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Sample records for differentiated neuronal cells

  1. Nrf2 promotes neuronal cell differentiation

    PubMed Central

    Zhao, Fei; Wu, Tongde; Lau, Alexandria; Jiang, Tao; Huang, Zheping; Wang, Xiao-Jun; Chen, Weimin; Wong, Pak Kin; Zhang, Donna D.

    2009-01-01

    The transcription factor Nrf2 has emerged as a master regulator for the endogenous antioxidant response, which is critical in defending cells against environmental insults and in maintaining intracellular redox balance. However, whether Nrf2 has any role in neuronal cell differentiation is largely unknown. In this report, we have examined the effects of Nrf2 on cell differentiation using a neuroblastoma cell line, SH-SY5Y. Retinoic acid (RA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), two well-studied inducers for neuronal differentiation, are able to induce Nrf2 and its target gene NAD(P)H quinone oxidoreductase 1 (NQO1) in a dose- and time- dependent manner. RA-induced Nrf2 up-regulation is accompanied by neurite outgrowth and an induction of two neuronal differentiation markers, neurofilament-M (NF-M) and microtubule-associated protein 2 (MAP-2). Overexpression of Nrf2 in SH-SY5Y cells promotes neuronal differentiation whereas inhibition of endogenous Nrf2 expression inhibited neuronal differentiation. More remarkably, the positive role of Nrf2 in neuronal differentiation was verified ex vivo in primary neuron culture. Primary neurons isolated from Nrf2-null mice showed a retarded progress in differentiation, compared to that from wild-type mice. Collectively, our data demonstrate a novel role for Nrf2 in promoting neuronal cell differentiation, which will open new perspectives for therapeutic uses of Nrf2 activators in patients with neurodegenerative diseases. PMID:19573594

  2. Phenazopyridine induces and synchronizes neuronal differentiation of embryonic stem cells.

    PubMed

    Suter, David M; Preynat-Seauve, Olivier; Tirefort, Diderik; Feki, Anis; Krause, Karl-Heinz

    2009-09-01

    Embryonic stem (ES) cells are powerful tools to understand mechanisms of neuronal differentiation and to engineer neurons for in vitro studies and cell therapy. We developed a screening approach to identify small organic molecules driving neuronal differentiation of ES cells. For this purpose, we used a lentivector carrying a dual luciferase reporter system to engineer an ES cell line which allowed us to screen for small organic molecules enhancing neuronal differentiation. One of them, phenazopyridine, was further analysed in human ES cells. Phenazopyridine: (i) enhanced neuronal differentiation, (ii) increased cell survival, (iii) decreased the amount of non-neuronal and undifferentiated cells and (iv) synchronized the cellular differentiation state. Phenazopyridine allowed the development of a differentiation protocol compatible with the generation of clinical grade neural precursors, which were able differentiate into different neuronal subtypes, astrocytes and oligodendrocytes. In summary, we describe a powerful approach to identify small molecules directing stem cell differentiation. This led to the establishment of a new application for an old drug and the development of a novel clinical grade protocol for neuronal differentiation of ES cells.

  3. Sambucus williamsii induced embryonic stem cells differentiated into neurons.

    PubMed

    Liu, Shih-Ping; Hsu, Chien-Yu; Fu, Ru-Huei; Huang, Yu-Chuen; Chen, Shih-Yin; Lin, Shinn-Zong; Shyu, Woei-Cherng

