gains of human chromosome 15q11.2-q13.3 (Dup15q syndrome or Dup15q) cause intellectual disability, epilepsy, developmental delay, hypotonia, speech impairments, and minor dysmorphic features. Dup15q syndrome is one of the most common and penetrant chromosomal abnormalities observed in individuals with autism spectrum disorder (ASD). Although ∼40 genes are located in the 15q11.2-q13.3 region, overexpression of the ubiquitin-protein E3A ligase (UBE3A) gene is thought to be the predominant molecular cause of the phenotypes observed in Dup15q syndrome. The UBE3A gene demonstrates maternal-specific expression in neurons and loss of maternal UBE3A causes Angelman syndrome, a neurodevelopmental disorder with some overlapping neurological features to Dup15q. To directly test the hypothesis that overexpression of UBE3A is an important underlying molecular cause of neurodevelopmental dysfunction, we developed and characterized a mouse overexpressing Ube3a isoform 2 in excitatory neurons. Ube3a isoform 2 is conserved between mouse and human and known to play key roles in neuronal function. Transgenic mice overexpressing Ube3a isoform 2 in excitatory forebrain neurons exhibited increased anxiety-like behaviors, learning impairments, and reduced seizure thresholds. However, these transgenic mice displayed normal social approach, social interactions, and repetitive motor stereotypies that are relevant to ASD. Reduced forebrain, hippocampus, striatum, amygdala, and cortical volume were also observed. Altogether, these findings show neuronal overexpression of Ube3a isoform 2 causes phenotypes translatable to neurodevelopmental disorders. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  10. Structure of the toxic core of α-synuclein from invisible crystals

    DOE PAGES

    Rodriguez, Jose A.; Ivanova, Magdalena I.; Sawaya, Michael R.; ...

    2015-09-09

    We report that the protein α-synuclein is the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson disease and other neurodegenerative pathologies. An 11-residue segment, which we term NACore, appears to be responsible for amyloid formation and cytotoxicity of human α-synuclein. Here we describe crystals of NACore that have dimensions smaller than the wavelength of visible light and thus are invisible by optical microscopy. As the crystals are thousands of times too small for structure determination by synchrotron X-ray diffraction, we use micro-electron diffraction to determine the structure at atomic resolution. The 1.4 Å resolution structure demonstrates thatmore » this method can determine previously unknown protein structures and here yields, to our knowledge, the highest resolution achieved by any cryo-electron microscopy method to date. The structure exhibits protofibrils built of pairs of face-to-face β-sheets. X-ray fibre diffraction patterns show the similarity of NACore to toxic fibrils of full-length α-synuclein. Finally, the NACore structure, together with that of a second segment, inspires a model for most of the ordered portion of the toxic, full-length α-synuclein fibril, presenting opportunities for the design of inhibitors of α-synuclein fibrils.« less

  11. Alpha-SNAP functions in insulin exocytosis from mature, but not immature secretory granules in pancreatic beta cells.

    PubMed

    Nakamichi, Y; Nagamatsu, S

    1999-06-24

    To explore alpha-SNAP function in insulin exocytosis from either immature or mature secretory granules in pancreatic beta cells, we studied the effects of overexpression of adenovirus-mediated wild-type alpha-SNAP and C-terminally deleted alpha-SNAP mutant (1-285) on newly synthesized proinsulin and insulin release by rat islets and MIN6 cells. Rat islets overexpressing alpha-SNAP and mutant alpha-SNAP were pulse-chased. Exocytosis from immature and mature insulin secretory granules was measured as fractional (%) labeled-proinsulin release immediately after the pulse-labeling and percentage labeled-insulin release after a 3-h chase period, respectively. There was no difference in percentage labeled-proinsulin release between the control and alpha-SNAP or mutant alpha-SNAP-overexpressed islets. Although percentage labeled-insulin release after a 3-h chase period was significantly increased in alpha-SNAP-overexpressed islets, it was decreased in mutant alpha-SNAP-overexpressed islets. Thus, the results demonstrated that alpha-SNAP overexpression in rat islets primarily increased exocytosis from mature, but not immature insulin secretory granules. On the other hand, in MIN6 cells, alpha-SNAP overexpression scarcely affected glucose-stimulated insulin release; therefore, we examined the effect of mutant alpha-SNAP overexpression as the dominant-negative inhibitor on the newly synthesized proinsulin/insulin release using the same protocol as in the rat islet experiments. alpha-SNAP mutant (1-285) overexpression in MIN6 cells decreased the percentage labeled insulin release from mature secretory granules, but not percentage labeled proinsulin release from immature secretory granules. Thus, our data demonstrate that alpha-SNAP functions mainly in the mature insulin secretory granules in pancreatic beta cells. Copyright 1999 Academic Press.

  12. Split T Cell Tolerance against a Self/Tumor Antigen: Spontaneous CD4+ but Not CD8+ T Cell Responses against p53 in Cancer Patients and Healthy Donors

    PubMed Central

    Tsuji, Takemasa; Matsuzaki, Junko; Ritter, Erika; Miliotto, Anthony; Ritter, Gerd; Odunsi, Kunle; Old, Lloyd J.; Gnjatic, Sacha

    2011-01-01

    Analyses of NY-ESO-1-specific spontaneous immune responses in cancer patients revealed that antibody and both CD4+ and CD8+ T cell responses were induced together in cancer patients. To explore whether such integrated immune responses are also spontaneously induced for other tumor antigens, we have evaluated antibody and T cell responses against self/tumor antigen p53 in ovarian cancer patients and healthy individuals. We found that 21% (64/298) of ovarian cancer patients but no healthy donors showed specific IgG responses against wild-type p53 protein. While none of 12 patients with high titer p53 antibody showed spontaneous p53-specific CD8+ T cell responses following a single in vitro sensitization, significant p53-specific IFN-γ producing CD4+ T cells were detected in 6 patients. Surprisingly, similar levels of p53-specific CD4+ T cells but not CD8+ T cells were also detected in 5/10 seronegative cancer patients and 9/12 healthy donors. Importantly, p53-specific CD4+ T cells in healthy donors originated from a CD45RA− antigen-experienced T cell population and recognized naturally processed wild-type p53 protein. These results raise the possibility that p53-specific CD4+ T cells reflect abnormalities in p53 occurring in normal individuals and that they may play a role in processes of immunosurveillance or immunoregulation of p53-related neoplastic events. PMID:21858191

  13. Neurofilament L gene is not a genetic factor of sporadic and familial Parkinson's disease.

    PubMed

    Rahner, Nils; Holzmann, Carsten; Krüger, Rejko; Schöls, Ludger; Berger, Klaus; Riess, Olaf

    2002-09-27

    Mutations in two genes, alpha-synuclein and parkin, have been identified as some rare causes for familial Parkinson's disease (PD). alpha-Synuclein and parkin protein have subsequently been identified in Lewy bodies (LB). To gain further insight into the pathogenesis of PD we investigated the role of neurofilament light (NF-L), another component of LB aggregation. A detailed mutation search of the NF-L gene in 328 sporadic and familial PD patients of German ancestry revealed three silent DNA changes (G163A, C224T, C487T) in three unrelated patients. Analysis of the promoter region of the NF-L gene identified a total of three base pair substitutions defining five haplotypes. Association studies based on these haplotypes revealed no significant differences between PD patients and 344 control individuals. Therefore, NF-L is unlikely to play a major role in the pathogenesis of PD.

  14. Structural Effects of Two Camelid Nanobodies Directed to Distinct C-Terminal Epitopes on α-Synuclein.

    PubMed

    El-Turk, Farah; Newby, Francisco N; De Genst, Erwin; Guilliams, Tim; Sprules, Tara; Mittermaier, Anthony; Dobson, Christopher M; Vendruscolo, Michele

    2016-06-07

    α-Synuclein is an intrinsically disordered protein whose aggregation is associated with Parkinson's disease and other related neurodegenerative disorders. Recently, two single-domain camelid antibodies (nanobodies) were shown to bind α-synuclein with high affinity. Herein, we investigated how these two nanobodies (NbSyn2 and NbSyn87), which are directed to two distinct epitopes within the C-terminal domain of α-synuclein, affect the conformational properties of this protein. Our results suggest that nanobody NbSyn2, which binds to the five C-terminal residues of α-synuclein (residues 136-140), does not disrupt the transient long-range interactions that generate a degree of compaction within the native structural ensemble of α-synuclein. In contrast, the data that we report indicate that NbSyn87, which targets a central region within the C-terminal domain (residues 118-128), has more substantial effects on the fluctuating secondary and tertiary structure of the protein. These results are consistent with the different effects that the two nanobodies have on the aggregation behavior of α-synuclein in vitro. Our findings thus provide new insights into the type of effects that nanobodies can have on the conformational ensemble of α-synuclein.

  15. Alpha-Synuclein Pathology in Sensory Nerve Terminals of the Upper Aerodigestive Tract of Parkinson's Disease Patients.

    PubMed

    Mu, Liancai; Chen, Jingming; Sobotka, Stanislaw; Nyirenda, Themba; Benson, Brian; Gupta, Fiona; Sanders, Ira; Adler, Charles H; Caviness, John N; Shill, Holly A; Sabbagh, Marwan; Samanta, Johan E; Sue, Lucia I; Beach, Thomas G

    2015-08-01

    Dysphagia is common in Parkinson's disease (PD) and causes significant morbidity and mortality. PD dysphagia has usually been explained as dysfunction of central motor control, much like other motor symptoms that are characteristic of the disease. However, PD dysphagia does not correlate with severity of motor symptoms nor does it respond to motor therapies. It is known that PD patients have sensory deficits in the pharynx, and that impaired sensation may contribute to dysphagia. However, the underlying cause of the pharyngeal sensory deficits in PD is not known. We hypothesized that PD dysphagia with sensory deficits may be due to degeneration of the sensory nerve terminals in the upper aerodigestive tract (UAT). We have previously shown that Lewy-type synucleinopathy (LTS) is present in the main pharyngeal sensory nerves of PD patients, but not in controls. In this study, the sensory terminals in UAT mucosa were studied to discern the presence and distribution of LTS. Whole-mount specimens (tongue-pharynx-larynx-upper esophagus) were obtained from 10 deceased human subjects with clinically diagnosed and neuropathologically confirmed PD (five with dysphagia and five without) and four age-matched healthy controls. Samples were taken from six sites and immunostained for phosphorylated α-synuclein (PAS). The results showed the presence of PAS-immunoreactive (PAS-ir) axons in all the PD subjects and in none of the controls. Notably, PD patients with dysphagia had more PAS-ir axons in the regions that are critical for initiating the swallowing reflex. These findings suggest that Lewy pathology affects mucosal sensory axons in specific regions of the UAT and may be related to PD dysphagia.

  16. Increased lipolysis and altered lipid homeostasis protect y-synuclein null mutant mice from diet-induced obesity

    USDA-ARS?s Scientific Manuscript database

    Synucleins are a family of homologous proteins principally known for their involvement in neurodegeneration. In neurons a-synuclein promotes assembly of SNARE complexes required for fusion of synaptic vesicles with the plasma membrane during neurotransmitter release. Y-synuclein is highly expressed ...

  17. Combustion-derived nanoparticles, the neuroenteric system, cervical vagus, hyperphosphorylated alpha synuclein and tau in young Mexico City residents.

    PubMed

    Calderón-Garcidueñas, Lilian; Reynoso-Robles, Rafael; Pérez-Guillé, Beatriz; Mukherjee, Partha S; Gónzalez-Maciel, Angélica

    2017-11-01

    Mexico City (MC) young residents are exposed to high levels of fine particulate matter (PM 2.5 ), have high frontal concentrations of combustion-derived nanoparticles (CDNPs), accumulation of hyperphosphorylated aggregated α-synuclein (α-Syn) and early Parkinson's disease (PD). Swallowed CDNPs have easy access to epithelium and submucosa, damaging gastrointestinal (GI) barrier integrity and accessing the enteric nervous system (ENS). This study is focused on the ENS, vagus nerves and GI barrier in young MC v clean air controls. Electron microscopy of epithelial, endothelial and neural cells and immunoreactivity of stomach and vagus to phosphorylated ɑ-synuclein Ser129 and Hyperphosphorylated-Tau (Htau) were evaluated and CDNPs measured in ENS. CDNPs were abundant in erythrocytes, unmyelinated submucosal, perivascular and intramuscular nerve fibers, ganglionic neurons and vagus nerves and associated with organelle pathology. ɑSyn and Htau were present in 25/27 MC gastric,15/26 vagus and 18/27 gastric and 2/26 vagus samples respectively. We strongly suggest CDNPs are penetrating and damaging the GI barrier and reaching preganglionic parasympathetic fibers and the vagus nerve. This work highlights the potential role of CDNPs in the neuroenteric hyperphosphorylated ɑ-Syn and tau pathology as seen in Parkinson and Alzheimer's diseases. Highly oxidative, ubiquitous CDNPs constitute a biologically plausible path into Parkinson's and Alzheimer's pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Patients with mild cognitive impairment have an abnormal upper-alpha event-related desynchronization/synchronization (ERD/ERS) during a task of temporal attention.

    PubMed

    Caravaglios, Giuseppe; Muscoso, Emma Gabriella; Di Maria, Giulia; Costanzo, Erminio

    2015-03-01

    There are several evidences indicating that an impairment in attention-executive functions is present in prodromal Alzheimer's disease and predict future global cognitive decline. In particular, the issue of temporal orienting of attention in patients with mild cognitive impairment (MCI) due to Alzheimer's disease has been overlooked. The present research aimed to explore whether subtle deficits of cortical activation are present in these patients early in the course of the disease. We studied the upper-alpha event-related synchronization/desynchronization phenomenon during a paradigm of temporal orientation of attention. MCI patients (n = 27) and healthy elderly controls (n = 15) performed a task in which periodically omitted tones had to be predicted and their virtual onset time had to be marked by pressing a button. Single-trial responses were measured, respectively, before and after the motor response. Then, upper-alpha responses were compared to upper-alpha power during eyes-closed resting state. The time course of the task was characterized by two different behavioral conditions: (1) a pre-event epoch, in which the subject awaited the virtual onset of the omitted tone, (2) a post-event epoch (after button pressing), in which the subject was in a post-motor response condition. The principal findings are: (1) during the waiting epoch, only healthy elderly had an upper-alpha ERD at the level of both temporal and posterior brain regions; (2) during the post-motor epoch, the aMCI patients had a weaker upper-alpha ERS on prefrontal regions; (3) only healthy elderly showed a laterality effect: (a) during the waiting epoch, the upper-alpha ERD was greater at the level of the right posterior-temporal lead; during the post-motor epoch, the upper alpha ERS was greater on the left prefrontal lead. The relevance of these findings is that the weaker upper-alpha response observed in aMCI patients is evident even if the accuracy of the behavioral performance (i.e., button

  19. p53-mediated inhibition of angiogenesis through up-regulation of a collagen prolyl hydroxylase.

    PubMed

    Teodoro, Jose G; Parker, Albert E; Zhu, Xiaochun; Green, Michael R

    2006-08-18

    Recent evidence suggests that antiangiogenic therapy is sensitive to p53 status in tumors, implicating a role for p53 in the regulation of angiogenesis. Here we show that p53 transcriptionally activates the alpha(II) collagen prolyl-4-hydroxylase [alpha(II)PH] gene, resulting in the extracellular release of antiangiogenic fragments of collagen type 4 and 18. Conditioned media from cells ectopically expressing either p53 or alpha(II)PH selectively inhibited growth of primary human endothelial cells. When expressed intracellularly or exogenously delivered, alpha(II)PH significantly inhibited tumor growth in mice. Our results reveal a genetic and biochemical linkage between the p53 tumor suppressor pathway and the synthesis of antiangiogenic collagen fragments.

  20. Metabolic Investigations of the Molecular Mechanisms Associated with Parkinson’s Disease

    PubMed Central

    Powers, Robert; Lei, Shulei; Anandhan, Annadurai; Marshall, Darrell D.; Worley, Bradley; Cerny, Ronald L.; Dodds, Eric D.; Huang, Yuting; Panayiotidis, Mihalis I.; Pappa, Aglaia; Franco, Rodrigo

    2017-01-01

    Parkinson’s disease (PD) is a neurodegenerative disorder characterized by fibrillar cytoplasmic aggregates of α-synuclein (i.e., Lewy bodies) and the associated loss of dopaminergic cells in the substantia nigra. Mutations in genes such as α-synuclein (SNCA) account for only 10% of PD occurrences. Exposure to environmental toxicants including pesticides and metals (e.g., paraquat (PQ) and manganese (Mn)) is also recognized as an important PD risk factor. Thus, aging, genetic alterations, and environmental factors all contribute to the etiology of PD. In fact, both genetic and environmental factors are thought to interact in the promotion of idiopathic PD, but the mechanisms involved are still unclear. In this study, we summarize our findings to date regarding the toxic synergistic effect between α-synuclein and paraquat treatment. We identified an essential role for central carbon (glucose) metabolism in dopaminergic cell death induced by paraquat treatment that is enhanced by the overexpression of α-synuclein. PQ “hijacks” the pentose phosphate pathway (PPP) to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. PQ also stimulated an increase in glucose uptake, the translocation of glucose transporters to the plasma membrane, and AMP-activated protein kinase (AMPK) activation. The overexpression of α-synuclein further stimulated an increase in glucose uptake and AMPK activity, but impaired glucose metabolism, likely directing additional carbon to the PPP to supply paraquat redox cycling. PMID:28538683

  1. Sestrin2 Protects Dopaminergic Cells against Rotenone Toxicity through AMPK-Dependent Autophagy Activation

    PubMed Central

    Hou, Yi-Sheng; Guan, Jun-Jie; Xu, Hai-Dong; Wu, Feng; Sheng, Rui

    2015-01-01

    Dysfunction of the autophagy-lysosomal pathway (ALP) and the ubiquitin-proteasome system (UPS) was thought to be an important pathogenic mechanism in synuclein pathology and Parkinson's disease (PD). In the present study, we investigated the role of sestrin2 in autophagic degradation of α-synuclein and preservation of cell viability in a rotenone-induced cellular model of PD. We speculated that AMP-activated protein kinase (AMPK) was involved in regulation of autophagy and protection of dopaminergic cells against rotenone toxicity by sestrin2. The results showed that both the mRNA and protein levels of sestrin2 were increased in a TP53-dependent manner in Mes 23.5 cells after treatment with rotenone. Genetic knockdown of sestrin2 compromised the autophagy induction in response to rotenone, while overexpression of sestrin2 increased the basal autophagy activity. Sestrin2 presumably enhanced autophagy in an AMPK-dependent fashion, as sestrin2 overexpression activated AMPK, and genetic knockdown of AMPK abrogated autophagy induction by rotenone. Restoration of AMPK activity by metformin after sestrin2 knockdown recovered the autophagy activity. Sestrin2 overexpression ameliorated α-synuclein accumulation, inhibited caspase 3 activation, and reduced the cytotoxicity of rotenone. These results suggest that sestrin2 upregulation attempts to maintain autophagy activity and suppress rotenone cytotoxicity through activation of AMPK, and that sestrin2 exerts a protective effect on dopaminergic cells. PMID:26031332

  2. 17{alpha}-Estradiol arrests cell cycle progression at G{sub 2}/M and induces apoptotic cell death in human acute leukemia Jurkat T cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jun, Do Youn; Park, Hae Sun; Kim, Jun Seok

    2008-09-15

    A pharmacological dose (2.5-10 {mu}M) of 17{alpha}-estradiol (17{alpha}-E{sub 2}) exerted a cytotoxic effect on human leukemias Jurkat T and U937 cells, which was not suppressed by the estrogen receptor (ER) antagonist ICI 182,780. Along with cytotoxicity in Jurkat T cells, several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, PARP degradation, and DNA fragmentation were induced. The cytotoxicity of 17{alpha}-E{sub 2} was not blocked by the anti-Fas neutralizing antibody ZB-4. While undergoing apoptosis, there was a remarkable accumulation of G{sub 2}/M cells with the upregulatoin of cdc2 kinase activity, which was reflected in the Thr56more » phosphorylation of Bcl-2. Dephosphorylation at Tyr15 and phosphorylation at Thr161 of cdc2, and significant increase in the cyclin B1 level were underlying factors for the cdc2 kinase activation. Whereas the 17{alpha}-E{sub 2}-induced apoptosis was completely abrogated by overexpression of Bcl-2 or by pretreatment with the pan-caspase inhibitor z-VAD-fmk, the accumulation of G{sub 2}/M cells significantly increased. The caspase-8 inhibitor z-IETD-fmk failed to influence 17{alpha}-E{sub 2}-mediated caspase-9 activation, but it markedly reduced caspase-3 activation and PARP degradation with the suppression of apoptosis, indicating the contribution of caspase-8; not as an upstream event of the mitochondrial cytochrome c release, but to caspase-3 activation. In the presence of hydroxyurea, which blocked the cell cycle progression at the G{sub 1}/S boundary, 17{alpha}-E{sub 2} failed to induce the G{sub 2}/M arrest as well as apoptosis. These results demonstrate that the cytotoxicity of 17{alpha}-E{sub 2} toward Jurkat T cells is attributable to apoptosis mainly induced in G{sub 2}/M-arrested cells, in an ER-independent manner, via a mitochondria-dependent caspase pathway regulated by Bcl-2.« less

  3. FLZ Attenuates α-Synuclein-Induced Neurotoxicity by Activating Heat Shock Protein 70.

    PubMed

    Bao, Xiu-Qi; Wang, Xiao-Liang; Zhang, Dan

    2017-01-01

    Parkinson's disease (PD) is the second most prevalent neurodegenerative disease. The pathology of PD is caused by progressive degeneration of dopaminergic neurons and is characterized by the presence of intracellular inclusions known as Lewy bodies, composed mainly of α-synuclein. Heat shock proteins (HSPs) are crucial in protein quality control in cells. HSP70 in particular prevents the aggregation of protein aggregation, such as α-synuclein, providing a degree of protection against PD. The compound FLZ has been shown to protect several PD models in previous studies and was reported as an HSP inducer to protect against MPP + -induced neurotoxicity, but the mechanism remains unclear. In this study, we investigated the effects of FLZ-mediated HSP70 induction in α-synuclein transgenic mice and cells. FLZ treatment alleviated motor dysfunction and improved dopaminergic neuronal function in α-synuclein transgenic mice. HSP70 protein expression and transcriptional activity were increased by FLZ treatment, eliciting a reduction of α-synuclein aggregation and associated toxicity. The inhibition of HSP70 by quercetin or HSP70 siRNA markedly attenuated the neuroprotective effects of FLZ, confirming that FLZ exerted a neuroprotective effect through HSP70. We revealed that FLZ directly bound to and increased the expression of Hip, a cochaperone of HSP70, which in turn enhanced HSP70 activity. In conclusion, we defined a critical role for HSP70 and its cochaperones activated by FLZ in preventing neurodegeneration and proposed that targeting the HSP70 system may represent a potential therapy for α-synuclein-related diseases, such as PD.

