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Sample records for mutation impairs trafficking

  1. Dynamin-2 mutations linked to Centronuclear Myopathy impair actin-dependent trafficking in muscle cells.

    PubMed

    González-Jamett, Arlek M; Baez-Matus, Ximena; Olivares, María José; Hinostroza, Fernando; Guerra-Fernández, Maria José; Vasquez-Navarrete, Jacqueline; Bui, Mai Thao; Guicheney, Pascale; Romero, Norma Beatriz; Bevilacqua, Jorge A; Bitoun, Marc; Caviedes, Pablo; Cárdenas, Ana M

    2017-07-04

    Dynamin-2 is a ubiquitously expressed GTP-ase that mediates membrane remodeling. Recent findings indicate that dynamin-2 also regulates actin dynamics. Mutations in dynamin-2 cause dominant centronuclear myopathy (CNM), a congenital myopathy characterized by progressive weakness and atrophy of skeletal muscles. However, the muscle-specific roles of dynamin-2 affected by these mutations remain elusive. Here we show that, in muscle cells, the GTP-ase activity of dynamin-2 is involved in de novo actin polymerization as well as in actin-mediated trafficking of the glucose transporter GLUT4. Expression of dynamin-2 constructs carrying CNM-linked mutations disrupted the formation of new actin filaments as well as the stimulus-induced translocation of GLUT4 to the plasma membrane. Similarly, mature muscle fibers isolated from heterozygous knock-in mice that harbor the dynamin-2 mutation p.R465W, an animal model of CNM, exhibited altered actin organization, reduced actin polymerization and impaired insulin-induced translocation of GLUT4 to the sarcolemma. Moreover, GLUT4 displayed aberrant perinuclear accumulation in biopsies from CNM patients carrying dynamin-2 mutations, further suggesting trafficking defects. These results suggest that dynamin-2 is a key regulator of actin dynamics and GLUT4 trafficking in muscle cells. Our findings also support a model in which impairment of actin-dependent trafficking contributes to the pathological mechanism in dynamin-2-associated CNM.

  2. Impaired prosaposin lysosomal trafficking in frontotemporal lobar degeneration due to progranulin mutations

    PubMed Central

    Zhou, Xiaolai; Sun, Lirong; Bracko, Oliver; Choi, Ji Whae; Jia, Yan; Nana, Alissa L.; Brady, Owen Adam; Hernandez, Jean C. Cruz; Nishimura, Nozomi; Seeley, William W.; Hu, Fenghua

    2017-01-01

    Haploinsufficiency of progranulin (PGRN) due to mutations in the granulin (GRN) gene causes frontotemporal lobar degeneration (FTLD), and complete loss of PGRN leads to a lysosomal storage disorder, neuronal ceroid lipofuscinosis (NCL). Accumulating evidence suggests that PGRN is essential for proper lysosomal function, but the precise mechanisms involved are not known. Here, we show that PGRN facilitates neuronal uptake and lysosomal delivery of prosaposin (PSAP), the precursor of saposin peptides that are essential for lysosomal glycosphingolipid degradation. We found reduced levels of PSAP in neurons both in mice deficient in PGRN and in human samples from FTLD patients due to GRN mutations. Furthermore, mice with reduced PSAP expression demonstrated FTLD-like pathology and behavioural changes. Thus, our data demonstrate a role of PGRN in PSAP lysosomal trafficking and suggest that impaired lysosomal trafficking of PSAP is an underlying disease mechanism for NCL and FTLD due to GRN mutations. PMID:28541286

  3. Leukoencephalopathy-causing CLCN2 mutations are associated with impaired Cl(-) channel function and trafficking.

    PubMed

    Gaitán-Peñas, Héctor; Apaja, Pirjo M; Arnedo, Tanit; Castellanos, Aida; Elorza-Vidal, Xabier; Soto, David; Gasull, Xavier; Lukacs, Gergely L; Estévez, Raúl

    2017-09-14

    Mutations in CLCN2 have been recently identified in patients suffering from a type of leukoencephalopathy involving intramyelinic oedema. Here, we characterised most of these mutations, which reduce the function of the chloride channel ClC-2 and impair its plasma membrane (PM) expression. Detailed biochemical and electrophysiological analyses of the Ala500Val mutation revealed that defective gating and increased cellular and PM turnover contributed to defective A500V-ClC-2 functional expression. Co-expression of the adhesion molecule GlialCAM, which forms a tertiary complex with ClC-2 and megalencephalic leukoencephalopathy with subcortical cyst 1 (MLC1), rescued the functional expression of the mutant by modifying its gating properties. GlialCAM also restored the PM levels of the channel by impeding its turnover at the PM. This rescue required ClC-2 localisation to cell-cell junctions, since a GlialCAM mutant with compromised junctional localisation failed to rescue the impaired stability of mutant ClC-2 at the PM. Wild-type, but not mutant ClC-2 was also stabilised by MLC1 overexpression. We suggest that leukodystrophy-causing CLCN2 mutations reduce the functional expression of ClC-2, which is partly counteracted by GlialCAM/MLC1-mediated increase in the gating and stability of the channel. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  4. Slitrk Missense Mutations Associated with Neuropsychiatric Disorders Distinctively Impair Slitrk Trafficking and Synapse Formation

    PubMed Central

    Kang, Hyeyeon; Han, Kyung Ah; Won, Seoung Youn; Kim, Ho Min; Lee, Young-Ho; Ko, Jaewon; Um, Ji Won

    2016-01-01

    Slit- and Trk-like (Slitrks) are a six-member family of synapse organizers that control excitatory and inhibitory synapse formation by forming trans-synaptic adhesions with LAR receptor protein tyrosine phosphatases (PTPs). Intriguingly, genetic mutations of Slitrks have been associated with a multitude of neuropsychiatric disorders. However, nothing is known about the neuronal and synaptic consequences of these mutations. Here, we report the structural and functional effects on synapses of various rare de novo mutations identified in patients with schizophrenia or Tourette syndrome. A number of single amino acid substitutions in Slitrk1 (N400I or T418S) or Slitrk4 (V206I or I578V) reduced their surface expression levels. These substitutions impaired glycosylation of Slitrks expressed in HEK293T cells, caused retention of Slitrks in the endoplasmic reticulum and cis-Golgi compartment in COS-7 cells and neurons, and abolished Slitrk binding to PTPδ. Furthermore, these substitutions eliminated the synapse-inducing activity of Slitrks, abolishing their functional effects on synapse density in cultured neurons. Strikingly, a valine-to-methionine mutation in Slitrk2 (V89M) compromised synapse formation activity in cultured neuron, without affecting surface transport, expression, or synapse-inducing activity in coculture assays. Similar deleterious effects were observed upon introduction of the corresponding valine-to-methionine mutation into Slitrk1 (V85M), suggesting that this conserved valine residue plays a key role in maintaining the synaptic functions of Slitrks. Collectively, these data indicate that inactivation of distinct cellular mechanisms caused by specific Slitrk dysfunctions may underlie Slitrk-associated neuropsychiatric disorders in humans, and provide a robust cellular readout for the development of knowledge-based therapies. PMID:27812321

  5. Slitrk Missense Mutations Associated with Neuropsychiatric Disorders Distinctively Impair Slitrk Trafficking and Synapse Formation.

    PubMed

    Kang, Hyeyeon; Han, Kyung Ah; Won, Seoung Youn; Kim, Ho Min; Lee, Young-Ho; Ko, Jaewon; Um, Ji Won

    2016-01-01

    Slit- and Trk-like (Slitrks) are a six-member family of synapse organizers that control excitatory and inhibitory synapse formation by forming trans-synaptic adhesions with LAR receptor protein tyrosine phosphatases (PTPs). Intriguingly, genetic mutations of Slitrks have been associated with a multitude of neuropsychiatric disorders. However, nothing is known about the neuronal and synaptic consequences of these mutations. Here, we report the structural and functional effects on synapses of various rare de novo mutations identified in patients with schizophrenia or Tourette syndrome. A number of single amino acid substitutions in Slitrk1 (N400I or T418S) or Slitrk4 (V206I or I578V) reduced their surface expression levels. These substitutions impaired glycosylation of Slitrks expressed in HEK293T cells, caused retention of Slitrks in the endoplasmic reticulum and cis-Golgi compartment in COS-7 cells and neurons, and abolished Slitrk binding to PTPδ. Furthermore, these substitutions eliminated the synapse-inducing activity of Slitrks, abolishing their functional effects on synapse density in cultured neurons. Strikingly, a valine-to-methionine mutation in Slitrk2 (V89M) compromised synapse formation activity in cultured neuron, without affecting surface transport, expression, or synapse-inducing activity in coculture assays. Similar deleterious effects were observed upon introduction of the corresponding valine-to-methionine mutation into Slitrk1 (V85M), suggesting that this conserved valine residue plays a key role in maintaining the synaptic functions of Slitrks. Collectively, these data indicate that inactivation of distinct cellular mechanisms caused by specific Slitrk dysfunctions may underlie Slitrk-associated neuropsychiatric disorders in humans, and provide a robust cellular readout for the development of knowledge-based therapies.

  6. AP1S3 Mutations Are Associated with Pustular Psoriasis and Impaired Toll-like Receptor 3 Trafficking

    PubMed Central

    Setta-Kaffetzi, Niovi; Simpson, Michael A.; Navarini, Alexander A.; Patel, Varsha M.; Lu, Hui-Chun; Allen, Michael H.; Duckworth, Michael; Bachelez, Hervé; Burden, A. David; Choon, Siew-Eng; Griffiths, Christopher E.M.; Kirby, Brian; Kolios, Antonios; Seyger, Marieke M.B.; Prins, Christa; Smahi, Asma; Trembath, Richard C.; Fraternali, Franca; Smith, Catherine H.; Barker, Jonathan N.; Capon, Francesca

    2014-01-01

    Adaptor protein complex 1 (AP-1) is an evolutionary conserved heterotetramer that promotes vesicular trafficking between the trans-Golgi network and the endosomes. The knockout of most murine AP-1 complex subunits is embryonically lethal, so the identification of human disease-associated alleles has the unique potential to deliver insights into gene function. Here, we report two founder mutations (c.11T>G [p.Phe4Cys] and c.97C>T [p.Arg33Trp]) in AP1S3, the gene encoding AP-1 complex subunit σ1C, in 15 unrelated individuals with a severe autoinflammatory skin disorder known as pustular psoriasis. Because the variants are predicted to destabilize the 3D structure of the AP-1 complex, we generated AP1S3-knockdown cell lines to investigate the consequences of AP-1 deficiency in skin keratinocytes. We found that AP1S3 silencing disrupted the endosomal translocation of the innate pattern-recognition receptor TLR-3 (Toll-like receptor 3) and resulted in a marked inhibition of downstream signaling. These findings identify pustular psoriasis as an autoinflammatory phenotype caused by defects in vesicular trafficking and demonstrate a requirement of AP-1 for Toll-like receptor homeostasis. PMID:24791904

  7. A dominant mutation in Snap25 causes impaired vesicle trafficking, sensorimotor gating, and ataxia in the blind-drunk mouse

    PubMed Central

    Jeans, Alexander F.; Oliver, Peter L.; Johnson, Reuben; Capogna, Marco; Vikman, Jenny; Molnár, Zoltán; Babbs, Arran; Partridge, Christopher J.; Salehi, Albert; Bengtsson, Martin; Eliasson, Lena; Rorsman, Patrik; Davies, Kay E.

    2007-01-01

    The neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is essential for synaptic vesicle exocytosis, but its study has been limited by the neonatal lethality of murine SNARE knockouts. Here, we describe a viable mouse line carrying a mutation in the b-isoform of neuronal SNARE synaptosomal-associated protein of 25 kDa (SNAP-25). The causative I67T missense mutation results in increased binding affinities within the SNARE complex, impaired exocytotic vesicle recycling and granule exocytosis in pancreatic β-cells, and a reduction in the amplitude of evoked cortical excitatory postsynaptic potentials. The mice also display ataxia and impaired sensorimotor gating, a phenotype which has been associated with psychiatric disorders in humans. These studies therefore provide insights into the role of the SNARE complex in both diabetes and psychiatric disease. PMID:17283335

  8. A dominant mutation in Snap25 causes impaired vesicle trafficking, sensorimotor gating, and ataxia in the blind-drunk mouse.

    PubMed

    Jeans, Alexander F; Oliver, Peter L; Johnson, Reuben; Capogna, Marco; Vikman, Jenny; Molnár, Zoltán; Babbs, Arran; Partridge, Christopher J; Salehi, Albert; Bengtsson, Martin; Eliasson, Lena; Rorsman, Patrik; Davies, Kay E

    2007-02-13

    The neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is essential for synaptic vesicle exocytosis, but its study has been limited by the neonatal lethality of murine SNARE knockouts. Here, we describe a viable mouse line carrying a mutation in the b-isoform of neuronal SNARE synaptosomal-associated protein of 25 kDa (SNAP-25). The causative I67T missense mutation results in increased binding affinities within the SNARE complex, impaired exocytotic vesicle recycling and granule exocytosis in pancreatic beta-cells, and a reduction in the amplitude of evoked cortical excitatory postsynaptic potentials. The mice also display ataxia and impaired sensorimotor gating, a phenotype which has been associated with psychiatric disorders in humans. These studies therefore provide insights into the role of the SNARE complex in both diabetes and psychiatric disease.

  9. Loss-of-function mutations in ATP6V0A2 impair vesicular trafficking, tropoelastin secretion and cell survival

    PubMed Central

    Hucthagowder, Vishwanathan; Morava, Eva; Kornak, Uwe; Lefeber, Dirk J.; Fischer, Björn; Dimopoulou, Aikaterini; Aldinger, Annika; Choi, Jiwon; Davis, Elaine C.; Abuelo, Dianne N.; Adamowicz, Maciej; Al-Aama, Jumana; Basel-Vanagaite, Lina; Fernandez, Bridget; Greally, Marie T.; Gillessen-Kaesbach, Gabriele; Kayserili, Hulya; Lemyre, Emmanuelle; Tekin, Mustafa; Türkmen, Seval; Tuysuz, Beyhan; Yüksel-Konuk, Berrin; Mundlos, Stefan; Van Maldergem, Lionel; Wevers, Ron A.; Urban, Zsolt

    2009-01-01

    Autosomal recessive cutis laxa type 2 (ARCL2), a syndrome of growth and developmental delay and redundant, inelastic skin, is caused by mutations in the a2 subunit of the vesicular ATPase H+-pump (ATP6V0A2). The goal of this study was to define the disease mechanisms that lead to connective tissue lesions in ARCL2. In a new cohort of 17 patients, DNA sequencing of ATP6V0A2 detected either homozygous or compound heterozygous mutations. Considerable allelic and phenotypic heterogeneity was observed, with a missense mutation of a moderately conserved residue p.P87L leading to unusually mild disease. Abnormal N- and/or mucin type O-glycosylation was observed in all patients tested. Premature stop codon mutations led to decreased ATP6V0A2 mRNA levels by destabilizing the mutant mRNA via the nonsense-mediated decay pathway. Loss of ATP6V0A2 either by siRNA knockdown or in ARCL2 cells resulted in distended Golgi cisternae, accumulation of abnormal lysosomes and multivesicular bodies. Immunostaining of ARCL2 cells showed the accumulation of tropoelastin (TE) in the Golgi and in large, abnormal intracellular and extracellular aggregates. Pulse–chase studies confirmed impaired secretion and increased intracellular retention of TE, and insoluble elastin assays showed significantly reduced extracellular deposition of mature elastin. Fibrillin-1 microfibril assembly and secreted lysyl oxidase activity were normal in ARCL2 cells. TUNEL staining demonstrated increased rates of apoptosis in ARCL2 cell cultures. We conclude that loss-of-function mutations in ATP6V0A2 lead to TE aggregation in the Golgi, impaired clearance of TE aggregates and increased apoptosis of elastogenic cells. PMID:19321599

  10. Impaired intracellular trafficking defines early Parkinson's disease.

    PubMed

    Hunn, Benjamin H M; Cragg, Stephanie J; Bolam, J Paul; Spillantini, Maria-Grazia; Wade-Martins, Richard

    2015-03-01

    Parkinson's disease (PD) is an insidious and incurable neurodegenerative disease, and represents a significant cost to individuals, carers, and ageing societies. It is defined at post-mortem by the loss of dopamine neurons in the substantia nigra together with the presence of Lewy bodies and Lewy neurites. We examine here the role of α-synuclein and other cellular transport proteins implicated in PD and how their aberrant activity may be compounded by the unique anatomy of the dopaminergic neuron. This review uses multiple lines of evidence from genetic studies, human tissue, induced pluripotent stem cells, and refined animal models to argue that prodromal PD can be defined as a disease of impaired intracellular trafficking. Dysfunction of the dopaminergic synapse heralds trafficking impairment.

  11. Mechanism of impaired microtubule-dependent peroxisome trafficking and oxidative stress in SPAST-mutated cells from patients with Hereditary Spastic Paraplegia

    PubMed Central

    Wali, Gautam; Sutharsan, Ratneswary; Fan, Yongjun; Stewart, Romal; Tello Velasquez, Johana; Sue, Carolyn M; Crane, Denis I.; Mackay-Sim, Alan

    2016-01-01

    Hereditary spastic paraplegia (HSP) is an inherited neurological condition that leads to progressive spasticity and gait abnormalities. Adult-onset HSP is most commonly caused by mutations in SPAST, which encodes spastin a microtubule severing protein. In olfactory stem cell lines derived from patients carrying different SPAST mutations, we investigated microtubule-dependent peroxisome movement with time-lapse imaging and automated image analysis. The average speed of peroxisomes in patient-cells was slower, with fewer fast moving peroxisomes than in cells from healthy controls. This was not because of impairment of peroxisome-microtubule interactions because the time-dependent saltatory dynamics of movement of individual peroxisomes was unaffected in patient-cells. Our observations indicate that average peroxisome speeds are less in patient-cells because of the lower probability of individual peroxisome interactions with the reduced numbers of stable microtubules: peroxisome speeds in patient cells are restored by epothilone D, a tubulin-binding drug that increases the number of stable microtubules to control levels. Patient-cells were under increased oxidative stress and were more sensitive than control-cells to hydrogen peroxide, which is primarily metabolised by peroxisomal catalase. Epothilone D also ameliorated patient-cell sensitivity to hydrogen-peroxide. Our findings suggest a mechanism for neurodegeneration whereby SPAST mutations indirectly lead to impaired peroxisome transport and oxidative stress. PMID:27229699

  12. Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation

    PubMed Central

    Payne, Aimee S.; Kelly, Edward J.; Gitlin, Jonathan D.

    1998-01-01

    Wilson disease is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase. To elucidate the function of the Wilson protein, wild-type and mutant Wilson cDNAs were expressed in a Menkes copper transporter-deficient mottled fibroblast cell line defective in copper export. Expression of the wild-type cDNA demonstrated trans-Golgi network localization and copper-dependent trafficking of the Wilson protein identical to previous observations for the endogenously expressed protein in hepatocytes. Furthermore, expression of the Wilson cDNA rescued the mottled phenotype as evidenced by a reduction in copper accumulation and restoration of cell viability. In contrast, expression of an H1069Q mutant Wilson cDNA did not rescue the mottled phenotype, and immunofluorescence studies showed that this mutant Wilson protein was localized in the endoplasmic reticulum. Consistent with these findings, pulse–chase analysis demonstrated a 5-fold decrease in the half-life of the H1069Q mutant as compared with the wild-type protein. Maintenance of these transfected cell lines at 28°C resulted in localization of the H1069Q protein in the trans-Golgi network, suggesting that a temperature-sensitive defect in protein folding followed by degradation constitutes the molecular basis of Wilson disease in patients harboring the H1069Q mutation. Taken together, these studies describe a tractable expression system for elucidating the function and localization of the copper-transporting ATPases in mammalian cells and provide compelling evidence that the Wilson protein can functionally substitute for the Menkes protein, supporting the concept that these proteins use common biochemical mechanisms to effect cellular copper homeostasis. PMID:9724794

  13. Pharmacological rescue of trafficking-impaired ATP-sensitive potassium channels

    PubMed Central

    Martin, Gregory M.; Chen, Pei-Chun; Devaraneni, Prasanna; Shyng, Show-Ling

    2013-01-01

    ATP-sensitive potassium (KATP) channels link cell metabolism to membrane excitability and are involved in a wide range of physiological processes including hormone secretion, control of vascular tone, and protection of cardiac and neuronal cells against ischemic injuries. In pancreatic β-cells, KATP channels play a key role in glucose-stimulated insulin secretion, and gain or loss of channel function results in neonatal diabetes or congenital hyperinsulinism, respectively. The β-cell KATP channel is formed by co-assembly of four Kir6.2 inwardly rectifying potassium channel subunits encoded by KCNJ11 and four sulfonylurea receptor 1 subunits encoded by ABCC8. Many mutations in ABCC8 or KCNJ11 cause loss of channel function, thus, congenital hyperinsulinism by hampering channel biogenesis and hence trafficking to the cell surface. The trafficking defects caused by a subset of these mutations can be corrected by sulfonylureas, KATP channel antagonists that have long been used to treat type 2 diabetes. More recently, carbamazepine, an anticonvulsant that is thought to target primarily voltage-gated sodium channels has been shown to correct KATP channel trafficking defects. This article reviews studies to date aimed at understanding the mechanisms by which mutations impair channel biogenesis and trafficking and the mechanisms by which pharmacological ligands overcome channel trafficking defects. Insight into channel structure-function relationships and therapeutic implications from these studies are discussed. PMID:24399968

  14. Carbamazepine as a novel small molecule corrector of trafficking-impaired ATP-sensitive potassium channels identified in congenital hyperinsulinism.

    PubMed

    Chen, Pei-Chun; Olson, Erik M; Zhou, Qing; Kryukova, Yelena; Sampson, Heidi M; Thomas, David Y; Shyng, Show-Ling

    2013-07-19

    ATP-sensitive potassium (KATP) channels consisting of sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to β-cell membrane potential. Mutations in SUR1 and Kir6.2 that impair channel trafficking to the cell surface lead to loss of channel function and congenital hyperinsulinism. We report that carbamazepine, an anticonvulsant, corrects the trafficking defects of mutant KATP channels previously identified in congenital hyperinsulinism. Strikingly, of the 19 SUR1 mutations examined, only those located in the first transmembrane domain of SUR1 responded to the drug. We show that unlike that reported for several other protein misfolding diseases, carbamazepine did not correct KATP channel trafficking defects by activating autophagy; rather, it directly improved the biogenesis efficiency of mutant channels along the secretory pathway. In addition to its effect on channel trafficking, carbamazepine also inhibited KATP channel activity. Upon subsequent removal of carbamazepine, however, the function of rescued channels was recovered. Importantly, combination of the KATP channel opener diazoxide and carbamazepine led to enhanced mutant channel function without carbamazepine washout. The corrector effect of carbamazepine on mutant KATP channels was also demonstrated in rat and human β-cells with an accompanying increase in channel activity. Our findings identify carbamazepine as a novel small molecule corrector that may be used to restore KATP channel expression and function in a subset of congenital hyperinsulinism patients.

  15. Carbamazepine as a Novel Small Molecule Corrector of Trafficking-impaired ATP-sensitive Potassium Channels Identified in Congenital Hyperinsulinism*

    PubMed Central

    Chen, Pei-Chun; Olson, Erik M.; Zhou, Qing; Kryukova, Yelena; Sampson, Heidi M.; Thomas, David Y.; Shyng, Show-Ling

    2013-01-01

    ATP-sensitive potassium (KATP) channels consisting of sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to β-cell membrane potential. Mutations in SUR1 and Kir6.2 that impair channel trafficking to the cell surface lead to loss of channel function and congenital hyperinsulinism. We report that carbamazepine, an anticonvulsant, corrects the trafficking defects of mutant KATP channels previously identified in congenital hyperinsulinism. Strikingly, of the 19 SUR1 mutations examined, only those located in the first transmembrane domain of SUR1 responded to the drug. We show that unlike that reported for several other protein misfolding diseases, carbamazepine did not correct KATP channel trafficking defects by activating autophagy; rather, it directly improved the biogenesis efficiency of mutant channels along the secretory pathway. In addition to its effect on channel trafficking, carbamazepine also inhibited KATP channel activity. Upon subsequent removal of carbamazepine, however, the function of rescued channels was recovered. Importantly, combination of the KATP channel opener diazoxide and carbamazepine led to enhanced mutant channel function without carbamazepine washout. The corrector effect of carbamazepine on mutant KATP channels was also demonstrated in rat and human β-cells with an accompanying increase in channel activity. Our findings identify carbamazepine as a novel small molecule corrector that may be used to restore KATP channel expression and function in a subset of congenital hyperinsulinism patients. PMID:23744072

  16. Mutant Huntingtin Impairs Axonal Trafficking in Mammalian Neurons In Vivo and In Vitro

    PubMed Central

    Trushina, Eugenia; Dyer, Roy B.; Badger, John D.; Ure, Daren; Eide, Lars; Tran, David D.; Vrieze, Brent T.; Legendre-Guillemin, Valerie; McPherson, Peter S.; Mandavilli, Bhaskar S.; Van Houten, Bennett; Zeitlin, Scott; McNiven, Mark; Aebersold, Ruedi; Hayden, Michael; Parisi, Joseph E.; Seeberg, Erling; Dragatsis, Ioannis; Doyle, Kelly; Bender, Anna; Chacko, Celin; McMurray, Cynthia T.

    2004-01-01

    Recent data in invertebrates demonstrated that huntingtin (htt) is essential for fast axonal trafficking. Here, we provide direct and functional evidence that htt is involved in fast axonal trafficking in mammals. Moreover, expression of full-length mutant htt (mhtt) impairs vesicular and mitochondrial trafficking in mammalian neurons in vitro and in whole animals in vivo. Particularly, mitochondria become progressively immobilized and stop more frequently in neurons from transgenic animals. These defects occurred early in development prior to the onset of measurable neurological or mitochondrial abnormalities. Consistent with a progressive loss of function, wild-type htt, trafficking motors, and mitochondrial components were selectively sequestered by mhtt in human Huntington's disease-affected brain. Data provide a model for how loss of htt function causes toxicity; mhtt-mediated aggregation sequesters htt and components of trafficking machinery leading to loss of mitochondrial motility and eventual mitochondrial dysfunction. PMID:15340079

  17. First missense mutation outside of SERAC1 lipase domain affecting intracellular cholesterol trafficking.

    PubMed

    Rodríguez-García, María Elena; Martín-Hernández, Elena; de Aragón, Ana Martínez; García-Silva, María Teresa; Quijada-Fraile, Pilar; Arenas, Joaquín; Martín, Miguel A; Martínez-Azorín, Francisco

    2016-01-01

    We report the clinical and genetic findings in a Spanish boy who presented MEGDEL syndrome, a very rare inborn error of metabolism. Whole-exome sequencing uncovered a new homozygous mutation in the serine active site containing 1 (SERAC1) gene, which is essential for both mitochondrial function and intracellular cholesterol trafficking. Functional studies in patient fibroblasts showed that p.D224G mutation affects the intracellular cholesterol trafficking. Only three missense mutations in this gene have been described before, being p.D224G the first missense mutation outside of the SERAC1 serine-lipase domain. Therefore, we conclude that the defect in cholesterol trafficking is not limited to alterations in this specific part of the protein.

  18. Endosomal accumulation of APP in wobbler motor neurons reflects impaired vesicle trafficking: implications for human motor neuron disease.

    PubMed

    Palmisano, Ralf; Golfi, Panagiota; Heimann, Peter; Shaw, Christopher; Troakes, Claire; Schmitt-John, Thomas; Bartsch, Jörg W

    2011-03-07

    The cause of sporadic amyotrophic lateral sclerosis (ALS) is largely unknown but hypotheses about disease mechanisms include oxidative stress, defective axonal transport, mitochondrial dysfunction and disrupted RNA processing. Whereas familial ALS is well represented by transgenic mutant SOD1 mouse models, the mouse mutant wobbler (WR) develops progressive motor neuron degeneration due to a point mutation in the Vps54 gene, and provides an animal model for sporadic ALS. VPS54 protein as a component of a protein complex is involved in vesicular Golgi trafficking; impaired vesicle trafficking might also be mechanistic in the pathogenesis of human ALS. In motor neurons of homozygous symptomatic WR mice, a massive number of endosomal vesicles significantly enlarged (up to 3 μm in diameter) were subjected to ultrastructural analysis and immunohistochemistry for the endosome-specific small GTPase protein Rab7 and for amyloid precursor protein (APP). Enlarged vesicles were neither detected in heterozygous WR nor in transgenic SOD1(G93A) mice; in WR motor neurons, numerous APP/Rab7-positive vesicles were observed which were mostly LC3-negative, suggesting they are not autophagosomes. We conclude that endosomal APP/Rab7 staining reflects impaired vesicle trafficking in WR mouse motor neurons. Based on these findings human ALS tissues were analysed for APP in enlarged vesicles and were detected in spinal cord motor neurons in six out of fourteen sporadic ALS cases. These enlarged vesicles were not detected in any of the familial ALS cases. Thus our study provides the first evidence for wobbler-like aetiologies in human ALS and suggests that the genes encoding proteins involved in vesicle trafficking should be screened for pathogenic mutations.

  19. Altered trafficking of mutated growth factor receptors and their associated molecules

    PubMed Central

    Kon, Shunsuke; Kobayashi, Nobuhide; Satake, Masanobu

    2014-01-01

    Ligand-stimulated receptor tyrosine kinases (RTKs) are phosphorylated/ubiquitinated, endocytosed and transported to the lysosomes via endosomes/multivesicular bodies, resulting in the attenuation of signal transmission. If this physiological mechanism of RTK signal downregulation is perturbed, signal transduction persists and may contribute to cellular transformation. This article presents several such examples. In some cases, endocytosis is impaired, and the activated RTK remains on the plasma membrane. In other cases, the activated RTK is endocytosed into endosomes/multivesicular bodies, but not subsequently sorted to the lysosomes for degradation. The latter cases indicate that even endocytosed RTKs can transmit signals. Transport of RTKs is accomplished via the formation and movement of membrane vesicles. Blockage or delay of endocytosis/trafficking can be caused by genetic alterations in the RTK itself or by mutations in CBL, Arf GAPs, or other components involved in internalization and vesicle transport. A survey of the literature indicates that, in some cases, even RTKs synthesized de novo can initiate signaling at the endoplasmic reticulum/Golgi before reaching the plasma membrane. The spectrum of molecules targeted by the signal is likely to be different between cell surface- and endoplasmic reticulum/Golgi-localized RTKs. PMID:25210647

  20. L1 syndrome mutations impair neuronal L1 function at different levels by divergent mechanisms.

    PubMed

    Schäfer, Michael K E; Nam, Yun-Chung; Moumen, Anice; Keglowich, Laura; Bouché, Elisabeth; Küffner, Mercedes; Bock, Hans H; Rathjen, Fritz G; Raoul, Cedric; Frotscher, Michael

    2010-10-01

    Mutations in the human L1CAM gene cause neurodevelopmental disorders collectively referred to as L1 syndrome. Here, we investigated cellular pathomechanisms underlying two L1 syndrome mutations, R184Q and W1036L. We demonstrate that these mutations cause partial endoplasmic reticulum (ER) retention of L1, reduce L1 cell surface expression, but do not induce ER stress in neuronal NSC-34 cells. We provide evidence that surface trafficking of mutated L1 is affected by defective sorting to ER exit sites and attenuated ER export. However, in differentiated neuronal cultures and long-term cultured hippocampal slices, the L1-R184Q protein is restricted to cell bodies, whereas L1-W1036L also aberrantly localizes to dendrites. These trafficking defects preclude axonal targeting of L1, thereby affecting L1-mediated axon growth and arborization. Our results indicate that L1 syndrome mutations impair neuronal L1 function at different levels, firstly by attenuating ER export and secondly by interfering with polarized neuronal trafficking. (c) 2010 Elsevier Inc. All rights reserved.

  1. M-CSF receptor mutations in hereditary diffuse leukoencephalopathy with spheroids impair not only kinase activity but also surface expression

    SciTech Connect

    Hiyoshi, Masateru; Hashimoto, Michihiro; Yukihara, Mamiko; Bhuyan, Farzana; Suzu, Shinya

    2013-11-01

    Highlights: •Many mutations were identified in Fms as a putative genetic cause of HDLS. •All of the mutations tested severely impair the kinase activity. •Most of the mutations also impair the trafficking to the cell surface. •These defects further suggest that HDLS is caused by a loss of Fms function. -- Abstract: The tyrosine kinase Fms, the cell surface receptor for M-CSF and IL-34, is critical for microglial proliferation and differentiation in the brain. Recently, a number of mutations have been identified in Fms as a putative genetic cause of hereditary diffuse leukoencephalopathy with spheroids (HDLS), implying an important role of microglial dysfunction in HDLS pathogenesis. In this study, we initially confirmed that 11 mutations, which reside within the ATP-binding or major tyrosine kinase domain, caused a severe impairment of ligand-induced Fms auto-phosphorylation. Intriguingly, we found that 10 of the 11 mutants also showed a weak cell surface expression, which was associated with a concomitant increase in the low molecular weight hypo-N-glycosylated immature gp130Fms-like species. Indeed, the mutant proteins heavily accumulated to the Golgi-like perinuclear regions. These results indicate that all of the Fms mutations tested severely impair the kinase activity and most of the mutations also impair the trafficking to the cell surface, further suggesting that HDLS is caused by the loss of Fms function.

  2. Endothelin-1 Impairs Glucose Transporter Trafficking via a Membrane-Based Mechanism

    PubMed Central

    Strawbridge, Andrew B.; Elmendorf, Jeffrey S.

    2008-01-01

    Endothelin-1 (ET-1) disrupts insulin-regulated glucose transporter GLUT4 trafficking. Since the negative consequence of chronic ET-1 exposure appears to be independent of signal disturbance along the insulin receptor substrate-1/phosphatidylinositol (PI) 3-kinase (PI3K)/Akt-2 pathway of insulin action, we tested if ET-1 altered GLUT4 regulation engaged by osmotic shock, a PI3K-independent stimulus that mimics insulin action. Regulation of GLUT4 by hyperosmotic stress was impaired by ET-1. Because of the mutual disruption of both insulin- and hyperosmolarity-stimulated GLUT4 translocation, we tested whether shared signaling and/or key phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated cytoskeletal events of GLUT4 trafficking were targets of ET-1. Both insulin and hyperosmotic stress signaling to Cbl were impaired by ET-1. Also, plasma membrane PIP2 and cortical actin levels were reduced in cells exposed to ET-1. Exogenous PIP2, but not PI 3,4,5-bisphosphate, restored actin structure, Cbl activation, and GLUT4 translocation. These data show that ET-1-induced PIP2/actin disruption impairs GLUT4 trafficking elicited by insulin and hyperosmolarity. In addition to showing for the first time the important role of PIP2-regulated cytoskeletal events in GLUT4 regulation by stimuli other than insulin, these studies reveal a novel function of PIP2/actin structure in signal transduction. PMID:16240321

  3. Endothelin-1 impairs glucose transporter trafficking via a membrane-based mechanism.

    PubMed

    Strawbridge, Andrew B; Elmendorf, Jeffrey S

    2006-03-01

    Endothelin-1 (ET-1) disrupts insulin-regulated glucose transporter GLUT4 trafficking. Since the negative consequence of chronic ET-1 exposure appears to be independent of signal disturbance along the insulin receptor substrate-1/phosphatidylinositol (PI) 3-kinase (PI3K)/Akt-2 pathway of insulin action, we tested if ET-1 altered GLUT4 regulation engaged by osmotic shock, a PI3K-independent stimulus that mimics insulin action. Regulation of GLUT4 by hyperosmotic stress was impaired by ET-1. Because of the mutual disruption of both insulin- and hyperosmolarity-stimulated GLUT4 translocation, we tested whether shared signaling and/or key phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated cytoskeletal events of GLUT4 trafficking were targets of ET-1. Both insulin and hyperosmotic stress signaling to Cbl were impaired by ET-1. Also, plasma membrane PIP2 and cortical actin levels were reduced in cells exposed to ET-1. Exogenous PIP2, but not PI 3,4,5-bisphosphate, restored actin structure, Cbl activation, and GLUT4 translocation. These data show that ET-1-induced PIP2/actin disruption impairs GLUT4 trafficking elicited by insulin and hyperosmolarity. In addition to showing for the first time the important role of PIP2-regulated cytoskeletal events in GLUT4 regulation by stimuli other than insulin, these studies reveal a novel function of PIP2/actin structure in signal transduction. 2005 Wiley-Liss, Inc.

  4. Impaired Leukocyte Trafficking and Skin Inflammatory Responses in Hamsters Lacking a Functional Circadian System

    PubMed Central

    Prendergast, Brian J.; Cable, Erin J.; Patel, Priyesh N.; Pyter, Leah M.; Onishi, Kenneth G.; Stevenson, Tyler J.; Ruby, Norman F.; Bradley, Sean P.

    2013-01-01

    The immune system is under strong circadian control, and circadian desynchrony is a risk factor for metabolic disorders, inflammatory responses and cancer. Signaling pathways that maintain circadian rhythms (CRs) in immune function in vivo, and the mechanisms by which circadian desynchrony impairs immune function, remain to be fully-identified. These experiments tested the hypothesis that the hypothalamic circadian pacemaker in the suprachiasmatic nucleus (SCN) drives CRs in the immune system, using a non-invasive model of SCN circadian arrhythmia. Robust CRs in blood leukocyte trafficking, with a peak during the early light phase (ZT4) and nadir in the early dark phase (ZT18), were absent in arrhythmic hamsters, as were CRs in spleen clock gene (per1, bmal1) expression, indicating that a functional pacemaker in the SCN is required for the generation of CRs in leukocyte trafficking and for driving peripheral clocks in secondary lymphoid organs. Pinealectomy was without effect on CRs in leukocyte trafficking, but abolished CRs in spleen clock gene expression, indicating that nocturnal melatonin secretion is necessary for communicating circadian time information to the spleen. CRs in trafficking of antigen presenting cells (CD11c+ dendritic cells) in the skin were abolished, and antigen-specific delayed-type hypersensitivity skin inflammatory responses were markedly impaired in arrhythmic hamsters. The SCN drives robust CRs in leukocyte trafficking and lymphoid clock gene expression; the latter of which is not expressed in the absence of melatonin. Robust entrainment of the circadian pacemaker provides a signal critical to diurnal rhythms in immunosurveilliance and optimal memory T-cell dependent immune responses. PMID:23474187

  5. BIN1 is Reduced and Cav1.2 Trafficking is Impaired in Human Failing Cardiomyocytes

    PubMed Central

    Hong, Ting-Ting; Smyth, James W.; Chu, Kevin Y.; Vogan, Jacob M.; Fong, Tina S.; Jensen, Brian C.; Fang, Kun; Halushka, Marc K.; Russell, Stuart D.; Colecraft, Henry; Hoopes, Charles W.; Ocorr, Karen; Chi, Neil C.; Shaw, Robin M.

    2011-01-01

    Background Heart failure is a growing epidemic and a typical aspect of heart failure pathophysiology is altered calcium transients. Normal cardiac calcium transients are initiated by Cav1.2 channels at cardiac T-tubules. BIN1 is a membrane scaffolding protein that causes Cav1.2 to traffic to T-tubules in healthy hearts. The mechanisms of Cav1.2 trafficking in heart failure are not known. Objective To study BIN1 expression and its effect on Cav1.2 trafficking in failing hearts. Methods Intact myocardium and freshly isolated cardiomyocytes from non-failing and end-stage failing human hearts were used to study BIN1 expression and Cav1.2 localization. To confirm Cav1.2 surface expression dependence on BIN1, patch clamp recordings were performed of Cav1.2 current in cell lines with and without trafficking competent BIN1. Also, in adult mouse cardiomyocytes, surface Cav1.2 and calcium transients were studied after shRNA mediated knockdown of BIN1. For a functional readout in intact heart, calcium transients and cardiac contractility were analyzed in a zebrafish model with morpholino mediated knockdown of BIN1. Results BIN1 expression is significantly decreased in failing cardiomyocytes at both mRNA (30% down) and protein (36% down) levels. Peripheral Cav1.2 is reduced 42% by imaging and biochemical T-tubule fraction of Cav1.2 is reduced 68%. Total calcium current is reduced 41% in a cell line expressing non-trafficking BIN1 mutant. In mouse cardiomyocytes, BIN1 knockdown decreases surface Cav1.2 and impairs calcium transients. In zebrafish hearts, BIN1 knockdown causes a 75% reduction in calcium transients and severe ventricular contractile dysfunction. Conclusions The data indicate that BIN1 is significantly reduced in human heart failure, and this reduction impairs Cav1.2 trafficking, calcium transients, and contractility. PMID:22138472

  6. Impaired leukocyte trafficking and skin inflammatory responses in hamsters lacking a functional circadian system.

    PubMed

    Prendergast, Brian J; Cable, Erin J; Patel, Priyesh N; Pyter, Leah M; Onishi, Kenneth G; Stevenson, Tyler J; Ruby, Norman F; Bradley, Sean P

    2013-08-01

    The immune system is under strong circadian control, and circadian desynchrony is a risk factor for metabolic disorders, inflammatory responses and cancer. Signaling pathways that maintain circadian rhythms (CRs) in immune function in vivo, and the mechanisms by which circadian desynchrony impairs immune function, remain to be fully identified. These experiments tested the hypothesis that the hypothalamic circadian pacemaker in the suprachiasmatic nucleus (SCN) drives CRs in the immune system, using a non-invasive model of SCN circadian arrhythmia. Robust CRs in blood leukocyte trafficking, with a peak during the early light phase (ZT4) and nadir in the early dark phase (ZT18), were absent in arrhythmic hamsters, as were CRs in spleen clock gene (per1, bmal1) expression, indicating that a functional pacemaker in the SCN is required for the generation of CRs in leukocyte trafficking and for driving peripheral clocks in secondary lymphoid organs. Pinealectomy was without effect on CRs in leukocyte trafficking, but abolished CRs in spleen clock gene expression, indicating that nocturnal melatonin secretion is necessary for communicating circadian time information to the spleen. CRs in trafficking of antigen presenting cells (CD11c(+) dendritic cells) in the skin were abolished, and antigen-specific delayed-type hypersensitivity skin inflammatory responses were markedly impaired in arrhythmic hamsters. The SCN drives robust CRs in leukocyte trafficking and lymphoid clock gene expression; the latter of which is not expressed in the absence of melatonin. Robust entrainment of the circadian pacemaker provides a signal critical to diurnal rhythms in immunosurveilliance and optimal memory T-cell dependent immune responses. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Astrocytic Pathological Calcium Homeostasis and Impaired Vesicle Trafficking in Neurodegeneration

    PubMed Central

    Vardjan, Nina; Verkhratsky, Alexej; Zorec, Robert

    2017-01-01

    Although the central nervous system (CNS) consists of highly heterogeneous populations of neurones and glial cells, clustered into diverse anatomical regions with specific functions, there are some conditions, including alertness, awareness and attention that require simultaneous, coordinated and spatially homogeneous activity within a large area of the brain. During such events, the brain, representing only about two percent of body mass, but consuming one fifth of body glucose at rest, needs additional energy to be produced. How simultaneous energy procurement in a relatively extended area of the brain takes place is poorly understood. This mechanism is likely to be impaired in neurodegeneration, for example in Alzheimer’s disease, the hallmark of which is brain hypometabolism. Astrocytes, the main neural cell type producing and storing glycogen, a form of energy in the brain, also hold the key to metabolic and homeostatic support in the central nervous system and are impaired in neurodegeneration, contributing to the slow decline of excitation-energy coupling in the brain. Many mechanisms are affected, including cell-to-cell signalling. An important question is how changes in cellular signalling, a process taking place in a rather short time domain, contribute to the neurodegeneration that develops over decades. In this review we focus initially on the slow dynamics of Alzheimer’s disease, and on the activity of locus coeruleus, a brainstem nucleus involved in arousal. Subsequently, we overview much faster processes of vesicle traffic and cytosolic calcium dynamics, both of which shape the signalling landscape of astrocyte-neurone communication in health and neurodegeneration. PMID:28208745

  8. Microglial beclin 1 regulates retromer trafficking and phagocytosis and is impaired in Alzheimer's disease.

    PubMed

    Lucin, Kurt M; O'Brien, Caitlin E; Bieri, Gregor; Czirr, Eva; Mosher, Kira I; Abbey, Rachelle J; Mastroeni, Diego F; Rogers, Joseph; Spencer, Brian; Masliah, Eliezer; Wyss-Coray, Tony

    2013-09-04

    Phagocytosis controls CNS homeostasis by facilitating the removal of unwanted cellular debris. Accordingly, impairments in different receptors or proteins involved in phagocytosis result in enhanced inflammation and neurodegeneration. While various studies have identified extrinsic factors that modulate phagocytosis in health and disease, key intracellular regulators are less understood. Here we show that the autophagy protein beclin 1 is required for efficient phagocytosis in vitro and in mouse brains. Furthermore, we show that beclin 1-mediated impairments in phagocytosis are associated with dysfunctional recruitment of retromer to phagosomal membranes, reduced retromer levels, and impaired recycling of phagocytic receptors CD36 and Trem2. Interestingly, microglia isolated from human Alzheimer's disease (AD) brains show significantly reduced beclin 1 and retromer protein levels. These findings position beclin 1 as a link between autophagy, retromer trafficking, and receptor-mediated phagocytosis and provide insight into mechanisms by which phagocytosis is regulated and how it may become impaired in AD.

  9. Congenital hypothyroidism mutations affect common folding and trafficking in the α/β-hydrolase fold proteins.

    PubMed

    De Jaco, Antonella; Dubi, Noga; Camp, Shelley; Taylor, Palmer

    2012-12-01

    The α/β-hydrolase fold superfamily of proteins is composed of structurally related members that, despite great diversity in their catalytic, recognition, adhesion and chaperone functions, share a common fold governed by homologous residues and conserved disulfide bridges. Non-synonymous single nucleotide polymorphisms within the α/β-hydrolase fold domain in various family members have been found for congenital endocrine, metabolic and nervous system disorders. By examining the amino acid sequence from the various proteins, mutations were found to be prevalent in conserved residues within the α/β-hydrolase fold of the homologous proteins. This is the case for the thyroglobulin mutations linked to congenital hypothyroidism. To address whether correct folding of the common domain is required for protein export, we inserted the thyroglobulin mutations at homologous positions in two correlated but simpler α/β-hydrolase fold proteins known to be exported to the cell surface: neuroligin3 and acetylcholinesterase. Here we show that these mutations in the cholinesterase homologous region alter the folding properties of the α/β-hydrolase fold domain, which are reflected in defects in protein trafficking, folding and function, and ultimately result in retention of the partially processed proteins in the endoplasmic reticulum. Accordingly, mutations at conserved residues may be transferred amongst homologous proteins to produce common processing defects despite disparate functions, protein complexity and tissue-specific expression of the homologous proteins. More importantly, a similar assembly of the α/β-hydrolase fold domain tertiary structure among homologous members of the superfamily is required for correct trafficking of the proteins to their final destination.

  10. A Novel Strategy Using Cardiac Sodium Channel Polymorphic Fragments to Rescue Trafficking Deficient SCN5A Mutations

    PubMed Central

    Shinlapawittayatorn, Krekwit; Dudash, Lynn A.; Du, Xi X.; Heller, Lisa; Poelzing, Steven; Ficker, Eckhard; Deschênes, Isabelle

    2011-01-01

    Background Brugada Syndrome (BrS) is associated with mutations in the cardiac sodium channel (Nav1.5). We previously reported that the function of a trafficking-deficient BrS Nav1.5 mutation, R282H, could be restored by co-expression with the sodium channel polymorphism H558R. Here, we tested the hypothesis that peptide fragments from Nav1.5, spanning the H558R-polymorphism, can be used to restore trafficking of trafficking-deficient BrS sodium channel mutations. Methods and Results Whole-cell patch-clamping revealed that co-transfection in HEK293 cells of the R282H channel with either the 40 or 20 amino acid cDNA fragments of Nav1.5 containing the H558R polymorphism restored trafficking of this mutant channel. Fluorescence Resonance Energy Transfer (FRET) suggested that the trafficking-deficient R282H channel was misfolded and this was corrected upon co-expression with R558-containing peptides which restored trafficking of the R282H channel. Importantly, we also expressed the peptide spanning the H558R-polymorphism with 8 additional BrS Nav1.5 mutations with reduced currents, and demonstrated that the peptide was able to restore significant sodium currents in 4 of them. Conclusions In the present study, we demonstrated that small peptides, spanning the H558R-polymorphism, are sufficient to restore trafficking defect of BrS-associated Nav1.5 mutations. Our findings suggest that it might be possible to use short-cDNA constructs as a novel strategy that is tailored to specific disease-causing mutants of BrS. PMID:21840964

  11. The CFTR trafficking mutation F508del inhibits the constitutive activity of SLC26A9.

    PubMed

    Bertrand, Carol A; Mitra, Shalini; Mishra, Sanjay K; Wang, Xiaohui; Zhao, Yu; Pilewski, Joseph M; Madden, Dean R; Frizzell, Raymond A

    2017-06-01

    Several members of the SLC26A family of anion transporters associate with CFTR, forming complexes in which CFTR and SLC26A functions are reciprocally regulated. These associations are thought to be facilitated by PDZ scaffolding interactions. CFTR has been shown to be positively regulated by NHERF-1, and negatively regulated by CAL in airway epithelia. However, it is unclear which PDZ-domain protein(s) interact with SLC26A9, a SLC26A family member found in airway epithelia. We have previously shown that primary, human bronchial epithelia (HBE) from non-CF donors exhibit constitutive anion secretion attributable to SLC26A9. However, constitutive anion secretion is absent in HBE from CF donors. We examined whether changes in SLC26A9 constitutive activity could be attributed to a loss of CFTR trafficking, and what role PDZ interactions played. HEK293 coexpressing SLC26A9 with the trafficking mutant F508del CFTR exhibited a significant reduction in constitutive current compared with cells coexpressing SLC26A9 and wt CFTR. We found that SLC26A9 exhibits complex glycosylation when coexpressed with F508del CFTR, but its expression at the plasma membrane is decreased. SLC26A9 interacted with both NHERF-1 and CAL, and its interaction with both significantly increased with coexpression of wt CFTR. However, coexpression with F508del CFTR only increased SLC26A9's interaction with CAL. Mutation of SLC26A9's PDZ motif decreased this association with CAL, and restored its constitutive activity. Correcting aberrant F508del CFTR trafficking in CF HBE with corrector VX-809 also restored SLC26A9 activity. We conclude that when SLC26A9 is coexpressed with F508del CFTR, its trafficking defect leads to a PDZ motif-sensitive intracellular retention of SLC26A9. Copyright © 2017 the American Physiological Society.

  12. The AQP2 mutation V71M causes nephrogenic diabetes insipidus in humans but does not impair the function of a bacterial homolog.

    PubMed

    Klein, Noreen; Kümmerer, Nadine; Hobernik, Dominika; Schneider, Dirk

    2015-01-01

    Several point mutations have been identified in human aquaporins, but their effects on the function of the respective aquaporins are mostly enigmatic. We analyzed the impact of the aquaporin 2 mutation V71M, which causes nephrogenic diabetes insipidus in humans, on aquaporin structure and activity, using the bacterial aquaglyceroporin GlpF as a model. Importantly, the sequence and structure around the V71M mutation is highly conserved between aquaporin 2 and GlpF. The V71M mutation neither impairs substrate flux nor oligomerization of the aquaglyceroporin. Therefore, the human aquaporin 2 mutant V71M is most likely active, but cellular trafficking is probably impaired.

  13. GRIN1 mutation associated with intellectual disability alters NMDA receptor trafficking and function.

    PubMed

    Chen, Wenjuan; Shieh, Christine; Swanger, Sharon A; Tankovic, Anel; Au, Margaret; McGuire, Marianne; Tagliati, Michele; Graham, John M; Madan-Khetarpal, Suneeta; Traynelis, Stephen F; Yuan, Hongjie; Pierson, Tyler Mark

    2017-02-23

    N-methyl-d-aspartate receptors (NMDARs) play important roles in brain development and neurological disease. We report two individuals with similar dominant de novo GRIN1 mutations (c.1858 G>A and c.1858 G>C; both p.G620R). Both individuals presented at birth with developmental delay and hypotonia associated with behavioral abnormalities and stereotypical movements. Recombinant NMDARs containing the mutant GluN1-G620R together with either GluN2A or GluN2B were evaluated for changes in their trafficking to the plasma membrane and their electrophysiological properties. GluN1-G620R/GluN2A complexes showed a mild reduction in trafficking, a ~2-fold decrease in glutamate and glycine potency, a strong decrease in sensitivity to Mg(2+) block, and a significant reduction of current responses to a maximal effective concentration of agonists. GluN1-G620R/GluN2B complexes showed significantly reduced delivery of protein to the cell surface associated with similarly altered electrophysiology. These results indicate these individuals may have suffered neurodevelopmental deficits as a result of the decreased presence of GluN1-G620R/GluN2B complexes on the neuronal surface during embryonic brain development and reduced current responses of GluN1-G620R-containing NMDARs after birth. These cases emphasize the importance of comprehensive functional characterization of de novo mutations and illustrates how a combination of several distinct features of NMDAR expression, trafficking and function can be present and influence phenotype.Journal of Human Genetics advance online publication, 23 February 2017; doi:10.1038/jhg.2017.19.

  14. Distinct phenotype of a Wilson disease mutation reveals a novel trafficking determinant in the copper transporter ATP7B

    PubMed Central

    Braiterman, Lelita T.; Murthy, Amrutha; Jayakanthan, Samuel; Nyasae, Lydia; Tzeng, Eric; Gromadzka, Grazyna; Woolf, Thomas B.; Lutsenko, Svetlana; Hubbard, Ann L.

    2014-01-01

    Wilson disease (WD) is a monogenic autosomal-recessive disorder of copper accumulation that leads to liver failure and/or neurological deficits. WD is caused by mutations in ATP7B, a transporter that loads Cu(I) onto newly synthesized cupro-enzymes in the trans-Golgi network (TGN) and exports excess copper out of cells by trafficking from the TGN to the plasma membrane. To date, most WD mutations have been shown to disrupt ATP7B activity and/or stability. Using a multidisciplinary approach, including clinical analysis of patients, cell-based assays, and computational studies, we characterized a patient mutation, ATP7BS653Y, which is stable, does not disrupt Cu(I) transport, yet renders the protein unable to exit the TGN. Bulky or charged substitutions at position 653 mimic the phenotype of the patient mutation. Molecular modeling and dynamic simulation suggest that the S653Y mutation induces local distortions within the transmembrane (TM) domain 1 and alter TM1 interaction with TM2. S653Y abolishes the trafficking-stimulating effects of a secondary mutation in the N-terminal apical targeting domain. This result indicates a role for TM1/TM2 in regulating conformations of cytosolic domains involved in ATP7B trafficking. Taken together, our experiments revealed an unexpected role for TM1/TM2 in copper-regulated trafficking of ATP7B and defined a unique class of WD mutants that are transport-competent but trafficking-defective. Understanding the precise consequences of WD-causing mutations will facilitate the development of advanced mutation-specific therapies. PMID:24706876

  15. Matrilin-3 mutations that cause chondrodysplasias interfere with protein trafficking while a mutation associated with hand osteoarthritis does not

    PubMed Central

    Otten, C; Wagener, R; Paulsson, M; Zaucke, F

    2005-01-01

    Several mutations in the extracellular matrix protein matrilin-3 cause a heterogeneous disease spectrum affecting skeletal tissues. We introduced three disease causing point mutations leading to single amino acid exchanges (R116W, T298M, C299S) in matrilin-3 and expressed the corresponding proteins in primary articular chondrocytes to elucidate pathogenic mechanisms at the cellular level. Expression levels, processing, and the secretion pattern of a mutation linked to hand osteoarthritis (T298M) were similar to the wildtype protein, whereas the two other mutants were poorly expressed and hardly detectable in supernatants of transiently transfected cells. Using immunofluorescence staining, we demonstrated that mutants R116W and C299S are retained and accumulate within the endoplasmatic reticulum (ER). Their further trafficking to the Golgi compartment seems to be disturbed, whereas T298M is secreted normally. In cells transfected with the wildtype and T298M constructs, a matrilin-3 containing filamentous network was formed surrounding the cells, whereas in the case of R116W and C299S such structures were completely absent. These observations are similar to those for mutations in the cartilage oligomeric matrix protein (COMP) leading to multiple epiphyseal dysplasia and pseudoachondroplasia suggesting that retention and accumulation of cartilage proteins in the ER might be a general mechanism involved in the pathogenesis of chondrodysplasias. PMID:16199550

  16. Pharmacological Correction of Trafficking Defects in ATP-sensitive Potassium Channels Caused by Sulfonylurea Receptor 1 Mutations.

    PubMed

    Martin, Gregory M; Rex, Emily A; Devaraneni, Prasanna; Denton, Jerod S; Boodhansingh, Kara E; DeLeon, Diva D; Stanley, Charles A; Shyng, Show-Ling

    2016-10-14

    ATP-sensitive potassium (KATP) channels play a key role in mediating glucose-stimulated insulin secretion by coupling metabolic signals to β-cell membrane potential. Loss of KATP channel function due to mutations in ABCC8 or KCNJ11, genes encoding the sulfonylurea receptor 1 (SUR1) or the inwardly rectifying potassium channel Kir6.2, respectively, results in congenital hyperinsulinism. Many SUR1 mutations prevent trafficking of channel proteins from the endoplasmic reticulum to the cell surface. Channel inhibitors, including sulfonylureas and carbamazepine, have been shown to correct channel trafficking defects. In the present study, we identified 13 novel SUR1 mutations that cause channel trafficking defects, the majority of which are amenable to pharmacological rescue by glibenclamide and carbamazepine. By contrast, none of the mutant channels were rescued by KATP channel openers. Cross-linking experiments showed that KATP channel inhibitors promoted interactions between the N terminus of Kir6.2 and SUR1, whereas channel openers did not, suggesting the inhibitors enhance intersubunit interactions to overcome channel biogenesis and trafficking defects. Functional studies of rescued mutant channels indicate that most mutants rescued to the cell surface exhibited WT-like sensitivity to ATP, MgADP, and diazoxide. In intact cells, recovery of channel function upon trafficking rescue by reversible sulfonylureas or carbamazepine was facilitated by the KATP channel opener diazoxide. Our study expands the list of KATP channel trafficking mutations whose function can be recovered by pharmacological ligands and provides further insight into the structural mechanism by which channel inhibitors correct channel biogenesis and trafficking defects. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Congenital Hyperinsulinism–Associated ABCC8 Mutations That Cause Defective Trafficking of ATP-Sensitive K+ Channels

    PubMed Central

    Yan, Fei-Fei; Lin, Yu-Wen; MacMullen, Courtney; Ganguly, Arupa; Stanley, Charles A.; Shyng, Show-Ling

    2008-01-01

    Congenital hyperinsulinism (CHI) is a disease characterized by persistent insulin secretion despite severe hypoglycemia. Mutations in the pancreatic ATP-sensitive K+ (KATP) channel proteins sulfonylurea receptor 1 (SUR1) and Kir6.2, encoded by ABCC8 and KCNJ11, respectively, is the most common cause of the disease. Many mutations in SUR1 render the channel unable to traffic to the cell surface, thereby reducing channel function. Previous studies have shown that for some SUR1 trafficking mutants, the defects could be corrected by treating cells with sulfonylureas or diazoxide. The purpose of this study is to identify additional mutations that cause channel biogenesis/trafficking defects and those that are amenable to rescue by pharmacological chaperones. Fifteen previously uncharacterized CHI-associated missense SUR1 mutations were examined for their biogenesis/trafficking defects and responses to pharmacological chaperones, using a combination of immunological and functional assays. Twelve of the 15 mutations analyzed cause reduction in cell surface expression of KATP channels by >50%. Sulfonylureas rescued a subset of the trafficking mutants. By contrast, diazoxide failed to rescue any of the mutants. Strikingly, the mutations rescued by sulfonylureas are all located in the first transmembrane domain of SUR1, designated as TMD0. All TMD0 mutants rescued to the cell surface by the sulfonylurea tolbutamide could be subsequently activated by metabolic inhibition on tolbutamide removal. Our study identifies a group of CHI-causing SUR1 mutations for which the resulting KATP channel trafficking and expression defects may be corrected pharmacologically to restore channel function. PMID:17575084

  18. Mutations of Vasopressin Receptor 2 Including Novel L312S Have Differential Effects on Trafficking

    PubMed Central

    Tiulpakov, Anatoly; White, Carl W.; Abhayawardana, Rekhati S.; See, Heng B.; Chan, Audrey S.; Seeber, Ruth M.; Heng, Julian I.; Dedov, Ivan; Pavlos, Nathan J.

    2016-01-01

    Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a genetic disease first described in 2 unrelated male infants with severe symptomatic hyponatremia. Despite undetectable arginine vasopressin levels, patients have inappropriately concentrated urine resulting in hyponatremia, hypoosmolality, and natriuresis. Here, we describe and functionally characterize a novel vasopressin type 2 receptor (V2R) gain-of-function mutation. An L312S substitution in the seventh transmembrane domain was identified in a boy presenting with water-induced hyponatremic seizures at the age of 5.8 years. We show that, compared with wild-type V2R, the L312S mutation results in the constitutive production of cAMP, indicative of the gain-of-function NSIAD profile. Interestingly, like the previously described F229V and I130N NSIAD-causing mutants, this appears to both occur in the absence of notable constitutive β-arrestin2 recruitment and can be reduced by the inverse agonist Tolvaptan. In addition, to understand the effect of various V2R substitutions on the full receptor “life-cycle,” we have used and further developed a bioluminescence resonance energy transfer intracellular localization assay using multiple localization markers validated with confocal microscopy. This allowed us to characterize differences in the constitutive and ligand-induced localization and trafficking profiles of the novel L312S mutation as well as for previously described V2R gain-of-function mutants (NSIAD; R137C and R137L), loss-of-function mutants (nephrogenic diabetes insipidus; R137H, R181C, and M311V), and a putative silent V266A V2R polymorphism. In doing so, we describe differences in trafficking between unique V2R substitutions, even at the same amino acid position, therefore highlighting the value of full and thorough characterization of receptor function beyond simple signaling pathway analysis. PMID:27355191

  19. An Eye on Trafficking Genes: Identification of Four Eye Color Mutations in Drosophila.

    PubMed

    Grant, Paaqua; Maga, Tara; Loshakov, Anna; Singhal, Rishi; Wali, Aminah; Nwankwo, Jennifer; Baron, Kaitlin; Johnson, Diana

    2016-10-13

    Genes that code for proteins involved in organelle biogenesis and intracellular trafficking produce products that are critical in normal cell function . Conserved orthologs of these are present in most or all eukaryotes, including Drosophila melanogaster Some of these genes were originally identified as eye color mutants with decreases in both types of pigments found in the fly eye. These criteria were used for identification of such genes, four eye color mutations that are not annotated in the genome sequence: chocolate, maroon, mahogany, and red Malpighian tubules were molecularly mapped and their genome sequences have been evaluated. Mapping was performed using deletion analysis and complementation tests. chocolate is an allele of the VhaAC39-1 gene, which is an ortholog of the Vacuolar H(+) ATPase AC39 subunit 1. maroon corresponds to the Vps16A gene and its product is part of the HOPS complex, which participates in transport and organelle fusion. red Malpighian tubule is the CG12207 gene, which encodes a protein of unknown function that includes a LysM domain. mahogany is the CG13646 gene, which is predicted to be an amino acid transporter. The strategy of identifying eye color genes based on perturbations in quantities of both types of eye color pigments has proven useful in identifying proteins involved in trafficking and biogenesis of lysosome-related organelles. Mutants of these genes can form the basis of valuable in vivo models to understand these processes.

  20. An Eye on Trafficking Genes: Identification of Four Eye Color Mutations in Drosophila

    PubMed Central

    Grant, Paaqua; Maga, Tara; Loshakov, Anna; Singhal, Rishi; Wali, Aminah; Nwankwo, Jennifer; Baron, Kaitlin; Johnson, Diana

    2016-01-01

    Genes that code for proteins involved in organelle biogenesis and intracellular trafficking produce products that are critical in normal cell function . Conserved orthologs of these are present in most or all eukaryotes, including Drosophila melanogaster. Some of these genes were originally identified as eye color mutants with decreases in both types of pigments found in the fly eye. These criteria were used for identification of such genes, four eye color mutations that are not annotated in the genome sequence: chocolate, maroon, mahogany, and red Malpighian tubules were molecularly mapped and their genome sequences have been evaluated. Mapping was performed using deletion analysis and complementation tests. chocolate is an allele of the VhaAC39-1 gene, which is an ortholog of the Vacuolar H+ ATPase AC39 subunit 1. maroon corresponds to the Vps16A gene and its product is part of the HOPS complex, which participates in transport and organelle fusion. red Malpighian tubule is the CG12207 gene, which encodes a protein of unknown function that includes a LysM domain. mahogany is the CG13646 gene, which is predicted to be an amino acid transporter. The strategy of identifying eye color genes based on perturbations in quantities of both types of eye color pigments has proven useful in identifying proteins involved in trafficking and biogenesis of lysosome-related organelles. Mutants of these genes can form the basis of valuable in vivo models to understand these processes. PMID:27558665

  1. Impaired trafficking and subcellular localization of a mutant lactase associated with congenital lactase deficiency.

    PubMed

    Behrendt, Marc; Keiser, Markus; Hoch, Melanie; Naim, Hassan Y

    2009-06-01

    Congenital lactase deficiency (CLD) is a cause of disaccharide intolerance and malabsorption characterized by watery diarrhea in infants fed breast milk or lactose-containing formulas. The molecular basis of CLD is unknown. Mutations in the coding region of the brush border enzyme lactase phlorizin hydrolase (LPH) were found to cause CLD in a study of 19 Finnish families. We analyzed the effects of one of these mutations, G1363S, on LPH folding, trafficking, and function. We introduced a mutation into the LPH complementary DNA that resulted in the amino acid substitution G1363S. The mutant gene was transiently expressed in COS-1 cells, and the effects were assessed at the protein, structural, and subcellular levels. The mutant protein LPH-G1363S was misfolded and could not exit the endoplasmic reticulum. Interestingly, the mutation creates an additional N-glycosylation site that is characteristic of a temperature-sensitive protein. The intracellular transport and enzymatic activity, but not correct folding, of LPH-G1363S were partially restored by expression at 20 degrees C. However, a form of LPH that contains the mutations G1363S and N1361A, which eliminates the N-glycosylation site, did not restore the features of wild-type LPH. Thus, the additional glycosyl group is not required for the LPH-G1363S defects. This is the first characterization, at the molecular and subcellular levels, of a mutant form of LPH that is involved in the pathogenesis of CLD. Mutant LPH accumulates predominantly in the endoplasmic reticulum but can partially mature at a permissive temperature; these features are unique for a protein involved in a carbohydrate malabsorption defect implicating LPH.

  2. A novel mutation in DDR2 causing spondylo-meta-epiphyseal dysplasia with short limbs and abnormal calcifications (SMED-SL) results in defective intra-cellular trafficking

    PubMed Central

    2014-01-01

    Background The rare autosomal genetic disorder, Spondylo-meta-epiphyseal dysplasia with short limbs and abnormal calcifications (SMED-SL), is reported to be caused by missense or splice site mutations in the human discoidin domain receptor 2 (DDR2) gene. Previously our group has established that trafficking defects and loss of ligand binding are the underlying cellular mechanisms of several SMED-SL causing mutations. Here we report the clinical characteristics of two siblings of consanguineous marriage with suspected SMED-SL and identification of a novel disease-causing mutation in the DDR2 gene. Methods Clinical evaluation and radiography were performed to evaluate the patients. All the coding exons and splice sites of the DDR2 gene were sequenced by Sanger sequencing. Subcellular localization of the mutated DDR2 protein was determined by confocal microscopy, deglycosylation assay and Western blotting. DDR2 activity was measured by collagen activation and Western analysis. Results In addition to the typical features of SMED-SL, one of the patients has an eye phenotype including visual impairment due to optic atrophy. DNA sequencing revealed a novel homozygous dinucleotide deletion mutation (c.2468_2469delCT) on exon 18 of the DDR2 gene in both patients. The mutation resulted in a frameshift leading to an amino acid change at position S823 and a predicted premature termination of translation (p.S823Cfs*2). Subcellular localization of the mutant protein was analyzed in mammalian cell lines, and it was found to be largely retained in the endoplasmic reticulum (ER), which was further supported by its N-glycosylation profile. In keeping with its cellular mis-localization, the mutant protein was found to be deficient in collagen-induced receptor activation, suggesting protein trafficking defects as the major cellular mechanism underlying the loss of DDR2 function in our patients. Conclusions Our results indicate that the novel mutation results in defective trafficking

  3. A novel mutation in DDR2 causing spondylo-meta-epiphyseal dysplasia with short limbs and abnormal calcifications (SMED-SL) results in defective intra-cellular trafficking.

    PubMed

    Al-Kindi, Adila; Kizhakkedath, Praseetha; Xu, Huifang; John, Anne; Sayegh, Abeer Al; Ganesh, Anuradha; Al-Awadi, Maha; Al-Anbouri, Lamya; Al-Gazali, Lihadh; Leitinger, Birgit; Ali, Bassam R

    2014-04-11

    The rare autosomal genetic disorder, Spondylo-meta-epiphyseal dysplasia with short limbs and abnormal calcifications (SMED-SL), is reported to be caused by missense or splice site mutations in the human discoidin domain receptor 2 (DDR2) gene. Previously our group has established that trafficking defects and loss of ligand binding are the underlying cellular mechanisms of several SMED-SL causing mutations. Here we report the clinical characteristics of two siblings of consanguineous marriage with suspected SMED-SL and identification of a novel disease-causing mutation in the DDR2 gene. Clinical evaluation and radiography were performed to evaluate the patients. All the coding exons and splice sites of the DDR2 gene were sequenced by Sanger sequencing. Subcellular localization of the mutated DDR2 protein was determined by confocal microscopy, deglycosylation assay and Western blotting. DDR2 activity was measured by collagen activation and Western analysis. In addition to the typical features of SMED-SL, one of the patients has an eye phenotype including visual impairment due to optic atrophy. DNA sequencing revealed a novel homozygous dinucleotide deletion mutation (c.2468_2469delCT) on exon 18 of the DDR2 gene in both patients. The mutation resulted in a frameshift leading to an amino acid change at position S823 and a predicted premature termination of translation (p.S823Cfs*2). Subcellular localization of the mutant protein was analyzed in mammalian cell lines, and it was found to be largely retained in the endoplasmic reticulum (ER), which was further supported by its N-glycosylation profile. In keeping with its cellular mis-localization, the mutant protein was found to be deficient in collagen-induced receptor activation, suggesting protein trafficking defects as the major cellular mechanism underlying the loss of DDR2 function in our patients. Our results indicate that the novel mutation results in defective trafficking of the DDR2 protein leading to loss of

  4. Pathogenic Mechanism of an Autism-Associated Neuroligin Mutation Involves Altered AMPA-Receptor Trafficking

    PubMed Central

    Chanda, Soham; Aoto, Jason; Lee, Sung-Jin; Wernig, Marius; Südhof, Thomas C.

    2015-01-01

    Neuroligins are postsynaptic cell-adhesion molecules that bind to presynaptic neurexins. Although the general synaptic role of neuroligins is undisputed, their specific functions at a synapse remain unclear, even controversial. Moreover, many neuroligin gene mutations were associated with autism, but the pathophysiological relevance of these mutations is often unknown, and their mechanisms of action uninvestigated. Here, we examine the synaptic effects of an autism-associated neuroligin-4 substitution (called R704C) which mutates a cytoplasmic arginine residue that is conserved in all neuroligins. We show that the R704C mutation, when introduced into neuroligin-3, enhances the interaction between neuroligin-3 and AMPA-receptors, increases AMPA-receptor internalization, and decreases postsynaptic AMPA-receptor levels. When introduced into neuroligin-4, conversely, the R704C mutation unexpectedly elevated AMPA-receptor mediated synaptic responses. These results suggest a general functional link between neuroligins and AMPA-receptors, indicate that both neuroligin-3 and -4 act at excitatory synapses but perform surprisingly distinct functions, and demonstrate that the R704C mutation significantly impairs the normal function of neuroligin-4, thereby validating its pathogenicity. PMID:25778475

  5. Functional Rescue of Trafficking-Impaired ABCB4 Mutants by Chemical Chaperones.

    PubMed

    Gordo-Gilart, Raquel; Andueza, Sara; Hierro, Loreto; Jara, Paloma; Alvarez, Luis

    2016-01-01

    Multidrug resistance protein 3 (MDR3, ABCB4) is a hepatocellular membrane protein that mediates biliary secretion of phosphatidylcholine. Null mutations in ABCB4 gene give rise to severe early-onset cholestatic liver disease. We have previously shown that the disease-associated mutations p.G68R, p.G228R, p.D459H, and p.A934T resulted in retention of ABCB4 in the endoplasmic reticulum, thus failing to target the plasma membrane. In the present study, we tested the ability of two compounds with chaperone-like activity, 4-phenylbutyrate and curcumin, to rescue these ABCB4 mutants by assessing their effects on subcellular localization, protein maturation, and phospholipid efflux capability. Incubation of transfected cells at a reduced temperature (30°C) or exposure to pharmacological doses of either 4-PBA or curcumin restored cell surface expression of mutants G228R and A934T. The delivery of these mutants to the plasma membrane was accompanied by a switch in the ratio of mature to inmature protein forms, leading to a predominant expression of the mature protein. This effect was due to an improvement in the maturation rate and not to the stabilization of the mature forms. Both mutants were also functionally rescued, displaying bile salt-dependent phospholipid efflux activity after addition of 4-PBA or curcumin. Drug-induced rescue was mutant specific, given neither 4-PBA nor curcumin had an effect on the ABCB4 mutants G68R and A934T. Collectively, these data indicate that the functionality of selected trafficking-defective ABCB4 mutants can be recovered by chemical chaperones through restoration of membrane localization, suggesting a potential treatment for patients carrying such mutations.

  6. Prevalence of mitochondrial gene mutations among hearing impaired patients

    PubMed Central

    Usami, S.; Abe, S.; Akita, J.; Namba, A.; Shinkawa, H.; Ishii, M.; Iwasaki, S.; Hoshino, T.; Ito, J.; Doi, K.; Kubo, T.; Nakagawa, T.; Komiyama, S.; Tono, T.; Komune, S.

    2000-01-01

    The frequency of three mitochondrial point mutations, 1555A→G, 3243A→G, and 7445A→G, known to be associated with hearing impairment, was examined using restriction fragment length polymorphism (RFLP) analysis in two Japanese groups: (1) 319 unrelated SNHL outpatients (including 21 with aminoglycoside antibiotic injection history), and (2) 140 cochlear implantation patients (including 22 with aminoglycoside induced hearing loss). Approximately 3% of the outpatients and 10% of the cochlear implantation patients had the 1555A→G mutation. The frequency was higher in the patients with a history of aminoglycoside injection (outpatient group 33%, cochlear implantation group 59%). One outpatient (0.314%) had the 3243A→G mutation, but no outpatients had the 7445A→G mutation and neither were found in the cochlear implantation group. The significance of the 1555A→G mutation, the most prevalent mitochondrial mutation found in this study of a hearing impaired population in Japan, among subjects with specific backgrounds, such as aminoglycoside induced hearing loss, is evident.


Keywords: mitochondria; point mutation; hearing impairment; frequencies PMID:10633132

  7. RCAN1 links impaired neurotrophin trafficking to aberrant development of the sympathetic nervous system in Down syndrome

    PubMed Central

    Patel, Ami; Yamashita, Naoya; Ascaño, Maria; Bodmer, Daniel; Boehm, Erica; Bodkin-Clarke, Chantal; Ryu, Yun Kyoung; Kuruvilla, Rejji

    2015-01-01

    Down syndrome is the most common chromosomal disorder affecting the nervous system in humans. To date, investigations of neural anomalies in Down syndrome have focused on the central nervous system, although dysfunction of the peripheral nervous system is a common manifestation. The molecular and cellular bases underlying peripheral abnormalities have remained undefined. Here, we report the developmental loss of sympathetic innervation in human Down syndrome organs and in a mouse model. We show that excess regulator of calcineurin 1 (RCAN1), an endogenous inhibitor of the calcineurin phosphatase that is triplicated in Down syndrome, impairs neurotrophic support of sympathetic neurons by inhibiting endocytosis of the nerve growth factor (NGF) receptor, TrkA. Genetically correcting RCAN1 levels in Down syndrome mice markedly improves NGF-dependent receptor trafficking, neuronal survival and innervation. These results uncover a critical link between calcineurin signalling, impaired neurotrophin trafficking and neurodevelopmental deficits in the peripheral nervous system in Down syndrome. PMID:26658127

  8. The HIV-1 protein Vpr impairs phagosome maturation by controlling microtubule-dependent trafficking

    PubMed Central

    Dumas, Audrey; Lê-Bury, Gabrielle; Marie-Anaïs, Florence; Herit, Floriane; Mazzolini, Julie; Guilbert, Thomas; Bourdoncle, Pierre; Russell, David G.; Benichou, Serge; Zahraoui, Ahmed

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) impairs major functions of macrophages but the molecular basis for this defect remains poorly characterized. Here, we show that macrophages infected with HIV-1 were unable to respond efficiently to phagocytic triggers and to clear bacteria. The maturation of phagosomes, defined by the presence of late endocytic markers, hydrolases, and reactive oxygen species, was perturbed in HIV-1–infected macrophages. We showed that maturation arrest occurred at the level of the EHD3/MICAL-L1 endosomal sorting machinery. Unexpectedly, we found that the regulatory viral protein (Vpr) was crucial to perturb phagosome maturation. Our data reveal that Vpr interacted with EB1, p150Glued, and dynein heavy chain and was sufficient to critically alter the microtubule plus end localization of EB1 and p150Glued, hence altering the centripetal movement of phagosomes and their maturation. Thus, we identify Vpr as a modulator of the microtubule-dependent endocytic trafficking in HIV-1–infected macrophages, leading to strong alterations in phagolysosome biogenesis. PMID:26504171

  9. Neuronal vesicular trafficking and release in age-related cognitive impairment.

    PubMed

    Deák, Ferenc

    2014-11-01

    Aging is a common major risk factor for many neurological disorders resulting in cognitive impairment and neurodegeneration including Parkinson's and Alzheimer's diseases. Novel results from the fields of molecular neuroscience and aging research provide evidence for a link between decline of various cognitive, executive functions and changes in neuronal mechanisms of intracellular trafficking and regulated vesicle release processes in the aging nervous system. In this Perspective, we review these recent findings and formulate a hypothesis on how cargo delivery to the synapses and the release of neurotrophic factors may be involved in maintaining learning and memory capabilities during healthy aging and present examples on how defects of those disrupt normal cognition. We provide an overview of emerging new concepts and approaches that will significantly advance our understanding of the aging brain and pathophysiology of dementia. This knowledge will be instrumental in defining drug targets and designing novel therapeutic strategies. © The Author 2014. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix.

    PubMed

    Ambrosi, Cinzia; Walker, Amy E; Depriest, Adam D; Cone, Angela C; Lu, Connie; Badger, John; Skerrett, I Martha; Sosinsky, Gina E

    2013-01-01

    Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased

  11. Analysis of Trafficking, Stability and Function of Human Connexin 26 Gap Junction Channels with Deafness-Causing Mutations in the Fourth Transmembrane Helix

    PubMed Central

    Ambrosi, Cinzia; Walker, Amy E.; DePriest, Adam D.; Cone, Angela C.; Lu, Connie; Badger, John; Skerrett, I. Martha; Sosinsky, Gina E.

    2013-01-01

    Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased

  12. Disruption of endocytic trafficking in frontotemporal dementia with CHMP2B mutations

    PubMed Central

    Urwin, Hazel; Authier, Astrid; Nielsen, Jorgen E.; Metcalf, Daniel; Powell, Caroline; Froud, Kristina; Malcolm, Denise S.; Holm, Ida; Johannsen, Peter; Brown, Jeremy; Fisher, Elizabeth M.C.; van der Zee, Julie; Bruyland, Marc; Van Broeckhoven, Christine; Collinge, John; Brandner, Sebastian; Futter, Clare; Isaacs, Adrian M.

    2010-01-01

    Mutations in CHMP2B cause frontotemporal dementia (FTD) in a large Danish pedigree, which is termed FTD linked to chromosome 3 (FTD-3), and also in an unrelated familial FTD patient. CHMP2B is a component of the ESCRT-III complex, which is required for function of the multivesicular body (MVB), an endosomal structure that fuses with the lysosome to degrade endocytosed proteins. We report a novel endosomal pathology in CHMP2B mutation-positive patient brains and also identify and characterize abnormal endosomes in patient fibroblasts. Functional studies demonstrate a specific disruption of endosome–lysosome fusion but not protein sorting by the MVB. We provide evidence for a mechanism for impaired endosome–lysosome fusion whereby mutant CHMP2B constitutively binds to MVBs and prevents recruitment of proteins necessary for fusion to occur, such as Rab7. The fusion of endosomes with lysosomes is required for neuronal function and the data presented therefore suggest a pathogenic mechanism for FTD caused by CHMP2B mutations. PMID:20223751

  13. Disruption of endocytic trafficking in frontotemporal dementia with CHMP2B mutations.

    PubMed

    Urwin, Hazel; Authier, Astrid; Nielsen, Jorgen E; Metcalf, Daniel; Powell, Caroline; Froud, Kristina; Malcolm, Denise S; Holm, Ida; Johannsen, Peter; Brown, Jeremy; Fisher, Elizabeth M C; van der Zee, Julie; Bruyland, Marc; Van Broeckhoven, Christine; Collinge, John; Brandner, Sebastian; Futter, Clare; Isaacs, Adrian M

    2010-06-01

    Mutations in CHMP2B cause frontotemporal dementia (FTD) in a large Danish pedigree, which is termed FTD linked to chromosome 3 (FTD-3), and also in an unrelated familial FTD patient. CHMP2B is a component of the ESCRT-III complex, which is required for function of the multivesicular body (MVB), an endosomal structure that fuses with the lysosome to degrade endocytosed proteins. We report a novel endosomal pathology in CHMP2B mutation-positive patient brains and also identify and characterize abnormal endosomes in patient fibroblasts. Functional studies demonstrate a specific disruption of endosome-lysosome fusion but not protein sorting by the MVB. We provide evidence for a mechanism for impaired endosome-lysosome fusion whereby mutant CHMP2B constitutively binds to MVBs and prevents recruitment of proteins necessary for fusion to occur, such as Rab7. The fusion of endosomes with lysosomes is required for neuronal function and the data presented therefore suggest a pathogenic mechanism for FTD caused by CHMP2B mutations.