    2015-01-01

    The pluripotent stem cells, including embryonic stem cells (ESCs), are capable of self-renewal and differentiation into any cell type, thus making them the focus of many clinical application studies. However, the efficiency of ESCs differentiated into neurons needs to improve. In this study, we tried to increase efficiently to a neural fate in the presence of various transitional Chinese medicines through a three-step differentiation strategy. From extracts of 10 transitional Chinese medicine candidates, we determined that Sambucus williamsii (SW) extract triggers the up-regulation of Nestin and Tuj1 (neuron cells markers) gene expression levels. After determining the different concentrations of SW extract, the number of neurons in the 200 μg/ml SW extract group was higher than the control, 50, 100, and 400 μg/ml SW extract groups. In addition, the number of neurons in the 200 μg/ml SW extract group was higher and higher after each time passage (three times). We also detected the Oct4, Sox2 (stem cells markers), Tuj1, and Nestin genes expression levels by RT-PCR. In the differentiated process, Oct4 and Sox2 genes decreased while the Tuj1 and Nestin genes expression levels increased. In summary, we demonstrated that SW could induce pluripotent stem cells differentiated into neurons. Thus, SW might become a powerful material for neurons-differentiating strategies.

  4. Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds

    NASA Astrophysics Data System (ADS)

    Hsu, Tzu-Chia; Liu, Kuang-Kai; Chang, Huan-Cheng; Hwang, Eric; Chao, Jui-I.

    2014-05-01

    Nanodiamond is a promising carbon nanomaterial developed for biomedical applications. Here, we show fluorescent nanodiamond (FND) with the biocompatible properties that can be used for the labeling and tracking of neuronal differentiation and neuron cells derived from embryonal carcinoma stem (ECS) cells. The fluorescence intensities of FNDs were increased by treatment with FNDs in both the mouse P19 and human NT2/D1 ECS cells. FNDs were taken into ECS cells; however, FNDs did not alter the cellular morphology and growth ability. Moreover, FNDs did not change the protein expression of stem cell marker SSEA-1 of ECS cells. The neuronal differentiation of ECS cells could be induced by retinoic acid (RA). Interestingly, FNDs did not affect on the morphological alteration, cytotoxicity and apoptosis during the neuronal differentiation. Besides, FNDs did not alter the cell viability and the expression of neuron-specific marker β-III-tubulin in these differentiated neuron cells. The existence of FNDs in the neuron cells can be identified by confocal microscopy and flow cytometry. Together, FND is a biocompatible and readily detectable nanomaterial for the labeling and tracking of neuronal differentiation process and neuron cells from stem cells.

  5. Integrating human stem cell expansion and neuronal differentiation in bioreactors

    PubMed Central

    Serra, Margarida; Brito, Catarina; Costa, Eunice M; Sousa, Marcos FQ; Alves, Paula M

    2009-01-01

    Background Human stem cells are cellular resources with outstanding potential for cell therapy. However, for the fulfillment of this application, major challenges remain to be met. Of paramount importance is the development of robust systems for in vitro stem cell expansion and differentiation. In this work, we successfully developed an efficient scalable bioprocess for the fast production of human neurons. Results The expansion of undifferentiated human embryonal carcinoma stem cells (NTera2/cl.D1 cell line) as 3D-aggregates was firstly optimized in spinner vessel. The media exchange operation mode with an inoculum concentration of 4 × 105 cell/mL was the most efficient strategy tested, with a 4.6-fold increase in cell concentration achieved in 5 days. These results were validated in a bioreactor where similar profile and metabolic performance were obtained. Furthermore, characterization of the expanded population by immunofluorescence microscopy and flow cytometry showed that NT2 cells maintained their stem cell characteristics along the bioreactor culture time. Finally, the neuronal differentiation step was integrated in the bioreactor process, by addition of retinoic acid when cells were in the middle of the exponential phase. Neurosphere composition was monitored and neuronal differentiation efficiency evaluated along the culture time. The results show that, for bioreactor cultures, we were able to increase significantly the neuronal differentiation efficiency by 10-fold while reducing drastically, by 30%, the time required for the differentiation process. Conclusion The culture systems developed herein are robust and represent one-step-forward towards the development of integrated bioprocesses, bridging stem cell expansion and differentiation in fully controlled bioreactors. PMID:19772662