  4. Parkin-mediated protection of dopaminergic neurons in a chronic MPTP-minipump mouse model of Parkinson disease.

    PubMed

    Yasuda, Toru; Hayakawa, Hideki; Nihira, Tomoko; Ren, Yong-Ri; Nakata, Yasuto; Nagai, Makiko; Hattori, Nobutaka; Miyake, Koichi; Takada, Masahiko; Shimada, Takashi; Mizuno, Yoshikuni; Mochizuki, Hideki

    2011-08-01

    Loss-of-function mutations in the ubiquitin ligase parkin are the major cause of recessively inherited early-onset Parkinson disease (PD). Impairment of parkin activity caused by nitrosative or dopamine-related modifications may also be responsible for the loss of dopaminergic (DA) neurons in sporadic PD. Previous studies have shown that viral vector-mediated delivery of parkin prevented DA neurodegeneration in several animal models, but little is known about the neuroprotective actions of parkin in vivo. Here, we investigated mechanisms of neuroprotection of overexpressed parkin in a modified long-term mouse model of PD using osmotic minipump administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Recombinant adeno-associated viral vector-mediated intranigral delivery of parkin prevented motor deficits and DA cell loss in the mice. Ser129-phosphorylated α-synuclein-immunoreactive cells were increased in the substantia nigra of parkin-treated mice. Moreover, delivery of parkin alleviated the MPTP-induced decrease of the active phosphorylated form of Akt. On the other hand, upregulation of p53 and mitochondrial alterations induced by chronic MPTP administration were barely suppressed by parkin. These results suggest that the neuroprotective actions of parkin may be impaired in severe PD.

  5. MIR517C inhibits autophagy and the epithelial-to-mesenchymal (-like) transition phenotype in human glioblastoma through KPNA2-dependent disruption of TP53 nuclear translocation

    PubMed Central

    Lu, Yuntao; Xiao, Limin; Liu, Yawei; Wang, Hai; Li, Hong; Zhou, Qiang; Pan, Jun; Lei, Bingxi; Huang, Annie; Qi, Songtao

    2015-01-01

    The epithelial-to-mesenchymal (-like) transition (EMT), a crucial embryonic development program, has been linked to the regulation of glioblastoma (GBM) progression and invasion. Here, we investigated the role of MIR517C/miR-517c, which belongs to the C19MC microRNA cluster identified in our preliminary studies, in the pathogenesis of GBM. We found that MIR517C was associated with improved prognosis in patients with GBM. Furthermore, following treatment with the autophagy inducer temozolomide (TMZ) and low glucose (LG), MIR517C degraded KPNA2 (karyopherin alpha 2 [RAG cohort 1, importin alpha 1]) and subsequently disturbed the nuclear translocation of TP53 in the GBM cell line U87 in vitro. Interestingly, this microRNA could inhibit autophagy and reduce cell migration and infiltration in U87 cells harboring wild-type (WT) TP53, but not in U251 cells harboring mutant (MU) TP53. Moreover, the expression of epithelial markers (i.e., CDH13/T-cadherin and CLDN1 [claudin 1]) increased, while the expression of mesenchymal markers (i.e., CDH2/N-cadherin, SNAI1/Snail, and VIM [vimentin]) decreased, indicating that the EMT status was blocked by MIR517C in U87 cells. Compared with MIR517C overexpression, MIR517C knockdown promoted infiltration of U87 cells to the surrounding structures in nude mice in vivo. The above phenotypic changes were also observed in TP53+/+ and TP53-/- HCT116 colon cancer cells. In summary, our study provided support for a link between autophagy and EMT status in WT TP53 GBM cells and provided evidence for the signaling pathway (MIR517C-KPNA2-cytoplasmic TP53) involved in attenuating autophagy and eliminating the increased migration and invasion during the EMT. PMID:26553592

  6. Overexpression of SKI Oncoprotein Leads to p53 Degradation through Regulation of MDM2 Protein Sumoylation*

    PubMed Central

    Ding, Boxiao; Sun, Yin; Huang, Jiaoti

    2012-01-01

    Protooncogene Ski was identified based on its ability to transform avian fibroblasts in vitro. In support of its oncogenic activity, SKI was found to be overexpressed in a variety of human cancers, although the exact molecular mechanism(s) responsible for its oncogenic activity is not fully understood. We found that SKI can negatively regulate p53 by decreasing its level through up-regulation of MDM2 activity, which is mediated by the ability of SKI to enhance sumoylation of MDM2. This stimulation of MDM2 sumoylation is accomplished through a direct interaction of SKI with SUMO-conjugating enzyme E2, Ubc9, resulting in enhanced thioester bond formation and mono-sumoylation of Ubc9. A mutant SKI defective in transformation fails to increase p53 ubiquitination and is unable to increase MDM2 levels and to increase mono-sumoylation of Ubc9, suggesting that the ability of SKI to enhance Ubc9 activity is essential for its transforming function. These results established a detailed molecular mechanism that underlies the ability of SKI to cause cellular transformation while unraveling a novel connection between sumoylation and tumorigenesis, providing potential new therapeutic targets for cancer. PMID:22411991

  7. The potential of zwitterionic nanoliposomes against neurotoxic alpha-synuclein aggregates in Parkinson's Disease.

    PubMed

    Aliakbari, Farhang; Mohammad-Beigi, Hossein; Rezaei-Ghaleh, Nasrollah; Becker, Stefan; Dehghani Esmatabad, Faezeh; Eslampanah Seyedi, Hadieh Alsadat; Bardania, Hassan; Tayaranian Marvian, Amir; Collingwood, Joanna F; Christiansen, Gunna; Zweckstetter, Markus; Otzen, Daniel E; Morshedi, Dina

    2018-05-17

    The protein α-synuclein (αSN) aggregates to form fibrils in neuronal cells of Parkinson's patients. Here we report on the effect of neutral (zwitterionic) nanoliposomes (NLPs), supplemented with cholesterol (NLP-Chol) and decorated with PEG (NLP-Chol-PEG), on αSN aggregation and neurotoxicity. Both NLPs retard αSN fibrillization in a concentration-independent fashion. They do so largely by increasing lag time (formation of fibrillization nuclei) rather than elongation (extension of existing nuclei). Interactions between neutral NLPs and αSN may locate to the N-terminus of the protein. This interaction can even perturb the interaction of αSN with negatively charged NLPs which induces an α-helical structure in αSN. This interaction was found to occur throughout the fibrillization process. Both NLP-Chol and NLP-Chol-PEG were shown to be biocompatible in vitro, and to reduce αSN neurotoxicity and reactive oxygen species (ROS) levels with no influence on intracellular calcium in neuronal cells, emphasizing a prospective role for NLPs in reducing αSN pathogenicity in vivo as well as utility as a vehicle for drug delivery.

  8. Atp13a2-deficient mice exhibit neuronal ceroid lipofuscinosis, limited α-synuclein accumulation and age-dependent sensorimotor deficits

    PubMed Central

    Schultheis, Patrick J.; Fleming, Sheila M.; Clippinger, Amy K.; Lewis, Jada; Tsunemi, Taiji; Giasson, Benoit; Dickson, Dennis W.; Mazzulli, Joseph R.; Bardgett, Mark E.; Haik, Kristi L.; Ekhator, Osunde; Chava, Anil Kumar; Howard, John; Gannon, Matt; Hoffman, Elizabeth; Chen, Yinhuai; Prasad, Vikram; Linn, Stephen C.; Tamargo, Rafael J.; Westbroek, Wendy; Sidransky, Ellen; Krainc, Dimitri; Shull, Gary E.

    2013-01-01

    Mutations in ATP13A2 (PARK9), encoding a lysosomal P-type ATPase, are associated with both Kufor–Rakeb syndrome (KRS) and neuronal ceroid lipofuscinosis (NCL). KRS has recently been classified as a rare genetic form of Parkinson's disease (PD), whereas NCL is a lysosomal storage disorder. Although the transport activity of ATP13A2 has not been defined, in vitro studies show that its loss compromises lysosomal function, which in turn is thought to cause neuronal degeneration. To understand the role of ATP13A2 dysfunction in disease, we disrupted its gene in mice. Atp13a2−/− and Atp13a2+/+ mice were tested behaviorally to assess sensorimotor and cognitive function at multiple ages. In the brain, lipofuscin accumulation, α-synuclein aggregation and dopaminergic pathology were measured. Behaviorally, Atp13a2−/− mice displayed late-onset sensorimotor deficits. Accelerated deposition of autofluorescent storage material (lipofuscin) was observed in the cerebellum and in neurons of the hippocampus and the cortex of Atp13a2−/− mice. Immunoblot analysis showed increased insoluble α-synuclein in the hippocampus, but not in the cortex or cerebellum. There was no change in the number of dopaminergic neurons in the substantia nigra or in striatal dopamine levels in aged Atp13a2−/− mice. These results show that the loss of Atp13a2 causes sensorimotor impairments, α-synuclein accumulation as occurs in PD and related synucleinopathies, and accumulation of lipofuscin deposits characteristic of NCL, thus providing the first direct demonstration that null mutations in Atp13a2 can cause pathological features of both diseases in the same organism. PMID:23393156

  9. O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease

    NASA Astrophysics Data System (ADS)

    Marotta, Nicholas P.; Lin, Yu Hsuan; Lewis, Yuka E.; Ambroso, Mark R.; Zaro, Balyn W.; Roth, Maxwell T.; Arnold, Don B.; Langen, Ralf; Pratt, Matthew R.

    2015-11-01

    Several aggregation-prone proteins associated with neurodegenerative diseases can be modified by O-linked N-acetyl-glucosamine (O-GlcNAc) in vivo. One of these proteins, α-synuclein, is a toxic aggregating protein associated with synucleinopathies, including Parkinson's disease. However, the effect of O-GlcNAcylation on α-synuclein is not clear. Here, we use synthetic protein chemistry to generate both unmodified α-synuclein and α-synuclein bearing a site-specific O-GlcNAc modification at the physiologically relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, while not affecting the membrane binding or bending properties of α-synuclein. O-GlcNAcylation is also shown to affect the phosphorylation of α-synuclein in vitro and block the toxicity of α-synuclein that was exogenously added to cells in culture. These results suggest that increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation.

  10. T1alpha/podoplanin shows raft-associated distribution in mouse lung alveolar epithelial E10 cells.

    PubMed

    Barth, Kathrin; Bläsche, Robert; Kasper, Michael

    2010-01-01

    T1alpha/(podoplanin) is abundantly expressed in the alveolar epithelial type I cells (ATI) of rodent and human lungs. Caveolin-1 is a classical primary structural protein of plasmalemal invaginations, so-called caveolae, which represent specialized lipid rafts, and which are particularly abundant in ATI cells. The biological functions of T1alpha in the alveolar epithelium are unknown. Here we report on the characteristics of raft domains in the microplicae/microvillar protrusions of ATI cells, which contain T1alpha. Detergent resistant membranes (DRMs) from cell lysates of the mouse epithelial ATI-like cell line E10 were prepared using different detergents followed by flotation in a sucrose gradient and tested by Western and dot blots with raft markers (caveolin-1, GM1) and nonraft markers (transferrin receptor, PDI and beta-Cop). Immunocytochemistry was employed for the localization of T1alpha in E10 cells and in situ in rat lungs. Our biochemical results showed that the solubility or insolubility of T1alpha and caveolin-1 differs in Triton X-100 and Lubrol WX, two distinct non-ionic detergents. Caveolin-1 was unsoluble in both detergents, whereas T1alpha was Triton X-100 soluble but Lubrol WX insoluble. Immunofluorescence double stainings revealed that both proteins were colocalized with GM1, while caveolin-1 and T1alpha were not colocalized in the plasma membrane. Cholesterol depletion modified the segregation of T1alpha in Lubrol WX DRMs. Cellular processes in ultrathin sections of cultured mouse E10 cells were immunogold positive. Immunoelectron microscopy (postembedding) of rat lung tissue revealed the preferential localization of T1alpha on apical microvillar protrusions of ATI cells. We conclude that T1alpha and caveolin-1 are located in distinct plasma membrane microdomains, which differ in their protein-lipid interactions. The raft-associated distribution of T1alpha may have an impact on a specific, not yet clarified function of this protein in the

  11. Theta and Alpha Oscillation Impairments in Autistic Spectrum Disorder Reflect Working Memory Deficit.

    PubMed

    Larrain-Valenzuela, Josefina; Zamorano, Francisco; Soto-Icaza, Patricia; Carrasco, Ximena; Herrera, Claudia; Daiber, Francisca; Aboitiz, Francisco; Billeke, Pablo

    2017-10-30

    A dysfunction in the excitatory-inhibitory (E/I) coordination in neuronal assembly has been proposed as a possible neurobiological mechanism of Autistic Spectrum Disorder (ASD). However, the potential impact of this mechanism in cognitive performance is not fully explored. Since the main consequence of E/I dysfunction is an impairment in oscillatory activity and its underlying cognitive computations, we assessed the electroencephalographic activity of ASD and typically developing (TD) subjects during a working-memory task. We found that ASD subjects committed more errors than TD subjects. Moreover, TD subjects demonstrated a parametric modulation in the power of alpha and theta band while ASD subjects did not demonstrate significant modulations. The preceding leads to significant differences between the groups in both the alpha power placed on the occipital cortex and the theta power placed on the left premotor and the right prefrontal cortex. The impaired theta modulation correlated with autistic symptoms. The results indicated that ASD may present an alteration in the recruitment of the oscillatory activity during working-memory, and this alteration could be related to the physiopathology of the disorder.

  12. Evaluation of the synuclein-y (SNCG) gene as a PPARy target in murine adipocytes, dorsal root ganglia somatosensory neurons, and human adipose tissue

    USDA-ARS?s Scientific Manuscript database

    Synuclein-gamma is highly expressed in both adipocytes and peripheral nervous system (PNS) somatosensory neurons. Its mRNA is induced during adipogenesis, increased in obese human white adipose tissue (WAT), may be coordinately regulated with leptin, and is decreased following treatment of murine 3T...

  13. Secretion of alpha 2-plasmin inhibitor is impaired by amino acid deletion in a small region of the molecule.

    PubMed

    Toyota, S; Hirosawa, S; Aoki, N

    1994-02-01

    Alpha 2-plasmin inhibitor (alpha 2PI) deficiency Okinawa results from defective secretion of the inhibitor from the liver and appears to be a direct consequence of the deletion of Glu137 in the amino acid sequence of alpha 2PI. To examine the effects of replacing the amino acid occupying position 137 or deleting its neighboring amino acid on alpha 2PI secretion, we used oligonucleotide-directed mutagenesis of alpha 2PI cDNA to change the codon specifying Glu137 or delete a codon specifying its neighboring amino acid. The effects were determined by pulse-chase experiments and by enzyme-linked immunosorbent assay of media from transiently transfected COS-7 cells. Replacement of Glu137 with an amino acid other than Cys had little effect on alpha 2PI secretion. In contrast, deletion of an amino acid in a region spanning a sequence of less than 30 amino acids including positions 127 and 137 severely impaired the secretion. The results suggest that structural integrity of the region, rather than its component amino acids, is important for the intracellular transport and secretion of alpha 2PI.

  14. Structures and Free Energy Landscapes of the Wild-Type and A30P Mutant-Type α-Synuclein Proteins with Dynamics

    PubMed Central

    2013-01-01

    The genetic missense A30P mutation of the wild-type α-synuclein protein results in the replacement of the 30th amino acid residue from alanine (Ala) to proline (Pro) and was initially found in the members of a German family who developed Parkinson’s disease. Even though the structures of these proteins have been measured before, detailed understanding about the structures and their relationships with free energy landscapes is lacking, which is of interest to provide insights into the pathogenic mechanism of Parkinson’s disease. We report the secondary and tertiary structures and conformational free energy landscapes of the wild-type and A30P mutant-type α-synuclein proteins in an aqueous solution environment via extensive parallel tempering molecular dynamics simulations along with thermodynamic calculations. In addition, we present the residual secondary structure component transition stabilities at the atomic level with dynamics in terms of free energy change calculations using a new strategy that we reported most recently. Our studies yield new interesting results; for instance, we find that the A30P mutation has local as well as long-range effects on the structural properties of the wild-type α-synuclein protein. The helical content at Ala18-Gly31 is less prominent in comparison to the wild-type α-synuclein protein. The β-sheet structure abundance decreases in the N-terminal region upon A30P mutation of the wild-type α-synuclein, whereas the NAC and C-terminal regions possess larger tendencies for β-sheet structure formation. Long-range intramolecular protein interactions are less abundant upon A30P mutation, especially between the NAC and C-terminal regions, which is linked to the less compact and less stable structures of the A30P mutant-type rather than the wild-type α-synuclein protein. Results including the usage of our new strategy for secondary structure transition stabilities show that the A30P mutant-type α-synuclein tendency toward

  15. Structures and free energy landscapes of the wild-type and A30P mutant-type α-synuclein proteins with dynamics.

    PubMed

    Wise-Scira, Olivia; Aloglu, Ahmet Kemal; Dunn, Aquila; Sakallioglu, Isin Tuna; Coskuner, Orkid

    2013-03-20

    The genetic missense A30P mutation of the wild-type α-synuclein protein results in the replacement of the 30th amino acid residue from alanine (Ala) to proline (Pro) and was initially found in the members of a German family who developed Parkinson's disease. Even though the structures of these proteins have been measured before, detailed understanding about the structures and their relationships with free energy landscapes is lacking, which is of interest to provide insights into the pathogenic mechanism of Parkinson's disease. We report the secondary and tertiary structures and conformational free energy landscapes of the wild-type and A30P mutant-type α-synuclein proteins in an aqueous solution environment via extensive parallel tempering molecular dynamics simulations along with thermodynamic calculations. In addition, we present the residual secondary structure component transition stabilities at the atomic level with dynamics in terms of free energy change calculations using a new strategy that we reported most recently. Our studies yield new interesting results; for instance, we find that the A30P mutation has local as well as long-range effects on the structural properties of the wild-type α-synuclein protein. The helical content at Ala18-Gly31 is less prominent in comparison to the wild-type α-synuclein protein. The β-sheet structure abundance decreases in the N-terminal region upon A30P mutation of the wild-type α-synuclein, whereas the NAC and C-terminal regions possess larger tendencies for β-sheet structure formation. Long-range intramolecular protein interactions are less abundant upon A30P mutation, especially between the NAC and C-terminal regions, which is linked to the less compact and less stable structures of the A30P mutant-type rather than the wild-type α-synuclein protein. Results including the usage of our new strategy for secondary structure transition stabilities show that the A30P mutant-type α-synuclein tendency toward

  16. Comparison of independent screens on differentially vulnerable motor neurons reveals alpha-synuclein as a common modifier in motor neuron diseases

    PubMed Central

    Kaifer, Kevin A.; Osman, Erkan Y.; Carella, Francesco; Tiberi, Ariana; Ross, Jolill; Pennetta, Giuseppa; Lorson, Christian L.