  14. Mutant Huntingtin Impairs Post-Golgi Trafficking to Lysosomes by Delocalizing Optineurin/Rab8 Complex from the Golgi Apparatus

    PubMed Central

    del Toro, Daniel; Alberch, Jordi; Lázaro-Diéguez, Francisco; Martín-Ibáñez, Raquel; Xifró, Xavier; Egea, Gustavo

    2009-01-01

    Huntingtin regulates post-Golgi trafficking of secreted proteins. Here, we studied the mechanism by which mutant huntingtin impairs this process. Colocalization studies and Western blot analysis of isolated Golgi membranes showed a reduction of huntingtin in the Golgi apparatus of cells expressing mutant huntingtin. These findings correlated with a decrease in the levels of optineurin and Rab8 in the Golgi apparatus that can be reverted by overexpression of full-length wild-type huntingtin. In addition, immunoprecipitation studies showed reduced interaction between mutant huntingtin and optineurin/Rab8. Cells expressing mutant huntingtin produced both an accumulation of clathrin adaptor complex 1 at the Golgi and an increase of clathrin-coated vesicles in the vicinity of Golgi cisternae as revealed by electron microscopy. Furthermore, inverse fluorescence recovery after photobleaching analysis for lysosomal-associated membrane protein-1 and mannose-6-phosphate receptor showed that the optineurin/Rab8-dependent post-Golgi trafficking to lysosomes was impaired in cells expressing mutant huntingtin or reducing huntingtin levels by small interfering RNA. Accordingly, these cells showed a lower content of cathepsin D in lysosomes, which led to an overall reduction of lysosomal activity. Together, our results indicate that mutant huntingtin perturbs post-Golgi trafficking to lysosomal compartments by delocalizing the optineurin/Rab8 complex, which, in turn, affects the lysosomal function. PMID:19144827

  15. Exome Sequence Data From Multigenerational Families Implicate AMPA Receptor Trafficking in Neurocognitive Impairment and Schizophrenia Risk.

    PubMed

    Kos, Mark Z; Carless, Melanie A; Peralta, Juan; Blackburn, August; Almeida, Marcio; Roalf, David; Pogue-Geile, Michael F; Prasad, Konasale; Gur, Ruben C; Nimgaonkar, Vishwajit; Curran, Joanne E; Duggirala, Ravi; Glahn, David C; Blangero, John; Gur, Raquel E; Almasy, Laura

    2016-03-01

    Schizophrenia is a mental disorder characterized by impairments in behavior, thought, and neurocognitive performance. We searched for susceptibility loci at a quantitative trait locus (QTL) previously reported for abstraction and mental flexibility (ABF), a cognitive function often compromised in schizophrenia patients and their unaffected relatives. Exome sequences were determined for 134 samples in 8 European American families from the original linkage study, including 25 individuals with schizophrenia or schizoaffective disorder. At chromosome 5q32-35.3, we analyzed 407 protein-altering variants for association with ABF and schizophrenia status. For replication, significant, Bonferroni-corrected findings were tested against cognitive traits in Mexican American families (n = 959), as well as interrogated for schizophrenia risk using GWAS results from the Psychiatric Genomics Consortium (PGC). From the gene SYNPO, rs6579797 (MAF = 0.032) shows significant associations with ABF (P = .015) and schizophrenia (P = .040), as well as jointly (P = .0027). In the Mexican American pedigrees, rs6579797 exhibits significant associations with IQ (P = .011), indicating more global effects on neurocognition. From the PGC results, other SYNPO variants were identified with near significant effects on schizophrenia risk, with a local linkage disequilibrium block displaying signatures of positive selection. A second missense variant within the QTL, rs17551608 (MAF = 0.19) in the gene WWC1, also displays a significant effect on schizophrenia in our exome sequences (P = .038). Remarkably, the protein products of SYNPO and WWC1 are interaction partners involved in AMPA receptor trafficking, a brain process implicated in synaptic plasticity. Our study reveals variants in these genes with significant effects on neurocognition and schizophrenia risk, identifying a potential pathogenic mechanism for schizophrenia spectrum disorders.

  16. Exome Sequence Data From Multigenerational Families Implicate AMPA Receptor Trafficking in Neurocognitive Impairment and Schizophrenia Risk

    PubMed Central

    Kos, Mark Z.; Carless, Melanie A.; Peralta, Juan; Blackburn, August; Almeida, Marcio; Roalf, David; Pogue-Geile, Michael F.; Prasad, Konasale; Gur, Ruben C.; Nimgaonkar, Vishwajit; Curran, Joanne E.; Duggirala, Ravi; Glahn, David C.; Blangero, John; Gur, Raquel E.; Almasy, Laura

    2016-01-01

    Schizophrenia is a mental disorder characterized by impairments in behavior, thought, and neurocognitive performance. We searched for susceptibility loci at a quantitative trait locus (QTL) previously reported for abstraction and mental flexibility (ABF), a cognitive function often compromised in schizophrenia patients and their unaffected relatives. Exome sequences were determined for 134 samples in 8 European American families from the original linkage study, including 25 individuals with schizophrenia or schizoaffective disorder. At chromosome 5q32–35.3, we analyzed 407 protein-altering variants for association with ABF and schizophrenia status. For replication, significant, Bonferroni-corrected findings were tested against cognitive traits in Mexican American families (n = 959), as well as interrogated for schizophrenia risk using GWAS results from the Psychiatric Genomics Consortium (PGC). From the gene SYNPO, rs6579797 (MAF = 0.032) shows significant associations with ABF (P = .015) and schizophrenia (P = .040), as well as jointly (P = .0027). In the Mexican American pedigrees, rs6579797 exhibits significant associations with IQ (P = .011), indicating more global effects on neurocognition. From the PGC results, other SYNPO variants were identified with near significant effects on schizophrenia risk, with a local linkage disequilibrium block displaying signatures of positive selection. A second missense variant within the QTL, rs17551608 (MAF = 0.19) in the gene WWC1, also displays a significant effect on schizophrenia in our exome sequences (P = .038). Remarkably, the protein products of SYNPO and WWC1 are interaction partners involved in AMPA receptor trafficking, a brain process implicated in synaptic plasticity. Our study reveals variants in these genes with significant effects on neurocognition and schizophrenia risk, identifying a potential pathogenic mechanism for schizophrenia spectrum disorders. PMID:26405221

  17. The KCNH2-IVS9-28A/G mutation causes aberrant isoform expression and hERG trafficking defect in cardiomyocytes derived from patients affected by Long QT Syndrome type 2.

    PubMed

    Mura, Manuela; Mehta, Ashish; Ramachandra, Chrishan J; Zappatore, Rita; Pisano, Federica; Ciuffreda, Maria Chiara; Barbaccia, Vincenzo; Crotti, Lia; Schwartz, Peter J; Shim, Winston; Gnecchi, Massimiliano

    2017-08-01

    Long QT Syndrome type 2 (LQT2) is caused by mutations in the KCNH2 gene that encodes for the α-subunit (hERG) of the ion channel conducting the rapid delayed rectifier potassium current (IKr). We have previously identified a disease causing mutation (IVS9-28A/G) in the branch point of the splicing of KCNH2 intron 9. However, the mechanism through which this mutation causes the disease is unknown. We generated human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from fibroblasts of two IVS9-28A/G mutation carriers. IVS9-28A/G iPSC-CMs showed prolonged repolarization time, mimicking what observed at the ECG level in the same patients. The expression of the full-length ERG1a isoform resulted reduced, whereas the C-terminally truncated ERG1aUSO isoform was upregulated in mutant iPSC-CMs, with consequent alteration of the physiological ERG1aUSO/ERG1a ratio. Importantly, we observed an impairment of hERG trafficking to the cell membrane. The severity of the alterations in hERG expression and trafficking correlated with the clinical severity of the disease in the two patients under study. Finally, we were able to revert the trafficking defect and reduce the repolarization duration in LQT2 iPSC-CMs using the proteasome inhibitor ALLN. Our results highlight the key role of the KCNH2 intron 9 branch point in the regulation of KCNH2 isoform expression and hERG channel function, and allow to categorize the IVS9-28A/G mutation as LQT2 class 2 mutation. These findings may result in a more personalized clinical management of IVS9-28A/G mutation carriers. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Mutations in Bruton's tyrosine kinase impair IgA responses.

    PubMed

    Mitsuiki, Noriko; Yang, Xi; Bartol, Sophinus J W; Grosserichter-Wagener, Christina; Kosaka, Yoshiyuki; Takada, Hidetoshi; Imai, Kohsuke; Kanegane, Hirokazu; Mizutani, Shuki; van der Burg, Mirjam; van Zelm, Menno C; Ohara, Osamu; Morio, Tomohiro

    2015-03-01

    X-linked agammaglobulinemia (XLA) is a primary immunodeficiency caused by mutations in Bruton's tyrosine kinase (BTK), and is characterized by markedly decreased numbers of blood B cells and an absence of all immunoglobulin isotypes. We performed whole exome sequencing in a male pediatric patient with dysgammaglobulinemia with IgA deficiency. Genetic analysis revealed a BTK missense mutation (Thr316Ala). To investigate whether a BTK mutation underlay this antibody deficiency with marked decrease of IgA in this patient, we performed functional analyses of B cells and phagocytes, and molecular analyses of somatic hypermutation and class switch recombination. The BTK missense mutation resulted in B cells with reduced BTK and high IgM expression. Equal proportions of CD19(low) and CD19(normal) fractions were observed, and both included naïve and memory B cells. Calcium influx and phospholipase Cγ2 phosphorylation upon IgM stimulation were marginally impaired in CD19(low), but not in CD19(+) B cells. Similar to XLA patients, IgA transcripts showed low SHM levels, whereas IgG transcripts were hardly affected. Our analyses suggest that the BTK mutation likely underlies the disease in this case, and that hypomorphic BTK mutations can result in normal circulating B cell numbers, but specifically impair IgA responses.

  19. An ancient founder mutation in PROKR2 impairs human reproduction.

    PubMed

    Avbelj Stefanija, Magdalena; Jeanpierre, Marc; Sykiotis, Gerasimos P; Young, Jacques; Quinton, Richard; Abreu, Ana Paula; Plummer, Lacey; Au, Margaret G; Balasubramanian, Ravikumar; Dwyer, Andrew A; Florez, Jose C; Cheetham, Timothy; Pearce, Simon H; Purushothaman, Radhika; Schinzel, Albert; Pugeat, Michel; Jacobson-Dickman, Elka E; Ten, Svetlana; Latronico, Ana Claudia; Gusella, James F; Dode, Catherine; Crowley, William F; Pitteloud, Nelly

    2012-10-01

    Congenital gonadotropin-releasing hormone (GnRH) deficiency manifests as absent or incomplete sexual maturation and infertility. Although the disease exhibits marked locus and allelic heterogeneity, with the causal mutations being both rare and private, one causal mutation in the prokineticin receptor, PROKR2 L173R, appears unusually prevalent among GnRH-deficient patients of diverse geographic and ethnic origins. To track the genetic ancestry of PROKR2 L173R, haplotype mapping was performed in 22 unrelated patients with GnRH deficiency carrying L173R and their 30 first-degree relatives. The mutation's age was estimated using a haplotype-decay model. Thirteen subjects were informative and in all of them the mutation was present on the same ~123 kb haplotype whose population frequency is ≤10%. Thus, PROKR2 L173R represents a founder mutation whose age is estimated at approximately 9000 years. Inheritance of PROKR2 L173R-associated GnRH deficiency was complex with highly variable penetrance among carriers, influenced by additional mutations in the other PROKR2 allele (recessive inheritance) or another gene (digenicity). The paradoxical identification of an ancient founder mutation that impairs reproduction has intriguing implications for the inheritance mechanisms of PROKR2 L173R-associated GnRH deficiency and for the relevant processes of evolutionary selection, including potential selective advantages of mutation carriers in genes affecting reproduction.

  20. A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

    PubMed Central

    Zhang, Michael L.; Sinner, Moritz F.; Dolmatova, Elena V.; Tucker, Nathan R.; McLellan, Micheal; Shea, Marisa A.; Milan, David J.; Lunetta, Kathryn L.; Benjamin, Emelia J.; Ellinor, Patrick T.

    2014-01-01

    Background Atrial fibrillation (AF) is the most common arrhythmia, and a recent genome-wide association study identified HCN4 as a novel AF susceptibility locus. HCN4 encodes for the cardiac pacemaker channel and HCN4 mutations are associated with familial sinus bradycardia and AF. Objective To determine whether novel variants in the coding region of HCN4 contribute to the susceptibility for AF. Methods We sequenced the coding region of HCN4 for novel variants from 527 cases with early-onset AF from the Massachusetts General Hospital AF Study and 443 referents from the Framingham Heart Study. We used site-directed mutagenesis, cellular electrophysiology, immunocytochemistry and confocal microscopy to functionally characterize novel variants. Results We found the frequency of novel coding HCN4 variants was 2-fold greater for individuals with AF (seven variants) compared to the referents (three variants). We determined that one, (p.Pro257Ser, located in the amino-terminus adjacent to the first transmembrane spanning domain) of the seven novel HCN4 variants in our AF cases did not traffick to cell membrane while the remaining six were not functionally different from wild type. Also, the three novel variants in our referents did not alter function compared to wild type. Co-expression studies showed that the p.Pro257Ser mutant channel failed to co-localize with the wild type HCN4 channel on the cell membrane. Conclusion Our findings are consistent with HCN4 haploinsufficiency as the likely mechanism for early-onset AF in the p.Pro257Ser carrier. PMID:24607718

  1. A vital region for human glycoprotein hormone trafficking revealed by an LHB mutation.

    PubMed

    Potorac, Iulia; Rivero-Müller, Adolfo; Trehan, Ashutosh; Kiełbus, Michał; Jozwiak, Krzysztof; Pralong, Francois; Hafidi, Aicha; Thiry, Albert; Ménagé, Jean-Jacques; Huhtaniemi, Ilpo; Beckers, Albert; Daly, Adrian F

    2016-12-01

    Glycoprotein hormones are complex hormonally active macromolecules. Luteinizing hormone (LH) is essential for the postnatal development and maturation of the male gonad. Inactivating Luteinizing hormone beta (LHB) gene mutations are exceptionally rare and lead to hypogonadism that is particularly severe in males. We describe a family with selective LH deficiency and hypogonadism in two brothers. DNA sequencing of LHB was performed and the effects of genetic variants on hormone function and secretion were characterized by mutagenesis studies, confocal microscopy and functional assays. A 20-year-old male from a consanguineous family had pubertal delay, hypogonadism and undetectable LH. A homozygous c.118_120del (p.Lys40del) mutation was identified in the patient and his brother, who subsequently had the same phenotype. Treatment with hCG led to pubertal development, increased circulating testosterone and spermatogenesis. Experiments in HeLa cells revealed that the mutant LH is retained intracellularly and showed diffuse cytoplasmic distribution. The mutated LHB heterodimerizes with the common alpha-subunit and can activate its receptor. Deletion of flanking glutamic acid residues at positions 39 and 41 impair LH to a similar extent as deletion of Lys40. This region is functionally important across all heterodimeric glycoprotein hormones, because deletion of the corresponding residues in hCG, follicle-stimulating hormone and thyroid-stimulating hormone beta-subunits also led to intracellular hormone retention. This novel LHB mutation results in hypogonadism due to intracellular sequestration of the hormone and reveals a discrete region in the protein that is crucial for normal secretion of all human glycoprotein hormones.

  2. Inherited human sex reversal due to impaired nucleocytoplasmic trafficking of SRY defines a male transcriptional threshold.

    PubMed

    Chen, Yen-Shan; Racca, Joseph D; Phillips, Nelson B; Weiss, Michael A

    2013-09-17

    Human testis determination is initiated by SRY (sex determining region on Y chromosome). Mutations in SRY cause gonadal dysgenesis with female somatic phenotype. Two subtle variants (V60L and I90M in the high-mobility group box) define inherited alleles shared by an XY sterile daughter and fertile father. Whereas specific DNA binding and bending are unaffected in a rat embryonic pre-Sertoli cell line, the variants exhibited selective defects in nucleocytoplasmic shuttling due to impaired nuclear import (V60L; mediated by Exportin-4) or export (I90M; mediated by chromosome region maintenance 1). Decreased shuttling limits nuclear accumulation of phosphorylated (activated) SRY, in turn reducing occupancy of DNA sites regulating Sertoli-cell differentiation [the testis-specific SRY-box 9 (Sox9) enhancer]. Despite distinct patterns of biochemical and cell-biological perturbations, V60L and I90M each attenuated Sox9 expression in transient transfection assays by twofold. Such attenuation was also observed in studies of V60A, a clinical variant associated with ovotestes and hence ambiguity between divergent cell fates. This shared twofold threshold is reminiscent of autosomal syndromes of transcription-factor haploinsufficiency, including XY sex reversal associated with mutations in SOX9. Our results demonstrate that nucleocytoplasmic shuttling of SRY is necessary for robust initiation of testicular development. Although also characteristic of ungulate orthologs, such shuttling is not conserved among rodents wherein impaired nuclear export of the high-mobility group box and import-dependent phosphorylation are compensated by a microsatellite-associated transcriptional activation domain. Human sex reversal due to subtle defects in the nucleocytoplasmic shuttling of SRY suggests that its transcriptional activity lies near the edge of developmental ambiguity.

  3. Inherited human sex reversal due to impaired nucleocytoplasmic trafficking of SRY defines a male transcriptional threshold

    PubMed Central

    Chen, Yen-Shan; Racca, Joseph D.; Phillips, Nelson B.; Weiss, Michael A.

    2013-01-01

    Human testis determination is initiated by SRY (sex determining region on Y chromosome). Mutations in SRY cause gonadal dysgenesis with female somatic phenotype. Two subtle variants (V60L and I90M in the high-mobility group box) define inherited alleles shared by an XY sterile daughter and fertile father. Whereas specific DNA binding and bending are unaffected in a rat embryonic pre-Sertoli cell line, the variants exhibited selective defects in nucleocytoplasmic shuttling due to impaired nuclear import (V60L; mediated by Exportin-4) or export (I90M; mediated by chromosome region maintenance 1). Decreased shuttling limits nuclear accumulation of phosphorylated (activated) SRY, in turn reducing occupancy of DNA sites regulating Sertoli-cell differentiation [the testis-specific SRY-box 9 (Sox9) enhancer]. Despite distinct patterns of biochemical and cell-biological perturbations, V60L and I90M each attenuated Sox9 expression in transient transfection assays by twofold. Such attenuation was also observed in studies of V60A, a clinical variant associated with ovotestes and hence ambiguity between divergent cell fates. This shared twofold threshold is reminiscent of autosomal syndromes of transcription-factor haploinsufficiency, including XY sex reversal associated with mutations in SOX9. Our results demonstrate that nucleocytoplasmic shuttling of SRY is necessary for robust initiation of testicular development. Although also characteristic of ungulate orthologs, such shuttling is not conserved among rodents wherein impaired nuclear export of the high-mobility group box and import-dependent phosphorylation are compensated by a microsatellite-associated transcriptional activation domain. Human sex reversal due to subtle defects in the nucleocytoplasmic shuttling of SRY suggests that its transcriptional activity lies near the edge of developmental ambiguity. PMID:24003159

  4. Overexpression of EphB2 in hippocampus rescues impaired NMDA receptors trafficking and cognitive dysfunction in Alzheimer model.

    PubMed

    Hu, Rui; Wei, Pan; Jin, Lu; Zheng, Teng; Chen, Wen-Yu; Liu, Xiao-Ya; Shi, Xiao-Dong; Hao, Jing-Ru; Sun, Nan; Gao, Can

    2017-03-30

    Alzheimer's disease (AD) is a progressive neurodegenerative disease, which affects more and more people. But there is still no effective treatment for preventing or reversing the progression of the disease. Soluble amyloid-beta (Aβ) oligomers, also known as Aβ-derived diffusible ligands (ADDLs) play an important role in AD. Synaptic activity and cognition critically depend on the function of glutamate receptors. Targeting N-methyl-D-aspartic acid (NMDA) receptors trafficking and its regulation is a new strategy for AD early treatment. EphB2 is a key regulator of synaptic localization of NMDA receptors. Aβ oligomers could bind to the fibronectin repeats domain of EphB2 and trigger EphB2 degradation in the proteasome. Here we identified that overexpression of EphB2 with lentiviral vectors in dorsal hippocampus improved impaired memory deficits and anxiety or depression-like behaviors in APPswe/PS1-dE9 (APP/PS1) transgenic mice. Phosphorylation and surface expression of GluN2B-containing NMDA receptors were also improved. Overexpression of EphB2 also rescued the ADDLs-induced depletion of the expression of EphB2 and GluN2B-containing NMDA receptors trafficking in cultured hippocampal neurons. These results suggest that improving the decreased expression of EphB2 and subsequent GluN2B-containing NMDA receptors trafficking in hippocampus may be a promising strategy for AD treatment.

  5. Progesterone Impairs Human Ether-a-go-go-related Gene (HERG) Trafficking by Disruption of Intracellular Cholesterol Homeostasis*

    PubMed Central

    Wu, Zhi-Yuan; Yu, De-Jie; Soong, Tuck Wah; Dawe, Gavin S.; Bian, Jin-Song

    2011-01-01

    The prolongation of QT intervals in both mothers and fetuses during the later period of pregnancy implies that higher levels of progesterone may regulate the function of the human ether-a-go-go-related gene (HERG) potassium channel, a key ion channel responsible for controlling the length of QT intervals. Here, we studied the effect of progesterone on the expression, trafficking, and function of HERG channels and the underlying mechanism. Treatment with progesterone for 24 h decreased the abundance of the fully glycosylated form of the HERG channel in rat neonatal cardiac myocytes and HERG-HEK293 cells, a cell line stably expressing HERG channels. Progesterone also concentration-dependently decreased HERG current density, but had no effect on voltage-gated L-type Ca2+ and K+ channels. Immunofluorescence microscopy and Western blot analysis show that progesterone preferentially decreased HERG channel protein abundance in the plasma membrane, induced protein accumulation in the dilated endoplasmic reticulum (ER), and increased the protein expression of C/EBP homologous protein, a hallmark of ER stress. Application of 2-hydroxypropyl-β-cyclodextrin (a sterol-binding agent) or overexpression of Rab9 rescued the progesterone-induced HERG trafficking defect and ER stress. Disruption of intracellular cholesterol homeostasis with simvastatin, imipramine, or exogenous application of cholesterol mimicked the effect of progesterone on HERG channel trafficking. Progesterone may impair HERG channel folding in the ER and/or block its trafficking to the Golgi complex by disrupting intracellular cholesterol homeostasis. Our findings may reveal a novel molecular mechanism to explain the QT prolongation and high risk of developing arrhythmias during late pregnancy. PMID:21525004

  6. Foxp2 mutations impair auditory-motor association learning.

    PubMed

    Kurt, Simone; Fisher, Simon E; Ehret, Günter

    2012-01-01

    Heterozygous mutations of the human FOXP2 transcription factor gene cause the best-described examples of monogenic speech and language disorders. Acquisition of proficient spoken language involves auditory-guided vocal learning, a specialized form of sensory-motor association learning. The impact of etiological Foxp2 mutations on learning of auditory-motor associations in mammals has not been determined yet. Here, we directly assess this type of learning using a newly developed conditioned avoidance paradigm in a shuttle-box for mice. We show striking deficits in mice heterozygous for either of two different Foxp2 mutations previously implicated in human speech disorders. Both mutations cause delays in acquiring new motor skills. The magnitude of impairments in association learning, however, depends on the nature of the mutation. Mice with a missense mutation in the DNA-binding domain are able to learn, but at a much slower rate than wild type animals, while mice carrying an early nonsense mutation learn very little. These results are consistent with expression of Foxp2 in distributed circuits of the cortex, striatum and cerebellum that are known to play key roles in acquisition of motor skills and sensory-motor association learning, and suggest differing in vivo effects for distinct variants of the Foxp2 protein. Given the importance of such networks for the acquisition of human spoken language, and the fact that similar mutations in human FOXP2 cause problems with speech development, this work opens up a new perspective on the use of mouse models for understanding pathways underlying speech and language disorders.

  7. Novel Mutations and Mutation Combinations of TMPRSS3 Cause Various Phenotypes in One Chinese Family with Autosomal Recessive Hearing Impairment

    PubMed Central

    Wang, Guo-Jian; Xu, Jin-Cao; Su, Yu

    2017-01-01

    Autosomal recessive hearing impairment with postlingual onset is rare. Exceptions are caused by mutations in the TMPRSS3 gene, which can lead to prelingual (DFNB10) as well as postlingual deafness (DFNB8). TMPRSS3 mutations can be classified as mild or severe, and the phenotype is dependent on the combination of TMPRSS3 mutations. The combination of two severe mutations leads to profound hearing impairment with a prelingual onset, whereas severe mutations in combination with milder TMPRSS3 mutations lead to a milder phenotype with postlingual onset. We characterized a Chinese family (number FH1523) with not only prelingual but also postlingual hearing impairment. Three mutations in TMPRSS3, one novel mutation c.36delC [p.(Phe13Serfs⁎12)], and two previously reported pathogenic mutations, c.916G>A (p.Ala306Thr) and c.316C>T (p.Arg106Cys), were identified. Compound heterozygous mutations of p.(Phe13Serfs⁎12) and p.Ala306Thr manifest as prelingual, profound hearing impairment in the patient (IV: 1), whereas the combination of p.Arg106Cys and p.Ala306Thr manifests as postlingual, milder hearing impairment in the patient (II: 2, II: 3, II: 5), suggesting that p.Arg106Cys mutation has a milder effect than p.(Phe13Serfs⁎12). We concluded that different combinations of TMPRSS3 mutations led to different hearing impairment phenotypes (DFNB8/DFNB10) in this family. PMID:28246597

  8. Mutations in dopachrome tautomerase (Dct) affect eumelanin/pheomelanin synthesis, but do not affect intracellular trafficking of the mutant protein

    PubMed Central

    2005-01-01

    Dopachrome tautomerase (Dct) is a type I membrane protein and an important regulatory enzyme that plays a pivotal role in the biosynthesis of melanin and in the rapid metabolism of its toxic intermediates. Dct-mutant melanocytes carrying the slaty or slaty light mutations were derived from the skin of newborn congenic C57BL/6J non-agouti black mice and were used to study the effect(s) of these mutations on the intracellular trafficking of Dct and on the pigmentation of the cells. Dct activity is 3-fold lower in slaty cells compared with non-agouti black melanocytes, whereas slaty light melanocytes have a surprisingly 28-fold lower Dct activity. Homology modelling of the active site of Dct suggests that the slaty mutation [R194Q (Arg194→Gln)] is located in the active site and may alter the ability of the enzyme to transform the substrate. Transmembrane prediction methods indicate that the slaty light mutation [G486R (Gly486→Arg)] may result in the sliding of the transmembrane domain towards the N-terminus, thus interfering with Dct function. Chemical analysis showed that both Dct mutations increase pheomelanin and reduce eumelanin produced by melanocytes in culture. Thus the enzymatic activity of Dct may play a role in determining whether the eumelanin or pheomelanin pathway is preferred for pigment biosynthesis. PMID:15960609

  9. Heterozygous MDR3 missense mutation associated with intrahepatic cholestasis of pregnancy: evidence for a defect in protein trafficking.

    PubMed

    Dixon, P H; Weerasekera, N; Linton, K J; Donaldson, O; Chambers, J; Egginton, E; Weaver, J; Nelson-Piercy, C; de Swiet, M; Warnes, G; Elias, E; Higgins, C F; Johnston, D G; McCarthy, M I; Williamson, C

    2000-05-01

    Intrahepatic cholestasis of pregnancy (ICP) is a liver disease of pregnancy with serious consequences for the mother and fetus. Two pedigrees have been reported with ICP in the mothers of children with a subtype of autosomal recessive progressive familial intrahepatic cholestasis (PFIC) with raised serum gamma-glutamyl transpeptidase (gamma-GT). Affected children have homozygous mutations in the MDR3 gene (also called ABCB4 ), and heterozygous mothers have ICP. More frequently, however, ICP occurs in women with no known family history of PFIC and the genetic basis of this disorder is unknown. We investigated eight women with ICP and raised serum gamma-GT, but with no known family history of PFIC. DNA sequence analysis revealed a C to A transversion in codon 546 in exon 14 of MDR3 in one patient, which results in the missense substitution of the wild-type alanine with an aspartic acid. We performed functional studies of this mutation introduced into MDR1, a closely related homologue of MDR3. Fluorescence activated cell sorting (FACS) and western analysis indicated that this missense mutation causes disruption of protein trafficking with a subsequent lack of functional protein at the cell surface. The demonstration of a heterozygous missense mutation in the MDR3 gene in a patient with ICP with no known family history of PFIC, analysed by functional studies, is a novel finding. This shows that MDR3 mutations are responsible for the additional phenotype of ICP in a subgroup of women with raised gamma-GT.

  10. Catecholamine stress alters neutrophil trafficking and impairs wound healing by β2-adrenergic receptor-mediated upregulation of IL-6.

    PubMed

    Kim, Min-Ho; Gorouhi, Farzam; Ramirez, Sandra; Granick, Jennifer L; Byrne, Barbara A; Soulika, Athena M; Simon, Scott I; Rivkah Isseroff, R

    2014-03-01

    Stress-induced hormones can alter the inflammatory response to tissue injury; however, the precise mechanism by which epinephrine influences inflammatory response and wound healing is not well defined. Here we demonstrate that epinephrine alters the neutrophil (polymorphonuclear leukocyte (PMN))-dependent inflammatory response to a cutaneous wound. Using noninvasive real-time imaging of genetically tagged PMNs in a murine skin wound, chronic, epinephrine-mediated stress was modeled by sustained delivery of epinephrine. Prolonged systemic exposure of epinephrine resulted in persistent PMN trafficking to the wound site via an IL-6-mediated mechanism, and this in turn impaired wound repair. Further, we demonstrate that β2-adrenergic receptor-dependent activation of proinflammatory macrophages is critical for epinephrine-mediated IL-6 production. This study expands our current understanding of stress hormone-mediated impairment of wound healing and provides an important mechanistic link to explain how epinephrine stress exacerbates inflammation via increased number and lifetime of PMNs.

  11. Visual impairment in FOXG1-mutated individuals and mice.

    PubMed

    Boggio, E M; Pancrazi, L; Gennaro, M; Lo Rizzo, C; Mari, F; Meloni, I; Ariani, F; Panighini, A; Novelli, E; Biagioni, M; Strettoi, E; Hayek, J; Rufa, A; Pizzorusso, T; Renieri, A; Costa, M

    2016-06-02

    The Forkead Box G1 (FOXG1 in humans, Foxg1 in mice) gene encodes for a DNA-binding transcription factor, essential for the development of the telencephalon in mammalian forebrain. Mutations in FOXG1 have been reported to be involved in the onset of Rett Syndrome, for which sequence alterations of MECP2 and CDKL5 are known. While visual alterations are not classical hallmarks of Rett syndrome, an increasing body of evidence shows visual impairment in patients and in MeCP2 and CDKL5 animal models. Herein we focused on the functional role of FOXG1 in the visual system of animal models (Foxg1(+/Cre) mice) and of a cohort of subjects carrying FOXG1 mutations or deletions. Visual physiology of Foxg1(+/Cre) mice was assessed by visually evoked potentials, which revealed a significant reduction in response amplitude and visual acuity with respect to wild-type littermates. Morphological investigation showed abnormalities in the organization of excitatory/inhibitory circuits in the visual cortex. No alterations were observed in retinal structure. By examining a cohort of FOXG1-mutated individuals with a panel of neuro-ophthalmological assessments, we found that all of them exhibited visual alterations compatible with high-level visual dysfunctions. In conclusion our data show that Foxg1 haploinsufficiency results in an impairment of mouse and human visual cortical function.

  12. MDMA impairs mitochondrial neuronal trafficking in a Tau- and Mitofusin2/Drp1-dependent manner.

    PubMed

    Barbosa, Daniel José; Serrat, Román; Mirra, Serena; Quevedo, Martí; Gómez de Barreda, Elena; Avila, Jesús; Fernandes, Eduarda; Bastos, Maria de Lourdes; Capela, João Paulo; Carvalho, Félix; Soriano, Eduardo

    2014-08-01

    Identification of the mechanisms by which drugs of abuse cause neuronal dysfunction is essential for understanding the biological bases of their acute and long-lasting effects in the brain. Here, we performed real-time functional experiments of axonal transport of mitochondria to explore the role of in situ mitochondrial dysfunction in 3,4-methylenedioxymethamphetamine (MDMA; "ecstasy")-related brain actions. We showed that MDMA dramatically reduced mitochondrial trafficking in hippocampal neurons in a Tau-dependent manner, in which glycogen synthase kinase 3β activity was implicated. Furthermore, we found that these trafficking abnormalities were rescued by over-expression of Mitofusin2 and dynamin-related protein 1, but not of Miro1. Given the relevance of mitochondrial targeting for neuronal function and neurotransmission, our data underscore a novel mechanism of action of MDMA that may contribute to our understanding of how this drug of abuse alters neuronal functioning.

  13. ATP7A trafficking and mechanisms underlying the distal motor neuropathy induced by mutations in ATP7A

    PubMed Central

    Yi, Ling; Kaler, Stephen

    2014-01-01

    Diverse mutations in the gene encoding the copper transporter ATP7A lead to X-linked recessive Menkes disease or occipital horn syndrome. Recently, two unique ATP7A mutations, T994I and P1386S, were shown to cause isolated adult-onset distal motor neuropathy. These mutations induce subtle defects in ATP7A intracellular trafficking resulting in preferential accumulation at the plasma membrane compared to wild-type ATP7A. Immunoprecipitation assays revealed abnormal interaction between ATP7AT994I and p97/VCP, a protein mutated in two autosomal dominant forms of motor neuron disease. Small-interfering RNA knockdown of p97/VCP corrected ATP7AT994I mislocalization. For ATP7AP1386S, flow cytometry documented that non-permeabilized fibroblasts bound a C-terminal ATP7A antibody, suggesting unstable insertion of the 8th transmembrane segment due to a helix-breaker effect of the amino acid substitution. This could sabotage interaction of ATP7AP1386S with adaptor protein complexes. These molecular events appear to selectively disturb normal motor neuron function and lead to neurologic illness that takes years and sometimes decades to develop. PMID:24754450

  14. Breast cancer-associated mutations in metalloprotease disintegrin ADAM12 interfere with the intracellular trafficking and processing of the protein.

    PubMed

    Dyczynska, Emilia; Syta, Emilia; Sun, Danqiong; Zolkiewska, Anna

    2008-06-01

    ADAM12 has recently emerged as a Candidate Cancer Gene in a comprehensive genetic analysis of human breast cancers. Three somatic mutations in ADAM12 were observed at significant frequencies in breast cancers: D301H, G479E and L792F. The first 2 of these mutations involve highly conserved residues in ADAM12, and our computational sequence analysis confirms that they may be cancer-related. We show that the corresponding mutations in mouse ADAM12 inhibit the proteolytic processing and activation of ADAM12 in NIH3T3, COS-7, CHO-K1 cells and in MCF-7 breast cancer cells. The D/H and G/E ADAM12 mutants exert a dominant-negative effect on the processing of the wild-type ADAM12. Immunofluorescence analysis and cell surface biotinylation experiments demonstrate that the D/H and G/E mutants are retained inside the cell and are not transported to the cell surface. Consequently, the D/H and G/E mutants, unlike the wild-type ADAM12, are not capable of shedding Delta-like l, a ligand for Notch receptor, at the cell surface, or of stimulating cell migration. Our results suggest that the breast cancer-associated mutations interfere with the intracellular trafficking of ADAM12 and result in loss of the functional ADAM12 at the cell surface.

  15. Partial nephrogenic diabetes insipidus caused by a novel AQP2 variation impairing trafficking of the aquaporin-2 water channel.

    PubMed

    Dollerup, Pia; Thomsen, Troels Møller; Nejsum, Lene N; Færch, Mia; Österbrand, Martin; Gregersen, Niels; Rittig, Søren; Christensen, Jane H; Corydon, Thomas J

    2015-12-29

    Autosomal dominant inheritance of congenital nephrogenic diabetes insipidus (CNDI) is rare and usually caused by variations in the AQP2 gene. We have investigated the genetic and molecular background underlying symptoms of diabetes insipidus (DI) in a Swedish family with autosomal dominant inheritance of the condition. The proband and her father were subjected to water deprivation testing and direct DNA sequencing of the coding regions of the AQP2 and AVP genes. Madin-Darby canine kidney (MDCK) cells stably expressing AQP2 variant proteins were generated by lentiviral gene delivery. Localization of AQP2 variant proteins in the cells under stimulated and unstimulated conditions was analyzed by means of immunostaining and confocal laser scanning microscopy. Intracellular trafficking of AQP2 variant proteins was studied using transient expression of mutant dynamin2-K44A-GFP protein and AQP2 variant protein phosphorylation levels were assessed by Western blotting analysis. Clinical and genetic data suggest that the proband and her father suffer from partial nephrogenic DI due to a variation (g.4807C > T) in the AQP2 gene. The variation results in substitution of arginine-254 to tryptophan (p.R254W) in AQP2. Analysis of MDCK cells stably expressing AQP2 variant proteins revealed disabled phosphorylation, impaired trafficking and intracellular accumulation of AQP2-R254W protein. Notably, blocking of the endocytic pathway demonstrated impairment of AQP2-R254W to reach the cell surface. Partial CNDI in the Swedish family is caused by an AQP2 variation that seems to disable the encoded AQP2-R254W protein to reach the subapical vesicle population as well as impairing its phosphorylation at S256. The AQP2-R254W protein is thus unable to reach the plasma membrane to facilitate AVP mediated urine concentration.

  16. Hippocampal and Cognitive Aging across the Lifespan: A Bioenergetic Shift Precedes and Increased Cholesterol Trafficking Parallels Memory Impairment

    PubMed Central

    Kadish, I; Thibault, O; Blalock, EM; Chen, K-C; Gant, JC; Porter, NM; Landfield, PW

    2009-01-01

    Multiple hippocampal processes and cognitive functions change with aging or Alzheimer’s disease, but the potential triggers of these aging cascades are not well understood. Here, we quantified hippocampal expression profiles and behavior across the adult lifespan, to identify early aging changes and changes that coincide with subsequent onset of cognitive impairment. Well-powered microarray analyses (N=49 arrays), immunohistochemistry and Morris spatial maze learning were used to study male F344 rats at 5 age points. Genes that changed with aging (by ANOVA) were assigned to one-of-four onset-age ranges based upon template pattern matching; functional pathways represented by these genes were identified statistically (Genome Ontology). In the earliest onset-age range (3–6 months-old), upregulation began for genes in lipid/protein catabolic and lysosomal pathways, indicating a shift in metabolic substrates, whereas downregulation began for lipid synthesis, GTP/ATP-dependent signaling and neural development genes. By 6–9 months-of-age, upregulation of immune/inflammatory cytokines was pronounced. Cognitive impairment first appeared in the Midlife range (9–12 months), and coincided and correlated primarily with midlife upregulation of genes associated with cholesterol trafficking (apolipoprotein E), myelinogenic and proteolytic/MHC antigen-presenting pathways. Immunolabeling revealed that cholesterol trafficking proteins were substantially increased in astrocytes, and that myelination increased with aging. Together, our data suggest a novel sequential model in which an early-adult metabolic shift, favoring lipid/ketone body oxidation, triggers inflammatory degradation of myelin and resultant excess cholesterol that, by midlife, activates cholesterol transport from astrocytes to remyelinating oligodendrocytes. These processes may damage structure and compete with neuronal pathways for bioenergetic resources, thereby impairing cognitive function. PMID:19211887

  17. Whole Genome Sequencing Identifies Novel Compound Heterozygous Lysosomal Trafficking Regulator Gene Mutations Associated with Autosomal Recessive Chediak-Higashi Syndrome

    PubMed Central

    Jin, Yaqiong; Zhang, Li; Wang, Senfen; Chen, Feng; Gu, Yang; Hong, Enyu; Yu, Yongbo; Ni, Xin; Guo, Yongli; Shi, Tieliu; Xu, Zigang

    2017-01-01

    Chediak–Higashi syndrome (CHS) is a rare autosomal recessive disease characterized by varying degrees of oculocutaneous albinism, recurrent infections, and a mild bleeding tendency, with late neurologic dysfunction. This syndrome is molecularly characterized by pathognomonic mutations in the LYST (lysosomal trafficking regulator). Using whole genome sequencing (WGS) we attempted to identify novel mutations of CHS based on a family of CHS with atypical symptoms. The two patients demonstrated a phenotypic constellation including partial oculocutaneous albinism, frequency upper respiratory infection or a marginal intelligence, without bleeding tendency and severe immunodeficiency. WGS revealed two compound LYST mutations including a maternally inherited chr1:235969126G > A (rs80338652) and a novel paternally inherited chr1: 235915327A > AT, associated with autosomal recessive CHS. These two variants fall in the coding regions of LYST, resulting in premature truncation of LYST due to R1104X/N2535KfsX2 induced incomplete translation. Notably, the heterozygous carriers (i.e. parents) were unaffected. Our finding also reveals decreased plasma serotonin levels in patients with CHS compared with unaffected individuals for the first time. The present study contributes to improved understanding of the causes of this disease and provides new ideas for possible treatments. PMID:28145517

  18. Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain.

    PubMed

    Poirier, Steve; Hamouda, Hocine Ait; Villeneuve, Louis; Demers, Annie; Mayer, Gaétan

    2016-01-01

    PCSK9 is a secreted ligand and negative post-translational regulator of low-density lipoprotein receptor (LDLR) in hepatocytes. Gain-of-function (GOF) or loss-of-function (LOF) mutations in PCSK9 are directly correlated with high or low plasma LDL-cholesterol levels, respectively. Therefore, PCSK9 is a prevailing lipid-lowering target to prevent coronary heart diseases and stroke. Herein, we fused monomeric fluorescent proteins to PCSK9 and LDLR to visualize their intra- and extracellular trafficking dynamics by live confocal microscopy. Fluorescence recovery after photobleaching (FRAP) showed that PCSK9 LOF R46L mutant and GOF mutations S127R and D129G, but not the LDLR high-affinity mutant D374Y, significantly accelerate PCSK9 exit from the endoplasmic reticulum (ER). Quantitative analysis of inverse FRAP revealed that only R46L presented a much slower trafficking from the trans-Golgi network (TGN) to the plasma membrane and a lower mobile fraction likely suggesting accumulation or delayed exit at the TGN as an underlying mechanism. While not primarily involved in LDLR binding, PCSK9 C-terminal domain (CTD) was found to be essential to induce LDLR degradation both upon its overexpression in cells or via the extracellular pathway. Our data revealed that PCSK9 CTD is required for the localization of PCSK9 at the TGN and increases its LDLR-mediated endocytosis. Interestingly, intracellular lysosomal targeting of PCSK9-ΔCTD was able to rescue its capacity to induce LDLR degradation emphasizing a role of the CTD in the sorting of PCSK9-LDLR complex towards late endocytic compartments. Finally, we validated our dual fluorescence system as a cell based-assay by preventing PCSK9 internalization using a PCSK9-LDLR blocking antibody, which may be expended to identify protein, peptide or small molecule inhibitors of PCSK9.

  19. Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain

    PubMed Central

    Villeneuve, Louis; Demers, Annie; Mayer, Gaétan

    2016-01-01

    PCSK9 is a secreted ligand and negative post-translational regulator of low-density lipoprotein receptor (LDLR) in hepatocytes. Gain-of-function (GOF) or loss-of-function (LOF) mutations in PCSK9 are directly correlated with high or low plasma LDL-cholesterol levels, respectively. Therefore, PCSK9 is a prevailing lipid-lowering target to prevent coronary heart diseases and stroke. Herein, we fused monomeric fluorescent proteins to PCSK9 and LDLR to visualize their intra- and extracellular trafficking dynamics by live confocal microscopy. Fluorescence recovery after photobleaching (FRAP) showed that PCSK9 LOF R46L mutant and GOF mutations S127R and D129G, but not the LDLR high-affinity mutant D374Y, significantly accelerate PCSK9 exit from the endoplasmic reticulum (ER). Quantitative analysis of inverse FRAP revealed that only R46L presented a much slower trafficking from the trans-Golgi network (TGN) to the plasma membrane and a lower mobile fraction likely suggesting accumulation or delayed exit at the TGN as an underlying mechanism. While not primarily involved in LDLR binding, PCSK9 C-terminal domain (CTD) was found to be essential to induce LDLR degradation both upon its overexpression in cells or via the extracellular pathway. Our data revealed that PCSK9 CTD is required for the localization of PCSK9 at the TGN and increases its LDLR-mediated endocytosis. Interestingly, intracellular lysosomal targeting of PCSK9-ΔCTD was able to rescue its capacity to induce LDLR degradation emphasizing a role of the CTD in the sorting of PCSK9-LDLR complex towards late endocytic compartments. Finally, we validated our dual fluorescence system as a cell based-assay by preventing PCSK9 internalization using a PCSK9-LDLR blocking antibody, which may be expended to identify protein, peptide or small molecule inhibitors of PCSK9. PMID:27280970

  20. Age-Dependent Cell Trafficking Defects in Draining Lymph Nodes Impair Adaptive Immunity and Control of West Nile Virus Infection

    PubMed Central

    Richner, Justin M.; Gmyrek, Grzegorz B.; Govero, Jennifer; Tu, Yizheng; van der Windt, Gerritje J. W.; Metcalf, Talibah U.; Haddad, Elias K.; Textor, Johannes; Miller, Mark J.; Diamond, Michael S.

    2015-01-01

    Impaired immune responses in the elderly lead to reduced vaccine efficacy and increased susceptibility to viral infections. Although several groups have documented age-dependent defects in adaptive immune priming, the deficits that occur prior to antigen encounter remain largely unexplored. Herein, we identify novel mechanisms for compromised adaptive immunity that occurs with aging in the context of infection with West Nile virus (WNV), an encephalitic flavivirus that preferentially causes disease in the elderly. An impaired IgM and IgG response and enhanced vulnerability to WNV infection during aging was linked to delayed germinal center formation in the draining lymph node (DLN). Adoptive transfer studies and two-photon intravital microscopy revealed a decreased trafficking capacity of donor naïve CD4+ T cells from old mice, which manifested as impaired T cell diapedesis at high endothelial venules and reduced cell motility within DLN prior to antigen encounter. Furthermore, leukocyte accumulation in the DLN within the first few days of WNV infection or antigen-adjuvant administration was diminished more generally in old mice and associated with a second aging-related defect in local cytokine and chemokine production. Thus, age-dependent cell-intrinsic and environmental defects in the DLN result in delayed immune cell recruitment and antigen recognition. These deficits compromise priming of early adaptive immune responses and likely contribute to the susceptibility of old animals to acute WNV infection. PMID:26204259

  1. Age-Dependent Cell Trafficking Defects in Draining Lymph Nodes Impair Adaptive Immunity and Control of West Nile Virus Infection.

    PubMed

    Richner, Justin M; Gmyrek, Grzegorz B; Govero, Jennifer; Tu, Yizheng; van der Windt, Gerritje J W; Metcalf, Talibah U; Haddad, Elias K; Textor, Johannes; Miller, Mark J; Diamond, Michael S

    2015-07-01

    Impaired immune responses in the elderly lead to reduced vaccine efficacy and increased susceptibility to viral infections. Although several groups have documented age-dependent defects in adaptive immune priming, the deficits that occur prior to antigen encounter remain largely unexplored. Herein, we identify novel mechanisms for compromised adaptive immunity that occurs with aging in the context of infection with West Nile virus (WNV), an encephalitic flavivirus that preferentially causes disease in the elderly. An impaired IgM and IgG response and enhanced vulnerability to WNV infection during aging was linked to delayed germinal center formation in the draining lymph node (DLN). Adoptive transfer studies and two-photon intravital microscopy revealed a decreased trafficking capacity of donor naïve CD4+ T cells from old mice, which manifested as impaired T cell diapedesis at high endothelial venules and reduced cell motility within DLN prior to antigen encounter. Furthermore, leukocyte accumulation in the DLN within the first few days of WNV infection or antigen-adjuvant administration was diminished more generally in old mice and associated with a second aging-related defect in local cytokine and chemokine production. Thus, age-dependent cell-intrinsic and environmental defects in the DLN result in delayed immune cell recruitment and antigen recognition. These deficits compromise priming of early adaptive immune responses and likely contribute to the susceptibility of old animals to acute WNV infection.

  2. The mixture of "ecstasy" and its metabolites impairs mitochondrial fusion/fission equilibrium and trafficking in hippocampal neurons, at in vivo relevant concentrations.

    PubMed

    Barbosa, Daniel José; Serrat, Romàn; Mirra, Serena; Quevedo, Martí; de Barreda, Elena Goméz; Àvila, Jesús; Ferreira, Luísa Maria; Branco, Paula Sério; Fernandes, Eduarda; Lourdes Bastos, Maria de; Capela, João Paulo; Soriano, Eduardo; Carvalho, Félix

    2014-06-01

    3,4-Methylenedioxymethamphetamine (MDMA; "ecstasy") is a potentially neurotoxic recreational drug of abuse. Though the mechanisms involved are still not completely understood, formation of reactive metabolites and mitochondrial dysfunction contribute to MDMA-related neurotoxicity. Neuronal mitochondrial trafficking, and their targeting to synapses, is essential for proper neuronal function and survival, rendering neurons particularly vulnerable to mitochondrial dysfunction. Indeed, MDMA-associated disruption of Ca(2+) homeostasis and ATP depletion have been described in neurons, thus suggesting possible MDMA interference on mitochondrial dynamics. In this study, we performed real-time functional experiments of mitochondrial trafficking to explore the role of in situ mitochondrial dysfunction in MDMA's neurotoxic actions. We show that the mixture of MDMA and six of its major in vivo metabolites, each compound at 10μM, impaired mitochondrial trafficking and increased the fragmentation of axonal mitochondria in cultured hippocampal neurons. Furthermore, the overexpression of mitofusin 2 (Mfn2) or dynamin-related protein 1 (Drp1) K38A constructs almost completely rescued the trafficking deficits caused by this mixture. Finally, in hippocampal neurons overexpressing a Mfn2 mutant, Mfn2 R94Q, with impaired fusion and transport properties, it was confirmed that a dysregulation of mitochondrial fission/fusion events greatly contributed to the reported trafficking phenotype. In conclusion, our study demonstrated, for the first time, that the mixture of MDMA and its metabolites, at concentrations relevant to the in vivo scenario, impaired mitochondrial trafficking and increased mitochondrial fragmentation in hippocampal neurons, thus providing a new insight in the context of "ecstasy"-induced neuronal injury.

  3. LMTK3 deficiency causes pronounced locomotor hyperactivity and impairs endocytic trafficking.

    PubMed

    Inoue, Takeshi; Hoshina, Naosuke; Nakazawa, Takanobu; Kiyama, Yuji; Kobayashi, Shizuka; Abe, Takaya; Yamamoto, Toshifumi; Manabe, Toshiya; Yamamoto, Tadashi

    2014-04-23

    LMTK3 belongs to the LMTK family of protein kinases that are predominantly expressed in the brain. Physiological functions of LMTK3 and other members of the LMTK family in the CNS remain unknown. In this study, we performed a battery of behavioral analyses using Lmtk3(-/-) mice and showed that these mice exhibit abnormal behaviors, including pronounced locomotor hyperactivity, reduced anxiety behavior, and decreased depression-like behavior. Concurrently, the dopamine metabolite levels and dopamine turnover rate are increased in the striata of Lmtk3(-/-) mice compared with wild-type controls. In addition, using cultured primary neurons from Lmtk3(-/-) mice, we found that LMTK3 is involved in the endocytic trafficking of N-methyl-d-aspartate receptors, a type of ionotropic glutamate receptor. Altered membrane traffic of the receptor in Lmtk3(-/-) neurons may underlie behavioral abnormalities in the mutant animals. Together, our data suggest that LMTK3 plays an important role in regulating locomotor behavior in mice.

  4. Fast track, dynein-dependent nuclear targeting of human immunodeficiency virus Vpr protein; impaired trafficking in a clinical isolate.

    PubMed

    Caly, Leon; Kassouf, Vicki T; Moseley, Gregory W; Diefenbach, Russell J; Cunningham, Anthony L; Jans, David A

    2016-02-12

    Nuclear import of the accessory protein Vpr is central to infection by human immunodeficiency virus (HIV). We previously identified the Vpr F72L mutation in a HIV-infected, long-term non-progressor, showing that it resulted in reduced Vpr nuclear accumulation and altered cytoplasmic localisation. Here we demonstrate for the first time that the effects of nuclear accumulation of the F72L mutation are due to impairment of microtubule-dependent-enhancement of Vpr nuclear import. We use high resolution imaging approaches including fluorescence recovery after photobleaching and other approaches to document interaction between Vpr and the dynein light chain protein, DYNLT1, and impaired interaction of the F72L mutant with DYNLT1. The results implicate MTs/DYNLT1 as drivers of Vpr nuclear import and HIV infection, with important therapeutic implications.

  5. Electrophysiological and trafficking defects of the SCN5A T353I mutation in Brugada syndrome are rescued by alpha-allocryptopine.

    PubMed

    Zhang, Jiancheng; Chen, Yu; Yang, Jie; Xu, Bin; Wen, Yi; Xiang, Guojian; Wei, Guoliang; Zhu, Chao; Xing, Yanwei; Li, Yang

    2015-01-05

    Brugada syndrome (BrS), which causes arrhythmias that lead to sudden cardiac death, is linked to loss-of-function mutations that affect sodium channels. Here, we investigate the rescue effect of alpha-allocryptopine (All) from Chinese herbal medicine in a T353I mutation of SCN5A, which combines trafficking abnormalities with Brugada syndrome. SCN5A-T353I expressed in HEK293 cells showed a small peak current (I(peak)) of only 59.6% of WT and an observably sustained current (I(sus)). We found that All strongly enhanced the I(peak) of the T353I channel by enhancing the plasma membrane (PM) expression of Nav1.5 and rescued defective trafficking after co-incubation with HEK293 cells that carry mutation channel 24 h. It is also beneficial to increase the I(peak) of the T353I mutation by All by prolonging the closed-state inactivation (CSI) process and shortening the recovery from inactivation of the T353I mutation. Interestingly, the I(sus) of T353I was significantly inhibited by All, which reduces the occurrence of LQT syndrome 3 (LQT3). We provide evidence that All can rescue the trafficking deficiencies and restore the cellular electrophysiological characteristics of SCN5A-T353I. This feature of All may benefit patients with the BrS-associated Nav1.5 channel and might have other potential therapeutic effects.

  6. Fast track, dynein-dependent nuclear targeting of human immunodeficiency virus Vpr protein; impaired trafficking in a clinical isolate

    SciTech Connect

    Caly, Leon; Kassouf, Vicki T.; Moseley, Gregory W.; Diefenbach, Russell J.; Cunningham, Anthony L.; Jans, David A.

    2016-02-12

    Nuclear import of the accessory protein Vpr is central to infection by human immunodeficiency virus (HIV). We previously identified the Vpr F72L mutation in a HIV-infected, long-term non-progressor, showing that it resulted in reduced Vpr nuclear accumulation and altered cytoplasmic localisation. Here we demonstrate for the first time that the effects of nuclear accumulation of the F72L mutation are due to impairment of microtubule-dependent-enhancement of Vpr nuclear import. We use high resolution imaging approaches including fluorescence recovery after photobleaching and other approaches to document interaction between Vpr and the dynein light chain protein, DYNLT1, and impaired interaction of the F72L mutant with DYNLT1. The results implicate MTs/DYNLT1 as drivers of Vpr nuclear import and HIV infection, with important therapeutic implications. - Highlights: • HIV-1 Vpr utilizes the microtubule network to traffic towards the nucleus. • Mechanism relies on interaction between Vpr and dynein light chain protein DYNLT1. • Long-term non-progressor derived mutation (F72L) impairs this interaction. • Key residues in the vicinity of F72 contribute to interaction with DYNLT1.

  7. A Mutation in the Vesicle-Trafficking Protein VAPB Causes Late-Onset Spinal Muscular Atrophy and Amyotrophic Lateral Sclerosis

    PubMed Central

    Nishimura, Agnes L.; Mitne-Neto, Miguel; Silva, Helga C. A.; Richieri-Costa, Antônio; Middleton, Susan; Cascio, Duilio; Kok, Fernando; Oliveira, João R. M.; Gillingwater, Tom; Webb, Jeanette; Skehel, Paul; Zatz, Mayana

    2004-01-01

    Motor neuron diseases (MNDs) are a group of neurodegenerative disorders with involvement of upper and/or lower motor neurons, such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), progressive bulbar palsy, and primary lateral sclerosis. Recently, we have mapped a new locus for an atypical form of ALS/MND (atypical amyotrophic lateral sclerosis [ALS8]) at 20q13.3 in a large white Brazilian family. Here, we report the finding of a novel missense mutation in the vesicle-associated membrane protein/synaptobrevin-associated membrane protein B (VAPB) gene in patients from this family. Subsequently, the same mutation was identified in patients from six additional kindreds but with different clinical courses, such as ALS8, late-onset SMA, and typical severe ALS with rapid progression. Although it was not possible to link all these families, haplotype analysis suggests a founder effect. Members of the vesicle-associated proteins are intracellular membrane proteins that can associate with microtubules and that have been shown to have a function in membrane transport. These data suggest that clinically variable MNDs may be caused by a dysfunction in intracellular membrane trafficking. PMID:15372378

  8. Catecholamine stress alters neutrophil trafficking and impairs wound healing by β2 adrenergic receptor mediated upregulation of IL-6

    PubMed Central

    Kim, Min-Ho; Gorouhi, Farzam; Ramirez, Sandra; Granick, Jennifer L.; Byrne, Barbara A.; Soulika, Athena M.; Simon, Scott I.; Isseroff, R. Rivkah.

    2013-01-01

    Stress-induced hormones can alter the inflammatory response to tissue injury, however, the precise mechanism by which epinephrine influences inflammatory response and wound healing is not well defined. Here we demonstrate that epinephrine alters the neutrophil (PMN)-dependent inflammatory response to a cutaneous wound. Using non-invasive real-time imaging of genetically-tagged PMNs in a murine skin wound, chronic, epinephrine-mediated stress was modeled by sustained delivery of epinephrine. Prolonged systemic exposure of epinephrine resulted in persistent PMN trafficking to the wound site via an IL-6 mediated mechanism, and this in turn impaired wound repair. Further, we demonstrate that β2 adrenergic receptor-dependent activation of pro-inflammatory macrophages is critical for epinephrine-mediated IL-6 production. This study expands our current understanding of stress hormone-mediated impairment of wound healing and provides an important mechanistic link to explain how epinephrine stress exacerbates inflammation via increased number and lifetime of PMNs. PMID:24121404

  9. Anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects.

    PubMed

    Bekerman, Elena; Neveu, Gregory; Shulla, Ana; Brannan, Jennifer; Pu, Szu-Yuan; Wang, Stanley; Xiao, Fei; Barouch-Bentov, Rina; Bakken, Russell R; Mateo, Roberto; Govero, Jennifer; Nagamine, Claude M; Diamond, Michael S; De Jonghe, Steven; Herdewijn, Piet; Dye, John M; Randall, Glenn; Einav, Shirit

    2017-02-27

    Global health is threatened by emerging viral infections, which largely lack effective vaccines or therapies. Targeting host pathways that are exploited by multiple viruses could offer broad-spectrum solutions. We previously reported that AAK1 and GAK, kinase regulators of the host adaptor proteins AP1 and AP2, are essential for hepatitis C virus (HCV) infection, but the underlying mechanism and relevance to other viruses or in vivo infections remained unknown. Here, we have discovered that AP1 and AP2 cotraffic with HCV particles in live cells. Moreover, we found that multiple viruses, including dengue and Ebola, exploit AAK1 and GAK during entry and infectious virus production. In cultured cells, treatment with sunitinib and erlotinib, approved anticancer drugs that inhibit AAK1 or GAK activity, or with more selective compounds inhibited intracellular trafficking of HCV and multiple unrelated RNA viruses with a high barrier to resistance. In murine models of dengue and Ebola infection, sunitinib/erlotinib combination protected against morbidity and mortality. We validated sunitinib- and erlotinib-mediated inhibition of AAK1 and GAK activity as an important mechanism of antiviral action. Additionally, we revealed potential roles for additional kinase targets. These findings advance our understanding of virus-host interactions and establish a proof of principle for a repurposed, host-targeted approach to combat emerging viruses.

  10. MILD CHOLESTEROL DEPLETION REDUCES AMYLOID-β PRODUCTION BY IMPAIRING APP TRAFFICKING TO THE CELL SURFACE

    PubMed Central

    Guardia-Laguarta, Cristina; Coma, Mireia; Pera, Marta; Clarimón, Jordi; Sereno, Lidia; Agulló, José M.; Molina-Porcel, Laura; Gallardo, Eduard; Deng, Amy; Berezovska, Oksana; Hyman, Bradley T.; Blesa, Rafael; Gómez-Isla, Teresa; Lleó, Alberto

    2009-01-01

    It has been suggested that cellular cholesterol levels can modulate the metabolism of the amyloid precursor protein (APP) but the underlying mechanism remains controversial. In the current study, we investigate in detail the relationship between cholesterol reduction, APP processing and γ-secretase function in cell culture studies. We found that mild membrane cholesterol reduction led to a decrease in Aβ40 and Aβ42 in different cell types. We did not detect changes in APP intracellular domain or Notch intracellular domain generation. Western blot analyses showed a cholesterol-dependent decrease in the APP C-terminal fragments and cell surface APP. Finally, we applied a fluorescence resonance energy transfer (FRET)-based technique to study APP-Presenilin 1 (PS1) interactions and lipid rafts in intact cells. Our data indicate that cholesterol depletion reduces association of APP into lipid rafts and disrupts APP-PS1 interaction. Taken together, our results suggest that mild membrane cholesterol reduction impacts the cleavage of APP upstream of γ-secretase and appears to be mediated by changes in APP trafficking and partitioning into lipid rafts. PMID:19457132

  11. Anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects

    PubMed Central

    Bekerman, Elena; Shulla, Ana; Brannan, Jennifer; Wang, Stanley; Barouch-Bentov, Rina; Bakken, Russell R.; Mateo, Roberto; Govero, Jennifer; Nagamine, Claude M.; Diamond, Michael S.; De Jonghe, Steven; Herdewijn, Piet; Dye, John M.; Randall, Glenn

    2017-01-01

    Global health is threatened by emerging viral infections, which largely lack effective vaccines or therapies. Targeting host pathways that are exploited by multiple viruses could offer broad-spectrum solutions. We previously reported that AAK1 and GAK, kinase regulators of the host adaptor proteins AP1 and AP2, are essential for hepatitis C virus (HCV) infection, but the underlying mechanism and relevance to other viruses or in vivo infections remained unknown. Here, we have discovered that AP1 and AP2 cotraffic with HCV particles in live cells. Moreover, we found that multiple viruses, including dengue and Ebola, exploit AAK1 and GAK during entry and infectious virus production. In cultured cells, treatment with sunitinib and erlotinib, approved anticancer drugs that inhibit AAK1 or GAK activity, or with more selective compounds inhibited intracellular trafficking of HCV and multiple unrelated RNA viruses with a high barrier to resistance. In murine models of dengue and Ebola infection, sunitinib/erlotinib combination protected against morbidity and mortality. We validated sunitinib- and erlotinib-mediated inhibition of AAK1 and GAK activity as an important mechanism of antiviral action. Additionally, we revealed potential roles for additional kinase targets. These findings advance our understanding of virus-host interactions and establish a proof of principle for a repurposed, host-targeted approach to combat emerging viruses. PMID:28240606

  12. Trafficking of alpha4* nicotinic receptors revealed by superecliptic phluorin: effects of a beta4 amyotrophic lateral sclerosis-associated mutation and chronic exposure to nicotine.

    PubMed

    Richards, Christopher I; Srinivasan, Rahul; Xiao, Cheng; Mackey, Elisha D W; Miwa, Julie M; Lester, Henry A

    2011-09-09

    We employed a pH-sensitive GFP analog, superecliptic phluorin, to observe aspects of nicotinic acetylcholine receptor (nAChR) trafficking to the plasma membrane (PM) in cultured mouse cortical neurons. The experiments exploit differences in the pH among endoplasmic reticulum (ER), trafficking vesicles, and the extracellular solution. The data confirm that few α4β4 nAChRs, but many α4β2 nAChRs, remain in neutral intracellular compartments, mostly the ER. We observed fusion events between nAChR-containing vesicles and PM; these could be quantified in the dendritic processes. We also studied the β4R348C polymorphism, linked to amyotrophic lateral sclerosis (ALS). This mutation depressed fusion rates of α4β4 receptor-containing vesicles with the PM by ∼2-fold, with only a small decrease in the number of nAChRs per vesicle. The mutation also decreased the number of ER exit sites, showing that the reduced receptor insertion results from a change at an early stage in trafficking. We confirm the previous report that the mutation leads to reduced agonist-induced currents; in the cortical neurons studied, the reduction amounts to 2-3-fold. Therefore, the reduced agonist-induced currents are caused by the reduced number of α4β4-containing vesicles reaching the membrane. Chronic nicotine exposure (0.2 μM) did not alter the PM insertion frequency or trafficking behavior of α4β4-laden vesicles. In contrast, chronic nicotine substantially increased the number of α4β2-containing vesicle fusions at the PM; this stage in α4β2 nAChR up-regulation is presumably downstream from increased ER exit. Superecliptic phluorin provides a tool to monitor trafficking dynamics of nAChRs in disease and addiction.

  13. Methylglyoxal impairs GLUT4 trafficking and leads to increased glucose uptake in L6 myoblasts.

    PubMed

    Engelbrecht, B; Mattern, Y; Scheibler, S; Tschoepe, D; Gawlowski, T; Stratmann, B

    2014-02-01

    Methylglyoxal (MG) is a highly reactive dicarbonyl compound derived mainly from glucose degradation pathways, but also from protein and fatty acid metabolism. MG modifies structure and function of different biomolecules and thus plays an important role in the pathogenesis of diabetic complications. Hyperglycemia-associated accumulation of MG might be associated with generation of oxidative stress and subsequently insulin resistance. Therefore, the effects of MG on insulin signaling and on translocation of glucose transporter 4 (GLUT4) were investigated in the rat skeletal muscle cell line L6-GLUT4myc stably expressing myc-tagged GLUT4. Twenty four-hour MG treatment resulted in elevated GLUT4 presentation on the surface of L6 myoblasts and in an increased uptake of glucose even without insulin stimulation. Exogenously added MG neither effected IRS-1 expression nor IRS-1 phosphorylation. A decreased expression of Akt1 but not Akt2 and concomitantly increased apoptosis were detected following MG treatment. To exclude that oxidative stress caused by MG treatment leads to increased GLUT4 translocation, effects of pretreatment with 2 antioxidants were investigated. The antioxidant and MG scavenger NAC prevented the MG-induced GLUT4 translocation. In contrast, tiron, a well-known antioxidant that does not exert MG-scavenger function, had no impact on MG-induced GLUT4 translocation supporting the hypothesis of a direct effect of MG on GLUT4 trafficking. In conclusion, prolonged treatment with MG augments GLUT4 level on the surface of L6 myoblasts, at least in part through a higher translocation of GLUT4 from the intracellular compartment as well as a reduction of GLUT4 internalization, resulting in increased glucose uptake.

  14. New Hyperekplexia Mutations Provide Insight into Glycine Receptor Assembly, Trafficking, and Activation Mechanisms*

    PubMed Central

    Bode, Anna; Wood, Sian-Elin; Mullins, Jonathan G. L.; Keramidas, Angelo; Cushion, Thomas D.; Thomas, Rhys H.; Pickrell, William O.; Drew, Cheney J. G.; Masri, Amira; Jones, Elizabeth A.; Vassallo, Grace; Born, Alfred P.; Alehan, Fusun; Aharoni, Sharon; Bannasch, Gerald; Bartsch, Marius; Kara, Bulent; Krause, Amanda; Karam, Elie G.; Matta, Stephanie; Jain, Vivek; Mandel, Hanna; Freilinger, Michael; Graham, Gail E.; Hobson, Emma; Chatfield, Sue; Vincent-Delorme, Catherine; Rahme, Jubran E.; Afawi, Zaid; Berkovic, Samuel F.; Howell, Owain W.; Vanbellinghen, Jean-François; Rees, Mark I.; Chung, Seo-Kyung; Lynch, Joseph W.

    2013-01-01

    Hyperekplexia is a syndrome of readily provoked startle responses, alongside episodic and generalized hypertonia, that presents within the first month of life. Inhibitory glycine receptors are pentameric ligand-gated ion channels with a definitive and clinically well stratified linkage to hyperekplexia. Most hyperekplexia cases are caused by mutations in the α1 subunit of the human glycine receptor (hGlyR) gene (GLRA1). Here we analyzed 68 new unrelated hyperekplexia probands for GLRA1 mutations and identified 19 mutations, of which 9 were novel. Electrophysiological analysis demonstrated that the dominant mutations p.Q226E, p.V280M, and p.R414H induced spontaneous channel activity, indicating that this is a recurring mechanism in hGlyR pathophysiology. p.Q226E, at the top of TM1, most likely induced tonic activation via an enhanced electrostatic attraction to p.R271 at the top of TM2, suggesting a structural mechanism for channel activation. Receptors incorporating p.P230S (which is heterozygous with p.R65W) desensitized much faster than wild type receptors and represent a new TM1 site capable of modulating desensitization. The recessive mutations p.R72C, p.R218W, p.L291P, p.D388A, and p.E375X precluded cell surface expression unless co-expressed with α1 wild type subunits. The recessive p.E375X mutation resulted in subunit truncation upstream of the TM4 domain. Surprisingly, on the basis of three independent assays, we were able to infer that p.E375X truncated subunits are incorporated into functional hGlyRs together with unmutated α1 or α1 plus β subunits. These aberrant receptors exhibit significantly reduced glycine sensitivity. To our knowledge, this is the first suggestion that subunits lacking TM4 domains might be incorporated into functional pentameric ligand-gated ion channel receptors. PMID:24108130

  15. Restoration of Proper Trafficking to the Cell Surface for Membrane Proteins Harboring Cysteine Mutations

    PubMed Central

    Lopez-Rodriguez, Angelica; Holmgren, Miguel

    2012-01-01

    A common phenotype for many genetic diseases is that the cell is unable to deliver full-length membrane proteins to the cell surface. For some forms of autism, hereditary spherocytosis and color blindness, the culprits are single point mutations to cysteine. We have studied two inheritable cysteine mutants of cyclic nucleotide-gated channels that produce achromatopsia, a common form of severe color blindness. By taking advantage of the reactivity of cysteine’s sulfhydryl group, we modified these mutants with chemical reagents that attach moieties with similar chemistries to the wild-type amino acids’ side chains. We show that these modifications restored proper delivery to the cell membrane. Once there, the channels exhibited normal functional properties. This strategy might provide a unique opportunity to assess the chemical nature of membrane protein traffic problems. PMID:23082193

  16. Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

    PubMed Central

    Burg, Elyssa D.; Platoshyn, Oleksandr; Tsigelny, Igor F.; Lozano-Ruiz, Beatriz; Rana, Brinda K.

    2010-01-01

    The activity of voltage-gated K+ (KV) channels plays an important role in regulating pulmonary artery smooth muscle cell (PASMC) contraction, proliferation, and apoptosis. The highly conserved NH2-terminal tetramerization domain (T1) of KV channels is important for proper channel assembly, association with regulatory KV β-subunits, and localization of the channel to the plasma membrane. We recently reported two nonsynonymous mutations (G182R and E211D) in the KCNA5 gene of patients with idiopathic pulmonary arterial hypertension, which localize to the T1 domain of KCNA5. To study the electrophysiological properties and expression patterns of the mutants compared with the wild-type (WT) channel in vitro, we transfected HEK-293 cells with WT KCNA5, G182R, E211D, or the double mutant G182R/E211D channel. The mutants form functional channels; however, whole cell current kinetic differences between WT and mutant channels exist. Steady-state inactivation curves of the G182R and G182R/E211D channels reveal accelerated inactivation; the mutant channels inactivated at more hyperpolarized potentials compared with the WT channel. Channel protein expression was also decreased by the mutations. Compared with the WT channel, which was present in its mature glycosylated form, the mutant channels are present in greater proportion in their immature form in HEK-293 cells. Furthermore, G182R protein level is greatly reduced in COS-1 cells compared with WT. Immunostaining data support the hypothesis that, while WT protein localizes to the plasma membrane, mutant protein is mainly retained in intracellular packets. Overall, these data support a role for the T1 domain in channel kinetics as well as in KCNA5 channel subcellular localization. PMID:20018952

  17. Mycobacterium tuberculosis Type VII Secreted Effector EsxH Targets Host ESCRT to Impair Trafficking

    PubMed Central

    Thompson, Victor; Sirisaengtaksin, Natalie; Wells, Ashley; Porto, Maura; Köster, Stefan; Penberthy, Kristen; Kubota, Yoshihisha; Dricot, Amelie; Rogan, Daniel; Vidal, Marc; Hill, David E.; Bean, Andrew J.; Philips, Jennifer A.

    2013-01-01

    Mycobacterium tuberculosis (Mtb) disrupts anti-microbial pathways of macrophages, cells that normally kill bacteria. Over 40 years ago, D'Arcy Hart showed that Mtb avoids delivery to lysosomes, but the molecular mechanisms that allow Mtb to elude lysosomal degradation are poorly understood. Specialized secretion systems are often used by bacterial pathogens to translocate effectors that target the host, and Mtb encodes type VII secretion systems (TSSSs) that enable mycobacteria to secrete proteins across their complex cell envelope; however, their cellular targets are unknown. Here, we describe a systematic strategy to identify bacterial virulence factors by looking for interactions between the Mtb secretome and host proteins using a high throughput, high stringency, yeast two-hybrid (Y2H) platform. Using this approach we identified an interaction between EsxH, which is secreted by the Esx-3 TSSS, and human hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). ESCRT has a well-described role in directing proteins destined for lysosomal degradation into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs), ensuring degradation of the sorted cargo upon MVB-lysosome fusion. Here, we show that ESCRT is required to deliver Mtb to the lysosome and to restrict intracellular bacterial growth. Further, EsxH, in complex with EsxG, disrupts ESCRT function and impairs phagosome maturation. Thus, we demonstrate a role for a TSSS and the host ESCRT machinery in one of the central features of tuberculosis pathogenesis. PMID:24204276

  18. Missense mutations near the N-glycosylation site of the A2 domain lead to various intracellular trafficking defects in coagulation factor VIII

    PubMed Central

    Wei, Wei; Zheng, Chunlei; Zhu, Min; Zhu, Xiaofan; Yang, Renchi; Misra, Saurav; Zhang, Bin

    2017-01-01

    Missense mutation is the most common mutation type in hemophilia. However, the majority of missense mutations remain uncharacterized. Here we characterize how hemophilia mutations near the unused N-glycosylation site of the A2 domain (N582) of FVIII affect protein conformation and intracellular trafficking. N582 is located in the middle of a short 310-helical turn (D580-S584), in which most amino acids have multiple hemophilia mutations. All 14 missense mutations found in this 310-helix reduced secretion levels of the A2 domain and full-length FVIII. Secreted mutants have decreased activities relative to WT FVIII. Selected mutations also lead to partial glycosylation of N582, suggesting that rapid folding of local conformation prevents glycosylation of this site in wild-type FVIII. Protease sensitivity, stability and degradation of the A2 domain vary among mutants, and between non-glycosylated and glycosylated species of the same mutant. Most of the mutants interact with the ER chaperone BiP, while only mutants with aberrant glycosylation interact with calreticulin. Our results show that the short 310-helix from D580 to S584 is critical for proper biogenesis of the A2 domain and FVIII, and reveal a range of molecular mechanisms by which FVIII missense mutations lead to moderate to severe hemophilia A. PMID:28327546

  19. Maternally inherited hearing impairment in a family with the mitochondrial DNA A7445G mutation.

    PubMed

    Hutchin, T P; Lench, N J; Arbuzova, S; Markham, A F; Mueller, R F

    2001-01-01

    Despite the increasing number of reports of families with hearing impairment and mitochondrial DNA (mtDNA) mutations, the frequency of these mutations as causes of non-syndromic sensorineural hearing impairment (NSSHI) remains unknown. Mutations such as A1555G, A7445G and 7472insC have been found in several unrelated families implying they are more frequent than initially thought. We describe a family with NSSHI due to the presence of the homoplasmic mtDNA A7445G mutation in the tRNASer(UCN) gene. This is the fourth such family described with this mutation, all of different genetic backgrounds. Our study also demonstrates the difficulties sometimes encountered in establishing mitochondrial inheritance of hearing impairment in some families.

  20. β-Amyloid (Aβ) Oligomers Impair Brain-derived Neurotrophic Factor Retrograde Trafficking by Down-regulating Ubiquitin C-terminal Hydrolase, UCH-L1*

    PubMed Central

    Poon, Wayne W.; Carlos, Anthony J.; Aguilar, Brittany L.; Berchtold, Nicole C.; Kawano, Crystal K.; Zograbyan, Vahe; Yaopruke, Tim; Shelanski, Michael; Cotman, Carl W.

    2013-01-01

    We previously found that BDNF-dependent retrograde trafficking is impaired in AD transgenic mouse neurons. Utilizing a novel microfluidic culture chamber, we demonstrate that Aβ oligomers compromise BDNF-mediated retrograde transport by impairing endosomal vesicle velocities, resulting in impaired downstream signaling driven by BDNF/TrkB, including ERK5 activation, and CREB-dependent gene regulation. Our data suggest that a key mechanism mediating the deficit involves ubiquitin C-terminal hydrolase L1 (UCH-L1), a deubiquitinating enzyme that functions to regulate cellular ubiquitin. Aβ-induced deficits in BDNF trafficking and signaling are mimicked by LDN (an inhibitor of UCH-L1) and can be reversed by increasing cellular UCH-L1 levels, demonstrated here using a transducible TAT-UCH-L1 strategy. Finally, our data reveal that UCH-L1 mRNA levels are decreased in the hippocampi of AD brains. Taken together, our data implicate that UCH-L1 is important for regulating neurotrophin receptor sorting to signaling endosomes and supporting retrograde transport. Further, our results support the idea that in AD, Aβ may down-regulate UCH-L1 in the AD brain, which in turn impairs BDNF/TrkB-mediated retrograde signaling, compromising synaptic plasticity and neuronal survival. PMID:23599427

  1. β-Amyloid (Aβ) oligomers impair brain-derived neurotrophic factor retrograde trafficking by down-regulating ubiquitin C-terminal hydrolase, UCH-L1.

    PubMed

    Poon, Wayne W; Carlos, Anthony J; Aguilar, Brittany L; Berchtold, Nicole C; Kawano, Crystal K; Zograbyan, Vahe; Yaopruke, Tim; Shelanski, Michael; Cotman, Carl W

    2013-06-07

    We previously found that BDNF-dependent retrograde trafficking is impaired in AD transgenic mouse neurons. Utilizing a novel microfluidic culture chamber, we demonstrate that Aβ oligomers compromise BDNF-mediated retrograde transport by impairing endosomal vesicle velocities, resulting in impaired downstream signaling driven by BDNF/TrkB, including ERK5 activation, and CREB-dependent gene regulation. Our data suggest that a key mechanism mediating the deficit involves ubiquitin C-terminal hydrolase L1 (UCH-L1), a deubiquitinating enzyme that functions to regulate cellular ubiquitin. Aβ-induced deficits in BDNF trafficking and signaling are mimicked by LDN (an inhibitor of UCH-L1) and can be reversed by increasing cellular UCH-L1 levels, demonstrated here using a transducible TAT-UCH-L1 strategy. Finally, our data reveal that UCH-L1 mRNA levels are decreased in the hippocampi of AD brains. Taken together, our data implicate that UCH-L1 is important for regulating neurotrophin receptor sorting to signaling endosomes and supporting retrograde transport. Further, our results support the idea that in AD, Aβ may down-regulate UCH-L1 in the AD brain, which in turn impairs BDNF/TrkB-mediated retrograde signaling, compromising synaptic plasticity and neuronal survival.

  2. The E693Δ (Osaka) mutation in amyloid precursor protein potentiates cholesterol-mediated intracellular amyloid β toxicity via its impaired cholesterol efflux.

    PubMed

    Nomura, Sachiko; Umeda, Tomohiro; Tomiyama, Takami; Mori, Hiroshi

    2013-12-01

    It has been shown that amyloid β (Aβ) secretion regulates cholesterol efflux from cells and that the E693Δ (Osaka) mutation in amyloid precursor protein (APP) promotes intracellular accumulation of Aβ and thus reduces its secretion. These findings led us to speculate that APP with the Osaka mutation (APPOSK ) might have a defect in cholesterol efflux and thus cause cellular malfunction. We therefore examined the effects of this mutation on intracellular cholesterol transport and efflux in cultured cells. Upon cholesterol loading, APPOSK -expressing cells exhibited higher levels of cellular cholesterol than wild-type APP-expressing cells, suggesting impaired cholesterol efflux. It is known that, after its internalization, cholesterol is transported from the endosomes to the endoplasmic reticulum (ER) and Golgi apparatus and then to the plasma membrane. In APPOSK -expressing cells, cholesterol accumulated with Aβ in the ER and Golgi apparatus and alone in endosomes/lysosomes. These results imply that the mutation-induced disturbance of Aβ trafficking from the ER to the plasma membrane affects cholesterol transport to cause cholesterol accumulation in the ER and Golgi apparatus and, consequently, in endosomes. Furthermore, we detected an enhanced mitochondrial accumulation of Aβ and cholesterol in APPOSK -expressing cells, and this was accompanied by an increase in the generation of reactive oxygen species (ROS). The present findings suggest that Aβ trafficking is important for intracellular cholesterol transport and efflux and that the Osaka mutation potentiates cholesterol-dependent exacerbation of intracellular Aβ toxicity, i.e. Aβ-induced ROS generation, by disturbing Aβ-mediated cholesterol efflux from the cell. Copyright © 2013 Wiley Periodicals, Inc.

  3. Homeostatic regulation of T cell trafficking by a B cell derived peptide is impaired in autoimmune and chronic inflammatory disease

    PubMed Central

    Apta, Bonita; Kuravi, Sahithi J.; Yates, Clara M.; Kennedy, Amy; Odedra, Arjun; Alassiri, Mohammed; Harrison, Matthew; Martin, Ashley; Barone, Francesca; Nayar, Saba; Hitchcock, Jessica R.; Cunningham, Adam F.; Raza, Karim; Filer, Andrew; Copland, David A.; Dick, Andrew D.; Robinson, Joseph; Kalia, Neena; Walker, Lucy S. K.; Buckley, Christopher D.; Nash, Gerard B.; Narendran, Parth; Rainger, G. Ed.

    2015-01-01

    During an inflammatory response, lymphocyte recruitment into tissue must be tightly controlled because dysregulated trafficking contributes to the pathogenesis of chronic disease. Here we show that during inflammation and in response to adiponectin, B cells tonically inhibit T cell trafficking by secreting a peptide (PEPITEM) proteolytically derived from 14.3.3.ζδ protein. PEPITEM binds cadherin-15 on endothelial cells, promoting synthesis and release of sphingosine-1 phosphate, which inhibits trafficking of T cells without affecting recruitment of other leukocytes. Expression of adiponectin receptors on B cells and adiponectin induced PEPITEM secretion wanes with age, implying immune senescence of the pathway. Additionally, these changes are evident in individuals with type-1-diabetes or rheumatoid arthritis, and circulating PEPITEM in patient serum is reduced compared to healthy age matched donors. In both diseases, tonic inhibition of T cell trafficking across inflamed endothelium is lost. Importantly, control of patient T cell trafficking is re-established by exogenous PEPITEM. Moreover, in animal models of peritonitis, hepatic I/R injury, Salmonella infection, Uveitis and Sjögren’s Syndrome, PEPITEM could reduce T cell recruitment into inflamed tissues. PMID:25894827

  4. The Salih ataxia mutation impairs Rubicon endosomal localization.

    PubMed

    Assoum, M; Salih, M A; Drouot, N; Hnia, K; Martelli, A; Koenig, M

    2013-12-01

    We previously described a new form of recessive ataxia, Salih ataxia, in a large consanguineous Saudi Arabian family with three affected children carrying a new identified mutation in the KIAA0226 gene (c.2624delC; p.Ala875ValfsX146) coding for Rubicon. The pathogenicity of such mutation remains to be identified. Hence, we address the cellular impact of Rubicon p.Ala875ValfsX146 on endosomal/lysosomal machinery on cultured cells. We confirm that Rubicon colocalizes with the late endosome marker Rab7 and demonstrate that it also colocalizes with LampI at lysosomes. The Salih ataxia mutation leads to a diffuse cytosolic distribution and mislocalized protein from the late endosomes, indicating that deletion of the diacylglycerol binding-like motif in the mutant protein interferes with normal Rubicon subcellular localization and confirming the pathogenicity of the mutation.