  6. Mouse bone marrow stromal cells differentiate to neuron-like cells upon inhibition of BMP signaling.

    PubMed

    Saxena, Monika; Prashar, Paritosh; Yadav, Prem Swaroop; Sen, Jonaki

    2016-01-01

    Bone marrow stromal cells (BMSCs) are a source of autologous stem cells that have the potential for undergoing differentiation into multiple cell types including neurons. Although the neuronal differentiation of mesenchymal stem cells has been studied for a long time, the molecular players involved are still not defined. Here we report that the genetic deletion of two members of the bone morphogenetic protein (Bmp) family, Bmp2 and Bmp4 in mouse BMSCs causes their differentiation into cells with neuron-like morphology. Surprisingly these cells expressed certain markers characteristic of both neuronal and glial cells. Based on this observation, we inhibited BMP signaling in mouse BMSCs through a brief exposure to Noggin protein which also led to their differentiation into cells expressing both neuronal and glial markers. Such cells seem to have the potential for further differentiation into subtypes of neuronal and glial cells and thus could be utilized for cell-based therapeutic applications.

  7. Hypergravity Stimulation Enhances PC12 Neuron-Like Cell Differentiation

    PubMed Central

    2015-01-01

    Altered gravity is a strong physical cue able to elicit different cellular responses, representing a largely uninvestigated opportunity for tissue engineering/regenerative medicine applications. Our recent studies have shown that both proliferation and differentiation of C2C12 skeletal muscle cells can be enhanced by hypergravity treatment; given these results, PC12 neuron-like cells were chosen to test the hypothesis that hypergravity stimulation might also affect the behavior of neuronal cells, in particular promoting an enhanced differentiated phenotype. PC12 cells were thus cultured under differentiating conditions for either 12 h or 72 h before being stimulated with different values of hypergravity (50 g and 150 g). Effects of hypergravity were evaluated at transcriptional level 1 h and 48 h after the stimulation, and at protein level 48 h from hypergravity exposure, to assess its influence on neurite development over increasing differentiation times. PC12 differentiation resulted strongly affected by the hypergravity treatments; in particular, neurite length was significantly enhanced after exposure to high acceleration values. The achieved results suggest that hypergravity might induce a faster and higher neuronal differentiation and encourage further investigations on the potential of hypergravity in the preparation of cellular constructs for regenerative medicine and tissue engineering purposes. PMID:25785273

  8. Hypergravity stimulation enhances PC12 neuron-like cell differentiation.

    PubMed

    Genchi, Giada Graziana; Cialdai, Francesca; Monici, Monica; Mazzolai, Barbara; Mattoli, Virgilio; Ciofani, Gianni

    2015-01-01

    Altered gravity is a strong physical cue able to elicit different cellular responses, representing a largely uninvestigated opportunity for tissue engineering/regenerative medicine applications. Our recent studies have shown that both proliferation and differentiation of C2C12 skeletal muscle cells can be enhanced by hypergravity treatment; given these results, PC12 neuron-like cells were chosen to test the hypothesis that hypergravity stimulation might also affect the behavior of neuronal cells, in particular promoting an enhanced differentiated phenotype. PC12 cells were thus cultured under differentiating conditions for either 12 h or 72 h before being stimulated with different values of hypergravity (50 g and 150 g). Effects of hypergravity were evaluated at transcriptional level 1 h and 48 h after the stimulation, and at protein level 48 h from hypergravity exposure, to assess its influence on neurite development over increasing differentiation times. PC12 differentiation resulted strongly affected by the hypergravity treatments; in particular, neurite length was significantly enhanced after exposure to high acceleration values. The achieved results suggest that hypergravity might induce a faster and higher neuronal differentiation and encourage further investigations on the potential of hypergravity in the preparation of cellular constructs for regenerative medicine and tissue engineering purposes.