    2017-01-01

    The term “motor neuron disease” encompasses a spectrum of disorders in which motor neurons are the primary pathological target. However, in both patients and animal models of these diseases, not all motor neurons are equally vulnerable, in that while some motor neurons are lost very early in disease, others remain comparatively intact, even at late stages. This creates a valuable system to investigate the factors that regulate motor neuron vulnerability. In this study, we aim to use this experimental paradigm to identify potential transcriptional modifiers. We have compared the transcriptome of motor neurons from healthy wild-type mice, which are differentially vulnerable in the childhood motor neuron disease Spinal Muscular Atrophy (SMA), and have identified 910 transcriptional changes. We have compared this data set with published microarray data sets on other differentially vulnerable motor neurons. These neurons were differentially vulnerable in the adult onset motor neuron disease Amyotrophic Lateral Sclerosis (ALS), but the screen was performed on the equivalent population of neurons from neurologically normal human, rat and mouse. This cross species comparison has generated a refined list of differentially expressed genes, including CELF5, Col5a2, PGEMN1, SNCA, Stmn1 and HOXa5, alongside a further enrichment for synaptic and axonal transcripts. As an in vivo validation, we demonstrate that the manipulation of a significant number of these transcripts can modify the neurodegenerative phenotype observed in a Drosophila line carrying an ALS causing mutation. Finally, we demonstrate that vector-mediated expression of alpha-synuclein (SNCA), a transcript decreased in selectively vulnerable motor neurons in all four screens, can extend life span, increase weight and decrease neuromuscular junction pathology in a mouse model of SMA. In summary, we have combined multiple data sets to identify transcripts, which are strong candidates for being phenotypic

  17. Initiation of autoimmunity to the p53 tumor suppressor protein by complexes of p53 and SV40 large T antigen

    PubMed Central

    1994-01-01

    Antinuclear antibodies (ANAs) reactive with a limited spectrum of nuclear antigens are characteristic of systemic lupus erythematosus (SLE) and other collagen vascular diseases, and are also associated with certain viral infections. The factors that initiate ANA production and determine ANA specificity are not well understood. In this study, high titer ANAs specific for the p53 tumor suppressor protein were induced in mice immunized with purified complexes of murine p53 and the Simian virus 40 large T antigen (SVT), but not in mice immunized with either protein separately. The autoantibodies to p53 in these mice were primarily of the IgG1 isotype, were not cross-reactive with SVT, and were produced at titers up to 1:25,000, without the appearance of other autoantibodies. The high levels of autoantibodies to p53 in mice immunized with p53/SVT complexes were transient, but low levels of the autoantibodies persisted. The latter may have been maintained by self antigen, since the anti-p53, but not the SVT, response in these mice could be boosted by immunizing with murine p53. Thus, once autoimmunity to p53 was established by immunizing with p53/SVT complexes, it could be maintained without a requirement for SVT. These data may be explained in at least two ways. First, altered antigen processing resulting from the formation of p53/SVT complexes might activate autoreactive T helper cells specific for cryptic epitopes of murine p53, driving anti-p53 autoantibody production. Alternatively, SVT- responsive T cells may provide intermolecular-intrastructural help to B cells specific for murine p53. In a second stage, these activated B cells might themselves process self p53, generating p53-responsive autoreactive T cells. The induction of autoantibodies during the course of an immune response directed against this naturally occurring complex of self and nonself antigens may be relevant to the generation of specific autoantibodies in viral infections, and may also have

  18. alpha(4)beta(7) independent pathway for CD8(+) T cell-mediated intestinal immunity to rotavirus.

    PubMed

    Kuklin, N A; Rott, L; Darling, J; Campbell, J J; Franco, M; Feng, N; Müller, W; Wagner, N; Altman, J; Butcher, E C; Greenberg, H B

    2000-12-01

    Rotavirus (RV), which replicates exclusively in cells of the small intestine, is the most important cause of severe diarrhea in young children worldwide. Using a mouse model, we show that expression of the intestinal homing integrin alpha(4)ss(7) is not essential for CD8(+) T cells to migrate to the intestine or provide immunity to RV. Mice deficient in ss7 expression (ss7(-/-)) and unable to express alpha(4)ss(7) integrin were found to clear RV as quickly as wild-type (wt) animals. Depletion of CD8(+) T cells in ss7(-/-) animals prolonged viral shedding, and transfer of immune ss7(-/-) CD8(+) T cells into chronically infected Rag-2-deficient mice resolved RV infection as efficiently as wt CD8(+) T cells. Paradoxically, alpha(4)ss(7)(hi) memory CD8(+) T cells purified from wt mice that had been orally immunized cleared RV more efficiently than alpha(4)ss(7)(low) CD8(+) T cells. We explained this apparent contradiction by demonstrating that expression of alpha(4)ss(7) on effector CD8(+) T cells depends upon the site of initial antigen exposure: oral immunization generates RV-specific CD8(+) T cells primarily of an alpha(4)ss(7)(hi) phenotype, but subcutaneous immunization yields both alpha(4)ss(7)(hi) and alpha(4)ss(7)(low) immune CD8(+) T cells with anti-RV effector capabilities. Thus, alpha(4)ss(7) facilitates normal intestinal immune trafficking to the gut, but it is not required for effective CD8(+) T cell immunity.

  19. MFTF-. cap alpha. + T progress report

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nelson, W.D.

    1985-04-01

    Early in FY 1983, several upgrades of the Mirror Fusion Test Facility (MFTF-B) at Lawrence Livermore National Laboratory (LLNL) were proposed to the fusion community. The one most favorably received was designated MFTF-..cap alpha..+T. The engineering design of this device, guided by LLNL, has been a principal activity of the Fusion Engineering Design Center during FY 1983. This interim progress report represents a snapshot of the device design, which was begun in FY 1983 and will continue for several years. The report is organized as a complete design description. Because it is an interim report, some parts are incomplete; theymore » will be supplied as the design study proceeds. As described in this report, MFTF-..cap alpha..+T uses existing facilities, many MFTF-B components, and a number of innovations to improve on the physics parameters of MFTF-B. It burns deuterium-tritium and has a central-cell Q of 2, a wall loading GAMMA/sub n/ of 2 MW/m/sup 2/ (with a central-cell insert module), and an availability of 10%. The machine is fully shielded, allows hands-on maintenance of components outside the vacuum vessel 24 h after shutdown, and has provisions for repair of all operating components.« less

  20. α-Synuclein oligomers and clinical implications for Parkinson disease

    PubMed Central

    Kalia, Lorraine V.; Kalia, Suneil K.; McLean, Pamela J.; Lozano, Andres M.; Lang, Anthony E.

    2012-01-01

    Protein aggregation within the central nervous system has been recognized as a defining feature of neurodegenerative diseases since the early 20th century. Since that time, there has been a growing list of neurodegenerative disorders, including Parkinson disease, which are characterized by inclusions of specific pathogenic proteins. This has led to the long-held dogma that these characteristic protein inclusions, which are composed of large insoluble fibrillar protein aggregates and visible by light microscopy, are responsible for cell death in these diseases. However, the correlation between protein inclusion formation and cytotoxicity is inconsistent suggesting another form of the pathogenic proteins may be contributing to neurodegeneration. There is emerging evidence implicating soluble oligomers, smaller protein aggregates not detectable by conventional microscopy, as potential culprits in the pathogenesis of neurodegenerative diseases. The protein α-synuclein is well recognized to contribute to the pathogenesis of Parkinson disease and is the major component of Lewy bodies and Lewy neurites. However, α-synuclein also forms oligomeric species with certain conformations being toxic to cells. The mechanisms by which these α-synuclein oligomers cause cell death are being actively investigated as they may provide new strategies for diagnosis and treatment of Parkinson disease and related disorders. Here we review the possible role of α-synuclein oligomers in cell death in Parkinson disease and discuss the potential clinical implications. PMID:23225525

  1. p53 elevation in human cells halt SV40 infection by inhibiting T-ag expression

    PubMed Central

    Drayman, Nir; Ben-nun-Shaul, Orly; Butin-Israeli, Veronika; Srivastava, Rohit; Rubinstein, Ariel M.; Mock, Caroline S.; Elyada, Ela; Ben-Neriah, Yinon; Lahav, Galit; Oppenheim, Ariella

    2016-01-01

    SV40 large T-antigen (T-ag) has been known for decades to inactivate the tumor suppressor p53 by sequestration and additional mechanisms. Our present study revealed that the struggle between p53 and T-ag begins very early in the infection cycle. We found that p53 is activated early after SV40 infection and defends the host against the infection. Using live cell imaging and single cell analyses we found that p53 dynamics are variable among individual cells, with only a subset of cells activating p53 immediately after SV40 infection. This cell-to-cell variabilty had clear consequences on the outcome of the infection. None of the cells with elevated p53 at the beginning of the infection proceeded to express T-ag, suggesting a p53-dependent decision between abortive and productive infection. In addition, we show that artificial elevation of p53 levels prior to the infection reduces infection efficiency, supporting a role for p53 in defending against SV40. We further found that the p53-mediated host defense mechanism against SV40 is not facilitated by apoptosis nor via interferon-stimulated genes. Instead p53 binds to the viral DNA at the T-ag promoter region, prevents its transcriptional activation by Sp1, and halts the progress of the infection. These findings shed new light on the long studied struggle between SV40 T-ag and p53, as developed during virus-host coevolution. Our studies indicate that the fate of SV40 infection is determined as soon as the viral DNA enters the nucleus, before the onset of viral gene expression. PMID:27462916

  2. [Study on serum p53 protein in cops in Guangzhou city].

    PubMed

    Zhu, Wen-Chang; Chen, Qing; Chu, Xin-Wei; Luo, Chen-Ling; Wu, Min; Wang, Ya-Xian; Chen, Si-Dong

    2003-10-01

    Serum p53 protein overexpression was detected in population exposed to traffic exhaust gas to study the relation between traffic exhaust gas and the increased risk in p53 gene mutation. Serum p53 protein expression was measured by enzyme-linked immunosorbent assay. Relationship between different types of job and serum p53 protein overexpression were studied by pearson Chi-square tests. Results on serum p53 protein overexpression on jobs outside of office (5.74%) were not significantly higher than jobs inside the office. However, it suggested that traffic police men (12.12%) working outside of office, with whose length of service longer than 30 years had a significant overexpression of serum p53 protein than the others (5.36%) whose length of service was less than 30 years (P < 0.05, OR = 2.43, 95% CI: 1.11 - 5.33). Overexpression rate of p53 protein appeared to be 6.89% in the group whose average weekly exposure hours were more than 40 hours, which was significant higher than the group whose exposed hours were less than 40 hours (P < 0.05, OR = 1.71, 95% CI: 1.03 - 2.81). The result suggested that traffic exhaust gas was likely to cause mutation of p53 gene and increasing the incidence of lung cancer.

  3. Solid-State NMR Structure of a Pathogenic Fibril of Full-Length Human α-Synuclein

    PubMed Central

    Tuttle, Marcus D.; Comellas, Gemma; Nieuwkoop, Andrew J.; Covell, Dustin J.; Berthold, Deborah A.; Kloepper, Kathryn D.; Courtney, Joseph M.; Kim, Jae K.; Barclay, Alexander M.; Kendall, Amy; Wan, William; Stubbs, Gerald; Schwieters, Charles D.; Lee, Virginia M. Y.; George, Julia M.; Rienstra, Chad M.

    2016-01-01

    Misfolded α-synuclein amyloid fibrils are the principal components of Lewy bodies and neurites, hallmarks of Parkinson’s disease (PD). Here we present a high-resolution structure of an α-synuclein fibril, in a form that induces robust pathology in primary neuronal culture, determined by solid-state NMR spectroscopy and validated by electron microscopy and X-ray fiber diffraction. Over 200 unique long-range distance restraints define a consensus structure with common amyloid features including parallel in-register β-sheets and hydrophobic core residues, but also substantial complexity, arising from diverse structural features: an intermolecular salt bridge, a glutamine ladder, close backbone interactions involving small residues, and several steric zippers stabilizing a novel, orthogonal Greek-key topology. These characteristics contribute to the robust propagation of this fibril form, as evidenced by structural similarity of early-onset PD mutants. The structure provides a framework for understanding the interactions of α-synuclein with other proteins and small molecules to diagnose and treat PD. PMID:27018801

  4. Derivation of mouse embryonic stem cell lines from tyrosine hydroxylase reporter mice crossed with a human SNCA transgenic mouse model of Parkinson's disease.

    PubMed

    Chumarina, Margarita; Azevedo, Carla; Bigarreau, Julie; Vignon, Clémentine; Kim, Kwang-Soo; Li, Jia-Yi; Roybon, Laurent

    2017-03-01

    Mouse embryonic stem cell (mESC) lines were derived by crossing heterozygous transgenic (tg) mice expressing green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase (TH) promoter, with homozygous alpha-synuclein (aSYN) mice expressing human mutant SNCA A53T under the control of the mouse Prion promoter (MoPrP), or wildtype (WT) mice. The expression of GFP and human aSYN was validated by immunocytochemistry in midbrain neuron cultures upon differentiation of mESC lines using stromal cell-derived inducing activity. These mESC lines can help to study the impact of human aSYN expression in neurons and oligodendrocytes, and also trace GFP-expressing midbrain neurons. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Recent advances in α-synuclein functions, advanced glycation, and toxicity: implications for Parkinson's disease.

    PubMed

    Guerrero, Erika; Vasudevaraju, P; Hegde, Muralidhar L; Britton, G B; Rao, K S

    2013-04-01

    The toxicity of α-synuclein in the neuropathology of Parkinson's disease which includes its hallmark aggregation has been studied scrupulously in the last decade. Although little is known regarding the normal functions of α-synuclein, its association with membrane phospholipids suggests its potential role in signaling pathways. Following extensive evidences for its nuclear localization, we and others recently demonstrated DNA binding activity of α-synuclein that modulates its conformation as well as aggregation properties. Furthermore, we also underscored the similarities among various amyloidogenic proteins involved in neurodegenerative diseases including amyloid beta peptides and tau. Our more recent studies show that α-synuclein is glycated and glycosylated both in vitro and in neurons, significantly affecting its folding, oligomeric, and DNA binding properties. Glycated α-synuclein causes increased genome damage both via its direct interaction with DNA and by increased generation of reactive oxygen species as glycation byproduct. In this review, we discuss the mechanisms of glycation and other posttranslational modifications of α-synuclein, including phosphorylation and nitration, and their role in neuronal death in Parkinson's disease.

  6. Monitoring in real time the effect of TLX overexpression on proliferation and migration of C6 cells.

    PubMed

    Li, G L; Fang, S H; Xu, B

    2017-01-01

    Orphan nuclear receptor TLX has been shown to play an essential role in regulating the self-renewal and proliferation of neural stem cells (NSCs). However, TLX overexpression in NSCs induces long-term NSC expansion and further leads to glioma initiation in mouse when combined with p53 mutations. Whether overexpression of TLX plays a role in glioma stem cell (GSC) proliferation and migration still remains largely unknown. In this study, we infected C6 cells, a special glioma cell line which is mainly composed of cancer stem cells(CSCs), with lentiviruses expressing GFP(LV-GFP) or GFP-T2A-TLX(LV-TLX) and then monitored cell proliferation and migration using the real-time analyzer system (RTCA, xCELLigence, Roche). We found that the cell index (CI) observed for the TLX overexpressing C6 cells showed a lower value than that of the LV-GFP transduced cells. And the MTT results correlated highly with the RTCA proliferation assessments. Furthermore, the expression of p21 was decreased while other downstream genes PTEN and p53 were not significantly changed in TLX overexpressing C6 cells . These findings strongly indicate that TLX overexpression has the ability to decrease the proliferating and migratory properties of C6 cells by targeting p21. Further, our results suggest that TLX overexpression may also have a similar inhibitory effect on GSC proliferation and migration.

  7. The NACP/synuclein gene: chromosomal assignment and screening for alterations in Alzheimer disease.

    PubMed

    Campion, D; Martin, C; Heilig, R; Charbonnier, F; Moreau, V; Flaman, J M; Petit, J L; Hannequin, D; Brice, A; Frebourg, T

    1995-03-20

    The major component of the vascular and plaque amyloid deposits in Alzheimer disease is the amyloid beta peptide (A beta). A second intrinsic component of amyloid, the NAC (non-A beta component of amyloid) peptide, has recently been identified, and its precursor protein was named NACP. A computer homology search allowed us to establish that the human NACP gene was homologous to the rat synuclein gene. We mapped the NACP/synuclein gene to chromosome 4 and cloned three alternatively spliced transcripts in lymphocytes derived from a normal subject. We analyzed by RT-PCR and direct sequencing the entire coding region of the NACP/synuclein gene in a group of patients with familial early onset Alzheimer disease. No mutation was found in 26 unrelated patients. Further studies are required to investigate the implication of the NACP/synuclein gene in Alzheimer disease.

  8. The 14alpha-Demethylasse(CYP51A1) Gene is Overexpressed in Venturia inaequalis Strains Resistant to Myclobutanil.

    PubMed

    Schnabel, G; Jones, A L

    2001-01-01

    ABSTRACT We identified the cytochrome P450 sterol 14alpha-demethylase (CYP51A1) gene from Venturia inaequalis and optional insertions located upstream from CYP51A1 and evaluated their potential role in conferring resistance to the sterol demethylation-inhibitor (DMI) fungicide my-clobutanil. The CYP51A1 gene was completely sequenced from one my-clobutanil sensitive (S) and two myclobutanil-resistant (R) strains. No nucleotide variation was found when the three sequences were aligned. Allele-specific polymerase chain reaction (PCR) analysis indicated that a previously described single base pair mutation that correlated with resistance to DMI fungicides in strains of other filamentous fungi was absent in 19 S and 32 R strains of V. inaequalis from Michigan and elsewhere. The sequencing results and PCR analyses suggest that resistance in these strains was not due to a mutation in the sterol demethylase target site for DMI fungicides. Expression of CYP51A1 was determined for strains from an orchard that had never been sprayed with DMI fungicides (baseline orchard), and the data provided a reference for evaluating the expression of strains collected from a research orchard and from three commercial Michigan apple orchards with a long history of DMI use and a high frequency of R strains. Overexpression of CYP51A1 was significantly higher in 9 of 11 R strains from the research orchard than in S strains from the baseline orchard. The high expression was correlated with the presence of a 553-bp insertion located upstream of CYP51A1. Overexpression of the CYP51A1 gene was also detected in eight of eight, five of nine, and nine of nine R strains from three commercial orchards, but the insertion was not detected in the majority of these strains. The results suggest that overexpression of the target-site CYP51A1 gene is an important mechanism of resistance in some field resistant strains of V. inaequalis, but other mechanisms of resistance also appear to exist.