  5. GATA2 germline mutations impair GATA2 transcription, causing haploinsufficiency: functional analysis of the p.Arg396Gln mutation.

    PubMed

    Cortés-Lavaud, Xabier; Landecho, Manuel F; Maicas, Miren; Urquiza, Leire; Merino, Juana; Moreno-Miralles, Isabel; Odero, María D

    2015-03-01

    Germline GATA2 mutations have been identified as the cause of familial syndromes with immunodeficiency and predisposition to myeloid malignancies. GATA2 mutations appear to cause loss of function of the mutated allele leading to haploinsufficiency; however, this postulate has not been experimentally validated as the basis of these syndromes. We hypothesized that mutations that are translated into abnormal proteins could affect the transcription of GATA2, triggering GATA2 deficiency. Chromatin immunoprecipitation and luciferase assays showed that the human GATA2 protein activates its own transcription through a specific region located at -2.4 kb, whereas the p.Thr354Met, p.Thr355del, and p.Arg396Gln germline mutations impair GATA2 promoter activation. Accordingly, GATA2 expression was decreased to ∼58% in a patient with p.Arg396Gln, compared with controls. p.Arg396Gln is the second most common mutation in these syndromes, and no previous functional analyses have been performed. We therefore analyzed p.Arg396Gln. Our data show that p.Arg396Gln is a loss-of-function mutation affecting DNA-binding ability and, as a consequence, it fails to maintain the immature characteristics of hematopoietic stem and progenitor cells, which could result in defects in this cell compartment. In conclusion, we show that human GATA2 binds to its own promoter, activating its transcription, and that the aforementioned mutations impair the transcription of GATA2. Our results indicate that they can affect other GATA2 target genes, which could partially explain the variability of symptoms in these diseases. Moreover, we show that p.Arg396Gln is a loss-of-function mutation, which is unable to retain the progenitor phenotype in cells where it is expressed.

  6. Ethanol metabolism by alcohol dehydrogenase or cytochrome P450 2E1 differentially impairs hepatic protein trafficking and growth hormone signaling.

    PubMed

    Doody, Erin E; Groebner, Jennifer L; Walker, Jetta R; Frizol, Brittnee M; Tuma, Dean J; Fernandez, David J; Tuma, Pamela L

    2017-09-01

    The liver metabolizes alcohol using alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1). Both enzymes metabolize ethanol into acetaldehyde, but CYP2E1 activity also results in the production of reactive oxygen species (ROS) that promote oxidative stress. We have previously shown that microtubules are hyperacetylated in ethanol-treated polarized, hepatic WIF-B cells and livers from ethanol fed rats. We have also shown that enhanced protein acetylation correlates with impaired clathrin-mediated endocytosis, constitutive secretion and nuclear translocation and that the defects are likely mediated by acetaldehyde. However, the roles of CYP2E1-generated metabolites and ROS in microtubule acetylation and these alcohol-induced impairments have not been examined. To determine if CYP2E1-mediated alcohol metabolism is required for enhanced acetylation and the trafficking defects, we co-incubated cells with ethanol and diallyl sulfide (a CYP2E1 inhibitor) or N-acetyl cysteine (an anti-oxidant). Both agents failed to prevent microtubule hyperacetylation in ethanol-treated cells and also failed to prevent impaired secretion or clathrin-mediated endocytosis. Somewhat surprisingly, both NAS and DAC prevented impaired STAT5B nuclear translocation. Further examination of microtubule-independent steps of the pathway revealed that Jak2/STAT5B activation by growth hormone (GH) was prevented by DAS and NAC. These results were confirmed in ethanol-exposed HepG2 cells expressing only ADH or CYP2E1. Using quantitative RT-PCR, we further determined that ethanol exposure led to blunted GH-mediated gene expression. In conclusion, we determined that alcohol-induced microtubule acetylation and associated defects in microtubule-dependent trafficking are mediated by ADH metabolism whereas impaired microtubule-independent Jak2/STAT5B activation is mediated by CYP2E1 activity. Copyright © 2017, American Journal of Physiology-Gastrointestinal and Liver Physiology.

  7. HLA-B27 Selects for Rare Escape Mutations that Significantly Impair Hepatitis C Virus Replication and Require Compensatory Mutations

    PubMed Central

    Neumann-Haefelin, Christoph; Oniangue-Ndza, Cesar; Kuntzen, Thomas; Schmidt, Julia; Nitschke, Katja; Sidney, John; Caillet-Saguy, Célia; Binder, Marco; Kersting, Nadine; Kemper, Michael W.; Power, Karen A.; Ingber, Susan; Reyor, Laura L.; Hills-Evans, Kelsey; Kim, Arthur Y.; Lauer, Georg M.; Lohmann, Volker; Sette, Alessandro; Henn, Matthew R.; Bressanelli, Stéphane; Thimme, Robert; Allen, Todd M.

    2011-01-01

    HLA-B27 is associated with spontaneous viral clearance in hepatitis C virus (HCV) infection. Viral escape within the immunodominant HLA-B27 restricted HCV-specific CD8+ T cell epitope NS5B2841-2849 (ARMILMTHF) has been shown to be limited by viral fitness costs as well as broad T cell cross-recognition, suggesting a potential mechanism of protection by HLA-B27. Here, we studied the subdominant HLA-B27 restricted epitope NS5B2936-2944 (GRAAICGKY) in order to further define the mechanisms of protection by HLA-B27. We identified a unique pattern of escape mutations within this epitope in a large cohort of HCV genotype 1a infected patients. The predominant escape mutations represented conservative substitutions at the main HLA-B27 anchor residue or a T cell receptor contact site, neither of which impaired viral replication capacity as assessed in a subgenomic HCV replicon system. In contrast, however, in a subset of HLA-B27+ subjects rare escape mutations arose at the HLA-B27 anchor residue R2937, which nearly abolished viral replication. Notably, these rare mutations only occurred in conjunction with the selection of two equally rare, and structurally proximal, upstream mutations. Co-expression of these upstream mutations with the rare escape mutations dramatically restored viral replication capacity from <5% to ≥70% of wild-type levels. Conclusion The selection of rare CTL escape mutations in this HLA-B27 restricted epitope dramatically impairs viral replicative fitness unless properly compensated. These data support a role for the targeting of highly-constrained regions by HLA-B27 in its ability to assert immune control of HCV and other highly variable pathogens. PMID:22006856

  8. Hypomorphic, homozygous mutations in Phosphoglucomutase 3 impair immunity and increase serum IgE levels

    PubMed Central

    Sassi, Atfa; Lazaroski, Sandra; Wu, Gang; Haslam, Stuart M.; Fliegauf, Manfred; Mellouli, Fethi; Patiroglu, Turkan; Unal, Ekrem; Ozdemir, Mehmet Akif; Jouhadi, Zineb; Khadir, Khadija; Ben-Khemis, Leila; Ben-Ali, Meriem; Ben-Mustapha, Imen; Borchani, Lamia; Pfeifer, Dietmar; Jakob, Thilo; Khemiri, Monia; Asplund, A. Charlotta; Gustafsson, Manuela O.; Lundin, Karin E.; Falk-Sörqvist, Elin; Moens, Lotte N.; Gungor, Hatice Eke; Engelhardt, Karin R.; Dziadzio, Magdalena; Stauss, Hans; Fleckenstein, Bernhard; Meier, Rebecca; Prayitno, Khairunnadiya; Maul-Pavicic, Andrea; Schaffer, Sandra; Rakhmanov, Mirzokhid; Henneke, Philipp; Kraus, Helene; Eibel, Hermann; Kölsch, Uwe; Nadifi, Sellama; Nilsson, Mats; Bejaoui, Mohamed; Schäffer, Alejandro A.; Edvard Smith, C. I.; Dell, Anne; Barbouche, Mohamed-Ridha; Grimbacher, Bodo

    2016-01-01

    Background Recurrent bacterial and fungal infections, eczema and elevated serum IgE levels characterize patients with the hyper-IgE syndrome (HIES). Known genetic causes for HIES are mutations in STAT3 and DOCK8, involved in signal transduction pathways. However, glycosylation defects have not been described in HIES. One crucial enzyme in the glycosylation pathway is Phosphoglucomutase 3 (PGM3), which catalyzes a key step in the synthesis of UDP-GlcNAc which is required for the biosynthesis of N-glycans. Objective To elucidate the genetic cause in HIES patients who do not carry mutations in STAT3 or DOCK8. Methods After establishing a linkage interval by SNP-chip genotyping and homozygosity mapping in two HIES families from Tunisia, mutational analysis was performed with selector-based, high-throughput sequencing. Protein expression was analyzed by Western blotting and glycosylation was profiled by mass spectrometry. Results Mutational analysis of candidate genes in a 11.9 Mb linkage region on chromosome 6 shared by two multiplex families identified two homozygous mutations in PGM3 which segregated with the disease status and followed a recessive inheritance trait. The mutations predict amino acid changes in Phosphoglucomutase-3; PGM3 (p.Glu340del and p.Leu83Ser). A third homozygous mutation (p.Asp502Tyr) and the p.Leu83Ser variant were identified in two other affected families, respectively. These hypomorphic mutations have impact on the biosynthetic reactions involving UDP-GlcNAc. Glycomic analysis revealed an aberrant glycosylation pattern in leukocytes demonstrated by a reduced level of tri-/tetra-antennary N-glycans. T cell proliferation and differentiation was impaired in patients. Most patients showed developmental delay and many had psychomotor retardation. Conclusion Impairment of PGM3 function leads to a novel primary (inborn) error of development and immunity, as biallelic hypomorphic mutations are associated with impaired glycosylation and a hyper

  9. Pharmacological correction of long QT-linked mutations in KCNH2 (hERG) increases the trafficking of Kv11.1 channels stored in the transitional endoplasmic reticulum

    PubMed Central

    Smith, Jennifer L.; Reloj, Allison R.; Nataraj, Parvathi S.; Bartos, Daniel C.; Schroder, Elizabeth A.; Moss, Arthur J.; Ohno, Seiko; Horie, Minoru; Anderson, Corey L.; January, Craig T.

    2013-01-01

    KCNH2 encodes Kv11.1 and underlies the rapidly activating delayed rectifier K+ current (IKr) in the heart. Loss-of-function KCNH2 mutations cause the type 2 long QT syndrome (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channels. Drugs that bind to Kv11.1 and block IKr (e.g., E-4031) can act as pharmacological chaperones to increase the trafficking and functional expression for most LQT2 channels (pharmacological correction). We previously showed that LQT2 channels are selectively stored in a microtubule-dependent compartment within the endoplasmic reticulum (ER). We tested the hypothesis that pharmacological correction promotes the trafficking of LQT2 channels stored in this compartment. Confocal analyses of cells expressing the trafficking-deficient LQT2 channel G601S showed that the microtubule-dependent ER compartment is the transitional ER. Experiments with E-4031 and the protein synthesis inhibitor cycloheximide suggested that pharmacological correction promotes the trafficking of G601S stored in this compartment. Treating cells in E-4031 or ranolazine (a drug that blocks IKr and has a short half-life) for 30 min was sufficient to cause pharmacological correction. Moreover, the increased functional expression of G601S persisted 4–5 h after drug washout. Coexpression studies with a dominant-negative form of Rab11B, a small GTPase that regulates Kv11.1 trafficking, prevented the pharmacological correction of G601S trafficking from the transitional ER. These data suggest that pharmacological correction quickly increases the trafficking of LQT2 channels stored in the transitional ER via a Rab11B-dependent pathway, and we conclude that the pharmacological chaperone activity of drugs like ranolazine might have therapeutic potential. PMID:23864605

  10. Pharmacological correction of long QT-linked mutations in KCNH2 (hERG) increases the trafficking of Kv11.1 channels stored in the transitional endoplasmic reticulum.

    PubMed

    Smith, Jennifer L; Reloj, Allison R; Nataraj, Parvathi S; Bartos, Daniel C; Schroder, Elizabeth A; Moss, Arthur J; Ohno, Seiko; Horie, Minoru; Anderson, Corey L; January, Craig T; Delisle, Brian P

    2013-11-01

    KCNH2 encodes Kv11.1 and underlies the rapidly activating delayed rectifier K(+) current (IKr) in the heart. Loss-of-function KCNH2 mutations cause the type 2 long QT syndrome (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channels. Drugs that bind to Kv11.1 and block IKr (e.g., E-4031) can act as pharmacological chaperones to increase the trafficking and functional expression for most LQT2 channels (pharmacological correction). We previously showed that LQT2 channels are selectively stored in a microtubule-dependent compartment within the endoplasmic reticulum (ER). We tested the hypothesis that pharmacological correction promotes the trafficking of LQT2 channels stored in this compartment. Confocal analyses of cells expressing the trafficking-deficient LQT2 channel G601S showed that the microtubule-dependent ER compartment is the transitional ER. Experiments with E-4031 and the protein synthesis inhibitor cycloheximide suggested that pharmacological correction promotes the trafficking of G601S stored in this compartment. Treating cells in E-4031 or ranolazine (a drug that blocks IKr and has a short half-life) for 30 min was sufficient to cause pharmacological correction. Moreover, the increased functional expression of G601S persisted 4-5 h after drug washout. Coexpression studies with a dominant-negative form of Rab11B, a small GTPase that regulates Kv11.1 trafficking, prevented the pharmacological correction of G601S trafficking from the transitional ER. These data suggest that pharmacological correction quickly increases the trafficking of LQT2 channels stored in the transitional ER via a Rab11B-dependent pathway, and we conclude that the pharmacological chaperone activity of drugs like ranolazine might have therapeutic potential.

  11. Trafficking defects and loss of ligand binding are the underlying causes of all reported DDR2 missense mutations found in SMED-SL patients.

    PubMed

    Ali, Bassam R; Xu, Huifang; Akawi, Nadia A; John, Anne; Karuvantevida, Noushad S; Langer, Ruth; Al-Gazali, Lihadh; Leitinger, Birgit

    2010-06-01

    Spondylo-meta-epiphyseal dysplasia (SMED) with short limbs and abnormal calcifications (SMED-SL) is a rare, autosomal recessive human growth disorder, characterized by disproportionate short stature, short limbs, short broad fingers, abnormal metaphyses and epiphyses, platyspondyly and premature calcifications. Recently, three missense mutations and one splice-site mutation in the DDR2 gene were identified as causative genetic defects for SMED-SL, but the underlying cellular and biochemical mechanisms were not explored. Here we report a novel DDR2 missense mutation, c.337G>A (p.E113K), that causes SMED-SL in two siblings in the United Arab Emirates. Another DDR2 missense mutation, c.2254C>T (p.R752C), matching one of the previously reported SMED-SL mutations, was found in a second affected family. DDR2 is a plasma membrane receptor tyrosine kinase that functions as a collagen receptor. We expressed DDR2 constructs with the identified point mutations in human cell lines and evaluated their localization and functional properties. We found that all SMED-SL missense mutants were defective in collagen-induced receptor activation and that the three previously reported mutants (p.T713I, p.I726R and p.R752C) were retained in the endoplasmic reticulum. The novel mutant (p.E113K), in contrast, trafficked normally, like wild-type DDR2, but failed to bind collagen. This finding is in agreement with our recent structural data identifying Glu113 as an important amino acid in the DDR2 ligand-binding site. Our data thus demonstrate that SMED-SL can result from at least two different loss-of-function mechanisms: namely defects in DDR2 targeting to the plasma membrane or the loss of its ligand-binding activity.

  12. Trafficking defects and loss of ligand binding are the underlying causes of all reported DDR2 missense mutations found in SMED-SL patients

    PubMed Central

    Ali, Bassam R.; Xu, Huifang; Akawi, Nadia A.; John, Anne; Karuvantevida, Noushad S.; Langer, Ruth; Al-Gazali, Lihadh; Leitinger, Birgit

    2010-01-01

    Spondylo-meta-epiphyseal dysplasia (SMED) with short limbs and abnormal calcifications (SMED-SL) is a rare, autosomal recessive human growth disorder, characterized by disproportionate short stature, short limbs, short broad fingers, abnormal metaphyses and epiphyses, platyspondyly and premature calcifications. Recently, three missense mutations and one splice-site mutation in the DDR2 gene were identified as causative genetic defects for SMED-SL, but the underlying cellular and biochemical mechanisms were not explored. Here we report a novel DDR2 missense mutation, c.337G>A (p.E113K), that causes SMED-SL in two siblings in the United Arab Emirates. Another DDR2 missense mutation, c.2254C>T (p.R752C), matching one of the previously reported SMED-SL mutations, was found in a second affected family. DDR2 is a plasma membrane receptor tyrosine kinase that functions as a collagen receptor. We expressed DDR2 constructs with the identified point mutations in human cell lines and evaluated their localization and functional properties. We found that all SMED-SL missense mutants were defective in collagen-induced receptor activation and that the three previously reported mutants (p.T713I, p.I726R and p.R752C) were retained in the endoplasmic reticulum. The novel mutant (p.E113K), in contrast, trafficked normally, like wild-type DDR2, but failed to bind collagen. This finding is in agreement with our recent structural data identifying Glu113 as an important amino acid in the DDR2 ligand-binding site. Our data thus demonstrate that SMED-SL can result from at least two different loss-of-function mechanisms: namely defects in DDR2 targeting to the plasma membrane or the loss of its ligand-binding activity. PMID:20223752

  13. Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation

    PubMed Central

    Valentin-Vega, Yasmine A.; Wang, Yong-Dong; Parker, Matthew; Patmore, Deanna M.; Kanagaraj, Anderson; Moore, Jennifer; Rusch, Michael; Finkelstein, David; Ellison, David W.; Gilbertson, Richard J.; Zhang, Jinghui; Kim, Hong Joo; Taylor, J. Paul

    2016-01-01

    DDX3X is a DEAD-box RNA helicase that has been implicated in multiple aspects of RNA metabolism including translation initiation and the assembly of stress granules (SGs). Recent genomic studies have reported recurrent DDX3X mutations in numerous tumors including medulloblastoma (MB), but the physiological impact of these mutations is poorly understood. Here we show that a consistent feature of MB-associated mutations is SG hyper-assembly and concomitant translation impairment. We used CLIP-seq to obtain a comprehensive assessment of DDX3X binding targets and ribosome profiling for high-resolution assessment of global translation. Surprisingly, mutant DDX3X expression caused broad inhibition of translation that impacted DDX3X targeted and non-targeted mRNAs alike. Assessment of translation efficiency with single-cell resolution revealed that SG hyper-assembly correlated precisely with impaired global translation. SG hyper-assembly and translation impairment driven by mutant DDX3X were rescued by a genetic approach that limited SG assembly and by deletion of the N-terminal low complexity domain within DDX3X. Thus, in addition to a primary defect at the level of translation initiation caused by DDX3X mutation, SG assembly itself contributes to global translation inhibition. This work provides mechanistic insights into the consequences of cancer-related DDX3X mutations, suggesting that globally reduced translation may provide a context-dependent survival advantage that must be considered as a possible contributor to tumorigenesis. PMID:27180681

  14. MYBPC1 mutations impair skeletal muscle function in zebrafish models of arthrogryposis.

    PubMed

    Ha, Kyungsoo; Buchan, Jillian G; Alvarado, David M; McCall, Kevin; Vydyanath, Anupama; Luther, Pradeep K; Goldsmith, Matthew I; Dobbs, Matthew B; Gurnett, Christina A

    2013-12-15

    Myosin-binding protein C1 (MYBPC1) is an abundant skeletal muscle protein that is expressed predominantly in slow-twitch muscle fibers. Human MYBPC1 mutations are associated with distal arthrogryposis type 1 and lethal congenital contracture syndrome type 4. As MYBPC1 function is incompletely understood, the mechanism by which human mutations result in contractures is unknown. Here, we demonstrate using antisense morpholino knockdown, that mybpc1 is required for embryonic motor activity and survival in a zebrafish model of arthrogryposis. Mybpc1 morphant embryos have severe body curvature, cardiac edema, impaired motor excitation and are delayed in hatching. Myofibril organization is selectively impaired in slow skeletal muscle and sarcomere numbers are greatly reduced in mybpc1 knockdown embryos, although electron microscopy reveals normal sarcomere structure. To evaluate the effects of human distal arthrogryposis mutations, mybpc1 mRNAs containing the corresponding human W236R and Y856H MYBPC1 mutations were injected into embryos. Dominant-negative effects of these mutations were suggested by the resultant mild bent body curvature, decreased motor activity, as well as impaired overall survival compared with overexpression of wild-type RNA. These results demonstrate a critical role for mybpc1 in slow skeletal muscle development and establish zebrafish as a tractable model of human distal arthrogryposis.

  15. MYBPC1 mutations impair skeletal muscle function in zebrafish models of arthrogryposis

    PubMed Central

    Ha, Kyungsoo; Buchan, Jillian G.; Alvarado, David M.; Mccall, Kevin; Vydyanath, Anupama; Luther, Pradeep K.; Goldsmith, Matthew I.; Dobbs, Matthew B.; Gurnett, Christina A.

    2013-01-01

    Myosin-binding protein C1 (MYBPC1) is an abundant skeletal muscle protein that is expressed predominantly in slow-twitch muscle fibers. Human MYBPC1 mutations are associated with distal arthrogryposis type 1 and lethal congenital contracture syndrome type 4. As MYBPC1 function is incompletely understood, the mechanism by which human mutations result in contractures is unknown. Here, we demonstrate using antisense morpholino knockdown, that mybpc1 is required for embryonic motor activity and survival in a zebrafish model of arthrogryposis. Mybpc1 morphant embryos have severe body curvature, cardiac edema, impaired motor excitation and are delayed in hatching. Myofibril organization is selectively impaired in slow skeletal muscle and sarcomere numbers are greatly reduced in mybpc1 knockdown embryos, although electron microscopy reveals normal sarcomere structure. To evaluate the effects of human distal arthrogryposis mutations, mybpc1 mRNAs containing the corresponding human W236R and Y856H MYBPC1 mutations were injected into embryos. Dominant-negative effects of these mutations were suggested by the resultant mild bent body curvature, decreased motor activity, as well as impaired overall survival compared with overexpression of wild-type RNA. These results demonstrate a critical role for mybpc1 in slow skeletal muscle development and establish zebrafish as a tractable model of human distal arthrogryposis. PMID:23873045

  16. GJB2 mutation spectrum in 2063 Chinese patients with nonsyndromic hearing impairment

    PubMed Central

    Dai, Pu; Yu, Fei; Han, Bing; Liu, Xuezhong; Wang, Guojian; Li, Qi; Yuan, Yongyi; Liu, Xin; Huang, Deliang; Kang, Dongyang; Zhang, Xin; Yuan, Huijun; Yao, Kun; Hao, Jinsheng; He, Jia; He, Yong; Wang, Youqin; Ye, Qing; Yu, Youjun; Lin, Hongyan; Liu, Lijia; Deng, Wei; Zhu, Xiuhui; You, Yiwen; Cui, Jinghong; Hou, Nongsheng; Xu, Xuehai; Zhang, Jin; Tang, Liang; Song, Rendong; Lin, Yongjun; Sun, Shuanzhu; Zhang, Ruining; Wu, Hao; Ma, Yuebing; Zhu, Shanxiang; Wu, Bai-lin; Han, Dongyi; Wong, Lee-Jun C

    2009-01-01

    Background Mutations in GJB2 are the most common molecular defects responsible for autosomal recessive nonsyndromic hearing impairment (NSHI). The mutation spectra of this gene vary among different ethnic groups. Methods In order to understand the spectrum and frequency of GJB2 mutations in the Chinese population, the coding region of the GJB2 gene from 2063 unrelated patients with NSHI was PCR amplified and sequenced. Results A total of 23 pathogenic mutations were identified. Among them, five (p.W3X, c.99delT, c.155_c.158delTCTG, c.512_c.513insAACG, and p.Y152X) are novel. Three hundred and seven patients carry two confirmed pathogenic mutations, including 178 homozygotes and 129 compound heterozygotes. One hundred twenty five patients carry only one mutant allele. Thus, GJB2 mutations account for 17.9% of the mutant alleles in 2063 NSHI patients. Overall, 92.6% (684/739) of the pathogenic mutations are frame-shift truncation or nonsense mutations. The four prevalent mutations; c.235delC, c.299_c.300delAT, c.176_c.191del16, and c.35delG, account for 88.0% of all mutantalleles identified. The frequency of GJB2 mutations (alleles) varies from 4% to 30.4% among different regions of China. It also varies among different sub-ethnic groups. Conclusion In some regions of China, testing of the three most common mutations can identify at least one GJB2 mutant allele in all patients. In other regions such as Tibet, the three most common mutations account for only 16% the GJB2 mutant alleles. Thus, in this region, sequencing of GJB2 would be recommended. In addition, the etiology of more than 80% of the mutant alleles for NSHI in China remains to be identified. Analysis of other NSHI related genes will be necessary. PMID:19366456

  17. The Dynamin Chemical Inhibitor Dynasore Impairs Cholesterol Trafficking and Sterol-Sensitive Genes Transcription in Human HeLa Cells and Macrophages

    PubMed Central

    Girard, Emmanuelle; Paul, Jean Louis; Fournier, Natalie; Beaune, Philippe; Johannes, Ludger

    2011-01-01

    Intracellular transport of cholesterol contributes to the regulation of cellular cholesterol homeostasis by mechanisms that are yet poorly defined. In this study, we characterized the impact of dynasore, a recently described drug that specifically inhibits the enzymatic activity of dynamin, a GTPase regulating receptor endocytosis and cholesterol trafficking. Dynasore strongly inhibited the uptake of low-density lipoprotein (LDL) in HeLa cells, and to a lower extent in human macrophages. In both cell types, dynasore treatment led to the abnormal accumulation of LDL and free cholesterol (FC) within the endolysosomal network. The measure of cholesterol esters (CE) further showed that the delivery of regulatory cholesterol to the endoplasmic reticulum (ER) was deficient. This resulted in the inhibition of the transcriptional control of the three major sterol-sensitive genes, sterol-regulatory element binding protein 2 (SREBP-2), 3-hydroxy-3-methyl-coenzymeA reductase (HMGCoAR), and low-density lipoprotein receptor (LDLR). The sequestration of cholesterol in the endolysosomal compartment impaired both the active and passive cholesterol efflux in HMDM. Our data further illustrate the importance of membrane trafficking in cholesterol homeostasis and validate dynasore as a new pharmacological tool to study the intracellular transport of cholesterol. PMID:22205993

  18. Rare Synaptogenesis-Impairing Mutations in SLITRK5 Are Associated with Obsessive Compulsive Disorder.

    PubMed

    Song, Minseok; Mathews, Carol A; Stewart, S Evelyn; Shmelkov, Sergey V; Mezey, Jason G; Rodriguez-Flores, Juan L; Rasmussen, Steven A; Britton, Jennifer C; Oh, Yong-Seok; Walkup, John T; Lee, Francis S; Glatt, Charles E

    2017-01-01

    Obsessive compulsive disorder (OCD) is substantially heritable, but few molecular genetic risk factors have been identified. Knockout mice lacking SLIT and NTRK-Like Family, Member 5 (SLITRK5) display OCD-like phenotypes including serotonin reuptake inhibitor-sensitive pathologic grooming, and corticostriatal dysfunction. Thus, mutations that impair SLITRK5 function may contribute to the genetic risk for OCD. We re-sequenced the protein-coding sequence of the human SLITRK5 gene (SLITRK5) in three hundred and seventy seven OCD subjects and compared rare non-synonymous mutations (RNMs) in that sample with similar mutations in the 1000 Genomes database. We also performed in silico assessments and in vitro functional synaptogenesis assays on the Slitrk5 mutations identified. We identified four RNM's among these OCD subjects. There were no significant differences in the prevalence or in silico effects of rare non-synonymous mutations in the OCD sample versus controls. Direct functional testing of recombinant SLITRK5 proteins found that all mutations identified in OCD subjects impaired synaptogenic activity whereas none of the pseudo-matched mutations identified in 1000 Genomes controls had significant effects on SLITRK5 function (Fisher's exact test P = 0.028). These results demonstrate that rare functional mutations in SLITRK5 contribute to the genetic risk for OCD in human populations. They also highlight the importance of biological characterization of allelic effects in understanding genotype-phenotype relationships as there were no statistical differences in overall prevalence or bioinformatically predicted effects of OCD case versus control mutations. Finally, these results converge with others to highlight the role of aberrant synaptic function in corticostriatal neurons in the pathophysiology of OCD.

  19. Rare Synaptogenesis-Impairing Mutations in SLITRK5 Are Associated with Obsessive Compulsive Disorder

    PubMed Central

    Mathews, Carol A.; Stewart, S. Evelyn; Shmelkov, Sergey V.; Mezey, Jason G.; Rodriguez-Flores, Juan L.; Rasmussen, Steven A.; Britton, Jennifer C.; Oh, Yong-Seok; Walkup, John T.; Lee, Francis S.; Glatt, Charles E.

    2017-01-01

    Obsessive compulsive disorder (OCD) is substantially heritable, but few molecular genetic risk factors have been identified. Knockout mice lacking SLIT and NTRK-Like Family, Member 5 (SLITRK5) display OCD-like phenotypes including serotonin reuptake inhibitor-sensitive pathologic grooming, and corticostriatal dysfunction. Thus, mutations that impair SLITRK5 function may contribute to the genetic risk for OCD. We re-sequenced the protein-coding sequence of the human SLITRK5 gene (SLITRK5) in three hundred and seventy seven OCD subjects and compared rare non-synonymous mutations (RNMs) in that sample with similar mutations in the 1000 Genomes database. We also performed in silico assessments and in vitro functional synaptogenesis assays on the Slitrk5 mutations identified. We identified four RNM’s among these OCD subjects. There were no significant differences in the prevalence or in silico effects of rare non-synonymous mutations in the OCD sample versus controls. Direct functional testing of recombinant SLITRK5 proteins found that all mutations identified in OCD subjects impaired synaptogenic activity whereas none of the pseudo-matched mutations identified in 1000 Genomes controls had significant effects on SLITRK5 function (Fisher’s exact test P = 0.028). These results demonstrate that rare functional mutations in SLITRK5 contribute to the genetic risk for OCD in human populations. They also highlight the importance of biological characterization of allelic effects in understanding genotype-phenotype relationships as there were no statistical differences in overall prevalence or bioinformatically predicted effects of OCD case versus control mutations. Finally, these results converge with others to highlight the role of aberrant synaptic function in corticostriatal neurons in the pathophysiology of OCD. PMID:28085938

  20. Study of modifiers factors associated to mitochondrial mutations in individuals with hearing impairment

    SciTech Connect

    Sousa de Moraes, Vanessa Cristine; Alexandrino, Fabiana; Andrade, Paula Baloni; Camara, Marilia Fontenele; Sartorato, Edi Lucia

    2009-04-03

    Hearing impairment is the most prevalent sensorial deficit in the general population. Congenital deafness occurs in about 1 in 1000 live births, of which approximately 50% has hereditary cause in development countries. Non-syndromic deafness can be caused by mutations in both nuclear and mitochondrial genes. Mutations in mtDNA have been associated with aminoglycoside-induced and non-syndromic deafness in many families worldwide. However, the nuclear background influences the phenotypic expression of these pathogenic mutations. Indeed, it has been proposed that nuclear modifier genes modulate the phenotypic manifestation of the mitochondrial A1555G mutation in the MTRNR1 gene. The both putative nuclear modifiers genes TRMU and MTO1 encoding a highly conserved mitochondrial related to tRNA modification. It has been hypothesizes that human TRMU and also MTO1 nuclear genes may modulate the phenotypic manifestation of deafness-associated mitochondrial mutations. The aim of this work was to elucidate the contribution of mitochondrial mutations, nuclear modifier genes mutations and aminoglycoside exposure in the deafness phenotype. Our findings suggest that the genetic background of individuals may play an important role in the pathogenesis of deafness-associated with mitochondrial mutation and aminoglycoside-induced.

  1. Loss of chloride channel ClC-5 impairs endocytosis by defective trafficking of megalin and cubilin in kidney proximal tubules.

    PubMed

    Christensen, Erik I; Devuyst, Olivier; Dom, Geneviève; Nielsen, Rikke; Van der Smissen, Patrick; Verroust, Pierre; Leruth, Michèle; Guggino, William B; Courtoy, Pierre J

    2003-07-08

    Loss of the renal endosome-associated chloride channel, ClC-5, in Dent's disease and knockout (KO) mice strongly inhibits endocytosis of filtered proteins by kidney proximal tubular cells (PTC). The underlying mechanism remains unknown. We therefore tested whether this endocytic failure could primarily reflect a loss of reabsorption by the multiligand receptors, megalin, and cubilin, caused by a trafficking defect. Impaired protein endocytosis in PTC of ClC-5 KO mice was demonstrated by (i) a major decreased uptake of injected 125I-beta 2-microglobulin, but not of the fluid-phase tracer, FITC-dextran, (ii) reduced labeling of endosomes by injected peroxidase and for the endogenous megalin/cubilin ligands, vitamin D- and retinol-binding proteins, and (iii) urinary appearance of low-molecular-weight proteins and the selective cubilin ligand, transferrin. Contrasting with preserved mRNA levels, megalin and cubilin abundance was significantly decreased in kidney extracts of KO mice. Percoll gradients resolving early and late endosomes (Rab5a, Rab7), brush border (villin, aminopeptidase M), and a dense peak comprising lysosomes (acid hydrolases) showed a disappearance of the brush border component for megalin and cubilin in KO mice. Quantitative ultrastructural immunogold labeling confirmed the overall decrease of megalin and cubilin in PTC and their selective loss at the brush border. In contrast, total contents of the rate-limiting endocytic catalysts, Rab5a and Rab7, were unaffected. Thus, impaired protein endocytosis caused by invalidation of ClC-5 primarily reflects a trafficking defect of megalin and cubilin in PTC.

  2. PLEKHM2 mutation leads to abnormal localization of lysosomes, impaired autophagy flux and associates with recessive dilated cardiomyopathy and left ventricular noncompaction

    PubMed Central

    Muhammad, Emad; Levitas, Aviva; Singh, Sonia R.; Braiman, Alex; Ofir, Rivka; Etzion, Sharon; Sheffield, Val C.; Etzion, Yoram; Carrier, Lucie; Parvari, Ruti

    2015-01-01

    Gene mutations, mostly segregating with a dominant mode of inheritance, are important causes of dilated cardiomyopathy (DCM), a disease characterized by enlarged ventricular dimensions, impaired cardiac function, heart failure and high risk of death. Another myocardial abnormality often linked to gene mutations is left ventricular noncompaction (LVNC) characterized by a typical diffuse spongy appearance of the left ventricle. Here, we describe a large Bedouin family presenting with a severe recessive DCM and LVNC. Homozygosity mapping and exome sequencing identified a single gene variant that segregated as expected and was neither reported in databases nor in Bedouin population controls. The PLEKHM2 cDNA2156_2157delAG variant causes the frameshift p.Lys645AlafsTer12 and/or the skipping of exon 11 that results in deletion of 30 highly conserved amino acids. PLEKHM2 is known to interact with several Rabs and with kinesin-1, affecting endosomal trafficking. Accordingly, patients' primary fibroblasts exhibited abnormal subcellular distribution of endosomes marked by Rab5, Rab7 and Rab9, as well as the Golgi apparatus. In addition, lysosomes appeared to be concentrated in the perinuclear region, and autophagy flux was impaired. Transfection of wild-type PLEKHM2 cDNA into patient's fibroblasts corrected the subcellular distribution of the lysosomes, supporting the causal effect of PLEKHM2 mutation. PLEKHM2 joins LAMP-2 and BAG3 as a disease gene altering autophagy resulting in an isolated cardiac phenotype. The association of PLEKHM2 mutation with DCM and LVNC supports the importance of autophagy for normal cardiac function. PMID:26464484

  3. A Novel Splice-Site Mutation in the GJB2 Gene Causing Mild Postlingual Hearing Impairment

    PubMed Central

    Gandía, Marta; del Castillo, Francisco J.; Rodríguez-Álvarez, Francisco J.; Garrido, Gema; Villamar, Manuela; Calderón, Manuela; Moreno-Pelayo, Miguel A.; Moreno, Felipe; del Castillo, Ignacio

    2013-01-01

    The DFNB1 subtype of autosomal recessive, nonsyndromic hearing impairment, caused by mutations affecting the GJB2 (connection-26) gene, is highly prevalent in most populations worldwide. DFNB1 hearing impairment is mostly severe or profound and usually appears before the acquisition of speech (prelingual onset), though a small number of hypomorphic missense mutations result in mild or moderate deafness of postlingual onset. We identified a novel GJB2 splice-site mutation, c. -22-2A>C, in three siblings with mild postlingual hearing impairment that were compound heterozygous for c. -22-2A>C and c.35delG. Reverse transcriptase-PCR experiments performed on total RNA extracted from saliva samples from one of these siblings confirmed that c. -22-2A>C abolished the acceptor splice site of the single GJB2 intron, resulting in the absence of normally processed transcripts from this allele. However, we did isolate transcripts from the c. -22-2A>C allele that keep an intact GJB2 coding region and that were generated by use of an alternative acceptor splice site previously unknown. The residual expression of wild-type connection-26 encoded by these transcripts probably underlies the mild severity and late onset of the hearing impairment of these subjects. PMID:24039984

  4. A neuroligin-4 missense mutation associated with autism impairs neuroligin-4 folding and endoplasmic reticulum export.

    PubMed

    Zhang, Chen; Milunsky, Jeff M; Newton, Stephanie; Ko, Jaewon; Zhao, Geping; Maher, Tom A; Tager-Flusberg, Helen; Bolliger, Marc F; Carter, Alice S; Boucard, Antony A; Powell, Craig M; Südhof, Thomas C

    2009-09-02

    Neuroligins (NLs) are postsynaptic cell-adhesion molecules essential for normal synapse function. Mutations in neuroligin-4 (NL4) (gene symbol: NLGN4) have been reported in some patients with autism spectrum disorder (ASD) and other neurodevelopmental impairments. However, the low frequency of NL4 mutations and the limited information about the affected patients and the functional consequences of their mutations cast doubt on the causal role of NL4 mutations in these disorders. Here, we describe two brothers with classical ASD who carry a single amino-acid substitution in NL4 (R87W). This substitution was absent from the brothers' asymptomatic parents, suggesting that it arose in the maternal germ line. R87 is conserved in all NL isoforms, and the R87W substitution is not observed in control individuals. At the protein level, the R87W substitution impaired glycosylation processing of NL4 expressed in HEK293 and COS cells, destabilized NL4, caused NL4 retention in the endoplasmic reticulum in non-neuronal cells and neurons, and blocked NL4 transport to the cell surface. As a result, the R87W substitution inactivated the synapse-formation activity of NL4 and abolished the functional effect of NL4 on synapse strength. Viewed together, these observations suggest that a point mutation in NL4 can cause ASD by a loss-of-function mechanism.

  5. A Neuroligin-4 Missense Mutation Associated With Autism Impairs Neuroligin-4 Folding and ER Export

    PubMed Central

    Zhang, Chen; Milunsky, Jeff M.; Newton, Stephanie; Ko, Jaewon; Zhao, Geping; Maher, Tom A.; Tager-Flusberg, Helen; Bolliger, Marc F.; S.Carter, Alice; Boucard, Antony; Powell, Craig M.; Südhof, Thomas C.

    2009-01-01

    Neuroligins (NLs) are postsynaptic cell-adhesion molecules essential for normal synapse function. Mutations in neuroligin-4 (NL4; gene symbol: NLGN4) have been reported in some patients with autism spectrum disorder (ASD) and other neurodevelopmental impairments. However, the low frequency of NL4 mutations, and the limited information about the affected patients and the functional consequences of their mutations, cast doubt on the causal role of NL4 mutations in these disorders. Here, we describe two brothers with classical ASD who carry a single amino acid substitution in NL4 (R87W). This substitution was absent from the brothers’ asymptomatic parents, suggesting that it arose in the maternal germline. R87 is conserved in all NL isoforms, and the R87W substitution is not observed in control individuals. At the protein level, the R87W-substitution impaired glycosylation processing of NL4 expressed in HEK293 and COS cells, destabilized NL4, caused NL4 retention in the endoplasmic reticulum in non-neuronal cells and neurons, and blocked NL4 transport to the cell-surface. As a result, the R87W-substitution inactivated the synapse-formation activity of NL4, and abolished the functional effect of NL4 on synapse strength. Viewed together, these observations suggest that a point mutation in NL4 can cause ASD by a loss-of-function mechanism. PMID:19726642

  6. Loss-of-Function Mutations of ILDR1 Cause Autosomal-Recessive Hearing Impairment DFNB42

    PubMed Central

    Borck, Guntram; Rehman, Atteeq Ur; Lee, Kwanghyuk; Pogoda, Hans-Martin; Kakar, Naseebullah; von Ameln, Simon; Grillet, Nicolas; Hildebrand, Michael S.; Ahmed, Zubair M.; Nürnberg, Gudrun; Ansar, Muhammad; Basit, Sulman; Javed, Qamar; Morell, Robert J.; Nasreen, Nabilah; Shearer, A. Eliot; Ahmad, Adeel; Kahrizi, Kimia; Shaikh, Rehan S.; Ali, Rana A.; Khan, Shaheen N.; Goebel, Ingrid; Meyer, Nicole C.; Kimberling, William J.; Webster, Jennifer A.; Stephan, Dietrich A.; Schiller, Martin R.; Bahlo, Melanie; Najmabadi, Hossein; Gillespie, Peter G.; Nürnberg, Peter; Wollnik, Bernd; Riazuddin, Saima; Smith, Richard J.H.; Ahmad, Wasim; Müller, Ulrich; Hammerschmidt, Matthias; Friedman, Thomas B.; Riazuddin, Sheikh; Leal, Suzanne M.; Ahmad, Jamil; Kubisch, Christian

    2011-01-01

    By using homozygosity mapping in a consanguineous Pakistani family, we detected linkage of nonsyndromic hearing loss to a 7.6 Mb region on chromosome 3q13.31-q21.1 within the previously reported DFNB42 locus. Subsequent candidate gene sequencing identified a homozygous nonsense mutation (c.1135G>T [p.Glu379X]) in ILDR1 as the cause of hearing impairment. By analyzing additional consanguineous families with homozygosity at this locus, we detected ILDR1 mutations in the affected individuals of 10 more families from Pakistan and Iran. The identified ILDR1 variants include missense, nonsense, frameshift, and splice-site mutations as well as a start codon mutation in the family that originally defined the DFNB42 locus. ILDR1 encodes the evolutionarily conserved immunoglobulin-like domain containing receptor 1, a putative transmembrane receptor of unknown function. In situ hybridization detected expression of Ildr1, the murine ortholog, early in development in the vestibule and in hair cells and supporting cells of the cochlea. Expression in hair cell- and supporting cell-containing neurosensory organs is conserved in the zebrafish, in which the ildr1 ortholog is prominently expressed in the developing ear and neuromasts of the lateral line. These data identify loss-of-function mutations of ILDR1, a gene with a conserved expression pattern pointing to a conserved function in hearing in vertebrates, as underlying nonsyndromic prelingual sensorineural hearing impairment. PMID:21255762

  7. Human Trafficking

    ERIC Educational Resources Information Center

    Wilson, David McKay

    2011-01-01

    The shadowy, criminal nature of human trafficking makes evaluating its nature and scope difficult. The U.S. State Department and anti-trafficking groups estimate that worldwide some 27 million people are caught in a form of forced servitude today. Public awareness of modern-day slavery is gaining momentum thanks to new abolitionist efforts. Among…

  8. Human Trafficking

    ERIC Educational Resources Information Center

    Wilson, David McKay

    2011-01-01

    The shadowy, criminal nature of human trafficking makes evaluating its nature and scope difficult. The U.S. State Department and anti-trafficking groups estimate that worldwide some 27 million people are caught in a form of forced servitude today. Public awareness of modern-day slavery is gaining momentum thanks to new abolitionist efforts. Among…

  9. De Novo Mutations in FOXP1 in Cases with Intellectual Disability, Autism, and Language Impairment

    PubMed Central

    Hamdan, Fadi F.; Daoud, Hussein; Rochefort, Daniel; Piton, Amélie; Gauthier, Julie; Langlois, Mathieu; Foomani, Gila; Dobrzeniecka, Sylvia; Krebs, Marie-Odile; Joober, Ridha; Lafrenière, Ronald G.; Lacaille, Jean-Claude; Mottron, Laurent; Drapeau, Pierre; Beauchamp, Miriam H.; Phillips, Michael S.; Fombonne, Eric; Rouleau, Guy A.; Michaud, Jacques L.

    2010-01-01

    Heterozygous mutations in FOXP2, which encodes a forkhead transcription factor, have been shown to cause developmental verbal dyspraxia and language impairment. FOXP2 and its closest homolog, FOXP1, are coexpressed in brain regions that are important for language and cooperatively regulate developmental processes, raising the possibility that FOXP1 may also be involved in developmental conditions that are associated with language impairment. In order to explore this possibility, we searched for mutations in FOXP1 in patients with intellectual disability (ID; mental retardation) and/or autism spectrum disorders (ASD). We first performed array-based genomic hybridization on sporadic nonsyndromic ID (NSID) (n = 30) or ASD (n = 80) cases. We identified a de novo intragenic deletion encompassing exons 4–14 of FOXP1 in a patient with NSID and autistic features. In addition, sequencing of all coding exons of FOXP1 in sporadic NSID (n = 110) or ASD (n = 135) cases, as well as in 570 controls, revealed the presence of a de novo nonsense mutation (c.1573C>T [p.R525X]) in the conserved forkhead DNA-binding domain in a patient with NSID and autism. Luciferase reporter assays showed that the p.R525X alteration disrupts the activity of the protein. Formal assessments revealed that both patients with de novo mutations in FOXP1 also show severe language impairment, mood lability with physical aggressiveness, and specific obsessions and compulsions. In conclusion, both FOXP1 and FOXP2 are associated with language impairment, but decrease of the former has a more global impact on brain development than that of the latter. PMID:20950788

  10. Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation

    PubMed Central

    Zhang, Leilei; He, Jie; Han, Bing; Lu, Linna; Fan, Jiayan; Zhang, He; Ge, Shengfang; Zhou, Yixiong; Jia, Renbing; Fan, Xianqun

    2016-01-01

    Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease. PMID:27570485

  11. Recessive mutations in POLR1C cause a leukodystrophy by impairing biogenesis of RNA polymerase III

    PubMed Central

    Thiffault, Isabelle; Wolf, Nicole I.; Forget, Diane; Guerrero, Kether; Tran, Luan T.; Choquet, Karine; Lavallée-Adam, Mathieu; Poitras, Christian; Brais, Bernard; Yoon, Grace; Sztriha, Laszlo; Webster, Richard I.; Timmann, Dagmar; van de Warrenburg, Bart P.; Seeger, Jürgen; Zimmermann, Alíz; Máté, Adrienn; Goizet, Cyril; Fung, Eva; van der Knaap, Marjo S.; Fribourg, Sébastien; Vanderver, Adeline; Simons, Cas; Taft, Ryan J.; Yates III, John R.; Coulombe, Benoit; Bernard, Geneviève

    2015-01-01

    A small proportion of 4H (Hypomyelination, Hypodontia and Hypogonadotropic Hypogonadism) or RNA polymerase III (POLR3)-related leukodystrophy cases are negative for mutations in the previously identified causative genes POLR3A and POLR3B. Here we report eight of these cases carrying recessive mutations in POLR1C, a gene encoding a shared POLR1 and POLR3 subunit, also mutated in some Treacher Collins syndrome (TCS) cases. Using shotgun proteomics and ChIP sequencing, we demonstrate that leukodystrophy-causative mutations, but not TCS mutations, in POLR1C impair assembly and nuclear import of POLR3, but not POLR1, leading to decreased binding to POLR3 target genes. This study is the first to show that distinct mutations in a gene coding for a shared subunit of two RNA polymerases lead to selective modification of the enzymes' availability leading to two different clinical conditions and to shed some light on the pathophysiological mechanism of one of the most common hypomyelinating leukodystrophies, POLR3-related leukodystrophy. PMID:26151409

  12. Mitotic impairment by doublecortin is diminished by doublecortin mutations found in patients.

    PubMed

    Couillard-Despres, Sebastien; Uyanik, Goekhan; Ploetz, Sonja; Karl, Claudia; Koch, Hartmut; Winkler, Juergen; Aigner, Ludwig

    2004-06-01

    Mutations in doublecortin ( DCX) affect the migration of neuronal precursor cells and cause subcortical band heterotopia and lissencephaly. DCX is known to bind and bundle microtubules; however, the impact of mutation on DCX function and its relation to the manifestation of DCX-associated disorders is still unclear. We analyzed the impact of DCX mutants on COS7 cell microtubule networks. We found that both mutant and wild type DCX are able to bind and bundle microtubules; however, mutants possess a decreased ability to perturb the mitotic machinery, to cause abnormal spindle orientation, and to impair mitotic progression. The magnitude of this decrease is proportional to the severity of the mutation-associated clinical symptoms, thereby providing a cell-based assay for the prognosis of DCX-associated neuronal migration disorders.

  13. Ciliopathies: The Trafficking Connection

    PubMed Central

    Madhivanan, Kayalvizhi; Aguilar, R. Claudio

    2014-01-01

    The primary cilium (PC) is a very dynamic hair-like membrane structure that assembles/disassembles in a cell-cycle dependent manner and is present in almost every cell type. Despite being continuous with the plasma membrane, a diffusion barrier located at the ciliary base confers the PC properties of a separate organelle with very specific characteristics and membrane composition. Therefore, vesicle trafficking is the major process by which components are acquired for cilium formation and maintenance. In fact, a system of specific sorting signals controls the right of cargo admission into the cilia. Disruption to the ciliary structure or its function leads to multi-organ diseases known as ciliopathies. These illnesses arise from a spectrum of mutations in any of the more than 50 loci linked to these conditions. Therefore, it is not surprising that symptom variability (specific manifestations and severity) among and within ciliopathies seems to be an emerging characteristic. Nevertheless, one can speculate that mutations occurring in genes whose products contribute to the overall vesicle trafficking to the PC (i.e., affecting cilia assembly) will lead to more severe symptoms, while those involved in the transport of specific cargoes will result in milder phenotypes. In this review, we summarize the trafficking mechanisms to the cilia and also provide a description of the trafficking defects observed in some ciliopathies which can be correlated to the severity of the pathology. PMID:25040720

  14. Point mutation in the HCN4 cardiac ion channel pore affecting synthesis, trafficking, and functional expression is associated with familial asymptomatic sinus bradycardia.

    PubMed

    Nof, Eyal; Luria, David; Brass, Dovrat; Marek, Dina; Lahat, Hadas; Reznik-Wolf, Haya; Pras, Elon; Dascal, Nathan; Eldar, Michael; Glikson, Michael

    2007-07-31

    The hyperpolarization-activated nucleotide-gated channel--HCN4 plays a major role in the diastolic depolarization of sinus atrial node cells. Mutant HCN4 channels have been found to be associated with inherited sinus bradycardia. Sixteen members of a family with sinus bradycardia were evaluated. Evaluation included a clinical questionnaire, 12-lead ECGs, Holter monitoring, echocardiography, and treadmill exercise testing. Eight family members (5 males) were classified as affected. All affected family members were asymptomatic with normal exercise capacity during long-term follow-up. Electrophysiological testing performed on 2 affected family members confirmed significant isolated sinus node dysfunction. Segregation analysis suggested autosomal-dominant inheritance. Direct sequencing of the exons encoding HCN4 revealed a missense mutation, G480R, in the ion channel pore domain in all affected family members. Function analysis, including expression of HCN4 wild-type and G480R in Xenopus oocytes and human embryonic kidney 293 cells, revealed that mutant channels were activated at more negative voltages compared with wild-type channels. Synthesis and expression of the wild-type and mutant HCN4 channel on the plasma membrane tested in human embryonic kidney 293 cells using biotinylation and Western blot analysis demonstrated a reduction in synthesis and a trafficking defect in mutant compared with wild-type channels. We describe an inherited, autosomal-dominant form of sinus node dysfunction caused by a missense mutation in the HCN4 ion channel pore. Despite its critical location, this mutation carries a favorable prognosis without the need for pacemaker implantation during long-term follow-up.

  15. A Novel Dominant Hyperekplexia Mutation Y705C Alters Trafficking and Biochemical Properties of the Presynaptic Glycine Transporter GlyT2*

    PubMed Central

    Giménez, Cecilio; Pérez-Siles, Gonzalo; Martínez-Villarreal, Jaime; Arribas-González, Esther; Jiménez, Esperanza; Núñez, Enrique; de Juan-Sanz, Jaime; Fernández-Sánchez, Enrique; García-Tardón, Noemí; Ibáñez, Ignacio; Romanelli, Valeria; Nevado, Julián; James, Victoria M.; Topf, Maya; Chung, Seo-Kyung; Thomas, Rhys H.; Desviat, Lourdes R.; Aragón, Carmen; Zafra, Francisco; Rees, Mark I.; Lapunzina, Pablo; Harvey, Robert J.; López-Corcuera, Beatriz

    2012-01-01

    Hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, producing hypertonia and apnea episodes. Although rare, this orphan disorder can have serious consequences, including sudden infant death. Dominant and recessive mutations in the human glycine receptor (GlyR) α1 gene (GLRA1) are the major cause of this disorder. However, recessive mutations in the presynaptic Na+/Cl−-dependent glycine transporter GlyT2 gene (SLC6A5) are rapidly emerging as a second major cause of startle disease. In this study, systematic DNA sequencing of SLC6A5 revealed a new dominant GlyT2 mutation: pY705C (c.2114A→G) in transmembrane domain 11, in eight individuals from Spain and the United Kingdom. Curiously, individuals harboring this mutation show significant variation in clinical presentation. In addition to classical hyperekplexia symptoms, some individuals had abnormal respiration, facial dysmorphism, delayed motor development, or intellectual disability. We functionally characterized this mutation using molecular modeling, electrophysiology, [3H]glycine transport, cell surface expression, and cysteine labeling assays. We found that the introduced cysteine interacts with the cysteine pair Cys-311–Cys-320 in the second external loop of GlyT2. This interaction impairs transporter maturation through the secretory pathway, reduces surface expression, and inhibits transport function. Additionally, Y705C presents altered H+ and Zn2+ dependence of glycine transport that may affect the function of glycinergic neurotransmission in vivo. PMID:22753417

  16. A Tbc1d1 (Ser231Ala)-knockin mutation partially impairs AICAR- but not exercise-induced muscle glucose uptake in mice.

    PubMed

    Chen, Qiaoli; Xie, Bingxian; Zhu, Sangsang; Rong, Ping; Sheng, Yang; Ducommun, Serge; Chen, Liang; Quan, Chao; Li, Min; Sakamoto, Kei; MacKintosh, Carol; Chen, Shuai; Wang, Hong Yu

    2017-02-01

    TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) is a Rab GTPase-activating protein (RabGAP) that has been implicated in regulating GLUT4 trafficking. TBC1D1 can be phosphorylated by the AMP-activated protein kinase (AMPK) on Ser(231), which consequently interacts with 14-3-3 proteins. Given the key role for AMPK in regulating insulin-independent muscle glucose uptake, we hypothesised that TBC1D1-Ser(231) phosphorylation and/or 14-3-3 binding may mediate AMPK-governed glucose homeostasis. Whole-body glucose homeostasis and muscle glucose uptake were assayed in mice bearing a Tbc1d1 (Ser231Ala)-knockin mutation or harbouring skeletal muscle-specific Ampkα1/α2 (also known as Prkaa1/2) double-knockout mutations in response to an AMPK-activating agent, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). Exercise-induced muscle glucose uptake and exercise capacity were also determined in the Tbc1d1 (Ser231Ala)-knockin mice. Skeletal muscle-specific deletion of Ampkα1/a2 in mice prevented AICAR-induced hypoglycaemia and muscle glucose uptake. The Tbc1d1 (Ser231Ala)-knockin mutation also attenuated the glucose-lowering effect of AICAR in mice. Glucose uptake and cell surface GLUT4 content were significantly lower in muscle isolated from the Tbc1d1 (Ser231Ala)-knockin mice upon stimulation with a submaximal dose of AICAR. However, this Tbc1d1 (Ser231Ala)-knockin mutation neither impaired exercise-induced muscle glucose uptake nor affected exercise capacity in mice. TBC1D1-Ser(231) phosphorylation and/or 14-3-3 binding partially mediates AMPK-governed glucose homeostasis and muscle glucose uptake in a context-dependent manner.

  17. De Novo Mutations in CHAMP1 Cause Intellectual Disability with Severe Speech Impairment

    PubMed Central

    Hempel, Maja; Cremer, Kirsten; Ockeloen, Charlotte W.; Lichtenbelt, Klaske D.; Herkert, Johanna C.; Denecke, Jonas; Haack, Tobias B.; Zink, Alexander M.; Becker, Jessica; Wohlleber, Eva; Johannsen, Jessika; Alhaddad, Bader; Pfundt, Rolph; Fuchs, Sigrid; Wieczorek, Dagmar; Strom, Tim M.; van Gassen, Koen L.I.; Kleefstra, Tjitske; Kubisch, Christian; Engels, Hartmut; Lessel, Davor

    2015-01-01

    CHAMP1 encodes a protein with a function in kinetochore-microtubule attachment and in the regulation of chromosome segregation, both of which are known to be important for neurodevelopment. By trio whole-exome sequencing, we have identified de novo deleterious mutations in CHAMP1 in five unrelated individuals affected by intellectual disability with severe speech impairment, motor developmental delay, muscular hypotonia, and similar dysmorphic features including short philtrum and a tented upper and everted lover lip. In addition to two frameshift and one nonsense mutations, we found an identical nonsense mutation, c.1192C>T (p.Arg398∗), in two affected individuals. All mutations, if resulting in a stable protein, are predicted to lead to the loss of the functionally important zinc-finger domains in the C terminus of the protein, which regulate CHAMP1 localization to chromosomes and the mitotic spindle, thereby providing a mechanistic understanding for their pathogenicity. We thus establish deleterious de novo mutations in CHAMP1 as a cause of intellectual disability. PMID:26340335

  18. A Meier-Gorlin syndrome mutation impairs the ORC1-nucleosome association.

    PubMed

    Zhang, Wei; Sankaran, Saumya; Gozani, Or; Song, Jikui

    2015-05-15

    Recent studies have identified several genetic mutations within the BAH domain of human Origin Recognition Complex subunit 1 (hORC1BAH), including the R105Q mutation, implicated in Meier-Gorlin Syndrome (MGS). However, the pathological role of the hORC1 R105Q mutation remains unclear. In this study, we have investigated the interactions of the hORC1BAH domain with histone H4K20me2, DNA, and the nucleosome core particle labeled with H4Kc20me2, a chemical analog of H4K20me2. Our study revealed a nucleosomal DNA binding site for hORC1BAH. The R105Q mutation reduces the hORC1BAH-DNA binding affinity, leading to impaired hORC1BAH-nucleosome interaction, which likely influences DNA replication initiation and MGS pathogenesis. This study provides an etiologic link between the hORC1 R105Q mutation and MGS.

  19. Mutations in MUSK causing congenital myasthenic syndrome impair MuSK–Dok-7 interaction

    PubMed Central

    Maselli, Ricardo A.; Arredondo, Juan; Cagney, Órla; Ng, Jarae J.; Anderson, Jennifer A.; Williams, Colette; Gerke, Bae J.; Soliven, Betty; Wollmann, Robert L.

    2010-01-01

    We describe a severe congenital myasthenic syndrome (CMS) caused by two missense mutations in the gene encoding the muscle specific receptor tyrosine kinase (MUSK). The identified MUSK mutations M605I and A727V are both located in the kinase domain of MuSK. Intracellular microelectrode recordings and microscopy studies of the neuromuscular junction conducted in an anconeus muscle biopsy revealed decreased miniature endplate potential amplitudes, reduced endplate size and simplification of secondary synaptic folds, which were consistent with postsynaptic deficit. The study also showed a striking reduction of the endplate potential quantal content, consistent with additional presynaptic failure. Expression studies in MuSK deficient myotubes revealed that A727V, which is located within the catalytic loop of the enzyme, caused severe impairment of agrin-dependent MuSK phosphorylation, aggregation of acetylcholine receptors (AChRs) and interaction of MuSK with Dok-7, an essential intracellular binding protein of MuSK. In contrast, M605I, resulted in only moderate impairment of agrin-dependent MuSK phosphorylation, aggregation of AChRs and interaction of MuSK with Dok-7. There was no impairment of interaction of mutants with either the low-density lipoprotein receptor-related protein, Lrp4 (a co-receptor of agrin) or with the mammalian homolog of the Drosophila tumorous imaginal discs (Tid1). Our findings demonstrate that missense mutations in MUSK can result in a severe form of CMS and indicate that the inability of MuSK mutants to interact with Dok-7, but not with Lrp4 or Tid1, is a major determinant of the pathogenesis of the CMS caused by MUSK mutations. PMID:20371544

  20. Mutations in MUSK causing congenital myasthenic syndrome impair MuSK-Dok-7 interaction.

    PubMed

    Maselli, Ricardo A; Arredondo, Juan; Cagney, Orla; Ng, Jarae J; Anderson, Jennifer A; Williams, Colette; Gerke, Bae J; Soliven, Betty; Wollmann, Robert L

    2010-06-15

    We describe a severe congenital myasthenic syndrome (CMS) caused by two missense mutations in the gene encoding the muscle specific receptor tyrosine kinase (MUSK). The identified MUSK mutations M605I and A727V are both located in the kinase domain of MuSK. Intracellular microelectrode recordings and microscopy studies of the neuromuscular junction conducted in an anconeus muscle biopsy revealed decreased miniature endplate potential amplitudes, reduced endplate size and simplification of secondary synaptic folds, which were consistent with postsynaptic deficit. The study also showed a striking reduction of the endplate potential quantal content, consistent with additional presynaptic failure. Expression studies in MuSK deficient myotubes revealed that A727V, which is located within the catalytic loop of the enzyme, caused severe impairment of agrin-dependent MuSK phosphorylation, aggregation of acetylcholine receptors (AChRs) and interaction of MuSK with Dok-7, an essential intracellular binding protein of MuSK. In contrast, M605I, resulted in only moderate impairment of agrin-dependent MuSK phosphorylation, aggregation of AChRs and interaction of MuSK with Dok-7. There was no impairment of interaction of mutants with either the low-density lipoprotein receptor-related protein, Lrp4 (a co-receptor of agrin) or with the mammalian homolog of the Drosophila tumorous imaginal discs (Tid1). Our findings demonstrate that missense mutations in MUSK can result in a severe form of CMS and indicate that the inability of MuSK mutants to interact with Dok-7, but not with Lrp4 or Tid1, is a major determinant of the pathogenesis of the CMS caused by MUSK mutations.

  1. Biallelic mutations in IRF8 impair human NK cell maturation and function

    PubMed Central

    Mace, Emily M.; Gunesch, Justin T.; Chinn, Ivan K.; Angelo, Laura S.; Maisuria, Sheetal; Keller, Michael D.; Togi, Sumihito; Watkin, Levi B.; LaRosa, David F.; Jhangiani, Shalini N.; Muzny, Donna M.; Stray-Pedersen, Asbjørg; Coban Akdemir, Zeynep; Smith, Jansen B.; Hernández-Sanabria, Mayra; Le, Duy T.; Hogg, Graham D.; Cao, Tram N.; Freud, Aharon G.; Szymanski, Eva P.; Collin, Matthew; Cant, Andrew J.; Gibbs, Richard A.; Holland, Steven M.; Caligiuri, Michael A.; Ozato, Keiko; Paust, Silke; Doody, Gina M.; Lupski, James R.; Orange, Jordan S.

    2016-01-01

    Human NK cell deficiencies are rare yet result in severe and often fatal disease, particularly as a result of viral susceptibility. NK cells develop from hematopoietic stem cells, and few monogenic errors that specifically interrupt NK cell development have been reported. Here we have described biallelic mutations in IRF8, which encodes an interferon regulatory factor, as a cause of familial NK cell deficiency that results in fatal and severe viral disease. Compound heterozygous or homozygous mutations in IRF8 in 3 unrelated families resulted in a paucity of mature CD56dim NK cells and an increase in the frequency of the immature CD56bright NK cells, and this impairment in terminal maturation was also observed in Irf8–/–, but not Irf8+/–, mice. We then determined that impaired maturation was NK cell intrinsic, and gene expression analysis of human NK cell developmental subsets showed that multiple genes were dysregulated by IRF8 mutation. The phenotype was accompanied by deficient NK cell function and was stable over time. Together, these data indicate that human NK cells require IRF8 for development and functional maturation and that dysregulation of this function results in severe human disease, thereby emphasizing a critical role for NK cells in human antiviral defense. PMID:27893462

  2. Biallelic mutations in IRF8 impair human NK cell maturation and function.

    PubMed

    Mace, Emily M; Bigley, Venetia; Gunesch, Justin T; Chinn, Ivan K; Angelo, Laura S; Care, Matthew A; Maisuria, Sheetal; Keller, Michael D; Togi, Sumihito; Watkin, Levi B; LaRosa, David F; Jhangiani, Shalini N; Muzny, Donna M; Stray-Pedersen, Asbjørg; Coban Akdemir, Zeynep; Smith, Jansen B; Hernández-Sanabria, Mayra; Le, Duy T; Hogg, Graham D; Cao, Tram N; Freud, Aharon G; Szymanski, Eva P; Savic, Sinisa; Collin, Matthew; Cant, Andrew J; Gibbs, Richard A; Holland, Steven M; Caligiuri, Michael A; Ozato, Keiko; Paust, Silke; Doody, Gina M; Lupski, James R; Orange, Jordan S

    2017-01-03

    Human NK cell deficiencies are rare yet result in severe and often fatal disease, particularly as a result of viral susceptibility. NK cells develop from hematopoietic stem cells, and few monogenic errors that specifically interrupt NK cell development have been reported. Here we have described biallelic mutations in IRF8, which encodes an interferon regulatory factor, as a cause of familial NK cell deficiency that results in fatal and severe viral disease. Compound heterozygous or homozygous mutations in IRF8 in 3 unrelated families resulted in a paucity of mature CD56dim NK cells and an increase in the frequency of the immature CD56bright NK cells, and this impairment in terminal maturation was also observed in Irf8-/-, but not Irf8+/-, mice. We then determined that impaired maturation was NK cell intrinsic, and gene expression analysis of human NK cell developmental subsets showed that multiple genes were dysregulated by IRF8 mutation. The phenotype was accompanied by deficient NK cell function and was stable over time. Together, these data indicate that human NK cells require IRF8 for development and functional maturation and that dysregulation of this function results in severe human disease, thereby emphasizing a critical role for NK cells in human antiviral defense.

  3. Mutation of a single threonine in the cytoplasmic NH2 terminus disrupts trafficking of renal betaine-GABA transporter 1 during hypertonic stress.

    PubMed

    Schweikhard, Eva S; Kempson, Stephen A; Ziegler, Christine; Burckhardt, Birgitta C

    2014-07-01

    Betaine is an important osmolyte and is, compared with other organs, much more abundant in the kidneys, where it enters cells in the medulla by betaine-GABA transporter 1 (BGT1) to balance osmoregulation in the countercurrent system. In wild-type (wt-)BGT1-expressing oocytes, GABA-mediated currents were diminished by preincubation of oocytes with 100 nM PMA or 5 μM dioctanoyl-sn-glycerol, activators of PKC, whereas the application of staurosporine before the application of dioctanoyl-sn-glycerol restored the response to GABA. Four potential phosphorylation sites on BGT1 were mutated to alanine by site-directed mutagenesis. Three mutants (T235A, S428A, and S564A) evoked GABA currents comparable in magnitude to currents observed in wt-BGT1-expressing oocytes, whereas GABA currents in T40A were barely detectable. Uptake of [(3)H]GABA was also determined in human embryonic kidney-293 cells expressing enhanced green fluorescent protein (EGFP)-tagged BGT1 with the same mutations. T235A, S428A, and S564A showed upregulation of GABA uptake after hypertonic stress and downregulation by PMA similar to EGFP-wt-BGT1. In contrast, T40A did not respond to either hypertonicity or PMA. Confocal microscopy of the EGFP-BGT1 mutants expressed in Madin-Darby canine kidney cells revealed that T40A was present in the cytoplasm after 24 h of hypertonic stress. whereas the other mutants and EGFP-wt-BGT1 were in the plasma membrane. All mutants, including T40A, comigrated with wt-BGT1 on Western blots, suggesting that they are full-length proteins. T40A, however, cannot be phosphorylated, as revealed using a specific anti-phosphoantibody, and, therefore, T40 may be important for the trafficking and insertion of BGT1 in the plasma membrane. Copyright © 2014 the American Physiological Society.

  4. Resistance mutations against dolutegravir in HIV integrase impair the emergence of resistance against reverse transcriptase inhibitors.

    PubMed

    Oliveira, Maureen; Mesplède, Thibault; Quashie, Peter K; Moïsi, Daniela; Wainberg, Mark A

    2014-03-27

    Among 1222 antiretroviral-naive patients who received dolutegravir (DTG) as part of first-line therapy, none has developed resistance against this compound after 48-96 weeks of follow-up. Moreover, only four occurrences of virological failure with resistance mutations have been documented in previously drug-experienced patients who received DTG as a first time integrase inhibitor as a component of a second-line regimen. The R263K integrase resistance mutation was observed in two of these individuals who received suboptimal background regimens. We have previously selected mutations at position R263K, G118R, H51Y, and E138K as being associated with low-level resistance to DTG. Now, we sought to investigate the facility with which resistance on the part of R263K-containing viruses might develop. We tested the ability of DTG-resistant viruses containing either the R263K or G118R and/or H51Y mutations to develop further resistance against several reverse transcriptase inhibitors during in-vitro selection experiments. Our results show that DTG-resistant viruses are impaired in their ability to acquire further resistance to each of nevirapine and lamivudine as a consequence of their relative inability to develop resistance mutations associated with these two compounds. Our findings provide an explanation for the fact that no individual has yet progressed to virological failure with resistance mutations associated with dolutegravir in clinical trials in which patients received dolutegravir together with an optimized background regimen.

  5. PRUNE is crucial for normal brain development and mutated in microcephaly with neurodevelopmental impairment.

    PubMed

    Zollo, Massimo; Ahmed, Mustafa; Ferrucci, Veronica; Salpietro, Vincenzo; Asadzadeh, Fatemeh; Carotenuto, Marianeve; Maroofian, Reza; Al-Amri, Ahmed; Singh, Royana; Scognamiglio, Iolanda; Mojarrad, Majid; Musella, Luca; Duilio, Angela; Di Somma, Angela; Karaca, Ender; Rajab, Anna; Al-Khayat, Aisha; Mohan Mohapatra, Tribhuvan; Eslahi, Atieh; Ashrafzadeh, Farah; Rawlins, Lettie E; Prasad, Rajniti; Gupta, Rashmi; Kumari, Preeti; Srivastava, Mona; Cozzolino, Flora; Kumar Rai, Sunil; Monti, Maria; Harlalka, Gaurav V; Simpson, Michael A; Rich, Philip; Al-Salmi, Fatema; Patton, Michael A; Chioza, Barry A; Efthymiou, Stephanie; Granata, Francesca; Di Rosa, Gabriella; Wiethoff, Sarah; Borgione, Eugenia; Scuderi, Carmela; Mankad, Kshitij; Hanna, Michael G; Pucci, Piero; Houlden, Henry; Lupski, James R; Crosby, Andrew H; Baple, Emma L

    2017-02-28

    PRUNE is a member of the DHH (Asp-His-His) phosphoesterase protein superfamily of molecules important for cell motility, and implicated in cancer progression. Here we investigated multiple families from Oman, India, Iran and Italy with individuals affected by a new autosomal recessive neurodevelopmental and degenerative disorder in which the cardinal features include primary microcephaly and profound global developmental delay. Our genetic studies identified biallelic mutations of PRUNE1 as responsible. Our functional assays of disease-associated variant alleles revealed impaired microtubule polymerization, as well as cell migration and proliferation properties, of mutant PRUNE. Additionally, our studies also highlight a potential new role for PRUNE during microtubule polymerization, which is essential for the cytoskeletal rearrangements that occur during cellular division and proliferation. Together these studies define PRUNE as a molecule fundamental for normal human cortical development and define cellular and clinical consequences associated with PRUNE mutation.

  6. PRUNE is crucial for normal brain development and mutated in microcephaly with neurodevelopmental impairment

    PubMed Central

    Zollo, Massimo; Ahmed, Mustafa; Ferrucci, Veronica; Salpietro, Vincenzo; Asadzadeh, Fatemeh; Carotenuto, Marianeve; Maroofian, Reza; Al-Amri, Ahmed; Singh, Royana; Scognamiglio, Iolanda; Mojarrad, Majid; Musella, Luca; Duilio, Angela; Di Somma, Angela; Karaca, Ender; Rajab, Anna; Al-Khayat, Aisha; Mohan Mohapatra, Tribhuvan; Eslahi, Atieh; Ashrafzadeh, Farah; Rawlins, Lettie E.; Prasad, Rajniti; Gupta, Rashmi; Kumari, Preeti; Srivastava, Mona; Cozzolino, Flora; Kumar Rai, Sunil; Monti, Maria; Harlalka, Gaurav V.; Simpson, Michael A.; Rich, Philip; Al-Salmi, Fatema; Patton, Michael A.; Chioza, Barry A.; Efthymiou, Stephanie; Granata, Francesca; Di Rosa, Gabriella; Wiethoff, Sarah; Borgione, Eugenia; Scuderi, Carmela; Mankad, Kshitij; Hanna, Michael G.; Pucci, Piero; Houlden, Henry; Lupski, James R.; Crosby, Andrew H.

    2017-01-01

    Abstract PRUNE is a member of the DHH (Asp-His-His) phosphoesterase protein superfamily of molecules important for cell motility, and implicated in cancer progression. Here we investigated multiple families from Oman, India, Iran and Italy with individuals affected by a new autosomal recessive neurodevelopmental and degenerative disorder in which the cardinal features include primary microcephaly and profound global developmental delay. Our genetic studies identified biallelic mutations of PRUNE1 as responsible. Our functional assays of disease-associated variant alleles revealed impaired microtubule polymerization, as well as cell migration and proliferation properties, of mutant PRUNE. Additionally, our studies also highlight a potential new role for PRUNE during microtubule polymerization, which is essential for the cytoskeletal rearrangements that occur during cellular division and proliferation. Together these studies define PRUNE as a molecule fundamental for normal human cortical development and define cellular and clinical consequences associated with PRUNE mutation. PMID:28334956

  7. GIGYF2 mutation in late-onset Parkinson's disease with cognitive impairment.

    PubMed

    Ruiz-Martinez, Javier; Krebs, Catharine E; Makarov, Vladimir; Gorostidi, Ana; Martí-Massó, Jose Félix; Paisán-Ruiz, Coro

    2015-10-01

    Although in the last two decades there has been considerable progress in understanding the genetic basis of Parkinson's disease (PD), the majority of PD is sporadic and its genetic causes are largely unknown. In an attempt to identify novel genetic causes of PD, whole-exome sequencing and subsequent analyses were performed in a family featuring late-onset PD with cognitive impairment. A novel genetic variant (p.Arg610Gly) in the GIGYF2 gene, previously known to be associated with PD, was identified as potential disease-causing mutation. The GIGYF2 p.Arg610Gly mutation situated in the GYF domain of the encoding protein was predicted to be pathogenic and to disrupt the GYF's ligand-binding abilities. Although further research is still required, this finding may shed light on the GIGYF2-associated mechanisms that lead to PD and suggests insulin dysregulation as a disease-specific mechanism for both PD and cognitive dysfunction.

  8. A Mutation in CABP2, Expressed in Cochlear Hair Cells, Causes Autosomal-Recessive Hearing Impairment

    PubMed Central

    Schrauwen, Isabelle; Helfmann, Sarah; Inagaki, Akira; Predoehl, Friederike; Tabatabaiefar, Mohammad Amin; Picher, Maria Magdalena; Sommen, Manou; Seco, Celia Zazo; Oostrik, Jaap; Kremer, Hannie; Dheedene, Annelies; Claes, Charlotte; Fransen, Erik; Chaleshtori, Morteza Hashemzadeh; Coucke, Paul; Lee, Amy; Moser, Tobias; Van Camp, Guy

    2012-01-01

    CaBPs are a family of Ca2+-binding proteins related to calmodulin and are localized in the brain and sensory organs, including the retina and cochlea. Although their physiological roles are not yet fully elucidated, CaBPs modulate Ca2+ signaling through effectors such as voltage-gated Cav Ca2+ channels. In this study, we identified a splice-site mutation (c.637+1G>T) in Ca2+-binding protein 2 (CABP2) in three consanguineous Iranian families affected by moderate-to-severe hearing loss. This mutation, most likely a founder mutation, probably leads to skipping of exon 6 and premature truncation of the protein (p.Phe164Serfs∗4). Compared with wild-type CaBP2, the truncated CaBP2 showed altered Ca2+ binding in isothermal titration calorimetry and less potent regulation of Cav1.3 Ca2+ channels. We show that genetic defects in CABP2 cause moderate-to-severe sensorineural hearing impairment. The mutation might cause a hypofunctional CaBP2 defective in Ca2+ sensing and effector regulation in the inner ear. PMID:22981119

  9. Hypomorphic mutation in mouse Nppc gene causes retarded bone growth due to impaired endochondral ossification

    SciTech Connect

    Tsuji, Takehito Kondo, Eri; Yasoda, Akihiro; Inamoto, Masataka; Kiyosu, Chiyo; Nakao, Kazuwa; Kunieda, Tetsuo

    2008-11-07

    Long bone abnormality (lbab/lbab) is a spontaneous mutant mouse characterized by dwarfism with shorter long bones. A missense mutation was reported in the Nppc gene, which encodes C-type natriuretic peptide (CNP), but it has not been confirmed whether this mutation is responsible for the dwarf phenotype. To verify that the mutation causes the dwarfism of lbab/lbab mice, we first investigated the effect of CNP in lbab/lbab mice. By transgenic rescue with chondrocyte-specific expression of CNP, the dwarf phenotype in lbab/lbab mice was completely compensated. Next, we revealed that CNP derived from the lbab allele retained only slight activity to induce cGMP production through its receptor. Histological analysis showed that both proliferative and hypertrophic zones of chondrocytes in the growth plate of lbab/lbab mice were markedly reduced. Our results demonstrate that lbab/lbab mice have a hypomorphic mutation in the Nppc gene that is responsible for dwarfism caused by impaired endochondral ossification.

  10. GJB2 (Connexin-26) mutations are not frequent among hearing impaired patients in east Greenland.

    PubMed

    Homøe, Preben; Koch, Anders; Rendtorff, Nanna Dahl; Lodahl, Marianne; Andersen, Ture; Andersen, Stig; Eiberg, Hans; Nielsen, Inge-Merete; Tranebjærg, Lisbeth

    2012-06-01

    Investigate genetic causes of HI among the Inuit populations in the Arctic with a high prevalence of hearing impairment (HI). A cross-sectional survey with population-based controls. Forty-five patients, with sensorineural or mixed HI and an available blood sample for GJB2 sequencing from DNA, were selected from 166 east Greenlanders by specialist audiology examination, including pure-tone air and bone conduction audiometry from 125 Hz to 8000 Hz. Controls were 108 east- and 109 west-Greenlanders. Forty-five patients with HI were included, 24 males and 21 females. Median age was 35 years (range: 5-76). The c.35delG allele frequency was 3.3%. One patient, homozygous for the c.35delG GJB2 mutation, had bilateral congenital profound HI. Another with mixed HI was heterozygous for the same mutation. Three were heterozygous for the p.V27I variant and one was heterozygous for the p.V153I variant. The frequency of the c.35delG mutation in the controls varied between 0.5% in west Greenland to 2.3% in east Greenland. The c.35delG GJB2 mutation occurs in Greenland with low frequency. We conclude the main causes behind the prevalence of HI in this population are chronic otitis media, noise traumas, and/or unidentified genetic causes.

  11. Dynein mutations associated with hereditary motor neuropathies impair mitochondrial morphology and function with age.

    PubMed

    Eschbach, Judith; Sinniger, Jérôme; Bouitbir, Jamal; Fergani, Anissa; Schlagowski, Anna-Isabel; Zoll, Joffrey; Geny, Bernard; René, Frédérique; Larmet, Yves; Marion, Vincent; Baloh, Robert H; Harms, Matthew B; Shy, Michael E; Messadeq, Nadia; Weydt, Patrick; Loeffler, Jean-Philippe; Ludolph, Albert C; Dupuis, Luc

    2013-10-01

    Mutations in the DYNC1H1 gene encoding for dynein heavy chain cause two closely related human motor neuropathies, dominant spinal muscular atrophy with lower extremity predominance (SMA-LED) and axonal Charcot-Marie-Tooth (CMT) disease, and lead to sensory neuropathy and striatal atrophy in mutant mice. Dynein is the molecular motor carrying mitochondria retrogradely on microtubules, yet the consequences of dynein mutations on mitochondrial physiology have not been explored. Here, we show that mouse fibroblasts bearing heterozygous or homozygous point mutation in Dync1h1, similar to human mutations, show profoundly abnormal mitochondrial morphology associated with the loss of mitofusin 1. Furthermore, heterozygous Dync1h1 mutant mice display progressive mitochondrial dysfunction in muscle and mitochondria progressively increase in size and invade sarcomeres. As a likely consequence of systemic mitochondrial dysfunction, Dync1h1 mutant mice develop hyperinsulinemia and hyperglycemia and progress to glucose intolerance with age. Similar defects in mitochondrial morphology and mitofusin levels are observed in fibroblasts from patients with SMA-LED. Last, we show that Dync1h1 mutant fibroblasts show impaired perinuclear clustering of mitochondria in response to mitochondrial uncoupling. Our results show that dynein function is required for the maintenance of mitochondrial morphology and function with aging and suggest that mitochondrial dysfunction contributes to dynein-dependent neurological diseases, such as SMA-LED.

  12. COPA mutations impair ER-Golgi transport causing hereditary autoimmune-mediated lung disease and arthritis

    PubMed Central

    Watkin, Levi B.; Jessen, Birthe; Wiszniewski, Wojciech; Vece, Timothy; Jan, Max; Sha, Youbao; Thamsen, Maike; Santos-Cortez, Regie L. P.; Lee, Kwanghyuk; Gambin, Tomasz; Forbes, Lisa; Law, Christopher S.; Stray-Petersen, Asbjørg; Cheng, Mickie H.; Mace, Emily M.; Anderson, Mark S.; Liu, Dongfang; Tang, Ling Fung; Nicholas, Sarah K.; Nahmod, Karen; Makedonas, George; Canter, Debra; Kwok, Pui-Yan; Hicks, John; Jones, Kirk D.; Penney, Samantha; Jhangiani, Shalini N.; Rosenblum, Michael D.; Dell, Sharon D.; Waterfield, Michael R.; Papa, Feroz R.; Muzny, Donna M.; Zaitlen, Noah; Leal, Suzanne M.; Gonzaga-Jauregui, Claudia; Boerwinkle, Eric; Eissa, N. Tony; Gibbs, Richard A.; Lupski, James R.; Orange, Jordan S.; Shum, Anthony K.