  9. Vascular Mural Cells Promote Noradrenergic Differentiation of Embryonic Sympathetic Neurons.

    PubMed

    Fortuna, Vitor; Pardanaud, Luc; Brunet, Isabelle; Ola, Roxana; Ristori, Emma; Santoro, Massimo M; Nicoli, Stefania; Eichmann, Anne

    2015-06-23

    The sympathetic nervous system controls smooth muscle tone and heart rate in the cardiovascular system. Postganglionic sympathetic neurons (SNs) develop in close proximity to the dorsal aorta (DA) and innervate visceral smooth muscle targets. Here, we use the zebrafish embryo to ask whether the DA is required for SN development. We show that noradrenergic (NA) differentiation of SN precursors temporally coincides with vascular mural cell (VMC) recruitment to the DA and vascular maturation. Blocking vascular maturation inhibits VMC recruitment and blocks NA differentiation of SN precursors. Inhibition of platelet-derived growth factor receptor (PDGFR) signaling prevents VMC differentiation and also blocks NA differentiation of SN precursors. NA differentiation is normal in cloche mutants that are devoid of endothelial cells but have VMCs. Thus, PDGFR-mediated mural cell recruitment mediates neurovascular interactions between the aorta and sympathetic precursors and promotes their noradrenergic differentiation.

  10. Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons

    SciTech Connect

    Park, Kyoung Ho; Yeo, Sang Won; Troy, Frederic A.

    2014-10-17

    Highlights: • PolySia expressed on neurons primarily during early stages of neuronal development. • PolySia–NCAM is expressed on neural stem cells from adult guinea pig spiral ganglion. • PolySia is a biomarker that modulates neuronal differentiation in inner ear stem cells. - Abstract: During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia–NCAMs) modulate cell–cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia–NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb’s to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell–cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders.

  11. Immediate differentiation of neuronal cells from stem/progenitor-like cells in the avian iris tissues.

    PubMed

    Matsushita, Tamami; Fujihara, Ai; Royall, Lars; Kagiwada, Satoshi; Kosaka, Mitsuko; Araki, Masasuke

    2014-06-01

    A simple culture method that was recently developed in our laboratory was applied to the chick iris tissues to characterize neural stem/progenitor-like cells. Iris tissue is a non-neuronal tissue and does not contain any neuronal cells. In the present study we isolated iris tissues from chick embryos just prior to hatching. The isolated iris pigmented epithelium (IPE) or the stroma was embedded in Matrigel and cultured in Dulbecco's MEM supplemented with either fetal bovine serum or the synthetic serum replacement solution B27. Within 24 h of culture, elongated cells with long processes extended out from the explants of both tissues and were positively stained for various neuronal markers such as transitin, Tuj-1 and acetylated tubulin. After a longer culture period, cells positive for photoreceptor markers like rhodopsin, iodopsin and visinin were found, suggesting that the iris tissues contain retinal stem/progenitor-like cells. Several growth factors were examined to determine their effects on neuronal differentiation. EGF was shown to dramatically enhance neuronal cell differentiation, particularly the elongation of neuronal fibers. The addition of exogenous FGF2, however, did not show any positive effects on neuronal differentiation, although FGF signaling inhibitor, SU5402, suppressed neuronal differentiation. The results show that neuronal stem/progenitor-like cells can differentiate into neuronal cells immediately after they are transferred into an appropriate environment. This process did not require any exogenous factors, suggesting that neural stem/progenitor-like cells are simply suppressed from neuronal differentiation within the tissue, and isolation from the tissue releases the cells from the suppression mechanism.

  12. Neuronal expression of pathological tau accelerates oligodendrocyte progenitor cell differentiation.

    PubMed

    Ossola, Bernardino; Zhao, Chao; Compston, Alastair; Pluchino, Stefano; Franklin, Robin J M; Spillantini, Maria Grazia