  9. CRLF2 over-expression is a poor prognostic marker in children with high risk T-cell acute lymphoblastic leukemia

    PubMed Central

    Palmi, Chiara; Savino, Angela M.; Silvestri, Daniela; Bronzini, Ilaria; Cario, Gunnar; Paganin, Maddalena; Buldini, Barbara; Galbiati, Marta; Muckenthaler, Martina U.; Bugarin, Cristina; Mina, Pamela Della; Nagel, Stefan; Barisone, Elena; Casale, Fiorina; Locatelli, Franco; Nigro, Luca Lo; Micalizzi, Concetta; Parasole, Rosanna; Pession, Andrea; Putti, Maria C.; Santoro, Nicola; Testi, Anna M.; Ziino, Ottavio; Kulozik, Andreas E.; Zimmermann, Martin; Schrappe, Martin; Villa, Antonello; Gaipa, Giuseppe; Basso, Giuseppe; Biondi, Andrea; Valsecchi, Maria G.; Stanulla, Martin; Conter, Valentino; te Kronnie, Geertruy; Cazzaniga, Giovanni

    2016-01-01

    Pediatric T-ALL patients have a worse outcome compared to BCP-ALL patients and they could benefit from new prognostic marker identification. Alteration of CRLF2 gene, a hallmark correlated with poor outcome in BCP-ALL, has not been reported in T-ALL. We analyzed CRLF2 expression in 212 T-ALL pediatric patients enrolled in AIEOP-BFM ALL2000 study in Italian and German centers. Seventeen out of 120 (14.2%) Italian patients presented CRLF2 mRNA expression 5 times higher than the median (CRLF2-high); they had a significantly inferior event-free survival (41.2%±11.9 vs. 68.9%±4.6, p=0.006) and overall survival (47.1%±12.1 vs. 73.8%±4.3, p=0.009) and an increased cumulative incidence of relapse/resistance (52.9%±12.1 vs. 26.2%±4.3, p=0.007) compared to CRLF2-low patients. The prognostic value of CRLF2 over-expression was validated in the German cohort. Of note, CRLF2 over-expression was associated with poor prognosis in the high risk (HR) subgroup where CRLF2-high patients were more frequently allocated. Interestingly, although in T-ALL CRLF2 protein was localized mainly in the cytoplasm, in CRLF2-high blasts we found a trend towards a stronger TSLP-induced pSTAT5 response, sensitive to the JAK inhibitor Ruxolitinib. In conclusion, CRLF2 over-expression is a poor prognostic marker identifying a subset of HR T-ALL patients that could benefit from alternative therapy, potentially targeting the CRLF2 pathway. PMID:27449287

  10. CRLF2 over-expression is a poor prognostic marker in children with high risk T-cell acute lymphoblastic leukemia.

    PubMed

    Palmi, Chiara; Savino, Angela M; Silvestri, Daniela; Bronzini, Ilaria; Cario, Gunnar; Paganin, Maddalena; Buldini, Barbara; Galbiati, Marta; Muckenthaler, Martina U; Bugarin, Cristina; Della Mina, Pamela; Nagel, Stefan; Barisone, Elena; Casale, Fiorina; Locatelli, Franco; Lo Nigro, Luca; Micalizzi, Concetta; Parasole, Rosanna; Pession, Andrea; Putti, Maria C; Santoro, Nicola; Testi, Anna M; Ziino, Ottavio; Kulozik, Andreas E; Zimmermann, Martin; Schrappe, Martin; Villa, Antonello; Gaipa, Giuseppe; Basso, Giuseppe; Biondi, Andrea; Valsecchi, Maria G; Stanulla, Martin; Conter, Valentino; Te Kronnie, Geertruy; Cazzaniga, Giovanni

    2016-09-13

    Pediatric T-ALL patients have a worse outcome compared to BCP-ALL patients and they could benefit from new prognostic marker identification. Alteration of CRLF2 gene, a hallmark correlated with poor outcome in BCP-ALL, has not been reported in T-ALL.We analyzed CRLF2 expression in 212 T-ALL pediatric patients enrolled in AIEOP-BFM ALL2000 study in Italian and German centers.Seventeen out of 120 (14.2%) Italian patients presented CRLF2 mRNA expression 5 times higher than the median (CRLF2-high); they had a significantly inferior event-free survival (41.2%±11.9 vs. 68.9%±4.6, p=0.006) and overall survival (47.1%±12.1 vs. 73.8%±4.3, p=0.009) and an increased cumulative incidence of relapse/resistance (52.9%±12.1 vs. 26.2%±4.3, p=0.007) compared to CRLF2-low patients. The prognostic value of CRLF2 over-expression was validated in the German cohort. Of note, CRLF2 over-expression was associated with poor prognosis in the high risk (HR) subgroup where CRLF2-high patients were more frequently allocated.Interestingly, although in T-ALL CRLF2 protein was localized mainly in the cytoplasm, in CRLF2-high blasts we found a trend towards a stronger TSLP-induced pSTAT5 response, sensitive to the JAK inhibitor Ruxolitinib.In conclusion, CRLF2 over-expression is a poor prognostic marker identifying a subset of HR T-ALL patients that could benefit from alternative therapy, potentially targeting the CRLF2 pathway.

  11. Transplantation of mesenchymal stem cells overexpressing IL10 attenuates cardiac impairments in rats with myocardial infarction.

    PubMed

    Meng, Xin; Li, Jianping; Yu, Ming; Yang, Jian; Zheng, Minjuan; Zhang, Jinzhou; Sun, Chao; Liang, Hongliang; Liu, Liwen

    2018-01-01

    Mesenchymal stem cell (MSC) has been well known to exert therapeutic potential for patients with myocardial infarction (MI). In addition, interleukin-10 (IL10) could attenuate MI through suppressing inflammation. Thus, the combination of MSC implantation with IL10 delivery may extend health benefits to ameliorate cardiac injury after MI. Here we established overexpression of IL10 in bone marrow-derived MSC through adenoviral transduction. Cell viability, apoptosis, and IL10 secretion under ischemic challenge in vitro were examined. In addition, MSC was transplanted into the injured hearts in a rat model of MI. Four weeks after the MI induction, MI, cardiac functions, apoptotic cells, and inflammation cytokines were assessed. In response to in vitro oxygen-glucose deprivation (OGD), IL10 overexpression in MSC (Ad.IL10-MSC) enhanced cell viability, decreased apoptosis, and increased IL10 secretion. Consistently, the implantation of Ad.IL10-MSCs into MI animals resulted in more reductions in myocardial infarct size, cardiac impairment, and cell apoptosis, compared to the individual treatments of either MSC or IL10 administration. Moreover, the attenuation of both systemic and local inflammations was most prominent for Ad.IL10-MSC treatment. IL10 overexpression and MSC may exert a synergistic anti-inflammatory effect to alleviate cardiac injury after MI. © 2017 Wiley Periodicals, Inc.

  12. Phenylbutyrate Up-regulates the DJ-1 Protein and Protects Neurons in Cell Culture and in Animal Models of Parkinson Disease*

    PubMed Central

    Zhou, Wenbo; Bercury, Kathryn; Cummiskey, Jessica; Luong, Nancy; Lebin, Jacob; Freed, Curt R.

    2011-01-01

    Parkinson disease is caused by the death of midbrain dopamine neurons from oxidative stress, abnormal protein aggregation, and genetic predisposition. In 2003, Bonifati et al. (23) found that a single amino acid mutation in the DJ-1 protein was associated with early-onset, autosomal recessive Parkinson disease (PARK7). The mutation L166P prevents dimerization that is essential for the antioxidant and gene regulatory activity of the DJ-1 protein. Because low levels of DJ-1 cause Parkinson, we reasoned that overexpression might stop the disease. We found that overexpression of DJ-1 improved tolerance to oxidative stress by selectively up-regulating the rate-limiting step in glutathione synthesis. When we imposed a different metabolic insult, A53T mutant α-synuclein, we found that DJ-1 turned on production of the chaperone protein Hsp-70 without affecting glutathione synthesis. After screening a number of small molecules, we have found that the histone deacetylase inhibitor phenylbutyrate increases DJ-1 expression by 300% in the N27 dopamine cell line and rescues cells from oxidative stress and mutant α-synuclein toxicity. In mice, phenylbutyrate treatment leads to a 260% increase in brain DJ-1 levels and protects dopamine neurons against 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) toxicity. In a transgenic mouse model of diffuse Lewy body disease, long-term administration of phenylbutyrate reduces α-synuclein aggregation in brain and prevents age-related deterioration in motor and cognitive function. We conclude that drugs that up-regulate DJ-1 gene expression may slow the progression of Parkinson disease by moderating oxidative stress and protein aggregation. PMID:21372141

  13. TNF-alpha induction of GM2 expression on renal cell carcinomas promotes T cell dysfunction.

    PubMed

    Raval, Gira; Biswas, Soumika; Rayman, Patricia; Biswas, Kaushik; Sa, Gaurisankar; Ghosh, Sankar; Thornton, Mark; Hilston, Cynthia; Das, Tanya; Bukowski, Ronald; Finke, James; Tannenbaum, Charles S

    2007-05-15

    Previous studies from our laboratory demonstrated the role of tumor-derived gangliosides as important mediators of T cell apoptosis, and hence, as one mechanism by which tumors evade immune destruction. In this study, we report that TNF-alpha secreted by infiltrating inflammatory cells and/or genetically modified tumors augments tumor-associated GM2 levels, which leads to T cell death and immune dysfunction. The conversion of weakly apoptogenic renal cell carcinoma (RCC) clones to lines that can induce T cell death requires 3-5 days of TNF-alpha pretreatment, a time frame paralleling that needed for TNF-alpha to stimulate GM2 accumulation by SK-RC-45, SK-RC-54, and SK-RC-13. RCC tumor cell lines permanently transfected with the TNF-alpha transgene are similarly toxic for T lymphocytes, which correlates with their constitutively elevated levels of GM2. TNF-alpha increases GM2 ganglioside expression by enhancing the mRNA levels encoding its synthetic enzyme, GM2 synthase, as demonstrated by both RT-PCR and Southern analysis. The contribution of GM2 gangliosides to tumor-induced T cell death was supported by the finding that anti-GM2 Abs significantly blocked T cell apoptosis mediated by TNF-alpha-treated tumor cells, and by the observation that small interfering RNA directed against TNF-alpha abrogated GM2 synthase expression by TNF-transfected SK-RC-45, diminished its GM2 accumulation, and inhibited its apoptogenicity for T lymphocytes. Our results indicate that TNF-alpha signaling promotes RCC-induced killing of T cells by stimulating the acquisition of a distinct ganglioside assembly in RCC tumor cells.

  14. Reduced TH expression and α-synuclein accumulation contribute towards nigrostriatal dysfunction in experimental hepatic encephalopathy.

    PubMed

    Suárez, Isabel; Bodega, Guillermo; Rubio, Miguel; Fernández, Benjamín

    2017-01-01

    The present work examines α-synuclein expression in the nigrostriatal system of a rat chronic hepatic encephalopathy model induced by portacaval anastomosis (PCA). There is evidence that dopaminergic dysfunction in disease conditions is strongly associated with such expression. Possible relationships among dopaminergic neurons, astroglial cells and α-synuclein expression were sought. Brain tissue samples from rats at 1 and 6 months post-PCA, and controls, were analysed immunohistochemically using antibodies against tyrosine hydroxylase (TH), α-synuclein, glial fibrillary acidic protein (GFAP) and ubiquitin (Ub). In the control rats, TH immunoreactivity was detected in the neuronal cell bodies and processes in the substantia nigra pars compacta (SNc). A dense TH-positive network of neurons was also seen in the striatum. In the PCA-exposed rats, however, a reduction in TH-positive neurons was seen at both 1 and 6 months in the SNc, as well as a reduction in TH-positive fibres in the striatum. This was coincident with the appearance of α-synuclein-immunoreactive neurons in the SNc; some of the TH-positive neurons also showed α-synuclein immunoreactivity. In addition, α-synuclein accumulation was seen in the SNc and striatum at both 1 and 6 months post-PCA, whereas α-synuclein was only mildly expressed in the nigrostriatal pathway of the controls. Astrogliosis was also seen following PCA, as revealed by increased GFAP expression from 1 month to 6 months post-PCA in both the SN and striatum. The astroglial activation level in the SN paralleled the reduced neuronal expression of TH throughout PCA exposure. α-synuclein accumulation following PCA may induce dopaminergic dysfunction via the downregulation of TH, as well as astroglial activation.

  15. Dopamine and α-synuclein dysfunction in Smad3 null mice

    PubMed Central

    2011-01-01

    Background Parkinson's disease (PD) is characterized by dopaminergic neurodegeneration in the substantia nigra (SN). Transforming growth factor-β1 (TGF-β1) levels increase in patients with PD, although the effects of this increment remain unclear. We have examined the mesostriatal system in adult mice deficient in Smad3, a molecule involved in the intracellular TGF-β1 signalling cascade. Results Striatal monoamine oxidase (MAO)-mediated dopamine (DA) catabolism to 3,4-dihydroxyphenylacetic acid (DOPAC) is strongly increased, promoting oxidative stress that is reflected by an increase in glutathione levels. Fewer astrocytes are detected in the ventral midbrain (VM) and striatal matrix, suggesting decreased trophic support to dopaminergic neurons. The SN of these mice has dopaminergic neuronal degeneration in its rostral portion, and the pro-survival Erk1/2 signalling is diminished in nigra dopaminergic neurons, not associated with alterations to p-JNK or p-p38. Furthermore, inclusions of α-synuclein are evident in selected brain areas, both in the perikaryon (SN and paralemniscal nucleus) or neurites (motor and cingulate cortices, striatum and spinal cord). Interestingly, these α-synuclein deposits are detected with ubiquitin and PS129-α-synuclein in a core/halo cellular distribution, which resemble those observed in human Lewy bodies (LB). Conclusions Smad3 deficiency promotes strong catabolism of DA in the striatum (ST), decrease trophic and astrocytic support to dopaminergic neurons and may induce α-synuclein aggregation, which may be related to early parkinsonism. These data underline a role for Smad3 in α-synuclein and DA homeostasis, and suggest that modulatory molecules of this signalling pathway should be evaluated as possible neuroprotective agents. PMID:21995845

  16. Overexpression of hypoxia/inflammatory markers in atherosclerotic carotid plaques.

    PubMed

    Luque, Ana; Turu, Marta; Juan-Babot, Oriol; Cardona, Pere; Font, Angels; Carvajal, Ana; Slevin, Mark; Iborra, Elena; Rubio, Francisco; Badimon, Lina; Krupinski, Jerzy

    2008-05-01

    Hypoxia, angiogenesis and inflammation leads to plaque progression and remodelling and may significantly contribute towards plaque rupture and subsequent cerebrovascular events. Our aim was to study, markers of hypoxia and inflammation previously identified by microarray analysis, in atherosclerotic carotid arteries with low to moderate stenosis. We hoped to describe different cellular populations expressing the studied markers. The location of selected inflammatory molecules obtained as vascular transplants from organ donors were analysed by immunohistochemistry with monoclonal and polyclonal antibodies. Paraffin-embedded sections were cut and probed with antibodies recognizing active B and T-lymphocytes (CD30), hypoxia-inducible factor-1alpha, endoglin (CD105), Interleukin-6 and C-reactive protein. We observed a notable overexpression of HIF-1alpha in inflammatory and hypoxic areas of carotid arteries in all types of lesions from type II-V taken from the patients with carotid stenosis less than 50%. This suggests that HIF-1alpha may have a putative role in atherosclerosis progression and angiogenesis. Dynamic changes in the non-occluding plaques may explain some of the clinical events in patients with low to moderate carotid stenosis.

  17. Increased Frequency of α-Synuclein in the Substantia Nigra in HIV Infection

    PubMed Central

    Khanlou, Negar; Moore, David J.; Chana, Gursharan; Cherner, Mariana; Lazzaretto, Deborah; Dawes, Sharron; Grant, Igor; Masliah, Eliezer; Everall, Ian P.

    2014-01-01

    The frequency of neurodegenerative markers among long surviving HIV infected individuals is unknown, therefore, the present study investigated the frequency of α-synuclein, β-amyloid and HIV-associated brain pathology in the brains of older HIV infected individuals. We examined the substantia nigra of 73 clinically well-characterized HIV infected individuals aged 50 to 76 years from the National NeuroAIDS Tissue Consortium. We also examined the frontal and temporal cortical regions of a subset of 36 individuals. The brain regions were examined for the presence of α-synuclein, β-amyloid and HIV-associated brain pathology. Neuritic α-synuclein expression was found in 16% (12/73) of the substantia nigra of the HIV+ cases and none of the older control cases (0/18). β-amyloid deposits were prevalent and found in nearly all of the HIV+ cases (35/36). Despite these increases of degenerative pathology, HIV-associated brain pathology was present in only 10% of cases. Among older HIV+ adults HIV-associated brain pathology does not appear elevated; however, the frequency of both α-synuclein and β-amyloid is higher than that found in older healthy persons. The increased prevalence of α-synuclein and β-amyloid in the brains of older HIV-infected individuals may predict an increased risk of developing neurodegenerative disease. PMID:19115126

  18. Human T-cell leukemia virus type-1-encoded protein HBZ represses p53 function by inhibiting the acetyltransferase activity of p300/CBP and HBO1

    PubMed Central

    Hoang, Kimson; Ankney, John A.; Nguyen, Stephanie T.; Rushing, Amanda W.; Polakowski, Nicholas; Miotto, Benoit; Lemasson, Isabelle

    2016-01-01

    Adult T-cell leukemia (ATL) is an often fatal malignancy caused by infection with the complex retrovirus, human T-cell Leukemia Virus, type 1 (HTLV-1). In ATL patient samples, the tumor suppressor, p53, is infrequently mutated; however, it has been shown to be inactivated by the viral protein, Tax. Here, we show that another HTLV-1 protein, HBZ, represses p53 activity. In HCT116 p53+/+ cells treated with the DNA-damaging agent, etoposide, HBZ reduced p53-mediated activation of p21/CDKN1A and GADD45A expression, which was associated with a delay in G2 phase-arrest. These effects were attributed to direct inhibition of the histone acetyltransferase (HAT) activity of p300/CBP by HBZ, causing a reduction in p53 acetylation, which has be linked to decreased p53 activity. In addition, HBZ bound to, and inhibited the HAT activity of HBO1. Although HBO1 did not acetylate p53, it acted as a coactivator for p53 at the p21/CDKN1A promoter. Therefore, through interactions with two separate HAT proteins, HBZ impairs the ability of p53 to activate transcription. This mechanism may explain how p53 activity is restricted in ATL cells that do not express Tax due to modifications of the HTLV-1 provirus, which accounts for a majority of patient samples. PMID:26625199

  19. The NACP/synuclein gene: Chromosomal assignment and screening for alterations in Alzheimer disease

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Campion, D.; Martin, C.; Charbonnier, F.

    1995-03-20

    The major component of the vascular and plaque amyloid deposits in Alzheimer disease is the amyloid {beta} peptide (A{beta}). A second intrinsic component of amyloid, the NAC (non-A{beta} component of amyloid) peptide, has recently been identified, and its precursor protein was named NACP. A computer homology search allowed us to establish that the human NACP gene was homologous to the rat synuclein gene. We mapped the NACP/synuclein gene to chromosome 4 and cloned three alternatively spliced transcripts in lymphocytes derived from a normal subject. We analyzed by RT-PCR and direct sequencing the entire coding region of the NACP/synuclein gene inmore » a group of patients with familial early onset Alzheimer disease. No mutation was found in 26 unrelated patients. Further studies are required to investigate the implication of the NACP/synuclein gene in Alzheimer disease. 21 refs., 3 tabs.« less

  20. p53 isoform Δ133p53 promotes efficiency of induced pluripotent stem cells and ensures genomic integrity during reprogramming.

    PubMed

    Gong, Lu; Pan, Xiao; Chen, Haide; Rao, Lingjun; Zeng, Yelin; Hang, Honghui; Peng, Jinrong; Xiao, Lei; Chen, Jun

    2016-11-22

    Human induced pluripotent stem (iPS) cells have great potential in regenerative medicine, but this depends on the integrity of their genomes. iPS cells have been found to contain a large number of de novo genetic alterations due to DNA damage response during reprogramming. Thus, to maintain the genetic stability of iPS cells is an important goal in iPS cell technology. DNA damage response can trigger tumor suppressor p53 activation, which ensures genome integrity of reprogramming cells by inducing apoptosis and senescence. p53 isoform Δ133p53 is a p53 target gene and functions to not only antagonize p53 mediated apoptosis, but also promote DNA double-strand break (DSB) repair. Here we report that Δ133p53 is induced in reprogramming. Knockdown of Δ133p53 results 2-fold decrease in reprogramming efficiency, 4-fold increase in chromosomal aberrations, whereas overexpression of Δ133p53 with 4 Yamanaka factors showes 4-fold increase in reprogamming efficiency and 2-fold decrease in chromosomal aberrations, compared to those in iPS cells induced only with 4 Yamanaka factors. Overexpression of Δ133p53 can inhibit cell apoptosis and promote DNA DSB repair foci formation during reprogramming. Our finding demonstrates that the overexpression of Δ133p53 not only enhances reprogramming efficiency, but also results better genetic quality in iPS cells.

  1. DRAGO (KIAA0247), a new DNA damage-responsive, p53-inducible gene that cooperates with p53 as oncosuppressor. [Corrected].

    PubMed

    Polato, Federica; Rusconi, Paolo; Zangrossi, Stefano; Morelli, Federica; Boeri, Mattia; Musi, Alberto; Marchini, Sergio; Castiglioni, Vittoria; Scanziani, Eugenio; Torri, Valter; Broggini, Massimo

    2014-04-01

    p53 influences genomic stability, apoptosis, autophagy, response to stress, and DNA damage. New p53-target genes could elucidate mechanisms through which p53 controls cell integrity and response to damage. DRAGO (drug-activated gene overexpressed, KIAA0247) was characterized by bioinformatics methods as well as by real-time polymerase chain reaction, chromatin immunoprecipitation and luciferase assays, time-lapse microscopy, and cell viability assays. Transgenic mice (94 p53(-/-) and 107 p53(+/-) mice on a C57BL/6J background) were used to assess DRAGO activity in vivo. Survival analyses were performed using Kaplan-Meier curves and the Mantel-Haenszel test. All statistical tests were two-sided. We identified DRAGO as a new p53-responsive gene induced upon treatment with DNA-damaging agents. DRAGO is highly conserved, and its ectopic overexpression resulted in growth suppression and cell death. DRAGO(-/-) mice are viable without macroscopic alterations. However, in p53(-/-) or p53(+/-) mice, the deletion of both DRAGO alleles statistically significantly accelerated tumor development and shortened lifespan compared with p53(-/-) or p53(+/-) mice bearing wild-type DRAGO alleles (p53(-/-), DRAGO(-/-) mice: hazard ratio [HR] = 3.25, 95% confidence interval [CI] = 1.7 to 6.1, P < .001; p53(+/-), DRAGO(-/-) mice: HR = 2.35, 95% CI = 1.3 to 4.0, P < .001; both groups compared with DRAGO(+/+) counterparts). DRAGO mRNA levels were statistically significantly reduced in advanced-stage, compared with early-stage, ovarian tumors, but no mutations were found in several human tumors. We show that DRAGO expression is regulated both at transcriptional-through p53 (and p73) and methylation-dependent control-and post-transcriptional levels by miRNAs. DRAGO represents a new p53-dependent gene highly regulated in human cells and whose expression cooperates with p53 in tumor suppressor functions.