    2015-01-01

    Advances in genomics have allowed unbiased genetic studies of human disease with unexpected insights into the molecular mechanisms of cellular immunity and autoimmunity1. We performed whole exome sequencing (WES) and targeted sequencing in patients with an apparent Mendelian syndrome of autoimmune disease characterized by high-titer autoantibodies, inflammatory arthritis and interstitial lung disease (ILD). In five families, we identified four unique deleterious variants in the Coatomer subunit alpha (COPA) gene all located within the same functional domain. We hypothesized that mutant COPA leads to a defect in intracellular transport mediated by coat protein complex I (COPI)2–4. We show that COPA variants impair binding of proteins targeted for retrograde Golgi to ER transport and demonstrate that expression of mutant COPA leads to ER stress and the upregulation of Th17 priming cytokines. Consistent with this pattern of cytokine expression, patients demonstrated a significant skewing of CD4+ T cells toward a T helper 17 (Th17) phenotype, an effector T cell population implicated in autoimmunity5,6. Our findings uncover an unexpected molecular link between a vesicular transport protein and a syndrome of autoimmunity manifested by lung and joint disease. These findings provide a unique opportunity to understand how alterations in cellular homeostasis caused by a defect in the intracellular trafficking pathway leads to the generation of human autoimmune disease. PMID:25894502

  13. TMEM240 mutations cause spinocerebellar ataxia 21 with mental retardation and severe cognitive impairment.

    PubMed

    Delplanque, Jérôme; Devos, David; Huin, Vincent; Genet, Alexandre; Sand, Olivier; Moreau, Caroline; Goizet, Cyril; Charles, Perrine; Anheim, Mathieu; Monin, Marie Lorraine; Buée, Luc; Destée, Alain; Grolez, Guillaume; Delmaire, Christine; Dujardin, Kathy; Dellacherie, Delphine; Brice, Alexis; Stevanin, Giovanni; Strubi-Vuillaume, Isabelle; Dürr, Alexandra; Sablonnière, Bernard

    2014-10-01

    Autosomal dominant cerebellar ataxia corresponds to a clinically and genetically heterogeneous group of neurodegenerative disorders that primarily affect the cerebellum. Here, we report the identification of the causative gene in spinocerebellar ataxia 21, an autosomal-dominant disorder previously mapped to chromosome 7p21.3-p15.1. This ataxia was firstly characterized in a large French family with slowly progressive cerebellar ataxia, accompanied by severe cognitive impairment and mental retardation in two young children. Following the recruitment of 12 additional young family members, linkage analysis enabled us to definitively map the disease locus to chromosome 1p36.33-p36.32. The causative mutation, (c.509C>T/p.P170L) in the transmembrane protein gene TMEM240, was identified by whole exome sequencing and then was confirmed by Sanger sequencing and co-segregation analyses. Index cases from 368 French families with autosomal-dominant cerebellar ataxia were also screened for mutations. In seven cases, we identified a range of missense mutations (c.509C>T/p.P170L, c.239C>T/p.T80M, c.346C>T/p.R116C, c.445G>A/p.E149K, c.511C>T/p.R171W), and a stop mutation (c.489C>G/p.Y163*) in the same gene. TMEM240 is a small, strongly conserved transmembrane protein of unknown function present in cerebellum and brain. Spinocerebellar ataxia 21 may be a particular early-onset disease associated with severe cognitive impairment.

  14. Autosomal recessive PGM3 mutations link glycosylation defects to atopy, immune deficiency, autoimmunity, and neurocognitive impairment

    PubMed Central

    Zhang, Yu; Yu, Xiaomin; Ichikawa, Mie; Lyons, Jonathan J.; Datta, Shrimati; Lamborn, Ian T.; Jing, Huie; Kim, Emily S.; Biancalana, Matthew; Wolfe, Lynne A.; DiMaggio, Thomas; Matthews, Helen F.; Kranick, Sarah M.; Stone, Kelly D.; Holland, Steven M.; Reich, Daniel S.; Hughes, Jason D.; Mehmet, Huseyin; McElwee, Joshua; Freeman, Alexandra F.; Freeze, Hudson H.; Su, Helen C.; Milner, Joshua D.

    2014-01-01

    Background Identifying genetic syndromes that lead to significant atopic disease can open new pathways for investigation and intervention in allergy. Objective To define a genetic syndrome of severe atopy, elevated serum IgE, immune deficiency, autoimmunity, and motor and neurocognitive impairment. Methods Eight patients from two families who had similar syndromic features were studied. Thorough clinical evaluations, including brain MRI and sensory evoked potentials, were performed. Peripheral lymphocyte flow cytometry, antibody responses, and T cell cytokine production were measured. Whole exome sequencing was performed to identify disease-causing mutations. Immunoblotting, qRT-PCR, enzymatic assays, nucleotide sugar and sugar phosphate analyses along with MALDI-TOF mass spectrometry of glycans were used to determine the molecular consequences of the mutations. Results Marked atopy and autoimmunity were associated with increased TH2 and TH17 cytokine production by CD4+ T cells. Bacterial and viral infection susceptibility were noted along with T cell lymphopenia, particularly of CD8+ T cells, and reduced memory B cells. Apparent brain hypomyelination resulted in markedly delayed evoked potentials and likely contributed to neurological abnormalities. Disease segregated with novel autosomal recessive mutations in a single gene, phosphoglucomutase 3 (PGM3). Although PGM3 protein expression was variably diminished, impaired function was demonstrated by decreased enzyme activity and reduced UDP-GlcNAc, along with decreased O- and N-linked protein glycosylation in patients’ cells. These results define a new Congenital Disorder of Glycosylation. Conclusions Autosomal recessive, hypomorphic PGM3 mutations underlie a disorder of severe atopy, immune deficiency, autoimmunity, intellectual disability and hypomyelination. PMID:24589341

  15. GJB2 and GJB6 mutations: genotypic and phenotypic correlations in a large cohort of hearing-impaired patients.

    PubMed

    Marlin, Sandrine; Feldmann, Delphine; Blons, Hélène; Loundon, Natalie; Rouillon, Isabelle; Albert, Sébastien; Chauvin, Pierre; Garabédian, Eréa-Noël; Couderc, Rémy; Odent, Sylvie; Joannard, Alain; Schmerber, Sébastien; Delobel, Bruno; Leman, Jacques; Journel, Hubert; Catros, Hélène; Lemarechal, Cédric; Dollfus, Hélène; Eliot, Marie-Madeleine; Delaunoy, Jean-Louis; David, Albert; Calais, Catherine; Drouin-Garraud, Valérie; Obstoy, Marie-Françoise; Goizet, Cyril; Duriez, Françoise; Fellmann, Florence; Hélias, Jocelyne; Vigneron, Jacqueline; Montaut, Bettina; Matin-Coignard, Dominique; Faivre, Laurence; Baumann, Clarisse; Lewin, Patricia; Petit, Christine; Denoyelle, Françoise

    2005-06-01

    To analyze the clinical features of hearing impairment and to search for correlations with the genotype in patients with DFNB1. Case series. Collaborative study in referral centers, institutional practice. Patients A total of 256 hearing-impaired patients selected on the basis of the presence of biallelic mutations in GJB2 or the association of 1 GJB2 mutation with the GJB6 deletion (GJB6-D13S1830)del. The prevalence of GJB2 mutations and the GJB6 deletion and audiometric phenotypes related to the most frequent genotypes. Twenty-nine different GJB2 mutations were identified. Allelic frequency of 35delG was 69%, and the other common mutations, 313del14, E47X, Q57X, and L90P, accounted for 2.6% to 2.9% of the variants. Concerning GJB6, (GJB6-D13S1830)del accounted for 5% of all mutated alleles and was observed in 25 of 93 compound heterozygous patients. Three novel GJB2 mutations, 355del9, V95M, and 573delCA, were identified. Hearing impairment was frequently less severe in compound heterozygotes 35delG/L90P and 35delG/N206S than in 35delG homozygotes. Moderate or mild hearing impairment was more frequent in patients with 1 or 2 noninactivating mutations than in patients with 2 inactivating mutations. Of 93 patients, hearing loss was stable in 73, progressive in 21, and fluctuant in 2. Progressive hearing loss was more frequent in patients with 1 or 2 noninactivating mutations than in those with 2 inactivating mutations. In 49 families, hearing loss was compared between siblings with similar genotypes, and variability in terms of severity was found in 18 families (37%). Genotype may affect deafness severity, but environmental and other genetic factors may also modulate the severity and evolution of GJB2-GJB6 deafness.

  16. TCTEX1D2 mutations underlie Jeune asphyxiating thoracic dystrophy with impaired retrograde intraflagellar transport

    PubMed Central

    Schmidts, Miriam; Hou, Yuqing; Cortés, Claudio R.; Mans, Dorus A.; Huber, Celine; Boldt, Karsten; Patel, Mitali; van Reeuwijk, Jeroen; Plaza, Jean-Marc; van Beersum, Sylvia E. C.; Yap, Zhi Min; Letteboer, Stef J. F.; Taylor, S. Paige; Herridge, Warren; Johnson, Colin A.; Scambler, Peter J.; Ueffing, Marius; Kayserili, Hulya; Krakow, Deborah; King, Stephen M.; Beales, Philip L.; Al-Gazali, Lihadh; Wicking, Carol; Cormier-Daire, Valerie; Roepman, Ronald; Mitchison, Hannah M.; Witman, George B.; Al-Turki, Saeed; Anderson, Carl; Anney, Richard; Antony, Dinu; Asimit, Jennifer; Ayub, Mohammad; Barrett, Jeff; Barroso, Inês; Bentham, Jamie; Bhattacharya, Shoumo; Blackwood, Douglas; Bobrow, Martin; Bochukova, Elena; Bolton, Patrick; Boustred, Chris; Breen, Gerome; Brion, Marie-Jo; Brown, Andrew; Calissano, Mattia; Carss, Keren; Chatterjee, Krishna; Chen, Lu; Cirak, Sebhattin; Clapham, Peter; Clement, Gail; Coates, Guy; Collier, David; Cosgrove, Catherine; Cox, Tony; Craddock, Nick; Crooks, Lucy; Curran, Sarah; Daly, Allan; Danecek, Petr; Smith, George Davey; Day-Williams, Aaron; Day, Ian; Durbin, Richard; Edkins, Sarah; Ellis, Peter; Evans, David; Farooqi, I. Sadaf; Fatemifar, Ghazaleh; Fitzpatrick, David; Flicek, Paul; Floyd, Jamie; Foley, A. Reghan; Franklin, Chris; Futema, Marta; Gallagher, Louise; Gaunt, Tom; Geschwind, Daniel; Greenwood, Celia; Grozeva, Detelina; Guo, Xiaosen; Gurling, Hugh; Hart, Deborah; Hendricks, Audrey; Holmans, Peter; Huang, Jie; Humphries, Steve E.; Hurles, Matt; Hysi, Pirro; Jackson, David; Jamshidi, Yalda; Jewell, David; Chris, Joyce; Kaye, Jane; Keane, Thomas; Kemp, John; Kennedy, Karen; Kent, Alastair; Kolb-Kokocinski, Anja; Lachance, Genevieve; Langford, Cordelia; Lee, Irene; Li, Rui; Li, Yingrui; Ryan, Liu; Lönnqvist, Jouko; Lopes, Margarida; MacArthur, Daniel G.; Massimo, Mangino; Marchini, Jonathan; Maslen, John; McCarthy, Shane; McGuffin, Peter; McIntosh, Andrew; McKechanie, Andrew; McQuillin, Andrew; Memari, Yasin; Metrustry, Sarah; Min, Josine; Moayyeri, Alireza; Morris, James; Muddyman, Dawn; Muntoni, Francesco; Northstone, Kate; O'Donovan, Michael; O'Rahilly, Stephen; Onoufriadis, Alexandros; Oualkacha, Karim; Owen, Michael; Palotie, Aarno; Panoutsopoulou, Kalliope; Parker, Victoria; Parr, Jeremy; Paternoster, Lavinia; Paunio, Tiina; Payne, Felicity; Perry, John; Pietilainen, Olli; Plagnol, Vincent; Quail, Michael A.; Quaye, Lydia; Raymond, Lucy; Rehnström, Karola; Brent Richards, J.; Ring, Sue; Ritchie, Graham R S; Savage, David B.; Schoenmakers, Nadia; Semple, Robert K.; Serra, Eva; Shihab, Hashem; Shin, So-Youn; Skuse, David; Small, Kerrin; Smee, Carol; Soler, Artigas María; Soranzo, Nicole; Southam, Lorraine; Spector, Tim; St Pourcain, Beate; St. Clair, David; Stalker, Jim; Surdulescu, Gabriela; Suvisaari, Jaana; Tachmazidou, Ioanna; Tian, Jing; Timpson, Nic; Tobin, Martin; Valdes, Ana; van Kogelenberg, Margriet; Vijayarangakannan, Parthiban; Wain, Louise; Walter, Klaudia; Wang, Jun; Ward, Kirsten; Wheeler, Ellie; Whittall, Ros; Williams, Hywel; Williamson, Kathy; Wilson, Scott G.; Wong, Kim; Whyte, Tamieka; ChangJiang, Xu; Zeggini, Eleftheria; Zhang, Feng; Zheng, Hou-Feng

    2015-01-01

    The analysis of individuals with ciliary chondrodysplasias can shed light on sensitive mechanisms controlling ciliogenesis and cell signalling that are essential to embryonic development and survival. Here we identify TCTEX1D2 mutations causing Jeune asphyxiating thoracic dystrophy with partially penetrant inheritance. Loss of TCTEX1D2 impairs retrograde intraflagellar transport (IFT) in humans and the protist Chlamydomonas, accompanied by destabilization of the retrograde IFT dynein motor. We thus define TCTEX1D2 as an integral component of the evolutionarily conserved retrograde IFT machinery. In complex with several IFT dynein light chains, it is required for correct vertebrate skeletal formation but may be functionally redundant under certain conditions. PMID:26044572

  17. Tyrosinase processing and intracellular trafficking is disrupted in mouse primary melanocytes carrying the underwhite (uw) mutation. A model for oculocutaneous albinism (OCA) type 4.

    PubMed

    Costin, Gertrude-E; Valencia, Julio C; Vieira, Wilfred D; Lamoreux, M Lynn; Hearing, Vincent J

    2003-08-01

    Oculocutaneous albinism (OCA) type 4 is a newly identified human autosomal recessive hypopigmentary disorder that disrupts pigmentation in the skin, hair and eyes. Three other forms of OCA have been previously characterized, each resulting from the aberrant processing and/or sorting of tyrosinase, the enzyme critical to pigment production in mammals. The disruption of tyrosinase trafficking occurs at the level of the endoplasmic reticulum (ER) in OCA1 and OCA3, but at the post-Golgi level in OCA2. The gene responsible for OCA4 is the human homologue of the mouse underwhite (uw) gene, which encodes the membrane-associated transporter protein (MATP). To characterize OCA4, we investigated the processing and sorting of melanogenic proteins in primary melanocytes derived from uw/uw mice and from wild-type mice. OCA4 melanocytes were found to be constantly secreted into the medium dark vesicles that contain tyrosinase and two other melanogenic enzymes, Tyrp1 (tyrosinase-related protein 1) and Dct (DOPAchrome tautomerase); this secretory process is not seen in wild-type melanocytes. Although tyrosinase was synthesized at comparable rates in wild-type and in uw-mutant melanocytes, tyrosinase activity in uw-mutant melanocytes was only about 20% of that found in wild-type melanocytes, and was enriched only about threefold in melanosomes compared with the ninefold enrichment in wild-type melanocytes. OCA4 melanocytes showed a marked difference from wild-type melanocytes in that tyrosinase was abnormally secreted from the cells, a process similar to that seen in OCA2 melanocytes, which results from a mutation of the pink-eyed dilution (P) gene. The P protein and MATP have 12 transmembrane regions and are predicted to function as transporters. Ultrastructural analysis shows that the vesicles secreted from OCA4 melanocytes are mostly early stage melanosomes. Taken together, our results show that in OCA4 melanocytes, tyrosinase processing and intracellular trafficking to the

  18. Mutation in E1, the ubiquitin activating enzyme, reduces Drosophila lifespan and results in motor impairment.

    PubMed

    Liu, Hsiu-Yu; Pfleger, Cathie M

    2013-01-01

    Neurodegenerative diseases cause tremendous suffering for those afflicted and their families. Many of these diseases involve accumulation of mis-folded or aggregated proteins thought to play a causal role in disease pathology. Ubiquitinated proteins are often found in these protein aggregates, and the aggregates themselves have been shown to inhibit the activity of the proteasome. These and other alterations in the Ubiquitin Pathway observed in neurodegenerative diseases have led to the question of whether impairment of the Ubiquitin Pathway on its own can increase mortality or if ongoing neurodegeneration alters Ubiquitin Pathway function as a side-effect. To address the role of the Ubiquitin Pathway in vivo, we studied loss-of-function mutations in the Drosophila Ubiquitin Activating Enzyme, Uba1 or E1, the most upstream enzyme in the Ubiquitin Pathway. Loss of only one functional copy of E1 caused a significant reduction in adult lifespan. Rare homozygous hypomorphic E1 mutants reached adulthood. These mutants exhibited further reduced lifespan and showed inappropriate Ras activation in the brain. Removing just one functional copy of Ras restored the lifespan of heterozygous E1 mutants to that of wild-type flies and increased the survival of homozygous E1 mutants. E1 homozygous mutants also showed severe motor impairment. Our findings suggest that processes that impair the Ubiquitin Pathway are sufficient to cause early mortality. Reduced lifespan and motor impairment are seen in the human disease X-linked Infantile Spinal Muscular Atrophy, which is associated with mutation in human E1 warranting further analysis of these mutants as a potential animal model for study of this disease.

  19. The Application of Next-Generation Sequencing for Mutation Detection in Autosomal-Dominant Hereditary Hearing Impairment.

    PubMed

    Gürtler, Nicolas; Röthlisberger, Benno; Ludin, Katja; Schlegel, Christoph; Lalwani, Anil K

    2017-07-01

    Identification of the causative mutation using next-generation sequencing in autosomal-dominant hereditary hearing impairment, as mutation analysis in hereditary hearing impairment by classic genetic methods, is hindered by the high heterogeneity of the disease. Two Swiss families with autosomal-dominant hereditary hearing impairment. Amplified DNA libraries for next-generation sequencing were constructed from extracted genomic DNA, derived from peripheral blood, and enriched by a custom-made sequence capture library. Validated, pooled libraries were sequenced on an Illumina MiSeq instrument, 300 cycles and paired-end sequencing. Technical data analysis was performed with SeqMonk, variant analysis with GeneTalk or VariantStudio. The detection of mutations in genes related to hearing loss by next-generation sequencing was subsequently confirmed using specific polymerase-chain-reaction and Sanger sequencing. Mutation detection in hearing-loss-related genes. The first family harbored the mutation c.5383+5delGTGA in the TECTA-gene. In the second family, a novel mutation c.2614-2625delCATGGCGCCGTG in the WFS1-gene and a second mutation TCOF1-c.1028G>A were identified. Next-generation sequencing successfully identified the causative mutation in families with autosomal-dominant hereditary hearing impairment. The results helped to clarify the pathogenic role of a known mutation and led to the detection of a novel one. NGS represents a feasible approach with great potential future in the diagnostics of hereditary hearing impairment, even in smaller labs.

  20. PX-RICS-deficient mice mimic autism spectrum disorder in Jacobsen syndrome through impaired GABAA receptor trafficking.

    PubMed

    Nakamura, Tsutomu; Arima-Yoshida, Fumiko; Sakaue, Fumika; Nasu-Nishimura, Yukiko; Takeda, Yasuko; Matsuura, Ken; Akshoomoff, Natacha; Mattson, Sarah N; Grossfeld, Paul D; Manabe, Toshiya; Akiyama, Tetsu

    2016-03-16

    Jacobsen syndrome (JBS) is a rare congenital disorder caused by a terminal deletion of the long arm of chromosome 11. A subset of patients exhibit social behavioural problems that meet the diagnostic criteria for autism spectrum disorder (ASD); however, the underlying molecular pathogenesis remains poorly understood. PX-RICS is located in the chromosomal region commonly deleted in JBS patients with autistic-like behaviour. Here we report that PX-RICS-deficient mice exhibit ASD-like social behaviours and ASD-related comorbidities. PX-RICS-deficient neurons show reduced surface γ-aminobutyric acid type A receptor (GABAAR) levels and impaired GABAAR-mediated synaptic transmission. PX-RICS, GABARAP and 14-3-3ζ/θ form an adaptor complex that interconnects GABAAR and dynein/dynactin, thereby facilitating GABAAR surface expression. ASD-like behavioural abnormalities in PX-RICS-deficient mice are ameliorated by enhancing inhibitory synaptic transmission with a GABAAR agonist. Our findings demonstrate a critical role of PX-RICS in cognition and suggest a causal link between PX-RICS deletion and ASD-like behaviour in JBS patients.

  1. Loss of cortical actin filaments in insulin-resistant skeletal muscle cells impairs GLUT4 vesicle trafficking and glucose transport

    PubMed Central

    McCarthy, Alicia M.; Spisak, Kristen O.; Brozinick, Joseph T.; Elmendorf, Jeffrey S.

    2008-01-01

    Study has demonstrated an essential role of cortical filamentous actin (F-actin) in insulin-regulated glucose uptake by skeletal muscle. Here, we tested whether perturbations in F-actin contributed to impaired insulin responsiveness provoked by hyperinsulinemia. In L6 myo-tubes stably expressing GLUT4 that carries an exofacial myc-epitope tag, acute insulin stimulation (20 min, 100 nM) increased GLUT4myc translocation and glucose uptake by ~2-fold. In contrast, a hyperinsulinemic state, induced by inclusion of 5 nM insulin in the medium for 12 h decreased the ability of insulin to stimulate these processes. Defects in insulin signaling did not readily account for the observed disruption. In contrast, hyperinsulinemia reduced cortical F-actin. This occurred concomitant with a loss of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2), a lipid involved in cytoskeletal regulation. Restoration of plasma membrane PIP2 in hyperinsulinemic cells restored F-actin and insulin responsiveness. Consistent with these in vitro observations suggesting that the hyperinsulinemic state negatively affects cortical F-actin structure, epitrochlearis skeletal muscle from insulin-resistant hyperinsulinemic Zucker fatty rats displayed a similar loss of F-actin structure compared with that in muscle from lean insulin-sensitive littermates. We propose that a component of insulin-induced insulin resistance in skeletal muscle involves defects in PIP2/F-actin structure essential for insulin-regulated glucose transport. PMID:16774991

  2. Loss of cortical actin filaments in insulin-resistant skeletal muscle cells impairs GLUT4 vesicle trafficking and glucose transport.

    PubMed

    McCarthy, Alicia M; Spisak, Kristen O; Brozinick, Joseph T; Elmendorf, Jeffrey S

    2006-11-01

    Study has demonstrated an essential role of cortical filamentous actin (F-actin) in insulin-regulated glucose uptake by skeletal muscle. Here, we tested whether perturbations in F-actin contributed to impaired insulin responsiveness provoked by hyperinsulinemia. In L6 myotubes stably expressing GLUT4 that carries an exofacial myc-epitope tag, acute insulin stimulation (20 min, 100 nM) increased GLUT4myc translocation and glucose uptake by approximately 2-fold. In contrast, a hyperinsulinemic state, induced by inclusion of 5 nM insulin in the medium for 12 h decreased the ability of insulin to stimulate these processes. Defects in insulin signaling did not readily account for the observed disruption. In contrast, hyperinsulinemia reduced cortical F-actin. This occurred concomitant with a loss of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), a lipid involved in cytoskeletal regulation. Restoration of plasma membrane PIP(2) in hyperinsulinemic cells restored F-actin and insulin responsiveness. Consistent with these in vitro observations suggesting that the hyperinsulinemic state negatively affects cortical F-actin structure, epitrochlearis skeletal muscle from insulin-resistant hyperinsulinemic Zucker fatty rats displayed a similar loss of F-actin structure compared with that in muscle from lean insulin-sensitive littermates. We propose that a component of insulin-induced insulin resistance in skeletal muscle involves defects in PIP(2)/F-actin structure essential for insulin-regulated glucose transport.

  3. PX-RICS-deficient mice mimic autism spectrum disorder in Jacobsen syndrome through impaired GABAA receptor trafficking

    PubMed Central

    Nakamura, Tsutomu; Arima-Yoshida, Fumiko; Sakaue, Fumika; Nasu-Nishimura, Yukiko; Takeda, Yasuko; Matsuura, Ken; Akshoomoff, Natacha; Mattson, Sarah N.; Grossfeld, Paul D.; Manabe, Toshiya; Akiyama, Tetsu

    2016-01-01

    Jacobsen syndrome (JBS) is a rare congenital disorder caused by a terminal deletion of the long arm of chromosome 11. A subset of patients exhibit social behavioural problems that meet the diagnostic criteria for autism spectrum disorder (ASD); however, the underlying molecular pathogenesis remains poorly understood. PX-RICS is located in the chromosomal region commonly deleted in JBS patients with autistic-like behaviour. Here we report that PX-RICS-deficient mice exhibit ASD-like social behaviours and ASD-related comorbidities. PX-RICS-deficient neurons show reduced surface γ-aminobutyric acid type A receptor (GABAAR) levels and impaired GABAAR-mediated synaptic transmission. PX-RICS, GABARAP and 14-3-3ζ/θ form an adaptor complex that interconnects GABAAR and dynein/dynactin, thereby facilitating GABAAR surface expression. ASD-like behavioural abnormalities in PX-RICS-deficient mice are ameliorated by enhancing inhibitory synaptic transmission with a GABAAR agonist. Our findings demonstrate a critical role of PX-RICS in cognition and suggest a causal link between PX-RICS deletion and ASD-like behaviour in JBS patients. PMID:26979507

  4. MARVELD2 (DFNB49) Mutations in the Hearing Impaired Central European Roma Population - Prevalence, Clinical Impact and the Common Origin

    PubMed Central

    Mátyás, Petra; Ficek, Andrej; Hučková, Miloslava; Sůrová, Martina; Šafka-Brožková, Dana; Anwar, Saima; Bene, Judit; Straka, Slavomír; Janicsek, Ingrid; Ahmed, Zubair M.; Seeman, Pavel; Melegh, Béla; Profant, Milan; Klimeš, Iwar; Riazuddin, Saima; Kádasi, Ľudevít; Gašperíková, Daniela

    2015-01-01

    Background In the present study we aimed: 1) To establish the prevalence and clinical impact of DFNB49 mutations in deaf Roma from 2 Central European countries (Slovakia and Hungary), and 2) to analyze a possible common origin of the c.1331+2T>C mutation among Roma and Pakistani mutation carriers identified in the present and previous studies. Methods We sequenced 6 exons of the MARVELD2 gene in a group of 143 unrelated hearing impaired Slovak Roma patients. Simultaneously, we used RFLP to detect the c.1331+2T>C mutation in 85 Hungarian deaf Roma patients, control groups of 702 normal hearing Romanies from both countries and 375 hearing impaired Slovak Caucasians. We analyzed the haplotype using 21 SNPs spanning a 5.34Mb around the mutation c.1331+2T>C. Results One pathogenic mutation (c.1331+2T>C) was identified in 12 homozygous hearing impaired Roma patients. Allele frequency of this mutation was higher in Hungarian (10%) than in Slovak (3.85%) Roma patients. The identified common haplotype in Roma patients was defined by 18 SNP markers (3.89 Mb). Fourteen common SNPs were also shared among Pakistani and Roma homozygotes. Biallelic mutation carriers suffered from prelingual bilateral moderate to profound sensorineural hearing loss. Conclusions We demonstrate different frequencies of the c.1331+2T>C mutation in hearing impaired Romanies from 3 Central European countries. In addition, our results provide support for the hypothesis of a possible common ancestor of the Slovak, Hungarian and Czech Roma as well as Pakistani deaf patients. Testing for the c.1331+2T>C mutation may be recommended in GJB2 negative Roma cases with early-onset sensorineural hearing loss. PMID:25885414

  5. Migraine Mutations Impair Hippocampal Learning Despite Enhanced Long-Term Potentiation

    PubMed Central

    Dilekoz, Ergin; Houben, Thijs; Eikermann-Haerter, Katharina; Balkaya, Mustafa; Lenselink, A. Mariette; Whalen, Michael J.; Spijker, Sabine; Ferrari, Michel D.; van den Maagdenberg, Arn M.J.M.

    2015-01-01

    To explain cognitive and memory difficulties observed in some familial hemiplegic migraine (FHM) patients, we examined hippocampal neurotransmission and plasticity in knock-in mice expressing the FHM type 1 (FHM1) R192Q gain-of function mutation in the CACNA1A gene that encodes the α1A subunit of neuronal CaV2.1 channels. We determined stimulus intensity–response curves for anterior commissure-evoked hippocampal CA1 field potentials in strata pyramidale and radiatum and assessed neuroplasticity by inducing long-term potentiation (LTP) and long-term depression (LTD) in anesthetized mice in vivo. We also studied learning and memory using contextual fear-conditioning, Morris water maze, and novel object recognition tests. Hippocampal field potentials were significantly enhanced in R192Q mice compared with wild-type controls. Stimulus intensity–response curves were shifted to the left and displayed larger maxima in the mutants. LTP was augmented by twofold in R192Q mice, whereas LTD was unchanged compared with wild-type mice. R192Q mice showed significant spatial memory deficits in contextual fear-conditioning and Morris water maze tests compared with wild-type controls. Novel object recognition was not impaired in R192Q mice; however, mice carrying the more severe S218L CACNA1A mutation showed marked deficits in this test, suggesting a genotype–phenotype relationship. Thus, whereas FHM1 gain-of-function mutations enhance hippocampal excitatory transmission and LTP, learning and memory are paradoxically impaired, providing a possible explanation for cognitive changes detected in FHM. Data suggest that abnormally enhanced plasticity can be as detrimental to efficient learning as reduced plasticity and highlight how genetically enhanced neuronal excitability may impact cognitive function. PMID:25716839

  6. RNASEH1 Mutations Impair mtDNA Replication and Cause Adult-Onset Mitochondrial Encephalomyopathy

    PubMed Central

    Reyes, Aurelio; Melchionda, Laura; Nasca, Alessia; Carrara, Franco; Lamantea, Eleonora; Zanolini, Alice; Lamperti, Costanza; Fang, Mingyan; Zhang, Jianguo; Ronchi, Dario; Bonato, Sara; Fagiolari, Gigliola; Moggio, Maurizio; Ghezzi, Daniele; Zeviani, Massimo

    2015-01-01

    Chronic progressive external ophthalmoplegia (CPEO) is common in mitochondrial disorders and is frequently associated with multiple mtDNA deletions. The onset is typically in adulthood, and affected subjects can also present with general muscle weakness. The underlying genetic defects comprise autosomal-dominant or recessive mutations in several nuclear genes, most of which play a role in mtDNA replication. Next-generation sequencing led to the identification of compound-heterozygous RNASEH1 mutations in two singleton subjects and a homozygous mutation in four siblings. RNASEH1, encoding ribonuclease H1 (RNase H1), is an endonuclease that is present in both the nucleus and mitochondria and digests the RNA component of RNA-DNA hybrids. Unlike mitochondria, the nucleus harbors a second ribonuclease (RNase H2). All affected individuals first presented with CPEO and exercise intolerance in their twenties, and these were followed by muscle weakness, dysphagia, and spino-cerebellar signs with impaired gait coordination, dysmetria, and dysarthria. Ragged-red and cytochrome c oxidase (COX)-negative fibers, together with impaired activity of various mitochondrial respiratory chain complexes, were observed in muscle biopsies of affected subjects. Western blot analysis showed the virtual absence of RNase H1 in total lysate from mutant fibroblasts. By an in vitro assay, we demonstrated that altered RNase H1 has a reduced capability to remove the RNA from RNA-DNA hybrids, confirming their pathogenic role. Given that an increasing amount of evidence indicates the presence of RNA primers during mtDNA replication, this result might also explain the accumulation of mtDNA deletions and underscores the importance of RNase H1 for mtDNA maintenance. PMID:26094573

  7. Biallelic mutations in CAD, impair de novo pyrimidine biosynthesis and decrease glycosylation precursors

    PubMed Central

    Ng, Bobby G.; Wolfe, Lynne A.; Ichikawa, Mie; Markello, Thomas; He, Miao; Tifft, Cynthia J.; Gahl, William A.; Freeze, Hudson H.

    2015-01-01

    In mitochondria, carbamoyl-phosphate synthetase 1 activity produces carbamoyl phosphate for urea synthesis, and deficiency results in hyperammonemia. Cytoplasmic carbamoyl-phosphate synthetase 2, however, is part of a tri-functional enzyme encoded by CAD; no human disease has been attributed to this gene. The tri-functional enzyme contains carbamoyl-phosphate synthetase 2 (CPS2), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities, which comprise the first three of six reactions required for de novo pyrimidine biosynthesis. Here we characterize an individual who is compound heterozygous for mutations in different domains of CAD. One mutation, c.1843-1G>A, results in an in-frame deletion of exon 13. The other, c.6071G>A, causes a missense mutation (p.Arg2024Gln) in a highly conserved residue that is essential for carbamoyl-phosphate binding. Metabolic flux studies showed impaired aspartate incorporation into RNA and DNA through the de novo synthesis pathway. In addition, CTP, UTP and nearly all UDP-activated sugars that serve as donors for glycosylation were decreased. Uridine supplementation rescued these abnormalities, suggesting a potential therapy for this new glycosylation disorder. PMID:25678555

  8. Prevalence and audiological profiles of GJB2 mutations in a large collective of hearing impaired patients.

    PubMed

    Burke, W F; Warnecke, A; Schöner-Heinisch, A; Lesinski-Schiedat, A; Maier, H; Lenarz, T

    2016-03-01

    Mutations in the GJB2 gene are known to represent the commonest cause of hereditary and congenital hearing loss. In this study, a complete sequencing of the GJB2 gene in a cohort of 506 patients from a single, large cochlear implant program in Europe was performed. Audiological testing for those patients who could actively participate was performed using pure tone audiometry (PTA). Those unable to undergo PTA were measured using click-auditory brainstem response (ABR). Data analysis was performed to determine genotype-phenotype correlations of the mutational status vs. audiological profiles and vs. age at the time of presentation. An overall prevalence of biallelic mutations of 13.4% was found for the total collective. When subsets of younger patients were examined, the prevalence increased to 27% of those up to age 18 and 35% of those up to age 5 at the time of testing, respectively. This increase was found to be highly significant (p < 0.001). Analysis of the mean PTA thresholds revealed a strong correlation between allele combination status and mean PTA (p = 0.021). The prevalence of simple heterozygotes was found to be approximately 10.1%, which is around 3.3 times the value expected in the general population. As GJB2 follows a recessive pattern of inheritance, the question arises as to why such a large fraction of simple heterozygotes was observed among the hearing impaired patients included in this study.

  9. The effect of nanoparticle size and NLS density on nuclear targeting in cancer and normal cells; impaired nuclear import and aberrant nanoparticle intracellular trafficking in glioma.

    PubMed

    Tammam, Salma N; Azzazy, Hassan M E; Lamprecht, Alf

    2017-02-27

    The cell nucleus is an interesting target in many diseases with particular interest in cancer. Previously, nuclear targeted small and large chitosan nanoparticles (S-NPs≈25nm, and L-NPs≈150nm respectively), modified with low, intermediate and high densities of NLS (L-NLS, I-NLS and H-NLS) were developed and assessed in L929 fibroblasts. However, to evade apoptosis and stimulate tumor growth cancer cells are capable of manipulating the nuclear-cytoplasmic transport on many levels, making NPs that are capable of nuclear targeting in normal cells incapable of doing so in cancer. For such reason, here, the nuclear delivery efficiency of S-NPs and L-NPs was assessed as a function of their NLS density in cancer and non-cancer cells. For S-NPs, in all cells tested, NLS was unnecessary for nuclear delivery; unmodified S-NPs showed higher nuclear delivery than NLS-S-NPs due to their ability to gain nuclear entry in a passive manner. For L-NPs, L-NLS-L-NPs showed ≈ 8.5, 33, 1.8 and 7.2 fold higher nuclear deliveries than H-NLS-L-NPs in L929 fibroblasts, primary human fibroblasts, HEK 293 and lung cancer cells, respectively. In glioma however, unmodified L-NPs showed highest nuclear delivery, whereas NLS-L-NPs were retained in the cytoplasm. Experiments conducted in the presence of inhibitors of the classical nuclear import pathway indicated that due to overexpression of importin α, classical nuclear import in glioma is impaired leading to aberrant NP intracellular trafficking and nuclear import.

  10. Expression and Dendritic Trafficking of BDNF-6 Splice Variant are Impaired in Knock-In Mice Carrying Human BDNF Val66Met Polymorphism

    PubMed Central

    Baj, Gabriele; Ieraci, Alessandro; Corna, Stefano; Musazzi, Laura; Lee, Francis S.; Tongiorgi, Enrico; Popoli, Maurizio

    2015-01-01

    Background: The human Val66Met polymorphism in brain-derived neurotrophic factor (BDNF), a key factor in neuroplasticity, synaptic function, and cognition, has been implicated in the pathophysiology of neuropsychiatric and neurodegenerative disorders. BDNF is encoded by multiple transcripts with distinct regulation and localization, but the impact of the Val66Met polymorphism on BDNF regulation remains unclear. Methods: In BDNF Val66Met knock-in mice, which recapitulate the phenotypic hallmarks of individuals carrying the BDNFMet allele, we measured expression levels, epigenetic changes at promoters, and dendritic trafficking of distinct BDNF transcripts using quantitative PCR, chromatin immunoprecipitation (ChIP), and in situ hybridization. Results: BDNF-4 and BDNF-6 transcripts were reduced in BDNFMet/Met mice, compared with BDNFVal/Val mice. ChIP for acetyl-histone H3, a marker of active gene transcription, and trimethyl-histone-H3-Lys27 (H3K27me3), a marker of gene repression, showed higher H3K27me3 binding to exon 5, 6, and 8 promoters in BDNFMet/Met. The H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) is involved in epigenetic regulation of BDNF expression, because in neuroblastoma cells BDNF expression was increased both by short interference RNA for EZH2 and incubation with 3-deazaneplanocin A, an inhibitor of EZH2. In situ hybridization for BDNF-2, BDNF-4, and BDNF-6 after pilocarpine treatment showed that BDNF-6 transcript was virtually absent from distal dendrites of the CA1 and CA3 regions in BDNFMet/Met mice, while no changes were found for BDNF-2 and BDNF-4. Conclusions: Impaired BDNF expression and dendritic targeting in BDNFMet/Met mice may contribute to reduced regulated secretion of BDNF at synapses, and may be a specific correlate of pathology in individuals carrying the Met allele. PMID:26108221

  11. Ribosomal Protein Mutations Result in Constitutive p53 Protein Degradation through Impairment of the AKT Pathway

    PubMed Central

    Hermkens, Dorien; Wlodarski, Marcin W.; Da Costa, Lydie; MacInnes, Alyson W.

    2015-01-01

    Mutations in ribosomal protein (RP) genes can result in the loss of erythrocyte progenitor cells and cause severe anemia. This is seen in patients with Diamond-Blackfan anemia (DBA), a pure red cell aplasia and bone marrow failure syndrome that is almost exclusively linked to RP gene haploinsufficiency. While the mechanisms underlying the cytopenia phenotype of patients with these mutations are not completely understood, it is believed that stabilization of the p53 tumor suppressor protein may induce apoptosis in the progenitor cells. In stark contrast, tumor cells from zebrafish with RP gene haploinsufficiency are unable to stabilize p53 even when exposed to acute DNA damage despite transcribing wild type p53 normally. In this work we demonstrate that p53 has a limited role in eliciting the anemia phenotype of zebrafish models of DBA. In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells. Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53. By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53. Our work indicates that the anemia phenotype of zebrafish models of DBA is dependent on factors other than p53, and may hold clinical significance for both DBA and the increasing number of cancers revealing spontaneous mutations in RP genes. PMID:26132763

  12. Retinitis Pigmentosa Mutations in Bad Response to Refrigeration 2 (Brr2) Impair ATPase and Helicase Activity.

    PubMed

    Ledoux, Sarah; Guthrie, Christine

    2016-06-03

    Brr2 is an RNA-dependent ATPase required to unwind the U4/U6 snRNA duplex during spliceosome assembly. Mutations within the ratchet helix of the Brr2 RNA binding channel result in a form of degenerative human blindness known as retinitis pigmentosa (RP). The biochemical consequences of these mutations on Brr2's RNA binding, helicase, and ATPase activity have not yet been characterized. Therefore, we identified the largest construct of Brr2 that is soluble in vitro, which truncates the first 247 amino acids of the N terminus (Δ247-Brr2), to characterize the effects of the RP mutations on Brr2 activity. The Δ247-Brr2 RP mutants exhibit a gradient of severity of weakened RNA binding, reduced helicase activity, and reduced ATPase activity compared with wild type Δ247-Brr2. The globular C-terminal Jab1/Mpn1-like domain of Prp8 increases the ability of Δ247-Brr2 to bind the U4/U6 snRNA duplex at high pH and increases Δ247-Brr2's RNA-dependent ATPase activity and the extent of RNA unwinding. However, this domain of Prp8 does not differentially affect the Δ247-Brr2 RP mutants compared with the wild type Δ247-Brr2. When stimulated by Prp8, wild type Δ247-Brr2 is able to unwind long stable duplexes in vitro, and even the RP mutants capable of binding RNA with tight affinity are incapable of fully unwinding short duplex RNAs. Our data suggest that the RP mutations within the ratchet helix impair Brr2 translocation through RNA helices.

  13. Pseudohypoparathyroidism, a novel mutation in the betagamma-contact region of Gsalpha impairs receptor stimulation.

    PubMed

    Farfel, Z; Iiri, T; Shapira, H; Roitman, A; Mouallem, M; Bourne, H R

    1996-08-16

    Pseudohypoparathyroidism, type Ia (PHP-Ia), is a dominantly inherited endocrine disorder characterized by resistance to hormones that act by stimulating adenylyl cyclase. It is caused by inheritance of an autosomal mutation that inactivates the alpha subunit (alphas) of Gs, the stimulatory regulator of adenylyl cyclase. In three members of a family, the PHP-Ia phenotype is associated with a mutation (R231H) that substitutes histidine for an arginine at position 231 in alphas. We assessed signaling function of alphas-WT versus alphas-R231H transiently transfected in HEK293 cells. Hormone receptor-dependent stimulation of cAMP accumulation in cells expressing alphas-R231H is reduced by approximately 75% in comparison to cAMP accumulation in cells expressing alphas-WT. A second mutation, alphas-R201C, inhibits the GTPase turnoff reaction of alphas, thus producing receptor-independent stimulation of cAMP accumulation. The double mutant, alphas-R231H/R201C, stimulates cAMP accumulation almost as well (approximately 80%) as does alphas-R201C itself, indicating that the R231H mutation selectively impairs receptor-dependent signaling. In three-dimensional structures of G protein heterotrimers, Arg-231 is located in a region, switch 2, that is thought to interact with the betagamma subunit rather than with the hormone receptor. Thus, the R231H phenotype suggests that switch 2 (perhaps in concert with betagamma) mediates G protein activation by receptors at a site distant from the receptor-G protein contact surface.

  14. Trafficking modulator TENin1 inhibits endocytosis, causes endomembrane protein accumulation at the pre-vacuolar compartment and impairs gravitropic response in Arabidopsis thaliana

    PubMed Central

    Paudyal, Rupesh; Jamaluddin, Adam; Warren, James P.; Doyle, Siamsa M.; Robert, Stéphanie; Warriner, Stuart L.; Baker, Alison

    2014-01-01

    Auxin gradients are established and maintained by polarized distribution of auxin transporters that undergo constitutive endocytic recycling from the PM (plasma membrane) and are essential for the gravitropic response in plants. The present study characterizes an inhibitor of endomembrane protein trafficking, TE1 (trafficking and endocytosis inhibitor 1/TENin1) that reduces gravitropic root bending in Arabidopsis thaliana seedlings. Short-term TE1 treatment causes accumulation of PM proteins, including the BR (brassinosteroid) receptor BRI1 (BR insensitive 1), PIP2a (PM intrinsic protein 2a) and the auxin transporter PIN2 (PIN-FORMED 2) in a PVC (pre-vacuolar related compartment), which is sensitive to BFA (Brefeldin A). This compound inhibits endocytosis from the PM and promotes trafficking to the vacuole, consistent with inhibition of retrieval of proteins to the TGN (trans-Golgi network) from the PVC and the PM. However, trafficking of newly synthesized proteins to the PM is unaffected. The short-term protein trafficking inhibition and long-term effect on plant growth and survival caused by TE1 were fully reversible upon drug washout. Structure–activity relationship studies revealed that only minor modifications were possible without loss of biological activity. Diversity in Arabidopsis ecotypes was also exploited to identify two Arabidopsis accessions that display reduced sensitivity to TE1. This compound and the resistant Arabidopsis accessions may be used as a resource in future studies to better understand endomembrane trafficking in plants. PMID:24654932

  15. Trafficking modulator TENin1 inhibits endocytosis, causes endomembrane protein accumulation at the pre-vacuolar compartment and impairs gravitropic response in Arabidopsis thaliana.

    PubMed

    Paudyal, Rupesh; Jamaluddin, Adam; Warren, James P; Doyle, Siamsa M; Robert, Stéphanie; Warriner, Stuart L; Baker, Alison

    2014-06-01

    Auxin gradients are established and maintained by polarized distribution of auxin transporters that undergo constitutive endocytic recycling from the PM (plasma membrane) and are essential for the gravitropic response in plants. The present study characterizes an inhibitor of endomembrane protein trafficking, TE1 (trafficking and endocytosis inhibitor 1/TENin1) that reduces gravitropic root bending in Arabidopsis thaliana seedlings. Short-term TE1 treatment causes accumulation of PM proteins, including the BR (brassinosteroid) receptor BRI1 (BR insensitive 1), PIP2a (PM intrinsic protein 2a) and the auxin transporter PIN2 (PIN-FORMED 2) in a PVC (pre-vacuolar related compartment), which is sensitive to BFA (Brefeldin A). This compound inhibits endocytosis from the PM and promotes trafficking to the vacuole, consistent with inhibition of retrieval of proteins to the TGN (trans-Golgi network) from the PVC and the PM. However, trafficking of newly synthesized proteins to the PM is unaffected. The short-term protein trafficking inhibition and long-term effect on plant growth and survival caused by TE1 were fully reversible upon drug washout. Structure-activity relationship studies revealed that only minor modifications were possible without loss of biological activity. Diversity in Arabidopsis ecotypes was also exploited to identify two Arabidopsis accessions that display reduced sensitivity to TE1. This compound and the resistant Arabidopsis accessions may be used as a resource in future studies to better understand endomembrane trafficking in plants.

  16. Rare compound heterozygosity involving dominant and recessive mutations of GJB2 gene in an assortative mating hearing impaired Indian family.

    PubMed

    Pavithra, Amritkumar; Chandru, Jayasankaran; Jeffrey, Justin Margret; Karthikeyen, N P; Srisailapathy, C R Srikumari

    2017-01-01

    Connexin 26 (Cx-26), a gap junction protein coded by GJB2 gene, plays a very important role in recycling of potassium ions, one of the vital steps in the mechanotransduction process of hearing. Mutations in the GJB2 gene have been associated with both autosomal recessive as well as dominant nonsyndromic hearing loss. As Cx-26 is linked with skin homeostasis, mutations in this gene are sometimes associated with syndromic forms of hearing loss showing skin anomalies. We report here a non consanguineous assortatively mating hearing impaired family with one of the hearing impaired partners, their hearing impaired sibling and hearing impaired offspring showing compound heterozygosity in the GJB2 gene, involving a dominant mutation p.R184Q and two recessive mutations p.Q124X and c.IVS 1+1G>A in a unique triallelic combination. To the best of our knowledge, this is the first report from India on p.R184Q mutation in the GJB2 gene associated with rare compound heterozygosity showing nonsyndromic presentation.

  17. Munc18-1 mutations that strongly impair SNARE-complex binding support normal synaptic transmission.

    PubMed

    Meijer, Marieke; Burkhardt, Pawel; de Wit, Heidi; Toonen, Ruud F; Fasshauer, Dirk; Verhage, Matthijs

    2012-05-02

    Synaptic transmission depends critically on the Sec1p/Munc18 protein Munc18-1, but it is unclear whether Munc18-1 primarily operates as a integral part of the fusion machinery or has a more upstream role in fusion complex assembly. Here, we show that point mutations in Munc18-1 that interfere with binding to the free Syntaxin1a N-terminus and strongly impair binding to assembled SNARE complexes all support normal docking, priming and fusion of synaptic vesicles, and normal synaptic plasticity in munc18-1 null mutant neurons. These data support a prevailing role of Munc18-1 before/during SNARE-complex assembly, while its continued association to assembled SNARE complexes is dispensable for synaptic transmission.

  18. Munc18-1 mutations that strongly impair SNARE-complex binding support normal synaptic transmission

    PubMed Central

    Meijer, Marieke; Burkhardt, Pawel; de Wit, Heidi; Toonen, Ruud F; Fasshauer, Dirk; Verhage, Matthijs

    2012-01-01

    Synaptic transmission depends critically on the Sec1p/Munc18 protein Munc18-1, but it is unclear whether Munc18-1 primarily operates as a integral part of the fusion machinery or has a more upstream role in fusion complex assembly. Here, we show that point mutations in Munc18-1 that interfere with binding to the free Syntaxin1a N-terminus and strongly impair binding to assembled SNARE complexes all support normal docking, priming and fusion of synaptic vesicles, and normal synaptic plasticity in munc18-1 null mutant neurons. These data support a prevailing role of Munc18-1 before/during SNARE-complex assembly, while its continued association to assembled SNARE complexes is dispensable for synaptic transmission. PMID:22446389

  19. KSR2 Mutations Are Associated with Obesity, Insulin Resistance, and Impaired Cellular Fuel Oxidation

    PubMed Central

    Pearce, Laura R.; Atanassova, Neli; Banton, Matthew C.; Bottomley, Bill; van der Klaauw, Agatha A.; Revelli, Jean-Pierre; Hendricks, Audrey; Keogh, Julia M.; Henning, Elana; Doree, Deon; Jeter-Jones, Sabrina; Garg, Sumedha; Bochukova, Elena G.; Bounds, Rebecca; Ashford, Sofie; Gayton, Emma; Hindmarsh, Peter C.; Shield, Julian P.H.; Crowne, Elizabeth; Barford, David; Wareham, Nick J.; O’Rahilly, Stephen; Murphy, Michael P.; Powell, David R.; Barroso, Ines; Farooqi, I. Sadaf

    2013-01-01

    Summary Kinase suppressor of Ras 2 (KSR2) is an intracellular scaffolding protein involved in multiple signaling pathways. Targeted deletion of Ksr2 leads to obesity in mice, suggesting a role in energy homeostasis. We explored the role of KSR2 in humans by sequencing 2,101 individuals with severe early-onset obesity and 1,536 controls. We identified multiple rare variants in KSR2 that disrupt signaling through the Raf-MEK-ERK pathway and impair cellular fatty acid oxidation and glucose oxidation in transfected cells; effects that can be ameliorated by the commonly prescribed antidiabetic drug, metformin. Mutation carriers exhibit hyperphagia in childhood, low heart rate, reduced basal metabolic rate and severe insulin resistance. These data establish KSR2 as an important regulator of energy intake, energy expenditure, and substrate utilization in humans. Modulation of KSR2-mediated effects may represent a novel therapeutic strategy for obesity and type 2 diabetes. PaperFlick PMID:24209692

  20. Impairment of immunity to Candida and Mycobacterium in humans with bi-allelic RORC mutations*

    PubMed Central

    Halwani, Rabih; Ma, Cindy S.; Wong, Natalie; Soudais, Claire; Henderson, Lauren A.; Marzouqa, Hiyam; Shamma, Jamal; Gonzalez, Marcela; Martinez-Barricarte, Rubén; Okada, Chizuru; Avery, Danielle T.; Latorre, Daniela; Deswarte, Caroline; Jabot-Hanin, Fabienne; Torrado, Egidio; Fountain, Jeffrey; Belkadi, Aziz; Itan, Yuval; Boisson, Bertrand; Migaud, Mélanie; Arlehamn, Cecilia S. Lindestam; Sette, Alessandro; Breton, Sylvain; McCluskey, James; Rossjohn, Jamie; de Villartay, Jean-Pierre; Moshous, Despina; Hambleton, Sophie; Latour, Sylvain; Arkwright, Peter D.; Picard, Capucine; Lantz, Olivier; Engelhard, Dan; Kobayashi, Masao; Abel, Laurent; Casanova, Jean-Laurent

    2015-01-01

    Human inborn errors of immunity mediated by the cytokines interleukin (IL)-17A/F underlie mucocutaneous candidiasis, whereas inborn errors of interferon (IFN)-γ immunity underlie mycobacterial disease. We report the discovery of bi-allelic RORC loss-of-function mutations in seven individuals from three kindreds of different ethnic origins with both candidiasis and mycobacteriosis. The lack of functional RORγ and RORγT isoforms resulted in the absence of IL-17A/F-producing T cells in these individuals, probably accounting for their chronic candidiasis. Unexpectedly, leukocytes from RORγ- and RORγT-deficient individuals also displayed an impaired IFN-γ response to Mycobacterium. This principally reflected profoundly defective IFN-γ production by circulating γδ T cells and CD4+CCR6+ CXCR3+ αβ T cells. In humans, both mucocutaneous immunity to Candida and systemic immunity to Mycobacterium require RORγ, or RORγT, or both. PMID:26160376

  1. KSR2 mutations are associated with obesity, insulin resistance, and impaired cellular fuel oxidation.

    PubMed

    Pearce, Laura R; Atanassova, Neli; Banton, Matthew C; Bottomley, Bill; van der Klaauw, Agatha A; Revelli, Jean-Pierre; Hendricks, Audrey; Keogh, Julia M; Henning, Elana; Doree, Deon; Jeter-Jones, Sabrina; Garg, Sumedha; Bochukova, Elena G; Bounds, Rebecca; Ashford, Sofie; Gayton, Emma; Hindmarsh, Peter C; Shield, Julian P H; Crowne, Elizabeth; Barford, David; Wareham, Nick J; O'Rahilly, Stephen; Murphy, Michael P; Powell, David R; Barroso, Ines; Farooqi, I Sadaf

    2013-11-07

    Kinase suppressor of Ras 2 (KSR2) is an intracellular scaffolding protein involved in multiple signaling pathways. Targeted deletion of Ksr2 leads to obesity in mice, suggesting a role in energy homeostasis. We explored the role of KSR2 in humans by sequencing 2,101 individuals with severe early-onset obesity and 1,536 controls. We identified multiple rare variants in KSR2 that disrupt signaling through the Raf-MEKERK pathway and impair cellular fatty acid oxidation and glucose oxidation in transfected cells; effects that can be ameliorated by the commonly prescribed antidiabetic drug, metformin. Mutation carriers exhibit hyperphagia in childhood, low heart rate, reduced basal metabolic rate and severe insulin resistance. These data establish KSR2 as an important regulator of energy intake, energy expenditure, and substrate utilization in humans. Modulation of KSR2-mediated effects may represent a novel therapeutic strategy for obesity and type 2 diabetes.

  2. LRRK2 delays degradative receptor trafficking by impeding late endosomal budding through decreasing Rab7 activity.

    PubMed

    Gómez-Suaga, Patricia; Rivero-Ríos, Pilar; Fdez, Elena; Blanca Ramírez, Marian; Ferrer, Isidro; Aiastui, Ana; López De Munain, Adolfo; Hilfiker, Sabine

    2014-12-20

    Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset autosomal dominant Parkinson's disease (PD), and sequence variations at the LRRK2 locus are associated with increased risk for sporadic PD. LRRK2 contains both GTPase and kinase domains flanked by protein interaction motifs, and mutations associated with familial PD have been described for both catalytic domains. LRRK2 has been implicated in diverse cellular processes, and recent evidence pinpoints to an important role for LRRK2 in modulating a variety of intracellular membrane trafficking pathways. However, the underlying mechanisms are poorly understood. Here, by studying the classical, well-understood, degradative trafficking pathway of the epidermal growth factor receptor (EGFR), we show that LRRK2 regulates endocytic membrane trafficking in an Rab7-dependent manner. Mutant LRRK2 expression causes a slight delay in early-to-late endosomal trafficking, and a pronounced delay in trafficking out of late endosomes, which become aberrantly elongated into tubules. This is accompanied by a delay in EGFR degradation. The LRRK2-mediated deficits in EGFR trafficking and degradation can be reverted upon coexpression of active Rab7 and of a series of proteins involved in bridging the EGFR to Rab7 on late endosomes. Effector pulldown assays indicate that pathogenic LRRK2 decreases Rab7 activity both in cells overexpressing LRRK2, as well as in fibroblasts from pathogenic mutant LRRK2 PD patients when compared with healthy controls. Together, these findings provide novel insights into a previously unknown regulation of Rab7 activity by mutant LRRK2 which impairs membrane trafficking at very late stages of the endocytic pathway. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  3. Disease-Causing SDHAF1 Mutations Impair Transfer of Fe-S Clusters to SDHB.

    PubMed

    Maio, Nunziata; Ghezzi, Daniele; Verrigni, Daniela; Rizza, Teresa; Bertini, Enrico; Martinelli, Diego; Zeviani, Massimo; Singh, Anamika; Carrozzo, Rosalba; Rouault, Tracey A

    2016-02-09

    SDHAF1 mutations cause a rare mitochondrial complex II (CII) deficiency, which manifests as infantile leukoencephalopathy with elevated levels of serum and white matter succinate and lactate. Here, we demonstrate that SDHAF1 contributes to iron-sulfur (Fe-S) cluster incorporation into the Fe-S subunit of CII, SDHB. SDHAF1 transiently binds to aromatic peptides of SDHB through an arginine-rich region in its C terminus and specifically engages a Fe-S donor complex, consisting of the scaffold, holo-ISCU, and the co-chaperone-chaperone pair, HSC20-HSPA9, through an LYR motif near its N-terminal domain. Pathogenic mutations of SDHAF1 abrogate binding to SDHB, which impairs biogenesis of holo-SDHB and results in LONP1-mediated degradation of SDHB. Riboflavin treatment was found to ameliorate the neurologic condition of patients. We demonstrate that riboflavin enhances flavinylation of SDHA and reduces levels of succinate and Hypoxia-Inducible Factor (HIF)-1α and -2α, explaining the favorable response of patients to riboflavin. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Mutations in LTBP4 Cause a Syndrome of Impaired Pulmonary, Gastrointestinal, Genitourinary, Musculoskeletal, and Dermal Development

    PubMed Central

    Urban, Zsolt; Hucthagowder, Vishwanathan; Schürmann, Nura; Todorovic, Vesna; Zilberberg, Lior; Choi, Jiwon; Sens, Carla; Brown, Chester W.; Clark, Robin D.; Holland, Kristen E.; Marble, Michael; Sakai, Lynn Y.; Dabovic, Branka; Rifkin, Daniel B.; Davis, Elaine C.

    2009-01-01

    We report recessive mutations in the gene for the latent transforming growth factor-β binding protein 4 (LTBP4) in four unrelated patients with a human syndrome disrupting pulmonary, gastrointestinal, urinary, musculoskeletal, craniofacial, and dermal development. All patients had severe respiratory distress, with cystic and atelectatic changes in the lungs complicated by tracheomalacia and diaphragmatic hernia. Three of the four patients died of respiratory failure. Cardiovascular lesions were mild, limited to pulmonary artery stenosis and patent foramen ovale. Gastrointestinal malformations included diverticulosis, enlargement, tortuosity, and stenosis at various levels of the intestinal tract. The urinary tract was affected by diverticulosis and hydronephrosis. Joint laxity and low muscle tone contributed to musculoskeletal problems compounded by postnatal growth delay. Craniofacial features included microretrognathia, flat midface, receding forehead, and wide fontanelles. All patients had cutis laxa. Four of the five identified LTBP4 mutations led to premature termination of translation and destabilization of the LTBP4 mRNA. Impaired synthesis and lack of deposition of LTBP4 into the extracellular matrix (ECM) caused increased transforming growth factor-β (TGF-β) activity in cultured fibroblasts and defective elastic fiber assembly in all tissues affected by the disease. These molecular defects were associated with blocked alveolarization and airway collapse in the lung. Our results show that coupling of TGF-β signaling and ECM assembly is essential for proper development and is achieved in multiple human organ systems by multifunctional proteins such as LTBP4. PMID:19836010

  5. Identification of a point mutation impairing the binding between aquaporin-4 and neuromyelitis optica autoantibodies.

    PubMed

    Pisani, Francesco; Mola, Maria Grazia; Simone, Laura; Rosito, Stefania; Alberga, Domenico; Mangiatordi, Giuseppe Felice; Lattanzi, Gianluca; Nicolotti, Orazio; Frigeri, Antonio; Svelto, Maria; Nicchia, Grazia Paola

    2014-10-31

    Neuromyelitis optica (NMO) is characterized by the presence of pathogenic autoantibodies (NMO-IgGs) against supra-molecular assemblies of aquaporin-4 (AQP4), known as orthogonal array of particles (OAPs). NMO-IgGs have a polyclonal origin and recognize different conformational epitopes involving extracellular AQP4 loops A, C, and E. Here we hypothesize a pivotal role for AQP4 transmembrane regions (TMs) in epitope assembly. On the basis of multialignment analysis, mutagenesis, NMO-IgG binding, and cytotoxicity assay, we have disclosed the key role of aspartate 69 (Asp(69)) of TM2 for NMO-IgG epitope assembly. Mutation of Asp(69) to histidine severely impairs NMO-IgG binding for 85.7% of the NMO patient sera analyzed here. Although Blue Native-PAGE, total internal reflection fluorescence microscopy, and water transport assays indicate that the OAP Asp(69) mutant is similar in structure and function to the wild type, molecular dynamic simulations have revealed that the D(69)H mutation has the effect of altering the structural rearrangements of extracellular loop A. In conclusion, Asp(69) is crucial for the spatial control of loop A, the particular molecular conformation of which enables the assembly of NMO-IgG epitopes. These findings provide additional clues for new strategies for NMO treatment and a wealth of information to better approach NMO pathogenesis.

  6. Identification of a Point Mutation Impairing the Binding between Aquaporin-4 and Neuromyelitis Optica Autoantibodies*

    PubMed Central

    Pisani, Francesco; Mola, Maria Grazia; Simone, Laura; Rosito, Stefania; Alberga, Domenico; Mangiatordi, Giuseppe Felice; Lattanzi, Gianluca; Nicolotti, Orazio; Frigeri, Antonio; Svelto, Maria; Nicchia, Grazia Paola

    2014-01-01

    Neuromyelitis optica (NMO) is characterized by the presence of pathogenic autoantibodies (NMO-IgGs) against supra-molecular assemblies of aquaporin-4 (AQP4), known as orthogonal array of particles (OAPs). NMO-IgGs have a polyclonal origin and recognize different conformational epitopes involving extracellular AQP4 loops A, C, and E. Here we hypothesize a pivotal role for AQP4 transmembrane regions (TMs) in epitope assembly. On the basis of multialignment analysis, mutagenesis, NMO-IgG binding, and cytotoxicity assay, we have disclosed the key role of aspartate 69 (Asp69) of TM2 for NMO-IgG epitope assembly. Mutation of Asp69 to histidine severely impairs NMO-IgG binding for 85.7% of the NMO patient sera analyzed here. Although Blue Native-PAGE, total internal reflection fluorescence microscopy, and water transport assays indicate that the OAP Asp69 mutant is similar in structure and function to the wild type, molecular dynamic simulations have revealed that the D69H mutation has the effect of altering the structural rearrangements of extracellular loop A. In conclusion, Asp69 is crucial for the spatial control of loop A, the particular molecular conformation of which enables the assembly of NMO-IgG epitopes. These findings provide additional clues for new strategies for NMO treatment and a wealth of information to better approach NMO pathogenesis. PMID:25239624

  7. Spontaneous mutation 7B-1 in tomato impairs blue light-induced stomatal opening.

    PubMed

    Hlavinka, Jan; Nauš, Jan; Fellner, Martin

    2013-08-01

    It was reported earlier that 7B-1 mutant in tomato (Solanum lycopersicum L.), an ABA overproducer, is defective in blue light (BL) signaling leading to BL-specific resistance to abiotic and biotic stresses. In this work, we examine responses of stomata to blue, red and white lights, fusicoccin, anion channel blockers (anthracene-9-carboxylic acid; 9-AC and niflumic acid; NIF) and ABA. Our results showed that the aperture of 7B-1 stomata does not increase in BL, suggesting that 7B-1 mutation impairs an element of BL signaling pathway involved in stomatal opening. Similar stomatal responses of 7B-1 and wild type (WT) to fusicoccin or 9-AC points out that activity of H(+)-ATPase and 9-AC-sensitive anion channels per se is not likely affected by the mutation. Since 9-AC restored stomatal opening of 7B-1 in BL, it seems that 9-AC and BL could block similar type of anion channels. The stomata of both genotypes did not respond to NIF neither in darkness nor in any light conditions tested. In light, 9-AC but not NIF restored stomatal opening inhibited by ABA in WT and 7B-1. We suggest that in comparison to WT, the activity of S-type anion channels in 7B-1 is more promoted by increased ABA content, and less reduced by BL, because of the mutant resistance to BL. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  8. Regulation of polycystin-1 ciliary trafficking by motifs at its C-terminus and polycystin-2 but not by cleavage at the GPS site.

    PubMed

    Su, Xuefeng; Wu, Maoqing; Yao, Gang; El-Jouni, Wassim; Luo, Chong; Tabari, Azadeh; Zhou, Jing

    2015-11-15

    Failure to localize membrane proteins to the primary cilium causes a group of diseases collectively named ciliopathies. Polycystin-1 (PC1, also known as PKD1) is a large ciliary membrane protein defective in autosomal dominant polycystic kidney disease (ADPKD). Here, we developed a large set of PC1 expression constructs and identified multiple sequences, including a coiled-coil motif in the C-terminal tail of PC1, regulating full-length PC1 trafficking to the primary cilium. Ciliary trafficking of wild-type and mutant PC1 depends on the dose of polycystin-2 (PC2, also known as PKD2), and the formation of a PC1-PC2 complex. Modulation of the ciliary trafficking module mediated by the VxP ciliary-targeting sequence and Arf4 and Asap1 does not affect the ciliary localization of full-length PC1. PC1 also promotes PC2 ciliary trafficking. PC2 mutations truncating its C-terminal tail but not those changing the VxP sequence to AxA or impairing the pore of the channel, leading to a dead channel, affect PC1 ciliary trafficking. Cleavage at the GPCR proteolytic site (GPS) of PC1 is not required for PC1 trafficking to cilia. We propose a mutually dependent model for the ciliary trafficking of PC1 and PC2, and that PC1 ciliary trafficking is regulated by multiple cis-acting elements. As all pathogenic PC1 mutations tested here are defective in ciliary trafficking, ciliary trafficking might serve as a functional read-out for ADPKD.

  9. Regulation of polycystin-1 ciliary trafficking by motifs at its C-terminus and polycystin-2 but not by cleavage at the GPS site

    PubMed Central

    Su, Xuefeng; Wu, Maoqing; Yao, Gang; El-Jouni, Wassim; Luo, Chong; Tabari, Azadeh; Zhou, Jing

    2015-01-01

    ABSTRACT Failure to localize membrane proteins to the primary cilium causes a group of diseases collectively named ciliopathies. Polycystin-1 (PC1, also known as PKD1) is a large ciliary membrane protein defective in autosomal dominant polycystic kidney disease (ADPKD). Here, we developed a large set of PC1 expression constructs and identified multiple sequences, including a coiled-coil motif in the C-terminal tail of PC1, regulating full-length PC1 trafficking to the primary cilium. Ciliary trafficking of wild-type and mutant PC1 depends on the dose of polycystin-2 (PC2, also known as PKD2), and the formation of a PC1–PC2 complex. Modulation of the ciliary trafficking module mediated by the VxP ciliary-targeting sequence and Arf4 and Asap1 does not affect the ciliary localization of full-length PC1. PC1 also promotes PC2 ciliary trafficking. PC2 mutations truncating its C-terminal tail but not those changing the VxP sequence to AxA or impairing the pore of the channel, leading to a dead channel, affect PC1 ciliary trafficking. Cleavage at the GPCR proteolytic site (GPS) of PC1 is not required for PC1 trafficking to cilia. We propose a mutually dependent model for the ciliary trafficking of PC1 and PC2, and that PC1 ciliary trafficking is regulated by multiple cis-acting elements. As all pathogenic PC1 mutations tested here are defective in ciliary trafficking, ciliary trafficking might serve as a functional read-out for ADPKD. PMID:26430213

  10. Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis through distinct mechanisms in Wilms tumors

    PubMed Central

    Rakheja, Dinesh; Chen, Kenneth S.; Liu, Yangjian; Shukla, Abhay A.; Schmid, Vanessa; Chang, Tsung-Cheng; Khokhar, Shama; Wickiser, Jonathan E.; Karandikar, Nitin J.; Malter, James S.; Mendell, Joshua T.; Amatruda, James F.

    2014-01-01

    Wilms tumor is the most common childhood kidney cancer. Here we report the whole-exome sequencing of 44 Wilms tumors, identifying missense mutations in the microRNA (miRNA)-processing enzymes DROSHA and DICER1 and novel mutations in MYCN, SMARCA4 and ARID1A. Examination of tumor miRNA expression, in vitro processing assays, and genomic editing in human cells demonstrate that DICER1 and DROSHA mutations influence miRNA processing through distinct mechanisms. DICER1 RNase IIIB mutations preferentially impair processing of miRNAs deriving from the 5′ arm of pre-miRNA hairpins, while DROSHA RNase IIIB mutations globally inhibit miRNA biogenesis through a dominant-negative mechanism. Both DROSHA and DICER1 mutations impair expression of tumor-suppressing miRNAs including the let-7 family, important regulators of MYCN, LIN28 and other Wilms tumor oncogenes. These results provide new insights into the mechanisms through which mutations in miRNA biogenesis components reprogram miRNA expression in human cancer and suggest that these defects define a distinct subclass of Wilms tumors. PMID:25190313

  11. ECEL1 mutation implicates impaired axonal arborization of motor nerves in the pathogenesis of distal arthrogryposis.

    PubMed

    Nagata, Kenichi; Kiryu-Seo, Sumiko; Tamada, Hiromi; Okuyama-Uchimura, Fumi; Kiyama, Hiroshi; Saido, Takaomi C

    2016-07-01

    The membrane-bound metalloprotease endothelin-converting enzyme-like 1 (ECEL1) has been newly identified as a causal gene of a specific type of distal arthrogryposis (DA). In contrast to most causal genes of DA, ECEL1 is predominantly expressed in neuronal cells, suggesting a unique neurogenic pathogenesis in a subset of DA patients with ECEL1 mutation. The present study analyzed developmental motor innervation and neuromuscular junction formation in limbs of the rodent homologue damage-induced neuronal endopeptidase (DINE)-deficient mouse. Whole-mount immunostaining was performed in DINE-deficient limbs expressing motoneuron-specific GFP to visualize motor innervation throughout the limb. Although DINE-deficient motor nerves displayed normal trajectory patterns from the spinal cord to skeletal muscles, they indicated impaired axonal arborization in skeletal muscles in the forelimbs and hindlimbs. Systematic examination of motor innervation in over 10 different hindlimb muscles provided evidence that DINE gene disruption leads to insufficient arborization of motor nerves after arriving at the skeletal muscle. Interestingly, the axonal arborization defect in foot muscles appeared more severe than in other hindlimb muscles, which was partially consistent with the proximal-distal phenotypic discordance observed in DA patients. Additionally, the number of innervated neuromuscular junction was significantly reduced in the severely affected DINE-deficient muscle. Furthermore, we generated a DINE knock-in (KI) mouse model with a pathogenic mutation, which was recently identified in DA patients. Axonal arborization defects were clearly detected in motor nerves of the DINE KI limb, which was identical to the DINE-deficient limb. Given that the encoded sequences, as well as ECEL1 and DINE expression profiles, are highly conserved between mouse and human, abnormal arborization of motor axons and subsequent failure of NMJ formation could be a primary cause of DA with ECEL1

  12. Gain-of-Function Mutations in RARB Cause Intellectual Disability with Progressive Motor Impairment.

    PubMed

    Srour, Myriam; Caron, Véronique; Pearson, Toni; Nielsen, Sarah B; Lévesque, Sébastien; Delrue, Marie-Ange; Becker, Troy A; Hamdan, Fadi F; Kibar, Zoha; Sattler, Shannon G; Schneider, Michael C; Bitoun, Pierre; Chassaing, Nicolas; Rosenfeld, Jill A; Xia, Fan; Desai, Sonal; Roeder, Elizabeth; Kimonis, Virginia; Schneider, Adele; Littlejohn, Rebecca Okashah; Douzgou, Sofia; Tremblay, André; Michaud, Jacques L

    2016-08-01

    Retinoic acid (RA) signaling plays a key role in the development and function of several systems in mammals. We previously discovered that the de novo mutations c.1159C>T (p.Arg387Cys) and c.1159C>A (p.Arg387Ser) in the RA Receptor Beta (RARB) gene cause microphthalmia and diaphragmatic hernia. However, the natural history of affected subjects beyond the prenatal or neonatal period was unknown. Here, we describe nine additional subjects with microphthalmia who have de novo mutations in RARB, including the previously described p.Arg387Cys as well as the novel c.887G>C (p.Gly296Ala) and c.638T>C (p.Leu213Pro). Moreover, we review the information on four previously reported cases. All subjects who survived the neonatal period (n = 10) displayed severe global developmental delay with progressive motor impairment due to spasticity and/or dystonia (with or without chorea). The majority of subjects also showed Chiari type I malformation and severe feeding difficulties. We previously found that p.Arg387Cys and p.Arg387Ser induce a gain-of-function. We show here that the p.Gly296Ala and p.Leu213Pro RARB mutations further promote the RA ligand-induced transcriptional activity by twofold to threefold over the wild-type receptor, also indicating a gain-of-function mechanism. These observations suggest that precise regulation of RA signaling is required for brain development and/or function in humans. © 2016 WILEY PERIODICALS, INC.

  13. FOCAL EXPRESSION OF MUTATED TAU IN ENTORHINAL CORTEX NEURONS OF RATS IMPAIRS SPATIAL WORKING MEMORY

    PubMed Central

    Ramirez, Julio J.; Poulton, Winona E.; Knelson, Erik; Barton, Cole; King, Michael A.; Klein, Ronald L.

    2010-01-01

    Entorhinal cortex neuropathology begins very early in Alzheimer's disease (AD), a disorder characterized by severe memory disruption. Indeed, loss of entorhinal volume is predictive of AD and two of the hallmark neuroanatomical markers of AD, amyloid plaques and neurofibrillary tangles (NFTs), are particularly prevalent in the entorhinal area of AD-afflicted brains. Gene transfer techniques were used to create a model neurofibrillary tauopathy by injecting a recombinant adeno-associated viral vector with a mutated human tau gene (P301L) into the entorhinal cortex of adult rats. The objective of the present investigation was to determine whether adult onset, spatially restricted tauopathy could be sufficient to reproduce progressive deficits in mnemonic function. Spatial memory on a Y-maze was tested for approximately three months post-surgery. Upon completion of behavioral testing the brains were assessed for expression of human tau and evidence of tauopathy. Rats injected with the tau vector became persistently impaired on the task after about six weeks of postoperative testing, whereas the control rats injected with a green fluorescent protein vector performed at criterion levels during that period. Histological analysis confirmed the presence of hyperphosphorylated tau and NFTs in the entorhinal cortex and neighboring retrohippocampal areas as well as limited synaptic degeneration of the perforant path. Thus, highly restricted vector-induced tauopathy in retrohippocampal areas is sufficient for producing progressive impairment in mnemonic ability in rats, successfully mimicking a key aspect of tauopathies such as AD. PMID:20727915

  14. Effects of naturally occurring G103D point mutation of AQP5 on its water permeability, trafficking and cellular localization in the submandibular gland of rats.

    PubMed

    Karabasil, Mileva Ratko; Hasegawa, Takahiro; Azlina, Ahmad; Purwanti, Nunuk; Yao, Chenjuan; Akamatsu, Tetsuya; Tomioka, Shigemasa; Hosoi, Kazuo

    2011-02-01

    AQPs (aquaporins) are water channel proteins that are expressed in almost all living things. In mammalians, 13 members of AQPs (AQP0-12) have been identified so far. AQP5 is known to be expressed mostly in the exocrine cells, including the salivary gland acinar cells. A naturally occurring point mutation (G308A, Gly103 > Asp103) was earlier found in the rat AQP5 gene [Murdiastuti, Purwanti, Karabasil, Li, Yao, Akamatsu, Kanamori and Hosoi (2006) Am. J. Physiol. 291, G1081-G1088]; in this mutant, the rate of initial saliva secretion under stimulated and unstimulated conditions is less than that for the wt (wild-type) animals. Here the mutant molecule was characterized in detail. Using the Xenopus oocyte system, we demonstrated the mutant AQP5 to have water permeability almost the same as that of the wt molecule. Mutant and wt AQP5s, tagged with GFP (green fluorescent protein; GFP-AQP5s) and expressed in polarized MDCK-II (Madin-Darby canine kidney II) cells, first appeared in the vesicular structure(s) in the cytoplasm, and were translocated to the upper plasma membrane or apical membrane during cultivation, with the mutant GFP-AQP5 being translocated less efficiently. Thapsigargin and H-89 both induced translocation in vitro of either molecule, whereas colchicine inhibited this activity; the fraction of cells showing apical localization of mutant GFP-AQP5 was less than that showing that of the wt molecule under any of the experimental conditions used. In the mutant SMG (submandibular gland) tissue, localization of AQP5 in the apical membrane of acinar cells was extremely reduced. Vesicular structures positive for AQP5 and present in the cytoplasm of the acinar cells were co-localized with LAMP2 (lysosome-associated membrane protein 2) or cathepsin D in the mutant gland, whereas such co-localizations were very rare in the wt gland, suggesting that the mutant molecules largely entered lysosomes for degradation. Replacement of highly conserved hydrophobic Gly103 with

  15. A novel biallelic splice site mutation of TECTA causes moderate to severe hearing impairment in an Algerian family.

    PubMed

    Behlouli, Asma; Bonnet, Crystel; Abdi, Samia; Hasbellaoui, Mokhtar; Boudjenah, Farid; Hardelin, Jean-Pierre; Louha, Malek; Makrelouf, Mohamed; Ammar-Khodja, Fatima; Zenati, Akila; Petit, Christine

    2016-08-01

    Congenital deafness is certainly one of the most common monogenic diseases in humans, but it is also one of the most genetically heterogeneous, which makes molecular diagnosis challenging in most cases. Whole-exome sequencing in two out of three Algerian siblings affected by recessively-inherited, moderate to severe sensorineural deafness allowed us to identify a novel splice donor site mutation (c.5272+1G > A) in the gene encoding α-tectorin, a major component of the cochlear tectorial membrane. The mutation was present at the homozygous state in the three affected siblings, and at the heterozygous state in their unaffected, consanguineous parents. To our knowledge, this is the first reported TECTA mutation leading to the DFNB21 form of hearing impairment among Maghrebian individuals suffering from congenital hearing impairment, which further illustrates the diversity of the genes involved in congenital deafness in the Maghreb.

  16. Mutations Affecting the SAND Domain of DEAF1 Cause Intellectual Disability with Severe Speech Impairment and Behavioral Problems

    PubMed Central

    Vulto-van Silfhout, Anneke T.; Rajamanickam, Shivakumar; Jensik, Philip J.; Vergult, Sarah; de Rocker, Nina; Newhall, Kathryn J.; Raghavan, Ramya; Reardon, Sara N.; Jarrett, Kelsey; McIntyre, Tara; Bulinski, Joseph; Ownby, Stacy L.; Huggenvik, Jodi I.; McKnight, G. Stanley; Rose, Gregory M.; Cai, Xiang; Willaert, Andy; Zweier, Christiane; Endele, Sabine; de Ligt, Joep; van Bon, Bregje W.M.; Lugtenberg, Dorien; de Vries, Petra F.; Veltman, Joris A.; van Bokhoven, Hans; Brunner, Han G.; Rauch, Anita; de Brouwer, Arjan P.M.; Carvill, Gemma L.; Hoischen, Alexander; Mefford, Heather C.; Eichler, Evan E.; Vissers, Lisenka E.L.M.; Menten, Björn; Collard, Michael W.; de Vries, Bert B.A.

    2014-01-01

    Recently, we identified in two individuals with intellectual disability (ID) different de novo mutations in DEAF1, which encodes a transcription factor with an important role in embryonic development. To ascertain whether these mutations in DEAF1 are causative for the ID phenotype, we performed targeted resequencing of DEAF1 in an additional cohort of over 2,300 individuals with unexplained ID and identified two additional individuals with de novo mutations in this gene. All four individuals had severe ID with severely affected speech development, and three showed severe behavioral problems. DEAF1 is highly expressed in the CNS, especially during early embryonic development. All four mutations were missense mutations affecting the SAND domain of DEAF1. Altered DEAF1 harboring any of the four amino acid changes showed impaired transcriptional regulation of the DEAF1 promoter. Moreover, behavioral studies in mice with a conditional knockout of Deaf1 in the brain showed memory deficits and increased anxiety-like behavior. Our results demonstrate that mutations in DEAF1 cause ID and behavioral problems, most likely as a result of impaired transcriptional regulation by DEAF1. PMID:24726472

  17. Mutations affecting the SAND domain of DEAF1 cause intellectual disability with severe speech impairment and behavioral problems.

    PubMed

    Vulto-van Silfhout, Anneke T; Rajamanickam, Shivakumar; Jensik, Philip J; Vergult, Sarah; de Rocker, Nina; Newhall, Kathryn J; Raghavan, Ramya; Reardon, Sara N; Jarrett, Kelsey; McIntyre, Tara; Bulinski, Joseph; Ownby, Stacy L; Huggenvik, Jodi I; McKnight, G Stanley; Rose, Gregory M; Cai, Xiang; Willaert, Andy; Zweier, Christiane; Endele, Sabine; de Ligt, Joep; van Bon, Bregje W M; Lugtenberg, Dorien; de Vries, Petra F; Veltman, Joris A; van Bokhoven, Hans; Brunner, Han G; Rauch, Anita; de Brouwer, Arjan P M; Carvill, Gemma L; Hoischen, Alexander; Mefford, Heather C; Eichler, Evan E; Vissers, Lisenka E L M; Menten, Björn; Collard, Michael W; de Vries, Bert B A

    2014-05-01

    Recently, we identified in two individuals with intellectual disability (ID) different de novo mutations in DEAF1, which encodes a transcription factor with an important role in embryonic development. To ascertain whether these mutations in DEAF1 are causative for the ID phenotype, we performed targeted resequencing of DEAF1 in an additional cohort of over 2,300 individuals with unexplained ID and identified two additional individuals with de novo mutations in this gene. All four individuals had severe ID with severely affected speech development, and three showed severe behavioral problems. DEAF1 is highly expressed in the CNS, especially during early embryonic development. All four mutations were missense mutations affecting the SAND domain of DEAF1. Altered DEAF1 harboring any of the four amino acid changes showed impaired transcriptional regulation of the DEAF1 promoter. Moreover, behavioral studies in mice with a conditional knockout of Deaf1 in the brain showed memory deficits and increased anxiety-like behavior. Our results demonstrate that mutations in DEAF1 cause ID and behavioral problems, most likely as a result of impaired transcriptional regulation by DEAF1.

  18. A novel mutation in the mitochondrial tRNA(Ser(UCN)) gene in a family with non-syndromic sensorineural hearing impairment.