    2016-03-01

    Oligodendrocyte progenitor cell (OPC) differentiation is an important therapeutic target to promote remyelination in multiple sclerosis (MS). We previously reported hyperphosphorylated and aggregated microtubule-associated protein tau in MS lesions, suggesting its involvement in axonal degeneration. However, the influence of pathological tau-induced axonal damage on the potential for remyelination is unknown. Therefore, we investigated OPC differentiation in human P301S tau (P301S-htau) transgenic mice, both in vitro and in vivo following focal demyelination. In 2-month-old P301S-htau mice, which show hyperphosphorylated tau in neurons, we found atrophic axons in the spinal cord in the absence of prominent axonal degeneration. These signs of early axonal damage were associated with microgliosis and an upregulation of IL-1β and TNFα. Following in vivo focal white matter demyelination we found that OPCs differentiated more efficiently in P301S-htau mice than wild type (Wt) mice. We also found an increased level of myelin basic protein within the lesions, which however did not translate into increased remyelination due to higher susceptibility of P301S-htau axons to demyelination-induced degeneration compared to Wt axons. In vitro experiments confirmed higher differentiation capacity of OPCs from P301S-htau mice compared with Wt mice-derived OPCs. Because the OPCs from P301S-htau mice do not ectopically express the transgene, and when isolated from newborn mice behave like Wt mice-derived OPCs, we infer that their enhanced differentiation capacity must have been acquired through microenvironmental priming. Our data suggest the intriguing concept that damaged axons may signal to OPCs and promote their differentiation in the attempt at rescue by remyelination.

  13. Neuronal expression of pathological tau accelerates oligodendrocyte progenitor cell differentiation.

    PubMed

    Ossola, Bernardino; Zhao, Chao; Compston, Alastair; Pluchino, Stefano; Franklin, Robin J M; Spillantini, Maria Grazia

    2016-03-01

    Oligodendrocyte progenitor cell (OPC) differentiation is an important therapeutic target to promote remyelination in multiple sclerosis (MS). We previously reported hyperphosphorylated and aggregated microtubule-associated protein tau in MS lesions, suggesting its involvement in axonal degeneration. However, the influence of pathological tau-induced axonal damage on the potential for remyelination is unknown. Therefore, we investigated OPC differentiation in human P301S tau (P301S-htau) transgenic mice, both in vitro and in vivo following focal demyelination. In 2-month-old P301S-htau mice, which show hyperphosphorylated tau in neurons, we found atrophic axons in the spinal cord in the absence of prominent axonal degeneration. These signs of early axonal damage were associated with microgliosis and an upregulation of IL-1β and TNFα. Following in vivo focal white matter demyelination we found that OPCs differentiated more efficiently in P301S-htau mice than wild type (Wt) mice. We also found an increased level of myelin basic protein within the lesions, which however did not translate into increased remyelination due to higher susceptibility of P301S-htau axons to demyelination-induced degeneration compared to Wt axons. In vitro experiments confirmed higher differentiation capacity of OPCs from P301S-htau mice compared with Wt mice-derived OPCs. Because the OPCs from P301S-htau mice do not ectopically express the transgene, and when isolated from newborn mice behave like Wt mice-derived OPCs, we infer that their enhanced differentiation capacity must have been acquired through microenvironmental priming. Our data suggest the intriguing concept that damaged axons may signal to OPCs and promote their differentiation in the attempt at rescue by remyelination. PMID:26576485

  14. cGMP modulates stem cells differentiation to neurons in brain in vivo.

    PubMed

    Gómez-Pinedo, U; Rodrigo, R; Cauli, O; Herraiz, S; Garcia-Verdugo, J-M; Pellicer, B; Pellicer, A; Felipo, V