  2. Disturbed P53-MDM2 Feedback Loop Contributes to Thoracic Aortic Dissection Formation and May be a Result of TRIM25 Overexpression.

    PubMed

    Gong, Bin; Wang, Zhiwei; Zhang, Min; Hu, Zhipeng; Ren, Zongli; Tang, Zheng; Jiang, Wanli; Cheng, Lianghao; Huang, Jun; Ren, Wei; Wang, Qingtao

    2017-04-01

    The development of thoracic aortic dissection (TAD) is attributed to a broad range of degenerative, genetic, structural, oxidative, apoptotic, and acquired disease states. In this study, we examined the role of the disturbed p53-MDM2 (murine double minute 2) feedback loop in the formation of TAD, and one of a potential feedback loop regulator, TRIM25 (tripartite motif protein-25). Surgical specimens of the aorta from TAD patients (n = 10) and controls (n = 10) were tested for α-smooth muscle actin (α-SMA), p53, MDM2, and TRIM25 by western blot, immunohistochemical staining, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), respectively. When compared with controls, western blot shows that the protein levels of p53, MDM2, and TRIM25 were increased significantly in the aortic media of TAD patients. qRT-PCR further verified that the mRNA expression of MDM2 and TRIM25 was also increased 6- and 4-folds, respectively, in the TAD media of the aortic wall. Immunohistochemistry results showed significantly decreased staining of α-SMA, smooth muscle cells, and more collagen deposition in the media of the aortic wall from patients with TAD. This study provided a new insight into the disturbed p53-MDM2 feedback loop in the pathogenesis of TAD, and this may be because of the TRIM25 overexpression. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Long-term oral kinetin does not protect against α-synuclein-induced neurodegeneration in rodent models of Parkinson's disease.

    PubMed

    Orr, Adam L; Rutaganira, Florentine U; de Roulet, Daniel; Huang, Eric J; Hertz, Nicholas T; Shokat, Kevan M; Nakamura, Ken

    2017-10-01

    Mutations in the mitochondrial kinase PTEN-induced putative kinase 1 (PINK1) cause Parkinson's disease (PD), likely by disrupting PINK1's kinase activity. Although the mechanism(s) underlying how this loss of activity causes degeneration remains unclear, increasing PINK1 activity may therapeutically benefit some forms of PD. However, we must first learn whether restoring PINK1 function prevents degeneration in patients harboring PINK1 mutations, or whether boosting PINK1 function can offer protection in more common causes of PD. To test these hypotheses in preclinical rodent models of PD, we used kinetin triphosphate, a small-molecule that activates both wild-type and mutant forms of PINK1, which affects mitochondrial function and protects neural cells in culture. We chronically fed kinetin, the precursor of kinetin triphosphate, to PINK1-null rats in which PINK1 was reintroduced into their midbrain, and also to rodent models overexpressing α-synuclein. The highest tolerated dose of oral kinetin increased brain levels of kinetin for up to 6 months, without adversely affecting the survival of nigrostriatal dopamine neurons. However, there was no degeneration of midbrain dopamine neurons lacking PINK1, which precluded an assessment of neuroprotection and raised questions about the robustness of the PINK1 KO rat model of PD. In two rodent models of α-synuclein-induced toxicity, boosting PINK1 activity with oral kinetin provided no protective effects. Our results suggest that oral kinetin is unlikely to protect against α-synuclein toxicity, and thus fail to provide evidence that kinetin will protect in sporadic models of PD. Kinetin may protect in cases of PINK1 deficiency, but this possibility requires a more robust PINK1 KO model that can be validated by proof-of-principle genetic correction in adult animals. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Memory Alteration Test to Detect Amnestic Mild Cognitive Impairment and Early Alzheimer's Dementia in Population with Low Educational Level.

    PubMed

    Custodio, Nilton; Lira, David; Herrera-Perez, Eder; Montesinos, Rosa; Castro-Suarez, Sheila; Cuenca-Alfaro, José; Valeriano-Lorenzo, Lucía

    2017-01-01

    Background/Aims : Short tests to early detection of the cognitive impairment are necessary in primary care setting, particularly in populations with low educational level. The aim of this study was to assess the performance of Memory Alteration Test (M@T) to discriminate controls, patients with amnestic Mild Cognitive Impairment (aMCI) and patients with early Alzheimer's Dementia (AD) in a sample of individuals with low level of education. Methods : Cross-sectional study to assess the performance of the M@T (study test), compared to the neuropsychological evaluation (gold standard test) scores in 247 elderly subjects with low education level from Lima-Peru. The cognitive evaluation included three sequential stages: (1) screening (to detect cases with cognitive impairment); (2) nosological diagnosis (to determinate specific disease); and (3) classification (to differentiate disease subtypes). The subjects with negative results for all stages were considered as cognitively normal (controls). The test performance was assessed by means of area under the receiver operating characteristic (ROC) curve. We calculated validity measures (sensitivity, specificity and correctly classified percentage), the internal consistency (Cronbach's alpha coefficient), and concurrent validity (Pearson's ratio coefficient between the M@T and Clinical Dementia Rating (CDR) scores). Results : The Cronbach's alpha coefficient was 0.79 and Pearson's ratio coefficient was 0.79 ( p < 0.01). The AUC of M@T to discriminate between early AD and aMCI was 99.60% (sensitivity = 100.00%, specificity = 97.53% and correctly classified = 98.41%) and to discriminate between aMCI and controls was 99.56% (sensitivity = 99.17%, specificity = 91.11%, and correctly classified = 96.99%). Conclusions : The M@T is a short test with a good performance to discriminate controls, aMCI and early AD in individuals with low level of education from urban settings.

  5. A sensitive assay reveals structural requirements for α-synuclein fibril growth

    PubMed Central

    Tsai, Christina; Bagchi, Devika P.; Engel, Laura A.; Sarezky, Jonathan; Kotzbauer, Paul T.

    2017-01-01

    The accumulation of α-synuclein (α-syn) fibrils in neuronal inclusions is the defining pathological process in Parkinson's disease (PD). A pathogenic role for α-syn fibril accumulation is supported by the identification of dominantly inherited α-syn (SNCA) gene mutations in rare cases of familial PD. Fibril formation involves a spontaneous nucleation event in which soluble α-syn monomers associate to form seeds, followed by fibril growth during which monomeric α-syn molecules sequentially associate with existing seeds. To better investigate this process, we developed sensitive assays that use the fluorescein arsenical dye FlAsH (fluorescein arsenical hairpin binder) to detect soluble oligomers and mature fibrils formed from recombinant α-syn protein containing an N-terminal bicysteine tag (C2-α-syn). Using seed growth by monomer association (SeGMA) assays to measure fibril growth over 3 h in the presence of C2-α-syn monomer, we observed that some familial PD-associated α-syn mutations (i.e. H50Q and A53T) greatly increased growth rates, whereas others (E46K, A30P, and G51D) decreased growth rates. Experiments with wild-type seeds extended by mutant monomer and vice versa revealed that single-amino acid differences between seed and monomer proteins consistently decreased growth rates. These results demonstrate that α-syn monomer association during fibril growth is a highly ordered process that can be disrupted by misalignment of individual amino acids and that only a subset of familial-PD mutations causes fibril accumulation through increased fibril growth rates. The SeGMA assays reported herein can be utilized to further elucidate structural requirements of α-syn fibril growth and to identify growth inhibitors as a potential therapeutic approach in PD. PMID:28373279

  6. Alpha-Synuclein Pathology in Sensory Nerve Terminals of the Upper Aerodigestive Tract of Parkinson’s Disease Patients

    PubMed Central

    Mu, Liancai; Chen, Jingming; Sobotka, Stanislaw; Nyirenda, Themba; Benson, Brian; Gupta, Fiona; Sanders, Ira; Adler, Charles H.; Caviness, John N.; Shill, Holly A.; Sabbagh, Marwan; Samanta, Johan E.; Sue, Lucia I.; Beach, Thomas G.

    2015-01-01

    Dysphagia is common in Parkinson’s disease (PD) and causes significant morbidity and mortality. PD dysphagia has usually been explained as dysfunction of central motor control, much like other motor symptoms that are characteristic of the disease. However, PD dysphagia does not correlate with severity of motor symptoms nor does it respond to motor therapies. It is known that PD patients have sensory deficits in the pharynx, and that impaired sensation may contribute to dysphagia. However, the underlying cause of the pharyngeal sensory deficits in PD is not known. We hypothesized that PD dysphagia with sensory deficits may be due to degeneration of the sensory nerve terminals in the upper aerodigestive tract (UAT). We have previously shown that Lewy-type synucleinopathy (LTS) is present in the main pharyngeal sensory nerves of PD patients, but not in controls. In this study, the sensory terminals in UAT mucosa were studied to discern the presence and distribution of LTS. Whole-mount specimens (tongue-pharynx-larynx-upper esophagus) were obtained from 10 deceased human subjects with clinically diagnosed and neuropathologically confirmed PD (five with dysphagia and five without) and four age-matched healthy controls. Samples were taken from six sites and immunostained for phosphorylated α-synuclein (PAS). The results showed the presence of PAS-immunoreactive (PAS-ir) axons in all the PD subjects and in none of the controls. Notably, PD patients with dysphagia had more PAS-ir axons in the regions that are critical for initiating the swallowing reflex. These findings suggest that Lewy pathology affects mucosal sensory axons in specific regions of the UAT and may be related to PD dysphagia. PMID:26041249

  7. Akt mediates 17beta-estradiol and/or estrogen receptor-alpha inhibition of LPS-induced tumor necresis factor-alpha expression and myocardial cell apoptosis by suppressing the JNK1/2-NFkappaB pathway.

    PubMed

    Liu, Chung-Jung; Lo, Jeng-Fan; Kuo, Chia-Hua; Chu, Chun-Hsien; Chen, Li-Ming; Tsai, Fuu-Jen; Tsai, Chang-Hai; Tzang, Bor-Show; Kuo, Wei-Wen; Huang, Chih-Yang

    2009-09-01

    Evidence shows that women have lower tumour necrosis factor-alpha (TNF-alpha) levels and lower incidences of heart dysfunction and sepsis-related morbidity and mortality. To identify the cardioprotective effects and precise cellular/molecular mechanisms behind estrogen and estrogen receptors (ERs), we investigated the effects of 17beta-estradiol (E(2)) and estrogen receptor alpha (ERalpha) on LPS-induced apoptosis by analyzing the activation of survival and death signalling pathways in doxycycline (Dox)-inducible Tet-On/ERalpha H9c2 myocardial cells and ERalpha-transfected primary cardiomyocytes overexpressing ERalpha. We found that LPS challenge activated JNK1/2, and then induced IkappaB degradation, NFkappaB activation, TNF-alpha up-regulation and subsequent myocardial apoptotic responses. In addition, treatments involving E(2), membrane-impermeable BSA-E(2) and/or Dox, which induces ERalpha overexpression, significantly inhibited LPS-induced apoptosis by suppressing LPS-up-regulated JNK1/2 activity, IkappaB degradation, NFkappaB activation and pro-apoptotic proteins (e.g. TNF-alpha, active caspases-8, t-Bid, Bax, released cytochrome c, active caspase-9, active caspase-3) in myocardial cells. However, the cardioprotective properties of E(2), BSA-E(2) and ERalpha overexpression to inhibit LPS-induced apoptosis and promote cell survival were attenuated by applying LY294002 (PI3K inhibitor) and PI3K siRNA. These findings suggest that E(2), BSA-E(2) and ERalpha expression exert their cardioprotective effects by inhibiting JNK1/2-mediated LPS-induced TNF-alpha expression and cardiomyocyte apoptosis through activation of Akt.

  8. Coordinated balancing of muscle oxidative metabolism through PGC-1{alpha} increases metabolic flexibility and preserves insulin sensitivity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Summermatter, Serge; Troxler, Heinz; Santos, Gesa

    2011-04-29

    Highlights: {yields} PGC-1{alpha} enhances muscle oxidative capacity. {yields} PGC-1{alpha} promotes concomitantly positive and negative regulators of lipid oxidation. {yields} Regulator abundance enhances metabolic flexibility and balances oxidative metabolism. {yields} Balanced oxidation prevents detrimental acylcarnitine and ROS generation. {yields} Absence of detrimental metabolites preserves insulin sensitivity -- Abstract: The peroxisome proliferator-activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}) enhances oxidative metabolism in skeletal muscle. Excessive lipid oxidation and electron transport chain activity can, however, lead to the accumulation of harmful metabolites and impair glucose homeostasis. Here, we investigated the effect of over-expression of PGC-1{alpha} on metabolic control and generation of insulin desensitizing agentsmore » in extensor digitorum longus (EDL), a muscle that exhibits low levels of PGC-1{alpha} in the untrained state and minimally relies on oxidative metabolism. We demonstrate that PGC-1{alpha} induces a strictly balanced substrate oxidation in EDL by concomitantly promoting the transcription of activators and inhibitors of lipid oxidation. Moreover, we show that PGC-1{alpha} enhances the potential to uncouple oxidative phosphorylation. Thereby, PGC-1{alpha} boosts elevated, yet tightly regulated oxidative metabolism devoid of side products that are detrimental for glucose homeostasis. Accordingly, PI3K activity, an early phase marker for insulin resistance, is preserved in EDL muscle. Our findings suggest that PGC-1{alpha} coordinately coactivates the simultaneous transcription of gene clusters implicated in the positive and negative regulation of oxidative metabolism and thereby increases metabolic flexibility. Thus, in mice fed a normal chow diet, over-expression of PGC-1{alpha} does not alter insulin sensitivity and the metabolic adaptations elicited by PGC-1{alpha} mimic the beneficial effects of endurance

  9. New Insights into the Mechanism of Inhibition of p53 by Simian Virus 40 Large T Antigen

    PubMed Central

    Sheppard, Hilary M.; Corneillie, Siska I.; Espiritu, Christine; Gatti, Andrea; Liu, Xuan

    1999-01-01

    Simian virus 40 (SV40) large tumor antigen (T antigen) has been shown to inhibit p53-dependent transcription by preventing p53 from binding to its cognate cis element. Data presented in this report provide the first direct functional evidence that T antigen, under certain conditions, may also repress p53-dependent transcription by a mechanism in which the transactivation domain of p53 is abrogated while DNA binding is unaffected. Specifically, p53 purified as a complex with T antigen from mouse cells was found to bind DNA as a transcriptionally inactive intact complex, while that purified from human cells was found to bind DNA independently of T antigen and could activate p53-dependent transcription. This difference in activity may be dependent on a different interaction of T antigen with mouse and human p53 and, in addition, on the presence of super T, which is found only in transformed rodent cells. These results suggest that subtle yet important differences exist between the inhibition of p53 by T antigen in mouse and human cells. The implications of this finding with respect to SV40-associated malignancies are discussed. PMID:10082540

  10. Transgenic mice overexpressing the extracellular domain of NCAM are impaired in working memory and cortical plasticity

    PubMed Central

    Brennaman, Leann H.; Kochlamazashvili, Gaga; Stoenica, Luminita; Nonneman, Randall J.; Moy, Sheryl S.; Schachner, Melitta; Dityatev, Alexander; Maness, Patricia F.

    2011-01-01

    The neural cell adhesion molecule, NCAM, is a pivotal regulator of neural development, with key roles in axonal and dendritic growth and synaptic plasticity. Alterations in NCAM expression or proteolytic cleavage have been linked to human neuropsychiatric disorders such as schizophrenia, bipolar disorder and Alzheimer’s disease, and may contribute to cognitive dysfunction. We have generated mice overexpressing the NCAM extracellular (EC) proteolytic cleavage fragment which has been reported to be increased in schizophrenic versus normal brains. These mice show impaired GABAergic innervation and reduced number of apical dendritic spines on pyramidal neurons in the prefrontal cortex (PFC). Here, these NCAM-EC transgenic mice were subjected to behavioral tasks and electrophysiological measurements to determine the impact of structural abnormalities in the PFC on synaptic and cognitive functions. NCAM-EC mice exhibited impaired working memory in a delayed non-match-to-sample task, which requires PFC function, but showed no differences in anxiety, olfactory abilities, or sociability. Transgenic mice displayed impaired long- and short-term potentiation in the PFC but normal synaptic plasticity in the hippocampus, suggesting that the abnormal synaptic innervation in NCAM-EC mice impairs PFC plasticity and alters working memory. These findings may have implications for cognitive dysfunctions observed in neuropsychiatric disorders. PMID:21515372

  11. Overexpression of the Mitochondrial T3 Receptor p43 Induces a Shift in Skeletal Muscle Fiber Types

    PubMed Central

    Casas, François; Pessemesse, Laurence; Grandemange, Stéphanie; Seyer, Pascal; Gueguen, Naïg; Baris, Olivier; Lepourry, Laurence; Cabello, Gérard; Wrutniak-Cabello, Chantal

    2008-01-01

    In previous studies, we have characterized a new hormonal pathway involving a mitochondrial T3 receptor (p43) acting as a mitochondrial transcription factor and consequently stimulating mitochondrial activity and mitochondrial biogenesis. We have established the involvement of this T3 pathway in the regulation of in vitro myoblast differentiation.We have generated mice overexpressing p43 under control of the human α-skeletal actin promoter. In agreement with the previous characterization of this promoter, northern-blot and western-blot experiments confirmed that after birth p43 was specifically overexpressed in skeletal muscle. As expected from in vitro studies, in 2-month old mice, p43 overexpression increased mitochondrial genes expression and mitochondrial biogenesis as attested by the increase of mitochondrial mass and mt-DNA copy number. In addition, transgenic mice had a body temperature 0.8°C higher than control ones and displayed lower plasma triiodothyronine levels. Skeletal muscles of transgenic mice were redder than wild-type animals suggesting an increased oxidative metabolism. In line with this observation, in gastrocnemius, we recorded a strong increase in cytochrome oxidase activity and in mitochondrial respiration. Moreover, we observed that p43 drives the formation of oxidative fibers: in soleus muscle, where MyHC IIa fibers were partly replaced by type I fibers; in gastrocnemius muscle, we found an increase in MyHC IIa and IIx expression associated with a reduction in the number of glycolytic fibers type IIb. In addition, we found that PGC-1α and PPARδ, two major regulators of muscle phenotype were up regulated in p43 transgenic mice suggesting that these proteins could be downstream targets of mitochondrial activity. These data indicate that the direct mitochondrial T3 pathway is deeply involved in the acquisition of contractile and metabolic features of muscle fibers in particular by regulating PGC-1α and PPARδ. PMID:18575627

  12. Data for a proteomic analysis of p53-independent induction of apoptosis by bortezomib

    PubMed Central

    Yerlikaya, Azmi; Okur, Emrah; Tarık Baykal, Ahmet; Acılan, Ceyda; Boyacı, İhsan; Ulukaya, Engin

    2014-01-01

    This data article contains data related to the research article entitled, “A proteomic analysis of p53-independent induction of apoptosis by bortezomib in 4T1 breast cancer cell line” by Yerlikaya et al. [1]. The research article presented 2-DE and nLC-MS/MS based proteomic analysis of proteasome inhibitor bortezomib-induced changes in the expression of cellular proteins. The report showed that GRP78 and TCEB2 were over-expressed in response to treatment with bortezomib for 24 h. In addition, the report demonstrated that Hsp70, the 26S proteasome non-ATPase regulatory subunit 14 and sequestosome 1 were increased at least 2 fold in p53-deficient 4T1 cells. The data here show for the first time the increased expressions of Card10, Dffb, Traf3 and Trp53bp2 in response to inhibition of the 26S proteasome. The information presented here also shows that both Traf1 and Xiap (a member of IAPs) are also downregulated simultaneously upon proteasomal inhibition. The increases in the level of Card10 and Trp53bp2 proteins were verified by Western blot analysis in response to varying concentrations of bortezomib for 24 h. PMID:26217687

  13. Genetically-induced Estrogen Receptor Alpha mRNA (Esr1) Overexpression Does Not Adversely Affect Fertility or Penile Development in Male Mice

    PubMed Central

    Heath, John; Abdelmageed, Yazeed; Braden, Tim D.; Williams, Carol S.; Williams, John W.; Paulose, Tessie; Hernandez-Ochoa, Isabel; Gupta, Rupesh; Flaws, Jodi A.; Goyal, Hari O.