    PubMed

    Hutchin, T P; Parker, M J; Young, I D; Davis, A C; Pulleyn, L J; Deeble, J; Lench, N J; Markham, A F; Mueller, R F

    2000-09-01

    We describe a family with non-syndromic sensorineural hearing impairment inherited in a manner consistent with maternal transmission. Affected members were found to have a novel heteroplasmic mtDNA mutation, T7510C, in the tRNA(Ser(UCN)) gene. This mutation was not found in 661 controls, is well conserved between species, and disrupts base pairing in the acceptor stem of the tRNA, making it the probable cause of hearing impairment in this family. Sequencing of the other mitochondrial tRNA genes did not show any other pathogenic mutations. Four other mutations causing hearing impairment have been reported in the tRNA(Ser(UCN)) gene, two having been shown to affect tRNA(Ser(UCN)) levels. With increasing numbers of reports of mtDNA mutations causing hearing impairment, screening for such mutations should be considered in all cases unless mitochondrial inheritance can be excluded for certain.

  19. Secretion-Positive LGI1 Mutations Linked to Lateral Temporal Epilepsy Impair Binding to ADAM22 and ADAM23 Receptors

    PubMed Central

    Dazzo, Emanuela; Belluzzi, Elisa; Malacrida, Sandro; Vitiello, Libero; Greggio, Elisa; Tosatto, Silvio C. E.

    2016-01-01

    Autosomal dominant lateral temporal epilepsy (ADTLE) is a focal epilepsy syndrome caused by mutations in the LGI1 gene, which encodes a secreted protein. Most ADLTE-causing mutations inhibit LGI1 protein secretion, and only a few secretion-positive missense mutations have been reported. Here we describe the effects of four disease-causing nonsynonymous LGI1 mutations, T380A, R407C, S473L, and R474Q, on protein secretion and extracellular interactions. Expression of LGI1 mutant proteins in cultured cells shows that these mutations do not inhibit protein secretion. This finding likely results from the lack of effects of these mutations on LGI1 protein folding, as suggested by 3D protein modelling. In addition, immunofluorescence and co-immunoprecipitation experiments reveal that all four mutations significantly impair interaction of LGI1 with the ADAM22 and ADAM23 receptors on the cell surface. These results support the existence of a second mechanism, alternative to inhibition of protein secretion, by which ADLTE-causing LGI1 mutations exert their loss-of-function effect extracellularly, and suggest that interactions of LGI1 with both ADAM22 and ADAM23 play an important role in the molecular mechanisms leading to ADLTE. PMID:27760137

  20. RUNX2 Mutation Impairs 1α,25-Dihydroxyvitamin D3 mediated Osteoclastogenesis in Dental Follicle Cells

    PubMed Central

    Wang, X. Z.; Sun, X. Y.; Zhang, C. Y.; Yang, X.; Yan, W. J.; Ge, L. H.; Zheng, S. G.

    2016-01-01

    Cleidocranial dysplasia (CCD), a skeletal disorder characterized by delayed permanent tooth eruption and other dental abnormalities, is caused by heterozygous RUNX2 mutations. As an osteoblast-specific transcription factor, RUNX2 plays a role in bone remodeling, tooth formation and tooth eruption. To investigate the crosstalk between RUNX2 and 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) in human dental follicle cells (hDFCs) during osteoclast formation, we established a co-culture system of hDFCs from CCD patient and healthy donors with peripheral blood mononuclear cells (PBMCs). Expression of the osteoclast-associated genes and the number of TRAP+ cells were reduced in CCD hDFCs, indicating its suppressed osteoclast-inductive ability, which was reflected by the downregulated RANKL/OPG ratio. In addition, 1α,25-(OH)2D3-stimulation elevated the expression of osteoclast-related genes, as well as RANKL mRNA levels and RANKL/OPG ratios in control hDFCs. Conversely, RUNX2 mutation abolished this 1α,25-(OH)2D3-induced RANKL gene activation and osteoclast formation in CCD hDFCs. Therefore, RUNX2 haploinsufficiency impairs dental follicle-induced osteoclast formation capacity through RANKL/OPG signaling, which may be partially responsible for delayed permanent tooth eruption in CCD patients. Furthermore, this abnormality was not rescued by 1α,25-(OH)2D3 application because 1α,25-(OH)2D3-induced RANKL activation in hDFCs is mediated principally via the RUNX2-dependent pathway. PMID:27068678

  1. Association between idiopathic hearing loss and mitochondrial DNA mutations: A study on 169 hearing-impaired subjects

    PubMed Central

    GUARAN, VALERIA; ASTOLFI, LAURA; CASTIGLIONE, ALESSANDRO; SIMONI, EDI; OLIVETTO, ELENA; GALASSO, MARCO; TREVISI, PATRIZIA; BUSI, MICOL; VOLINIA, STEFANO; MARTINI, ALESSANDRO

    2013-01-01

    Mutations in mitochondrial DNA (mtDNA) have been shown to be an important cause of sensorineural hearing loss (SNHL). In this study, we performed a clinical and genetic analysis of 169 hearing-impaired patients and some of their relatives suffering from idiopathic SNHL, both familial and sporadic. The analysis of four fragments of their mtDNA identified several polymorphisms, the well known pathogenic mutation, A1555G, and some novel mutations in different genes, implying changes in the aminoacidic sequence. A novel sporadic mutation in 12S rRNA (MT-RNR1), not previously reported in the literature, was found in a case of possible aminoglycoside-induced progressive deafness. PMID:23969527

  2. Economics of human trafficking.

    PubMed

    Wheaton, Elizabeth M; Schauer, Edward J; Galli, Thomas V

    2010-01-01

    Because freedom of choice and economic gain are at the heart of productivity, human trafficking impedes national and international economic growth. Within the next 10 years, crime experts expect human trafficking to surpass drug and arms trafficking in its incidence, cost to human well-being, and profitability to criminals (Schauer and Wheaton, 2006: 164-165). The loss of agency from human trafficking as well as from modern slavery is the result of human vulnerability (Bales, 2000: 15). As people become vulnerable to exploitation and businesses continually seek the lowest-cost labour sources, trafficking human beings generates profit and a market for human trafficking is created. This paper presents an economic model of human trafficking that encompasses all known economic factors that affect human trafficking both across and within national borders. We envision human trafficking as a monopolistically competitive industry in which traffickers act as intermediaries between vulnerable individuals and employers by supplying differentiated products to employers. In the human trafficking market, the consumers are employers of trafficked labour and the products are human beings. Using a rational-choice framework of human trafficking we explain the social situations that shape relocation and working decisions of vulnerable populations leading to human trafficking, the impetus for being a trafficker, and the decisions by employers of trafficked individuals. The goal of this paper is to provide a common ground upon which policymakers and researchers can collaborate to decrease the incidence of trafficking in humans.

  3. A nonsynonymous mutation in the WFS1 gene in a Finnish family with age-related hearing impairment.

    PubMed

    Kytövuori, Laura; Hannula, Samuli; Mäki-Torkko, Elina; Sorri, Martti; Majamaa, Kari

    2017-09-28

    Wolfram syndrome (WS) is caused by recessive mutations in the Wolfram syndrome 1 (WFS1) gene. Sensorineural hearing impairment (HI) is a frequent feature in WS and, furthermore, certain mutations in WFS1 cause nonsyndromic dominantly inherited low-frequency sensorineural HI. These two phenotypes are clinically distinct indicating that WFS1 is a reasonable candidate for genetic studies in patients with other phenotypes of HI. Here we have investigated, whether the variation in WFS1 has a pathogenic role in age-related hearing impairment (ARHI). WFS1 gene was investigated in a population sample of 518 Finnish adults born in 1938-1949 and representing variable hearing phenotypes. Identified variants were evaluated with respect to pathogenic potential. A rare mutation predicted to be pathogenic was found in a family with many members with impaired hearing. Twenty members were recruited to a segregation study and a detailed clinical examination. Heterozygous p.Tyr528His variant segregated completely with late-onset HI in which hearing deteriorated first at high frequencies and progressed to mid and low frequencies later in life. We report the first mutation in the WFS1 gene causing late-onset HI with audiogram configurations typical for ARHI. Monogenic forms of ARHI are rare and our results add WFS1 to the short list of such genes. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Meta-analysis of SHANK Mutations in Autism Spectrum Disorders: a gradient of severity in cognitive impairments.

    PubMed

    Leblond, Claire S; Nava, Caroline; Polge, Anne; Gauthier, Julie; Huguet, Guillaume; Lumbroso, Serge; Giuliano, Fabienne; Stordeur, Coline; Depienne, Christel; Mouzat, Kevin; Pinto, Dalila; Howe, Jennifer; Lemière, Nathalie; Durand, Christelle M; Guibert, Jessica; Ey, Elodie; Toro, Roberto; Peyre, Hugo; Mathieu, Alexandre; Amsellem, Frédérique; Rastam, Maria; Gillberg, I Carina; Rappold, Gudrun A; Holt, Richard; Monaco, Anthony P; Maestrini, Elena; Galan, Pilar; Heron, Delphine; Jacquette, Aurélia; Afenjar, Alexandra; Rastetter, Agnès; Brice, Alexis; Devillard, Françoise; Assouline, Brigitte; Laffargue, Fanny; Lespinasse, James; Chiesa, Jean; Rivier, François; Bonneau, Dominique; Regnault, Beatrice; Zelenika, Diana; Delepine, Marc; Lathrop, Mark; Sanlaville, Damien; Schluth-Bolard, Caroline; Edery, Patrick; Perrin, Laurence; Tabet, Anne Claude; Schmeisser, Michael J; Boeckers, Tobias M; Coleman, Mary; Sato, Daisuke; Szatmari, Peter; Scherer, Stephen W; Rouleau, Guy A; Betancur, Catalina; Leboyer, Marion; Gillberg, Christopher; Delorme, Richard; Bourgeron, Thomas

    2014-09-01

    SHANK genes code for scaffold proteins located at the post-synaptic density of glutamatergic synapses. In neurons, SHANK2 and SHANK3 have a positive effect on the induction and maturation of dendritic spines, whereas SHANK1 induces the enlargement of spine heads. Mutations in SHANK genes have been associated with autism spectrum disorders (ASD), but their prevalence and clinical relevance remain to be determined. Here, we performed a new screen and a meta-analysis of SHANK copy-number and coding-sequence variants in ASD. Copy-number variants were analyzed in 5,657 patients and 19,163 controls, coding-sequence variants were ascertained in 760 to 2,147 patients and 492 to 1,090 controls (depending on the gene), and, individuals carrying de novo or truncating SHANK mutations underwent an extensive clinical investigation. Copy-number variants and truncating mutations in SHANK genes were present in ∼1% of patients with ASD: mutations in SHANK1 were rare (0.04%) and present in males with normal IQ and autism; mutations in SHANK2 were present in 0.17% of patients with ASD and mild intellectual disability; mutations in SHANK3 were present in 0.69% of patients with ASD and up to 2.12% of the cases with moderate to profound intellectual disability. In summary, mutations of the SHANK genes were detected in the whole spectrum of autism with a gradient of severity in cognitive impairment. Given the rare frequency of SHANK1 and SHANK2 deleterious mutations, the clinical relevance of these genes remains to be ascertained. In contrast, the frequency and the penetrance of SHANK3 mutations in individuals with ASD and intellectual disability-more than 1 in 50-warrant its consideration for mutation screening in clinical practice.

  5. Meta-analysis of SHANK Mutations in Autism Spectrum Disorders: A Gradient of Severity in Cognitive Impairments

    PubMed Central

    Leblond, Claire S.; Nava, Caroline; Polge, Anne; Gauthier, Julie; Huguet, Guillaume; Lumbroso, Serge; Giuliano, Fabienne; Stordeur, Coline; Depienne, Christel; Mouzat, Kevin; Pinto, Dalila; Howe, Jennifer; Lemière, Nathalie; Durand, Christelle M.; Guibert, Jessica; Ey, Elodie; Toro, Roberto; Peyre, Hugo; Mathieu, Alexandre; Amsellem, Frédérique; Rastam, Maria; Gillberg, I. Carina; Rappold, Gudrun A.; Holt, Richard; Monaco, Anthony P.; Maestrini, Elena; Galan, Pilar; Heron, Delphine; Jacquette, Aurélia; Afenjar, Alexandra; Rastetter, Agnès; Brice, Alexis; Devillard, Françoise; Assouline, Brigitte; Laffargue, Fanny; Lespinasse, James; Chiesa, Jean; Rivier, François; Bonneau, Dominique; Regnault, Beatrice; Zelenika, Diana; Delepine, Marc; Lathrop, Mark; Sanlaville, Damien; Schluth-Bolard, Caroline; Edery, Patrick; Perrin, Laurence; Tabet, Anne Claude; Schmeisser, Michael J.; Boeckers, Tobias M.; Coleman, Mary; Sato, Daisuke; Szatmari, Peter; Scherer, Stephen W.; Rouleau, Guy A.; Betancur, Catalina; Leboyer, Marion; Gillberg, Christopher

    2014-01-01

    SHANK genes code for scaffold proteins located at the post-synaptic density of glutamatergic synapses. In neurons, SHANK2 and SHANK3 have a positive effect on the induction and maturation of dendritic spines, whereas SHANK1 induces the enlargement of spine heads. Mutations in SHANK genes have been associated with autism spectrum disorders (ASD), but their prevalence and clinical relevance remain to be determined. Here, we performed a new screen and a meta-analysis of SHANK copy-number and coding-sequence variants in ASD. Copy-number variants were analyzed in 5,657 patients and 19,163 controls, coding-sequence variants were ascertained in 760 to 2,147 patients and 492 to 1,090 controls (depending on the gene), and, individuals carrying de novo or truncating SHANK mutations underwent an extensive clinical investigation. Copy-number variants and truncating mutations in SHANK genes were present in ∼1% of patients with ASD: mutations in SHANK1 were rare (0.04%) and present in males with normal IQ and autism; mutations in SHANK2 were present in 0.17% of patients with ASD and mild intellectual disability; mutations in SHANK3 were present in 0.69% of patients with ASD and up to 2.12% of the cases with moderate to profound intellectual disability. In summary, mutations of the SHANK genes were detected in the whole spectrum of autism with a gradient of severity in cognitive impairment. Given the rare frequency of SHANK1 and SHANK2 deleterious mutations, the clinical relevance of these genes remains to be ascertained. In contrast, the frequency and the penetrance of SHANK3 mutations in individuals with ASD and intellectual disability—more than 1 in 50—warrant its consideration for mutation screening in clinical practice. PMID:25188300

  6. E2F1 somatic mutation within miRNA target site impairs gene regulation in colorectal cancer.

    PubMed

    Lopes-Ramos, Camila M; Barros, Bruna P; Koyama, Fernanda C; Carpinetti, Paola A; Pezuk, Julia; Doimo, Nayara T S; Habr-Gama, Angelita; Perez, Rodrigo O; Parmigiani, Raphael B

    2017-01-01

    Genetic studies have largely concentrated on the impact of somatic mutations found in coding regions, and have neglected mutations outside of these. However, 3' untranslated regions (3' UTR) mutations can also disrupt or create miRNA target sites, and trigger oncogene activation or tumor suppressor inactivation. We used next-generation sequencing to widely screen for genetic alterations within predicted miRNA target sites of oncogenes associated with colorectal cancer, and evaluated the functional impact of a new somatic mutation. Target sequencing of 47 genes was performed for 29 primary colorectal tumor samples. For 71 independent samples, Sanger methodology was used to screen for E2F1 mutations in miRNA predicted target sites, and the functional impact of these mutations was evaluated by luciferase reporter assays. We identified germline and somatic alterations in E2F1. Of the 100 samples evaluated, 3 had germline alterations at the MIR205-5p target site, while one had a somatic mutation at MIR136-5p target site. E2F1 gene expression was similar between normal and tumor tissues bearing the germline alteration; however, expression was increased 4-fold in tumor tissue that harbored a somatic mutation compared to that in normal tissue. Luciferase reporter assays revealed both germline and somatic alterations increased E2F1 activity relative to wild-type E2F1. We demonstrated that somatic mutation within E2F1:MIR136-5p target site impairs miRNA-mediated regulation and leads to increased gene activity. We conclude that somatic mutations that disrupt miRNA target sites have the potential to impact gene regulation, highlighting an important mechanism of oncogene activation.

  7. IMMUNODEFICIENCIES. Impairment of immunity to Candida and Mycobacterium in humans with bi-allelic RORC mutations.

    PubMed

    Okada, Satoshi; Markle, Janet G; Deenick, Elissa K; Mele, Federico; Averbuch, Dina; Lagos, Macarena; Alzahrani, Mohammed; Al-Muhsen, Saleh; Halwani, Rabih; Ma, Cindy S; Wong, Natalie; Soudais, Claire; Henderson, Lauren A; Marzouqa, Hiyam; Shamma, Jamal; Gonzalez, Marcela; Martinez-Barricarte, Rubén; Okada, Chizuru; Avery, Danielle T; Latorre, Daniela; Deswarte, Caroline; Jabot-Hanin, Fabienne; Torrado, Egidio; Fountain, Jeffrey; Belkadi, Aziz; Itan, Yuval; Boisson, Bertrand; Migaud, Mélanie; Arlehamn, Cecilia S Lindestam; Sette, Alessandro; Breton, Sylvain; McCluskey, James; Rossjohn, Jamie; de Villartay, Jean-Pierre; Moshous, Despina; Hambleton, Sophie; Latour, Sylvain; Arkwright, Peter D; Picard, Capucine; Lantz, Olivier; Engelhard, Dan; Kobayashi, Masao; Abel, Laurent; Cooper, Andrea M; Notarangelo, Luigi D; Boisson-Dupuis, Stéphanie; Puel, Anne; Sallusto, Federica; Bustamante, Jacinta; Tangye, Stuart G; Casanova, Jean-Laurent

    2015-08-07

    Human inborn errors of immunity mediated by the cytokines interleukin-17A and interleukin-17F (IL-17A/F) underlie mucocutaneous candidiasis, whereas inborn errors of interferon-γ (IFN-γ) immunity underlie mycobacterial disease. We report the discovery of bi-allelic RORC loss-of-function mutations in seven individuals from three kindreds of different ethnic origins with both candidiasis and mycobacteriosis. The lack of functional RORγ and RORγT isoforms resulted in the absence of IL-17A/F-producing T cells in these individuals, probably accounting for their chronic candidiasis. Unexpectedly, leukocytes from RORγ- and RORγT-deficient individuals also displayed an impaired IFN-γ response to Mycobacterium. This principally reflected profoundly defective IFN-γ production by circulating γδ T cells and CD4(+)CCR6(+)CXCR3(+) αβ T cells. In humans, both mucocutaneous immunity to Candida and systemic immunity to Mycobacterium require RORγ, RORγT, or both.

  8. Registered report: IDH mutation impairs histone demethylation and results in a block to cell differentiation.

    PubMed

    Richarson, Adam D; Scott, David A; Zagnitko, Olga; Aza-Blanc, Pedro; Chang, Chih-Cheng; Russler-Germain, David A

    2016-03-11

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from "IDH mutation impairs histone demethylation and results in a block to cell differentiation" by Lu and colleagues, published in Nature in 2012 (Lu et al., 2012). The experiments that will be replicated are those reported in Figures 1B, 2A, 2B, 2D and 4D. Lu and colleagues demonstrated that expression of mutant forms of IDH1 or IDH2 caused global increases in histone methylation and increased levels of 2 hydroxyglutarate (Figure 1B). This was correlated with a block in differentiation (Figures 2A, B and D). This effect appeared to be mediated by the histone demethylase KDM4C (Figure 4D). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Scienceand Science Exchange, and the results of the replications will be published by eLife.

  9. Functional analysis of human CNGA3 mutations associated with colour blindness suggests impaired surface expression of channel mutants A3(R427C) and A3(R563C).

    PubMed

    Koeppen, Katja; Reuter, Peggy; Kohl, Susanne; Baumann, Britta; Ladewig, Thomas; Wissinger, Bernd

    2008-05-01

    Mutations in the CNGA3 gene have been associated with complete and incomplete forms of total colour blindness (achromatopsia), a disorder characterized by reduced visual acuity, lack of colour discrimination, photophobia and nystagmus. CNGA3 encodes the A-subunit of the cone photoreceptor cyclic nucleotide-gated (CNG) channel, an essential component of the phototransduction cascade. Here we report the identification of three new CNGA3 mutations in patients with achromatopsia. To assess the pathogenicity of these newly identified and four previously reported mutations, mutant CNGA3 channels were heterologously expressed in a human embryonic kidney cell line (HEK293 cells) and functionally analysed using calcium imaging. Channels with the mutations R427C and R563C showed a response in imaging experiments and were subsequently characterized in-depth with the patch-clamp technique. The mutant channels were analysed as homooligomers and also as heterooligomers with the wild-type B-subunit present in native channels. Overall, cyclic guanosine monophosphate (cGMP) maximum currents of mutant channels were profoundly reduced in homo- and heteromers. Treatment with the chemical chaperone glycerol effectively increased macroscopic currents, presumably by enhancing surface expression of mutant channels as confirmed by immunocytochemistry. These results suggest decreased channel density in the cell membrane due to impaired folding or trafficking of the channel protein as the main pathogenic effect of the mutations R427C and R563C. Moreover, A3(R427C) homomers showed distinctly increased cGMP and cyclic adenosine monophosphate (cAMP) sensitivities as well as cAMP fractional currents that were raised to over 90% of cGMP maximum currents. Co-expression of A3(R427C) with the B3 subunit compensated for most of these aberrant properties, apart from the reduced cGMP maximum currents.

  10. A novel de novo dominant negative mutation in DNM1L impairs mitochondrial fission and presents as childhood epileptic encephalopathy.

    PubMed

    Fahrner, Jill A; Liu, Raymond; Perry, Michael Scott; Klein, Jessica; Chan, David C

    2016-08-01

    DNM1L encodes dynamin-related protein 1 (DRP1/DLP1), a key component of the mitochondrial fission machinery that is essential for proper functioning of the mammalian brain. Previously reported probands with de novo missense mutations in DNM1L presented in the first year of life with severe encephalopathy and refractory epilepsy, with several dying within the first several weeks after birth. In contrast, we report identical novel missense mutations in DNM1L in two unrelated probands who experienced normal development for several years before presenting with refractory focal status epilepticus and subsequent rapid neurological decline. We expand the phenotype of DNM1L-related mitochondrial fission defects, reveal common unique clinical characteristics and imaging findings, and compare the cellular impact of this novel mutation to the previously reported A395D lethal variant. We demonstrate that our R403C mutation, which resides in the assembly region of DRP1, acts by a dominant-negative mechanism and reduces oligomerization, mitochondrial fission activity, and mitochondrial recruitment of DRP1, but to a lesser extent compared to the A395D mutation. In contrast to the initial report of neonatal lethality resulting from DNM1L mutation and DRP1 dysfunction, our results show that milder DRP1 impairment is compatible with normal early development and subsequently results in a distinct set of neurological findings. In addition, we identify a common pathogenic mechanism whereby DNM1L mutations impair mitochondrial fission. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. Mutational analysis of the intramembranous H10 loop of yeast Nhx1 reveals a critical role in ion homoeostasis and vesicle trafficking.

    PubMed

    Mukherjee, Sanchita; Kallay, Laura; Brett, Christopher L; Rao, Rajini

    2006-08-15

    Yeast Nhx1 [Na+(K+)/H+ exchanger 1] is an intracellular Na+(K+)/H+ exchanger, localizing to the late endosome where it is important for ion homoeostasis and vesicle trafficking. Phylogenetic analysis of NHE (Na+/H+ exchanger) sequences has identified orthologous proteins, including HsNHE6 (human NHE6), HsNHE7 and HsNHE9 of unknown physiological role. These appear distinct from well-studied mammalian plasma membrane isoforms (NHE1-NHE5). To explore the differences between plasma membrane and intracellular NHEs and understand the link between ion homoeostasis and vesicle trafficking, we examined the consequence of replacing residues in the intramembranous H10 loop of Nhx1 between transmembrane segments 9 and 10. The critical role for the carboxy group of Glu355 in ion transport is consistent with the invariance of this residue in all NHEs. Surprisingly, residues specifically conserved in the intracellular isoforms (such as Phe357 and Tyr361) could not be replaced with closely similar residues (leucine and phenylalanine) found in the plasma membrane isoforms without loss of function, revealing unexpected side chain specificity. The trafficking phenotypes of all Nhx1 mutants, including hygromycin-sensitivity and missorting of carboxypeptidase Y, were found to directly correlate with pH homoeostasis defects and could be proportionately corrected by titration with weak base. The present study demonstrates the importance of the H10 loop of the NHE family, highlights the differences between plasma membrane and intracellular isoforms and shows that trafficking defects are tightly coupled with pH homoeostasis.

  12. Mutational analysis of the intramembranous H10 loop of yeast Nhx1 reveals a critical role in ion homoeostasis and vesicle trafficking

    PubMed Central

    Mukherjee, Sanchita; Kallay, Laura; Brett, Christopher L.; Rao, Rajini

    2006-01-01

    Yeast Nhx1 [Na+(K+)/H+ exchanger 1] is an intracellular Na+(K+)/H+ exchanger, localizing to the late endosome where it is important for ion homoeostasis and vesicle trafficking. Phylogenetic analysis of NHE (Na+/H+ exchanger) sequences has ident-ified orthologous proteins, including HsNHE6 (human NHE6), HsNHE7 and HsNHE9 of unknown physiological role. These appear distinct from well-studied mammalian plasma membrane isoforms (NHE1–NHE5). To explore the differences between plasma membrane and intracellular NHEs and understand the link between ion homoeostasis and vesicle trafficking, we examined the consequence of replacing residues in the intramembranous H10 loop of Nhx1 between transmembrane segments 9 and 10. The critical role for the carboxy group of Glu355 in ion transport is consistent with the invariance of this residue in all NHEs. Surprisingly, residues specifically conserved in the intracellular isoforms (such as Phe357 and Tyr361) could not be replaced with closely similar residues (leucine and phenylalanine) found in the plasma membrane isoforms without loss of function, revealing unexpected side chain specificity. The trafficking phenotypes of all Nhx1 mutants, including hygromycin-sensitivity and missorting of carboxypeptidase Y, were found to directly correlate with pH homoeostasis defects and could be proportionately corrected by titration with weak base. The present study demonstrates the importance of the H10 loop of the NHE family, highlights the differences between plasma membrane and intracellular isoforms and shows that trafficking defects are tightly coupled with pH homoeostasis. PMID:16671892

  13. Mutations of the gene encoding otogelin are a cause of autosomal-recessive nonsyndromic moderate hearing impairment.

    PubMed

    Schraders, Margit; Ruiz-Palmero, Laura; Kalay, Ersan; Oostrik, Jaap; del Castillo, Francisco J; Sezgin, Orhan; Beynon, Andy J; Strom, Tim M; Pennings, Ronald J E; Zazo Seco, Celia; Oonk, Anne M M; Kunst, Henricus P M; Domínguez-Ruiz, María; García-Arumi, Ana M; del Campo, Miguel; Villamar, Manuela; Hoefsloot, Lies H; Moreno, Felipe; Admiraal, Ronald J C; del Castillo, Ignacio; Kremer, Hannie

    2012-11-02

    Already 40 genes have been identified for autosomal-recessive nonsyndromic hearing impairment (arNSHI); however, many more genes are still to be identified. In a Dutch family segregating arNSHI, homozygosity mapping revealed a 2.4 Mb homozygous region on chromosome 11 in p15.1-15.2, which partially overlapped with the previously described DFNB18 locus. However, no putative pathogenic variants were found in USH1C, the gene mutated in DFNB18 hearing impairment. The homozygous region contained 12 additional annotated genes including OTOG, the gene encoding otogelin, a component of the tectorial membrane. It is thought that otogelin contributes to the stability and strength of this membrane through interaction or stabilization of its constituent fibers. The murine orthologous gene was already known to cause hearing loss when defective. Analysis of OTOG in the Dutch family revealed a homozygous 1 bp deletion, c.5508delC, which leads to a shift in the reading frame and a premature stop codon, p.Ala1838ProfsX31. Further screening of 60 unrelated probands from Spanish arNSHI families detected compound heterozygous OTOG mutations in one family, c.6347C>T (p.Pro2116Leu) and c. 6559C>T (p.Arg2187X). The missense mutation p.Pro2116Leu affects a highly conserved residue in the fourth von Willebrand factor type D domain of otogelin. The subjects with OTOG mutations have a moderate hearing impairment, which can be associated with vestibular dysfunction. The flat to shallow "U" or slightly downsloping shaped audiograms closely resembled audiograms of individuals with recessive mutations in the gene encoding α-tectorin, another component of the tectorial membrane. This distinctive phenotype may represent a clue to orientate the molecular diagnosis.

  14. Trafficking-Competent KCNQ1 Variably Influences the Function of HERG Long QT Alleles

    PubMed Central

    Hayashi, Kenshi; Shuai, Wen; Sakamoto, Yuichiro; Higashida, Haruhiro; Yamagishi, Masakazu; Kupershmidt, Sabina

    2010-01-01

    Background Mutations in the KCNQ1 and HERG genes cause the Long QT Syndromes, LQTS1 and LQTS2, due to reductions in the cardiac repolarizing IKs and IKr currents, respectively. It was previously reported that KCNQ1 co-expression modulates HERG function by enhancing membrane expression of HERG, and that the two proteins co-immunoprecipitate, and co-localize in myocytes. In vivo studies in genetically modified rabbits also support a HERG-KCNQ1 interaction. Objective We sought to determine whether KCNQ1 influences the current characteristics of HERG genetic variants. Methods Expression of HERG and KCNQ1 wild type (WT) and mutant channels in heterologous systems, combined with whole cell patch clamp analysis and biochemistry. Results Supporting the notion that KCNQ1 needs to be trafficking competent to influence HERG function, we found that although the tail current density of HERG expressed in CHO cells was approximately doubled by WT KCNQ1 co-expression, it was not altered in the presence of the trafficking-defective KCNQ1T587M variant. Activation and deactivation kinetics of HERG variants were not altered. The HERGM124T variant, previously shown to be mildly impaired functionally, was restored to WT levels by KCNQ1-WT but not KCNQ1T587M co-expression. The tail current densities of the severely trafficking-impaired HERGG601S and HERGF805C variants were only slightly improved by KCNQ1 co-expression. The trafficking competent, but incompletely processed HERGN598Q, and a mutation in the selectivity filter, HERGG628S, were not improved by KCNQ1 co-expression. Conclusions These findings suggest a functional co-dependence of HERG on KCNQ1 during channel biogenesis. Moreover, KCNQ1 variably modulates LQTS2 mutations with distinct underlying pathologies. PMID:20348026

  15. Germline mitochondrial DNA mutations aggravate ageing and can impair brain development.

    PubMed

    Ross, Jaime M; Stewart, James B; Hagström, Erik; Brené, Stefan; Mourier, Arnaud; Coppotelli, Giuseppe; Freyer, Christoph; Lagouge, Marie; Hoffer, Barry J; Olson, Lars; Larsson, Nils-Göran

    2013-09-19

    Ageing is due to an accumulation of various types of damage, and mitochondrial dysfunction has long been considered to be important in this process. There is substantial sequence variation in mammalian mitochondrial DNA (mtDNA), and the high mutation rate is counteracted by different mechanisms that decrease maternal transmission of mutated mtDNA. Despite these protective mechanisms, it is becoming increasingly clear that low-level mtDNA heteroplasmy is quite common and often inherited in humans. We designed a series of mouse mutants to investigate the extent to which inherited mtDNA mutations can contribute to ageing. Here we report that maternally transmitted mtDNA mutations can induce mild ageing phenotypes in mice with a wild-type nuclear genome. Furthermore, maternally transmitted mtDNA mutations lead to anticipation of reduced fertility in mice that are heterozygous for the mtDNA mutator allele (PolgA(wt/mut)) and aggravate premature ageing phenotypes in mtDNA mutator mice (PolgA(mut/mut)). Unexpectedly, a combination of maternally transmitted and somatic mtDNA mutations also leads to stochastic brain malformations. Our findings show that a pre-existing mutation load will not only allow somatic mutagenesis to create a critically high total mtDNA mutation load sooner but will also increase clonal expansion of mtDNA mutations to enhance the normally occurring mosaic respiratory chain deficiency in ageing tissues. Our findings suggest that maternally transmitted mtDNA mutations may have a similar role in aggravating aspects of normal human ageing.

  16. Congenital afibrinogenemia: identification and expression of a missense mutation in FGB impairing fibrinogen secretion.

    PubMed

    Vu, Dung; Bolton-Maggs, Paula H B; Parr, Jeremy R; Morris, Michael A; de Moerloose, Philippe; Neerman-Arbez, Marguerite

    2003-12-15

    Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by complete absence of detectable fibrinogen. We previously identified the first causative mutations for this disease: a homozygous deletion of approximately 11 kb of the fibrinogen alpha-chain gene (FGA). Subsequent studies revealed that the great majority of afibrinogenemia mutations are localized in FGA, but mutations were also found in FGG and FGB. Apart from 3 missense mutations identified in the C-terminal portion of FGB, all fibrinogen gene mutations responsible for afibrinogenemia are null. In this study, a young boy with afibrinogenemia was found to be a compound heterozygote for 2 mutations in FGB: an N-terminal nonsense mutation W47X (exon 2) and a missense mutation (G444S, exon 8). Coexpression of the FGB G444S mutant cDNA in combination with wild-type FGA and FGG cDNAs demonstrated that fibrinogen molecules containing the mutant beta chain are able to assemble but are not secreted into the media, confirming the pathogenic nature of the identified mutation.

  17. COL4A2 mutations impair COL4A1 and COL4A2 secretion and cause hemorrhagic stroke.

    PubMed

    Jeanne, Marion; Labelle-Dumais, Cassandre; Jorgensen, Jeff; Kauffman, W Berkeley; Mancini, Grazia M; Favor, Jack; Valant, Valerie; Greenberg, Steven M; Rosand, Jonathan; Gould, Douglas B

    2012-01-13

    Collagen, type IV, alpha 1 (COL4A1) and alpha 2 (COL4A2) form heterotrimers and are abundant components of basement membranes, including those of the cerebral vasculature. COL4A1 mutations are an increasingly recognized cause of multisystem disorders, including highly penetrant cerebrovascular disease and intracerebral hemorrhage (ICH). Because COL4A1 and COL4A2 are structurally and functionally associated, we hypothesized that variants in COL4A2 would also cause ICH. We sequence COL4A2 in 96 patients with ICH and identify three rare, nonsynonymous coding variants in four patients that are not present in a cohort of 144 ICH-free individuals. All three variants change evolutionarily conserved amino acids. Using a cellular assay, we show that these putative mutations cause intracellular accumulation of COL4A1 and COL4A2 at the expense of their secretion, which supports their pathogenecity. Furthermore, we show that Col4a2 mutant mice also have completely penetrant ICH and that mutations in mouse and human lead to retention of COL4A1 and COL4A2 within the endoplasmic reticulum (ER). Importantly, two of the three putative mutations found in patients trigger ER stress and activate the unfolded protein response. The identification of putative COL4A2 mutations that might contribute to ICH in human patients provides insight into the pathogenic mechanisms of this disease. Our data suggest that COL4A2 mutations impair COL4A1 and COL4A2 secretion and can also result in cytotoxicity. Finally, our findings suggest that, collectively, mutations in COL4A1 and COL4A2 contribute to sporadic cases of ICH.

  18. TACI mutations and impaired B-cell function in subjects with CVID and healthy heterozygotes.

    PubMed

    Martinez-Gallo, Monica; Radigan, Lin; Almejún, María Belén; Martínez-Pomar, Natalia; Matamoros, Núria; Cunningham-Rundles, Charlotte

    2013-02-01

    Mutations in the gene coding for the transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) are found in 8% to 10% of subjects with common variable immunodeficiency (CVID). Although heterozygous mutations may coincide with immunodeficiency in a few families, most mutation-bearing relatives are not hypogammaglobulinemic. Thus, the role of TACI mutations in producing the immune defect remains unclear. This study examined the expression and function of TACI mutations in healthy heterozygous relatives. We examined the surface and intracellular expression of TACI protein in EBV-transformed B cells of patients and relatives with mutations in 7 families, binding of a proliferation-inducing ligand, and secretion of IgG and IgA by ligand-activated B cells. We tested whether Toll-like receptor 9 agonists increased TACI expression and whether an agonistic anti-TACI antibody could induce activation-induced cytidine deaminase mRNA in those with mutations. Intracellular and extracellular TACI expression was defective for B cells of all subjects with mutations, including subjects with CVID and relatives. Although Toll-like receptor 9 triggering normally up-regulates B-cell TACI expression, this was defective for all subjects with mutations. Triggering TACI by an agonistic antibody showed loss of activation-induced cytidine deaminase mRNA induction in all mutation-bearing B cells. However, ligand-induced IgG and IgA production was normal for healthy relatives but not for subjects with CVID. Thus, B cells of relatives of subjects with CVID who have mutations in TACI but normal immune globulin levels still have detectable in vitro B-cell defects. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  19. The ER-Membrane Transport System Is Critical for Intercellular Trafficking of the NSm Movement Protein and Tomato Spotted Wilt Tospovirus

    PubMed Central

    Feng, Zhike; Xue, Fan; Xu, Min; Chen, Xiaojiao; Zhao, Wenyang; Garcia-Murria, Maria J.; Mingarro, Ismael; Liu, Yong; Huang, Ying; Jiang, Lei; Zhu, Min; Tao, Xiaorong

    2016-01-01

    Plant viruses move through plasmodesmata to infect new cells. The plant endoplasmic reticulum (ER) is interconnected among cells via the ER desmotubule in the plasmodesma across the cell wall, forming a continuous ER network throughout the entire plant. This ER continuity is unique to plants and has been postulated to serve as a platform for the intercellular trafficking of macromolecules. In the present study, the contribution of the plant ER membrane transport system to the intercellular trafficking of the NSm movement protein and Tomato spotted wilt tospovirus (TSWV) is investigated. We showed that TSWV NSm is physically associated with the ER membrane in Nicotiana benthamiana plants. An NSm-GFP fusion protein transiently expressed in single leaf cells was trafficked into neighboring cells. Mutations in NSm that impaired its association with the ER or caused its mis-localization to other subcellular sites inhibited cell-to-cell trafficking. Pharmacological disruption of the ER network severely inhibited NSm-GFP trafficking but not GFP diffusion. In the Arabidopsis thaliana mutant rhd3 with an impaired ER network, NSm-GFP trafficking was significantly reduced, whereas GFP diffusion was not affected. We also showed that the ER-to-Golgi secretion pathway and the cytoskeleton transport systems were not involved in the intercellular trafficking of TSWV NSm. Importantly, TSWV cell-to-cell spread was delayed in the ER-defective rhd3 mutant, and this reduced viral infection was not due to reduced replication. On the basis of robust biochemical, cellular and genetic analysis, we established that the ER membrane transport system serves as an important direct route for intercellular trafficking of NSm and TSWV. PMID:26863622

  20. The cross-talk of LDL-cholesterol with cell motility: Insights from the Niemann Pick Type C1 mutation and altered integrin trafficking

    PubMed Central

    Hoque, Monira; Rentero, Carles; Conway, James R; Murray, Rachael Z; Timpson, Paul; Enrich, Carlos; Grewal, Thomas

    2015-01-01

    Cholesterol is considered indispensible for the recruitment and functioning of integrins in focal adhesions for cell migration. However, the physiological cholesterol pools that control integrin trafficking and focal adhesion assembly remain unclear. Using Niemann Pick Type C1 (NPC) mutant cells, which accumulate Low Density lipoprotein (LDL)-derived cholesterol in late endosomes (LE), several recent studies indicate that LDL-cholesterol has multiple roles in regulating focal adhesion dynamics. Firstly, targeting of endocytosed LDL-cholesterol from LE to focal adhesions controls their formation at the leading edge of migrating cells. Other newly emerging literature suggests that this may be coupled to vesicular transport of integrins, Src kinase and metalloproteases from the LE compartment to focal adhesions. Secondly, our recent work identified LDL-cholesterol as a key factor that determines the distribution and ability of several Soluble NSF Attachment Protein (SNAP) Receptor (SNARE) proteins, key players in vesicle transport, to control integrin trafficking to the cell surface and extracellular matrix (ECM) secretion. Collectively, dietary, genetic and pathological changes in cholesterol metabolism may link with efficiency and speed of integrin and ECM cell surface delivery in metastatic cancer cells. This commentary will summarize how direct and indirect pathways enable LDL-cholesterol to modulate cell motility. PMID:26366834

  1. Adult-onset focal expression of mutated human tau in the hippocampus impairs spatial working memory of rats.

    PubMed

    Mustroph, Martina L; King, Michael A; Klein, Ronald L; Ramirez, Julio J

    2012-07-15

    Tauopathy in the hippocampus is one of the earliest cardinal features of Alzheimer's disease (AD), a condition characterized by progressive memory impairments. In fact, density of tau neurofibrillary tangles (NFTs) in the hippocampus strongly correlates with severity of cognitive impairments in AD. In the present study, we employed a somatic cell gene transfer technique to create a rodent model of tauopathy by injecting a recombinant adeno-associated viral vector with a mutated human tau gene (P301L) into the hippocampus of adult rats. The P301L mutation is causal for frontotemporal dementia with parkinsonism-17 (FTDP-17), but it has been used for studying memory effects characteristic of AD in transgenic mice. To ascertain if P301L-induced mnemonic deficits are persistent, animals were tested for 6 months. It was hypothesized that adult-onset, spatially restricted tau expression in the hippocampus would produce progressive spatial working memory deficits on a learned alternation task. Rats injected with the tau vector exhibited persistent impairments on the hippocampal-dependent task beginning at about 6 weeks post-transduction compared to rats injected with a green fluorescent protein vector. Histological analysis of brains for expression of human tau revealed hyperphosphorylated human tau and NFTs in the hippocampus in experimental animals only. Thus, adult-onset, vector-induced tauopathy spatially restricted to the hippocampus progressively impaired spatial working memory in rats. We conclude that the model faithfully reproduces histological and behavioral findings characteristic of dementing tauopathies. The rapid onset of sustained memory impairment establishes a preclinical model particularly suited to the development of potential tauopathy therapeutics.

  2. Impairment of autophagosome-lysosome fusion in the buff mutant mice with the VPS33AD251E mutation

    PubMed Central

    Zhen, Yuanli; Li, Wei

    2015-01-01

    The HOPS (homotypic fusion and protein sorting) complex functions in endocytic and autophagic pathways in both lower eukaryotes and mammalian cells through its involvement in fusion events between endosomes and lysosomes or autophagosomes and lysosomes. However, the differential molecular mechanisms underlying these fusion processes are largely unknown. Buff (bf) is a mouse mutant that carries an Asp251-to-Glu point mutation (D251E) in the VPS33A protein, a tethering protein and a core subunit of the HOPS complex. Bf mice showed impaired spontaneous locomotor activity, motor learning, and autophagic activity. Although the gross anatomy of the brain was apparently normal, the number of Purkinje cells was significantly reduced. Furthermore, we found that fusion between autophagosomes and lysosomes was defective in bf cells without compromising the endocytic pathway. The direct association of mutant VPS33AD251E with the autophagic SNARE complex, STX17 (syntaxin 17)-VAMP8-SNAP29, was enhanced. In addition, the VPS33AD251E mutation enhanced interactions with other HOPS subunits, namely VPS41, VPS39, VPS18, and VPS11, except for VPS16. Reduction of the interactions between VPS33AY440D and several other HOPS subunits led to decreased association with STX17. These results suggest that the VPS33AD251E mutation plays dual roles by increasing the HOPS complex assembly and its association with the autophagic SNARE complex, which selectively affects the autophagosome-lysosome fusion that impairs basal autophagic activity and induces Purkinje cell loss. PMID:26259518

  3. Fluconazole-induced long QT syndrome via impaired human ether-a-go-go-related gene (hERG) protein trafficking in rabbits.

    PubMed

    Wang, Jinli; Wang, Guan; Quan, Xiaoqing; Ruan, Lei; Liu, Yang; Ruan, Yanfei; Liu, Nian; Zhang, Cuntai; Bai, Rong

    2017-07-01

    hERG protein trafficking deficiency has long been known in drug-induced long QT syndrome (LQTS). However, validated evidence from in vivo data kept scanty. Our goal was to investigate the proarrhythmic action of fluconazole and its underlying mechanism in an animal model. Twenty female Japanese long-eared white rabbits were randomly distributed into a control group and a fluconazole group for a chronic 2-week treatment. The control group was treated with 0.5% sodium carboxymethylcellulose (CMCNa), and the fluconazole group was treated with fluconazole. Electrocardiograms (ECGs) were recorded during the experimental period. Isolated arterially perfused left ventricular wedge preparations from the rabbits were made 2 weeks after treatment, and the arrhythmia events, the transmural ECG, and action potential from both the endocardium and epicardium were recorded. The changes in hERG protein expression were measured by western blot. The fluconazole group showed a longer QT interval 1 week after treatment (P < 0.05) and a higher arrhythmia occurrence 2 weeks after treatment (P < 0.05) than the control group. The fluconazole group also showed a longer transmural dispersion of repolarization and a higher occurrence of life-threatening torsades de pointes in arterially perfused left ventricular preparations. Furthermore, western blot analysis showed that the density of mature hERG protein was lower in the fluconazole group than that in the control group. Fluconazole can prolong the QT interval and possess proarrhythmic activity due to its inhibition of hERG protein trafficking in our experimental model. These findings may impact the clinical potential of fluconazole in humans.

  4. Impaired mitochondrial respiration and decreased fatigue resistance followed by severe muscle weakness in skeletal muscle of mitochondrial DNA mutator mice.

    PubMed

    Yamada, Takashi; Ivarsson, Niklas; Hernández, Andrés; Fahlström, Andreas; Cheng, Arthur J; Zhang, Shi-Jin; Bruton, Joseph D; Ulfhake, Brun; Westerblad, Håkan

    2012-12-01

    Mitochondrial dysfunction can drastically impair muscle function, with weakness and exercise intolerance as key symptoms. Here we examine the time course of development of muscle dysfunction in a mouse model of premature ageing induced by defective proofreading function of mitochondrial DNA (mtDNA) polymerase (mtDNA mutator mouse). Isolated fast-twitch muscles and single muscle fibres from young (3-5 months) and end-stage (11 months) mtDNA mutator mice were compared to age-matched control mice. Force and free myoplasmic [Ca(2+)] ([Ca(2+)](i)) were measured under resting conditions and during fatigue induced by repeated tetani. Muscles of young mtDNA mutator mice displayed no weakness in the rested state, but had lower force and [Ca(2+)](i) than control mice during induction of fatigue. Muscles of young mtDNA mutator mice showed decreased activities of citrate synthase and β-hydroxyacyl-coenzyme A dehydrogenase, reduced expression of cytochrome c oxidase, and decreased expression of triggers of mitochondrial biogenesis (PGC-1α, PPARα, AMPK). Muscles from end-stage mtDNA mutator mice showed weakness under resting conditions with markedly decreased tetanic [Ca(2+)](i), force per cross-sectional area and protein expression of the sarcoplasmic reticulum Ca(2+) pump (SERCA1). In conclusion, fast-twitch muscles of prematurely ageing mtDNA mutator mice display a sequence of deleterious mitochondrial-to-nucleus signalling with an initial decrease in oxidative capacity, which was not counteracted by activation of signalling to increase mitochondrial biogenesis. This was followed by severe muscle weakness in the end stage. These results have implication for normal ageing and suggest that decreased mitochondrial oxidative capacity due to a sedentary lifestyle may predispose towards muscle weakness developing later in life.

  5. Aggregate-prone desmin mutations impair mitochondrial calcium uptake in primary myotubes.

    PubMed

    Smolina, Natalia; Bruton, Joseph; Sjoberg, Gunnar; Kostareva, Anna; Sejersen, Thomas

    2014-10-01

    Desmin, being a major intermediate filament of mature muscle cell, interacts with mitochondria within the cell and participates in mitochondria proper localization. The goal of the present study was to assess the effect of aggregate-prone and non-aggregate-prone desmin mutations on mitochondrial calcium uptake. Primary murine satellite cells were transduced with lentiviruses carrying desmin in wild type or mutant form, and were induced to differentiate into myotubes. Four mutations resulting in different degree of desmin aggregates formation were analyzed. Tail domain mutation Asp399Tyr has the mildest impact on desmin filament polymerization, rod domain mutation Ala357Pro causes formation of large aggregates composed of filamentous material, and Leu345Pro and Leu370Pro are considered to be the most severest in their impact on desmin polymerization and structure. For mitochondrial calcium measurement cells were loaded with rhod 2-AM. We found that aggregate-prone mutations significantly decreased [Ca(2+)]mit, whereas non-aggregate-prone mutations did not decrease [Ca(2+)]mit. Moreover aggregate-prone desmin mutations resulted in increased resting cytosolic [Ca(2+)]. However this increase was not accompanied by any alterations in sarcoplasmic reticulum calcium release. We suggest that the observed decline in [Ca(2+)]mit was due to desmin aggregate accumulation resulting in the loss of desmin mitochondria interactions.

  6. Mutations in the Primer Grip of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Impair Proviral DNA Synthesis and Virion Maturation

    PubMed Central

    Yu, Qiang; Ottmann, Michele; Pechoux, Christine; Le Grice, Stuart; Darlix, Jean-Luc

    1998-01-01

    This report describes the effects of mutating highly conserved residues in the primer grip domain of human immunodeficiency virus type 1 reverse transcriptase (RT) on virus formation and infectivity. Among a series of RT mutant viruses, three (M230A, L234D, and W239A) were found to be noninfectious or very poorly infectious. Our data indicate that these mutations in RT caused severe defects in proviral DNA synthesis. Interestingly, assembly and maturation of mutant virus M230A were similar to those of the wild type, while mutants L234D and W239A showed impaired maturation. The immature morphology of RT mutants L234D and W239A is due at least in part to premature cleavage of the gag-pol precursor, prior to virion budding, indicating that intracellular stability of Pr160gag-pol is of key importance during virus assembly. PMID:9696874

  7. Axonal transport of TDP-43 mRNA granules in neurons is impaired by ALS-causing mutations

    PubMed Central

    Carrasco, Monica A.; Williams, Luis A.; Winborn, Christina S.; Han, Steve S. W.; Kiskinis, Evangelos; Winborn, Brett; Freibaum, Brian D.; Kanagaraj, Anderson; Clare, Alison J.; Badders, Nisha M.; Bilican, Bilada; Chaum, Edward; Chandran, Siddharthan; Shaw, Christopher E.; Eggan, Kevin C.; Maniatis, Tom; Taylor, J. Paul

    2014-01-01

    Summary The RNA binding protein TDP-43 regulates RNA metabolism at multiple levels, including transcription, RNA splicing, and mRNA stability. TDP-43 is a major component of the cytoplasmic inclusions characteristic of amyotrophic lateral sclerosis and some types of frontotemporal lobar degeneration. The importance of TDP-43 in disease is underscored by the fact that dominant missense mutations are sufficient to cause disease, although the role of TDP-43 in pathogenesis is unknown. Here we show that TDP-43 forms cytoplasmic mRNP granules that undergo bidirectional, microtubule-dependent transport in neurons in vitro and in vivo and facilitate delivery of target mRNA to distal neuronal compartments. TDP-43 mutations impair this mRNA transport function in vivo and in vitro, including in stem cell-derived motor neurons from ALS patients bearing any one of three different TDP-43 ALS-causing mutations. Thus, TDP43 mutations that cause ALS lead to partial loss of a novel cytoplasmic function of TDP-43. PMID:24507191

  8. Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis

    PubMed Central

    Zhang, Yi; Nikolovski, Nino; Sorieul, Mathias; Vellosillo, Tamara; McFarlane, Heather E.; Dupree, Ray; Kesten, Christopher; Schneider, René; Driemeier, Carlos; Lathe, Rahul; Lampugnani, Edwin; Yu, Xiaolan; Ivakov, Alexander; Doblin, Monika S.; Mortimer, Jenny C.; Brown, Steven P.; Persson, Staffan; Dupree, Paul

    2016-01-01

    As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus. PMID:27277162

  9. Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis.

    PubMed

    Zhang, Yi; Nikolovski, Nino; Sorieul, Mathias; Vellosillo, Tamara; McFarlane, Heather E; Dupree, Ray; Kesten, Christopher; Schneider, René; Driemeier, Carlos; Lathe, Rahul; Lampugnani, Edwin; Yu, Xiaolan; Ivakov, Alexander; Doblin, Monika S; Mortimer, Jenny C; Brown, Steven P; Persson, Staffan; Dupree, Paul

    2016-06-09

    As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus.

  10. A Mutation in SNAP29, Coding for a SNARE Protein Involved in Intracellular Trafficking, Causes a Novel Neurocutaneous Syndrome Characterized by Cerebral Dysgenesis, Neuropathy, Ichthyosis, and Palmoplantar Keratoderma

    PubMed Central

    Sprecher, Eli; Ishida-Yamamoto, Akemi; Mizrahi-Koren, Mordechai; Rapaport, Debora; Goldsher, Dorit; Indelman, Margarita; Topaz, Orit; Chefetz, Ilana; Keren, Hanni; O’Brien, Timothy J.; Bercovich, Dani; Shalev, Stavit; Geiger, Dan; Bergman, Reuven; Horowitz, Mia; Mandel, Hanna

    2005-01-01

    Neurocutaneous syndromes represent a vast, largely heterogeneous group of disorders characterized by neurological and dermatological manifestations, reflecting the common embryonic origin of epidermal and neural tissues. In the present report, we describe a novel neurocutaneous syndrome characterized by cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK syndrome). Using homozygosity mapping in two large families, we localized the disease gene to 22q11.2 and identified, in all patients, a 1-bp deletion in SNAP29, which codes for a SNARE protein involved in vesicle fusion. SNAP29 expression was decreased in the skin of the patients, resulting in abnormal maturation of lamellar granules and, as a consequence, in mislocation of epidermal lipids and proteases. These data underscore the importance of vesicle trafficking regulatory mechanisms for proper neuroectodermal differentiation. PMID:15968592

  11. The deubiquitinating enzyme USP8 promotes trafficking and degradation of the chemokine receptor 4 at the sorting endosome.

    PubMed

    Berlin, Ilana; Higginbotham, Katherine M; Dise, Rebecca S; Sierra, Maria I; Nash, Piers D

    2010-11-26

    Reversible ubiquitination orchestrated by the opposition of ubiquitin ligases and deubiquitinating enzymes mediates endocytic trafficking of cell surface receptors for lysosomal degradation. Ubiquitin-specific protease 8 (USP8) has previously been implicated in endocytosis of several receptors by virtue of their deubiquitination. The present study explores an indirect role for USP8 in cargo trafficking through its regulation of the chemokine receptor 4 (CXCR4). Contrary to the effects of USP8 loss on enhanced green fluorescent protein, we find that USP8 depletion stabilizes CXCR4 on the cell surface and attenuates receptor degradation without affecting its ubiquitination status. In the presence of ligand, diminished CXCR4 turnover is accompanied by receptor accumulation on enlarged early endosomes and leads to enhancement of phospho-ERK signaling. Perturbation in CXCR4 trafficking, resulting from USP8 inactivation, occurs at the ESCRT-0 checkpoint, and catalytic mutation of USP8 specifically targeted to the ESCRT-0 complex impairs the spatial and temporal organization of the sorting endosome. USP8 functionally opposes the ubiquitin ligase AIP4 with respect to ESCRT-0 ubiquitination, thereby promoting trafficking of CXCR4. Collectively, our findings demonstrate a functional cooperation between USP8, AIP4, and the ESCRT-0 machinery at the early sorting phase of CXCR4 and underscore the versatility of USP8 in shaping trafficking events at the early-to-late endosome transition.

  12. Autosomal recessive phosphoglucomutase 3 (PGM3) mutations link glycosylation defects to atopy, immune deficiency, autoimmunity, and neurocognitive impairment.

    PubMed

    Zhang, Yu; Yu, Xiaomin; Ichikawa, Mie; Lyons, Jonathan J; Datta, Shrimati; Lamborn, Ian T; Jing, Huie; Kim, Emily S; Biancalana, Matthew; Wolfe, Lynne A; DiMaggio, Thomas; Matthews, Helen F; Kranick, Sarah M; Stone, Kelly D; Holland, Steven M; Reich, Daniel S; Hughes, Jason D; Mehmet, Huseyin; McElwee, Joshua; Freeman, Alexandra F; Freeze, Hudson H; Su, Helen C; Milner, Joshua D

    2014-05-01

    Identifying genetic syndromes that lead to significant atopic disease can open new pathways for investigation and intervention in allergy. We sought to define a genetic syndrome of severe atopy, increased serum IgE levels, immune deficiency, autoimmunity, and motor and neurocognitive impairment. Eight patients from 2 families with similar syndromic features were studied. Thorough clinical evaluations, including brain magnetic resonance imaging and sensory evoked potentials, were performed. Peripheral lymphocyte flow cytometry, antibody responses, and T-cell cytokine production were measured. Whole-exome sequencing was performed to identify disease-causing mutations. Immunoblotting, quantitative RT-PCR, enzymatic assays, nucleotide sugar, and sugar phosphate analyses, along with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry of glycans, were used to determine the molecular consequences of the mutations. Marked atopy and autoimmunity were associated with increased T(H)2 and T(H)17 cytokine production by CD4(+) T cells. Bacterial and viral infection susceptibility were noted along with T-cell lymphopenia, particularly of CD8(+) T cells, and reduced memory B-cell numbers. Apparent brain hypomyelination resulted in markedly delayed evoked potentials and likely contributed to neurologic abnormalities. Disease segregated with novel autosomal recessive mutations in a single gene, phosphoglucomutase 3 (PGM3). Although PGM3 protein expression was variably diminished, impaired function was demonstrated by decreased enzyme activity and reduced uridine diphosphate-N-acetyl-D-glucosamine, along with decreased O- and N-linked protein glycosylation in patients' cells. These results define a new congenital disorder of glycosylation. Autosomal recessive hypomorphic PGM3 mutations underlie a disorder of severe atopy, immune deficiency, autoimmunity, intellectual disability, and hypomyelination. Published by Mosby, Inc.

  13. CHCHD10 mutations promote loss of mitochondrial cristae junctions with impaired mitochondrial genome maintenance and inhibition of apoptosis.

    PubMed

    Genin, Emmanuelle C; Plutino, Morgane; Bannwarth, Sylvie; Villa, Elodie; Cisneros-Barroso, Eugenia; Roy, Madhuparna; Ortega-Vila, Bernardo; Fragaki, Konstantina; Lespinasse, Françoise; Pinero-Martos, Estefania; Augé, Gaëlle; Moore, David; Burté, Florence; Lacas-Gervais, Sandra; Kageyama, Yusuke; Itoh, Kie; Yu-Wai-Man, Patrick; Sesaki, Hiromi; Ricci, Jean-Ehrland; Vives-Bauza, Cristofol; Paquis-Flucklinger, Véronique

    2016-01-01

    CHCHD10-related diseases include mitochondrial DNA instability disorder, frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) clinical spectrum, late-onset spinal motor neuropathy (SMAJ), and Charcot-Marie-Tooth disease type 2 (CMT2). Here, we show that CHCHD10 resides with mitofilin, CHCHD3 and CHCHD6 within the "mitochondrial contact site and cristae organizing system" (MICOS) complex. CHCHD10 mutations lead to MICOS complex disassembly and loss of mitochondrial cristae with a decrease in nucleoid number and nucleoid disorganization. Repair of the mitochondrial genome after oxidative stress is impaired in CHCHD10 mutant fibroblasts and this likely explains the accumulation of deleted mtDNA molecules in patient muscle. CHCHD10 mutant fibroblasts are not defective in the delivery of mitochondria to lysosomes suggesting that impaired mitophagy does not contribute to mtDNA instability. Interestingly, the expression of CHCHD10 mutant alleles inhibits apoptosis by preventing cytochrome c release.

  14. Role of LRRK2 in the regulation of dopamine receptor trafficking

    PubMed Central

    Sanna, Simona; Taymans, Jean Marc; Morari, Michele; Brugnoli, Alberto; Frassineti, Martina; Masala, Alessandra; Esposito, Sonia; Galioto, Manuela; Valle, Cristiana; Carri, Maria Teresa; Biosa, Alice; Greggio, Elisa; Crosio, Claudia

    2017-01-01

    Mutations in LRRK2 play a critical role in both familial and sporadic Parkinson’s disease (PD). Up to date, the role of LRRK2 in PD onset and progression remains largely unknown. However, experimental evidence highlights a critical role of LRRK2 in the control of vesicle trafficking that in turn may regulate different aspects of neuronal physiology. We have analyzed the role of LRRK2 in regulating dopamine receptor D1 (DRD1) and D2 (DRD2) trafficking. DRD1 and DRD2 are the most abundant dopamine receptors in the brain. They differ in structural, pharmacological and biochemical properties, as well as in localization and internalization mechanisms. Our results indicate that disease-associated mutant G2019S LRRK2 impairs DRD1 internalization, leading to an alteration in signal transduction. Moreover, the mutant forms of LRRK2 affect receptor turnover by decreasing the rate of DRD2 trafficking from the Golgi complex to the cell membrane. Collectively, our findings are consistent with the conclusion that LRRK2 influences the motility of neuronal vesicles and the neuronal receptor trafficking. These findings have important implications for the complex role that LRRK2 plays in neuronal physiology and the possible pathological mechanisms that may lead to neuronal death in PD. PMID:28582422

  15. The Sorting Receptor SorCS1 Regulates Trafficking of Neurexin and AMPA Receptors

    PubMed Central

    Savas, Jeffrey N.; Ribeiro, Luís F.; Wierda, Keimpe D.; Wright, Rebecca; DeNardo, Laura A.; Rice, Heather C.; Chamma, Ingrid; Wang, Yi-Zhi; Zemla, Roland; Lavallée-Adam, Mathieu; Vennekens, Kristel M.; O'Sullivan, Matthew L.; Antonios, Joseph K.; Hall, Elizabeth A.; Thoumine, Olivier; Attie, Alan D.; Ghosh, Anirvan; Yates, John R.; de Wit, Joris

    2015-01-01

    The formation, function, and plasticity of synapses require dynamic changes in synaptic receptor composition. Here we identify the sorting receptor SorCS1 as a key regulator of synaptic receptor trafficking. Four independent proteomic analyses identify the synaptic adhesion molecule neurexin and the AMPA glutamate receptor (AMPAR) as major proteins sorted by SorCS1. SorCS1 localizes to early and recycling endosomes and regulates neurexin and AMPAR surface trafficking. Surface proteome analysis of SorCS1-deficient neurons shows decreased surface levels of these, and additional, receptors. Quantitative in vivo analysis of SorCS1 knockout synaptic proteomes identifies SorCS1 as a global trafficking regulator and reveals decreased levels of receptors regulating adhesion and neurotransmission, including neurexins and AMPARs. Consequently, glutamatergic transmission at SorCS1–deficient synapses is reduced due to impaired AMPAR surface expression. SORCS1 mutations have been associated with autism and Alzheimer's disease, suggesting that perturbed receptor trafficking contributes to defects in synaptic composition and function underlying synaptopathies. PMID:26291160

  16. Pathogenic SYNGAP1 mutations impair cognitive development by disrupting maturation of dendritic spine synapses.

    PubMed

    Clement, James P; Aceti, Massimiliano; Creson, Thomas K; Ozkan, Emin D; Shi, Yulin; Reish, Nicholas J; Almonte, Antoine G; Miller, Brooke H; Wiltgen, Brian J; Miller, Courtney A; Xu, Xiangmin; Rumbaugh, Gavin

    2012-11-09

    Mutations that cause intellectual disability (ID) and autism spectrum disorder (ASD) are commonly found in genes that encode for synaptic proteins. However, it remains unclear how mutations that disrupt synapse function impact intellectual ability. In the SYNGAP1 mouse model of ID/ASD, we found that dendritic spine synapses develop prematurely during the early postnatal period. Premature spine maturation dramatically enhanced excitability in the developing hippocampus, which corresponded with the emergence of behavioral abnormalities. Inducing SYNGAP1 mutations after critical developmental windows closed had minimal impact on spine synapse function, whereas repairing these pathogenic mutations in adulthood did not improve behavior and cognition. These data demonstrate that SynGAP protein acts as a critical developmental repressor of neural excitability that promotes the development of life-long cognitive abilities. We propose that the pace of dendritic spine synapse maturation in early life is a critical determinant of normal intellectual development.

  17. Pathogenic SYNGAP1 mutations impair cognitive development by disrupting the maturation of dendritic spine synapses

    PubMed Central

    Clement, James P.; Aceti, Massimiliano; Creson, Thomas K.; Ozkan, Emin D.; Shi, Yulin; Reish, Nicholas J.; Almonte, Antoine G.; Miller, Brooke H.; Wiltgen, Brian J.; Miller, Courtney A.; Xu, Xiangmin; Rumbaugh, Gavin

    2012-01-01

    SUMMARY Mutations that cause Intellectual Disability (ID) and Autism Spectrum Disorder (ASD) are commonly found in genes that encode for synaptic proteins. However, it remains unclear how mutations that disrupt synapse function impact intellectual ability. In the SYNGAP1 mouse model of ID/ASD, we found that dendritic spine synapses develop prematurely during the early postnatal period. Premature spine maturation dramatically enhanced excitability in the developing hippocampus, which corresponded with the emergence of behavioral abnormalities. Inducing SYNGAP1 mutations after critical developmental windows closed had minimal impact on spine synapse function, while repairing these pathogenic mutations in adulthood did not improve behavior and cognition. These data demonstrate that SynGAP protein acts as a critical developmental repressor of neural excitability that promotes the development of life-long cognitive abilities. We propose that the pace of dendritic spine synapse maturation in early life is a critical determinant of normal intellectual development. PMID:23141534

  18. Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis

    PubMed Central

    Liu, Luyan; Okada, Satoshi; Kong, Xiao-Fei; Kreins, Alexandra Y.; Cypowyj, Sophie; Abhyankar, Avinash; Toubiana, Julie; Itan, Yuval; Audry, Magali; Nitschke, Patrick; Masson, Cécile; Toth, Beata; Flatot, Jérome; Migaud, Mélanie; Chrabieh, Maya; Kochetkov, Tatiana; Bolze, Alexandre; Borghesi, Alessandro; Toulon, Antoine; Hiller, Julia; Eyerich, Stefanie; Eyerich, Kilian; Gulácsy, Vera; Chernyshova, Ludmyla; Chernyshov, Viktor; Bondarenko, Anastasia; María Cortés Grimaldo, Rosa; Blancas-Galicia, Lizbeth; Madrigal Beas, Ileana Maria; Roesler, Joachim; Magdorf, Klaus; Engelhard, Dan; Thumerelle, Caroline; Burgel, Pierre-Régis; Hoernes, Miriam; Drexel, Barbara; Seger, Reinhard; Kusuma, Theresia; Jansson, Annette F.; Sawalle-Belohradsky, Julie; Belohradsky, Bernd; Jouanguy, Emmanuelle; Bustamante, Jacinta; Bué, Mélanie; Karin, Nathan; Wildbaum, Gizi; Bodemer, Christine; Lortholary, Olivier; Fischer, Alain; Blanche, Stéphane; Al-Muhsen, Saleh; Reichenbach, Janine; Kobayashi, Masao; Rosales, Francisco Espinosa; Lozano, Carlos Torres; Kilic, Sara Sebnem; Oleastro, Matias; Etzioni, Amos; Traidl-Hoffmann, Claudia; Renner, Ellen D.; Abel, Laurent; Picard, Capucine; Maródi, László; Boisson-Dupuis, Stéphanie

    2011-01-01

    Chronic mucocutaneous candidiasis disease (CMCD) may be caused by autosomal dominant (AD) IL-17F deficiency or autosomal recessive (AR) IL-17RA deficiency. Here, using whole-exome sequencing, we identified heterozygous germline mutations in STAT1 in 47 patients from 20 kindreds with AD CMCD. Previously described heterozygous STAT1 mutant alleles are loss-of-function and cause AD predisposition to mycobacterial disease caused by impaired STAT1-dependent cellular responses to IFN-γ. Other loss-of-function STAT1 alleles cause AR predisposition to intracellular bacterial and viral diseases, caused by impaired STAT1-dependent responses to IFN-α/β, IFN-γ, IFN-λ, and IL-27. In contrast, the 12 AD CMCD-inducing STAT1 mutant alleles described here are gain-of-function and increase STAT1-dependent cellular responses to these cytokines, and to cytokines that predominantly activate STAT3, such as IL-6 and IL-21. All of these mutations affect the coiled-coil domain and impair the nuclear dephosphorylation of activated STAT1, accounting for their gain-of-function and dominance. Stronger cellular responses to the STAT1-dependent IL-17 inhibitors IFN-α/β, IFN-γ, and IL-27, and stronger STAT1 activation in response to the STAT3-dependent IL-17 inducers IL-6 and IL-21, hinder the development of T cells producing IL-17A, IL-17F, and IL-22. Gain-of-function STAT1 alleles therefore cause AD CMCD by impairing IL-17 immunity. PMID:21727188

  19. A novel missense mutation in ACTG1 causes dominant deafness in a Norwegian DFNA20/26 family, but ACTG1 mutations are not frequent among families with hereditary hearing impairment.

    PubMed

    Rendtorff, Nanna D; Zhu, Mei; Fagerheim, Toril; Antal, Torben L; Jones, MaryPat; Teslovich, Tanya M; Gillanders, Elizabeth M; Barmada, Michael; Teig, Erik; Trent, Jeffrey M; Friderici, Karen H; Stephan, Dietrich A; Tranebjaerg, Lisbeth

    2006-10-01

    The gamma-actin gene (ACTG1) encodes a major cytoskeletal protein of the sensory hair cells of the cochlea. Recently, mutations in ACTG1 were found to cause autosomal dominant, progressive, sensorineural hearing impairment linked to the DFNA20/26 locus on chromosome 17q25.3 in four American families and in one Dutch family. We report here the linkage of autosomal dominant, progressive, sensorineural hearing impairment in a large Norwegian family to the DFNA20/26 locus. Sequencing of ACTG1 identified a novel missense mutation (c.1109T>C; p.V370A) segregating with the hearing loss. Functional analysis in yeast showed that the p.V370A mutation restricts cell growth at elevated temperature or under hyperosmolar stress. Molecular modelling suggested that the p.V370A mutation modestly alters a site for protein-protein interaction in gamma-actin and thereby modestly alters gamma-actin-based cytoskeletal structures. Nineteen Norwegian and Danish families with autosomal, dominant hearing impairment were analyzed for mutations in ACTG1 by sequencing, but no disease-associated mutations were identified. Finally, a long-term follow-up of the hearing loss progression associated with the p.V370A mutation in ACTG1 is provided. The present study expands our understanding of the genotype-phenotype relationship of this deafness gene and provides a sensitive and simple functional assay for missense mutations in this gene, which may assist future molecular diagnosis of autosomal-dominant hearing impairment. Finally, the present results do not indicate that mutations in ACTG1 are a frequent cause of autosomal-dominant postlingual sensorineural hearing impairment in Norway nor Denmark.

  20. From Function to Phenotype: Impaired DNA Binding and Clustering Correlates with Clinical Severity in Males with Missense Mutations in MECP2

    PubMed Central

    Sheikh, Taimoor I.; Ausió, Juan; Faghfoury, Hannah; Silver, Josh; Lane, Jane B.; Eubanks, James H.; MacLeod, Patrick; Percy, Alan K.; Vincent, John B.

    2016-01-01

    Mutations in the MECP2 gene cause Rett syndrome (RTT). MeCP2 binds to chromocentric DNA through its methyl CpG-binding domain (MBD) to regulate gene expression. In heterozygous females the variable phenotypic severity is modulated by non-random X-inactivation, thus making genotype-phenotype comparisons unreliable. However, genotype-phenotype correlations in males with hemizygousMECP2 mutations can provide more accurate insights in to the true biological effect of specific mutations. Here, we compared chromatin organization and binding dynamics for twelve MeCP2 missense mutations (including two novel and the five most common MBD missense RTT mutations) and identifiedacorrelation with phenotype in hemizygous males. We observed impaired interaction of MeCP2-DNA for mutations around the MBD-DNA binding interface, and defective chromatin clustering for distal MBD mutations. Furthermore, binding and mobility dynamics show a gradient of impairment depending on the amino acid properties and tertiary structure within the MBD. Interestingly, a wide range of phenotypic/clinical severity, ranging from neonatal encephalopathy to mild psychiatric abnormalities were observed and all are consistent with our functional/molecular results. Overall, clinical severity showed a direct correlation with the functional impairment of MeCP2. These mechanistic and phenotypic correlations of MeCP2 mutations will enable improved and individualized diagnostics, and may lead to personalized therapeutic interventions. PMID:27929079

  1. MARVELD2 (DFNB49) mutations in the hearing impaired Central European Roma population--prevalence, clinical impact and the common origin.

    PubMed

    Mašindová, Ivica; Šoltýsová, Andrea; Varga, Lukáš; Mátyás, Petra; Ficek, Andrej; Hučková, Miloslava; Sůrová, Martina; Šafka-Brožková, Dana; Anwar, Saima; Bene, Judit; Straka, Slavomír; Janicsek, Ingrid; Ahmed, Zubair M; Seeman, Pavel; Melegh, Béla; Profant, Milan; Klimeš, Iwar; Riazuddin, Saima; Kádasi, Ľudevít; Gašperíková, Daniela

    2015-01-01

    In the present study we aimed: 1) To establish the prevalence and clinical impact of DFNB49 mutations in deaf Roma from 2 Central European countries (Slovakia and Hungary), and 2) to analyze a possible common origin of the c.1331+2T>C mutation among Roma and Pakistani mutation carriers identified in the present and previous studies. We sequenced 6 exons of the MARVELD2 gene in a group of 143 unrelated hearing impaired Slovak Roma patients. Simultaneously, we used RFLP to detect the c.1331+2T>C mutation in 85 Hungarian deaf Roma patients, control groups of 702 normal hearing Romanies from both countries and 375 hearing impaired Slovak Caucasians. We analyzed the haplotype using 21 SNPs spanning a 5.34Mb around the mutation c.1331+2T>C. One pathogenic mutation (c.1331+2T>C) was identified in 12 homozygous hearing impaired Roma patients. Allele frequency of this mutation was higher in Hungarian (10%) than in Slovak (3.85%) Roma patients. The identified common haplotype in Roma patients was defined by 18 SNP markers (3.89 Mb). Fourteen common SNPs were also shared among Pakistani and Roma homozygotes. Biallelic mutation carriers suffered from prelingual bilateral moderate to profound sensorineural hearing loss. We demonstrate different frequencies of the c.1331+2T>C mutation in hearing impaired Romanies from 3 Central European countries. In addition, our results provide support for the hypothesis of a possible common ancestor of the Slovak, Hungarian and Czech Roma as well as Pakistani deaf patients. Testing for the c.1331+2T>C mutation may be recommended in GJB2 negative Roma cases with early-onset sensorineural hearing loss.

  2. Mitochondrial impairment observed in fibroblasts from South African Parkinson's disease patients with parkin mutations.

    PubMed

    van der Merwe, Celia; Loos, Ben; Swart, Chrisna; Kinnear, Craig; Henning, Franclo; van der Merwe, Lize; Pillay, Komala; Muller, Nolan; Zaharie, Dan; Engelbrecht, Lize; Carr, Jonathan; Bardien, Soraya

    2014-05-02

    Parkinson's disease (PD), defined as a neurodegenerative disorder, is characterized by the loss of dopaminergic neurons in the substantia nigra in the midbrain. Loss-of-function mutations in the parkin gene are a major cause of autosomal recessive, early-onset PD. Parkin has been implicated in the maintenance of healthy mitochondria, although previous studies show conflicting findings regarding mitochondrial abnormalities in fibroblasts from patients harboring parkin-null mutations. The aim of the present study was to determine whether South African PD patients with parkin mutations exhibit evidence for mitochondrial dysfunction. Fibroblasts were cultured from skin biopsies obtained from three patients with homozygous parkin-null mutations, two heterozygous mutation carriers and two wild-type controls. Muscle biopsies were obtained from two of the patients. The muscle fibers showed subtle abnormalities such as slightly swollen mitochondria in focal areas of the fibers and some folding of the sarcolemma. Although no differences in the degree of mitochondrial network branching were found in the fibroblasts, ultrastructural abnormalities were observed including the presence of electron-dense vacuoles. Moreover, decreased ATP levels which are consistent with mitochondrial dysfunction were observed in the patients' fibroblasts compared to controls. Remarkably, these defects did not manifest in one patient, which may be due to possible compensatory mechanisms. These results suggest that parkin-null patients exhibit features of mitochondrial dysfunction. Involvement of mitochondria as a key role player in PD pathogenesis will have important implications for the design of new and more effective therapies.

  3. Autism-Associated Insertion Mutation (InsG) of Shank3 Exon 21 Causes Impaired Synaptic Transmission and Behavioral Deficits.

    PubMed

    Speed, Haley E; Kouser, Mehreen; Xuan, Zhong; Reimers, Jeremy M; Ochoa, Christine F; Gupta, Natasha; Liu, Shunan; Powell, Craig M

    2015-07-01

    SHANK3 (also known as PROSAP2) is a postsynaptic scaffolding protein at excitatory synapses in which mutations and deletions have been implicated in patients with idiopathic autism, Phelan-McDermid (aka 22q13 microdeletion) syndrome, and other neuropsychiatric disorders. In this study, we have created a novel mouse model of human autism caused by the insertion of a single guanine nucleotide into exon 21 (Shank3(G)). The resulting frameshift causes a premature STOP codon and loss of major higher molecular weight Shank3 isoforms at the synapse. Shank3(G/G) mice exhibit deficits in hippocampus-dependent spatial learning, impaired motor coordination, altered response to novelty, and sensory processing deficits. At the cellular level, Shank3(G/G) mice also exhibit impaired hippocampal excitatory transmission and plasticity as well as changes in baseline NMDA receptor-mediated synaptic responses. This work identifies clear alterations in synaptic function and behavior in a novel, genetically accurate mouse model of autism mimicking an autism-associated insertion mutation. Furthermore, these findings lay the foundation for future studies aimed to validate and study region-selective and temporally selective genetic reversal studies in the Shank3(G/G) mouse that was engineered with such future experiments in mind.

  4. Autism-Associated Insertion Mutation (InsG) of Shank3 Exon 21 Causes Impaired Synaptic Transmission and Behavioral Deficits

    PubMed Central

    Speed, Haley E.; Kouser, Mehreen; Xuan, Zhong; Reimers, Jeremy M.; Ochoa, Christine F.; Gupta, Natasha; Liu, Shunan

    2015-01-01

    SHANK3 (also known as PROSAP2) is a postsynaptic scaffolding protein at excitatory synapses in which mutations and deletions have been implicated in patients with idiopathic autism, Phelan–McDermid (aka 22q13 microdeletion) syndrome, and other neuropsychiatric disorders. In this study, we have created a novel mouse model of human autism caused by the insertion of a single guanine nucleotide into exon 21 (Shank3G). The resulting frameshift causes a premature STOP codon and loss of major higher molecular weight Shank3 isoforms at the synapse. Shank3G/G mice exhibit deficits in hippocampus-dependent spatial learning, impaired motor coordination, altered response to novelty, and sensory processing deficits. At the cellular level, Shank3G/G mice also exhibit impaired hippocampal excitatory transmission and plasticity as well as changes in baseline NMDA receptor-mediated synaptic responses. This work identifies clear alterations in synaptic function and behavior in a novel, genetically accurate mouse model of autism mimicking an autism-associated insertion mutation. Furthermore, these findings lay the foundation for future studies aimed to validate and study region-selective and temporally selective genetic reversal studies in the Shank3G/G mouse that was engineered with such future experiments in mind. PMID:26134648

  5. Mutations in the passenger polypeptide can affect its partitioning between mitochondria and cytoplasm: mutations can impair the mitochondrial import of DsRed.

    PubMed

    Pastukh, Viktoriya; Shokolenko, Inna N; Wilson, Glenn L; Alexeyev, Mikhail F

    2008-06-01

    In this study, we report that the partitioning between mitochondria and cytoplasm of two variants, mCherry and DsRed Express (DRE), of the red fluorescent protein, DsRed, fused to one of the six matrix targeting sequences (MTSs) can be affected by both MTS and amino acid substitutions in DsRed. Of the six MTSs tested, MTSs from superoxide dismutase and DNA polymerase gamma failed to direct mCherry, but not DRE to mitochondria. By evaluating a series of chimeras between mCherry and DRE fused to the MTS of superoxide dismutase, we attribute the differences in the mitochondrial partitioning to differences in the primary amino acid sequence of the passenger polypeptide. The impairment of mitochondrial partitioning closely parallels the number of mCherry-specific mutations, and is not specific to mutations located in any particular region of the polypeptide. These observations suggest that both MTS and the passenger polypeptide affect the efficiency of mitochondrial import and provide a rationale for the observed diversity in the primary amino acid sequences of natural MTSs.

  6. Progressive hereditary hearing impairment caused by a MYO6 mutation resembles presbyacusis.

    PubMed

    Oonk, A M M; Leijendeckers, J M; Lammers, E M; Weegerink, N J D; Oostrik, J; Beynon, A J; Huygen, P L M; Kunst, H P M; Kremer, H; Snik, A F M; Pennings, R J E

    2013-05-01

    Since deafness is the most common sensorineural disorder in humans, better understanding of the underlying causes is necessary to improve counseling and rehabilitation. A Dutch family with autosomal dominantly inherited sensorineural hearing loss was clinically and genetically assessed. The MYO6 gene was selected to be sequenced because of similarities with other, previously described DFNA22 phenotypes and a pathogenic c.3610C > T (p.R1204W) mutation was found to co-segregate with the disease. This missense mutation results in a flat configured audiogram with a mild hearing loss, which becomes severe to profound and gently to steeply downsloping later in life. The age-related typical audiograms (ARTA) constructed for this family resemble presbyacusis. Speech audiometry and results of loudness scaling support the hypothesis that the phenotype of this specific MYO6 mutation mimics presbyacusis.

  7. Impaired growth and intracranial calcifications in autosomal dominant hypocalcemia caused by a GNA11 mutation.

    PubMed

    Tenhola, Sirpa; Voutilainen, Raimo; Reyes, Monica; Toiviainen-Salo, Sanna; Jüppner, Harald; Mäkitie, Outi

    2016-09-01

    Autosomal dominant hypocalcemia (ADH) is characterized by hypocalcemia and inappropriately low PTH concentrations. ADH type 1 is caused by activating mutations in the calcium-sensing receptor (CASR), a G-protein-coupled receptor signaling through α11 (Gα11) and αq (Gαq) subunits. Heterozygous activating mutations in GNA11, the gene encoding Gα11, underlie ADH type 2. This study describes disease characteristics in a family with ADH caused by a gain-of-function mutation in GNA11. A three-generation family with seven members (3 adults, 4 children) presenting with ADH. Biochemical parameters of calcium metabolism, clinical, genetic and brain imaging findings were analyzed. Sanger sequencing revealed a heterozygous GNA11 missense mutation (c.1018G>A, p.V340M) in all seven hypocalcemic subjects, but not in the healthy family members (n=4). The adult patients showed clinical symptoms of hypocalcemia, while the children were asymptomatic. Plasma ionized calcium ranged from 0.95 to 1.14mmol/L, yet plasma PTH was inappropriately low for the degree of hypocalcemia. Serum 25OHD was normal. Despite hypocalcemia 1,25(OH)2D and urinary calcium excretion were inappropriately in the reference range. None of the patients had nephrocalcinosis. Two adults and one child (of the two MRI scanned children) had distinct intracranial calcifications. All affected subjects had short stature (height s.d. scores ranging from -3.4 to -2.3 vs -0.5 in the unaffected children). The identified GNA11 mutation results in biochemical abnormalities typical for ADH. Additional features, including short stature and early intracranial calcifications, cosegregated with the mutation. These findings may indicate a wider role for Gα11 signaling besides calcium regulation. © 2016 European Society of Endocrinology.