    2010-02-17

    During brain development neural stem cells may differentiate to neurons or to other cell types. The aim of this work was to assess the role of cGMP (cyclic GMP) in the modulation of differentiation of neural stem cells to neurons or non-neuronal cells. cGMP in brain of fetuses was reduced to 46% of controls by treating pregnant rats with nitroarginine-methylester (L-NAME) and was restored by co-treatment with sildenafil.Reducing cGMP during brain development leads to reduced differentiation of stem cells to neurons and increased differentiation to non-neuronal cells. The number of neurons in the prefrontal cortex originated from stem cells proliferating on gestational day 14 was 715+/-14/mm(2) in control rats and was reduced to 440+/-29/mm(2) (61% of control) in rats treated with L-NAME. In rats exposed to L-NAME plus sildenafil, differentiation to neurons was completely normalized, reaching 683+/-11 neurons/mm(2). In rats exposed to sildenafil alone the number of cells labelled with bromodeoxyuridine (BrdU) and NeuN was 841+/-16/mm(2). In prefrontal cortex of control rats 48% of the neural stem cells proliferating in gestational day 14 differentiate to neurons, but only 24% in rats exposed to L-NAME. This was corrected by sildenafil, 40% of cells differentiate to neurons. Similar results were obtained for neurons proliferating during all developmental period. Treatment with L-NAME did not reduce the total number of cells labelled with BrdU, further supporting that L-NAME reduces selectively the differentiation of stem cells to neurons. Similar results were obtained in hippocampus. Treatment with L-NAME reduced the differentiation of neural stem cells to neurons, although the effect was milder than in prefrontal cortex. These results support that cGMP modulates the fate of neural stem cells in brain in vivo and suggest that high cGMP levels promote its differentiation to neurons while reduced cGMP levels promote differentiation to non-neuronal cells.

  15. A high throughput embryonic stem cell screen identifies Oct-2 as a bifunctional regulator of neuronal differentiation

    PubMed Central

    Theodorou, Elias; Dalembert, George; Heffelfinger, Christopher; White, Eric; Weissman, Sherman; Corcoran, Lynn; Snyder, Michael

    2009-01-01

    Neuronal differentiation is a complex process that involves a plethora of regulatory steps. To identify transcription factors that influence neuronal differentiation we developed a high throughput screen using embryonic stem (ES) cells. Seven-hundred human transcription factor clones were stably introduced into mouse ES (mES) cells and screened for their ability to induce neuronal differentiation of mES cells. Twenty-four factors that are capable of inducing neuronal differentiation were identified, including four known effectors of neuronal differentiation, 11 factors with limited evidence of involvement in regulating neuronal differentiation, and nine novel factors. One transcription factor, Oct-2, was studied in detail and found to be a bifunctional regulator: It can either repress or induce neuronal differentiation, depending on the particular isoform. Ectopic expression experiments demonstrate that isoform Oct-2.4 represses neuronal differentiation, whereas Oct-2.2 activates neuron formation. Consistent with a role in neuronal differentiation, Oct-2.2 expression is induced during differentiation, and cells depleted of Oct-2 and its homolog Oct-1 have a reduced capacity to differentiate into neurons. Our results reveal a number of transcription factors potentially important for mammalian neuronal differentiation, and indicate that Oct-2 may serve as a binary switch to repress differentiation in precursor cells and induce neuronal differentiation later during neuronal development. PMID:19270158

  16. Bone morphogenetic protein 4 stimulates neuronal differentiation of neuronal stem cells through the ERK pathway

    PubMed Central

    Moon, Byoung-San; Yoon, Ju-Yong; Kim, Mi-Yeon; Lee, Sang-Hun; Choi, Thomas

    2009-01-01

    Bone morphogenic protein 4 (BMP4), a member of the TGF-β superfamily, induced neural differentiation of neural stem cells (NSCs) grown in a medium containing basic fibroblast growth factor (bFGF). The Ras protein level and the activities of the downstream ERKs were increased by transfection of BMP4 or treatment with recombinant BMP4. The effects of BMP4, including activation of the Ras-ERK pathway and induction of the neuron marker β-tubulin type III (Tuj1), were blocked by co-treatment of the BMP4 antagonist, noggin. The roles of the Ras-ERK pathway in neuronal differentiation by BMP4 were revealed by measuring the effect of the ERK pathway inhibition by dominant negative Ras or PD98059, the MEK specific inhibitor. BMP4 is a transcriptional target of Wnt/β-catenin signaling, and both the mRNA and protein levels of BMP4 were increased by treatment of valproic acid (VPA), a chemical inhibitor of glycogen synthase kinase 3β (GSK3β) activating the Wnt/β-catenin pathway. The BMP4-mimicking effects of VPA, activation of the Ras-ERK pathway and induction of Tuj1, also were blocked by noggin. These results indicate the potential therapeutic usage of VPA as a replacement for BMP4. PMID:19287192

  17. Efficient neuronal differentiation and maturation of human pluripotent stem cells encapsulated in 3D microfibrous scaffolds.