    2011-01-01

    Previously, we reported that estrogen receptor alpha mRNA (Esr1) or protein (ESR1) overexpression resulting from neonatal exposure to estrogens in rats was associated with infertility and mal-developed penis characterized by reduced length and weight and abnormal accumulation of fat cells. The objective of this study was to determine if mutant male mice overexpressing Esr1 are naturally infertile or have reduced fertility and/or develop abnormal penis. The fertility parameters, including fertility and fecundity indices, numbers of days from the day of cohabitation to the day of delivery, and numbers of pups per female, were not altered from controls, as a result of Esr1 overexpression. Likewise, penile morphology, including the length, weight, and diameter and os penis development, was not altered from controls. Conversely, weights of the seminal vesicles and bulbospongiosus and levator ani (BS/LA) muscles were significantly (P < 0.05) lower as compared to controls; however, the weight of the testis, the morphology of the testis and epididymis, and the plasma and testicular testosterone concentration were not different from controls. Hence, the genetically-induced Esr1 overexpression alone, without an exogenous estrogen exposure during the neonatal period, is unable to adversely affect the development of the penis as well as other male reproductive organs, except limited, but significant, reductions in weights of the seminal vesicles and BS/LA muscles. PMID:20930192

  14. Memory Alteration Test to Detect Amnestic Mild Cognitive Impairment and Early Alzheimer’s Dementia in Population with Low Educational Level

    PubMed Central

    Custodio, Nilton; Lira, David; Herrera-Perez, Eder; Montesinos, Rosa; Castro-Suarez, Sheila; Cuenca-Alfaro, José; Valeriano-Lorenzo, Lucía

    2017-01-01

    Background/Aims: Short tests to early detection of the cognitive impairment are necessary in primary care setting, particularly in populations with low educational level. The aim of this study was to assess the performance of Memory Alteration Test (M@T) to discriminate controls, patients with amnestic Mild Cognitive Impairment (aMCI) and patients with early Alzheimer’s Dementia (AD) in a sample of individuals with low level of education. Methods: Cross-sectional study to assess the performance of the M@T (study test), compared to the neuropsychological evaluation (gold standard test) scores in 247 elderly subjects with low education level from Lima-Peru. The cognitive evaluation included three sequential stages: (1) screening (to detect cases with cognitive impairment); (2) nosological diagnosis (to determinate specific disease); and (3) classification (to differentiate disease subtypes). The subjects with negative results for all stages were considered as cognitively normal (controls). The test performance was assessed by means of area under the receiver operating characteristic (ROC) curve. We calculated validity measures (sensitivity, specificity and correctly classified percentage), the internal consistency (Cronbach’s alpha coefficient), and concurrent validity (Pearson’s ratio coefficient between the M@T and Clinical Dementia Rating (CDR) scores). Results: The Cronbach’s alpha coefficient was 0.79 and Pearson’s ratio coefficient was 0.79 (p < 0.01). The AUC of M@T to discriminate between early AD and aMCI was 99.60% (sensitivity = 100.00%, specificity = 97.53% and correctly classified = 98.41%) and to discriminate between aMCI and controls was 99.56% (sensitivity = 99.17%, specificity = 91.11%, and correctly classified = 96.99%). Conclusions: The M@T is a short test with a good performance to discriminate controls, aMCI and early AD in individuals with low level of education from urban settings. PMID:28878665

  15. Frequent mutations in the p53 tumor suppressor gene in human leukemia T-cell lines.

    PubMed Central

    Cheng, J; Haas, M

    1990-01-01

    Human T-cell leukemia and T-cell acute lymphoblastic leukemia cell lines were studied for alterations in the p53 tumor suppressor gene. Southern blot analysis of 10 leukemic T-cell lines revealed no gross genomic deletions or rearrangements. Reverse transcription-polymerase chain reaction analysis of p53 mRNA indicated that all 10 lines produced p53 mRNA of normal size. By direct sequencing of polymerase chain reaction-amplified cDNA, we detected 11 missense and nonsense point mutations in 5 of the 10 leukemic T-cell lines studied. The mutations are primarily located in the evolutionarily highly conserved regions of the p53 gene. One of the five cell lines in which a mutation was detected possesses a homozygous point mutation in both p53 alleles, while the other four cell lines harbor from two to four different point mutations. An allelic study of two of the lines (CEM, A3/Kawa) shows that the two missense mutations found in each line are located on separate alleles, thus both alleles of the p53 gene may have been functionally inactivated by two different point mutations. Since cultured leukemic T-cell lines represent a late, fully tumorigenic stage of leukemic T cells, mutation of both (or more) alleles of the p53 gene may reflect the selection of cells possessing an increasingly tumorigenic phenotype, whether the selection took place in vivo or in vitro. Previously, we have shown that the HSB-2 T-cell acute lymphoblastic leukemia cell line had lost both alleles of the retinoblastoma tumor suppressor gene. Taken together, our data show that at least 6 of 10 leukemic T-cell lines examined may have lost the normal function of a known tumor suppressor gene, suggesting that this class of genes serves a critical role in the generation of fully tumorigenic leukemic T cells. Images PMID:2144611

  16. α - synuclein under the magnifying glass. Insights from atomistic and coarse-grain simulations

    NASA Astrophysics Data System (ADS)

    Ilie, Ioana M.; Nayar, Divya; den Otter, Wouter K.; van der Vegt, Nico F. A.; Briels, Wim J.; University of Twente Collaboration; University of Darmstadt Collaboration

    Neurodegenerative diseases are linked to the accumulation of misfolded intrinsically disordered proteins in the brain. Here, we use both all-atom and coarse-grain simulations to explore the intricate dynamics and the aggregation of α-synuclein, the protein implicated in Parkinson's disease. We explore the free energy landscapes of α-synuclein by using Molecular Dynamics simulations and extract information on the structure of the protein as well as on its binding affinities. Next, to study the aggregation, we proceed with representing α-synuclein as a chain of deformable particles that can adapt their geometry, binding affinities and can rearrange into different disordered and ordered structures. We use Brownian Dynamics to simulate the translational and rotational motions of the particles, as well as their interaction properties. The simulations show valuable insight into the internal dynamics of α-synuclein and the formation of ordered and disordered aggregates. In addition, the study is extended to investigate the attachment and folding of a protein to a fiber.

  17. DRAGO (KIAA0247), a New DNA Damage–Responsive, p53-Inducible Gene That Cooperates With p53 as Oncosupprossor

    PubMed Central

    Polato, Federica; Rusconi, Paolo

    2014-01-01

    Background p53 influences genomic stability, apoptosis, autophagy, response to stress, and DNA damage. New p53-target genes could elucidate mechanisms through which p53 controls cell integrity and response to damage. Methods DRAGO (drug-activated gene overexpressed, KIAA0247) was characterized by bioinformatics methods as well as by real-time polymerase chain reaction, chromatin immunoprecipitation and luciferase assays, time-lapse microscopy, and cell viability assays. Transgenic mice (94 p53−/− and 107 p53+/− mice on a C57BL/6J background) were used to assess DRAGO activity in vivo. Survival analyses were performed using Kaplan–Meier curves and the Mantel–Haenszel test. All statistical tests were two-sided. Results We identified DRAGO as a new p53-responsive gene induced upon treatment with DNA-damaging agents. DRAGO is highly conserved, and its ectopic overexpression resulted in growth suppression and cell death. DRAGO−/− mice are viable without macroscopic alterations. However, in p53−/− or p53+/− mice, the deletion of both DRAGO alleles statistically significantly accelerated tumor development and shortened lifespan compared with p53−/− or p53+/− mice bearing wild-type DRAGO alleles (p53−/−, DRAGO−/− mice: hazard ratio [HR] = 3.25, 95% confidence interval [CI] = 1.7 to 6.1, P < .001; p53+/−, DRAGO−/− mice: HR = 2.35, 95% CI = 1.3 to 4.0, P < .001; both groups compared with DRAGO+/+ counterparts). DRAGO mRNA levels were statistically significantly reduced in advanced-stage, compared with early-stage, ovarian tumors, but no mutations were found in several human tumors. We show that DRAGO expression is regulated both at transcriptional—through p53 (and p73) and methylation-dependent control—and post-transcriptional levels by miRNAs. Conclusions DRAGO represents a new p53-dependent gene highly regulated in human cells and whose expression cooperates with p53 in tumor suppressor functions. PMID:24652652

  18. [The effect of Foxc2 overexpression on the osteogenic properties of C3H10T1/2 cells].

    PubMed

    Wang, Min-Jiao; Si, Jia-Wen; Li, Hong-Liang; Ouyang, Ning-Juan; Shen, Guo-Fang

    2016-08-01

    To investigate the effect of Foxc2 overexpression on osteogenic and adipogenic differentiation of C3H10T1/2 cells. C3H10T1/2 cells were transfected with plenti-Foxc2 and selected with puromycin for stable clones. The expression of Foxc2 was determined by real-time PCR and Western blot. Cell proliferation was detected by CCK-8 kit. Cell cycle and apoptosis were detected by flow cytometry. The level of osteogenic biomarkers Runx2, OPN, OCN and adipogenic biomarker PPARγ were quantified by real-time PCR and Western blot. Alkaline phosphatase (ALP) staining and oil red staining were conducted to evaluate the effect of Foxc2 overexpression on osteogenic and adipogenic differentiation. Statistical analysis was performed using SPSS 17.0 software package. C3H10T1/2-Foxc2 cell line was successfully constructed and verified by direct sequencing and Foxc2 overexpression in vitro. Cell proliferation was reduced and cell cycle was blocked in G1/G0 phase. Enhanced ALP staining and reduced oil red staining were observed in C3H10T1/2-Foxc2 cells as compared with the control. Foxc2 overexpression up-regulated Runx2, OPN, OCN during osteogenic differentiation and down-regulated PPARγduring adipogenic differentiation. C3H10T1/2 cell line stably expressing Foxc2 gene was successfully established, cell proliferation was reduced, osteogenesis biomarkers were up-regulated during the osteogenesis by overexpression Foxc2, PPARγwas down-regulated during adipogenesis.

  19. Synthetic Proteins and Peptides for the Direct Interrogation of α-Synuclein Posttranslational Modifications

    PubMed Central

    Pratt, Matthew R.; Abeywardana, Tharindumala; Marotta, Nicholas P.

    2015-01-01

    α-Synuclein is the aggregation-prone protein associated with Parkinson’s disease (PD) and related neurodegenerative diseases. Complicating both its biological functions and toxic aggregation are a variety of posttranslational modifications. These modifications have the potential to either positively or negatively affect α-synuclein aggregation, raising the possibility that the enzymes that add or remove these modifications could be therapeutic targets in PD. Synthetic protein chemistry is uniquely positioned to generate site-specifically and homogeneously modified proteins for biochemical study. Here, we review the application of synthetic peptides and proteins towards understanding the effects of α-synuclein posttranslational modifications. PMID:26120904

  20. Pure versus combined Merkel cell carcinomas: immunohistochemical evaluation of cellular proteins (p53, Bcl-2, and c-kit) reveals significant overexpression of p53 in combined tumors.

    PubMed

    Lai, Jonathan H; Fleming, Kirsten E; Ly, Thai Yen; Pasternak, Sylvia; Godlewski, Marek; Doucette, Steve; Walsh, Noreen M

    2015-09-01

    Merkel cell polyomavirus is of oncogenic significance in approximately 80% of Merkel cell carcinomas. Morphological subcategories of the tumor differ in regard to viral status, the rare combined type being uniformly virus negative and the predominant pure type being mainly virus positive. Indications that different biological subsets of the tumor exist led us to explore this diversity. In an Eastern Canadian cohort of cases (75 patients; mean age, 76 years [range, 43-91]; male/female ratio, 43:32; 51 [68%] pure and 24 [34%] combined tumors), we semiquantitatively compared the immunohistochemical expression of 3 cellular proteins (p53, Bcl-2, and c-kit) in pure versus combined groups. Viral status was known in a subset of cases. The significant overexpression of p53 in the combined group (mean [SD], 153.8 [117.8] versus 121.6 [77.9]; P = .01) and the increased epidermal expression of this protein (p53 patches) in the same group lend credence to a primary etiologic role for sun damage in these cases. Expression of Bcl-2 and c-kit did not differ significantly between the 2 morphological groups. A relative increase in c-kit expression was significantly associated with a virus-negative status (median [interquartile range], 100 [60-115] versus 70 [0-100]; P = .03). Emerging data reveal divergent biological pathways in Merkel cell carcinoma, each with a characteristic immunohistochemical profile. Virus-positive tumors (all pure) exhibit high retinoblastoma protein and low p53 expression, whereas virus-negative cases (few pure and all combined) show high p53 and relatively high c-kit expression. The potential biological implications of this dichotomy call for consistent stratification of these tumors in future studies. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Evidence of native α-synuclein conformers in the human brain.

    PubMed

    Gould, Neal; Mor, Danielle E; Lightfoot, Richard; Malkus, Kristen; Giasson, Benoit; Ischiropoulos, Harry

    2014-03-14

    α-Synuclein aggregation is central to the pathogenesis of several brain disorders. However, the native conformations and functions of this protein in the human brain are not precisely known. The native state of α-synuclein was probed by gel filtration coupled with native gradient gel separation, an array of antibodies with non-overlapping epitopes, and mass spectrometry. The existence of metastable conformers and stable monomer was revealed in the human brain.

  2. Persistent short-term memory defects following sleep deprivation in a drosophila model of Parkinson disease.

    PubMed

    Seugnet, Laurent; Galvin, James E; Suzuki, Yasuko; Gottschalk, Laura; Shaw, Paul J

    2009-08-01

    Parkinson disease (PD) is the second most common neurodegenerative disorder in the United States. It is associated with motor deficits, sleep disturbances, and cognitive impairment. The pathology associated with PD and the effects of sleep deprivation impinge, in part, upon common molecular pathways suggesting that sleep loss may be particularly deleterious to the degenerating brain. Thus we investigated the long-term consequences of sleep deprivation on shortterm memory using a Drosophila model of Parkinson disease. Transgenic strains of Drosophila melanogaster. Using the GAL4-UAS system, human alpha-synuclein was expressed throughout the nervous system of adult flies. Alpha-synuclein expressing flies (alpha S flies) and the corresponding genetic background controls were sleep deprived for 12 h at age 16 days and allowed to recover undisturbed for at least 3 days. Short-term memory was evaluated using aversive phototaxis suppression. Dopaminergic systems were assessed using mRNA profiling and immunohistochemistry. MEASURMENTS AND RESULTS: When sleep deprived at an intermediate stage of the pathology, alpha S flies showed persistent short-term memory deficits that lasted > or = 3 days. Cognitive deficits were not observed in younger alpha S flies nor in genetic background controls. Long-term impairments were not associated with accelerated loss of dopaminergic neurons. However mRNA expression of the dopamine receptors dDA1 and DAMB were significantly increased in sleep deprived alpha S flies. Blocking D1-like receptors during sleep deprivation prevented persistent shortterm memory deficits. Importantly, feeding flies the polyphenolic compound curcumin blocked long-term learning deficits. These data emphasize the importance of sleep in a degenerating/reorganizing brain and shows that pathological processes induced by sleep deprivation can be dissected at the molecular and cellular level using Drosophila genetics.

  3. The influence of SV40 immortalization of human fibroblasts on p53-dependent radiation responses

    NASA Technical Reports Server (NTRS)

    Kohli, M.; Jorgensen, T. J.

    1999-01-01

    The simian virus 40 large tumor antigen (SV40 Tag) has been ascribed many functions critical to viral propagation, including binding to the mammalian tumor suppressor p53. Recent studies have demonstrated that SV40-transformed murine cells have functional p53. The status of p53 in SV40-immortalized human cells, however, has not been characterized. We have found that in response to ionizing radiation, p53-dependent p21 transactivation activity is present, albeit reduced, in SV40-immortalized cells and that this activity can be further reduced with either dominant negative p53 expression or higher SV40 Tag expression. Furthermore, overexpression of p53 in SV40-immortalized ataxia-telangiectasia (A-T) cells restores p53-dependent p21 induction to typical A-T levels. All SV40-immortalized cell lines exhibited an absence of G1 arrest. Moreover, all SV40-immortalized cell lines exhibited increased apoptosis relative to primary cells in response to ionizing radiation, suggesting that SV40 immortalization results in a unique phenotype with regard to DNA damage responses. Copyright 1999 Academic Press.

  4. Opposing behavioural alterations in male and female transgenic TGF alpha mice: association with tumour susceptibility.

    PubMed Central

    Hilakivi-Clarke, L. A.; Arora, P. K.; Clarke, R.; Wright, A.; Lippman, M. E.; Dickson, R. B.

    1993-01-01

    Psychosocial factors are thought to influence risk and survival from cancer. We have previously studied specific behaviours in transgenic male CD-1 MT42 mice, which overexpress the gene encoding human transforming growth factor alpha (TGF alpha) in multiple tissues, and which develop a high incidence of spontaneous hepatocellular carcinoma. The male TGF alpha mice spent a lengthened time immobile in the swim test, were highly aggressive, had increased plasma levels of 17 beta-estradiol (E2), and reduced natural killer (NK) cell activity. The female transgenic MT42 TGF alpha mice do not develop an increased rate of tumours at any site. We hypothesised that if the alterations in male TGF alpha mice are associated with their development of hepatocellular carcinomas, female TGF alpha should not show these alterations. The data in the present study indicate that female TGF alpha mice display shortened immobility in the swim test, suggesting an improved ability to cope with stress, and appear less aggressive in the resident-intruder test than non-transgenic female CD-1 mice. The female TGF alpha mice also exhibit a 3-fold increase in the plasma levels of E2, and a 3-fold increase in NK cell activity. These findings suggest that the elevated expression of TGF alpha in the transgenic mice is associated with gender-specific behavioural alterations, and the development of spontaneous hepatocellular tumours in the males. Furthermore, TGF alpha alters hormonal and immune parameters similarly in both sexes. It remains to be determined whether the development of hepatocarcinoma in the male TGF alpha animals is associated with an impaired ability to cope with stress and elevated aggressive tendencies and/or whether manipulations leading to an impaired ability to cope with stress will promote tumourigenesis in female TGF alpha mice. PMID:8494695

  5. 46 CFR 32.53-1 - Application-T/ALL.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... system that uses crude oil washing. (4) An existing product carrier of 20,000 deadweight tons (metric) or... REQUIREMENTS Inert Gas System § 32.53-1 Application—T/ALL. (a) Except as provided in paragraphs (b) and (c) of... tons (metric) or more or combination carrier of 50,000 DWT tons (metric) or more, that has a keel...

  6. 46 CFR 32.53-1 - Application-T/ALL.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... system that uses crude oil washing. (4) An existing product carrier of 20,000 deadweight tons (metric) or... REQUIREMENTS Inert Gas System § 32.53-1 Application—T/ALL. (a) Except as provided in paragraphs (b) and (c) of... tons (metric) or more or combination carrier of 50,000 DWT tons (metric) or more, that has a keel...

  7. 46 CFR 32.53-1 - Application-T/ALL.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... system that uses crude oil washing. (4) An existing product carrier of 20,000 deadweight tons (metric) or... REQUIREMENTS Inert Gas System § 32.53-1 Application—T/ALL. (a) Except as provided in paragraphs (b) and (c) of... tons (metric) or more or combination carrier of 50,000 DWT tons (metric) or more, that has a keel...

  8. 46 CFR 32.53-1 - Application-T/ALL.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... system that uses crude oil washing. (4) An existing product carrier of 20,000 deadweight tons (metric) or... REQUIREMENTS Inert Gas System § 32.53-1 Application—T/ALL. (a) Except as provided in paragraphs (b) and (c) of... tons (metric) or more or combination carrier of 50,000 DWT tons (metric) or more, that has a keel...

  9. 46 CFR 32.53-1 - Application-T/ALL.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... system that uses crude oil washing. (4) An existing product carrier of 20,000 deadweight tons (metric) or... REQUIREMENTS Inert Gas System § 32.53-1 Application—T/ALL. (a) Except as provided in paragraphs (b) and (c) of... tons (metric) or more or combination carrier of 50,000 DWT tons (metric) or more, that has a keel...

  10. Inhibition of NAMPT pathway by FK866 activates the function of p53 in HEK293T cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Thakur, Basant Kumar, E-mail: thakur.basant@mh-hannover.de; Department of Molecular Hematopoiesis, Hannover Medical School, Carl Neuberg Str-1, 30625 Hannover; Dittrich, Tino

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer In 293T cells, p53 is considered to be inactive due to its interaction with the large T-antigen. Black-Right-Pointing-Pointer Acetylation of p53 at lysine 382 is important for its functional activation. Black-Right-Pointing-Pointer First evidence to document the presence of a functional p53 in 293T cells. Black-Right-Pointing-Pointer Inhibition of NAMPT/SIRT pathway by FK866 in 293T cells increases the functional activity of p53. Black-Right-Pointing-Pointer This activation of p53 involves reversible acetylation of p53 at lysine 382. -- Abstract: Inactivation of p53 protein by endogenous and exogenous carcinogens is involved in the pathogenesis of different human malignancies. In cancer associated with SV-40more » DNA tumor virus, p53 is considered to be non-functional mainly due to its interaction with the large T-antigen. Using the 293T cell line (HEK293 cells transformed with large T antigen) as a model, we provide evidence that p53 is one of the critical downstream targets involved in FK866-mediated killing of 293T cells. A reduced rate of apoptosis and an increased number of cells in S-phase was accompanied after knockdown of p53 in these cells. Inhibition of NAMPT by FK866, or inhibition of SIRT by nicotinamide decreased proliferation and triggered death of 293T cells involving the p53 acetylation pathway. Additionally, knockdown of p53 attenuated the effect of FK866 on cell proliferation, apoptosis, and cell cycle arrest. The data presented here shed light on two important facts: (1) that p53 in 293T cells is active in the presence of FK866, an inhibitor of NAMPT pathway; (2) the apoptosis induced by FK866 in 293T cells is associated with increased acetylation of p53 at Lys382, which is required for the functional activity of p53.« less

  11. Memory impairment, oxidative damage and apoptosis induced by space radiation: ameliorative potential of alpha-lipoic acid.