  8. Impaired growth and intracranial calcifications in autosomal dominant hypocalcemia caused by a GNA11 mutation

    PubMed Central

    Tenhola, Sirpa; Voutilainen, Raimo; Reyes, Monica; Toiviainen-Salo, Sanna; Jüppner, Harald; Mäkitie, Outi

    2016-01-01

    Objective Autosomal dominant hypocalcemia (ADH) is characterized by hypocalcemia and inappropriately low PTH concentrations. ADH type 1 is caused by activating mutations in the calcium-sensing receptor (CASR), a G-protein-coupled receptor signaling through α11 (Gα11) and αq (Gαq) subunits. Heterozygous activating mutations in GNA11, the gene encoding Gα11, underlie ADH type 2. This study describes disease characteristics in a family with ADH caused by a presumed gain-of-function mutation in GNA11. Design A three-generation family with seven members (3 adults, 4 children) presenting with ADH. Methods Biochemical parameters of calcium metabolism, clinical, genetic and brain imaging findings were analyzed. Results Sanger sequencing revealed a heterozygous GNA11 missense mutation (c.1018G>A, p.V340M) in all seven hypocalcemic subjects, but not in the healthy family members (n = 4). The adult patients showed clinical symptoms of hypocalcemia, while the children were asymptomatic. Plasma ionized calcium ranged from 0.95 to 1.14 mmol/L, yet plasma PTH was inappropriately low for the degree of hypocalcemia. Serum 25OHD was normal. Despite hypocalcemia 1,25(OH)2D and urinary calcium excretion were inappropriately in the reference range. None of the patients had nephrocalcinosis. Two adults and one child (of the two MRI scanned children) had distinct intracranial calcifications. All affected subjects had short stature (height s.d. scores ranging from −3.4 to −2.3 vs −0.5 in the unaffected children). Conclusions The identified GNA11 mutation results in biochemical abnormalities typical for ADH. Additional features, including short stature and early intracranial calcifications, cosegregated with the mutation. These findings may indicate a wider role for Gα11 signaling besides calcium regulation. PMID:27334330

  9. Putative Breast Cancer Driver Mutations in TBX3 Cause Impaired Transcriptional Repression

    PubMed Central

    Fischer, Kathrin; Pflugfelder, Gert O.

    2015-01-01

    The closely related T-box transcription factors TBX2 and TBX3 are frequently overexpressed in melanoma and various types of human cancers, in particular, breast cancer. The overexpression of TBX2 and TBX3 can have several cellular effects, among them suppression of senescence, promotion of epithelial–mesenchymal transition, and invasive cell motility. In contrast, loss of function of TBX3 and most other human T-box genes causes developmental haploinsufficiency syndromes. Stephens and colleagues (1), by exome sequencing of breast tumor samples, identified five different mutations in TBX3, all affecting the DNA-binding T-domain. One in-frame deletion of a single amino acid, p.N212delN, was observed twice. Due to the clustering of these mutations to the T-domain and for statistical reasons, TBX3 was inferred to be a driver gene in breast cancer. Since mutations in the T-domain generally cause loss of function and because the tumorigenic action of TBX3 has generally been attributed to overexpression, we determined whether the putative driver mutations had loss- or gain-of-function properties. We tested two in-frame deletions, one missense, and one frameshift mutant protein for DNA-binding in vitro, and for target gene repression in cell culture. In addition, we performed an in silico analysis of somatic TBX mutations in breast cancer, collected in The Cancer Genome Atlas (TCGA). Both the experimental and the in silico analysis indicate that the observed mutations predominantly cause loss of TBX3 function. PMID:26579496

  10. Mitochondrial impairment observed in fibroblasts from South African Parkinson’s disease patients with parkin mutations

    SciTech Connect

    Merwe, Celia van der; Loos, Ben; Swart, Chrisna; Kinnear, Craig; Merwe, Lize van der; Pillay, Komala; Muller, Nolan; Zaharie, Dan; Engelbrecht, Lize; Carr, Jonathan; and others

    2014-05-02

    Highlights: • Mitochondrial dysfunction observed in patients with parkin-null mutations. • Mitochondrial ATP levels were decreased. • Electron-dense vacuoles were observed in the patients. • Mitochondria from muscle biopsies appeared within normal limits. • One patient did not show these defects possibly due to compensatory mechanisms. - Abstract: Parkinson’s disease (PD), defined as a neurodegenerative disorder, is characterized by the loss of dopaminergic neurons in the substantia nigra in the midbrain. Loss-of-function mutations in the parkin gene are a major cause of autosomal recessive, early-onset PD. Parkin has been implicated in the maintenance of healthy mitochondria, although previous studies show conflicting findings regarding mitochondrial abnormalities in fibroblasts from patients harboring parkin-null mutations. The aim of the present study was to determine whether South African PD patients with parkin mutations exhibit evidence for mitochondrial dysfunction. Fibroblasts were cultured from skin biopsies obtained from three patients with homozygous parkin-null mutations, two heterozygous mutation carriers and two wild-type controls. Muscle biopsies were obtained from two of the patients. The muscle fibers showed subtle abnormalities such as slightly swollen mitochondria in focal areas of the fibers and some folding of the sarcolemma. Although no differences in the degree of mitochondrial network branching were found in the fibroblasts, ultrastructural abnormalities were observed including the presence of electron-dense vacuoles. Moreover, decreased ATP levels which are consistent with mitochondrial dysfunction were observed in the patients’ fibroblasts compared to controls. Remarkably, these defects did not manifest in one patient, which may be due to possible compensatory mechanisms. These results suggest that parkin-null patients exhibit features of mitochondrial dysfunction. Involvement of mitochondria as a key role player in PD

  11. [Hereditary sensorineural hearing impairment and macrothrombocytopenia: a rare MYH9 gene mutation].

    PubMed

    Böttcher, A; Knecht, R; Busch, C-J; Lörincz, B B; Dalchow, C V

    2013-02-01

    We report on a rare case of an exon 16 mutation of the MYH9 gene in a 23-year-old woman. This gene encodes for non-muscular myosin IIA, which acts as a cytoskeletal contractile protein in diverse cell types. This disorder led to sensorineural hearing loss, macrothrombocytopenia, and proteinuria. MYH9 gene mutation can lead to diverse organ manifestation like pre-senile cataract or renal failure which are progressive in course. Due to the current lack of causal treatment, diagnostic steps, advice for follow-up examinations and symptomatic therapy approaches are presented.

  12. ATP2C1 gene mutations in Hailey–Hailey disease and possible roles of SPCA1 isoforms in membrane trafficking

    PubMed Central

    Micaroni, M; Giacchetti, G; Plebani, R; Xiao, G G; Federici, L

    2016-01-01

    ATP2C1 gene codes for the secretory pathway Ca2+/Mn2+-ATPase pump type 1 (SPCA1) localizing at the golgi apparatus. Mutations on the human ATP2C1 gene, causing decreased levels of the SPCA1 expression, have been identified as the cause of the Hailey–Hailey disease, a rare skin disorder. In the last few years, several mutations have been described, and here we summarize how they are distributed along the gene and how missense mutations affect protein expression. SPCA1 is expressed in four different isoforms through alternative splicing of the ATP2C1 gene and none of these isoforms is differentially affected by any of these mutations. However, a better understanding of the tissue specific expression of the isoforms, their localization along the secretory pathway, their specific binding partners and the role of the C-terminal tail making isoforms different from each other, will be future goals of the research in this field. PMID:27277681

  13. A novel ENG mutation causing impaired co-translational processing of endoglin associated with hereditary hemorrhagic telangiectasia.

    PubMed

    Suzuki, Atsuo; Nakashima, Daisuke; Miyawaki, Yuhri; Fujita, Junko; Maki, Asuka; Fujimori, Yuta; Takagi, Akira; Murate, Takashi; Teranishi, Masaaki; Matsushita, Tadashi; Saito, Hidehiko; Kojima, Tetsuhito

    2012-05-01

    Hereditary hemorrhagic telangiectasia (HHT) is an inherited autosomal dominant vascular dysplasia caused by mutations in mainly the endoglin gene (ENG) or activin-like kinase receptor 1 (ALK1) gene (ACVRL1). We investigated the molecular basis of HHT in a Japanese patient, and identified a novel missense mutation in ENG (c.38T>A, p.Leu13Gln) located in the signal peptide's hydrophobic core, but not in ACVRL1. In experiments in COS-1 cells, the Leu13Gln (L13Q) mutant endoglin appeared to be expressed as a precursor form, probably due to impaired protein processing. Flow cytometry analyses of the COS-1 cells transiently expressing recombinant endoglins revealed that the wild-type endoglin was detected on the cell surface, but the L13Q mutant was not. We also analyzed expression patterns of the recombinant endoglins by immunofluorescent staining, and found that the wild-type co-localized with the endoplasmic reticulum (ER), but the L13Q mutant did not. These results implied that the L13Q mutant endoglin fails to insert into the ER, probably due to destruction of the hydrophobic core structure in the signal peptide to be recognized by signal recognition particles. Thus, the Leu13 in the signal peptide of endoglin might be essential for correct protein processing through the ER and cell-surface expression. Taken together, the novel c.38T>A mutation in ENG would impair co-translational processing of the endoglin, and could be responsible for HHT in this patient. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. A deleterious Nav1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

    PubMed Central

    Sun, Yishan; Paşca, Sergiu P; Portmann, Thomas; Goold, Carleton; Worringer, Kathleen A; Guan, Wendy; Chan, Karen C; Gai, Hui; Vogt, Daniel; Chen, Ying-Jiun J; Mao, Rong; Chan, Karrie; Rubenstein, John LR; Madison, Daniel V; Hallmayer, Joachim; Froehlich-Santino, Wendy M; Bernstein, Jonathan A; Dolmetsch, Ricardo E

    2016-01-01

    Dravet Syndrome is an intractable form of childhood epilepsy associated with deleterious mutations in SCN1A, the gene encoding neuronal sodium channel Nav1.1. Earlier studies using human induced pluripotent stem cells (iPSCs) have produced mixed results regarding the importance of Nav1.1 in human inhibitory versus excitatory neurons. We studied a Nav1.1 mutation (p.S1328P) identified in a pair of twins with Dravet Syndrome and generated iPSC-derived neurons from these patients. Characterization of the mutant channel revealed a decrease in current amplitude and hypersensitivity to steady-state inactivation. We then differentiated Dravet-Syndrome and control iPSCs into telencephalic excitatory neurons or medial ganglionic eminence (MGE)-like inhibitory neurons. Dravet inhibitory neurons showed deficits in sodium currents and action potential firing, which were rescued by a Nav1.1 transgene, whereas Dravet excitatory neurons were normal. Our study identifies biophysical impairments underlying a deleterious Nav1.1 mutation and supports the hypothesis that Dravet Syndrome arises from defective inhibitory neurons. DOI: http://dx.doi.org/10.7554/eLife.13073.001 PMID:27458797

  15. Opinions of hearing parents about the causes of hearing impairment of their children with biallelic GJB2 mutations.

    PubMed

    Solovyev, Aisen V; Dzhemileva, Lilya U; Posukh, Olga L; Barashkov, Nikolay A; Bady-Khoo, Marita S; Lobov, Semen L; Popova, Natalya Yu; Romanov, Georgii P; Sazonov, Nikolay N; Bondar, Alexander A; Morozov, Igor V; Tomsky, Mikhail I; Fedorova, Sardana A; Khusnutdinova, Elza K

    2017-03-21

    Hereditary hearing impairment (HI) caused by recessive GJB2 mutations is a frequent sensory disorder. The results of the molecular-based studies of HI are widely used in various genetic test systems. However, the ethical aspects are less described than the genetic aspects. The concerns expressed by individuals from groups with genetic risks must be included in the counseling of patients and their families. For evaluation of subjective opinions of hearing parents about the presumed causes of HI of their children, we analyze the cohort of parents having children with confirmed hereditary HI caused by biallelic recessive GJB2 mutations (in a homozygous or a compound heterozygous state). This study included 70 deaf children with HI due to mutations in the GJB2 gene and 91 questionnaires about the presumed causes of their deafness filled by their parents. Most of the parents at 78% (CI 68.4-85.4%) attributed their children's HI to "non-hereditary" causes and 22% (CI 14.7-31.6%) to "hereditary" causes (p < 0.05). Therefore, the prior opinions of the parents did not correspond to positive GJB2 genetic testing results. The subjective opinions of parents are probably partly based on family history, since respondents with deaf relatives in their pedigree more likely supposed hereditary causes for HI in their children than the respondents without deaf relatives (p < 0.001).

  16. VCP is essential for mitochondrial quality control by PINK1/Parkin and this function is impaired by VCP mutations

    PubMed Central

    Kim, Nam Chul; Tresse, Emilie; Kolaitis, Regina M.; Molliex, Amandine; Thomas, Ruth E.; Alami, Nael H.; Wang, Bo; Joshi, Aashish; Smith, Rebecca B.; Ritson, Gillian P.; Winborn, Brett J.; Moore, Jennifer; Lee, Joo-Yong; Yao, Tso-Pang; Pallanck, Leo; Kundu, Mondira; Taylor, J. Paul

    2013-01-01

    Mutations in VCP cause multisystem degeneration impacting the nervous system, muscle, and/or bone. Patients may present with ALS, Parkinsonism, frontotemporal dementia, myopathy, Paget’s disease or a combination of these. The disease mechanism is unknown. We developed a Drosophila model of VCP mutation-dependent degeneration. The phenotype is reminiscent of PINK1 and parkin mutants, including a pronounced mitochondrial defect. Indeed, VCP interacts genetically with the PINK1/parkin pathway in vivo. Paradoxically, VCP complements PINK1 deficiency but not parkin deficiency. The basis of this paradox is resolved by mechanistic studies in vitro showing that VCP recruitment to damaged mitochondria requires Parkin-mediated ubiquitination of mitochondrial targets. VCP recruitment coincides temporally with mitochondrial fission, and VCP is required for proteasome-dependent degradation of Mitofusins in vitro and in vivo. Further, VCP and its adaptor Npl4/Ufd1 are required for clearance of damaged mitochondria via the PINK1/Parkin pathway, and this is impaired by pathogenic mutations in VCP. PMID:23498974

  17. DNMT3A R882 mutations promote anthracycline resistance in acute myeloid leukemia through impaired nucleosome remodeling

    PubMed Central

    Guryanova, Olga A.; Shank, Kaitlyn; Spitzer, Barbara; Luciani, Luisa; Koche, Richard P.; Garrett-Bakelman, Francine E.; Ganzel, Chezi; Durham, Benjamin H.; Mohanty, Abhinita; Hoermann, Gregor; Rivera, Sharon A.; Chramiec, Alan G.; Pronier, Elodie; Bastian, Lennart; Keller, Matthew D.; Tovbin, Daniel; Loizou, Evangelia; Weinstein, Abby R.; Gonzalez, Adriana Rodriguez; Lieu, Yen; Rowe, Jacob M.; Pastore, Friederike; McKenney, Anna Sophia; Krivtsov, Andrei V.; Sperr, Wolfgang R.; Cross, Justin R.; Mason, Christopher E.; Tallman, Martin S.; Arcila, Maria E.; Abdel-Wahab, Omar; Armstrong, Scott A.; Kubicek, Stefan; Staber, Philipp B.; Gönen, Mithat; Paietta, Elisabeth M.; Melnick, Ari M.; Nimer, Stephen D.; Mukherjee, Siddhartha; Levine, Ross L.

    2017-01-01

    Although the majority of acute myeloid leukemia (AML) patients initially respond to chemotherapy, many patients subsequently relapse; the mechanistic basis for AML persistence following chemotherapy has not been delineated. Recurrent somatic mutations in DNA methyltransferase 3A (DNMT3A), most frequently at arginine 882 (DNMT3Amut), are observed in AML1–3 and in individuals with clonal hematopoiesis in the absence of leukemic transformation4,5. DNMT3Amut AML patients have an inferior outcome when treated with standard-dose daunorubicin-based induction chemotherapy6,7, suggesting that DNMT3Amut cells persist and drive relapse8. Here we show that Dnmt3amut induces hematopoietic stem cell (HSC) expansion, cooperates with Flt3ITD and Npm1c to induce AML in vivo, and promotes resistance to anthracycline chemotherapy. In AML patients, DNMT3AR882 mutations predict for minimal residual disease (MRD), underscoring their role in AML chemoresistance. DNMT3Amut cells show impaired nucleosome eviction and chromatin remodeling in response to anthracyclines, resulting from attenuated recruitment of histone chaperone SPT-16 following anthracycline exposure. This defect leads to an inability to sense and repair DNA torsional stress, which results in increased mutagenesis. Our studies identify a critical role for DNMT3AR882 mutations in driving AML chemoresistance, and highlight the importance of chromatin remodeling in response to cytotoxic chemotherapy. PMID:27841873

  18. A deleterious Nav1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients.

    PubMed

    Sun, Yishan; Paşca, Sergiu P; Portmann, Thomas; Goold, Carleton; Worringer, Kathleen A; Guan, Wendy; Chan, Karen C; Gai, Hui; Vogt, Daniel; Chen, Ying-Jiun J; Mao, Rong; Chan, Karrie; Rubenstein, John Lr; Madison, Daniel V; Hallmayer, Joachim; Froehlich-Santino, Wendy M; Bernstein, Jonathan A; Dolmetsch, Ricardo E

    2016-07-26

    Dravet Syndrome is an intractable form of childhood epilepsy associated with deleterious mutations in SCN1A, the gene encoding neuronal sodium channel Nav1.1. Earlier studies using human induced pluripotent stem cells (iPSCs) have produced mixed results regarding the importance of Nav1.1 in human inhibitory versus excitatory neurons. We studied a Nav1.1 mutation (p.S1328P) identified in a pair of twins with Dravet Syndrome and generated iPSC-derived neurons from these patients. Characterization of the mutant channel revealed a decrease in current amplitude and hypersensitivity to steady-state inactivation. We then differentiated Dravet-Syndrome and control iPSCs into telencephalic excitatory neurons or medial ganglionic eminence (MGE)-like inhibitory neurons. Dravet inhibitory neurons showed deficits in sodium currents and action potential firing, which were rescued by a Nav1.1 transgene, whereas Dravet excitatory neurons were normal. Our study identifies biophysical impairments underlying a deleterious Nav1.1 mutation and supports the hypothesis that Dravet Syndrome arises from defective inhibitory neurons.

  19. SLC30A10 is a cell surface-localized manganese efflux transporter, and parkinsonism-causing mutations block its intracellular trafficking and efflux activity.

    PubMed

    Leyva-Illades, Dinorah; Chen, Pan; Zogzas, Charles E; Hutchens, Steven; Mercado, Jonathan M; Swaim, Caleb D; Morrisett, Richard A; Bowman, Aaron B; Aschner, Michael; Mukhopadhyay, Somshuvra

    2014-10-15

    Manganese (Mn) is an essential metal, but elevated cellular levels are toxic and may lead to the development of an irreversible parkinsonian-like syndrome that has no treatment. Mn-induced parkinsonism generally occurs as a result of exposure to elevated Mn levels in occupational or environmental settings. Additionally, patients with compromised liver function attributable to diseases, such as cirrhosis, fail to excrete Mn and may develop Mn-induced parkinsonism in the absence of exposure to elevated Mn. Recently, a new form of familial parkinsonism was reported to occur as a result of mutations in SLC30A10. The cellular function of SLC30A10 and the mechanisms by which mutations in this protein cause parkinsonism are unclear. Here, using a combination of mechanistic and functional studies in cell culture, Caenorhabditis elegans, and primary midbrain neurons, we show that SLC30A10 is a cell surface-localized Mn efflux transporter that reduces cellular Mn levels and protects against Mn-induced toxicity. Importantly, mutations in SLC30A10 that cause familial parkinsonism blocked the ability of the transporter to traffic to the cell surface and to mediate Mn efflux. Although expression of disease-causing SLC30A10 mutations were not deleterious by themselves, neurons and worms expressing these mutants exhibited enhanced sensitivity to Mn toxicity. Our results provide novel insights into the mechanisms involved in the onset of a familial form of parkinsonism and highlight the possibility of using enhanced Mn efflux as a therapeutic strategy for the potential management of Mn-induced parkinsonism, including that occurring as a result of mutations in SLC30A10.

  20. SLC30A10 Is a Cell Surface-Localized Manganese Efflux Transporter, and Parkinsonism-Causing Mutations Block Its Intracellular Trafficking and Efflux Activity

    PubMed Central

    Leyva-Illades, Dinorah; Chen, Pan; Zogzas, Charles E.; Hutchens, Steven; Mercado, Jonathan M.; Swaim, Caleb D.; Morrisett, Richard A.; Bowman, Aaron B.

    2014-01-01

    Manganese (Mn) is an essential metal, but elevated cellular levels are toxic and may lead to the development of an irreversible parkinsonian-like syndrome that has no treatment. Mn-induced parkinsonism generally occurs as a result of exposure to elevated Mn levels in occupational or environmental settings. Additionally, patients with compromised liver function attributable to diseases, such as cirrhosis, fail to excrete Mn and may develop Mn-induced parkinsonism in the absence of exposure to elevated Mn. Recently, a new form of familial parkinsonism was reported to occur as a result of mutations in SLC30A10. The cellular function of SLC30A10 and the mechanisms by which mutations in this protein cause parkinsonism are unclear. Here, using a combination of mechanistic and functional studies in cell culture, Caenorhabditis elegans, and primary midbrain neurons, we show that SLC30A10 is a cell surface-localized Mn efflux transporter that reduces cellular Mn levels and protects against Mn-induced toxicity. Importantly, mutations in SLC30A10 that cause familial parkinsonism blocked the ability of the transporter to traffic to the cell surface and to mediate Mn efflux. Although expression of disease-causing SLC30A10 mutations were not deleterious by themselves, neurons and worms expressing these mutants exhibited enhanced sensitivity to Mn toxicity. Our results provide novel insights into the mechanisms involved in the onset of a familial form of parkinsonism and highlight the possibility of using enhanced Mn efflux as a therapeutic strategy for the potential management of Mn-induced parkinsonism, including that occurring as a result of mutations in SLC30A10. PMID:25319704

  1. Clinical or ATPase domain mutations in ABCD4 disrupt the interaction between the vitamin B12-trafficking proteins ABCD4 and LMBD1.

    PubMed

    Fettelschoss, Victoria; Burda, Patricie; Sagné, Corinne; Coelho, David; De Laet, Corinne; Lutz, Seraina; Suormala, Terttu; Fowler, Brian; Pietrancosta, Nicolas; Gasnier, Bruno; Bornhauser, Beat; Froese, D Sean; Baumgartner, Matthias R

    2017-07-14

    Vitamin B12 (cobalamin (Cbl)), in the cofactor forms methyl-Cbl and adenosyl-Cbl, is required for the function of the essential enzymes methionine synthase and methylmalonyl-CoA mutase, respectively. Cbl enters mammalian cells by receptor-mediated endocytosis of protein-bound Cbl followed by lysosomal export of free Cbl to the cytosol and further processing to these cofactor forms. The integral membrane proteins LMBD1 and ABCD4 are required for lysosomal release of Cbl, and mutations in the genes LMBRD1 and ABCD4 result in the cobalamin metabolism disorders cblF and cblJ. We report a new (fifth) patient with the cblJ disorder who presented at 7 days of age with poor feeding, hypotonia, methylmalonic aciduria, and elevated plasma homocysteine and harbored the mutations c.1667_1668delAG [p.Glu556Glyfs*27] and c.1295G>A [p.Arg432Gln] in the ABCD4 gene. Cbl cofactor forms are decreased in fibroblasts from this patient but could be rescued by overexpression of either ABCD4 or, unexpectedly, LMBD1. Using a sensitive live-cell FRET assay, we demonstrated selective interaction between ABCD4 and LMBD1 and decreased interaction when ABCD4 harbored the patient mutations p.Arg432Gln or p.Asn141Lys or when artificial mutations disrupted the ATPase domain. Finally, we showed that ABCD4 lysosomal targeting depends on co-expression of, and interaction with, LMBD1. These data broaden the patient and mutation spectrum of cblJ deficiency, establish a sensitive live-cell assay to detect the LMBD1-ABCD4 interaction, and confirm the importance of this interaction for proper intracellular targeting of ABCD4 and cobalamin cofactor synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. NADP(+)-IDH Mutations Promote Hypersuccinylation that Impairs Mitochondria Respiration and Induces Apoptosis Resistance.

    PubMed

    Li, Feng; He, Xiadi; Ye, Dingwei; Lin, Yan; Yu, Hongxiu; Yao, Cuifang; Huang, Lei; Zhang, Jianong; Wang, Fang; Xu, Sha; Wu, Xiaohui; Liu, Lixia; Yang, Chen; Shi, Jiaqi; He, Xiaoyang; Liu, Jie; Qu, Yuanyuan; Guo, Fushen; Zhao, Jianyuan; Xu, Wei; Zhao, Shimin

    2015-11-19

    Elucidating the tumorigenic mechanism of R-2-hydroxyglutarate (R-2HG) is critical for determining how NADP(+)-IDH mutations cause cancer. Here we report that R-2HG induces cancerous metabolism and apoptosis resistance through promoting hypersuccinylation. By competitive inhibition of the mitochondrial tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH), R-2HG preferentially induced succinyl-CoA accumulation and hypersuccinylation in the mitochondria. IDH1 mutation-bearing glioma samples and cells were hypersuccinylated in the mitochondria. IDH1 mutation or SDH inactivation resulted in hypersuccinylation, causing respiration inhibition and inducing cancerous metabolism and mitochondrial depolarization. These mitochondrial dysfunctions induced BCL-2 accumulation at the mitochondrial membrane, leading to apoptosis resistance of hypersuccinylated cells. Relief of hypersuccinylation by overexpressing the desuccinylase SIRT5 or supplementing glycine rescued mitochondrial dysfunctions, reversed BCL-2 accumulation, and slowed the oncogenic growth of hypersuccinylated IDH1(R132C)-harboring HT1080 cells. Thus, R-2HG-induced hypersuccinylation contributes to the tumorigenicity of NADP(+)-IDH mutations, suggesting the potential of hypersuccinylation inhibition as an intervention for hypersuccinylation-related tumors.

  3. adPEO mutations in ANT1 impair ADP-ATP translocation in muscle mitochondria.

    PubMed

    Kawamata, Hibiki; Tiranti, Valeria; Magrané, Jordi; Chinopoulos, Christos; Manfredi, Giovanni

    2011-08-01

    Mutations in the heart and muscle isoform of adenine nucleotide translocator 1 (ANT1) are associated with autosomal-dominant progressive external opthalmoplegia (adPEO) clinically characterized by exercise intolerance, ptosis and muscle weakness. The pathogenic mechanisms underlying the mitochondrial myopathy caused by ANT1 mutations remain largely unknown. In yeast, expression of ANT1 carrying mutations corresponding to the human adPEO ones causes a wide range of mitochondrial abnormalities. However, functional studies of ANT1 mutations in mammalian cells are lacking, because they have been hindered by the fact that ANT1 expression leads to apoptotic cell death in commonly utilized replicating cell lines. Here, we successfully express functional ANT1 in differentiated mouse myotubes, which naturally contain high levels of ANT1, without causing cell death. We demonstrate, for the first time in these disease-relevant mammalian cells, that mutant human ANT1 causes dominant mitochondrial defects characterized by decreased ADP-ATP exchange function and abnormal translocator reversal potential. These abnormalities are not due to ANT1 loss of function, because knocking down Ant1 in myotubes causes functional changes different from ANT1 mutants. Under certain physiological conditions, mitochondria consume ATP to maintain membrane potential by reversing the ADP-ATP transport. The modified properties of mutant ANT1 can be responsible for disease pathogenesis in adPEO, because exchange reversal occurring at higher than normal membrane potential can cause excessive energy depletion and nucleotide imbalance in ANT1 mutant muscle cells.

  4. adPEO mutations in ANT1 impair ADP–ATP translocation in muscle mitochondria

    PubMed Central

    Kawamata, Hibiki; Tiranti, Valeria; Magrané, Jordi; Chinopoulos, Christos; Manfredi, Giovanni

    2011-01-01

    Mutations in the heart and muscle isoform of adenine nucleotide translocator 1 (ANT1) are associated with autosomal-dominant progressive external opthalmoplegia (adPEO) clinically characterized by exercise intolerance, ptosis and muscle weakness. The pathogenic mechanisms underlying the mitochondrial myopathy caused by ANT1 mutations remain largely unknown. In yeast, expression of ANT1 carrying mutations corresponding to the human adPEO ones causes a wide range of mitochondrial abnormalities. However, functional studies of ANT1 mutations in mammalian cells are lacking, because they have been hindered by the fact that ANT1 expression leads to apoptotic cell death in commonly utilized replicating cell lines. Here, we successfully express functional ANT1 in differentiated mouse myotubes, which naturally contain high levels of ANT1, without causing cell death. We demonstrate, for the first time in these disease-relevant mammalian cells, that mutant human ANT1 causes dominant mitochondrial defects characterized by decreased ADP–ATP exchange function and abnormal translocator reversal potential. These abnormalities are not due to ANT1 loss of function, because knocking down Ant1 in myotubes causes functional changes different from ANT1 mutants. Under certain physiological conditions, mitochondria consume ATP to maintain membrane potential by reversing the ADP–ATP transport. The modified properties of mutant ANT1 can be responsible for disease pathogenesis in adPEO, because exchange reversal occurring at higher than normal membrane potential can cause excessive energy depletion and nucleotide imbalance in ANT1 mutant muscle cells. PMID:21586654

  5. Novel IL1RAPL1 mutations associated with intellectual disability impair synaptogenesis

    PubMed Central

    Ramos-Brossier, Mariana; Montani, Caterina; Lebrun, Nicolas; Gritti, Laura; Martin, Christelle; Seminatore-Nole, Christine; Toussaint, Aurelie; Moreno, Sarah; Poirier, Karine; Dorseuil, Olivier; Chelly, Jamel; Hackett, Anna; Gecz, Jozef; Bieth, Eric; Faudet, Anne; Heron, Delphine; Kooy, Frank; Loeys, Bart; Humeau, Yann; Sala, Carlo; Billuart, Pierre

    2015-01-01

    Mutations in interleukin-1 receptor accessory protein like 1 (IL1RAPL1) gene have been associated with non-syndromic intellectual disability and autism spectrum disorder. This protein interacts with synaptic partners like PSD-95 and PTPδ, regulating the formation and function of excitatory synapses. The aim of this work is to characterize the synaptic consequences of three IL1RAPL1 mutations, two novel causing the deletion of exon 6 (Δex6) and one point mutation (C31R), identified in patients with intellectual disability. Using immunofluorescence and electrophysiological recordings we examined the effects of IL1RAPL1 mutants over-expression on synapse formation and function in cultured rodent hippocampal neurons. Δex6 but not C31R mutation leads to IL1RAPL1 protein instability and mislocalization within dendrites. Analysis of different markers of excitatory synapses and sEPSC recording revealed that both mutants fail to induce pre- and post-synaptic differentiation, contrary to WT IL1RAPL1 protein. Cell aggregation and immunoprecipitation assays in HEK293 cells showed a reduction of the interaction between IL1RAPL1 mutants and PTPδ that could explain the observed synaptogenic defect in neurons. However, these mutants do not affect all cellular signaling since their over-expression still activates JNK pathway. We conclude that both mutations described in this study lead to a partial loss of function of the IL1RAPL1 protein through different mechanisms. Our work highlights the important function of the trans-synaptic PTPδ/ IL1RAPL1 interaction in synaptogenesis and as such, in intellectual disability in the patients. PMID:25305082

  6. Novel IL1RAPL1 mutations associated with intellectual disability impair synaptogenesis.

    PubMed

    Ramos-Brossier, Mariana; Montani, Caterina; Lebrun, Nicolas; Gritti, Laura; Martin, Christelle; Seminatore-Nole, Christine; Toussaint, Aurelie; Moreno, Sarah; Poirier, Karine; Dorseuil, Olivier; Chelly, Jamel; Hackett, Anna; Gecz, Jozef; Bieth, Eric; Faudet, Anne; Heron, Delphine; Frank Kooy, R; Loeys, Bart; Humeau, Yann; Sala, Carlo; Billuart, Pierre

    2015-02-15

    Mutations in interleukin-1 receptor accessory protein like 1 (IL1RAPL1) gene have been associated with non-syndromic intellectual disability (ID) and autism spectrum disorder. This protein interacts with synaptic partners like PSD-95 and PTPδ, regulating the formation and function of excitatory synapses. The aim of this work was to characterize the synaptic consequences of three IL1RAPL1 mutations, two novel causing the deletion of exon 6 (Δex6) and one point mutation (C31R), identified in patients with ID. Using immunofluorescence and electrophysiological recordings, we examined the effects of IL1RAPL1 mutant over-expression on synapse formation and function in cultured rodent hippocampal neurons. Δex6 but not C31R mutation leads to IL1RAPL1 protein instability and mislocalization within dendrites. Analysis of different markers of excitatory synapses and sEPSC recording revealed that both mutants fail to induce pre- and post-synaptic differentiation, contrary to WT IL1RAPL1 protein. Cell aggregation and immunoprecipitation assays in HEK293 cells showed a reduction of the interaction between IL1RAPL1 mutants and PTPδ that could explain the observed synaptogenic defect in neurons. However, these mutants do not affect all cellular signaling because their over-expression still activates JNK pathway. We conclude that both mutations described in this study lead to a partial loss of function of the IL1RAPL1 protein through different mechanisms. Our work highlights the important function of the trans-synaptic PTPδ/IL1RAPL1 interaction in synaptogenesis and as such in ID in the patients.

  7. D184E mutation in aquaporin-4 gene impairs water permeability and links to deafness.

    PubMed

    Nicchia, G P; Ficarella, R; Rossi, A; Giangreco, I; Nicolotti, O; Carotti, A; Pisani, F; Estivill, X; Gasparini, P; Svelto, M; Frigeri, A

    2011-12-01

    Aquaporins (AQPs) play a physiological role in several organs and tissues, and their alteration is associated with disorders of water regulation. The identification of molecular interactions, which are crucial in determining the rate of water flux through the channel, is of pivotal role for the discovery of molecules able to target those interactions and therefore to be used for pathologies ascribable to an altered AQP-dependent water balance. In the present study, a mutational screening of human aquaporin-4 (AQP4) gene was performed on subjects with variable degrees of hearing loss. One heterozygous missense mutation was identified in a Spanish sporadic case, leading to an Asp/Glu amino acid substitution at position 184 (D184E). A BLAST analysis revealed that the amino acid D184 is conserved across species, consistently with a crucial role in the structure/function of AQP4 water channels. The mutation induces a significant reduction in water permeability as measured by the Xenopus laevis oocytes swelling assay and by the use of mammalian cells by total internal reflection microscopy. By Western blot, immunofluorescence and 2D Blue Native/SDS-PAGE we show that the reduction in water permeability is not ascribable to a reduced expression of AQP4 mutant protein or to its incorrect plasma membrane targeting and aggregation into orthogonal arrays of particles. Molecular dynamics simulation provided a molecular explanation of the mechanism whereby the mutation induces a loss of function of the channel. Substituting glutamate for aspartate affects the mobility of the D loop, which acquires a higher propensity to equilibrate in a "closed conformation", thus affecting the rate of water flux. We speculate that this mutation, combined with other genetic defects or concurrently with certain environmental stimuli, could confer a higher susceptibility to deafness.

  8. Dravet Syndrome and "SCN1A" Gene Mutation Related-Epilepsies: Cognitive Impairment and Its Determinants

    ERIC Educational Resources Information Center

    Guerrini, Renzo; Falchi, Melania

    2011-01-01

    Some studies have demonstrated that cognitive decline occurs in Dravet syndrome, starting shortly after the onset of seizures, rapidly progressing and then plateauing within a few years. It is unclear whether children that develop the syndrome had entirely normal cognitive skills before seizure onset, since subtle impairment easily escapes…

  9. Dravet Syndrome and "SCN1A" Gene Mutation Related-Epilepsies: Cognitive Impairment and Its Determinants

    ERIC Educational Resources Information Center

    Guerrini, Renzo; Falchi, Melania

    2011-01-01

    Some studies have demonstrated that cognitive decline occurs in Dravet syndrome, starting shortly after the onset of seizures, rapidly progressing and then plateauing within a few years. It is unclear whether children that develop the syndrome had entirely normal cognitive skills before seizure onset, since subtle impairment easily escapes…

  10. Functional characterization of an AQP0 missense mutation, R33C, that causes dominant congenital lens cataract, reveals impaired cell-to-cell adhesion

    PubMed Central

    Kumari, Sindhu S.; Gandhi, Jason; Mustehsan, Mohammed H.; Eren, Semih; Varadaraj, Kulandaiappan

    2013-01-01

    Aquaporin 0 (AQP0) performs dual functions in the lens fiber cells, as a water pore and as a cell-to-cell adhesion molecule. Mutations in AQP0 cause severe lens cataract in both humans and mice. An arginine to cysteine missense mutation at amino acid 33 (R33C) produced congenital autosomal dominant cataract in a Chinese family for five generations. We re-created this mutation in wild type (WT-AQP0) human AQP0 cDNA by site-directed mutagenesis, and cloned and expressed the mutant AQP0 (AQP0-R33C) in heterologous expression systems. Mutant AQP0-R33C showed proper trafficking and membrane localization like WT-AQP0. Functional studies conducted in Xenopus oocytes showed no significant difference (P>0.05) in water permeability between AQP0-R33C and WT-AQP0. However, the cell-to-cell adhesion property of AQP0-R33C was significantly reduced (P< 0.001) compared to that of WT-AQP0, indicated by cell aggregation and cell-to-cell adhesion assays. Scrape-loading assay using Lucifer Yellow dye showed reduction in cell-to-cell adhesion affecting gap junction coupling (P< 0.001). The data provided suggest that this mutation might not have caused significant alterations in protein folding since there was no obstruction in protein trafficking or water permeation. Reduction in cell-to-cell adhesion and development of cataract suggest that the conserved positive charge of Extracellular Loop A may play an important role in bringing fiber cells closer. The proposed schematic models illustrate that cell-to-cell adhesion elicited by AQP0 is vital for lens transparency and homeostasis. PMID:24120416

  11. The ataxia3 mutation in the N-terminal cytoplasmic domain of sodium channel Nav1.6 disrupts intracellular trafficking

    PubMed Central

    Sharkey, Lisa M.; Cheng, X-Y; Drews, Valerie; Buchner, David A.; Jones, Julie M.; Justice, Monica J.; Waxman, Stephen G.; Dib-Hajj, Sulayman D.; Meisler, Miriam H.

    2009-01-01

    The ENU-induced neurological mutant ataxia3 was mapped to distal mouse chromosome 15. Sequencing of the positional candidate gene Scn8a encoding the sodium channel Nav1.6 identified a T>C transition in exon 1 resulting in the amino acid substitution p.S21P near the N-terminus of the channel. The cytoplasmic N-terminal region is evolutionarily conserved but its function has not been well characterized. ataxia3 homozygotes exhibit a severe disorder that includes ataxia, tremor, and juvenile lethality. Unlike Scn8a null mice, they retain partial hind limb function. The mutant transcript is stable but protein abundance is reduced and the mutant channel is not detected in its usual site of concentration at nodes of Ranvier. In whole cell patch-clamp studies of transfected ND7/23 cells which were maintained at 37°C, the mutant channel did not produce sodium current, and function was not restored by co-expression of β1 and β2 subunits. However, when tranfected cells were maintained at 30°C, the mutant channel generated voltage-dependent inward sodium currents with an average peak current density comparable to wildtype, demonstrating recovery of channel activity. Immunohistochemistry of primary cerebellar granule cells from ataxia3 mice demonstrated that the mutant protein is retained in the cis-Golgi. This trafficking defect can account for the low level of Nav1.6-S21P at nodes of Ranvier in vivo and at the surface of transfected cells. The data demonstrate that the cytoplasmic N-terminal domain of the sodium channel is required for anterograde transport from the Golgi complex to the plasma membrane. PMID:19261867

  12. Germline Mutations in FAN1 Cause Hereditary Colorectal Cancer by Impairing DNA Repair.

    PubMed

    Seguí, Nuria; Mina, Leonardo B; Lázaro, Conxi; Sanz-Pamplona, Rebeca; Pons, Tirso; Navarro, Matilde; Bellido, Fernando; López-Doriga, Adriana; Valdés-Mas, Rafael; Pineda, Marta; Guinó, Elisabet; Vidal, August; Soto, José Luís; Caldés, Trinidad; Durán, Mercedes; Urioste, Miguel; Rueda, Daniel; Brunet, Joan; Balbín, Milagros; Blay, Pilar; Iglesias, Silvia; Garré, Pilar; Lastra, Enrique; Sánchez-Heras, Ana Beatriz; Valencia, Alfonso; Moreno, Victor; Pujana, Miguel Ángel; Villanueva, Alberto; Blanco, Ignacio; Capellá, Gabriel; Surrallés, Jordi; Puente, Xose S; Valle, Laura

    2015-09-01

    Identification of genes associated with hereditary cancers facilitates management of patients with family histories of cancer. We performed exome sequencing of DNA from 3 individuals from a family with colorectal cancer who met the Amsterdam criteria for risk of hereditary nonpolyposis colorectal cancer. These individuals had mismatch repair-proficient tumors and each carried nonsense variant in the FANCD2/FANCI-associated nuclease 1 gene (FAN1), which encodes a nuclease involved in DNA inter-strand cross-link repair. We sequenced FAN1 in 176 additional families with histories of colorectal cancer and performed in vitro functional analyses of the mutant forms of FAN1 identified. We detected FAN1 mutations in approximately 3% of families who met the Amsterdam criteria and had mismatch repair-proficient cancers with no previously associated mutations. These findings link colorectal cancer predisposition to the Fanconi anemia DNA repair pathway, supporting the connection between genome integrity and cancer risk.

  13. A mutation in FRIZZLED2 impairs Wnt signaling and causes autosomal dominant omodysplasia

    PubMed Central

    Saal, Howard M.; Prows, Cynthia A.; Guerreiro, Iris; Donlin, Milene; Knudson, Luke; Sund, Kristen L.; Chang, Ching-Fang; Brugmann, Samantha A.; Stottmann, Rolf W.

    2015-01-01

    Autosomal dominant omodysplasia is a rare skeletal dysplasia characterized by short humeri, radial head dislocation, short first metacarpals, facial dysmorphism and genitourinary anomalies. We performed next-generation whole-exome sequencing and comparative analysis of a proband with omodysplasia, her unaffected parents and her affected daughter. We identified a de novo mutation in FRIZZLED2 (FZD2) in the proband and her daughter that was not found in unaffected family members. The FZD2 mutation (c.1644G>A) changes a tryptophan residue at amino acid 548 to a premature stop (p.Trp548*). This altered protein is still produced in vitro, but we show reduced ability of this mutant form of FZD2 to interact with its downstream target DISHEVELLED. Furthermore, expressing the mutant form of FZD2 in vitro is not able to facilitate the cellular response to canonical Wnt signaling like wild-type FZD2. We therefore conclude that the FRIZZLED2 mutation is a de novo, novel cause for autosomal dominant omodysplasia. PMID:25759469

  14. Bordetella parapertussis Survives the Innate Interaction with Human Neutrophils by Impairing Bactericidal Trafficking inside the Cell through a Lipid Raft-Dependent Mechanism Mediated by the Lipopolysaccharide O Antigen

    PubMed Central

    Gorgojo, Juan; Lamberti, Yanina; Valdez, Hugo; Harvill, Eric T.

    2012-01-01

    Whooping cough is a reemerging disease caused by two closely related pathogens, Bordetella pertussis and Bordetella parapertussis. The incidence of B. parapertussis in whooping cough cases has been increasing since the introduction of acellular pertussis vaccines containing purified antigens that are common to both strains. Recently published results demonstrated that these vaccines do not protect against B. parapertussis due to the presence of the O antigen on the bacterial surface that impairs antibody access to shared antigens. We have investigated the effect of the lack of opsonization of B. parapertussis on the outcome of its interaction with human neutrophils (polymorphonuclear leukocytes [PMNs]). In the absence of opsonic antibodies, PMN interaction with B. parapertussis resulted in nonbactericidal trafficking upon phagocytosis. A high percentage of nonopsonized B. parapertussis was found in nonacidic lysosome marker (lysosome-associated membrane protein [LAMP])-negative phagosomes with access to the host cell-recycling pathway of external nutrients, allowing bacterial survival as determined by intracellular CFU counts. The lipopolysaccharide (LPS) O antigen was found to be involved in directing B. parapertussis to PMN lipid rafts, eventually determining the nonbactericidal fate inside the PMN. IgG opsonization of B. parapertussis drastically changed this interaction by not only inducing efficient PMN phagocytosis but also promoting PMN bacterial killing. These data provide new insights into the immune mechanisms of hosts against B. parapertussis and document the crucial importance of opsonic antibodies in immunity to this pathogen. PMID:23027528

  15. A mutation of Ikbkg causes immune deficiency without impairing degradation of IκBα

    PubMed Central

    Siggs, Owen M.; Berger, Michael; Krebs, Philippe; Arnold, Carrie N.; Eidenschenk, Celine; Huber, Christoph; Pirie, Elaine; Smart, Nora G.; Khovananth, Kevin; Xia, Yu; McInerney, Gerald; Karlsson Hedestam, Gunilla B.; Nemazee, David; Beutler, Bruce

    2010-01-01

    Null alleles of the gene encoding NEMO (NF-κB essential modulator) are lethal in hemizygous mice and men, whereas hypomorphic alleles typically cause a syndrome of immune deficiency and ectodermal dysplasia. Here we describe an allele of Ikbkg in mice that impaired Toll-like receptor signaling, lymph node formation, development of memory and regulatory T cells, and Ig production, but did not cause ectodermal dysplasia. Degradation of IκBα, which is considered a primary requirement for NEMO-mediated immune signaling, occurred normally in response to Toll-like receptor stimulation, yet ERK phosphorylation and NF-κB p65 nuclear translocation were severely impaired. This selective loss of function highlights the immunological importance of NEMO-regulated pathways beyond IκBα degradation, and offers a biochemical explanation for rare immune deficiencies in man. PMID:20133626

  16. Impaired neuronal KCC2 function by biallelic SLC12A5 mutations in migrating focal seizures and severe developmental delay

    PubMed Central

    Saitsu, Hirotomo; Watanabe, Miho; Akita, Tenpei; Ohba, Chihiro; Sugai, Kenji; Ong, Winnie Peitee; Shiraishi, Hideaki; Yuasa, Shota; Matsumoto, Hiroshi; Beng, Khoo Teik; Saitoh, Shinji; Miyatake, Satoko; Nakashima, Mitsuko; Miyake, Noriko; Kato, Mitsuhiro; Fukuda, Atsuo; Matsumoto, Naomichi

    2016-01-01

    Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the early-onset epileptic syndromes characterized by migrating polymorphous focal seizures. Whole exome sequencing (WES) in ten sporadic and one familial case of EIMFS revealed compound heterozygous SLC12A5 (encoding the neuronal K+-Cl− co-transporter KCC2) mutations in two families: c.279 + 1G > C causing skipping of exon 3 in the transcript (p.E50_Q93del) and c.572 C >T (p.A191V) in individuals 1 and 2, and c.967T > C (p.S323P) and c.1243 A > G (p.M415V) in individual 3. Another patient (individual 4) with migrating multifocal seizures and compound heterozygous mutations [c.953G > C (p.W318S) and c.2242_2244del (p.S748del)] was identified by searching WES data from 526 patients and SLC12A5-targeted resequencing data from 141 patients with infantile epilepsy. Gramicidin-perforated patch-clamp analysis demonstrated strongly suppressed Cl− extrusion function of E50_Q93del and M415V mutants, with mildly impaired function of A191V and S323P mutants. Cell surface expression levels of these KCC2 mutants were similar to wildtype KCC2. Heterologous expression of two KCC2 mutants, mimicking the patient status, produced a significantly greater intracellular Cl− level than with wildtype KCC2, but less than without KCC2. These data clearly demonstrated that partially disrupted neuronal Cl− extrusion, mediated by two types of differentially impaired KCC2 mutant in an individual, causes EIMFS. PMID:27436767

  17. Muscular Dystrophy Mutations Impair the Nuclear Envelope Emerin Self-assembly Properties.

    PubMed

    Herrada, Isaline; Samson, Camille; Velours, Christophe; Renault, Louis; Östlund, Cecilia; Chervy, Pierre; Puchkov, Dmytro; Worman, Howard J; Buendia, Brigitte; Zinn-Justin, Sophie

    2015-12-18

    More than 100 genetic mutations causing X-linked Emery-Dreifuss muscular dystrophy have been identified in the gene encoding the integral inner nuclear membrane protein emerin. Most mutations are nonsense or frameshift mutations that lead to the absence of emerin in cells. Only very few cases are due to missense or short in-frame deletions. Molecular mechanisms explaining the corresponding emerin variants' loss of function are particularly difficult to identify because of the mostly intrinsically disordered state of the emerin nucleoplasmic region. We now demonstrate that this EmN region can be produced as a disordered monomer, as revealed by nuclear magnetic resonance, but rapidly self-assembles in vitro. Increases in concentration and temperature favor the formation of long curvilinear filaments with diameters of approximately 10 nm, as observed by electron microscopy. Assembly of these filaments can be followed by fluorescence through Thioflavin-T binding and by Fourier-transform Infrared spectrometry through formation of β-structures. Analysis of the assembly properties of five EmN variants reveals that del95-99 and Q133H impact filament assembly capacities. In cells, these variants are located at the nuclear envelope, but the corresponding quantities of emerin-emerin and emerin-lamin proximities are decreased compared to wild-type protein. Furthermore, variant P183H favors EmN aggregation in vitro, and variant P183T provokes emerin accumulation in cytoplasmic foci in cells. Substitution of residue Pro183 might systematically favor oligomerization, leading to emerin aggregation and mislocalization in cells. Our results suggest that emerin self-assembly is necessary for its proper function and that a loss of either the protein itself or its ability to self-assemble causes muscular dystrophy.

  18. R4496C RyR2 mutation impairs atrial and ventricular contractility

    PubMed Central

    Coppini, Raffaele; Scellini, Beatrice; Ferrara, Claudia; Pioner, Josè Manuel; Mazzoni, Luca; Priori, Silvia; Cerbai, Elisabetta; Tesi, Chiara; Poggesi, Corrado

    2016-01-01

    Ryanodine receptor (RyR2) is the major Ca2+ channel of the cardiac sarcoplasmic reticulum (SR) and plays a crucial role in the generation of myocardial force. Changes in RyR2 gating properties and resulting increases in its open probability (Po) are associated with Ca2+ leakage from the SR and arrhythmias; however, the effects of RyR2 dysfunction on myocardial contractility are unknown. Here, we investigated the possibility that a RyR2 mutation associated with catecholaminergic polymorphic ventricular tachycardia, R4496C, affects the contractile function of atrial and ventricular myocardium. We measured isometric twitch tension in left ventricular and atrial trabeculae from wild-type mice and heterozygous transgenic mice carrying the R4496C RyR2 mutation and found that twitch force was comparable under baseline conditions (30°C, 2 mM [Ca2+]o, 1 Hz). However, the positive inotropic responses to high stimulation frequency, 0.1 µM isoproterenol, and 5 mM [Ca2+]o were decreased in R4496C trabeculae, as was post-rest potentiation. We investigated the mechanisms underlying inotropic insufficiency in R4496C muscles in single ventricular myocytes. Under baseline conditions, the amplitude of the Ca2+ transient was normal, despite the reduced SR Ca2+ content. Under inotropic challenge, however, R4496C myocytes were unable to boost the amplitude of Ca2+ transients because they are incapable of properly increasing the amount of Ca2+ stored in the SR because of a larger SR Ca2+ leakage. Recovery of force in response to premature stimuli was faster in R4496C myocardium, despite the unchanged rates of recovery of L-type Ca2+ channel current (ICa-L) and SR Ca2+ content in single myocytes. A faster recovery from inactivation of the mutant R4496C channels could explain this behavior. In conclusion, changes in RyR2 channel gating associated with the R4496C mutation could be directly responsible for the alterations in both ventricular and atrial contractility. The increased RyR2 Po

  19. Homozygous NOTCH3 null mutation and impaired NOTCH3 signaling in recessive early-onset arteriopathy and cavitating leukoencephalopathy

    PubMed Central

    Pippucci, Tommaso; Maresca, Alessandra; Magini, Pamela; Cenacchi, Giovanna; Donadio, Vincenzo; Palombo, Flavia; Papa, Valentina; Incensi, Alex; Gasparre, Giuseppe; Valentino, Maria Lucia; Preziuso, Carmela; Pisano, Annalinda; Ragno, Michele; Liguori, Rocco; Giordano, Carla; Tonon, Caterina; Lodi, Raffaele; Parmeggiani, Antonia; Carelli, Valerio; Seri, Marco

    2015-01-01

    Notch signaling is essential for vascular physiology. Neomorphic heterozygous mutations in NOTCH3, one of the four human NOTCH receptors, cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Hypomorphic heterozygous alleles have been occasionally described in association with a spectrum of cerebrovascular phenotypes overlapping CADASIL, but their pathogenic potential is unclear. We describe a patient with childhood-onset arteriopathy, cavitating leukoencephalopathy with cerebral white matter abnormalities presented as diffuse cavitations, multiple lacunar infarctions and disseminated microbleeds. We identified a novel homozygous c.C2898A (p.C966*) null mutation in NOTCH3 abolishing NOTCH3 expression and causing NOTCH3 signaling impairment. NOTCH3 targets acting in the regulation of arterial tone (KCNA5) or expressed in the vasculature (CDH6) were downregulated. Patient's vessels were characterized by smooth muscle degeneration as in CADASIL, but without deposition of granular osmiophilic material (GOM), the CADASIL hallmark. The heterozygous parents displayed similar but less dramatic trends in decrease in the expression of NOTCH3 and its targets, as well as in vessel degeneration. This study suggests a functional link between NOTCH3 deficiency and pathogenesis of vascular leukoencephalopathies. PMID:25870235

  20. Mutations Impairing GSK3-Mediated MAF Phosphorylation Cause Cataract, Deafness, Intellectual Disability, Seizures, and a Down Syndrome-like Facies

    PubMed Central

    Niceta, Marcello; Stellacci, Emilia; Gripp, Karen W.; Zampino, Giuseppe; Kousi, Maria; Anselmi, Massimiliano; Traversa, Alice; Ciolfi, Andrea; Stabley, Deborah; Bruselles, Alessandro; Caputo, Viviana; Cecchetti, Serena; Prudente, Sabrina; Fiorenza, Maria T.; Boitani, Carla; Philip, Nicole; Niyazov, Dmitriy; Leoni, Chiara; Nakane, Takaya; Keppler-Noreuil, Kim; Braddock, Stephen R.; Gillessen-Kaesbach, Gabriele; Palleschi, Antonio; Campeau, Philippe M.; Lee, Brendan H.L.; Pouponnot, Celio; Stella, Lorenzo; Bocchinfuso, Gianfranco; Katsanis, Nicholas; Sol-Church, Katia; Tartaglia, Marco

    2015-01-01

    Transcription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing development. PMID:25865493

  1. Mutations in the NB-ARC Domain of I-2 That Impair ATP Hydrolysis Cause Autoactivation1[OA

    PubMed Central

    Tameling, Wladimir I.L.; Vossen, Jack H.; Albrecht, Mario; Lengauer, Thomas; Berden, Jan A.; Haring, Michel A.; Cornelissen, Ben J.C.; Takken, Frank L.W.

    2006-01-01

    Resistance (R) proteins in plants confer specificity to the innate immune system. Most R proteins have a centrally located NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain. For two tomato (Lycopersicon esculentum) R proteins, I-2 and Mi-1, we have previously shown that this domain acts as an ATPase module that can hydrolyze ATP in vitro. To investigate the role of nucleotide binding and hydrolysis for the function of I-2 in planta, specific mutations were introduced in conserved motifs of the NB-ARC domain. Two mutations resulted in autoactivating proteins that induce a pathogen-independent hypersensitive response upon expression in planta. These mutant forms of I-2 were found to be impaired in ATP hydrolysis, but not in ATP binding, suggesting that the ATP- rather than the ADP-bound state of I-2 is the active form that triggers defense signaling. In addition, upon ADP binding, the protein displayed an increased affinity for ADP suggestive of a change of conformation. Based on these data, we propose that the NB-ARC domain of I-2, and likely of related R proteins, functions as a molecular switch whose state (on/off) depends on the nucleotide bound (ATP/ADP). PMID:16489136

  2. An ALS disease mutation in Cdc48/p97 impairs 20S proteasome binding and proteolytic communication.

    PubMed

    Barthelme, Dominik; Jauregui, Ruben; Sauer, Robert T

    2015-09-01

    Cdc48 (also known as p97 or VCP) is an essential and highly abundant, double-ring AAA+ ATPase, which is ubiquitous in archaea and eukaryotes. In archaea, Cdc48 ring hexamers play a direct role in quality control by unfolding and translocating protein substrates into the degradation chamber of the 20S proteasome. Whether Cdc48 and 20S cooperate directly in protein degradation in eukaryotic cells is unclear. Two regions of Cdc48 are important for 20S binding, the pore-2 loop at the bottom of the D2 AAA+ ring and a C-terminal tripeptide. Here, we identify an aspartic acid in the pore-2 loop as an important element in 20S recognition. Importantly, mutation of this aspartate in human Cdc48 has been linked to familial amyotrophic lateral sclerosis (ALS). In archaeal or human Cdc48 variants, we find that mutation of this pore-2 residue impairs 20S binding and proteolytic communication but does not affect the stability of the hexamer or rates of ATP hydrolysis and protein unfolding. These results suggest that human Cdc48 interacts functionally with the 20S proteasome.

  3. Mutations Impairing GSK3-Mediated MAF Phosphorylation Cause Cataract, Deafness, Intellectual Disability, Seizures, and a Down Syndrome-like Facies.

    PubMed

    Niceta, Marcello; Stellacci, Emilia; Gripp, Karen W; Zampino, Giuseppe; Kousi, Maria; Anselmi, Massimiliano; Traversa, Alice; Ciolfi, Andrea; Stabley, Deborah; Bruselles, Alessandro; Caputo, Viviana; Cecchetti, Serena; Prudente, Sabrina; Fiorenza, Maria T; Boitani, Carla; Philip, Nicole; Niyazov, Dmitriy; Leoni, Chiara; Nakane, Takaya; Keppler-Noreuil, Kim; Braddock, Stephen R; Gillessen-Kaesbach, Gabriele; Palleschi, Antonio; Campeau, Philippe M; Lee, Brendan H L; Pouponnot, Celio; Stella, Lorenzo; Bocchinfuso, Gianfranco; Katsanis, Nicholas; Sol-Church, Katia; Tartaglia, Marco

    2015-05-07

    Transcription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing development.

  4. Point mutation impairs centromeric CENH3 loading and induces haploid plants

    PubMed Central

    Karimi-Ashtiyani, Raheleh; Ishii, Takayoshi; Niessen, Markus; Stein, Nils; Heckmann, Stefan; Gurushidze, Maia; Banaei-Moghaddam, Ali Mohammad; Fuchs, Jörg; Schubert, Veit; Koch, Kerstin; Weiss, Oda; Demidov, Dmitri; Schmidt, Klaus; Kumlehn, Jochen; Houben, Andreas

    2015-01-01

    The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called “CENP-A”) is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923–937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wild-type A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest. PMID:26294252

  5. Point mutation impairs centromeric CENH3 loading and induces haploid plants.

    PubMed

    Karimi-Ashtiyani, Raheleh; Ishii, Takayoshi; Niessen, Markus; Stein, Nils; Heckmann, Stefan; Gurushidze, Maia; Banaei-Moghaddam, Ali Mohammad; Fuchs, Jörg; Schubert, Veit; Koch, Kerstin; Weiss, Oda; Demidov, Dmitri; Schmidt, Klaus; Kumlehn, Jochen; Houben, Andreas

    2015-09-08

    The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called "CENP-A") is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923-937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wild-type A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest.

  6. Violacein induces death of RAS-mutated metastatic melanoma by impairing autophagy process.

    PubMed

    Gonçalves, Paola R; Rocha-Brito, Karin J P; Fernandes, Maruska R N; Abrantes, Julia L; Durán, Nelson; Ferreira-Halder, Carmen V

    2016-10-01

    Treatment of metastatic melanoma still remains a challenge, since in advanced stage it is refractory to conventional treatments. Most patients with melanoma have either B-RAF or N-RAS mutations, and these oncogenes lead to activation of the RAS-RAF-MEK-ERK and AKT signal pathway, keeping active the proliferation and survival pathways in the cell. Therefore, the identification of small molecules that block metastatic cell proliferation and induce cell death is needed. Violacein, a pigment produced by Chromobacterium violaceum found in Amazon River, has been used by our group as a biotool for scrutinizing signaling pathways associated with proliferation, survival, aggressiveness, and resistance of cancer cells. In the present study, we demonstrate that violacein diminished the viability of RAS- and RAF-mutated melanoma cells (IC50 value ∼500 nM), and more important, this effect was not abolished after treatment medium removal. Furthermore, violacein was able to reduce significantly the invasion capacity of metastatic melanoma cells in 3D culture. In the molecular context, we have shown for the first time that violacein causes a strong drop on histone deacetylase 6 expression, a proliferating activator, in melanoma cells. Besides, an inhibition of AXL and AKT was detected. All these molecular events propitiate an inhibition of autophagy, and consequently, melanoma cell death by apoptosis.

  7. Simultaneous impairment of neuronal and metabolic function of mutated gephyrin in a patient with epileptic encephalopathy.

    PubMed

    Dejanovic, Borislav; Djémié, Tania; Grünewald, Nora; Suls, Arvid; Kress, Vanessa; Hetsch, Florian; Craiu, Dana; Zemel, Matthew; Gormley, Padhraig; Lal, Dennis; Myers, Candace T; Mefford, Heather C; Palotie, Aarno; Helbig, Ingo; Meier, Jochen C; De Jonghe, Peter; Weckhuysen, Sarah; Schwarz, Guenter

    2015-12-01

    Synaptic inhibition is essential for shaping the dynamics of neuronal networks, and aberrant inhibition plays an important role in neurological disorders. Gephyrin is a central player at inhibitory postsynapses, directly binds and organizes GABAA and glycine receptors (GABAARs and GlyRs), and is thereby indispensable for normal inhibitory neurotransmission. Additionally, gephyrin catalyzes the synthesis of the molybdenum cofactor (MoCo) in peripheral tissue. We identified a de novo missense mutation (G375D) in the gephyrin gene (GPHN) in a patient with epileptic encephalopathy resembling Dravet syndrome. Although stably expressed and correctly folded, gephyrin-G375D was non-synaptically localized in neurons and acted dominant-negatively on the clustering of wild-type gephyrin leading to a marked decrease in GABAAR surface expression and GABAergic signaling. We identified a decreased binding affinity between gephyrin-G375D and the receptors, suggesting that Gly375 is essential for gephyrin-receptor complex formation. Surprisingly, gephyrin-G375D was also unable to synthesize MoCo and activate MoCo-dependent enzymes. Thus, we describe a missense mutation that affects both functions of gephyrin and suggest that the identified defect at GABAergic synapses is the mechanism underlying the patient's severe phenotype.

  8. Tau proteins harboring neurodegeneration-linked mutations impair kinesin translocation in vitro.

    PubMed

    Yu, Dezhi; LaPointe, Nichole E; Guzman, Elmer; Pessino, Veronica; Wilson, Leslie; Feinstein, Stuart C; Valentine, Megan T

    2014-01-01

    We tested the hypothesis that mutant tau proteins that cause neurodegeneration and dementia differentially alter kinesin translocation along microtubules (MTs) relative to normal tau in vitro. We employed complementary in vitro motility assays using purified recombinant kinesin, purified recombinant tau, and purified bovine brain α:β tubulin to isolate interactions among these components without any contribution by cellular regulatory mechanisms. We found that kinesin translocates slower along MTs assembled by any of three independent tau mutants (4-repeat P301L tau, 4-repeat ΔN296 tau, and 4-repeat R406W tau) relative to its translocation rate along MTs assembled by normal, 4-repeat wild type (WT) tau. Moreover, the R406W mutation exhibited isoform specific effects; while kinesin translocation along 4-repeat R406W tau assembled MTs is slower than along MTs assembled by 4-repeat WT tau, the R406W mutation had no effect in the 3-repeat tau context. These data provide strong support for the notion that aberrant modulation of kinesin translocation is a component of tau-mediated neuronal cell death and dementia. Finally, we showed that assembling MTs with taxol before coating them with mutant tau obscured effects of the mutant tau that were readily apparent using more physiologically relevant MTs assembled with tau alone, raising important issues regarding the use of taxol as an experimental reagent and novel insights into therapeutic mechanisms of taxol action.

  9. Mutations that impair a posttranscriptional step in expression of HLA-A and -B antigens.

    PubMed Central

    DeMars, R; Rudersdorf, R; Chang, C; Petersen, J; Strandtmann, J; Korn, N; Sidwell, B; Orr, H T

    1985-01-01

    Mutations can interfere with posttranscriptional expression of the HLA-A and -B genes. B-lymphoblastoid cells that contain one copy of the major histocompatibility complex (MHC) were subjected to mutagenesis and immunoselection for MHC antigen-loss mutants. Some mutations partially reduced surface expression of HLA-A and eliminated HLA-B expression concurrently, although the HLA-A and -B genes were present and transcribed. Antigen expression was fully restored in hybrids of these mutants with other B-lymphoblastoid cells. Therefore, normal cell surface expression of the HLA-A and -B antigens on B lymphoblasts requires (i) execution of at least one trans-active step in the production of the antigens after transcription of the HLA-A and -B genes or (ii) association of the class I antigens with other molecules. DNA analysis of one mutant suggests the possibility that a locus required for the normal expression of the HLA-A and -B antigens is located between the MHC complement genes and the HLA-DP alpha II locus. Images PMID:3906658

  10. Mutations in PGAP3 Impair GPI-Anchor Maturation, Causing a Subtype of Hyperphosphatasia with Mental Retardation

    PubMed Central

    Howard, Malcolm F.; Murakami, Yoshiko; Pagnamenta, Alistair T.; Daumer-Haas, Cornelia; Fischer, Björn; Hecht, Jochen; Keays, David A.; Knight, Samantha J.L.; Kölsch, Uwe; Krüger, Ulrike; Leiz, Steffen; Maeda, Yusuke; Mitchell, Daphne; Mundlos, Stefan; Phillips, John A.; Robinson, Peter N.; Kini, Usha; Taylor, Jenny C.; Horn, Denise; Kinoshita, Taroh; Krawitz, Peter M.

    2014-01-01

    Glycosylphophatidylinositol (GPI)-anchored proteins play important roles in many biological processes, and mutations affecting proteins involved in the synthesis of the GPI anchor are reported to cause a wide spectrum of intellectual disabilities (IDs) with characteristic additional phenotypic features. Here, we describe a total of five individuals (from three unrelated families) in whom we identified mutations in PGAP3, encoding a protein that is involved in GPI-anchor maturation. Three siblings in a consanguineous Pakistani family presented with profound developmental delay, severe ID, no speech, psychomotor delay, and postnatal microcephaly. A combination of autozygosity mapping and exome sequencing identified a 13.8 Mb region harboring a homozygous c.275G>A (p.Gly92Asp) variant in PGAP3 region 17q11.2–q21.32. Subsequent testing showed elevated serum alkaline phosphatase (ALP), a GPI-anchored enzyme, in all three affected children. In two unrelated individuals in a cohort with developmental delay, ID, and elevated ALP, we identified compound-heterozygous variants c.439dupC (p.Leu147Profs∗16) and c.914A>G (p.Asp305Gly) and homozygous variant c.314C>G (p.Pro105Arg). The 1 bp duplication causes a frameshift and nonsense-mediated decay. Further evidence supporting pathogenicity of the missense mutations c.275G>A, c.314C>G, and c.914A>G was provided by the absence of the variants from ethnically matched controls, phylogenetic conservation, and functional studies on Chinese hamster ovary cell lines. Taken together with recent data on PGAP2, these results confirm the importance of the later GPI-anchor remodelling steps for normal neuronal development. Impairment of PGAP3 causes a subtype of hyperphosphatasia with ID, a congenital disorder of glycosylation that is also referred to as Mabry syndrome. PMID:24439110

  11. Mutations in PGAP3 impair GPI-anchor maturation, causing a subtype of hyperphosphatasia with mental retardation.

    PubMed

    Howard, Malcolm F; Murakami, Yoshiko; Pagnamenta, Alistair T; Daumer-Haas, Cornelia; Fischer, Björn; Hecht, Jochen; Keays, David A; Knight, Samantha J L; Kölsch, Uwe; Krüger, Ulrike; Leiz, Steffen; Maeda, Yusuke; Mitchell, Daphne; Mundlos, Stefan; Phillips, John A; Robinson, Peter N; Kini, Usha; Taylor, Jenny C; Horn, Denise; Kinoshita, Taroh; Krawitz, Peter M

    2014-02-06

    Glycosylphophatidylinositol (GPI)-anchored proteins play important roles in many biological processes, and mutations affecting proteins involved in the synthesis of the GPI anchor are reported to cause a wide spectrum of intellectual disabilities (IDs) with characteristic additional phenotypic features. Here, we describe a total of five individuals (from three unrelated families) in whom we identified mutations in PGAP3, encoding a protein that is involved in GPI-anchor maturation. Three siblings in a consanguineous Pakistani family presented with profound developmental delay, severe ID, no speech, psychomotor delay, and postnatal microcephaly. A combination of autozygosity mapping and exome sequencing identified a 13.8 Mb region harboring a homozygous c.275G>A (p.Gly92Asp) variant in PGAP3 region 17q11.2-q21.32. Subsequent testing showed elevated serum alkaline phosphatase (ALP), a GPI-anchored enzyme, in all three affected children. In two unrelated individuals in a cohort with developmental delay, ID, and elevated ALP, we identified compound-heterozygous variants c.439dupC (p.Leu147Profs(∗)16) and c.914A>G (p.Asp305Gly) and homozygous variant c.314C>G (p.Pro105Arg). The 1 bp duplication causes a frameshift and nonsense-mediated decay. Further evidence supporting pathogenicity of the missense mutations c.275G>A, c.314C>G, and c.914A>G was provided by the absence of the variants from ethnically matched controls, phylogenetic conservation, and functional studies on Chinese hamster ovary cell lines. Taken together with recent data on PGAP2, these results confirm the importance of the later GPI-anchor remodelling steps for normal neuronal development. Impairment of PGAP3 causes a subtype of hyperphosphatasia with ID, a congenital disorder of glycosylation that is also referred to as Mabry syndrome. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  12. Calmodulin is essential for cardiac IKS channel gating and assembly: impaired function in long-QT mutations.

    PubMed

    Shamgar, Liora; Ma, Lijuan; Schmitt, Nicole; Haitin, Yoni; Peretz, Asher; Wiener, Reuven; Hirsch, Joel; Pongs, Olaf; Attali, Bernard

    2006-04-28

    The slow IKS K+ channel plays a major role in repolarizing the cardiac action potential and consists of the assembly of KCNQ1 and KCNE1 subunits. Mutations in either KCNQ1 or KCNE1 genes produce the long-QT syndrome, a life-threatening ventricular arrhythmia. Here, we show that long-QT mutations located in the KCNQ1 C terminus impair calmodulin (CaM) binding, which affects both channel gating and assembly. The mutations produce a voltage-dependent macroscopic inactivation and dramatically alter channel assembly. KCNE1 forms a ternary complex with wild-type KCNQ1 and Ca(2+)-CaM that prevents inactivation, facilitates channel assembly, and mediates a Ca(2+)-sensitive increase of IKS-current, with a considerable Ca(2+)-dependent left-shift of the voltage-dependence of activation. Coexpression of KCNQ1 or IKS channels with a Ca(2+)-insensitive CaM mutant markedly suppresses the currents and produces a right shift in the voltage-dependence of channel activation. KCNE1 association to KCNQ1 long-QT mutants significantly improves mutant channel expression and prevents macroscopic inactivation. However, the marked right shift in channel activation and the subsequent decrease in current amplitude cannot restore normal levels of IKS channel activity. Our data indicate that in healthy individuals, CaM binding to KCNQ1 is essential for correct channel folding and assembly and for conferring Ca(2+)-sensitive IKS-current stimulation, which increases the cardiac repolarization reserve and hence prevents the risk of ventricular arrhythmias.

  13. A targeted mutation at the known collagenase cleavage site in mouse type I collagen impairs tissue remodeling

    PubMed Central

    1995-01-01

    Degradation of type I collagen, the most abundant collagen, is initiated by collagenase cleavage at a highly conserved site between Gly775 and Ile776 of the alpha 1 (I) chain. Mutations at or around this site render type I collagen resistant to collagenase digestion in vitro. We show here that mice carrying a collagenase-resistant mutant Col1a-1 transgene die late in embryo-genesis, ascribable to overexpression of the transgene, since the same mutation introduced into the endogenous Col1a-1 gene by gene targeting permitted normal development of mutant mice to young adulthood. With increasing age, animals carrying the targeted mutation developed marked fibrosis of the dermis similar to that in human scleroderma. Postpartum involution of the uterus in the mutant mice was also impaired, with persistence of collagenous nodules in the uterine wall. Although type I collagen from the homozygous mutant mice was resistant to cleavage by human or rat fibroblast collagenases at the helical site, only the rat collagenase cleaved collagen trimers at an additional, novel site in the nonhelical N-telopeptide domain. Our results suggest that cleavage by murine collagenase at the N-telopeptide site could account for resorption of type I collagen during embryonic and early adult life. During intense collagen resorption, however, such as in the immediate postpartum uterus and in the dermis later in life, cleavage at the helical site is essential for normal collagen turnover. Thus, type I collagen is degraded by at least two differentially controlled mechanisms involving collagenases with distinct, but overlapping, substrate specificities. PMID:7790374

  14. Homozygous SLC6A17 mutations cause autosomal-recessive intellectual disability with progressive tremor, speech impairment, and behavioral problems.

    PubMed

    Iqbal, Zafar; Willemsen, Marjolein H; Papon, Marie-Amélie; Musante, Luciana; Benevento, Marco; Hu, Hao; Venselaar, Hanka; Wissink-Lindhout, Willemijn M; Vulto-van Silfhout, Anneke T; Vissers, Lisenka E L M; de Brouwer, Arjan P M; Marouillat, Sylviane; Wienker, Thomas F; Ropers, Hans Hilger; Kahrizi, Kimia; Nadif Kasri, Nael; Najmabadi, Hossein; Laumonnier, Frédéric; Kleefstra, Tjitske; van Bokhoven, Hans

    2015-03-05

    We report on Dutch and Iranian families with affected individuals who present with moderate to severe intellectual disability and additional phenotypes including progressive tremor, speech impairment, and behavioral problems in certain individuals. A combination of exome sequencing and homozygosity mapping revealed homozygous mutations c.484G>A (p.Gly162Arg) and c.1898C>G (p.Pro633Arg) in SLC6A17. SLC6A17 is predominantly expressed in the brain, encodes a synaptic vesicular transporter of neutral amino acids and glutamate, and plays an important role in the regulation of glutamatergic synapses. Prediction programs and 3D modeling suggest that the identified mutations are deleterious to protein function. To directly test the functional consequences, we investigated the neuronal subcellular localization of overexpressed wild-type and mutant variants in mouse primary hippocampal neuronal cells. Wild-type protein was present in soma, axons, dendrites, and dendritic spines. p.Pro633Arg altered SLC6A17 was found in soma and proximal dendrites but did not reach spines. p.Gly162Arg altered SLC6A17 showed a normal subcellular distribution but was associated with an abnormal neuronal morphology mainly characterized by the loss of dendritic spines. In summary, our genetic findings implicate homozygous SLC6A17 mutations in autosomal-recessive intellectual disability, and their pathogenic role is strengthened by genetic evidence and in silico and in vitro functional analyses. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  15. Asp120Asn mutation impairs the catalytic activity of NDM-1 metallo-β-lactamase: experimental and computational study.

    PubMed

    Chen, Jiao; Chen, Hui; Zhu, Tong; Zhou, Dandan; Zhang, Fang; Lao, Xingzhen; Zheng, Heng

    2014-04-14

    New Delhi metallo-β-lactamase-1 (NDM-1) has attracted extensive attention in recent years for its high activity for hydrolyzing almost all β-lactam antibiotics. Like other metallo-β-lactamases (MβLs), NDM-1 features an invariant Asp120 that ligates the zinc ion (ZN2) in the active site. Previous studies showed that substitutions of Asp120 with residues such as Ala, Ser, Asn and Glu dramatically impaired the MβL (BcII, IMP-1, L1) activity, but no consensus about the exact role of Asp120 has reached. Here we constructed D120N mutant of NDM-1 by site-directed mutagenesis. The replacement of Asp120 with Asn, which has much weaker metal ligating capabilities than Asp, severely impaired the lactamase activity without abolishing the ZN2 site. Molecular dynamics simulations suggested that the ZN1-ZN2 distance increased because of mutation, leading to a rearrangement of the active site, including the bridging OH(-). Thereby, the Mulliken charges of ZN1 and ZN2 redistributed, especially for ZN2, which might be the major cause of the impaired activity. Reducing the point charges of Asp120 carboxyl oxygens weakened the ionic interactions between Asp120 and ZN2, and the positions of the zinc ions were also changed as a result. It is proposed that Asp120 acts as a strong ZN2 ligand, positioning ZN2 for catalytically important interactions with the substrate, stabilizing the negatively charged amide nitrogen of the hydrolyzed intermediate, and more importantly, orienting the ZN-bound OH(-) for nucleophilic attacks and protonation. These functions are of general importance for catalyzing β-lactam antibiotics by NDM-1 as well as other MβLs.

  16. IDH mutation impairs histone demethylation and results in a block to cell differentiation.

    PubMed

    Lu, Chao; Ward, Patrick S; Kapoor, Gurpreet S; Rohle, Dan; Turcan, Sevin; Abdel-Wahab, Omar; Edwards, Christopher R; Khanin, Raya; Figueroa, Maria E; Melnick, Ari; Wellen, Kathryn E; O'Rourke, Donald M; Berger, Shelley L; Chan, Timothy A; Levine, Ross L; Mellinghoff, Ingo K; Thompson, Craig B

    2012-02-15

    Recurrent mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 have been identified in gliomas, acute myeloid leukaemias (AML) and chondrosarcomas, and share a novel enzymatic property of producing 2-hydroxyglutarate (2HG) from α-ketoglutarate. Here we report that 2HG-producing IDH mutants can prevent the histone demethylation that is required for lineage-specific progenitor cells to differentiate into terminally differentiated cells. In tumour samples from glioma patients, IDH mutations were associated with a distinct gene expression profile enriched for genes expressed in neural progenitor cells, and this was associated with increased histone methylation. To test whether the ability of IDH mutants to promote histone methylation contributes to a block in cell differentiation in non-transformed cells, we tested the effect of neomorphic IDH mutants on adipocyte differentiation in vitro. Introduction of either mutant IDH or cell-permeable 2HG was associated with repression of the inducible expression of lineage-specific differentiation genes and a block to differentiation. This correlated with a significant increase in repressive histone methylation marks without observable changes in promoter DNA methylation. Gliomas were found to have elevated levels of similar histone repressive marks. Stable transfection of a 2HG-producing mutant IDH into immortalized astrocytes resulted in progressive accumulation of histone methylation. Of the marks examined, increased H3K9 methylation reproducibly preceded a rise in DNA methylation as cells were passaged in culture. Furthermore, we found that the 2HG-inhibitable H3K9 demethylase KDM4C was induced during adipocyte differentiation, and that RNA-interference suppression of KDM4C was sufficient to block differentiation. Together these data demonstrate that 2HG can inhibit histone demethylation and that inhibition of histone demethylation can be sufficient to block the differentiation of non-transformed cells.

  17. The murine equivalent of the A181E TACI mutation associated with common variable immunodeficiency severely impairs B-cell function

    PubMed Central

    Lee, John J.; Rauter, Ingrid; Garibyan, Lilit; Ozcan, Esra; Sannikova, Tatyana; Dillon, Stacey R.; Cruz, Anthony C.; Siegel, Richard M.; Bram, Richard; Jabara, Haifa

    2009-01-01

    TNFRSF13B, which encodes TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor), is mutated in 10% of patients with common variable immune deficiency (CVID). One of the 2 most common TACI mutations in CVID, A181E, introduces a negative charge into the transmembrane domain. To define the consequence of the A181E mutation on TACI function, we studied the effect of its murine equivalent, mTACI A144E, on TACI signaling in transfected cells and on TACI function in transgenic mice. The mTACI A144E mutant, like its human TACI A181E counterpart, was expressed on the surface of 293T transfectants and was able to bind ligand, but exhibited impaired constitutive and ligand-induced NFκB signaling. In addition, constitutive and ligand-induced clustering of the intracellular domain was deficient for A144E as measured by fluorescence resonance energy transfer. Transgenic mice expressing the A144E mutant on TACI−/− background had low serum IgA levels and significantly impaired antibody responses to the type II T-independent antigen TNP-Ficoll. B cells from A144E transgenic mice were impaired in their capacity to proliferate and secrete IgG1 and IgA in response to TACI ligation. These results suggest that mTACI A144E mutation and its human counterpart, A181E, disrupt TACI signaling and impair TACI-dependent B-cell functions. PMID:19605846

  18. Impaired dense core vesicle maturation in Caenorhabditis elegans mutants lacking Rab2.

    PubMed

    Edwards, Stacey L; Charlie, Nicole K; Richmond, Janet E; Hegermann, Jan; Eimer, Stefan; Miller, Kenneth G

    2009-09-21

    Despite a key role for dense core vesicles (DCVs) in neuronal function, there are major gaps in our understanding of DCV biogenesis. A genetic screen for Caenorhabditis elegans mutants with behavioral defects consistent with impaired DCV function yielded five mutations in UNC-108 (Rab2). A genetic analysis showed that unc-108 mutations impair a DCV function unrelated to neuropeptide release that, together with neuropeptide release, fully accounts for the role of DCVs in locomotion. An electron microscopy analysis of DCVs in unc-108 mutants, coupled with quantitative imaging of DCV cargo proteins, revealed that Rab2 acts in cell somas during DCV maturation to prevent the loss of soluble and membrane cargo. In Rab2 null mutants, two thirds of these cargoes move to early endosomes via a PI(3)P-dependent trafficking pathway, whereas aggregated neuropeptides are unaffected. These results reveal how neurons solve a challenging trafficking problem using the most highly conserved animal Rab.

  19. Methods of Assessing STING Activation and Trafficking.

    PubMed

    Pokatayev, Vladislav; Yan, Nan

    2017-01-01

    The signaling adapter protein STING is crucial for the host immune response to cytosolic DNA and cyclic dinucleotides. Under basal conditions, STING resides on the endoplasmic reticulum (ER ) , but upon activation, it traffics through secretory pathway to cytoplasmic vesicles, where STING activates downstream immune signaling. Classical STING activation and trafficking are triggered by binding of cyclic dinucleotide ligands. STING signaling can also be activated by gain-of-function mutations that lead to constitutive trafficking of STING. These gain-of-function mutations are associated with several human diseases such as STING-associated vasculopathy with onset in infancy (SAVI), systemic lupus erythematosus (SLE), or familial chilblain lupus (FCL). This dynamic activation pathway presents a challenge to study. We describe methods here for measuring ligand-dependent and ligand-independent activation of STING signaling in HEK293T cells. We also describe a retroviral-based reconstitution assay to study STING protein trafficking and activation in immune competent cells such as mouse embryonic fibroblasts (MEF), which avoids the use of plasmid DNA. These methods will expedite research regarding STING trafficking and signaling dynamics in the settings of infection and autoimmune diseases.