    PubMed

    Manda, Kailash; Ueno, Megumi; Anzai, Kazunori

    2008-03-05

    Exposure to high-energy particle radiation (HZE) may cause oxidative stress and cognitive impairment in the same manner that seen in aged mice. This phenomenon has raised the concerns about the safety of an extended manned mission into deep space where a significant portion of the radiation burden would come from HZE particle radiation. The present study aimed at investigating the role of alpha-lipoic acid against space radiation-induced oxidative stress and antioxidant status in cerebellum and its correlation with cognitive dysfunction. We observed spontaneous motor activities and spatial memory task of mice using pyroelectric infrared sensor and programmed video tracking system, respectively. Whole body irradiation of mice with high-LET (56)Fe beams (500 MeV/nucleon, 1.5 Gy) substantially impaired the reference memory at 30 day post-irradiation; however, no significant effect was observed on motor activities of mice. Acute intraperitoneal treatment of mice with alpha-lipoic acid prior to irradiation significantly attenuated such memory dysfunction. Radiation-induced apoptotic damage in cerebellum was examined using a neuronal-specific terminal deoxynucleotidyl transferase-mediated nick end-labeling method (NeuroTACS). Radiation-induced apoptotic and necrotic cell death of granule cells and Purkinje cells were inhibited significantly by alpha-lipoic acid pretreatment. Alpha-lipoic acid pretreatment exerted a very high magnitude of protection against radiation-induced augmentation of DNA damage (comet tail movement and serum 8-OHdG), lipid proxidation products (MDA+HAE) and protein carbonyls in mice cerebellum. Further, radiation-induced decline of non-protein sulfhydryl (NP-SH) contents of cerebellum and plasma ferric reducing power (FRAP) was also inhibited by alpha-lipoic acid pre-treatment. Results clearly indicate that alpha-lipoic acid is a potent neuroprotective antioxidant. Moreover, present finding also support the idea suggesting the cerebellar

  12. Monomeric and fibrillar α-synuclein exert opposite effects on the catalytic cycle that promotes the proliferation of Aβ42 aggregates

    PubMed Central

    Chia, Sean; Habchi, Johnny; Lattanzi, Veronica; Dobson, Christopher M.; Knowles, Tuomas P. J.; Vendruscolo, Michele

    2017-01-01

    The coaggregation of the amyloid-β peptide (Aβ) and α-synuclein is commonly observed in a range of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. The complex interplay between Aβ and α-synuclein has led to seemingly contradictory results on whether α-synuclein promotes or inhibits Aβ aggregation. Here, we show how these conflicts can be rationalized and resolved by demonstrating that different structural forms of α-synuclein exert different effects on Aβ aggregation. Our results demonstrate that whereas monomeric α-synuclein blocks the autocatalytic proliferation of Aβ42 (the 42-residue form of Aβ) fibrils, fibrillar α-synuclein catalyses the heterogeneous nucleation of Aβ42 aggregates. It is thus the specific balance between the concentrations of monomeric and fibrillar α-synuclein that determines the outcome of the Aβ42 aggregation reaction. PMID:28698377

  13. The molecular chaperone alphaA-crystallin enhances lens epithelial cell growth and resistance to UVA stress.

    PubMed

    Andley, U P; Song, Z; Wawrousek, E F; Bassnett, S

    1998-11-20

    alphaA-Crystallin (alphaA) is a member of the small heat shock protein (sHSP) family and has the ability to prevent denatured proteins from aggregating in vitro. Lens epithelial cells express relatively low levels of alphaA, but in differentiated fiber cells, alphaA is the most abundant soluble protein. The lenses of alphaA-knock-out mice develop opacities at an early age, implying a critical role for alphaA in the maintenance of fiber cell transparency. However, the function of alpha-crystallin in the lens epithelium is unknown. To investigate the physiological function of alphaA in lens epithelial cells, we used the following two systems: alphaA knock-out (alphaA(-/-)) mouse lens epithelial cells and human lens epithelial cells that overexpress alphaA. The growth rate of alphaA(-/-) mouse lens epithelial cells was reduced by 50% compared with wild type cells. Cell cycle kinetics, measured by fluorescence-activated cell sorter analysis of propidium iodide-stained cells, indicated a relative deficiency of alphaA(-/-) cells in the G2/M phases. Exposure of mouse lens epithelial cells to physiological levels of UVA resulted in an increase in the number of apoptotic cells in the cultures. Four hours after irradiation the fraction of apoptotic cells in the alphaA(-/-) cultures was increased 40-fold over wild type. In cells lacking alphaA, UVA exposure modified F-actin, but actin was protected in cells expressing alphaA. Stably transfected cell lines overexpressing human alphaA were generated by transfecting extended life span human lens epithelial cells with the mammalian expression vector construct pCI-neoalphaA. Cells overexpressing alphaA were resistant to UVA stress, as determined by clonogenic survival. alphaA remained cytoplasmic after exposure to either UVA or thermal stress indicating that, unlike other sHSPs, the protective effect of alphaA was not associated with its relocalization to the nucleus. These results indicate that alphaA has important cellular

  14. Decreased Expression of α-Synuclein, Nogo-A and UCH-L1 in Patients with Schizophrenia: A Preliminary Serum Study

    PubMed Central

    Cetin, İhsan; Turan, Şenol; Sağlam, Tarık; Yıldız, Nazım; Duran, Alaattin

    2017-01-01

    Objective α-synuclein, Nogo-A and Ubiquitin C-terminal hydrolase L1 (UCH-L1) have neuromodulatory roles for human brain. Therefore, abnormalities of these molecules are associated with neuropsychiatric disorders. Although some serum studies in the other disorders have been made, serum study of α-synuclein, Nogo-A and UCH-L1 is not present in patients with schizophrenia and healthy controls. Therefore, our aim was to compare serum levels of α-synuclein, Nogo-A and UCH-L1 of the patients with schizophrenia and healthy controls. Methods Forty-four patients with schizophrenia who is followed by psychotic disorders unit, and 40 healthy control were included in this study. Socio-demographic form and Positive and Negative Syndrome Scale (PANSS) was applied to patients, and sociodemographic form was applied to control group. Fasting bloods were collected and the serum levels of α-synuclein, Nogo-A and UCH-L1 were measured by ELISA method. Results Serum α-synuclein [patient: 12.73 (5.18–31.84) ng/mL; control: 41.77 (15.12–66.98) ng/mL], Nogo-A [patient: 33.58 (3.09–77.26) ng/mL; control: 286.05 (136.56–346.82) ng/mL] and UCH-L1 [patient: 5.26 (1.64–10.87) ng/mL; control: 20.48 (11.01–20.81) ng/mL] levels of the patients with schizophrenia were significianly lower than healthy controls (p<0.001). Conclusion Our study results added new evidence for explaining the etiopathogenesis of schizophrenia on the basis of neurochemical markers. PMID:28539953

  15. p53 mutation and expression in lymphoma.

    PubMed Central

    Adamson, D. J.; Thompson, W. D.; Dawson, A. A.; Bennett, B.; Haites, N. E.

    1995-01-01

    Mutation and abnormal expression of p53 was studied in 38 lymphomas [five Hodgkin's disease and 33 non-Hodgkin's lymphoma (NHL)]. CM1 polyclonal antibody was used to detect overexpression of p53. Three missense mutations were characterised in three cases of NHL after screening exons 5-8 of p53 of all the tumours with single-strand conformation polymorphism (SSCP) analysis. Only two out of three tumours with a missense mutation showed abnormal expression of p53 as measured by CM1. Conversely, seven out of nine tumours with positive CM1 staining had no point mutation demonstrated. Overexpression of p53 in the cases of NHL occurred in three out of twenty four low-grade tumours and five out of nine high-grade tumours (Kiel classification). The results suggest that abnormalities of p53 are commoner in high-grade than low-grade NHL, and that positive immunocytochemistry cannot be used to determine which tumours have mutations of p53. Images Figure 1 Figure 2 PMID:7599045

  16. VHL deletion impairs mammary alveologenesis but is not sufficient for mammary tumorigenesis.

    PubMed

    Seagroves, Tiffany N; Peacock, Danielle L; Liao, Debbie; Schwab, Luciana P; Krueger, Robin; Handorf, Charles R; Haase, Volker H; Johnson, Randall S

    2010-05-01

    Overexpression of hypoxia inducible factor-1 (HIF-1)alpha, which is common in most solid tumors, correlates with poor prognosis and high metastatic risk in breast cancer patients. Because HIF-1alpha protein stability is tightly controlled by the tumor suppressor von Hippel-Lindau (VHL), deletion of VHL results in constitutive HIF-1alpha expression. To determine whether VHL plays a role in normal mammary gland development, and if HIF-1alpha overexpression is sufficient to initiate breast cancer, Vhl was conditionally deleted in the mammary epithelium using the Cre/loxP system. During first pregnancy, loss of Vhl resulted in decreased mammary epithelial cell proliferation and impaired alveolar differentiation; despite these phenotypes, lactation was sufficient to support pup growth. In contrast, in multiparous dams, Vhl(-/-) mammary glands exhibited a progressive loss of alveolar epithelium, culminating in lactation failure. Deletion of Vhl in the epithelium also impacted the mammary stroma, as there was increased microvessel density accompanied by hemorrhage and increased immune cell infiltration. However, deletion of Vhl was not sufficient to induce mammary tumorigenesis in dams bred continuously for up to 24 months of age. Moreover, co-deletion of Hif1a could not rescue the Vhl(-/-)-dependent phenotype as dams were unable to successfully lactate during the first lactation. These results suggest that additional VHL-regulated genes besides HIF1A function to maintain the proliferative and regenerative potential of the breast epithelium.

  17. A blood-brain barrier (BBB) disrupter is also a potent α-synuclein (α-syn) aggregation inhibitor: a novel dual mechanism of mannitol for the treatment of Parkinson disease (PD).

    PubMed

    Shaltiel-Karyo, Ronit; Frenkel-Pinter, Moran; Rockenstein, Edward; Patrick, Christina; Levy-Sakin, Michal; Schiller, Abigail; Egoz-Matia, Nirit; Masliah, Eliezer; Segal, Daniel; Gazit, Ehud

    2013-06-14

    The development of disease-modifying therapy for Parkinson disease has been a main drug development challenge, including the need to deliver the therapeutic agents to the brain. Here, we examined the ability of mannitol to interfere with the aggregation process of α-synuclein in vitro and in vivo in addition to its blood-brain barrier-disrupting properties. Using in vitro studies, we demonstrated the effect of mannitol on α-synuclein aggregation. Although low concentration of mannitol inhibited the formation of fibrils, high concentration significantly decreased the formation of tetramers and high molecular weight oligomers and shifted the secondary structure of α-synuclein from α-helical to a different structure, suggesting alternative potential pathways for aggregation. When administered to a Parkinson Drosophila model, mannitol dramatically corrected its behavioral defects and reduced the amount of α-synuclein aggregates in the brains of treated flies. In the mThy1-human α-synuclein transgenic mouse model, a decrease in α-synuclein accumulation was detected in several brain regions following treatment, suggesting that mannitol promotes α-synuclein clearance in the cell bodies. It appears that mannitol has a general neuroprotective effect in the transgenic treated mice, which includes the dopaminergic system. We therefore suggest mannitol as a basis for a dual mechanism therapeutic agent for the treatment of Parkinson disease.

  18. Interferons alpha and gamma induce p53-dependent and p53-independent apoptosis, respectively.

    PubMed

    Porta, Chiara; Hadj-Slimane, Reda; Nejmeddine, Mohamed; Pampin, Mathieu; Tovey, Michael G; Espert, Lucile; Alvarez, Sandra; Chelbi-Alix, Mounira K

    2005-01-20

    Type I interferon (IFN) enhances the transcription of the tumor suppressor gene p53. To elucidate the molecular mechanism mediating IFN-induced apoptosis, we analysed programmed cell death in response to type I (IFNalpha) or type II (IFNgamma) treatment in relation to p53 status. In two cell lines (MCF-7, SKNSH), IFNalpha, but not IFNgamma, enhanced apoptosis in a p53-dependent manner. Furthermore, only IFNalpha upregulated p53 as well as p53 target genes (Noxa, Mdm2 and CD95). The apoptotic response to IFNalpha decreased in the presence of ZB4, an anti-CD95 antibody, suggesting that CD95 is involved in this process. When p53 was inactivated by the E6 viral protein or the expression of a p53 mutant, IFNalpha-induced apoptosis and p53 target genes upregulation were abrogated. Altogether these results demonstrate that p53 plays a pivotal role in the IFNalpha-induced apoptotic response. IFNalpha-induced PML was unable to recruit p53 into nuclear bodies and its downregulation by siRNA did not alter CD95 expression. In contrast, IFNgamma-induced apoptosis is p53-independent. CD95 and IFN-regulatory factor 1 (IRF1) are directly upregulated by this cytokine. Apoptotic response to IFNgamma is decreased in the presence of ZB4 and strongly diminished by IRF1 siRNA, implicating both CD95 and IRF1 in IFNgamma-induced apoptotic response. Taken together, these results show that in two different cell lines, IFNalpha and IFNgamma, induce p53-dependent -independent apoptosis, respectively.

  19. Nucleolus-derived mediators in oncogenic stress response and activation of p53-dependent pathways.

    PubMed

    Stępiński, Dariusz

    2016-08-01

    Rapid growth and division of cells, including tumor ones, is correlated with intensive protein biosynthesis. The output of nucleoli, organelles where translational machineries are formed, depends on a rate of particular stages of ribosome production and on accessibility of elements crucial for their effective functioning, including substrates, enzymes as well as energy resources. Different factors that induce cellular stress also often lead to nucleolar dysfunction which results in ribosome biogenesis impairment. Such nucleolar disorders, called nucleolar or ribosomal stress, usually affect cellular functioning which in fact is a result of p53-dependent pathway activation, elicited as a response to stress. These pathways direct cells to new destinations such as cell cycle arrest, damage repair, differentiation, autophagy, programmed cell death or aging. In the case of impaired nucleolar functioning, nucleolar and ribosomal proteins mediate activation of the p53 pathways. They are also triggered as a response to oncogenic factor overexpression to protect tissues and organs against extensive proliferation of abnormal cells. Intentional impairment of any step of ribosome biosynthesis which would direct the cells to these destinations could be a strategy used in anticancer therapy. This review presents current knowledge on a nucleolus, mainly in relation to cancer biology, which is an important and extremely sensitive element of the mechanism participating in cellular stress reaction mediating activation of the p53 pathways in order to counteract stress effects, especially cancer development.

  20. A special repertoire of alpha:beta T cells in neonatal mice.

    PubMed Central

    Bogue, M; Candéias, S; Benoist, C; Mathis, D

    1991-01-01

    According to several functional criteria, the mature thymocytes of neonatal and adult mice are distinctly different. We wondered whether these differences in function might have a structural correlate: do neonates have a distinct repertoire of alpha:beta T cells? In this study, we have exploited the power of polymerase chain reaction technology to generate large numbers of T cell receptor sequences from sorted thymocyte populations from newborn and adult mice. The newborn-derived sequences show very few N nucleotide additions, usually the major source of diversity in T cell receptors. Most interestingly, the paucity of N insertions appears to be exaggerated by selection events that operate during T cell differentiation in the thymus. The significance of these results is largely: (i) that they parallel recent findings on the B cell repertoire in neonates, raising questions about the reactivities specified by such a special repertoire; and (ii) that they suggest a means to 'tag' T cells exported perinatally, allowing one to test the premise that autoreactive T cells derive preferentially from the newborn repertoire. Images PMID:1834457

  1. Immunotherapy targeting α-synuclein, with relevance for future treatment of Parkinson's disease and other Lewy body disorders.

    PubMed

    Lindström, Veronica; Ihse, Elisabet; Fagerqvist, Therese; Bergström, Joakim; Nordström, Eva; Möller, Christer; Lannfelt, Lars; Ingelsson, Martin

    2014-01-01

    Immunotherapy targeting α-synuclein has evolved as a potential therapeutic strategy for neurodegenerative diseases, such as Parkinson's disease, and initial studies on cellular and animal models have shown promising results. α-synuclein vaccination of transgenic mice reduced the number of brain inclusions, whereas passive immunization studies demonstrated that antibodies against the C-terminus of α-synuclein can pass the blood-brain barrier and affect the pathology. In addition, preliminary evidence suggests that transgenic mice treated with an antibody directed against α-synuclein oligomers/protofibrils resulted in reduced levels of such species in the CNS. The underlying mechanisms of immunotherapy are not yet fully understood, but may include antibody-mediated clearance of pre-existing aggregates, prevention of protein propagation between cells and microglia-dependent protein clearance. Thus, immunotherapy targeting α-synuclein holds promise, but needs to be further developed as a future disease-modifying treatment in Parkinson's disease and other α-synucleinopathies.

  2. The nuclear accumulation of alpha-synuclein is mediated by importin alpha and promotes neurotoxicity by accelerating the cell cycle.

    PubMed

    Ma, Kai-Li; Song, Lian-Kun; Yuan, Yu-He; Zhang, Ying; Han, Ning; Gao, Kai; Chen, Nai-Hong

    2014-07-01

    α-Synuclein (α-syn), a 14 kDa pre-synaptic protein, is widely involved in the Parkinson's disease (PD) pathogenesis. Recent studies have shown that the nuclear accumulation of α-syn might have a toxic effect. The main purpose of the present study was to explore which amino acid residues in α-syn are associated with its nuclear accumulation, the molecule(s) mediated the nuclear import of α-syn, and the role of α-syn accumulated in the nucleus. It has been noted that the nuclear import of α-syn may be mediated by importin α and that both the amino acid residues 1-60 and 103-140 of α-syn were indispensable for its nuclear import. After imported into the nucleus, the accumulated α-syn played a toxic role in both the PC12 cells and the C57 mice. Furthermore, α-syn-nuclear localization signal-injected mice showed behavioral symptoms associated with PD. Further studies performed in vitro showed that the toxicity of α-syn in the nucleus might be due to an interference of the cell cycle. Thus, it can be concluded that α-syn can accumulate in nucleus, which is mediated by importin α, and promote neurotoxicity by accelerating the cell cycle. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Sirt1 overexpression suppresses fluoride-induced p53 acetylation to alleviate fluoride toxicity in ameloblasts responsible for enamel formation.

    PubMed

    Suzuki, Maiko; Ikeda, Atsushi; Bartlett, John D

    2018-03-01

    Low-dose fluoride is an effective caries prophylactic, but high-dose fluoride is an environmental health hazard that causes skeletal and dental fluorosis. Treatments to prevent fluorosis and the molecular pathways responsive to fluoride exposure remain to be elucidated. Previously we showed that fluoride activates SIRT1 as an adaptive response to protect cells. Here, we demonstrate that fluoride induced p53 acetylation (Ac-p53) [Lys379], which is a SIRT1 deacetylation target, in ameloblast-derived LS8 cells in vitro and in enamel organ in vivo. Here we assessed SIRT1 function on fluoride-induced Ac-p53 formation using CRISPR/Cas9-mediated Sirt1 knockout (LS8 Sirt/KO ) cells or CRISPR/dCas9/SAM-mediated Sirt1 overexpressing (LS8 Sirt1/over ) cells. NaF (5 mM) induced Ac-p53 formation and increased cell cycle arrest via Cdkn1a/p21 expression in Wild-type (WT) cells. However, fluoride-induced Ac-p53 was suppressed by the SIRT1 activator resveratrol (50 µM). Without fluoride, Ac-p53 persisted in LS8 Sirt/KO cells, whereas it decreased in LS8 Sirt1/over . Fluoride-induced Ac-p53 formation was also suppressed in LS8 Sirt1/over cells. Compared to WT cells, fluoride-induced Cdkn1a/p21 expression was elevated in LS8 Sirt/KO and these cells were more susceptible to fluoride-induced growth inhibition. In contrast, LS8 Sirt1/over cells were significantly more resistant. In addition, fluoride-induced cytochrome-c release and caspase-3 activation were suppressed in LS8 Sirt1/over cells. Fluoride induced expression of the DNA double strand break marker γH2AX in WT cells and this was augmented in LS8 Sirt1/KO cells, but was attenuated in LS8 Sirt1/over cells. Our results suggest that SIRT1 deacetylates Ac-p53 to mitigate fluoride-induced cell growth inhibition, mitochondrial damage, DNA damage and apoptosis. This is the first report implicating Ac-p53 in fluoride toxicity.