  20. Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin.

    PubMed

    Zhou, Xiaolai; Sun, Lirong; Bastos de Oliveira, Francisco; Qi, Xiaoyang; Brown, William J; Smolka, Marcus B; Sun, Ying; Hu, Fenghua

    2015-09-14

    Mutations in the progranulin (PGRN) gene have been linked to two distinct neurodegenerative diseases, frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL). Accumulating evidence suggests a critical role of PGRN in lysosomes. However, how PGRN is trafficked to lysosomes is still not clear. Here we report a novel pathway for lysosomal delivery of PGRN. We found that prosaposin (PSAP) interacts with PGRN and facilitates its lysosomal targeting in both biosynthetic and endocytic pathways via the cation-independent mannose 6-phosphate receptor and low density lipoprotein receptor-related protein 1. PSAP deficiency in mice leads to severe PGRN trafficking defects and a drastic increase in serum PGRN levels. We further showed that this PSAP pathway is independent of, but complementary to, the previously identified PGRN lysosomal trafficking mediated by sortilin. Collectively, our results provide new understanding on PGRN trafficking and shed light on the molecular mechanisms behind FTLD and NCL caused by PGRN mutations.

  1. Prevalence and Evolutionary Origins of the del(GJB6-D13S1830) Mutation in the DFNB1 Locus in Hearing-Impaired Subjects: a Multicenter Study

    PubMed Central

    del Castillo, Ignacio; Moreno-Pelayo, Miguel A.; del Castillo, Francisco J.; Brownstein, Zippora; Marlin, Sandrine; Adina, Quint; Cockburn, David J.; Pandya, Arti; Siemering, Kirby R.; Chamberlin, G. Parker; Ballana, Ester; Wuyts, Wim; Maciel-Guerra, Andréa Trevas; Álvarez, Araceli; Villamar, Manuela; Shohat, Mordechai; Abeliovich, Dvorah; Dahl, Hans-Henrik M.; Estivill, Xavier; Gasparini, Paolo; Hutchin, Tim; Nance, Walter E.; Sartorato, Edi L.; Smith, Richard J. H.; Van Camp, Guy; Avraham, Karen B.; Petit, Christine; Moreno, Felipe

    2003-01-01

    Mutations in GJB2, the gene encoding connexin-26 at the DFNB1 locus on 13q12, are found in as many as 50% of subjects with autosomal recessive, nonsyndromic prelingual hearing impairment. However, genetic diagnosis is complicated by the fact that 10%–50% of affected subjects with GJB2 mutations carry only one mutant allele. Recently, a deletion truncating the GJB6 gene (encoding connexin-30), near GJB2 on 13q12, was shown to be the accompanying mutation in ∼50% of these deaf GJB2 heterozygotes in a cohort of Spanish patients, thus becoming second only to 35delG at GJB2 as the most frequent mutation causing prelingual hearing impairment in Spain. Here, we present data from a multicenter study in nine countries that shows that the deletion is present in most of the screened populations, with higher frequencies in France, Spain, and Israel, where the percentages of unexplained GJB2 heterozygotes fell to 16.0%–20.9% after screening for the del(GJB6-D13S1830) mutation. Our results also suggest that additional mutations remain to be identified, either in DFNB1 or in other unlinked genes involved in epistatic interactions with GJB2. Analysis of haplotypes associated with the deletion revealed a founder effect in Ashkenazi Jews and also suggested a common founder for countries in Western Europe. These results have important implications for the diagnosis and counseling of families with DFNB1 deafness. PMID:14571368

  2. Impaired p32 regulation caused by the lymphoma-prone RECQ4 mutation drives mitochondrial dysfunction

    PubMed Central

    Wang, Jiin-Tarng; Xu, Xiaohua; Alontaga, Aileen Y.; Chen, Yuan; Liu, Yilun

    2014-01-01

    Mitochondrial DNA (mtDNA) encodes genes important for ATP biogenesis. Therefore, changes in mtDNA copy number will have profound consequences on cell survival and proliferation. RECQ4 DNA helicase plays important roles in both nuclear and mtDNA synthesis. However, the mechanism that balances the distribution of RECQ4 in the nucleus and mitochondria is unknown. Here, we show that RECQ4 forms protein complexes with Protein Phosphatase 2A (PP2A), nucleophosmin (NPM) and mitochondrial p32 in different cellular compartments. Critically, the interaction with p32 negatively controls the transport of both RECQ4 and its chromatin-associated replication factor MCM10 from the nucleus to mitochondria. Amino acids (Ala420-Ala463), which are deleted in the most common cancer-induced RECQ4 mutation, are required for the interaction with p32. Hence, this RECQ4 mutant, which is no longer regulated by p32 and is enriched in the mitochondria, interacts with the mitochondrial replication helicase PEO1 and induces abnormally high levels of mtDNA synthesis. PMID:24746816

  3. Mutations of Cytosolic Loop Residues Impair Assembly and Maturation of α7 Nicotinic Acetylcholine Receptors

    PubMed Central

    Mukherjee, Jayanta; Kuryatov, Alexander; Moss, Stephen J.; Lindstrom, Jon M.; Anand, Rene

    2009-01-01

    Mechanisms that regulate early events in the biogenesis of the α7 nicotinic acetylcholine receptor (α7 AChR) are not well understood. Data presented here show that single amino acid mutations in the cytoplasmic loop of the α7 AChR, between position 335 and 343, abolish or attenuate expression of mature pentameric α7 AChRs in both human embryonic kidney tsA201 (HEK) and neuronal SH-SY5Y cells. Although the number of mature α7 AChRs is increased significantly in the presence of the chaperone protein RIC-3 in HEK cells, sucrose gradient sedimentation reveals that the vast majority of α7 subunits are aggregated or improperly assembled. Transfection of α7 AChRs in SH-SY5Y cells, which endogenously express the α7 AChR, results in a much larger fraction of subunits assembled into mature AChRs. Thus, efficient assembly of α7 AChRs is influenced by several regions of the large cytoplasmic domain, as well perhaps by other parts of its structure, and requires as yet unknown factors not required by other AChR subtypes. PMID:19627445

  4. High association of T1858-G1896 precore mutations with impaired base pairing and high hepatitis B virus DNA levels in HBeAg-negative chronically infected patients.

    PubMed

    Castelain, Sandrine; Descamps, Véronique; Brochot, Etienne; Helle, François; Duverlie, Gilles; Nguyen-Khac, Eric; François, Catherine

    2017-07-01

    The progression of liver disease in hepatitis B virus (HBV) infection is fostered by active virus replication. Mutations in the basal core promoter (BCP) and precore (PC) regions of the HBV genome are known to have an impact on viral replication. The aim of the present study was to assess the correlation of mutation profiles in the BCP and PC regions with the viral load in HBeAg-negative chronically infected patients. The HBV genotype, BCP/PC mutations, serum HBV DNA levels, and associated serological markers were analyzed in 92 HBeAg-negative chronically infected patients. Sequence analysis of the BCP and PC regions revealed variability of 19% and 24.1%, respectively. This variability was primarily associated with five critical positions (1753, 1762, 1764, 1896 and 1899). An elevated HBV viral load (>20,000 IU/ml) was classically correlated with F2-F4 liver fibrosis, elevated serum alanine aminotransferase levels, 1762/1764 and 1753 combination mutations, and surprisingly, with an 1858T-1896G double mutation that impairs base pairing at the base of the bulge in the ε encapsidation signal. An analysis of covariance confirmed the independent nature of the relationship between the 1858T-1896G double mutation and the HBV viral load. In conclusion, independently of conventional parameters, this study demonstrates that a high serum HBV DNA level was also associated with PC 1858-1896 mutations. These BCP/PC mutations may have important clinical implications as predictive factors for HBV DNA increase.

  5. Mutation of β-glucosidase 2 causes glycolipid storage disease and impaired male fertility

    PubMed Central

    Yildiz, Yildiz; Matern, Heidrun; Thompson, Bonne; Allegood, Jeremy C.; Warren, Rebekkah L.; Ramirez, Denise M.O.; Hammer, Robert E.; Hamra, F. Kent; Matern, Siegfried; Russell, David W.

    2006-01-01

    β-Glucosidase 2 (GBA2) is a resident enzyme of the endoplasmic reticulum thought to play a role in the metabolism of bile acid–glucose conjugates. To gain insight into the biological function of this enzyme and its substrates, we generated mice deficient in GBA2 and found that these animals had normal bile acid metabolism. Knockout males exhibited impaired fertility. Microscopic examination of sperm revealed large round heads (globozoospermia), abnormal acrosomes, and defective mobility. Glycolipids, identified as glucosylceramides by mass spectrometry, accumulated in the testes, brains, and livers of the knockout mice but did not cause obvious neurological symptoms, organomegaly, or a reduction in lifespan. Recombinant GBA2 hydrolyzed glucosylceramide to glucose and ceramide; the same reaction catalyzed by the β-glucosidase acid 1 (GBA1) defective in subjects with the Gaucher’s form of lysosomal storage disease. We conclude that GBA2 is a glucosylceramidase whose loss causes accumulation of glycolipids and an endoplasmic reticulum storage disease. PMID:17080196

  6. The iodide-transport-defect-causing mutation R124H: a δ-amino group at position 124 is critical for maturation and trafficking of the Na+/I- symporter.

    PubMed

    Paroder, Viktoriya; Nicola, Juan P; Ginter, Christopher S; Carrasco, Nancy

    2013-08-01

    Na(+)/I(-) symporter (NIS)-mediated active accumulation of I(-) in thyrocytes is a key step in the biosynthesis of the iodine-containing thyroid hormones T3 and T4. Several NIS mutants have been identified as a cause of congenital I(-) transport defect (ITD), and their investigation has yielded valuable mechanistic information on NIS. Here we report novel findings derived from the thorough characterization of the ITD-causing mutation R124H, located in the second intracellular loop (IL-2). R124H NIS is incompletely glycosylated and colocalizes with endoplasmic reticulum (ER)-resident protein markers. As a result, R124H NIS is not targeted to the plasma membrane and therefore does not mediate any I(-) transport in transfected COS-7 cells. Strikingly, however, the mutant is intrinsically active, as revealed by its ability to mediate I(-) transport in membrane vesicles. Of all the amino acid substitutions we carried out at position 124 (K, D, E, A, W, N and Q), only Gln restored targeting of NIS to the plasma membrane and NIS activity, suggesting a key structural role for the δ-amino group of R124 in the transporter's maturation and cell surface targeting. Using our NIS homology model based on the structure of the Vibrio parahaemolyticus Na(+)/galactose symporter, we propose an interaction between the δ-amino group of either R or Q124 and the thiol group of C440, located in IL-6. We conclude that the interaction between IL-2 and IL-6 is critical for the local folding required for NIS maturation and plasma membrane trafficking.

  7. Heterozygous TBK1 mutations impair TLR3 immunity and underlie herpes simplex encephalitis of childhood

    PubMed Central

    Herman, Melina; Ciancanelli, Michael; Ou, Yi-Hung; Lorenzo, Lazaro; Klaudel-Dreszler, Maja; Pauwels, Elodie; Sancho-Shimizu, Vanessa; Pérez de Diego, Rebeca; Abhyankar, Avinash; Israelsson, Elisabeth; Guo, Yiqi; Cardon, Annabelle; Rozenberg, Flore; Lebon, Pierre; Tardieu, Marc; Heropolitańska-Pliszka, Edyta; Chaussabel, Damien; White, Michael A.; Abel, Laurent; Zhang, Shen-Ying

    2012-01-01

    Childhood herpes simplex virus-1 (HSV-1) encephalitis (HSE) may result from single-gene inborn errors of TLR3 immunity. TLR3-dependent induction of IFN-α/β or IFN-λ is crucial for protective immunity against primary HSV-1 infection in the central nervous system (CNS). We describe here two unrelated children with HSE carrying different heterozygous mutations (D50A and G159A) in TBK1, the gene encoding TANK-binding kinase 1, a kinase at the crossroads of multiple IFN-inducing signaling pathways. Both mutant TBK1 alleles are loss-of-function but through different mechanisms: protein instability (D50A) or a loss of kinase activity (G159A). Both are also associated with an autosomal-dominant (AD) trait but by different mechanisms: haplotype insufficiency (D50A) or negative dominance (G159A). A defect in polyinosinic-polycytidylic acid–induced TLR3 responses can be detected in fibroblasts heterozygous for G159A but not for D50A TBK1. Nevertheless, viral replication and cell death rates caused by two TLR3-dependent viruses (HSV-1 and vesicular stomatitis virus) were high in fibroblasts from both patients, and particularly so in G159A TBK1 fibroblasts. These phenotypes were rescued equally well by IFN-α2b. Moreover, the IFN responses to the TLR3-independent agonists and viruses tested were maintained in both patients’ peripheral blood mononuclear cells and fibroblasts. The narrow, partial cellular phenotype thus accounts for the clinical phenotype of these patients being limited to HSE. These data identify AD partial TBK1 deficiency as a new genetic etiology of childhood HSE, indicating that TBK1 is essential for the TLR3- and IFN-dependent control of HSV-1 in the CNS. PMID:22851595

  8. A Common Mutation in DEFB126 Causes Impaired Sperm Function and Subfertility

    PubMed Central

    Tollner, Theodore L.; Venners, Scott A.; Hollox, Edward J.; Yudin, Ashley I.; Liu, Xue; Tang, Genfu; Xing, Houxun; Kays, Robert J.; Lau, Tsang; Overstreet, James W.; Xu, Xiping; Bevins, Charles L.; Cherr, Gary N.

    2013-01-01

    A glycosylated polypeptide, β-defensin 126 (DEFB126), derived from the epididymis and adsorbed onto the sperm surface, has been implicated in immunoprotection and efficient movement of sperm in mucosal fluids of the female reproductive tract. Here, we report a sequence variant in DEFB126 that has a 2-nucleotide deletion in the open reading frame, which generates a non-stop mRNA. The allele frequency of this variant sequence is high in both a European (0.47) and a Chinese (0.45) population cohort. Binding of the Agaricus bisporus lectin to the sperm surface glycocalyx was significantly lower in men with the homozygous variant (del/del) genotype than in those with either a del/wt or wt/wt genotype, suggesting an altered sperm glycocalyx with fewer O-linked oligosaccharides in del/del men. Moreover, sperm from the del/del donors exhibited an 84% reduction in the rate of penetration of a hyaluronic acid (HA) gel, a surrogate for cervical mucus, compared to the other genotypes. This reduction in sperm performance in HA gels was not a result of decreased progressive motility (average curvilinear velocity) or morphological deficits. However, DEFB126 genotype and lectin binding were highly correlated with performance in the penetration assays. In a prospective cohort study of newly married couples who were trying to conceive by natural means, couples were less likely to become pregnant and took longer to achieve a live birth if the male partner was homozygous for the variant sequence. This common sequence variation in DEFB126, and its apparent cause of impaired reproductive function, provides an opportunity to better understand, clinically evaluate, and possibly treat human infertility. PMID:21775668

  9. Mutation in MTO1 involved in tRNA modification impairs mitochondrial RNA metabolism in the yeast Saccharomyces cerevisiae.

    PubMed

    Wang, Xinjian; Yan, Qingfeng; Guan, Min-Xin

    2009-06-01

    The yeast MTO1 gene encodes an evolutionarily conserved protein for the biosynthesis of the 5-carboxymethylaminomethyl group of cmnm(5)s(2)U in the wobble position of mitochondrial tRNA. However, mto1 null mutant expressed the respiratory deficient phenotype only when coupled with the C1409G mutation of mitochondrial 15S rRNA. To further understand the role of MTO1 in mitochondrial RNA metabolism, the yeast mto1 null mutants carrying either wild-type (P(S)) or 15S rRNA C1409G allele (P(R)) have been characterized by examining the steady-state levels, aminoacylation capacity of mitochondrial tRNA, mitochondrial gene expression and petite formation. The steady-state levels of tRNA(Lys), tRNA(Glu), tRNA(Gln), tRNA(Leu), tRNA(Gly), tRNA(Arg) and tRNA(Phe) were decreased significantly while those of tRNA(Met) and tRNA(His) were not affected in the mto1 strains carrying the P(S) allele. Strikingly, the combination of the mto1 and C1409G mutations gave rise to the synthetic phenotype for some of the tRNAs, especially in tRNA(Lys), tRNA(Met) and tRNA(Phe). Furthermore, the mto1 strains exhibited a marked reduction in the aminoacylation levels of mitochondrial tRNA(Lys), tRNA(Leu), tRNA(Arg) but almost no effect in those of tRNA(His). In addition, the steady-state levels of mitochondrial COX1, COX2, COX3, ATP6 and ATP9 mRNA were markedly decreased in mto1 strains. These data strongly indicate that unmodified tRNA caused by the deletion of MTO1 gene caused the instability of mitochondrial tRNAs and mRNAs and an impairment of aminoacylation of mitochondrial tRNAs. Consequently, the deletion of MTO1 gene acts in synergy with the 15S rRNA C1409G mutation, leading to the loss of COX1 synthesis and subsequent respiratory deficient phenotype.

  10. ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis.

    PubMed

    Cardona-López, Ximena; Cuyas, Laura; Marín, Elena; Rajulu, Charukesi; Irigoyen, María Luisa; Gil, Erica; Puga, María Isabel; Bligny, Richard; Nussaume, Laurent; Geldner, Niko; Paz-Ares, Javier; Rubio, Vicente

    2015-09-01

    Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life.

  11. ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis

    PubMed Central

    Cardona-López, Ximena; Cuyas, Laura; Marín, Elena; Irigoyen, María Luisa; Gil, Erica; Puga, María Isabel; Bligny, Richard; Nussaume, Laurent; Geldner, Niko; Paz-Ares, Javier

    2015-01-01

    Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life. PMID:26342016

  12. Disrupting vesicular trafficking at the endosome attenuates transcriptional activation by Gcn4.

    PubMed

    Zhang, Fan; Gaur, Naseem A; Hasek, Jiri; Kim, Soon-ja; Qiu, Hongfang; Swanson, Mark J; Hinnebusch, Alan G

    2008-11-01

    The late endosome (MVB) plays a key role in coordinating vesicular transport of proteins between the Golgi complex, vacuole/lysosome, and plasma membrane. We found that deleting multiple genes involved in vesicle fusion at the MVB (class C/D vps mutations) impairs transcriptional activation by Gcn4, a global regulator of amino acid biosynthetic genes, by decreasing the ability of chromatin-bound Gcn4 to stimulate preinitiation complex assembly at the promoter. The functions of hybrid activators with Gal4 or VP16 activation domains are diminished in class D mutants as well, suggesting a broader defect in activation. Class E vps mutations, which impair protein sorting at the MVB, also decrease activation by Gcn4, provided they elicit rapid proteolysis of MVB cargo proteins in the aberrant late endosome. By contrast, specifically impairing endocytic trafficking from the plasma membrane, or vesicular transport to the vacuole, has a smaller effect on Gcn4 function. Thus, it appears that decreasing cargo proteins in the MVB through impaired delivery or enhanced degradation, and not merely the failure to transport cargo properly to the vacuole or downregulate plasma membrane proteins by endocytosis, is required to attenuate substantially transcriptional activation by Gcn4.

  13. [The mutation spectrum of the GJB2 gene in Belarussian patients with hearing loss. Results of pilot genetic screening of hearing impairment in newborns].

    PubMed

    Bliznets, E A; Marcul', D N; Khorov, O G; Markova, T G; Poliakov, A V

    2014-02-01

    A total of 111 unrelated probands and their 8 sibs from Grodno oblast (Belarus) with bilateral isolated sensorineural hearing impairment were studied for the presence of mutations in the connexin 26--GJB2gene. Mutations were detected in 51 probands (46% of the sample). A significantly higher frequency of the GJB2gene mutations was observed in familial cases of the disease with the autosomal recessive type of inheritance (in 78% of families). Detected peculiarities of the GJB2 gene mutation spectrum demonstrated that use of the algorithm, which was developed for Russian patients, is optimal for the molecular study of patients from Be- larus. In the sample of patients with hearing loss, the highest (among other similar samples studied in the world) allele frequency of c.313_326de114 mutation (7% out of all pathological GJB2 alleles) was registered; Polish origin of this deletion was suggested. It was demonstrated that detection of the GJB2 gene mutation on only one patient's chromosome is insufficient to confirm a molecular genetic diagnosis of hearing loss of the DFNB1 genetic type (autosomal recessive hearing loss caused by the GJB2 gene mutations). Pilot screening in the presence of GJB2 gene mutations in newborns from Grodno oblast was conducted. The material from 235 children was studied during the screening; nine heterozygous carriers of the mutation were found. The c.35delG mutation was detected in a homozygous state in a single newborn (hearing loss of moderate severity was subsequently audiologically confirmed in this child).

  14. Adenylate cyclase 1 (ADCY1) mutations cause recessive hearing impairment in humans and defects in hair cell function and hearing in zebrafish

    PubMed Central

    Santos-Cortez, Regie Lyn P.; Lee, Kwanghyuk; Giese, Arnaud P.; Ansar, Muhammad; Amin-Ud-Din, Muhammad; Rehn, Kira; Wang, Xin; Aziz, Abdul; Chiu, Ilene; Hussain Ali, Raja; Smith, Joshua D.; Shendure, Jay; Bamshad, Michael; Nickerson, Deborah A.; Ahmed, Zubair M.; Ahmad, Wasim; Riazuddin, Saima; Leal, Suzanne M.

    2014-01-01

    Cyclic AMP (cAMP) production, which is important for mechanotransduction within the inner ear, is catalyzed by adenylate cyclases (AC). However, knowledge of the role of ACs in hearing is limited. Previously, a novel autosomal recessive non-syndromic hearing impairment locus DFNB44 was mapped to chromosome 7p14.1-q11.22 in a consanguineous family from Pakistan. Through whole-exome sequencing of DNA samples from hearing-impaired family members, a nonsense mutation c.3112C>T (p.Arg1038*) within adenylate cyclase 1 (ADCY1) was identified. This stop-gained mutation segregated with hearing impairment within the family and was not identified in ethnically matched controls or within variant databases. This mutation is predicted to cause the loss of 82 amino acids from the carboxyl tail, including highly conserved residues within the catalytic domain, plus a calmodulin-stimulation defect, both of which are expected to decrease enzymatic efficiency. Individuals who are homozygous for this mutation had symmetric, mild-to-moderate mixed hearing impairment. Zebrafish adcy1b morphants had no FM1-43 dye uptake and lacked startle response, indicating hair cell dysfunction and gross hearing impairment. In the mouse, Adcy1 expression was observed throughout inner ear development and maturation. ADCY1 was localized to the cytoplasm of supporting cells and hair cells of the cochlea and vestibule and also to cochlear hair cell nuclei and stereocilia. Ex vivo studies in COS-7 cells suggest that the carboxyl tail of ADCY1 is essential for localization to actin-based microvilli. These results demonstrate that ADCY1 has an evolutionarily conserved role in hearing and that cAMP signaling is important to hair cell function within the inner ear. PMID:24482543

  15. Homozygosity for the V37I GJB2 mutation in fifteen probands with mild to moderate sensorineural hearing impairment: further confirmation of pathogenicity and haplotype analysis in Asian populations.

    PubMed

    Gallant, Emily; Francey, Lauren; Tsai, Ellen A; Berman, Micah; Zhao, Yaru; Fetting, Heather; Kaur, Maninder; Deardorff, Matthew A; Wilkens, Alisha; Clark, Dinah; Hakonarson, Hakon; Rehm, Heidi L; Krantz, Ian D

    2013-09-01

    Hearing impairment affects 1 in 650 newborns, making it the most common congenital sensory impairment. Autosomal recessive nonsyndromic sensorineural hearing impairment (ARNSHI) comprises 80% of familial hearing impairment cases. Mutations in GJB2 account for a significant number of ARNSHI (and up to 50% of documented recessive (e.g., more than 1 affected sibling) hearing impairment in some populations). Mutations in the GJB2 gene are amongst the most common causes of hearing impairment in populations of various ethnic backgrounds. Two mutations of this gene, 35delG and 167delT, account for the majority of reported mutations in Caucasian populations, especially those of Mediterranean and Ashkenazi Jewish background. The 235delC mutation is most prevalent in East Asian populations. Some mutations are of less well-characterized significance. The V37I missense mutation, common in Asian populations, was initially described as a polymorphism and later as a potentially pathogenic mutation. We report here on 15 unrelated individuals with ARNSHI and homozygosity for the V37I GJB2 missense mutation. Nine individuals are of Chinese ancestry, two are of unspecified Asian descent, one is of Japanese descent, one individual is of Vietnamese ancestry, one of Philippine background and one of Italian and Cuban/Caucasian background. Homozygosity for the V37I GJB2 mutation may be a more common pathogenic missense mutation in Asian populations, resulting in mild to moderate sensorineural hearing impairment. We report a presumed haplotype block specific to East Asian individuals with the V37I mutation encompassing the GJB2 gene that may account for the high prevalence in East Asian populations.

  16. Correspondence regarding Ballana et al., "Mitochondrial 12S rRNA gene mutations affect RNA secondary structure and lead to variable penetrance in hearing impairment".

    PubMed

    Abreu-Silva, R S; Batissoco, A C; Lezirovitz, K; Romanos, J; Rincon, D; Auricchio, M T B M; Otto, P A; Mingroni-Netto, R C

    2006-05-12

    Ballana et al. [E. Ballana, E. Morales, R. Rabionet, B. Montserrat, M. Ventayol, O. Bravo, P. Gasparini, X. Estivill, Mitochondrial 12S rRNA gene mutations affect RNA secondary structure and lead to variable penetrance in hearing impairment, Biochem. Biophys. Res. Commun. 341 (2006) 950-957] detected a T1291C mutation segregating in a Cuban pedigree with hearing impairment. They interpreted it as probably pathogenic, based on family history, RNA conformation prediction and its absence in a control group of 95 Spanish subjects. We screened a sample of 203 deaf subjects and 300 hearing controls (110 "European-Brazilians" and 190 "African-Brazilians") for the mitochondrial mutations A1555G and T1291C. Five deaf subjects had the T1291C substitution, three isolated cases and two familial cases. In the latter, deafness was paternally inherited or segregated with the A1555G mutation. This doesn't support the hypothesis of T1291C mutation being pathogenic. Two "African-Brazilian" controls also had the T1291C substitution. Six of the seven T1291C-carriers (five deaf and two controls) had mitochondrial DNA of African origin, belonging to macrohaplogroup L1/L2. Therefore, these data point to T1291C substitution as most probably an African non-pathogenic polymorphism.

  17. Disease-associated missense mutations in the EVH1 domain disrupt intrinsic WASp function causing dysregulated actin dynamics and impaired dendritic cell migration

    PubMed Central

    Metelo, Joao; Bouma, Gerben; Moulding, Dale; Fritzsche, Marco; Vernay, Bertrand; Charras, Guillaume; Cory, Giles O. C.; Thrasher, Adrian J.; Burns, Siobhan O.

    2013-01-01

    Wiskott Aldrich syndrome (WAS), an X-linked immunodeficiency, results from loss-of-function mutations in the human hematopoietic cytoskeletal regulator gene WAS. Many missense mutations in the Ena Vasp homology1 (EVH1) domain preserve low-level WAS protein (WASp) expression and confer a milder clinical phenotype. Although disrupted binding to WASp-interacting protein (WIP) leads to enhanced WASp degradation in vivo, the intrinsic function of EVH1-mutated WASp is poorly understood. In the present study, we show that, despite mediating enhanced actin polymerization compared with wild-type WASp in vitro, EVH1 missense mutated proteins did not support full biologic function in cells, even when levels were restored by forced overexpression. Podosome assembly was aberrant and associated with dysregulated lamellipodia formation and impaired persistence of migration. At sites of residual podosome-associated actin polymerization, localization of EVH1-mutated proteins was preserved even after deletion of the entire domain, implying that WIP-WASp complex formation is not absolutely required for WASp localization. However, retention of mutant proteins in podosomes was significantly impaired and associated with reduced levels of WASp tyrosine phosphorylation. Our results indicate that the EVH1 domain is important not only for WASp stability, but also for intrinsic biologic activity in vivo. PMID:23160469

  18. A Novel Mutation in Isoform 3 of the Plasma Membrane Ca2+ Pump Impairs Cellular Ca2+ Homeostasis in a Patient with Cerebellar Ataxia and Laminin Subunit 1α Mutations*

    PubMed Central

    Calì, Tito; Lopreiato, Raffaele; Shimony, Joshua; Vineyard, Marisa; Frizzarin, Martina; Zanni, Ginevra; Zanotti, Giuseppe; Brini, Marisa; Shinawi, Marwan; Carafoli, Ernesto

    2015-01-01

    The particular importance of Ca2+ signaling to neurons demands its precise regulation within their cytoplasm. Isoform 3 of the plasma membrane Ca2+ ATPase (the PMCA3 pump), which is highly expressed in brain and cerebellum, plays an important role in the regulation of neuronal Ca2+. A genetic defect of the PMCA3 pump has been described in one family with X-linked congenital cerebellar ataxia. Here we describe a novel mutation in the ATP2B3 gene in a patient with global developmental delay, generalized hypotonia and cerebellar ataxia. The mutation (a R482H replacement) impairs the Ca2+ ejection function of the pump. It reduces the ability of the pump expressed in model cells to control Ca2+ transients generated by cell stimulation and impairs its Ca2+ extrusion function under conditions of low resting cytosolic Ca2+ as well. In silico analysis of the structural effect of the mutation suggests a reduced stabilization of the portion of the pump surrounding the mutated residue in the Ca2+-bound state. The patient also carries two missense mutations in LAMA1, encoding laminin subunit 1α. On the basis of the family pedigree of the patient, the presence of both PMCA3 and laminin subunit 1α mutations appears to be necessary for the development of the disease. Considering the observed defect in cellular Ca2+ homeostasis and the previous finding that PMCAs act as digenic modulators in Ca2+-linked pathologies, the PMCA3 dysfunction along with LAMA1 mutations could act synergistically to cause the neurological phenotype. PMID:25953895

  19. A Novel Mutation in Isoform 3 of the Plasma Membrane Ca2+ Pump Impairs Cellular Ca2+ Homeostasis in a Patient with Cerebellar Ataxia and Laminin Subunit 1α Mutations.

    PubMed

    Calì, Tito; Lopreiato, Raffaele; Shimony, Joshua; Vineyard, Marisa; Frizzarin, Martina; Zanni, Ginevra; Zanotti, Giuseppe; Brini, Marisa; Shinawi, Marwan; Carafoli, Ernesto

    2015-06-26

    The particular importance of Ca(2+) signaling to neurons demands its precise regulation within their cytoplasm. Isoform 3 of the plasma membrane Ca(2+) ATPase (the PMCA3 pump), which is highly expressed in brain and cerebellum, plays an important role in the regulation of neuronal Ca(2+). A genetic defect of the PMCA3 pump has been described in one family with X-linked congenital cerebellar ataxia. Here we describe a novel mutation in the ATP2B3 gene in a patient with global developmental delay, generalized hypotonia and cerebellar ataxia. The mutation (a R482H replacement) impairs the Ca(2+) ejection function of the pump. It reduces the ability of the pump expressed in model cells to control Ca(2+) transients generated by cell stimulation and impairs its Ca(2+) extrusion function under conditions of low resting cytosolic Ca(2+) as well. In silico analysis of the structural effect of the mutation suggests a reduced stabilization of the portion of the pump surrounding the mutated residue in the Ca(2+)-bound state. The patient also carries two missense mutations in LAMA1, encoding laminin subunit 1α. On the basis of the family pedigree of the patient, the presence of both PMCA3 and laminin subunit 1α mutations appears to be necessary for the development of the disease. Considering the observed defect in cellular Ca(2+) homeostasis and the previous finding that PMCAs act as digenic modulators in Ca(2+)-linked pathologies, the PMCA3 dysfunction along with LAMA1 mutations could act synergistically to cause the neurological phenotype. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Vigorous physical activity impairs myocardial function in patients with arrhythmogenic right ventricular cardiomyopathy and in mutation positive family members.

    PubMed

    Saberniak, Jørg; Hasselberg, Nina E; Borgquist, Rasmus; Platonov, Pyotr G; Sarvari, Sebastian I; Smith, Hans-Jørgen; Ribe, Margareth; Holst, Anders G; Edvardsen, Thor; Haugaa, Kristina H

    2014-12-01

    Exercise increases risk of ventricular arrhythmia in subjects with arrhythmogenic right ventricular cardiomyopathy (ARVC). We aimed to investigate the impact of exercise on myocardial function in ARVC subjects. We included 110 subjects (age 42 ± 17 years), 65 ARVC patients and 45 mutation-positive family members. Athletes were defined as subjects with ≥4 h vigorous exercise/week [≥1440 metabolic equivalents (METs × minutes/week)] during a minimum of 6 years. Athlete definition was fulfilled in 37/110 (34%) subjects. We assessed right ventricular (RV) and left ventricular (LV) myocardial function by echocardiography, and by magnetic resonance imaging (MRI). The RV function by RV fractional area change (FAC), RV global longitudinal strain (GLS) by echocardiography, and RV ejection fraction (EF) by MRI was reduced in athletes compared with non-athletes (FAC 34 ± 9% vs. 40 ± 11%, RVGLS -18.3 ± 6.1% vs. -22.0 ± 4.8%, RVEF 32 ± 8% vs. 43 ± 10%, all P < 0.01). LV function by LVEF and LVGLS was reduced in athletes compared with non-athletes (LVEF by echocardiography 50 ± 10% vs. 57 ± 5%, LVEF by MRI 46 ± 6% vs. 53 ± 8%, and LVGLS -16.7 ± 4.2% vs. -19.4 ± 2.9%, all P < 0.01). The METs × minutes/week correlated with reduced RV and LV function by echocardiography and MRI (all P < 0.01). The LVEF by MRI was also reduced in subgroups of athlete index patients (46 ± 7% vs. 54 ± 10%, P = 0.02) and in athlete family members (47 ± 3% vs. 52 ± 6%, P < 0.05). Athletes showed reduced biventricular function compared with non-athletes in ARVC patients and in mutation-positive family members. The amount and intensity of exercise activity was associated with impaired LV and RV function. Exercise may aggravate and accelerate myocardial dysfunction in ARVC. © 2014 The Authors. European Journal of Heart Failure published by John Wiley & Sons Ltd on behalf of European

  1. A selective histone deacetylase-6 inhibitor improves BDNF trafficking in hippocampal neurons from Mecp2 knockout mice: implications for Rett syndrome

    PubMed Central

    Xu, Xin; Kozikowski, Alan P.; Pozzo-Miller, Lucas

    2014-01-01

    Rett syndrome (RTT) is a neurodevelopmental disorder caused by loss-of-function mutations in the transcriptional modulator methyl-CpG-binding protein 2 (MECP2). One of the most prominent gene targets of MeCP2 is brain-derived neurotrophic factor (Bdnf), a potent modulator of activity-dependent synaptic development, function and plasticity. Dysfunctional BDNF signaling has been demonstrated in several pathophysiological mechanisms of RTT disease progression. To evaluate whether the dynamics of BDNF trafficking is affected by Mecp2 deletion, we analyzed movements of BDNF tagged with yellow fluorescent protein (YFP) in cultured hippocampal neurons by time-lapse fluorescence imaging. We found that both anterograde and retrograde vesicular trafficking of BDNF-YFP are significantly impaired in Mecp2 knockout hippocampal neurons. Selective inhibitors of histone deacetylase 6 (HDAC6) show neuroprotective effects in neurodegenerative diseases and stimulate microtubule-dependent vesicular trafficking of BDNF-containing dense core vesicles. Here, we show that the selective HDAC6 inhibitor Tubastatin-A increased the velocity of BDNF-YFP vesicles in Mecp2 knockout neurons in both directions by increasing α–tubulin acetylation. Tubastatin-A also restored activity-dependent BDNF release from Mecp2 knockout neurons to levels comparable to those shown by wildtype neurons. These findings demonstrate that a selective HDAC6 inhibitor is a potential pharmacological strategy to reverse cellular and synaptic impairments in RTT resulting from impaired BDNF signaling. PMID:24639629

  2. A Point Mutation in the Gene for Asparagine-Linked Glycosylation 10B (Alg10b) Causes Nonsyndromic Hearing Impairment in Mice (Mus musculus)

    PubMed Central

    Probst, Frank J.; Corrigan, Rebecca R.; del Gaudio, Daniela; Salinger, Andrew P.; Lorenzo, Isabel; Gao, Simon S.; Chiu, Ilene; Xia, Anping

    2013-01-01

    The study of mouse hearing impairment mutants has led to the identification of a number of human hearing impairment genes and has greatly furthered our understanding of the physiology of hearing. The novel mouse mutant neurological/sensory 5 (nse5) demonstrates a significantly reduced or absent startle response to sound and is therefore a potential murine model of human hearing impairment. Genetic analysis of 500 intercross progeny localized the mutant locus to a 524 kilobase (kb) interval on mouse chromosome 15. A missense mutation in a highly-conserved amino acid was found in the asparagine-linked glycosylation 10B gene (Alg10b), which is within the critical interval for the nse5 mutation. A 20.4 kb transgene containing a wildtype copy of the Alg10b gene rescued the mutant phenotype in nse5/nse5 homozygous animals, confirming that the mutation in Alg10b is responsible for the nse5/nse5 mutant phenotype. Homozygous nse5/nse5 mutants had abnormal auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAEs), and cochlear microphonics (CMs). Endocochlear potentials (EPs), on the other hand, were normal. ABRs and DPOAEs also confirmed the rescue of the mutant nse5/nse5 phenotype by the wildtype Alg10b transgene. These results suggested a defect in the outer hair cells of mutant animals, which was confirmed by histologic analysis. This is the first report of mutation in a gene involved in the asparagine (N)-linked glycosylation pathway causing nonsyndromic hearing impairment, and it suggests that the hearing apparatus, and the outer hair cells in particular, are exquisitely sensitive to perturbations of the N-linked glycosylation pathway. PMID:24303013

  3. A point mutation in the gene for asparagine-linked glycosylation 10B (Alg10b) causes nonsyndromic hearing impairment in mice (Mus musculus).

    PubMed

    Probst, Frank J; Corrigan, Rebecca R; Del Gaudio, Daniela; Salinger, Andrew P; Lorenzo, Isabel; Gao, Simon S; Chiu, Ilene; Xia, Anping; Oghalai, John S; Justice, Monica J

    2013-01-01

    The study of mouse hearing impairment mutants has led to the identification of a number of human hearing impairment genes and has greatly furthered our understanding of the physiology of hearing. The novel mouse mutant neurological/sensory 5 (nse5) demonstrates a significantly reduced or absent startle response to sound and is therefore a potential murine model of human hearing impairment. Genetic analysis of 500 intercross progeny localized the mutant locus to a 524 kilobase (kb) interval on mouse chromosome 15. A missense mutation in a highly-conserved amino acid was found in the asparagine-linked glycosylation 10B gene (Alg10b), which is within the critical interval for the nse5 mutation. A 20.4 kb transgene containing a wildtype copy of the Alg10b gene rescued the mutant phenotype in nse5/nse5 homozygous animals, confirming that the mutation in Alg10b is responsible for the nse5/nse5 mutant phenotype. Homozygous nse5/nse5 mutants had abnormal auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAEs), and cochlear microphonics (CMs). Endocochlear potentials (EPs), on the other hand, were normal. ABRs and DPOAEs also confirmed the rescue of the mutant nse5/nse5 phenotype by the wildtype Alg10b transgene. These results suggested a defect in the outer hair cells of mutant animals, which was confirmed by histologic analysis. This is the first report of mutation in a gene involved in the asparagine (N)-linked glycosylation pathway causing nonsyndromic hearing impairment, and it suggests that the hearing apparatus, and the outer hair cells in particular, are exquisitely sensitive to perturbations of the N-linked glycosylation pathway.

  4. Impairment of brain and muscle energy metabolism detected by magnetic resonance spectroscopy in hereditary spastic paraparesis type 28 patients with DDHD1 mutations.

    PubMed

    Liguori, Rocco; Giannoccaro, Maria Pia; Arnoldi, Alessia; Citterio, Andrea; Tonon, Caterina; Lodi, Raffaele; Bresolin, Nereo; Bassi, Maria Teresa

    2014-09-01

    Mutations in DDHD1 gene have been associated with the SPG28 subtype of Hereditary Spastic Paraparesis (HSP). Clinical phenotype includes axonal neuropathy, distal sensory loss, and cerebellar eye movement disturbances. We screened 96 index subjects from recessive HSP families for mutation and identified one family with two sibs carrying mutations in DDHD1 gene. Clinical, neuropsychological, and neuroimaging studies were performed, including MR spectroscopy of brain and muscle of the two mutated patients. Two novel heterozygous mutations in DDHD1 were found in the affected members of one family, with clinical features overlapping the SPG28 subtype. Of note, MR spectroscopy of brain and muscle in these patients indicated a mild deficit of brain energy metabolism in the oldest and most severely affected patient, while an impairment of energy metabolism was found in the skeletal muscle of both patients. Unlike the DDHD2 mutated patients, no evidence of lipid accumulation in the brain was found. Our data along with those previously reported suggest a dysfunction in the OXPHOS system possibly due to mitochondrial lipid content modification, which could be a central mechanism in the pathogenesis of SPG28.

  5. Mutation of histidine residues in CP47 leads to destabilization of the photosystem II complex and to impairment of light energy transfer.

    PubMed

    Shen, G; Eaton-Rye, J J; Vermaas, W F

    1993-05-18

    Site-directed mutagenesis has been used to change conserved histidine residues in hydrophobic regions of the photosystem II chlorophyll-binding protein CP47 in the cyanobacterium Synechocystis sp. PCC 6803. Nine mutants with one, four mutants with two, and four mutants with three His mutations in CP47 have been generated and characterized. Mutation of any one of seven different His residues to Tyr leads to slower photoautotrophic growth and apparent destabilization of the PS II complex. Mutations introduced into multiple His residues in one mutant exhibited a cumulative effect. Replacing His by Asn leads to a much smaller effect than observed upon mutation to Tyr. This is consistent with the hypothesis that the mutated His residues are chlorophyll ligands: Asn can substitute as chlorophyll ligand, whereas Tyr cannot. Further evidence supporting a role of the mutated His residues in chlorophyll binding comes from measurements of the light intensity needed to half-saturate oxygen evolution. All His mutants with impaired PS II function needed higher light intensities for half-saturation than wild type. A possible explanation for this decrease in antenna efficiency in the mutants is a loss of the Mg in the chlorophyll due to a loss of the fifth ligand, and thus the formation of a pheophytin molecule in the antenna. We conclude that conserved His residues in hydrophobic regions of CP47 indeed are chlorophyll ligands and that these ligands are important for PS II stability as well as efficient antenna function.

  6. Postnatal microcephaly and pain insensitivity due to a de novo heterozygous DNM1L mutation causing impaired mitochondrial fission and function.

    PubMed

    Sheffer, Ruth; Douiev, Liza; Edvardson, Simon; Shaag, Avraham; Tamimi, Khaled; Soiferman, Devorah; Meiner, Vardiella; Saada, Ann

    2016-06-01

    An emerging class of mitochondrial disorders is caused by mutations in nuclear genes affecting mitochondrial dynamics and function. One of these is the DNM1L gene encoding the dynamin-related protein 1 (DRP1), which is pivotal in the mitochondrial fission process. Here, we describe a patient with a novel dominant-negative, de novo DNM1L mutation, which expands the clinical spectrum. The patient reported here exhibits a chronic neurological disorder, characterized by postnatal microcephaly, developmental delay, and pain insensitivity. Muscle biopsy disclosed decreased respiratory chain complex IV activity. Exome sequencing showed a de novo heterozygous c.1084G>A (p.G362S) mutation. Subsequent studies of patient skin fibroblasts showed markedly impaired mitochondrial fission and a partial respiratory chain defect while peroxisomal morphology remained intact. Human foreskin fibroblasts over-expressing the mutant DNM1L gene displayed aberrant mitochondrial morphology. © 2016 Wiley Periodicals, Inc.

  7. Effects of hemochromatosis and transferrin gene mutations on peripheral iron dyshomeostasis in mild cognitive impairment and Alzheimer's and Parkinson's diseases

    PubMed Central

    Mariani, S.; Ventriglia, M.; Simonelli, I.; Spalletta, G.; Bucossi, S.; Siotto, M.; Assogna, F.; Melgari, J. M.; Vernieri, F.; Squitti, R.

    2013-01-01

    Deregulation of iron metabolism has been observed in patients with neurodegenerative diseases. We have carried out a molecular analysis investigating the interaction between iron specific gene variants [transferrin (TF, P589S), hemochromatosis (HFE) C282Y and (H63D)], iron biochemical variables [iron, Tf, ceruloplasmin (Cp), Cp:Tf ratio and % of Tf saturation (% Tf-sat)] and apolipoprotein E (APOE) gene variants in 139 Alzheimer's disease (AD), 27 Mild Cognitive Impairment (MCI), 78 Parkinson's disease (PD) patients and 139 healthy controls to investigate mechanisms of iron regulation or toxicity. No difference in genetic variant distributions between patients and controls was found in our Italian sample, but the stratification for the APOEε4 allele revealed that among the APOEε4 carriers was higher the frequency of those carriers of at least a mutated TF P589S allele. Decreased Tf in both AD and MCI and increased Cp:Tf ratio in AD vs. controls were detected. A multinomial logistic regression model revealed that increased iron and Cp:Tf ratio and being man instead of woman increased the risk of having PD, that increased values of Cp:Tf ratio corresponded to a 4-fold increase of the relative risk of having MCI, while higher Cp levels were protective for PD and MCI. Our study has some limitations: the small size of the samples, one ethnic group considered, the rarity of some alleles which prevent the statistical power of some genetic analysis. Even though they need confirmation in larger cohorts, our data suggest the hypothesis that deregulation of iron metabolism, in addition to other factors, has some effect on the PD disease risk. PMID:23935582

  8. Naturally occurring mutation affecting the MyD88-binding site of TNFRSF13B impairs triggering of class switch recombination

    PubMed Central

    Almejun, Maria B.; Cols, Montserrat; Zelazko, Marta; Oleastro, Matias; Cerutti, Andrea; Oppezzo, Pablo; Cunningham-Rundles, Charlotte; Danielian, Silvia

    2013-01-01

    Mutations in the transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) were previously found to be associated with hypogammaglobulinemia in humans. It has been shown that proliferation inducing ligand (APRIL) elicits class switch recombination (CSR) by inducing recruitment of MyD88 to a TACI highly conserved cytoplasmic domain (THC). We have identified a patient with hypogammaglobulinemia carrying a missense mutation (S231R) predicted to affect the THC. Aiming to evaluate the relevance of this novel mutation of TACI in CSR induction, we tested the ability of TACI, TLR9, or/and CD40 ligands to trigger CSR in naive B cells and B-cell lines carrying S231R. IgG secretion was impaired when triggered by TACI or/and TLR9 ligands on S231R-naive B cells. Likewise, these stimuli induced less expression of activation-induced cytidine deaminase, I(γ)1-C(μ), and I(γ)1-C(μ), while induction by optimal CD40 stimulation was indistinguishable from controls. These cells also showed an impaired cooperation between TACI and TLR9 pathways, as well as a lack of APRIL-mediated enhancement of CD40 activation in suboptimal conditions. Finally, after APRIL ligation, S231R-mutated TACI failed to colocalize with MyD88. Collectively, these results highlight the requirement of an intact MyD88-binding site in TACI to trigger CSR. PMID:23225259

  9. Prevalence of the A1555G (12S rRNA) and tRNASer(UCN) mitochondrial mutations in hearing-impaired Brazilian patients.

    PubMed

    Abreu-Silva, R S; Lezirovitz, K; Braga, M C C; Spinelli, M; Pirana, S; Della-Rosa, V A; Otto, P A; Mingroni-Netto, R C

    2006-02-01

    Mitochondrial mutations are responsible for at least 1% of the cases of hereditary deafness, but the contribution of each mutation has not yet been defined in African-derived or native American genetic backgrounds. A total of 203 unselected hearing-impaired patients were screened for the presence of the mitochondrial mutation A1555G in the 12S rRNA gene and mutations in the tRNASer(UCN) gene in order to assess their frequency in the ethnically admixed Brazilian population. We found four individuals with A1555G mutation (2%), which is a frequency similar to those reported for European-derived populations in unselected samples. On the other hand, complete sequencing of the tRNASer(UCN) did not reveal reported pathogenic substitutions, namely A7445G, 7472insC, T7510C, or T7511C. Instead, other rare substitutions were found such as T1291C, A7569G, and G7444A. To evaluate the significance of these findings, 110 "European-Brazilians" and 190 "African-Brazilians" unrelated hearing controls were screened. The T1291C, A7569G and G7444A substitutions were each found in about 1% (2/190) of individuals of African ancestry, suggesting that they are probably polymorphic. Our results indicate that screening for the A1555G mutation is recommended among all Brazilian deaf patients, while testing for mutations in the tRNASer(UCN) gene should be considered only when other frequent deafness-causing mutations have been excluded or in the presence of a maternal transmission pattern.

  10. Mutations in the amino-terminal domain of the human androgen receptor may be associated with partial androgen insensitivity and impaired transactivation in vitro.

    PubMed

    Holterhus, P-M; Werner, R; Struve, D; Hauffa, B P; Schroeder, C; Hiort, O

    2005-09-01

    The majority of genetic variations in the androgen receptor (AR) gene are point mutations leading to impairment of the DNA- or hormone-binding domains. The N-terminus encoded by the first exon of the AR-gene usually harbors disruptive mutations associated with complete androgen insensitivity syndrome (CAIS) while missense mutations related with partial androgen insensitivity syndrome (PAIS) are seemingly rare. We present a 46,XY male with scrotal hypospadias in whom we detected a S432 F point mutation within the N-terminus. Transient transfections of an AR expression plasmid carrying the S432 F mutation using Chinese Hamster Ovary (CHO) cells revealed a significant partial reduction in transactivation of the co-transfected androgen responsive (ARE) (2)TATA luciferase reporter gene thus confirming PAIS. In two further 46, XY patients with slight to moderate virilization defects, we detected an S411 N mutation, and a 9 base pair deletion leading to the loss of amino acids 409 to 411 (L-A-S), respectively. These mutations did not compromise AR-function under the chosen experimental settings. The S432 F-patient supports particular significance of the AR-N-terminus for mild forms of AIS while the functional role of the two further mutations remains unclear. The N-terminus is a species-specific AR-domain possibly also involved in contributing to target tissue selectivity of AR-actions via mediating co-regulator interactions. Therefore, mild molecular defects of the AR-N-terminus may not necessarily inhibit general transactivation properties using currently established reporter gene models.

  11. Sex Trafficking of Minors.

    PubMed

    Moore, Jessica L; Kaplan, Dana M; Barron, Christine E

    2017-04-01

    Sex trafficking is an increasingly recognized global health crisis affecting every country and region in the world. Domestic minor sex trafficking is a subset of commercial sexual exploitation of children, defined as engagement of minors (<18 years of age) in sexual acts for items of value (eg, food, shelter, drugs, money) involving children victimized within US borders. These involved youth are at risk for serious immediate and long-term physical and mental health consequences. Continued efforts are needed to improve preventive efforts, identification, screening, appropriate interventions, and subsequent resource provision for victimized and high-risk youth.

  12. Mitochondrial peptidase IMMP2L mutation causes early onset of age-associated disorders and impairs adult stem cell self-renewal

    PubMed Central

    George, Sunil K.; Jiao, Yan; Bishop, Colin E.; Lu, Baisong

    2011-01-01

    Summary Mitochondrial reactive oxygen species (ROS) are proposed to play a central role in aging and age-associated disorders, although direct in vivo evidence is lacking. We recently generated a mouse mutant with mutated Inner Mitochondrial Membrane Peptidase 2-like (Immp2l) gene, which impairs the signal peptide sequence processing of mitochondrial proteins cytochrome c1 and glycerol phosphate dehydrogenase 2. The mitochondria from mutant mice generate elevated levels of superoxide ion and cause impaired fertility in both sexes. Here we design experiments to examine the effects of excessive mitochondrial ROS generation on health span. We show that Immp2l mutation increases oxidative stress in multiple organs such as the brain and the kidney, although expression of superoxide dismutases in these tissues of the mutants is also increased. The mutants show multiple aging-associated phenotypes, including wasting, sarcopenia, loss of subcutaneous fat, kyphosis and ataxia, with female mutants showing earlier onset and more severe age-associated disorders than male mutants. The loss of body weight and fat was unrelated to food intake. Adipose derived stromal cells (ADSC) from mutant mice showed impaired proliferation capability, formed significantly less and smaller colonies in colony formation assays, although they retained adipogenic differentiation capability in vitro. This functional impairment was accompanied by increased levels of oxidative stress. Our data showed that mitochondrial ROS is the driving force of accelerated aging and suggested that ROS damage to adult stem cells could be one of the mechanisms for age-associated disorders. PMID:21332923

  13. Molecular Pathology of Genetic Epilepsies Associated with GABAA Receptor Subunit Mutations

    PubMed Central

    Macdonald, Robert L; Kang, Jing-Qiong

    2009-01-01

    Mutations in ligand-gated ion channel genes associated with idiopathic generalized epilepsies have been reported in excitatory acetylcholine receptor α4 and β2 subunit genes linked to autosomal dominant nocturnal frontal lobe epilepsy and in inhibitory GABAA receptor α1, β3, γ2, and δ subunit genes associated with childhood absence epilepsy, juvenile myoclonic epilepsy, pure febrile seizures, generalized epilepsy with febrile seizures plus, and generalized epilepsy with tonic–clonic seizures. Recent studies suggest that these mutations alter receptor function or biogenesis, including impaired receptor subunit messenger RNA stability, receptor subunit protein folding and stability, receptor assembly, and receptor trafficking. PMID:19396344

  14. A mutation in the insulin receptor gene that impairs transport of the receptor to the plasma membrane and causes insulin-resistant diabetes.

    PubMed Central

    Accili, D; Frapier, C; Mosthaf, L; McKeon, C; Elbein, S C; Permutt, M A; Ramos, E; Lander, E; Ullrich, A; Taylor, S I

    1989-01-01

    Insulin binds to a receptor on the cell surface, thereby triggering a biological response within the target cell. Mutations in the insulin receptor gene can render the cell resistant to the biological action of insulin. We have studied a family in which two sisters have a genetic form of insulin-resistant diabetes mellitus. The technique of homozygosity mapping has been used to demonstrate that the mutation causing diabetes in this consanguineous family is genetically linked to the insulin receptor gene. The two insulin-resistant sisters are homozygous for a mutation encoding substitution of valine for phenylalanine at position 382 in the alpha-subunit of the insulin receptor. Transfection of mutant insulin receptor cDNA into NIH3T3 cells demonstrated that the Val382 mutation impaired post-translational processing and retarded transport of the insulin receptor to the plasma membrane. Thus, the mutation causes insulin resistance by decreasing the number of insulin receptors on the surface of the patients' cells. Images PMID:2573522

  15. Barcoding of GPCR trafficking and signaling through the various trafficking roadmaps by compartmentalized signaling networks.

    PubMed

    Bahouth, Suleiman W; Nooh, Mohammed M

    2017-08-01

    Proper signaling by G protein coupled receptors (GPCR) is dependent on the specific repertoire of transducing, enzymatic and regulatory kinases and phosphatases that shape its signaling output. Activation and signaling of the GPCR through its cognate G protein is impacted by G protein-coupled receptor kinase (GRK)-imprinted "barcodes" that recruit β-arrestins to regulate subsequent desensitization, biased signaling and endocytosis of the GPCR. The outcome of agonist-internalized GPCR in endosomes is also regulated by sequence motifs or "barcodes" within the GPCR that mediate its recycling to the plasma membrane or retention and eventual degradation as well as its subsequent signaling in endosomes. Given the vast number of diverse sequences in GPCR, several trafficking mechanisms for endosomal GPCR have been described. The majority of recycling GPCR, are sorted out of endosomes in a "sequence-dependent pathway" anchored around a type-1 PDZ-binding module found in their C-tails. For a subset of these GPCR, a second "barcode" imprinted onto specific GPCR serine/threonine residues by compartmentalized kinase networks was required for their efficient recycling through the "sequence-dependent pathway". Mutating the serine/threonine residues involved, produced dramatic effects on GPCR trafficking, indicating that they played a major role in setting the trafficking itinerary of these GPCR. While endosomal SNX27, retromer/WASH complexes and actin were required for efficient sorting and budding of all these GPCR, additional proteins were required for GPCR sorting via the second "barcode". Here we will review recent developments in GPCR trafficking in general and the human β1-adrenergic receptor in particular across the various trafficking roadmaps. In addition, we will discuss the role of GPCR trafficking in regulating endosomal GPCR signaling, which promote biochemical and physiological effects that are distinct from those generated by the GPCR signal transduction pathway

  16. Emerging role of Cdc42-specific guanine nucleotide exchange factors as regulators of membrane trafficking in health and disease.

    PubMed

    Egorov, M V; Polishchuk, R S

    2017-04-01

    It is widely accepted that the Golgi complex operates as a main sorting station in the biosynthetic pathway. On the other hand, the Golgi complex harbors numerous signaling molecules that generate the platform for the coordination of the transduction of specific signals and of membrane transport events. A part of these processes, which require the complex integration of transport-, cytoskeleton- and polarity-associated mechanisms, is tightly regulated by molecular machineries comprising guanine nucleotide exchange factors (GEF) and their down-stream effectors, such as the small GTPase Cdc42. Dysfunction of several Cdc42-specific GEFs has been shown to cause a number of human diseases, which are associated with impaired intracellular trafficking at the level of the Golgi complex as well as in other compartments. Here we briefly overview how mutations in Cdc42-specific GEFs have an impact on the organization of intracellular trafficking fluxes and how such trafficking aberrations could be associated with a number of human disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. C-Terminal Di-leucine Motif of Dopamine D1 Receptor Plays an Important Role in Its Plasma Membrane Trafficking

    PubMed Central

    Guo, Yan; Jose, Pedro A.

    2011-01-01

    The dopamine D1 receptor (D1R), a G protein-coupled receptor, plays a critical role in regulating blood pressure through its actions on renal hemodynamics and epithelial ion transport, which are highly linked to its intracellular trafficking. In this study, we generated a series of C-terminal mutants of D1R that were tagged with or without enhanced yellow fluorescent protein, and analyzed the consequences of these mutants on the plasma membrane trafficking of D1R and cyclic AMP response to D1R stimulation. D1R with mutations within the endocytic recycling signal (amino acid residues 360–382) continued to be functional, albeit decreased relative to wild-type D1R. Mutation of the palmitoylation site (347C>S) of D1R did not impair its trafficking to the plasma membrane, but abolished its ability to increase cyclic AMP accumulation. In contrast, replacement of di-leucines (344–345L>A) by alanines resulted in the retention of D1R in the early endosome, decreased its glycosylation, and prevented its targeting to the plasma membrane. Our studies suggest that di-L motif at the C-terminus of D1R is critical for the glycosylation and cell surface targeting of D1R. PMID:22206002

  18. The Startle Disease Mutation E103K Impairs Activation of Human Homomeric α1 Glycine Receptors by Disrupting an Intersubunit Salt Bridge across the Agonist Binding Site*

    PubMed Central

    Safar, Fatemah; Hurdiss, Elliot; Erotocritou, Marios; Greiner, Timo; Irvine, Mark W.; Fang, Guangyu; Jane, David; Yu, Rilei; Dämgen, Marc A.

    2017-01-01

    Glycine receptors (GlyR) belong to the pentameric ligand-gated ion channel (pLGIC) superfamily and mediate fast inhibitory transmission in the vertebrate CNS. Disruption of glycinergic transmission by inherited mutations produces startle disease in man. Many startle mutations are in GlyRs and provide useful clues to the function of the channel domains. E103K is one of few startle mutations found in the extracellular agonist binding site of the channel, in loop A of the principal side of the subunit interface. Homology modeling shows that the side chain of Glu-103 is close to that of Arg-131, in loop E of the complementary side of the binding site, and may form a salt bridge at the back of the binding site, constraining its size. We investigated this hypothesis in recombinant human α1 GlyR by site-directed mutagenesis and functional measurements of agonist efficacy and potency by whole cell patch clamp and single channel recording. Despite its position near the binding site, E103K causes hyperekplexia by impairing the efficacy of glycine, its ability to gate the channel once bound, which is very high in wild type GlyR. Mutating Glu-103 and Arg-131 caused various degrees of loss-of-function in the action of glycine, whereas mutations in Arg-131 enhanced the efficacy of the slightly bigger partial agonist sarcosine (N-methylglycine). The effects of the single charge-swapping mutations of these two residues were largely rescued in the double mutant, supporting the possibility that they interact via a salt bridge that normally constrains the efficacy of larger agonist molecules. PMID:28174298

  19. PAPSS2 Deficiency Causes Androgen Excess via Impaired DHEA Sulfation—In Vitro and in Vivo Studies in a Family Harboring Two Novel PAPSS2 Mutations

    PubMed Central

    Oostdijk, Wilma; Idkowiak, Jan; Mueller, Jonathan W.; House, Philip J.; Taylor, Angela E.; O'Reilly, Michael W.; Hughes, Beverly A.; de Vries, Martine C.; Kant, Sarina G.; Santen, Gijs W. E.; Verkerk, Annemieke J. M. H.; Uitterlinden, André G.; Wit, Jan M.; Losekoot, Monique

    2015-01-01

    Context: PAPSS2 (PAPS synthase 2) provides the universal sulfate donor PAPS (3′-phospho-adenosine-5′-phosphosulfate) to all human sulfotransferases, including SULT2A1, responsible for sulfation of the crucial androgen precursor dehydroepiandrosterone (DHEA). Impaired DHEA sulfation is thought to increase the conversion of DHEA toward active androgens, a proposition supported by the previous report of a girl with inactivating PAPSS2 mutations who presented with low serum DHEA sulfate and androgen excess, clinically manifesting with premature pubarche and early-onset polycystic ovary syndrome. Patients and Methods: We investigated a family harboring two novel PAPSS2 mutations, including two compound heterozygous brothers presenting with disproportionate short stature, low serum DHEA sulfate, but normal serum androgens. Patients and parents underwent a DHEA challenge test comprising frequent blood sampling and urine collection before and after 100 mg DHEA orally, with subsequent analysis of DHEA sulfation and androgen metabolism by mass spectrometry. The functional impact of the mutations was investigated in silico and in vitro. Results: We identified a novel PAPSS2 frameshift mutation, c.1371del, p.W462Cfs*3, resulting in complete disruption, and a novel missense mutation, c.809G>A, p.G270D, causing partial disruption of DHEA sulfation. Both patients and their mother, who was heterozygous for p.W462Cfs*3, showed increased 5α-reductase activity at baseline and significantly increased production of active androgens after DHEA intake. The mother had a history of oligomenorrhea and chronic anovulation that required clomiphene for ovulation induction. Conclusions: We provide direct in vivo evidence for the significant functional impact of mutant PAPSS2 on DHEA sulfation and androgen activation. Heterozygosity for PAPSS2 mutations can be associated with a phenotype resembling polycystic ovary syndrome. PMID:25594860

  20. Mutations in the Transmembrane Natriuretic Peptide Receptor NPR-B Impair Skeletal Growth and Cause Acromesomelic Dysplasia, Type Maroteaux

    PubMed Central

    Bartels, Cynthia F.; Bükülmez, Hülya; Padayatti, Pius; Rhee, David K.; van Ravenswaaij-Arts, Conny; Pauli, Richard M.; Mundlos, Stefan; Chitayat, David; Shih, Ling-Yu; Al-Gazali, Lihadh I.; Kant, Sarina; Cole, Trevor; Morton, Jenny; Cormier-Daire, Valérie; Faivre, Laurence; Lees, Melissa; Kirk, Jeremy; Mortier, Geert R.; Leroy, Jules; Zabel, Bernhard; Kim, Chong Ae; Crow, Yanick; Braverman, Nancy E.; van den Akker, Focco; Warman, Matthew L.

    2004-01-01

    The homodimeric transmembrane receptor natriuretic peptide receptor B (NPR-B [also known as guanylate cyclase B, GC-B, and GUC2B]; gene name NPR2) produces cytoplasmic cyclic GMP from GTP on binding its extracellular ligand, C-type natriuretic peptide (CNP). CNP has previously been implicated in the regulation of skeletal growth in transgenic and knockout mice. The autosomal recessive skeletal dysplasia known as “acromesomelic dysplasia, type Maroteaux” (AMDM) maps to an interval that contains NPR2. We sequenced DNA from 21 families affected by AMDM and found 4 nonsense mutations, 4 frameshift mutations, 2 splice-site mutations, and 11 missense mutations. Molecular modeling was used to examine the putative protein change brought about by each missense mutation. Three missense mutations were tested in a functional assay and were found to have markedly deficient guanylyl cyclase activity. We also found that obligate carriers of NPR2 mutations have heights that are below the mean for matched controls. We conclude that, although NPR-B is expressed in a number of tissues, its major role is in the regulation of skeletal growth. PMID:15146390

  1. AIP mutations impair AhR signaling in pituitary adenoma patients fibroblasts and in GH3 cells.

    PubMed

    Lecoq, Anne-Lise; Viengchareun, Say; Hage, Mirella; Bouligand, Jérôme; Young, Jacques; Boutron, Audrey; Zizzari, Philippe; Lombès, Marc; Chanson, Philippe; Kamenický, Peter

    2016-05-01

    Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene predispose humans to pituitary adenomas through unknown molecular mechanisms. The best-known interacting partner of AIP is the aryl hydrocarbon receptor (AhR), a transcription factor that mediates the effects of xenobiotics implicated in carcinogenesis. As 75% of AIP mutations disrupt the physical and/or functional interaction with AhR, we postulated that the tumorigenic potential of AIP mutations might result from altered AhR signaling. We evaluated the impact of AIP mutations on the AhR signaling pathway, first in fibroblasts from AIP-mutated patients with pituitary adenomas, by comparison with fibroblasts from healthy subjects, then in transfected pituitary GH3 cells. The AIP protein level in mutated fibroblasts was about half of that in cells from healthy subjects, but AhR expression was unaffected. Gene expression analyses showed significant modifications in the expression of the AhR target genes CYP1B1 and AHRR in AIP-mutated fibroblasts, both before and after stimulation with the endogenous AhR ligand kynurenine. Kynurenine increased Cyp1b1 expression to a greater extent in GH3 cells overexpressing wild type compared with cells expressing mutant AIP Knockdown of endogenous Aip in these cells attenuated Cyp1b1 induction by the AhR ligand. Both mutant AIP expression and knockdown of endogenous Aip affected the kynurenine-dependent GH secretion of GH3 cells. This study of human fibroblasts bearing endogenous heterozygous AIP mutations and transfected pituitary GH3 cells shows that AIP mutations affect the AIP protein level and alter AhR transcriptional activity in a gene- and tissue-dependent manner.

  2. Ras trafficking, localization and compartmentalized signalling.

    PubMed

    Prior, Ian A; Hancock, John F

    2012-04-01

    Ras proteins are proto-oncogenes that are frequently mutated in human cancers. Three closely related isoforms, HRAS, KRAS and NRAS, are expressed in all cells and have overlapping but distinctive functions. Recent work has revealed how differences between the Ras isoforms in their trafficking, localization and protein-membrane orientation enable signalling specificity to be determined. We review the various strategies used to characterize compartmentalized Ras localization and signalling. Localization is an important contextual modifier of signalling networks and insights from the Ras system are of widespread relevance for researchers interested in signalling initiated from membranes.

  3. Impaired natural killer cell functions in patients with signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations.

    PubMed

    Tabellini, Giovanna; Vairo, Donatella; Scomodon, Omar; Tamassia, Nicola; Ferraro, Rosalba Monica; Patrizi, Ornella; Gasperini, Sara; Soresina, Annarosa; Giardino, Giuliana; Pignata, Claudio; Lougaris, Vassilios; Plebani, Alessandro; Dotta, Laura; Cassatella, Marco A; Parolini, Silvia; Badolato, Raffaele

    2017-08-01

    Gain-of-function (GOF) mutations affecting the coiled-coil domain or the DNA-binding domain of signal transducer and activator of transcription 1 (STAT1) cause chronic mucocutaneous candidiasis disease. This condition is characterized by fungal and bacterial infections caused by impaired generation of TH17 cells; meanwhile, some patients with chronic mucocutaneous candidiasis disease might also have viral or intracellular pathogen infections. We sought to investigate the effect of STAT1 GOF mutations on the functioning of natural killer (NK) cells. Because STAT1 is involved in the signaling response to several cytokines, we studied NK cell functional activities and STAT1 signaling in 8 patients with STAT1 GOF mutations. Functional analysis of NK cells shows a significant impairment of cytolytic and degranulation activities in patients with STAT1 GOF mutations. Moreover, NK cells from these patients display lower production of IFN-γ in response to IL-15 and reduced proliferation after stimulation with IL-2 or IL-15, suggesting that STAT5 signaling is affected. In addition, signaling studies demonstrate that the increased phosphorylation of STAT1 in response to IFN-α is associated with detectable activation of STAT1 and increased STAT1 binding to the interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) promoter in response to IL-15, whereas STAT5 phosphorylation and DNA binding to IL-2 receptor α (IL2RA) are reduced or not affected in response to the same cytokine. These observations suggest that persistent activation of STAT1 might affect NK cell proliferation and functional activities. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  4. A lethal de novo mutation in the middle domain of the dynamin-related GTPase Drp1 impairs higher order assembly and mitochondrial division.

    PubMed

    Chang, Chuang-Rung; Manlandro, Cara Marie; Arnoult, Damien; Stadler, Julia; Posey, Ammon E; Hill, R Blake; Blackstone, Craig

    2010-10-15

    Mitochondria dynamically fuse and divide within cells, and the proper balance of fusion and fission is necessary for normal mitochondrial function, morphology, and distribution. Drp1 is a dynamin-related GTPase required for mitochondrial fission in mammalian cells. It harbors four distinct domains: GTP-binding, middle, insert B, and GTPase effector. A lethal mutation (A395D) within the Drp1 middle domain was reported in a neonate with microcephaly, abnormal brain development, optic atrophy, and lactic acidemia (Waterham, H. R., Koster, J., van Roermund, C. W., Mooyer, P. A., Wanders, R. J., and Leonard, J. V. (2007) N. Engl. J. Med. 356, 1736-1741). Mitochondria within patient-derived fibroblasts were markedly elongated, but the molecular mechanisms underlying these findings were not demonstrated. Because the middle domain is particularly important for the self-assembly of some dynamin superfamily proteins, we tested the hypothesis that this A395D mutation, and two other middle domain mutations (G350D, G363D) were important for Drp1 tetramerization, higher order assembly, and function. Although tetramerization appeared largely intact, each of these mutations compromised higher order assembly and assembly-dependent stimulation of Drp1 GTPase activity. Moreover, mutant Drp1 proteins exhibited impaired localization to mitochondria, indicating that this higher order assembly is important for mitochondrial recruitment, retention, or both. Overexpression of these middle domain mutants markedly inhibited mitochondrial division in cells. Thus, the Drp1 A395D lethal defect likely resulted in impaired higher order assembly of Drp1 at mitochondria, leading to decreased fission, elongated mitochondria, and altered cellular distribution of mitochondria.

  5. Impaired mechanical response of an EDMD mutation leads to motility phenotypes that are repaired by loss of prenylation.

    PubMed

    Zuela, Noam; Zwerger, Monika; Levin, Tal; Medalia, Ohad; Gruenbaum, Yosef

    2016-05-01

    There are roughly 14 distinct heritable autosomal dominant diseases associated with mutations in lamins A/C, including Emery-Dreifuss muscular dystrophy (EDMD). The mechanical model proposes that the lamin mutations change the mechanical properties of muscle nuclei, leading to cell death and tissue deterioration. Here, we developed an experimental protocol that analyzes the effect of disease-linked lamin mutations on the response of nuclei to mechanical strain in living Caenorhabditis elegans We found that the EDMD mutation L535P disrupts the nuclear mechanical response specifically in muscle nuclei. Inhibiting lamin prenylation rescued the mechanical response of the EDMD nuclei, reversed the muscle phenotypes and led to normal motility. The LINC complex and emerin were also required to regulate the mechanical response of C. elegans nuclei. This study provides evidence to support the mechanical model and offers a potential future therapeutic approach towards curing EDMD. © 2016. Published by The Company of Biologists Ltd.

  6. A novel homozygous mutation in FGFR3 causes tall stature, severe lateral tibial deviation, scoliosis, hearing impairment, camptodactyly, and arachnodactyly.

    PubMed

    Makrythanasis, Periklis; Temtamy, Samia; Aglan, Mona S; Otaify, Ghada A; Hamamy, Hanan; Antonarakis, Stylianos E

    2014-08-01

    Most reported mutations in the FGFR3 gene are dominant activating mutations that cause a variety of short-limbed bone dysplasias including achondroplasia and syndromic craniosynostosis. We report the phenotype and underlying molecular abnormality in two brothers, born to first cousin parents. The clinical picture is characterized by tall stature and severe skeletal abnormalities leading to inability to walk, with camptodactyly, arachnodactyly, and scoliosis. Whole exome sequencing revealed a homozygous novel missense mutation in the FGFR3 gene in exon 12 (NM_000142.4:c.1637C>A: p.(Thr546Lys)). The variant is found in the kinase domain of the protein and is predicted to be pathogenic. It is located near a known hotspot for hypochondroplasia. This is the first report of a homozygous loss-of-function mutation in FGFR3 in human that results in a skeletal overgrowth syndrome. © 2014 WILEY PERIODICALS, INC.

  7. Mitochondrial peptidase IMMP2L mutation causes early onset of age-associated disorders and impairs adult stem cell self-renewal.

    PubMed

    George, Sunil K; Jiao, Yan; Bishop, Colin E; Lu, Baisong

    2011-08-01

    Mitochondrial reactive oxygen species (ROS) are proposed to play a central role in aging and age-associated disorders, although direct in vivo evidence is lacking. We recently generated a mouse mutant with mutated inner mitochondrial membrane peptidase 2-like (Immp2l) gene, which impairs the signal peptide sequence processing of mitochondrial proteins cytochrome c1 and glycerol phosphate dehydrogenase 2. The mitochondria from mutant mice generate elevated levels of superoxide ion and cause impaired fertility in both sexes. Here, we design experiments to examine the effects of excessive mitochondrial ROS generation on health span. We show that Immp2l mutation increases oxidative stress in multiple organs such as the brain and the kidney, although expression of superoxide dismutases in these tissues of the mutants is also increased. The mutants show multiple aging-associated phenotypes, including wasting, sarcopenia, loss of subcutaneous fat, kyphosis, and ataxia, with female mutants showing earlier onset and more severe age-associated disorders than male mutants. The loss of body weight and fat was unrelated to food intake. Adipose-derived stromal cells (ADSC) from mutant mice showed impaired proliferation capability, formed significantly less and smaller colonies in colony formation assays, although they retained adipogenic differentiation capability in vitro. This functional impairment was accompanied by increased levels of oxidative stress. Our data showed that mitochondrial ROS is the driving force of accelerated aging and suggested that ROS damage to adult stem cells could be one of the mechanisms for age-associated disorders. © 2011 The Authors. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

  8. Mutations of factor H impair regulation of surface-bound C3b by three mechanisms in atypical hemolytic uremic syndrome.

    PubMed

    Lehtinen, Markus J; Rops, Angelique L; Isenman, David E; van der Vlag, Johan; Jokiranta, T Sakari

    2009-06-05

    Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy associated with mutations in complement proteins, most frequently in the main plasma alternative pathway regulator factor H (FH). The hotspot for the FH mutations is in domains 19-20 (FH19-20) that are indispensable for FH activity on C3b bound covalently to host cells. In aHUS, down-regulation of cell-bound C3b by FH is impaired, but it is not clear whether this is due to an altered FH binding to surface-bound C3b or to cell surface structures. To explore the molecular pathogenesis of aHUS we tested binding of 14 FH19-20 point mutants to C3b and its C3d fragment, mouse glomerular endothelial cells (mGEnC-1), and heparin. The cell binding correlated well, but not fully, with heparin binding and the cell binding site was overlapping but distinct from the C3b/C3d binding site that was shown to extend to domain 19. Our results show that aHUS-associated FH19-20 mutants have different combinations of three primary defects: impaired binding to C3b/C3d, impaired binding to the mGEnC-1 cells/heparin, and, as a novel observation, an enhanced mGEnC-1 cell or heparin binding. We propose a model of the molecular pathogenesis of aHUS where all three mechanisms lead eventually to impaired control of C3b on the endothelial cell surfaces. Based on the results with the aHUS patient mutants and the overlap in FH19-20 binding sites for mGEnC-1/heparin and C3b/C3d we conclude that binding of FH19-20 to C3b/C3d is essential for target discrimination by the alternative pathway.

  9. NPC1, intracellular cholesterol trafficking and atherosclerosis.

    PubMed

    Yu, Xiao-Hua; Jiang, Na; Yao, Ping-Bo; Zheng, Xi-Long; Cayabyab, Francisco S; Tang, Chao-Ke

    2014-02-15

    Post-lysosomal cholesterol trafficking is an important, but poorly understood process that is essential to maintain lipid homeostasis. Niemann-Pick type C1 (NPC1), an integral membrane protein on the limiting membrane of late endosome/lysosome (LE/LY), is known to accept cholesterol from NPC2 and then mediate cholesterol transport from LE/LY to endoplasmic reticulum (ER) and plasma membrane in a vesicle- or oxysterol-binding protein (OSBP)-related protein 5 (ORP5)-dependent manner. Mutations in the NPC1 gene can be found in the majority of NPC patients, who accumulate massive amounts of cholesterol and other lipids in the LE/LY due to a defect in intracellular lipid trafficking. Liver X receptor (LXR) is the major positive regulator of NPC1 expression. Atherosclerosis is the pathological basis of coronary heart disease, one of the major causes of death worldwide. NPC1 has been shown to play a critical role in the atherosclerotic progression. In this review, we have summarized the role of NPC1 in regulating intracellular cholesterol trafficking and atherosclerosis.

  10. Impairment of mitochonrial tRNAIle processing by a novel mutation associated with chronic progressive external ophthalmoplegia

    PubMed Central

    Schaller, A; Desetty, R; Hahn, D; Jackson, C B; Nuoffer, J-M; Gallati, S; Levinger, L

    2011-01-01

    We report a sporadic case of chronic progressive external ophthalmoplegia associated with ragged red fibers. The patient presented with enlarged mitochondria with deranged internal architecture and crystalline inclusions. Biochemical studies showed reduced activities of complex I, III and IV in skeletal muscle. Molecular genetic analysis of all mitochondrial tRNAs revealed a G to A transition at nt 4308; the G is a highly conserved nucleotide that participates in a GC base-pair in the T-stem of mammalian mitochondrial tRNAIle. The mutation was detected at a high level (approx. 50%) in muscle but not in blood. The mutation co-segregated with the phenotype, as the mutation was absent from blood and muscle in the patient’s healthy mother. Functional characterisation of the mutation revealed a six-fold reduced rate of tRNAIle precursor 3’ end maturation in vitro by tRNAse Z. Furthermore, the mutated tRNAIle displays local structural differences from wild-type. These results suggest that structural perturbations reduce efficiency of tRNAIle precursor 3’ end processing and contribute to the molecular pathomechanism of this mutation. PMID:21292040

  11. The Arrhythmogenic Calmodulin p.Phe142Leu Mutation Impairs C-domain Ca2+ Binding but Not Calmodulin-dependent Inhibition of the Cardiac Ryanodine Receptor.

    PubMed

    Søndergaard, Mads Toft; Liu, Yingjie; Larsen, Kamilla Taunsig; Nani, Alma; Tian, Xixi; Holt, Christian; Wang, Ruiwu; Wimmer, Reinhard; Van Petegem, Filip; Fill, Michael; Chen, S R Wayne; Overgaard, Michael Toft

    2017-01-27

    A number of point mutations in the intracellular Ca(2+)-sensing protein calmodulin (CaM) are arrhythmogenic, yet their underlying mechanisms are not clear. These mutations generally decrease Ca(2+) binding to CaM and impair inhibition of CaM-regulated Ca(2+) channels like the cardiac Ca(2+) release channel (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca(2+) binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start-Met), which markedly reduces CaM Ca(2+) binding. Surprisingly, CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca(2+) release in HEK293 cells compared with CaM-WT. Furthermore, CaM-F142L enhanced CaM-dependent RyR2 inhibition at the single channel level compared with CaM-WT. This is in stark contrast to the actions of arrhythmogenic CaM mutations N54I, D96V, N98S, and D130G, which all diminish CaM-dependent RyR2 inhibition. Thermodynamic analysis showed that apoCaM-F142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover, NMR spectra revealed that the CaM-F142L-CaMBD interaction is structurally different from that of CaM-WT at low Ca(2+) These data indicate a distinct interaction between CaM-F142L and the RyR2 CaMBD, which may explain the stronger CaM-dependent RyR2 inhibition by CaM-F142L, despite its reduced Ca(2+) binding. Collectively, these results add to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca(2+) release by manipulating the CaM-RyR2 interaction. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. The Arrhythmogenic Calmodulin p.Phe142Leu Mutation Impairs C-domain Ca2+ Binding but Not Calmodulin-dependent Inhibition of the Cardiac Ryanodine Receptor*

    PubMed Central

    Liu, Yingjie; Larsen, Kamilla Taunsig; Nani, Alma; Tian, Xixi; Holt, Christian; Wang, Ruiwu; Fill, Michael

    2017-01-01

    A number of point mutations in the intracellular Ca2+-sensing protein calmodulin (CaM) are arrhythmogenic, yet their underlying mechanisms are not clear. These mutations generally decrease Ca2+ binding to CaM and impair inhibition of CaM-regulated Ca2+ channels like the cardiac Ca2+ release channel (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca2+ binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start-Met), which markedly reduces CaM Ca2+ binding. Surprisingly, CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca2+ release in HEK293 cells compared with CaM-WT. Furthermore, CaM-F142L enhanced CaM-dependent RyR2 inhibition at the single channel level compared with CaM-WT. This is in stark contrast to the actions of arrhythmogenic CaM mutations N54I, D96V, N98S, and D130G, which all diminish CaM-dependent RyR2 inhibition. Thermodynamic analysis showed that apoCaM-F142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover, NMR spectra revealed that the CaM-F142L-CaMBD interaction is structurally different from that of CaM-WT at low Ca2+. These data indicate a distinct interaction between CaM-F142L and the RyR2 CaMBD, which may explain the stronger CaM-dependent RyR2 inhibition by CaM-F142L, despite its reduced Ca2+ binding. Collectively, these results add to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca2+ release by manipulating the CaM-RyR2 interaction. PMID:27927985

  13. Catalytic Function of PLA2G6 Is Impaired by Mutations Associated with Infantile Neuroaxonal Dystrophy but Not Dystonia-Parkinsonism

    PubMed Central

    Engel, Laura A.; Jing, Zheng; O'Brien, Daniel E.; Sun, Mengyang; Kotzbauer, Paul T.

    2010-01-01

    Background Mutations in the PLA2G6 gene have been identified in autosomal recessive neurodegenerative diseases classified as infantile neuroaxonal dystrophy (INAD), neurodegeneration with brain iron accumulation (NBIA), and dystonia-parkinsonism. These clinical syndromes display two significantly different disease phenotypes. NBIA and INAD are very similar, involving widespread neurodegeneration that begins within the first 1–2 years of life. In contrast, patients with dystonia-parkinsonism present with a parkinsonian movement disorder beginning at 15 to 30 years of age. The PLA2G6 gene encodes the PLA2G6 enzyme, also known as group VIA calcium-independent phospholipase A2, which has previously been shown to hydrolyze the sn-2 acyl chain of phospholipids, generating free fatty acids and lysophospholipids. Methodology/Principal Findings We produced purified recombinant wildtype (WT) and mutant human PLA2G6 proteins and examined their catalytic function using in vitro assays with radiolabeled lipid substrates. We find that human PLA2G6 enzyme hydrolyzes both phospholipids and lysophospholipids, releasing free fatty acids. Mutations associated with different disease phenotypes have different effects on catalytic activity. Mutations associated with INAD/NBIA cause loss of enzyme activity, with mutant proteins exhibiting less than 20% of the specific activity of WT protein in both lysophospholipase and phospholipase assays. In contrast, mutations associated with dystonia-parkinsonism do not impair catalytic activity, and two mutations produce a significant increase in specific activity for phospholipid but not lysophospholipid substrates. Conclusions/Significance These results indicate that different alterations in PLA2G6 function produce the different disease phenotypes of NBIA/INAD and dystonia-parkinsonism. INAD/NBIA is caused by loss of the ability of PLA2G6 to catalyze fatty acid release from phospholipids, which predicts accumulation of PLA2G6 phospholipid

  14. Catalytic function of PLA2G6 is impaired by mutations associated with infantile neuroaxonal dystrophy but not dystonia-parkinsonism.

    PubMed

    Engel, Laura A; Jing, Zheng; O'Brien, Daniel E; Sun, Mengyang; Kotzbauer, Paul T

    2010-09-23

    Mutations in the PLA2G6 gene have been identified in autosomal recessive neurodegenerative diseases classified as infantile neuroaxonal dystrophy (INAD), neurodegeneration with brain iron accumulation (NBIA), and dystonia-parkinsonism. These clinical syndromes display two significantly different disease phenotypes. NBIA and INAD are very similar, involving widespread neurodegeneration that begins within the first 1-2 years of life. In contrast, patients with dystonia-parkinsonism present with a parkinsonian movement disorder beginning at 15 to 30 years of age. The PLA2G6 gene encodes the PLA2G6 enzyme, also known as group VIA calcium-independent phospholipase A(2), which has previously been shown to hydrolyze the sn-2 acyl chain of phospholipids, generating free fatty acids and lysophospholipids. We produced purified recombinant wildtype (WT) and mutant human PLA2G6 proteins and examined their catalytic function using in vitro assays with radiolabeled lipid substrates. We find that human PLA2G6 enzyme hydrolyzes both phospholipids and lysophospholipids, releasing free fatty acids. Mutations associated with different disease phenotypes have different effects on catalytic activity. Mutations associated with INAD/NBIA cause loss of enzyme activity, with mutant proteins exhibiting less than 20% of the specific activity of WT protein in both lysophospholipase and phospholipase assays. In contrast, mutations associated with dystonia-parkinsonism do not impair catalytic activity, and two mutations produce a significant increase in specific activity for phospholipid but not lysophospholipid substrates. These results indicate that different alterations in PLA2G6 function produce the different disease phenotypes of NBIA/INAD and dystonia-parkinsonism. INAD/NBIA is caused by loss of the ability of PLA2G6 to catalyze fatty acid release from phospholipids, which predicts accumulation of PLA2G6 phospholipid substrates and provides a mechanistic explanation for the accumulation

  15. Proliferation and ovarian hormone signaling are impaired in normal breast tissues from women with BRCA1 mutations: benefit of a progesterone receptor modulator treatment as a breast cancer preventive strategy in women with inherited BRCA1 mutations

    PubMed Central

    Communal, Laudine; Courtin, Aurélie; Mourra, Najat; Lahlou, Najiba; Le Guillou, Morwenna; de Jotemps, Muriel Perrault; Chauvet, Marie-Pierre; Chaouat, Marc; Pujol, Pascal; Feunteun, Jean; Delaloge, Suzette; Forgez, Patricia; Gompel, Anne

    2016-01-01

    Women with inherited BRCA1 mutations have an elevated risk (40-80%) for developing breast and ovarian cancers. Reproductive history has been reported to alter this risk, suggesting a relationship between ovarian hormone signaling and BRCA1-related tumor development. BRCA1 interactions with estrogen receptor (ER) and progesterone receptor (PR) signaling were previously described in human breast cancer cell lines and mouse models. However, few studies have examined the effect of ovarian hormone regulation in normal human breast tissues bearing a heterozygous BRCA1 mutation. This study compares the proliferation level (Ki67) and the expression of ER, PR, and of the PR target gene, fatty acid synthase (FASN), in histologically normal breast tissues from women with BRCA1 mutations (BRCA1+/mut, n=23) or without BRCA1 mutations (BRCA1+/+, n=28). BRCA1+/mut tissues showed an increased proliferation and impaired hormone receptor expression with a marked loss of the PR isoform, PR-B. Responses to estradiol and progesterone treatments in BRCA1+/mut and BRCA1+/+ breast tissues were studied in a mouse xenograft model, and showed that PR and FASN expression were deregulated in BRCA1+/mut breast tissues. Progesterone added to estradiol treatment increased the proliferation in a subset of BRCA1+/mut breast tissues. The PR inhibitor, ulipristal acetate (UPA), was able to reverse this aberrant progesterone-induced proliferation. This study suggests that a subset of women with BRCA1 mutations could be candidates for a UPA treatment as a preventive breast cancer strategy. PMID:27246982

  16. Engineered mutations in fibrillin-1 leading to Marfan syndrome act at the protein, cellular and organismal levels.

    PubMed

    Zeyer, Karina A; Reinhardt, Dieter P

    2015-01-01

    Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and non-elastic tissues. They are multi-domain proteins, containing primarily calcium binding epidermal growth factor-like (cbEGF) domains and 8-cysteine/transforming growth factor-beta binding protein-like (TB) domains. Mutations in the fibrillin-1 gene give rise to Marfan syndrome, a connective tissue disorder with clinical complications in the cardiovascular, skeletal, ocular and other organ systems. Here, we review the consequences of engineered Marfan syndrome mutations in fibrillin-1 at the protein, cellular and organismal levels. Representative point mutations associated with Marfan syndrome in affected individuals have been introduced and analyzed in recombinant fibrillin-1 fragments. Those mutations affect fibrillin-1 on a structural and functional level. Mutations which impair folding of cbEGF domains can affect protein trafficking. Protein folding disrupted by some mutations can lead to defective secretion in mutant fibrillin-1 fragments, whereas fragments with other Marfan mutations are secreted normally. Many Marfan mutations render fibrillin-1 more susceptible to proteolysis. There is also evidence that some mutations affect heparin binding. Few mutations have been further analyzed in mouse models. An extensively studied mouse model of Marfan syndrome expresses mouse fibrillin-1 with a missense mutation (p.C1039G). The mice display similar characteristics to human patients with Marfan syndrome. Overall, the analyses of engineered mutations leading to Marfan syndrome provide important insights into the pathogenic molecular mechanisms exerted by mutated fibrillin-1.

  17. Health implications of human trafficking.

    PubMed

    Richards, Tiffany A

    2014-01-01

    Freedom is arguably the most cherished right in the United States. But each year, approximately 14,500 to 17,500 women, men and children are trafficked into the United States for the purposes of forced labor or sexual exploitation. Human trafficking has significant effects on both physical and mental health. This article describes the features of human trafficking, its physical and mental health effects and the vital role nurses can play in providing care to this vulnerable population.

  18. Women trafficking: causes, concerns, care!

    PubMed

    Khowaja, Shaneela Sadaruddin; Tharani, Ambreen Jawed; Agha, Ajmal; Karamaliani, Rozina Sherali

    2012-08-01

    Pakistan is both a country of origin and destination as far as women trafficking is concerned. Poverty, gender discrimination, lack of education, and ignorance about legal rights are some of the underlying causes. Available data suggest several areas of concern, like, for instance: direct health effects, maladaptive coping leading to the use of illicit drugs, and inaccessibility to healthcare facilities. Therefore, numerous interventions would be required at three levels: the prevention of trafficking, the protection of victims and the prosecution of the traffickers.

  19. Identification of cancer-associated missense mutations in hace1 that impair cell growth control and Rac1 ubiquitylation

    PubMed Central

    Andrio, Emilie; Lotte, Romain; Hamaoui, Daniel; Cherfils, Jacqueline; Doye, Anne; Daugaard, Mads; Sorensen, Poul H.; Bost, Frédéric; Ruimy, Raymond; Mettouchi, Amel; Lemichez, Emmanuel

    2017-01-01

    The E3 ubiquitin ligase HACE1 is a potent tumor suppressor that controls cell proliferation and ubiquitylates the small GTPase Rac1 to target it to proteasomal degradation. Whether and how the activity of HACE1 is regulated by the N-terminal ankyrin (ANK) and the middle (MID) domains is ill defined. Here, we identified in the version 64 of the Catalogue of Somatic Mutations in Cancer (COSMIC) 13 missense mutations of hace1 located outside the HECT domain, and found that all lead to defective control of cell proliferation. In addition, several mutations located in the ankyrin domain displayed a dramatic reduction in Rac1 ubiquitylation associated with a decrease of colony formation in soft agar. 3D structure modelling of the 7 ankyrin-repeats coupled to functional analysis identified a surface epitope centered on one of the mutated residue, Gly-175, which is critical for controlling Rac1 binding and ubiquitylation. We also identified a role for the MID domain in conferring the specificity of association of HACE1 to the active form of Rac1. Our study of the functional interplay between HACE1 and Rac1 in cancer thus sheds a new light on the molecular mechanism of Rac1 ubiquitylation by HACE1 and the impact of its cancer-associated mutations in cell proliferation. PMID:28317937

  20. Hypofibrinogenaemia caused by a novel FGG missense mutation (W253C) in the γ chain globular domain impairing fibrinogen secretion

    PubMed Central

    Vu, D; de Moerloose, P; Batorova, A; Lazur, J; Palumbo, L; Neerman-Arbez, M

    2005-01-01

    Background: Inherited disorders of fibrinogen are rare and affect either the quantity (hypofibrinogenaemia and afibrinogenaemia) or the quality of the circulating fibrinogen (dysfibrinogenaemia). Extensive allelic heterogeneity has been found for all three disorders: in congenital afibrinogenaemia >30 mutations, the majority in FGA, have been identified in homozygosity or in compound heterozygosity. Several mutations have also been identified in patients with hypofibrinogenaemia; many of these are heterozygous carriers of afibrinogenaemia null mutations. Objective: To report the case of a patient from Slovakia diagnosed with hypofibrinogenaemia characterised by fibrinogen concentrations of around 0.7 g/l. Results: The patient was found to be heterozygous for a novel missense mutation W253C (W227C in the mature protein) in the C-terminal globular domain of the fibrinogen γ chain. Co-expression of the W253C FGG mutant cDNA (fibrinogen Bratislava) in combination with wild-type FGA and FGB cDNAs showed that fibrinogen molecules containing the mutant γ chain can assemble intracellularly but are not secreted into the media, confirming the causative nature of the identified mutation. Conclusions: Current analysis of fibrinogen Bratislava indicates that the domains important for the processes of hexamer assembly and hexamer secretion should not be considered as strictly restricted to one or other fibrinogen chain. PMID:16141000

  1. Impaired Acid Catalysis by Mutation of a Protein Loop Hinge Residue in a YopH Mutant Revealed by Crystal Structures

    SciTech Connect

    Brandao, T.; Robinson, H; Johnson, S; Hengge, A

    2009-01-01

    Catalysis by the Yersinia protein-tyrosine phosphatase YopH is significantly impaired by the mutation of the conserved Trp354 residue to Phe. Though not a catalytic residue, this Trp is a hinge residue in a conserved flexible loop (the WPD-loop) that must close during catalysis. To learn why this seemingly conservative mutation reduces catalysis by 2 orders of magnitude, we have solved high-resolution crystal structures for the W354F YopH in the absence and in the presence of tungstate and vanadate. Oxyanion binding to the P-loop in W354F is analogous to that observed in the native enzyme. However, the WPD-loop in the presence of oxyanions assumes a half-closed conformation, in contrast to the fully closed state observed in structures of the native enzyme. This observation provides an explanation for the impaired general acid catalysis observed in kinetic experiments with Trp mutants. A 1.4 Angstroms structure of the W354F mutant obtained in the presence of vanadate reveals an unusual divanadate species with a cyclic [VO]2 core, which has precedent in small molecules but has not been previously reported in a protein crystal structure.

  2. Age-Related Hearing Impairment (ARHI) Associated with GJB2 Single Mutation IVS1+1G>A in the Yakut Population Isolate in Eastern Siberia

    PubMed Central

    Pshennikova, Vera G.; Solovyev, Aisen V.; Klarov, Leonid A.; Solovyeva, Natalya A.; Kozhevnikov, Andrei A.; Vasilyeva, Lena M.; Fedotova, Elvira E.; Pak, Maria V.; Lekhanova, Sargylana N.; Zakharova, Elena V.; Savvinova, Kyunney E.; Gotovtsev, Nyurgun N.; Rafailo, Adyum M.; Luginov, Nikolay V.; Alexeev, Anatoliy N.; Posukh, Olga L.; Dzhemileva, Lilya U.; Khusnutdinova, Elza K.; Fedorova, Sardana A.

    2014-01-01

    Age-Related Hearing Impairment (ARHI) is one of the frequent sensory disorders registered in 50% of individuals over 80 years. ARHI is a multifactorial disorder due to environmental and poor-known genetic components. In this study, we present the data on age-related hearing impairment of 48 heterozygous carriers of mutation IVS1+1G>A (GJB2 gene) and 97 subjects with GJB2 genotype wt/wt in the Republic of Sakha/Yakutia (Eastern Siberia, Russia). This subarctic territory was found as the region with the most extensive accumulation of mutation IVS1+1G>A in the world as a result of founder effect in the unique Yakut population isolate. The GJB2 gene resequencing and detailed audiological analysis in the frequency range 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 kHz were performed in all examined subjects that allowed to investigate genotype-phenotype correlations between the presence of single mutation IVS1+1G>A and hearing of subjects from examined groups. We revealed the linear correlation between increase of average hearing thresholds at speech frequencies (PTA0.5,1.0,2.0,4.0 kHz) and age of individuals with GJB2 genotype IVS1+1G>A/wt (rs = 0.499, p = 0.006860 for males and rs = 0.427, p = 0.000277 for females). Moreover, the average hearing thresholds on high frequency (8.0 kHz) in individuals with genotype IVS1+1G>A/wt (both sexes) were significantly worse than in individuals with genotype wt/wt (p<0.05). Age of hearing loss manifestation in individuals with genotype IVS1+1G>A/wt was estimated to be ∼40 years (rs = 0.504, p = 0.003). These findings demonstrate that the single IVS1+1G>A mutation (GJB2) is associated with age-related hearing impairment (ARHI) of the IVS1+1G>A carriers in the Yakuts. PMID:24959830

  3. Age-Related Hearing Impairment (ARHI) associated with GJB2 single mutation IVS1+1G>A in the Yakut population isolate in Eastern Siberia.

    PubMed

    Barashkov, Nikolay A; Teryutin, Fedor M; Pshennikova, Vera G; Solovyev, Aisen V; Klarov, Leonid A; Solovyeva, Natalya A; Kozhevnikov, Andrei A; Vasilyeva, Lena M; Fedotova, Elvira E; Pak, Maria V; Lekhanova, Sargylana N; Zakharova, Elena V; Savvinova, Kyunney E; Gotovtsev, Nyurgun N; Rafailo, Adyum M; Luginov, Nikolay V; Alexeev, Anatoliy N; Posukh, Olga L; Dzhemileva, Lilya U; Khusnutdinova, Elza K; Fedorova, Sardana A

    2014-01-01

    Age-Related Hearing Impairment (ARHI) is one of the frequent sensory disorders registered in 50% of individuals over 80 years. ARHI is a multifactorial disorder due to environmental and poor-known genetic components. In this study, we present the data on age-related hearing impairment of 48 heterozygous carriers of mutation IVS1+1G>A (GJB2 gene) and 97 subjects with GJB2 genotype wt/wt in the Republic of Sakha/Yakutia (Eastern Siberia, Russia). This subarctic territory was found as the region with the most extensive accumulation of mutation IVS1+1G>A in the world as a result of founder effect in the unique Yakut population isolate. The GJB2 gene resequencing and detailed audiological analysis in the frequency range 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 kHz were performed in all examined subjects that allowed to investigate genotype-phenotype correlations between the presence of single mutation IVS1+1G>A and hearing of subjects from examined groups. We revealed the linear correlation between increase of average hearing thresholds at speech frequencies (PTA0.5,1.0,2.0,4.0 kHz) and age of individuals with GJB2 genotype IVS1+1G>A/wt (rs = 0.499, p = 0.006860 for males and rs = 0.427, p = 0.000277 for females). Moreover, the average hearing thresholds on high frequency (8.0 kHz) in individuals with genotype IVS1+1G>A/wt (both sexes) were significantly worse than in individuals with genotype wt/wt (p<0.05). Age of hearing loss manifestation in individuals with genotype IVS1+1G>A/wt was estimated to be ∼40 years (rs = 0.504, p = 0.003). These findings demonstrate that the single IVS1+1G>A mutation (GJB2) is associated with age-related hearing impairment (ARHI) of the IVS1+1G>A carriers in the Yakuts.

  4. Amino-terminal cysteine residues differentially influence RGS4 protein plasma membrane targeting, intracellular trafficking, and function.

    PubMed

    Bastin, Guillaume; Singh, Kevin; Dissanayake, Kaveesh; Mighiu, Alexandra S; Nurmohamed, Aliya; Heximer, Scott P

    2012-08-17

    Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization.

  5. Microcephaly, intellectual impairment, bilateral vesicoureteral reflux, distichiasis, and glomuvenous malformations associated with a 16q24.3 contiguous gene deletion and a Glomulin mutation.

    PubMed

    Butler, Matthew G; Dagenais, Susan L; Garcia-Perez, José L; Brouillard, Pascal; Vikkula, Miikka; Strouse, Peter; Innis, Jeffrey W; Glover, Thomas W

    2012-04-01

    Two hereditary syndromes, lymphedema-distichiasis (LD) syndrome and blepharo-chelio-dontic (BCD) syndrome include the aberrant growth of eyelashes from the meibomian glands, known as distichiasis. LD is an autosomal dominant syndrome primarily characterized by distichiasis and the onset of lymphedema usually during puberty. Mutations in the forkhead transcription factor FOXC2 are the only known cause of LD. BCD syndrome consists of autosomal dominant abnormalities of the eyelid, lip, and teeth, and the etiology remains unknown. In this report, we describe a proband that presented with distichiasis, microcephaly, bilateral grade IV vesicoureteral reflux requiring ureteral re-implantation, mild intellectual impairment and apparent glomuvenous malformations (GVM). Distichiasis was present in three generations of the proband's maternal side of the family. The GVMs were severe in the proband, and maternal family members exhibited lower extremity varicosities of variable degree. A GLMN (glomulin) gene mutation was identified in the proband that accounts for the observed GVMs; no other family member could be tested. TIE2 sequencing revealed no mutations. In the proband, an additional submicroscopic 265 kb contiguous gene deletion was identified in 16q24.3, located 609 kb distal to the FOXC2 locus, which was inherited from the proband's mother. The deletion includes the C16ORF95, FBXO31, MAP1LC3B, and ZCCHC14 loci and 115 kb of a gene desert distal to FOXC2 and FOXL1. Thus, it is likely that the microcephaly, distichiasis, vesicoureteral, and intellectual impairment in this family may be caused by the deletion of one or more of these genes and/or deletion of distant cis-regulatory elements of FOXC2 expression.

  6. Microcephaly, Intellectual Impairment, Bilateral Vesicoureteral Reflux, Distichiasis and Glomuvenous Malformations Associated with a 16q24.3 Contiguous Gene Deletion and a Glomulin Mutation

    PubMed Central

    Butler, Matthew G.; Dagenais, Susan L.; Garcia-Perez, José L.; Brouillard, Pascal; Vikkula, Miikka; Strouse, Peter; Innis, Jeffrey W.; Glover, Thomas W.

    2012-01-01

    Two hereditary syndromes, lymphedema-distichiasis syndrome (LD) and blepharo-chelio-dontic (BCD) syndrome include the aberrant growth of eyelashes from the meibomian glands, known as distichiasis. LD is an autosomal dominant syndrome primarily characterized by distichiasis and the onset of lymphedema usually during puberty. Mutations in the forkhead transcription factor FOXC2 are the only known cause of LD. BCD syndrome consists of autosomal dominant abnormalities of the eyelid, lip, and teeth, and the etiology remains unknown. In this report, we describe a proband that presented with distichiasis, microcephaly, bilateral grade IV vesicoureteral reflux requiring ureteral re-implantation, mild intellectual impairment and apparent glomuvenous malformations. Distichiasis was present in three generations of the proband’s maternal side of the family. The glomuvenous malformations were severe in the proband, and maternal family members exhibited lower extremity varicosities of variable degree. A GLMN (glomulin) gene mutation was identified in the proband that accounts for the observed glomuvenous malformations; no other family member could be tested. TIE2 sequencing revealed no mutations. In the proband, an additional submicroscopic 265 kb contiguous gene deletion was identified in 16q24.3, located 609 kb distal to the FOXC2 locus, which was inherited from the proband’s mother. The deletion includes the C16ORF95, FBXO31, MAP1LC3B, and ZCCHC14 loci and 115 kb of a gene desert distal to FOXC2 and FOXL1. Thus, it is likely that the microcephaly, distichiasis, vesicoureteral and intellectual impairment in this family may be caused by the deletion of one or more of these genes and/or deletion of distant cis-regulatory elements of FOXC2 expression. PMID:22407726

  7. ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function

    PubMed Central

    Murakami, Tetsuro; Qamar, Seema; Lin, Julie Qiaojin; Schierle, Gabriele S. Kaminski; Rees, Eric; Miyashita, Akinori; Costa, Ana R.; Dodd, Roger B.; Chan, Fiona T.S.; Michel, Claire H.; Kronenberg-Versteeg, Deborah; Li, Yi; Yang, Seung-Pil; Wakutani, Yosuke; Meadows, William; Ferry, Rodylyn Rose; Dong, Liang; Tartaglia, Gian Gaetano; Favrin, Giorgio; Lin, Wen-Lang; Dickson, Dennis W.; Zhen, Mei; Ron, David; Schmitt-Ulms, Gerold; Fraser, Paul E.; Shneider, Neil A.; Holt, Christine; Vendruscolo, Michele; Kaminski, Clemens F.; St George-Hyslop, Peter

    2015-01-01

    Summary The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins. PMID:26526393

  8. ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function.

    PubMed

    Murakami, Tetsuro; Qamar, Seema; Lin, Julie Qiaojin; Schierle, Gabriele S Kaminski; Rees, Eric; Miyashita, Akinori; Costa, Ana R; Dodd, Roger B; Chan, Fiona T S; Michel, Claire H; Kronenberg-Versteeg, Deborah; Li, Yi; Yang, Seung-Pil; Wakutani, Yosuke; Meadows, William; Ferry, Rodylyn Rose; Dong, Liang; Tartaglia, Gian Gaetano; Favrin, Giorgio; Lin, Wen-Lang; Dickson, Dennis W; Zhen, Mei; Ron, David; Schmitt-Ulms, Gerold; Fraser, Paul E; Shneider, Neil A; Holt, Christine; Vendruscolo, Michele; Kaminski, Clemens F; St George-Hyslop, Peter

    2015-11-18

    The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins.

  9. Role of adaptor proteins and clathrin in the trafficking of human kidney anion exchanger 1 (kAE1) to the cell surface.

    PubMed

    Junking, Mutita; Sawasdee, Nunghathai; Duangtum, Natapol; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

    2014-07-01

    Kidney anion exchanger 1 (kAE1) plays an important role in acid-base homeostasis by mediating chloride/bicarbornate (Cl-/HCO3-) exchange at the basolateral membrane of α-intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease - distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans-Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral-related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non-polarized kidney cells. By using RNA interference, co-immunoprecipitation, yellow fluorescent protein-based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin (but not AP-1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral-related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid-secreting α-intercalated cells.

  10. SLC6A3 coding variant Ala559Val found in two autism probands alters dopamine transporter function and trafficking.

    PubMed

    Bowton, E; Saunders, C; Reddy, I A; Campbell, N G; Hamilton, P J; Henry, L K; Coon, H; Sakrikar, D; Veenstra-VanderWeele, J M; Blakely, R D; Sutcliffe, J; Matthies, H J G; Erreger, K; Galli, A

    2014-10-14

    Emerging evidence associates dysfunction in the dopamine (DA) transporter (DAT) with the pathophysiology of autism spectrum disorder (ASD). The human DAT (hDAT; SLC6A3) rare variant with an Ala to Val substitution at amino acid 559 (hDAT A559V) was previously reported in individuals with bipolar disorder or attention-deficit hyperactivity disorder (ADHD). We have demonstrated that this variant is hyper-phosphorylated at the amino (N)-terminal serine (Ser) residues and promotes an anomalous DA efflux phenotype. Here, we report the novel identification of hDAT A559V in two unrelated ASD subjects and provide the first mechanistic description of its impaired trafficking phenotype. DAT surface expression is dynamically regulated by DAT substrates including the psychostimulant amphetamine (AMPH), which causes hDAT trafficking away from the plasma membrane. The integrity of DAT trafficking directly impacts DA transport capacity and therefore dopaminergic neurotransmission. Here, we show that hDAT A559V is resistant to AMPH-induced cell surface redistribution. This unique trafficking phenotype is conferred by altered protein kinase C β (PKCβ) activity. Cells expressing hDAT A559V exhibit constitutively elevated PKCβ activity, inhibition of which restores the AMPH-induced hDAT A559V membrane redistribution. Mechanistically, we link the inability of hDAT A559V to traffic in response to AMPH to the phosphorylation of the five most distal DAT N-terminal Ser. Mutation of these N-terminal Ser to Ala restores AMPH-induced trafficking. Furthermore, hDAT A559V has a diminished ability to transport AMPH, and therefore lacks AMPH-induced DA efflux. Pharmacological inhibition of PKCβ or Ser to Ala substitution in the hDAT A559V background restores AMPH-induced DA efflux while promoting intracellular AMPH accumulation. Although hDAT A559V is a rare variant, it has been found in multiple probands with neuropsychiatric disorders associated with imbalances in DA neurotransmission

  11. Cognitive impairment in the preclinical stage of dementia in FTD-3 CHMP2B mutation carriers: a longitudinal prospective study.

    PubMed

    Stokholm, Jette; Teasdale, Thomas W; Johannsen, Peter; Nielsen, Jorgen E; Nielsen, Troels Tolstrup; Isaacs, Adrian; Brown, Jerry M; Gade, Anders

    2013-02-01

    A longitudinal study spanning over 8 years and including 17 asymptomatic individuals with CHMP2B mutations was conducted to assess the earliest neuropsychological changes in autosomal dominant neurodegenerative disease frontotemporal dementia (FTD) linked to chromosome 3 (FTD-3). Subjects were assessed with neuropsychological tests in 2002, 2005 and 2010. Cross-sectional analyses showed that the mutation carriers scored lower on tests of psychomotor speed, working memory, executive functions and verbal memory than a control group consisting of not-at-risk family members and spouses. Longitudinal analyses showed a gradual decline in psychomotor speed, working memory capacity and global executive measures in the group of non-demented mutation carriers that was not found in the control group. In contrast, there were no significant group differences in domain scores on memory or visuospatial functions. On an individual level the cognitive changes over time varied considerably. Subjects with CHMP2B mutation show cognitive changes dominated by executive dysfunctions, years before they fulfil diagnostic criteria of FTD. However, there is great heterogeneity in the individual cognitive trajectories.

  12. Rab 11 regulates constitutive dopamine transporter trafficking and function in N2A neuroblastoma cells.

    PubMed

    Furman, Cheryse A; Lo, Charles B; Stokes, Stephanie; Esteban, Jose A; Gnegy, Margaret E

    2009-09-29

    The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission which undergoes constitutive and substrate-mediated trafficking to and from the membrane. Although, considerable research has been done to elucidate the regulation of substrate-stimulated DAT trafficking, less is known about which trafficking proteins are involved in constitutive DAT trafficking. Rab proteins are GTPases known to regulate the trafficking of proteins to and from specific endocytic compartments. Rabs 8 and 11, in particular, are involved in trafficking proteins from intracellular compartments to the plasma membrane. In this study, we sought to determine whether Rabs 8 and 11 would modulate DAT activity and trafficking in N2A neuroblastoma cells. We used Rab mutations known to confer constitutively active or dominant negative activity of these proteins to investigate the role of Rab activity in constitutive DAT trafficking and function. We found that constitutively active Rab 11 upregulates DAT function and surface expression while neither the constitutively active nor the dominant negative mutant of Rab 8 had any effect on DA uptake. Furthermore, immunofluorescence experiments revealed that dominant negative Rab 11 overexpression results in decreased surface DAT indicating a necessary function of Rab 11 in DAT trafficking to the plasma membrane. These data show for the first time a functional role of Rab proteins in the constitutive recycling of DAT to the plasma membrane.

  13. Impairment of Cargo Transportation Caused by gbf1 Mutation Disrupts Vascular Integrity and Causes Hemorrhage in Zebrafish Embryos.

    PubMed

    Chen, Jing; Wu, Xiaotong; Yao, Likun; Yan, Lu; Zhang, Lin; Qiu, Juhui; Liu, Xingfeng; Jia, Shunji; Meng, Anming

    2017-02-10

    ADP-ribosylation factor GTPases are activated by guanine nucleotide exchange factors including Gbf1 (Golgi brefeldin A-resistant factor 1) and play important roles in regulating organelle structure and cargo-selective vesicle trafficking. However, the developmental role of Gbf1 in vertebrates remains elusive. In this study, we report the zebrafish mutant line tsu3994 that arises from N-ethyl-N-nitrosourea (ENU)-mediated mutagenesis and is characterized by prominent intracerebral and trunk hemorrhage. The mutant embryos develop hemorrhage accompanied by fewer pigments and shorter caudal fin at day 2 of development. The hemorrhage phenotype is caused by vascular breakage in a cell autonomous fashion. Positional cloning identifies a T → G nucleotide substitution in the 23rd exon of the gbf1 locus, resulting in a leucine → arginine substitution (L1246R) in the HDS2 domain. The mutant phenotype is mimicked by gbf1 knockouts and morphants, suggesting a nature of loss of function. Experimental results in mammalian cells show that the mutant form Gbf1(L1246R) is unable to be recruited to the Golgi apparatus and fails to activate Arf1 for recruiting COPI complex. The hemorrhage in tsu3994 mutants can be prevented partially and temporally by treating with the endoplasmic reticulum stress/apoptosis inhibitor tauroursodeoxycholic acid or by knocking down the proapoptotic gene baxb Therefore, endothelial endoplasmic reticulum stress and subsequent apoptosis induced by gbf1 deficiency may account for the vascular collapse and hemorrhage.

  14. Ceramidase Regulates Synaptic Vesicle Exocytosis and Trafficking

    PubMed Central

    Rohrbough, Jeffrey; Rushton, Emma; Palanker, Laura; Woodruff, Elvin; Matthies, Heinrich J. G.; Acharya, Usha; Acharya, Jairaj K.; Broadie, Kendal

    2009-01-01

    A screen for Drosophila synaptic dysfunction mutants identified slug-a-bed (slab). The slab gene encodes ceramidase, a central enzyme in sphingolipid metabolism and regulation. Sphingolipids are major constituents of lipid rafts, membrane domains with roles in vesicle trafficking, and signaling pathways. Null slab mutants arrest as fully developed embryos with severely reduced movement. The SLAB protein is widely expressed in different tissues but enriched in neurons at all stages of development. Targeted neuronal expression of slab rescues mutant lethality, demonstrating the essential neuronal function of the protein. C5-ceramide applied to living preparations is rapidly accumulated at neuromuscular junction (NMJ) synapses dependent on the SLAB expression level, indicating that synaptic sphingolipid trafficking and distribution is regulated by SLAB function. Evoked synaptic currents at slab mutant NMJs are reduced by 50–70%, whereas postsynaptic glutamate-gated currents are normal, demonstrating a specific presynaptic impairment. Hypertonic saline-evoked synaptic vesicle fusion is similarly impaired by 50–70%, demonstrating a loss of readily releasable vesicles. In addition, FM1-43 dye uptake is reduced in slab mutant presynaptic terminals, indicating a smaller cycling vesicle pool. Ultrastructural analyses of mutants reveal a normal vesicle distribution clustered and docked at active zones, but fewer vesicles in reserve regions, and a twofold to threefold increased incidence of vesicles linked together and tethered at the plasma membrane. These results indicate that SLAB ceramidase function controls presynaptic terminal sphingolipid composition to regulate vesicle fusion and trafficking, and thus the strength and reliability of synaptic transmission. PMID:15356190

  15. Structure of Human B12 Trafficking Protein CblD Reveals Molecular Mimicry and Identifies a New Subfamily of Nitro-FMN Reductases.

    PubMed

    Yamada, Kazuhiro; Gherasim, Carmen; Banerjee, Ruma; Koutmos, Markos

    2015-12-04

    In mammals, B12 (or cobalamin) is an essential cofactor required by methionine synthase and methylmalonyl-CoA mutase. A complex intracellular pathway supports the assimilation of cobalamin into its active cofactor forms and delivery to its target enzymes. MMADHC (the methylmalonic aciduria and homocystinuria type D protein), commonly referred to as CblD, is a key chaperone involved in intracellular cobalamin trafficking, and mutations in CblD cause methylmalonic aciduria and/or homocystinuria. Herein, we report the first crystal structure of the globular C-terminal domain of human CblD, which is sufficient for its interaction with MMADHC (the methylmalonic aciduria and homocystinuria type C protein), or CblC, and for supporting the cytoplasmic cobalamin trafficking pathway. CblD contains an α+β fold that is structurally reminiscent of the nitro-FMN reductase superfamily. Two of the closest structural relatives of CblD are CblC, a multifunctional enzyme important for cobalamin trafficking, and the activation domain of methionine synthase. CblD, CblC, and the activation domain of methionine synthase share several distinguishing features and, together with two recently described corrinoid-dependent reductive dehalogenases, constitute a new subclass within the nitro-FMN reductase superfamily. We demonstrate that CblD enhances oxidation of cob(II)alamin bound to CblC and that disease-causing mutations in CblD impair the kinetics of this reaction. The striking structural similarity of CblD to CblC, believed to be contiguous in the cobalamin trafficking pathway, suggests the co-option of molecular mimicry as a strategy for achieving its function.

  16. Structure of Human B12 Trafficking Protein CblD Reveals Molecular Mimicry and Identifies a New Subfamily of Nitro-FMN Reductases*

    PubMed Central

    Yamada, Kazuhiro; Gherasim, Carmen; Banerjee, Ruma; Koutmos, Markos

    2015-01-01

    In mammals, B12 (or cobalamin) is an essential cofactor required by methionine synthase and methylmalonyl-CoA mutase. A complex intracellular pathway supports the assimilation of cobalamin into its active cofactor forms and delivery to its target enzymes. MMADHC (the methylmalonic aciduria and homocystinuria type D protein), commonly referred to as CblD, is a key chaperone involved in intracellular cobalamin trafficking, and mutations in CblD cause methylmalonic aciduria and/or homocystinuria. Herein, we report the first crystal structure of the globular C-terminal domain of human CblD, which is sufficient for its interaction with MMADHC (the methylmalonic aciduria and homocystinuria type C protein), or CblC, and for supporting the cytoplasmic cobalamin trafficking pathway. CblD contains an α+β fold that is structurally reminiscent of the nitro-FMN reductase superfamily. Two of the closest structural relatives of CblD are CblC, a multifunctional enzyme important for cobalamin trafficking, and the activation domain of methionine synthase. CblD, CblC, and the activation domain of methionine synthase share several distinguishing features and, together with two recently described corrinoid-dependent reductive dehalogenases, constitute a new subclass within the nitro-FMN reductase superfamily. We demonstrate that CblD enhances oxidation of cob(II)alamin bound to CblC and that disease-causing mutations in CblD impair the kinetics of this reaction. The striking structural similarity of CblD to CblC, believed to be contiguous in the cobalamin trafficking pathway, suggests the co-option of molecular mimicry as a strategy for achieving its function. PMID:26364851

  17. Alteration of the proteostasis network of plant cells promotes the post-endoplasmic reticulum trafficking of recombinant mutant (L444P) human β-glucocerebrosidase

    PubMed Central

    Babajani, Gholamreza; Kermode, Allison R

    2014-01-01

    Gaucher disease is a prevalent lysosomal storage disease characterized by a deficiency in the activity of lysosomal acid β-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45). One of the most prevalent disease-causing mutations in humans is a L444P missense mutation in the GCase protein, which results in its disrupted folding in the endoplasmic reticulum (ER) and impaired post-ER trafficking. To determine whether the post-ER trafficking of this severely malfolded protein can be restored, we expressed the mutant L444P GCase as a recombinant protein in transgenic tobacco (Nicotiana tabacum L. cv Bright Yellow 2 [BY2]) cells, in which the GCase variant was equipped with a plant signal peptide to allow for secretion upon rescued trafficking out of the ER. The recombinant L444P mutant GCase was retained in the plant endoplasmic reticulum (ER). Kifunensine and Eeyarestatin I, both inhibitors of ER-associated degradation (ERAD), and the proteostasis regulators, celastrol and MG-132, increased the steady-state levels of the mutant protein inside the plant cells and further promoted the post-ER trafficking of L444P GCase, as indicated by endoglycosidase-H sensitivity- and secretion- analyses. Transcript profiling of genes encoding ER-molecular chaperones, ER stress responsive proteins, and cytoplasmic heat shock response proteins, revealed insignificant or only very modest changes in response to the ERAD inhibitors and proteostasis regulators. An exception was the marked response to celastrol which reduced the steady-state levels of cytoplasmic HSP90 transcripts and protein. As HSP90 participates in the targeting of misfolded proteins to the proteasome pathway, its down-modulation in response to celastrol may partly account for the mechanism of improved homeostasis of L444P GCase mediated by this triterpene. PMID:24713615

  18. Depolarized inactivation overcomes impaired activation to produce DRG neuron hyperexcitability in a Nav1.7 mutation in a patient with distal limb pain.

    PubMed

    Huang, Jianying; Yang, Yang; Dib-Hajj, Sulayman D; van Es, Michael; Zhao, Peng; Salomon, Jody; Drenth, Joost P H; Waxman, Stephen G

    2014-09-10

    Sodium channel Nav1.7, encoded by SCN9A, is expressed in DRG neurons and regulates their excitability. Genetic and functional studies have established a critical contribution of Nav1.7 to human pain disorders. We have now characterized a novel Nav1.7 mutation (R1279P) from a female human subject with distal limb pain, in which depolarized fast inactivation overrides impaired activation to produce hyperexcitability and spontaneous firing in DRG neurons. Whole-cell voltage-clamp recordings in human embryonic kidney (HEK) 293 cells demonstrated that R1279P significantly depolarizes steady-state fast-, slow-, and closed-state inactivation. It accelerates deactivation, decelerates inactivation, and facilitates repriming. The mutation increases ramp currents in response to slow depolarizations. Our voltage-clamp analysis showed that R1279P depolarizes channel activation, a change that was supported by our multistate structural modeling. Because this mutation confers both gain-of-function and loss-of-function attributes on the Nav1.7 channel, we tested the impact of R1279P expression on DRG neuron excitability. Current-clamp studies reveal that R1279P depolarizes resting membrane potential, decreases current threshold, and increases firing frequency of evoked action potentials within small DRG neurons. The populations of spontaneously firing and repetitively firing neurons were increased by expressing R1279P. These observations indicate that the dominant proexcitatory gating changes associated with this mutation, including depolarized steady-state fast-, slow-, and closed-state inactivation, faster repriming, and larger ramp currents, override the depolarizing shift of activation, to produce hyperexcitability and spontaneous firing of nociceptive neurons that underlie pain.

  19. ZC4H2 mutations are associated with arthrogryposis multiplex congenita and intellectual disability through impairment of central and peripheral synaptic plasticity.

    PubMed

    Hirata, Hiromi; Nanda, Indrajit; van Riesen, Anne; McMichael, Gai; Hu, Hao; Hambrock, Melanie; Papon, Marie-Amélie; Fischer, Ute; Marouillat, Sylviane; Ding, Can; Alirol, Servane; Bienek, Melanie; Preisler-Adams, Sabine; Grimme, Astrid; Seelow, Dominik; Webster, Richard; Haan, Eric; MacLennan, Alastair; Stenzel, Werner; Yap, Tzu Ying; Gardner, Alison; Nguyen, Lam Son; Shaw, Marie; Lebrun, Nicolas; Haas, Stefan A; Kress, Wolfram; Haaf, Thomas; Schellenberger, Elke; Chelly, Jamel; Viot, Géraldine; Shaffer, Lisa G; Rosenfeld, Jill A; Kramer, Nancy; Falk, Rena; El-Khechen, Dima; Escobar, Luis F; Hennekam, Raoul; Wieacker, Peter; Hübner, Christoph; Ropers, Hans-Hilger; Gecz, Jozef; Schuelke, Markus; Laumonnier, Frédéric; Kalscheuer, Vera M

    2013-05-02

    Arthrogryposis multiplex congenita (AMC) is caused by heterogeneous pathologies leading to multiple antenatal joint contractures through fetal akinesia. Understanding the pathophysiology of this disorder is important for clinical care of the affected individuals and genetic counseling of the families. We thus aimed to establish the genetic basis of an AMC subtype that is associated with multiple dysmorphic features and intellectual disability (ID). We used haplotype analysis, next-generation sequencing, array comparative genomic hybridization, and chromosome breakpoint mapping to identify the pathogenic mutations in families and simplex cases. Suspected disease variants were verified by cosegregation analysis. We identified disease-causing mutations in the zinc-finger gene ZC4H2 in four families affected by X-linked AMC plus ID and one family affected by cerebral palsy. Several heterozygous females were also affected, but to a lesser degree. Furthermore, we found two ZC4H2 deletions and one rearrangement in two female and one male unrelated simplex cases, respectively. In mouse primary hippocampal neurons, transiently produced ZC4H2 localized to the postsynaptic compartment of excitatory synapses, and the altered protein influenced dendritic spine density. In zebrafish, antisense-morpholino-mediated zc4h2 knockdown caused abnormal swimming and impaired α-motoneuron development. All missense mutations identified herein failed to rescue the swimming defect of zebrafish morphants. We conclude that ZC4H2 point mutations, rearrangements, and small deletions cause a clinically variable broad-spectrum neurodevelopmental disorder of the central and peripheral nervous systems in both familial and simplex cases of both sexes. Our results highlight the importance of ZC4H2 for genetic testing of individuals presenting with ID plus muscle weakness and minor or major forms of AMC.

  20. ZC4H2 Mutations Are Associated with Arthrogryposis Multiplex Congenita and Intellectual Disability through Impairment of Central and Peripheral Synaptic Plasticity

    PubMed Central

    Hirata, Hiromi; Nanda, Indrajit; van Riesen, Anne; McMichael, Gai; Hu, Hao; Hambrock, Melanie; Papon, Marie-Amélie; Fischer, Ute; Marouillat, Sylviane; Ding, Can; Alirol, Servane; Bienek, Melanie; Preisler-Adams, Sabine; Grimme, Astrid; Seelow, Dominik; Webster, Richard; Haan, Eric; MacLennan, Alastair; Stenzel, Werner; Yap, Tzu Ying; Gardner, Alison; Nguyen, Lam Son; Shaw, Marie; Lebrun, Nicolas; Haas, Stefan A.; Kress, Wolfram; Haaf, Thomas; Schellenberger, Elke; Chelly, Jamel; Viot, Géraldine; Shaffer, Lisa G.; Rosenfeld, Jill A.; Kramer, Nancy; Falk, Rena; El-Khechen, Dima; Escobar, Luis F.; Hennekam, Raoul; Wieacker, Peter; Hübner, Christoph; Ropers, Hans-Hilger; Gecz, Jozef; Schuelke, Markus; Laumonnier, Frédéric; Kalscheuer, Vera M.

    2013-01-01

    Arthrogryposis multiplex congenita (AMC) is caused by heterogeneous pathologies leading to multiple antenatal joint contractures through fetal akinesia. Understanding the pathophysiology of this disorder is important for clinical care of the affected individuals and genetic counseling of the families. We thus aimed to establish the genetic basis of an AMC subtype that is associated with multiple dysmorphic features and intellectual disability (ID). We used haplotype analysis, next-generation sequencing, array comparative genomic hybridization, and chromosome breakpoint mapping to identify the pathogenic mutations in families and simplex cases. Suspected disease variants were verified by cosegregation analysis. We identified disease-causing mutations in the zinc-finger gene ZC4H2 in four families affected by X-linked AMC plus ID and one family affected by cerebral palsy. Several heterozygous females were also affected, but to a lesser degree. Furthermore, we found two ZC4H2 deletions and one rearrangement in two female and one male unrelated simplex cases, respectively. In mouse primary hippocampal neurons, transiently produced ZC4H2 localized to the postsynaptic compartment of excitatory synapses, and the altered protein influenced dendritic spine density. In zebrafish, antisense-morpholino-mediated zc4h2 knockdown caused abnormal swimming and impaired α-motoneuron development. All missense mutations identified herein failed to rescue the swimming defect of zebrafish morphants. We conclude that ZC4H2 point mutations, rearrangements, and small deletions cause a clinically variable broad-spectrum neurodevelopmental disorder of the central and peripheral nervous systems in both familial and simplex cases of both sexes. Our results highlight the importance of ZC4H2 for genetic testing of individuals presenting with ID plus muscle weakness and minor or major forms of AMC. PMID:23623388

  1. Disease-causing mutations affecting surface residues of mitochondrial glutaryl-CoA dehydrogenase impair stability, heteromeric complex formation and mitochondria architecture.

    PubMed

    Schmiesing, Jessica; Lohmöller, Benjamin; Schweizer, Michaela; Tidow, Henning; Gersting, Søren W; Muntau, Ania C; Braulke, Thomas; Mühlhausen, Chris

    2017-02-01

    The neurometabolic disorder glutaric aciduria type 1 (GA1) is caused by mutations in the GCDH gene encoding the mitochondrial matrix protein glutaryl-CoA dehydrogenase (GCDH), which forms homo- and heteromeric complexes. Twenty percent of all pathogenic mutations affect single amino acid residues on the surface of GCDH resulting in a severe clinical phenotype. We report here on heterologous expression studies of 18 missense mutations identified in GA1 patients affecting surface amino acids. Western blot and pulse chase experiments revealed that the stability of half of the GCDH mutants was significantly reduced. In silico analyses showed that none of the mutations impaired the 3D structure of GCDH. Immunofluorescence co-localisation studies in HeLa cells demonstrated that all GCDH mutants were correctly translocated into mitochondria. Surprisingly, the expression of p.Arg88Cys GCDH as well as further substitutions by alanine, lysine, or methionine but not histidine or leucine resulted in the disruption of mitochondrial architecture forming longitudinal structures composed of stacks of cristae and partial loss of the outer mitochondrial membrane. The expression of mitochondrial fusion or fission proteins was not affected in these cells. Bioluminescence resonance energy transfer analyses revealed that all GCDH mutants exhibit an increased binding affinity to electron transfer flavoprotein beta, whereas only p.Tyr155His GCDH showed a reduced interaction with dihydrolipoamide succinyl transferase. Our data underscore the impact of GCDH protein interactions mediated by amino acid residues on the surface of GCDH required for proper enzymatic activity. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Malignant hyperthermia-associated mutations in the S2-S3 cytoplasmic loop of type 1 ryanodine receptor calcium channel impair calcium-dependent inactivation.

    PubMed

    Gomez, Angela C; Holford, Timothy W; Yamaguchi, Naohiro

    2016-11-01

    Channel activities of skeletal muscle ryanodine receptor (RyR1) are activated by micromolar Ca(2+) and inactivated by higher (∼1 mM) Ca(2+) To gain insight into a mechanism underlying Ca(2+)-dependent inactivation of RyR1 and its relationship with skeletal muscle diseases, we constructed nine recombinant RyR1 mutants carrying malignant hyperthermia or centronuclear myopathy-associated mutations and determined RyR1 channel activities by [(3)H]ryanodine binding assay. These mutations are localized in or near the RyR1 domains which are responsible for Ca(2+)-dependent inactivation of RyR1. Four RyR1 mutations (F4732D, G4733E, R4736W, and R4736Q) in the cytoplasmic loop between the S2 and S3 transmembrane segments (S2-S3 loop) greatly reduced Ca(2+)-dependent channel inactivation. Activities of these mutant channels were suppressed at 10-100 μM Ca(2+), and the suppressions were relieved by 1 mM Mg(2+) The Ca(2+)- and Mg(2+)-dependent regulation of S2-S3 loop RyR1 mutants are similar to those of the cardiac isoform of RyR (RyR2) rather than wild-type RyR1. Two mutations (T4825I and H4832Y) in the S4-S5 cytoplasmic loop increased Ca(2+) affinities for channel activation and decreased Ca(2+) affinities for inactivation, but impairment of Ca(2+)-dependent inactivation was not as prominent as those of S2-S3 loop mutants. Three mutations (T4082M, S4113L, and N4120Y) in the EF-hand domain showed essentially the same Ca(2+)-dependent channel regulation as that of wild-type RyR1. The results suggest that nine RyR1 mutants associated with skeletal muscle diseases were differently regulated by Ca(2+) and Mg(2+) Four malignant hyperthermia-associated RyR1 mutations in the S2-S3 loop conferred RyR2-type Ca(2+)- and Mg(2+)-dependent channel regulation. Copyright © 2016 the American Physiological Society.

  3. TBC1D24 mutation associated with focal epilepsy, cognitive impairment and a distinctive cerebro-cerebellar malformation.

    PubMed

    Afawi, Zaid; Mandelstam, Simone; Korczyn, Amos D; Kivity, Sara; Walid, Simri; Shalata, Adel; Oliver, Karen L; Corbett, Mark; Gecz, Jozef; Berkovic, Samuel F; Jackson, Graeme D

    2013-07-01

    We describe the clinical and radiological features of a family with a homozygous mutation in TBC1D24. The phenotype comprised onset of focal seizures at 2 months with prominent eye-blinking, facial and limb jerking with an oral sensory aura. These were controllable with medication but persisted into adult life. Associated features were mild to moderate intellectual disability and cerebellar features. MRI showed subtle cortical thickening with cerebellar atrophy and high signal confined to the ansiform lobule. The disorder is allelic with familial infantile myoclonic epilepsy, where intellect and neurologic examination are normal, highlighting the phenotypic variation with mutations of TBC1D24. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Cysteine34 of the cytoplasmic tail of the cation-dependent mannose 6- phosphate receptor is reversibly palmitoylated and required for normal trafficking and lysosomal enzyme sorting

    PubMed Central

    1996-01-01

    We have examined whether the two cysteine residues (Cys30 and Cys34) in the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor are palmitoylated via thioesters and whether these residues influence the biologic function of the receptor. To do this, mouse L cells expressing wild-type and mutant receptors were analyzed by metabolic labeling with [3H]palmitate, immunoprecipitation, and SDS- PAGE. Both Cys30 and Cys34 were found to be sites of palmitoylation and together they accounted for the total palmitoylation of the receptor. The palmitate rapidly turned over with a half-life of approximately 2 h compared to a half-life of greater than 40 h for the protein. Mutation of Cys34 to Ala resulted in the gradual accumulation of the receptor in dense lysosomes and the total loss of cathepsin D sorting function in the Golgi. A Cys30 to Ala mutation had no biologic consequences, showing the importance of Cys34. Mutation of amino acids 35-39 to alanines impaired palmitoylation of Cys30 and Cys34 and resulted in abnormal receptor trafficking to lysosomes and loss of cathepsin D sorting. These data suggest that palmitoylation of Cys30 and Cys34 leads to anchoring of this region of the cytoplasmic tail to the lipid bilayer. Anchoring via Cys34 is essential for the normal trafficking and lysosomal enzyme sorting function of the receptor. PMID:8647889

  5. Impaired riboflavin transport due to missense mutations in SLC52A2 causes Brown-Vialetto-Van Laere syndrome.

    PubMed

    Haack, Tobias B; Makowski, Christine; Yao, Yoshiaki; Graf, Elisabeth; Hempel, Maja; Wieland, Thomas; Tauer, Ulrike; Ahting, Uwe; Mayr, Johannes A; Freisinger, Peter; Yoshimatsu, Hiroki; Inui, Ken; Strom, Tim M; Meitinger, Thomas; Yonezawa, Atsushi; Prokisch, Holger

    2012-11-01

    Brown-Vialetto-Van Laere syndrome (BVVLS [MIM 211530]) is a rare neurological disorder characterized by infancy onset sensorineural deafness and ponto-bulbar palsy. Mutations in SLC52A3 (formerly C20orf54), coding for riboflavin transporter 2 (hRFT2), have been identified as the molecular genetic correlate in several individuals with BVVLS. Exome sequencing of just one single case revealed that compound heterozygosity for two pathogenic mutations in the SLC52A2 gene coding for riboflavin transporter 3 (hRFT3), another member of the riboflavin transporter family, is also associated with BVVLS. Overexpression studies confirmed that the gene products of both mutant alleles have reduced riboflavin transport activities. While mutations in SLC52A3 cause decreased plasma riboflavin levels, concordant with a role of SLC52A3 in riboflavin uptake from food, the SLC52A2-mutant individual had normal plasma riboflavin concentrations, a finding in line with a postulated function of SLC52A2 in riboflavin uptake from blood into target cells. Our results contribute to the understanding of human riboflavin metabolism and underscore its role in the pathogenesis of BVVLS, thereby providing a rational basis for a high-dose riboflavin treatment.

  6. Impaired hematopoietic differentiation of RUNX1-mutated induced pluripotent stem cells derived from FPD/AML patients.

    PubMed

    Sakurai, M; Kunimoto, H; Watanabe, N; Fukuchi, Y; Yuasa, S; Yamazaki, S; Nishimura, T; Sadahira, K; Fukuda, K; Okano, H; Nakauchi, H; Morita, Y; Matsumura, I; Kudo, K; Ito, E; Ebihara, Y; Tsuji, K; Harada, Y; Harada, H; Okamoto, S; Nakajima, H

    2014-12-01

    Somatic mutation of RUNX1 is implicated in various hematological malignancies, including myelodysplastic syndrome and acute myeloid leukemia (AML), and previous studies using mouse models disclosed its critical roles in hematopoiesis. However, the role of RUNX1 in human hematopoiesis has never been tested in experimental settings. Familial platelet disorder (FPD)/AML is an autosomal dominant disorder caused by germline mutation of RUNX1, marked by thrombocytopenia and propensity to acute leukemia. To investigate the physiological function of RUNX1 in human hematopoiesis and pathophysiology of FPD/AML, we derived induced pluripotent stem cells (iPSCs) from three distinct FPD/AML pedigrees (FPD-iPSCs) and examined their defects in hematopoietic differentiation. By in vitro differentiation assays, FPD-iPSCs were clearly defective in the emergence of hematopoietic progenitors and differentiation of megakaryocytes, and overexpression of wild-type (WT)-RUNX1 reversed most of these phenotypes. We further demonstrated that overexpression of mutant-RUNX1 in WT-iPSCs did not recapitulate the phenotype of FPD-iPSCs, showing that the mutations were of loss-of-function type. Taken together, this study demonstrated that haploinsufficient RUNX1 allele imposed cell-intrinsic defects on hematopoietic differentiation in human experimental settings and revealed differential impacts of RUNX1 dosage on human and murine megakaryopoiesis. FPD-iPSCs will be a useful tool to investigate mutant RUNX1-mediated molecular processes in hematopoiesis and leukemogenesis.

  7. A Homozygous [Cys25]PTH(1-84) Mutation That Impairs PTH/PTHrP Receptor Activation Defines a Novel Form of Hypoparathyroidism.

    PubMed

    Lee, Sihoon; Mannstadt, Michael; Guo, Jun; Kim, Seul Min; Yi, Hyon-Seung; Khatri, Ashok; Dean, Thomas; Okazaki, Makoto; Gardella, Thomas J; Jüppner, Harald

    2015-10-01

    Hypocalcemia and hyperphosphatemia are encountered in idiopathic hypoparathyroidism (IHP) and pseudohypoparathyroidism type Ib (PHP1B). In contrast to PHP1B, which is caused by resistance toward parathyroid hormone (PTH), the genetic defects leading to IHP impair production of this important regulator of mineral ion homeostasis. So far, only five PTH mutations were shown to cause IHP, each of which is located in the hormone's pre-pro leader segment and thus impair hormone secretion. In three siblings affected by IHP, we now identified a homozygous arginine-to-cysteine mutation at position 25 (R25C) of the mature PTH(1-84) polypeptide; heterozygous family members are healthy. Depending on the assay used for evaluating these patients, plasma PTH levels were either low or profoundly elevated, thus leading to ambiguities regarding the underlying diagnosis, namely IHP or PHP1B. Consistent with increased PTH levels, recombinant [Cys25]PTH(1-84) and wild-type PTH(1-84) were secreted equally well by transfected COS-7 cells. However, synthetic [Cys25]PTH(1-34) was found to have a lower binding affinity for the PTH receptor type-1 (PTH1R) than PTH(1-34) and consequently a lower efficiency for stimulating cAMP formation in cells expressing this receptor. Consistent with these in vitro findings, long-term infusion of [Cys25]PTH(1-34) resulted only in minimal calcemic and phosphaturic responses, despite readily detectable levels of [Cys25]PTH(1-34) in plasma. The mineral ion abnormalities observed in the three IHP patients are thus most likely caused by the inherited homozygous missense PTH mutation, which reduces bioactivity of the secreted hormone. Based on these findings, screening for PTH(1-84) mutations should be considered when clinical and laboratory findings are consistent with PHP1B, but GNAS methylation changes have been excluded. Differentiating between IHP and PHP1B has considerable implications for genetic counseling, therapy, and long-term outcome because

  8. A Homozygous [Cys25]PTH(1-84) Mutation That Impairs PTH/PTHrP Receptor Activation Defines a Novel Form of Hypoparathyroidism

    PubMed Central

    Lee, Sihoon; Mannstadt, Michael; Guo, Jun; Kim, Seul Min; Yi, Hyon-Seung; Khatri, Ashok; Dean, Thomas; Okazaki, Makoto; Gardella, Thomas J; Jüppner, Harald

    2015-01-01

    Hypocalcemia and hyperphosphatemia are encountered in idiopathic hypoparathyroidism (IHP) and pseudohypoparathyroidism type Ib (PHP1B). In contrast to PHP1B, which is caused by resistance toward parathyroid hormone (PTH), the genetic defects leading to IHP impair production of this important regulator of mineral ion homeostasis. So far, only five PTH mutations were shown to cause IHP, each of which is located in the hormone’s pre-pro leader segment and thus impair hormone secretion. In three siblings affected by IHP, we now identified a homozygous arginine-to-cysteine mutation at position 25 (R25C) of the mature PTH(1-84) polypeptide; heterozygous family members are healthy. Depending on the assay used for evaluating these patients, plasma PTH levels were either low or profoundly elevated, thus leading to ambiguities regarding the underlying diagnosis, namely IHP or PHP1B. Consistent with increased PTH levels, recombinant [Cys25]PTH(1-84) and wild-type PTH(1-84) were secreted equally well by transfected COS-7 cells. However, synthetic [Cys25]PTH(1-34) was found to have a lower binding affinity for the PTH receptor type-1 (PTH1R) than PTH(1-34) and consequently a lower efficiency for stimulating cAMP formation in cells expressing this receptor. Consistent with these in vitro findings, long-term infusion of [Cys25]PTH(1-34) resulted only in minimal calcemic and phosphaturic responses, despite readily detectable levels of [Cys25]PTH(1-34) in plasma. The mineral ion abnormalities observed in the three IHP patients are thus most likely caused by the inherited homozygous missense PTH mutation, which reduces bioactivity of the secreted hormone. Based on these findings, screening for PTH(1-84) mutations should be considered when clinical and laboratory findings are consistent with PHP1B, but GNAS methylation changes have been excluded. Differentiating between IHP and PHP1B has considerable implications for genetic counseling, therapy, and long-term outcome because

  9. A central role for vesicle trafficking in epithelial neoplasia: Intracellular highways to carcinogenesis

    PubMed Central

    Goldenring, James R.

    2014-01-01

    Epithelial cell carcinogenesis involves the loss of polarity, alteration of polarized protein presentation, dynamic cell morphology changes, increased proliferation and increased cell motility and invasion. Elements of membrane vesicle trafficking underlie all of these processes. Specific membrane trafficking regulators, including Rab small GTPases, through the coordinated dynamics of intracellular trafficking along cytoskeletal pathways, determine cell surface presentation of proteins and overall function of both differentiated and neoplastic cells. While mutations in vesicle trafficking proteins may not be direct drivers of transformation, elements of the machinery of vesicle movement play critical roles in the phenotypes of neoplastic cells. Therefore, the regulators of membrane vesicle trafficking decisions are critical mediators of the full spectrum of cell physiologies driving cancer cell biology, including initial loss of polarity, invasion and metastasis. Targeting of these fundamental intracellular processes may provide important points for manipulation of cancer cell behaviour. PMID:24108097

  10. A central role for vesicle trafficking in epithelial neoplasia: intracellular highways to carcinogenesis.

    PubMed

    Goldenring, James R

    2013-11-01

    Epithelial cell carcinogenesis involves the loss of cell polarity, alteration of polarized protein presentation, dynamic cell morphology changes, increased proliferation, and increased cell motility and invasion. Membrane vesicle trafficking underlies all of these processes. Specific membrane trafficking regulators, including RAB small GTPases, through the coordinated dynamics of intracellular trafficking along cytoskeletal pathways, determine the cell surface presentation of proteins and the overall function of both differentiated and neoplastic cells. Although mutations in vesicle trafficking proteins may not be direct drivers of transformation, components of the machinery of vesicle movement have crucial roles in the phenotypes of neoplastic cells. Therefore, the regulators of membrane vesicle trafficking decisions are essential mediators of the full range of cell physiologies that drive cancer cell biology, including initial loss of cell polarity, invasion and metastasis. Targeting of these fundamental intracellular processes may permit the manipulation of cancer cell behaviour.

  11. Loss-of-function mutations in IGSF1 cause an X-linked syndrome of central hypothyroidism and testicular enlargement

    PubMed Central

    Sun, Yu; Bak, Beata; Schoenmakers, Nadia; van Trotsenburg, A.S. Paul; Oostdijk, Wilma; Voshol, Peter; Cambridge, Emma; White, Jacqueline K.; le Tissier, Paul; Gharavy, S. Neda Mousavy; Martinez-Barbera, Juan P.; Stokvis-Brantsma, Wilhelmina H.; Vulsma, Thomas; Kempers, Marlies J.; Persani, Luca; Campi, Irene; Bonomi, Marco; Beck-Peccoz, Paolo; Zhu, Hongdong; Davis, Timothy M.E.; Hokken-Koelega, Anita C.S.; Del Blanco, Daria Gorbenko; Rangasami, Jayanti J.; Ruivenkamp, Claudia A.L.; Laros, Jeroen F.J.; Kriek, Marjolein; Kant, Sarina G.; Bosch, Cathy A.J.; Biermasz, Nienke R.; Appelman-Dijkstra, Natasha M.; Corssmit, Eleonora P.; Hovens, Guido C.J.; Pereira, Alberto M.; den Dunnen, Johan T.; Wade, Michael G.; Breuning, Martijn H.; Hennekam, Raoul C.; Chatterjee, Krishna; Dattani, Mehul T.; Wit, Jan M.; Bernard, Daniel J.

    2012-01-01

    Congenital central hypothyroidism occurs either in isolation or in conjunction with other pituitary hormone deficits. Using exome and candidate gene sequencing, we identified eight distinct mutations and two deletions in IGSF1 in males from eleven unrelated families with central hypothyroidism, testicular enlargement, and variably low prolactin concentrations. IGSF1 is a membrane glycoprotein highly expressed in the anterior pituitary gland and the identified mutations impair its trafficking to the cell surface in heterologous cells. Igsf1-deficient male mice show diminished pituitary and serum thyroid-stimulating hormone (TSH) concentrations, reduced pituitary thyrotropin-releasing hormone (TRH) receptor expression, decreased triiodothyronine concentrations, and increased body mass. Collectively, our observations delineate a novel X-linked disorder in which loss-of-function mutations in IGSF1 cause central hypothyroidism, likely secondary to an associated impairment in pituitary TRH signaling. PMID:23143598

  12. Mutations of Factor H Impair Regulation of Surface-bound C3b by Three Mechanisms in Atypical Hemolytic Uremic Syndrome*

    PubMed Central

    Lehtinen, Markus J.; Rops, Angelique L.; Isenman, David E.; van der Vlag, Johan; Jokiranta, T. Sakari

    2009-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy associated with mutations in complement proteins, most frequently in the main plasma alternative pathway regulator factor H (FH). The hotspot for the FH mutations is in domains 19–20 (FH19–20) that are indispensable for FH activity on C3b bound covalently to host cells. In aHUS, down-regulation of cell-bound C3b by FH is impaired, but it is not clear whether this is due to an altered FH binding to surface-bound C3b or to cell surface structures. To explore the molecular pathogenesis of aHUS we tested binding of 14 FH19–20 point mutants to C3b and its C3d fragment, mouse glomerular endothelial cells (mGEnC-1), and heparin. The cell binding correlated well, but not fully, with heparin binding and the cell binding site was overlapping but distinct from the C3b/C3d binding site that was shown to extend to domain 19. Our results show that aHUS-associated FH19–20 mutants have different combinations of three primary defects: impaired binding to C3b/C3d, impaired binding to the mGEnC-1 cells/heparin, and, as a novel observation, an enhanced mGEnC-1 cell or heparin binding. We propose a model of the molecular pathogenesis of aHUS where all three mechanisms lead eventually to impaired control of C3b on the endothelial cell surfaces. Based on the results with the aHUS patient mutants and the overlap in FH19–20 binding sites for mGEnC-1/heparin and C3b/C3d we conclude that binding of FH19–20 to C3b/C3d is essential for target discrimination by the alternative pathway. PMID:19351878

  13. Mitochondrial trafficking in neurons.

    PubMed

    Schwarz, Thomas L

    2013-06-01

    Neurons, perhaps more than any other cell type, depend on mitochondrial trafficking for their survival. Recent studies have elucidated a motor/adaptor complex on the mitochondrial surface that is shared between neurons and other animal cells. In addition to kinesin and dynein, this complex contains the proteins Miro (also called RhoT1/2) and milton (also called TRAK1/2) and is responsible for much, although not necessarily all, mitochondrial movement. Elucidation of the complex has permitted inroads for understanding how this movement is regulated by a variety of intracellular signals, although many mysteries remain. Regulating mitochondrial movement can match energy demand to energy supply throughout the extraordinary architecture of these cells and can control the clearance and replenishing of mitochondria in the periphery. Because the extended axons of neurons contain uniformly polarized microtubules, they have been useful for studying mitochondrial motility in conjunction with biochemical assays in many cell types.

  14. A mutation in the Proteosomal Regulatory Particle AAA-ATPase-3 in Arabidopsis impairs the light-specific hypocotyl elongation response elicited by a glutamate receptor agonist, BMAA.

    PubMed

    Brenner, Eric D; Feinberg, Philip; Runko, Suzan; Coruzzi, Gloria M

    2009-07-01

    BMAA is a cycad-derived glutamate receptor agonist that causes a two- to three-fold increase in hypocotyl elongation on Arabidopsis seedlings grown in the light. To probe the role of plant glutamate receptors and their downstream mediators, we utilized a previously described genetic screen to identify a novel, BMAA insensitive morphology (bim) mutant, bim409. The normal BMAA-induced hypocotyl elongation response observed on wild-type seedlings grown in the light is impaired in the bim409 mutant. This BMAA-induced phenotype is light-specific, as the bim409 mutant exhibits normal hypocotyl elongation in etiolated (dark grown) plants (+ or - BMAA). The mutation in bim409 was identified to be in a gene encoding the Proteosomal Regulatory Particle AAA-ATPase-3 (RPT3). Possible roles of the proteosome in Glu-mediated signaling in plants is discussed.

  15. 31 CFR 536.311 - Narcotics trafficking.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false Narcotics trafficking. 536.311 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING SANCTIONS REGULATIONS General Definitions § 536.311 Narcotics trafficking. The term narcotics trafficking means any activity undertaken...

  16. 31 CFR 536.311 - Narcotics trafficking.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false Narcotics trafficking. 536.311 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING SANCTIONS REGULATIONS General Definitions § 536.311 Narcotics trafficking. The term narcotics trafficking means any activity undertaken...

  17. 31 CFR 536.311 - Narcotics trafficking.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false Narcotics trafficking. 536.311 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING SANCTIONS REGULATIONS General Definitions § 536.311 Narcotics trafficking. The term narcotics trafficking means any activity undertaken...

  18. 31 CFR 536.311 - Narcotics trafficking.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 31 Money and Finance: Treasury 3 2010-07-01 2010-07-01 false Narcotics trafficking. 536.311... OF FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING SANCTIONS REGULATIONS General Definitions § 536.311 Narcotics trafficking. The term narcotics trafficking means any activity...

  19. 31 CFR 536.311 - Narcotics trafficking.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Narcotics trafficking. 536.311 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING SANCTIONS REGULATIONS General Definitions § 536.311 Narcotics trafficking. The term narcotics trafficking means any activity undertaken...

  20. Hypertension-causing Mutations in Cullin3 Protein Impair RhoA Protein Ubiquitination and Augment the Association with Substrate Adaptors*

    PubMed Central

    Ibeawuchi, Stella-Rita C.; Agbor, Larry N.; Quelle, Frederick W.; Sigmund, Curt D.

    2015-01-01

    Cullin-Ring ubiquitin ligases regulate protein turnover by promoting the ubiquitination of substrate proteins, targeting them for proteasomal degradation. It has been shown previously that mutations in Cullin3 (Cul3) causing deletion of 57 amino acids encoded by exon 9 (Cul3Δ9) cause hypertension. Moreover, RhoA activity contributes to vascular constriction and hypertension. We show that ubiquitination and degradation of RhoA is dependent on Cul3 in HEK293T cells in which Cul3 expression is ablated by either siRNA or by CRISPR-Cas9 genome editing. The latter was used to generate a Cul3-null cell line (HEK293TCul3KO). When expressed in these cells, Cul3Δ9 supported reduced ubiquitin ligase activity toward RhoA compared with equivalent levels of wild-type Cul3 (Cul3WT). Consistent with its reduced activity, binding of Cul3Δ9 to the E3 ubiquitin ligase Rbx1 and neddylation of Cul3Δ9 were impaired significantly compared with Cul3WT. Conversely, Cul3Δ9 bound to substrate adaptor proteins more efficiently than Cul3WT. Cul3Δ9 also forms unstable dimers with Cul3WT, disrupting dimers of Cul3WT complexes that are required for efficient ubiquitination of some substrates. Indeed, coexpression of Cul3WT and Cul3Δ9 in HEK293TCul3KO cells resulted in a decrease in the active form of Cul3WT. We conclude that Cul3Δ9-associated ubiquitin ligase activity toward RhoA is impaired and suggest that Cul3Δ9 mutations may act dominantly by sequestering substrate adaptors and disrupting Cul3WT complexes. PMID:26100637

  1. A point mutation in the human Slo1 channel that impairs its sensitivity to omega-3 docosahexaenoic acid.

    PubMed

    Hoshi, Toshinori; Xu, Rong; Hou, Shangwei; Heinemann, Stefan H; Tian, Yutao

    2013-11-01

    Long-chain polyunsaturated omega-3 fatty acids such as docosahexaenoic acid (DHA) at nanomolar concentrations reversibly activate human large-conductance Ca(2+)- and voltage-gated K(+) (Slo1 BK) channels containing auxiliary β1 or β4 subunits in cell-free patches. Here we examined the action of DHA on the Slo1 channel without any auxiliary subunit and sought to elucidate the biophysical mechanism and the molecular determinants of the DHA sensitivity. Measurements of ionic currents through human Slo1 (hSlo1) channels reveal that the stimulatory effect of DHA does not require activation of the voltage or Ca(2+) sensors. Unlike gating of the hSlo1 channel, that of the Drosophila melanogaster Slo1 (dSlo1) channel is unaltered by DHA. Our mutagenesis study based on the differential responses of human and dSlo1 channels to DHA pinpoints that Y318 near the cytoplasmic end of S6 in the hSlo1 channel is a critical determinant of the stimulatory action of DHA. The mutation Y318S in hSlo1, which replaces Y with S as found in dSlo1, greatly diminishes the channel's response to DHA with a 22-carbon chain whether β1 or β4 is absent or present. However, the responses to α-linolenic acid, an omegea-3 fatty acid with an 18-carbon chain, and to arachidonic acid, an omega-6 fatty acid with a 20-carbon chain, remain unaffected by the mutation. Y318 in the S6 segment of hSlo1 is thus an important determinant of the electrophysiological response of the channel to DHA. Furthermore, the mutation Y318S may prove to be useful in dissecting out the complex lipid-mediated modulation of Slo1 BK channels.

  2. A point mutation in the human Slo1 channel that impairs its sensitivity to omega-3 docosahexaenoic acid

    PubMed Central

    Xu, Rong; Hou, Shangwei; Heinemann, Stefan H.; Tian, Yutao

    2013-01-01

    Long-chain polyunsaturated omega-3 fatty acids such as docosahexaenoic acid (DHA) at nanomolar concentrations reversibly activate human large-conductance Ca2+- and voltage-gated K+ (Slo1 BK) channels containing auxiliary β1 or β4 subunits in cell-free patches. Here we examined the action of DHA on the Slo1 channel without any auxiliary subunit and sought to elucidate the biophysical mechanism and the molecular determinants of the DHA sensitivity. Measurements of ionic currents through human Slo1 (hSlo1) channels reveal that the stimulatory effect of DHA does not require activation of the voltage or Ca2+ sensors. Unlike gating of the hSlo1 channel, that of the Drosophila melanogaster Slo1 (dSlo1) channel is unaltered by DHA. Our mutagenesis study based on the differential responses of human and dSlo1 channels to DHA pinpoints that Y318 near the cytoplasmic end of S6 in the hSlo1 channel is a critical determinant of the stimulatory action of DHA. The mutation Y318S in hSlo1, which replaces Y with S as found in dSlo1, greatly diminishes the channel’s response to DHA with a 22-carbon chain whether β1 or β4 is absent or present. However, the responses to α-linolenic acid, an omegea-3 fatty acid with an 18-carbon chain, and to arachidonic acid, an omega-6 fatty acid with a 20-carbon chain, remain unaffected by the mutation. Y318 in the S6 segment of hSlo1 is thus an important determinant of the electrophysiological response of the channel to DHA. Furthermore, the mutation Y318S may prove to be useful in dissecting out the complex lipid-mediated modulation of Slo1 BK channels. PMID:24127525

  3. Impaired secretion of carboxyl-terminal truncated factor VII due to an F7 nonsense mutation associated with FVII deficiency.

    PubMed

    Tanaka, Ryoko; Nakashima, Daisuke; Suzuki, Atsuo; Miyawaki, Yuhri; Fujimori, Yuta; Yamada, Takayuki; Takagi, Akira; Murate, Takashi; Yamamoto, Koji; Katsumi, Akira; Matsushita, Tadashi; Naoe, Tomoki; Kojima, Tetsuhito

    2010-03-01

    Factor VII (FVII) is a vitamin K-dependent glycoprotein secreted into the blood circulation from hepatic cells. We investigated the molecular basis of the congenital FVII deficiency found in a Japanese patient. We analyzed the F7 gene of the patient, who was diagnosed with a FVII deficiency at pregnancy. We expressed a carboxyl-terminal truncated FVII (Arg462X FVII) corresponding to the identified mutation in CHO-K1 cells. To study roles of the carboxyl-terminus in the secretion of FVII, we also expressed a series of recombinant FVIIs deleted of limited numbers of carboxyl-terminal amino acids (462Arg-466Pro). We identified a nonsense mutation (c.1384C>T: p.Arg462X) in F7, leading to a lack of five amino acids in the carboxyl-terminus. In expression experiments, Arg462X FVII was undetectable not only by Western blotting, but also by ELISA. A Western blot analysis of the truncated FVIIs revealed that all mutants were expressed in the cells the same as the wild type, but were secreted into the culture medium in lesser amounts than the wild type depending on the length of the deletion, which was confirmed by ELISA. Arg462X FVII did not colocalize with the Golgi on immunofluorescence staining, suggesting that it might be retained in the ER and degraded in the cell. The carboxyl-terminal amino acids of FVII play an important role in its secretion, and the p.Arg462X mutation was likely to have caused the FVII deficiency in this patient. (c) 2009 Elsevier Ltd. All rights reserved.

  4. Mutation E169K in junctophilin-2 causes atrial fibrillation due to impaired RyR2 stabilization

    PubMed Central

    Voigt, Niels; Garbino, Alejandro; Dixit, Sayali S.; Landstrom, Andrew P.; Li, Na; Wang, Qiongling; Olivotto, Iacopo; Dobrev, Dobromir; Ackerman, Michael J.; Wehrens, Xander H.T.

    2013-01-01

    Objectives To study the role of junctophilin 2 (JPH2) in atrial fibrillation (AF). Background JPH2 is believed to have an important role in sarcoplasmic reticulum (SR) Ca2+ handling and modulation of ryanodine receptor Ca2+ channels (RyR2). Whereas defective RyR2-mediated Ca2+ release contributes to the pathogenesis of AF, nothing is known about the potential role of JPH2 in atrial arrhythmias. Methods Screening 203 unrelated hypertrophic cardiomyopathy patients uncovered a novel JPH2 missense mutation (E169K) in 2 patients with juvenile-onset paroxysmal AF (pAF). Pseudo-knockin (PKI) mouse models were generated to determine the molecular defects underlying the development of AF caused by this JPH2 mutation. Results PKI mice expressing E169K mutant JPH2 exhibited a higher incidence of inducible AF compared with wildtype (WT)-PKI mice, while A399S-PKI mice expressing a HCM-linked JPH2 mutation not associated with atrial arrhythmias were not significantly different from WT-PKI. E169K-PKI but not A399A-PKI atrial cardiomyocytes showed an increased incidence of abnormal SR Ca2+ release events. These changes were attributed to reduced binding of E169KJPH2 to RyR2. Atrial JPH2 levels in WT-JPH2 transgenic, nontransgenic, and JPH2 knockdown mice correlated negatively with the incidence of pacing-induced AF. Ca2+ spark frequency in atrial myocytes and the open probability of single RyR2 channels from JPH2 knockdown mice was significantly reduced by a small JPH2-mimicking oligopeptide. Moreover, patients with pAF had reduced atrial JPH2 levels per RyR2 channel compared to sinus rhythm patients, and an increased frequency of spontaneous Ca2+ release events. Conclusions Our data suggest a novel mechanism by which reduced JPH2-mediated stabilization of RyR2 due to loss-of-function mutation or reduced JPH2:RyR2 ratios can promote SR Ca2+ leak and atrial arrhythmias, representing a potential novel therapeutic target for AF. PMID:23973696

  5. The Parkinson's Disease-Associated Mutation LRRK2-G2019S Impairs Synaptic Plasticity in Mouse Hippocampus

    PubMed Central

    Sweet, Eric S.; Saunier-Rebori, Bernadette

    2015-01-01

    Parkinson's disease (PD) is a major movement disorder characterized by the loss of dopamine neurons and formation of Lewy bodies. Clinical and pathological evidence indicates that multiple brain regions are affected in PD in a spatiotemporal manner and are associated with a variety of motor and nonmotor symptoms, including disturbances in mood, executive function, and memory. The common PD-associated gene for leucine-rich repeat kinase, leucine-rich repeat kinase 2 (LRRK2), is highly expressed in brain regions that are involved with nonmotor functions, including the neocortex and hippocampus, but whether mutant LRRK2 contributes to neuronal dysfunction in these regions is unknown. Here, we use bacterial artificial chromosome transgenic mouse models of LRRK2 to explore potential nonmotor mechanisms of PD. Through electrophysiological analysis of the Schaffer collateral–CA1 synapse in dorsal hippocampus, we find that overexpression of LRRK2-G2019S increases basal synaptic efficiency through a postsynaptic mechanism, and disrupts long-term depression. Furthermore, these effects of the G2019S mutation are age dependent and can be normalized by acute inhibition of LRRK2 kinase activity. In contrast, overexpression of wild-type LRRK2 has no effect under the same conditions, suggesting a specific phenotype for the G2019S mutation. These results identify a pathogenic function of LRRK2 in the hippocampus that may contribute to nonmotor symptoms of PD. SIGNIFICANCE STATEMENT Parkinson's disease (PD) is among the most common neurological diseases and is best known for its adverse effects on brain regions that control motor function, resulting in tremor, rigidity, and gait abnormalities. Less well appreciated are the psychiatric symptoms experienced by many PD patients, including depression and memory loss, which do not respond well to currently available treatments for PD. Here, we describe functional effects of a common PD-linked mutation of leucine-rich repeat kinase 2 in

  6. A novel ADIPOQ mutation (p.M40K) impairs assembly of high-molecular-weight adiponectin and is associated with early-onset obesity and metabolic syndrome.

    PubMed

    Bueno, Ana Carolina; Sun, Kai; Martins, Clarissa Silva; Elias Junior, Jorge; Miranda, Wallace; Tao, Caroline; Foss-Freitas, Maria Cristina; Barbieri, Marco Antonio; Bettiol, Heloísa; de Castro, Margaret; Scherer, Philipp E; Antonini, Sonir R

    2014-04-01

    affected family members presented features of metabolic syndrome. The newly identified ADIPOQ p.M40K mutation associates with severe cardiometabolic dysfunction due to the impairment of high-molecular weight adiponectin complex formation.

  7. Modulation of dendritic spine development and plasticity by BDNF and vesicular trafficking: fundamental roles in neurodevelopmental disorders associated with mental retardation and autism.

    PubMed

    Chapleau, Christopher A; Larimore, Jennifer L; Theibert, Anne; Pozzo-Miller, Lucas

    2009-09-01

    The process of axonal and dendritic development establishes the synaptic circuitry of the central nervous system (CNS) and is the result of interactions between intrinsic molecular factors and the external environment. One growth factor that has a compelling function in neuronal development is the neurotrophin brain-derived neurotrophic factor (BDNF). BDNF participates in axonal and dendritic differentiation during embryonic stages of neuronal development, as well as in the formation and maturation of dendritic spines during postnatal development. Recent studies have also implicated vesicular trafficking of BDNF via secretory vesicles, and both secretory and endosomal trafficking of vesicles containing synaptic proteins, such as neurotransmitter and neurotrophin receptors, in the regulation of axonal and dendritic differentiation, and in dendritic spine morphogenesis. Several genes that are either mutated or deregulated in neurodevelopmental disorders associated with mental retardation have now been identified, and several mouse models of these disorders have been generated and characterized. Interestingly, abnormalities in dendritic and synaptic structure are consistently observed in human neurodevelopmental disorders associated with mental retardation, and in mouse models of these disorders as well. Abnormalities in dendritic and synaptic differentiation are thought to underlie altered synaptic function and network connectivity, thus contributing to the clinical outcome. Here, we review the roles of BDNF and vesicular trafficking in axonal and dendritic differentiation in the context of dendritic and axonal morphological impairments commonly observed in neurodevelopmental disorders associated with mental retardation.

  8. The V499G/Y501H mutation impairs fast motor kinetics of prestin and has significan