  4. Tetraspanin CD151 regulates alpha6beta1 integrin adhesion strengthening

    NASA Technical Reports Server (NTRS)

    Lammerding, Jan; Kazarov, Alexander R.; Huang, Hayden; Lee, Richard T.; Hemler, Martin E.

    2003-01-01

    The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including alpha6beta1. To probe strength of alpha6beta1-dependent adhesion to laminin-1, defined forces (0-1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells. For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time. In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening. Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-alpha6 antibody were all unaffected by CD151 mutation. Hence, CD151 plays a key role in selectively strengthening alpha6beta1 integrin-mediated adhesion to laminin-1.

  5. Study of T53 engine vibration

    NASA Technical Reports Server (NTRS)

    Walter, T. J.

    1978-01-01

    Vibration characteristics for overhauled T53 engines, including rejection rate, principal sources of vibration, and normal procedures taken by the overhaul center to reduce engine vibration are summarized. Analytical and experimental data were compared to determine the engine's dynamic response to unbalance forces with results showing that the engine operates through bending critical speeds. Present rigid rotor balancing techniques are incapable of compensating for the flexible rotor unbalance. A comparison of typical test cell and aircraft vibration levels disclosed significant differences in the engine's dynamic response. A probable spline shift phenomenon was uncovered and investigated. Action items to control costs and reduce vibration levels were identified from analytical and experimental studies.

  6. The critical role of Nramp1 in degrading α-synuclein oligomers in microglia under iron overload condition.

    PubMed

    Wu, Kuo-Chen; Liou, Horng-Huei; Kao, Yu-Han; Lee, Chih-Yu; Lin, Chun-Jung

    2017-08-01

    Oligomeric α-synuclein is a key mediator in the pathogenesis of Parkinson's disease (PD) and is mainly cleared by autophagy-lysosomal pathway, whose dysfunction results in the accumulation and cell-to-cell transmission of α-synuclein. In this study, concomitant with the accumulation of iron and oligomeric α-synuclein, higher expression of a lysosomal iron transporter, natural resistance-associated macrophage protein-1 (Nramp1), was observed in microglia in post-mortem striatum of sporadic PD patients. Using Nramp1-deficient macrophage (RAW264.7) and microglial (BV-2) cells as in-vitro models, iron exposure significantly reduced the degradation rate of the administered human α-synuclein oligomers, which can be restored by the expression of the wild-type, but not mutant (D543N), Nramp1. Likewise, under iron overload condition, mice with functional Nramp1 (DBA/2 and C57BL/6 congenic mice carrying functional Nramp1) had a better ability to degrade infused human α-synuclein oligomers than mice with nonfunctional Nramp1 (C57BL/6) in the brain and microglia. The interplay between iron and Nramp1 exhibited parallel effects on the clearance of α-synuclein and the activity of lysosomal cathepsin D in vitro and in vivo. Collectively, these findings suggest that the function of Nramp1 contributes to microglial degradation of oligomeric α-synuclein under iron overload condition and may be implicated in the pathogenesis of PD. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. The Cleavage Effect of Mesenchymal Stem Cell and Its Derived Matrix Metalloproteinase‐2 on Extracellular α‐Synuclein Aggregates in Parkinsonian Models

    PubMed Central

    Oh, Se Hee; Kim, Ha Na; Park, Hyun Jung; Shin, Jin Young; Kim, Dong Yeol

    2016-01-01

    Abstract Ample evidence has suggested that extracellular α‐synuclein aggregates would play key roles in the pathogenesis and progression of Parkinsonian disorders (PDs). In the present study, we investigated whether mesenchymal stem cells (MSCs) and their derived soluble factors could exert neuroprotective effects via proteolysis of extracellular α‐synuclein. When preformed α‐synuclein aggregates were incubated with MSC‐conditioned medium, α‐synuclein aggregates were disassembled, and insoluble and oligomeric forms of α‐synuclein were markedly decreased, thus leading to a significant increase in neuronal viability. In an animal study, MSC or MSC‐conditioned medium treatment decreased the expression of α‐synuclein oligomers and the induction of pathogenic α‐synuclein with an attenuation of apoptotic cell death signaling. Furthermore, we identified that matrix metalloproteinase‐2 (MMP‐2), a soluble factor derived from MSCs, played an important role in the degradation of extracellular α‐synuclein. Our data demonstrated that MSCs and their derived MMP‐2 exert neuroprotective properties through proteolysis of aggregated α‐synuclein in PD‐related microenvironments. Stem Cells Translational Medicine 2017;6:949–961 PMID:28297586

  8. In vivo regulation of gene transcription by alpha- and gamma-Tocopherol in murine T lymphocytes

    USDA-ARS?s Scientific Manuscript database

    Of the 8 different analogues (alpha-, beta-, gamma-, delta-tocopherols and tocotrienols) designated as vitamin E, alpha-tocopherol (a-T) has been mostly studied, together with gamma-tocopherol (g-T) which is abundant in the US diet. We compared the effect of dietary supplementation with adequate or ...

  9. Phospholipase D1 downregulation by α-synuclein: Implications for neurodegeneration in Parkinson's disease.

    PubMed

    Conde, Melisa A; Alza, Natalia P; Iglesias González, Pablo A; Scodelaro Bilbao, Paola G; Sánchez Campos, Sofía; Uranga, Romina M; Salvador, Gabriela A

    2018-06-01

    We have previously shown that phospholipase D (PLD) pathways have a role in neuronal degeneration; in particular, we found that PLD activation is associated with synaptic injury induced by oxidative stress. In the present study, we investigated the effect of α-synuclein (α-syn) overexpression on PLD signaling. Wild Type (WT) α-syn was found to trigger the inhibition of PLD1 expression as well as a decrease in ERK1/2 phosphorylation and expression levels. Moreover, ERK1/2 subcellular localization was shown to be modulated by WT α-syn in a PLD1-dependent manner. Indeed, PLD1 inhibition was found to alter the neurofilament network and F-actin distribution regardless of the presence of WT α-syn. In line with this, neuroblastoma cells expressing WT α-syn exhibited a degenerative-like phenotype characterized by a marked reduction in neurofilament light subunit (NFL) expression and the rearrangement of the F-actin organization, compared with either the untransfected or the empty vector-transfected cells. The gain of function of PLD1 through the overexpression of its active form had the effect of restoring NFL expression in WT α-syn neurons. Taken together, our findings reveal an unforeseen role for α-syn in PLD regulation: PLD1 downregulation may constitute an early mechanism in the initial stages of WT α-syn-triggered neurodegeneration. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Suppression of p53 Activity through the Cooperative Action of Ski and Histone Deacetylase SIRT1*

    PubMed Central

    Inoue, Yasumichi; Iemura, Shun-ichiro; Natsume, Tohru; Miyazawa, Keiji; Imamura, Takeshi

    2011-01-01

    Ski was originally identified as an oncogene based on the fact that Ski overexpression transformed chicken and quail embryo fibroblasts. Consistent with these proposed oncogenic roles, Ski is overexpressed in various human tumors. However, whether and how Ski functions in mammalian tumorigenesis has not been fully investigated. Here, we show that Ski interacts with p53 and attenuates the biological functions of p53. Ski overexpression attenuated p53-dependent transactivation, whereas Ski knockdown enhanced the transcriptional activity of p53. Interestingly, Ski bound to the histone deacetylase SIRT1 and stabilized p53-SIRT1 interaction to promote p53 deacetylation, which subsequently decreased the DNA binding activity of p53. Consistent with the ability of Ski to inactivate p53, overexpressing Ski desensitized cells to genotoxic drugs and Nutlin-3, a small-molecule antagonist of Mdm2 that stabilizes p53 and activates the p53 pathway, whereas knocking down Ski increased the cellular sensitivity to these agents. These results indicate that Ski negatively regulates p53 and suggest that the p53-Ski-SIRT1 axis is an attractive target for cancer therapy. PMID:21149449

  11. Polo-like Kinase 2 (PLK2) Phosphorylates α-Synuclein at Serine 129 in Central Nervous System*S⃞

    PubMed Central

    Inglis, Kelly J.; Chereau, David; Brigham, Elizabeth F.; Chiou, San-San; Schöbel, Susanne; Frigon, Normand L.; Yu, Mei; Caccavello, Russell J.; Nelson, Seth; Motter, Ruth; Wright, Sarah; Chian, David; Santiago, Pamela; Soriano, Ferdie; Ramos, Carla; Powell, Kyle; Goldstein, Jason M.; Babcock, Michael; Yednock, Ted; Bard, Frederique; Basi, Guriqbal S.; Sham, Hing; Chilcote, Tamie J.; McConlogue, Lisa; Griswold-Prenner, Irene; Anderson, John P.

    2009-01-01

    Several neurological diseases, including Parkinson disease and dementia with Lewy bodies, are characterized by the accumulation of α-synuclein phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for this phosphorylation have been the subject of intense investigation. Here we submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible kinase or SNK) is a principle contributor to α-synuclein phosphorylation at Ser-129 in neurons. PLK2 directly phosphorylates α-synuclein at Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases inhibited α-synuclein phosphorylation both in primary cortical cell cultures and in mouse brain in vivo. Finally, specific knockdown of PLK2 expression by transduction with short hairpin RNA constructs or by knock-out of the plk2 gene reduced p-Ser-129 levels. These results indicate that PLK2 plays a critical role in α-synuclein phosphorylation in central nervous system. PMID:19004816

  12. A case of alpha-fetoprotein-producing esophageal adenocarcinoma.

    PubMed

    Chen, Yi-Yu; Hsu, Wen-Hung; Hu, Huang-Ming; Wu, Deng-Chyang; Lin, Wen-Yi

    2013-02-01

    Alpha-fetoprotein is a well-known tumor marker in the screening and follow-up of hepatocellular carcinoma. In Taiwanese society, a high prevalence of hepatitis and hepatoma and elevation of alpha-fetoprotein associated with liver function impairment usually suggested clinics undertake further examination for liver or genital tumor. We report the case of 45-year-old man who was found to have an alpha-fetoprotein-producing esophageal adenocarcinoma with an initial presentation of liver function impairment and rapid elevation of alpha-fetoprotein. Esophageal cancer was diagnosed via endoscope and a biopsy proved the presence of adenocarcinoma. A small endoscopic biopsy specimen failed to identify the alpha-fetoprotein positive tumor cell. Esophagectomy was performed and histopathological study of surgical specimen revealed grade II adenocarcinoma with regional metastatic lymphadenopathy. Immunohistochemical study was focal positive for alpha-fetoprotein. Serum alpha-fetoprotein declined transiently after esophagectomy and fluctuation of alpha-fetoprotein level was noted during the treatment with adjuvant chemotherapy. Finally, 19 months after the operation, the patient died due to multiple organ metastases with multiple organ failure. Thus, a small specimen for upper endoscopy may not be sufficient in the presence of alpha-fetoprotein-producing adenocarcinoma. Monitoring of serum alpha-fetoprotein may be useful in the evaluation and follow-up of esophageal alpha-fetoprotein-producing adenocarcinoma. Copyright © 2012. Published by Elsevier B.V.

  13. Modulation of α-synuclein fibrillization by ring-fused 2-pyridones: templation and inhibition involve oligomers with different structure.

    PubMed

    Horvath, Istvan; Sellstedt, Magnus; Weise, Christoph; Nordvall, Lina-Maria; Krishna Prasad, G; Olofsson, Anders; Larsson, Göran; Almqvist, Fredrik; Wittung-Stafshede, Pernilla

    2013-04-15

    In a recent study we discovered that a ring-fused 2-pyridone compound triggered fibrillization of a key protein in Parkinson's disease, α-synuclein. To reveal how variations in compound structure affect protein aggregation, we now prepared a number of strategic analogs and tested their effects on α-synuclein amyloid fiber formation in vitro. We find that, in contrast to the earlier templating effect, some analogs inhibit α-synuclein fibrillization. For both templating and inhibiting compounds, the key species formed in the reactions are α-synuclein oligomers that contain compound. Despite similar macroscopic appearance, the templating and inhibiting oligomers are distinctly different in secondary structure content. When the inhibitory oligomers are added in seed amounts, they inhibit fresh α-synuclein aggregation reactions. Our study demonstrates that small chemical changes to the same central fragment can result in opposite effects on protein aggregation. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. T-DM1, a novel antibody–drug conjugate, is highly effective against primary HER2 overexpressing uterine serous carcinoma in vitro and in vivo

    PubMed Central

    English, Diana P; Bellone, Stefania; Schwab, Carlton L; Bortolomai, Ileana; Bonazzoli, Elena; Cocco, Emiliano; Buza, Natalia; Hui, Pei; Lopez, Salvatore; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E; Rutherford, Thomas J; Santin, Alessandro D

    2014-01-01

    Amplification of c-erbB2 has been reported in over 30% of uterine serous carcinoma (USC) and found to confer poor survival because of high proliferation and increased resistance to therapy. In this study, we evaluated for the first time Trastuzumab emtansine (T-DM1), a novel antibody–drug conjugate, against multiple epidermal growth factor receptor-2 (HER2)-positive USC cells in vitro followed by developing a supportive in vivo model. Fifteen primary USC cell lines were assessed by immunohistochemistry (IHC) and flow cytometry for HER2 protein expression. C-erbB2 gene amplification was evaluated using fluorescent in situ hybridization. Sensitivity to T-DM1 and trastuzumab (T)-induced antibody-dependent cell-mediated cytotoxicity was evaluated in 5-h chromium release assays. T-DM1 and T cytostatic and apoptotic activities were evaluated using flow-cytometry-based proliferation assays. In vivo activity of T-DM1 versus T in USC xenografts in SCID mice was also evaluated. High levels of HER2 protein overexpression and HER2 gene amplification were detected in 33% of USC cell lines. T-DM1 was considerably more effective than trastuzumab in inhibiting cell proliferation and in causing apoptosis (P = 0.004) of USC showing HER2 overexpression. Importantly, T-DM1 was highly active at reducing tumor formation in vivo in USC xenografts overexpressing HER2 (P = 0.04) and mice treated with TDM-1 had significantly longer survival when compared to T-treated mice and control mice (P ≤ 0.0001). T-DM1 shows promising antitumor effect in HER2-positive USC cell lines and USC xenografts and its activity is significantly higher when compared to T. T-DM1 may represent a novel treatment option for HER2-positive USC patients with disease refractory to trastuzumab and traditional chemotherapy. PMID:24890382

  15. Mechanisms of alpha-Synuclein Aggregation and Toxicity

    DTIC Science & Technology

    2006-09-01

    Zhang, P. St George- [36] Y . Zhang, J. Gao, K.K. Chung, H. Huang, V.L. Dawson, T.M. Hyslop , Mutation of the conserved N-terminal cysteine (Cys92) of...pathies. The incidence of NFTs in Parkinson’s disease is much greater than in an age-matched population (Boller et al., 1980 ). Tau-immunoreactive Lewy...nervous system. J. Cell Biol. 101, 1371–1378. Boller, F., Mizutani, T., Roessmann, U., Gambetti, P., 1980 . Parkinson disease, dementia, and Alzheimer

  16. Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance.

    PubMed

    Xu, Sheng; Sun, Bin; Wang, Rong; He, Jia; Xia, Bing; Xue, Yong; Wang, Ren

    2017-08-19

    The phytoremediation by using of green plants in the removal of environmental pollutant is an environment friendly, green technology that is cost effective and energetically inexpensive. By using Agrobacterium-mediated gene transfer, we generated transgenic Arabidopsis plants ectopically expressing mercuric transport protein gene (merT) from Pseudomonas alcaligenes. Compared with wild-type (WT) plants, overexpressing PamerT in Arabidopsis enhanced the tolerance to HgCl 2 . Further results showed that the enhanced total activities or corresponding transcripts of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (POD) were observed in transgenic Arabidopsis under HgCl 2 stress. These results were confirmed by the alleviation of oxidative damage, as indicated by the decrease of thiobarbituric acid reactive substances (TBARS) contents and reactive oxygen species (ROS) accumulation. In addition, localization analysis of PaMerT in Arabidopsis protoplast showed that it is likely to be associated with vacuole. In all, PamerT increased mercury (Hg) tolerance in transgenic Arabidopsis, and decreased production of Hg-induced ROS, thereby protecting plants from oxidative damage. The present study has provided further evidence that bacterial MerT plays an important role in the plant tolerance to HgCl 2 and in reducing the production of ROS induced by HgCl 2 . Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Structural and functional properties of prefibrillar α-synuclein oligomers.

    PubMed

    Pieri, Laura; Madiona, Karine; Melki, Ronald

    2016-04-14

    The deposition of fibrillar alpha-synuclein (α-syn) within inclusions (Lewy bodies and Lewy neurites) in neurons and glial cells is a hallmark of synucleinopathies. α-syn populates a variety of assemblies ranging from prefibrillar oligomeric species to fibrils whose specific contribution to neurodegeneration is still unclear. Here, we compare the specific structural and biological properties of distinct soluble prefibrillar α-syn oligomers formed either spontaneously or in the presence of dopamine and glutaraldehyde. We show that both on-fibrillar assembly pathway and distinct dopamine-mediated and glutaraldehyde-cross-linked α-syn oligomers are only slightly effective in perturbing cell membrane integrity and inducing cytotoxicity, while mature fibrils exhibit the highest toxicity. In contrast to low-molecular weight and unstable oligomers, large stable α-syn oligomers seed the aggregation of soluble α-syn within reporter cells although to a lesser extent than mature α-syn fibrils. These oligomers appear elongated in shape. Our findings suggest that α-syn oligomers represent a continuum of species ranging from unstable low molecular weight particles to mature fibrils via stable elongated oligomers composed of more than 15 α-syn monomers that possess seeding capacity.

  18. Structural and functional properties of prefibrillar α-synuclein oligomers

    PubMed Central

    Pieri, Laura; Madiona, Karine; Melki, Ronald

    2016-01-01

    The deposition of fibrillar alpha-synuclein (α-syn) within inclusions (Lewy bodies and Lewy neurites) in neurons and glial cells is a hallmark of synucleinopathies. α-syn populates a variety of assemblies ranging from prefibrillar oligomeric species to fibrils whose specific contribution to neurodegeneration is still unclear. Here, we compare the specific structural and biological properties of distinct soluble prefibrillar α-syn oligomers formed either spontaneously or in the presence of dopamine and glutaraldehyde. We show that both on-fibrillar assembly pathway and distinct dopamine-mediated and glutaraldehyde-cross-linked α-syn oligomers are only slightly effective in perturbing cell membrane integrity and inducing cytotoxicity, while mature fibrils exhibit the highest toxicity. In contrast to low-molecular weight and unstable oligomers, large stable α-syn oligomers seed the aggregation of soluble α-syn within reporter cells although to a lesser extent than mature α-syn fibrils. These oligomers appear elongated in shape. Our findings suggest that α-syn oligomers represent a continuum of species ranging from unstable low molecular weight particles to mature fibrils via stable elongated oligomers composed of more than 15 α-syn monomers that possess seeding capacity. PMID:27075649

  19. Cold denaturation of α-synuclein amyloid fibrils.

    PubMed

    Ikenoue, Tatsuya; Lee, Young-Ho; Kardos, József; Saiki, Miyu; Yagi, Hisashi; Kawata, Yasushi; Goto, Yuji

    2014-07-21

    Although amyloid fibrils are associated with numerous pathologies, their conformational stability remains largely unclear. Herein, we probe the thermal stability of various amyloid fibrils. α-Synuclein fibrils cold-denatured to monomers at 0-20 °C and heat-denatured at 60-110 °C. Meanwhile, the fibrils of β2-microglobulin, Alzheimer's Aβ1-40/Aβ1-42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in stability at low temperature. A comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in fibril cores contributed to the cold denaturation of α-synuclein fibrils. We propose that although cold-denaturation is common to both native proteins and misfolded fibrillar states, the main-chain dominated amyloid structures may explain amyloid-specific cold denaturation arising from the unfavorable burial of charged side-chains in fibril cores. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. S100A4 interacts with p53 in the nucleus and promotes p53 degradation.

    PubMed

    Orre, L M; Panizza, E; Kaminskyy, V O; Vernet, E; Gräslund, T; Zhivotovsky, B; Lehtiö, J

    2013-12-05

    S100A4 is a small calcium-binding protein that is commonly overexpressed in a range of different tumor types, and it is widely accepted that S100A4 has an important role in the process of cancer metastasis. In vitro binding assays has shown that S100A4 interacts with the tumor suppressor protein p53, indicating that S100A4 may have additional roles in tumor development. In the present study, we show that endogenous S100A4 and p53 interact in complex samples, and that the interaction increases after inhibition of MDM2-dependent p53 degradation using Nutlin-3A. Further, using proximity ligation assay, we show that the interaction takes place in the cell nucleus. S100A4 knockdown experiments in two p53 wild-type cell lines, A549 and HeLa, resulted in stabilization of p53 protein, indicating that S100A4 is promoting p53 degradation. Finally, we demonstrate that S100A4 knockdown leads to p53-dependent cell cycle arrest and increased cisplatin-induced apoptosis. Thus, our data add a new layer to the oncogenic properties of S100A4 through its inhibition of p53-dependent processes.