    PubMed

    Lu, Hong Fang; Lim, Sze-Xian; Leong, Meng Fatt; Narayanan, Karthikeyan; Toh, Rebecca P K; Gao, Shujun; Wan, Andrew C A

    2012-12-01

    Developing an efficient culture system for controlled human pluripotent stem cell (hPSC) differentiation into selected lineages is a major challenge in realizing stem cell-based clinical applications. Here, we report the use of chitin-alginate 3D microfibrous scaffolds, previously developed for hPSC propagation, to support efficient neuronal differentiation and maturation under chemically defined culture conditions. When treated with neural induction medium containing Noggin/retinoic acid, the encapsulated cells expressed much higher levels of neural progenitor markers SOX1 and PAX6 than those in other treatment conditions. Immunocytochemisty analysis confirmed that the majority of the differentiated cells were nestin-positive cells. Subsequently transferring the scaffolds to neuronal differentiation medium efficiently directed these encapsulated neural progenitors into mature neurons, as detected by RT-PCR and positive immunostaining for neuron markers βIII tubulin and MAP2. Furthermore, flow cytometry confirmed that >90% βIII tubulin-positive neurons was achieved for three independent iPSC and hESC lines, a differentiation efficiency much higher than previously reported. Implantation of these terminally differentiated neurons into SCID mice yielded successful neural grafts comprising MAP2 positive neurons, without tumorigenesis, suggesting a potential safe cell source for regenerative medicine. These results bring us one step closer toward realizing large-scale production of stem cell derivatives for clinical and translational applications. PMID:22998816

  18. Differentiation patterns of mouse embryonic stem cells and induced pluripotent stem cells into neurons.

    PubMed

    Nakamura, Mai; Kamishibahara, Yu; Kitazawa, Ayako; Kawaguchi, Hideo; Shimizu, Norio

    2016-05-01

    Mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have the ability to differentiate in vitro into various cell lineages including neurons. The differentiation of these cells into neurons has potential applications in regenerative medicine. Previously, we reported that a chick dorsal root ganglion (DRG)-conditioned medium (CM) promoted the differentiation of mouse ES and iPS cells into neurons. Here, we used real-time PCR to investigate the differentiation patterns of ES and iPS cells into neurons when DRG-CM was added. DRG-CM promoted the expression levels of βIII-tubulin gene (a marker of postmitotic neurons) in ES and iPS cells. ES cells differentiated into neurons faster than iPS cells, and the maximum peaks of gene expression involved in motor, sensory, and dopaminergic neurons were different. Rho kinase (ROCK) inhibitors could be very valuable at numerous stages in the production and use of stem cells in basic research and eventual cell-based therapies. Thus, we investigated whether the addition of a ROCK inhibitor Y-27632 and DRG-CM on the basis of the differentiation patterns promotes the neuronal differentiation of ES cells. When the ROCK inhibitor was added to the culture medium at the initial stages of cultivation, it stimulated the neuronal differentiation of ES cells more strongly than that stimulated by DRG-CM. Moreover, the combination of the ROCK inhibitor and DRG-CM promoted the neuronal differentiation of ES cells when the ROCK inhibitor was added to the culture medium at day 3. The ROCK inhibitor may be useful for promoting neuronal differentiation of ES cells. PMID:25354731

  19. Cellular Zinc Homeostasis Contributes to Neuronal Differentiation in Human Induced Pluripotent Stem Cells.

    PubMed

    Pfaender, Stefanie; Föhr, Karl; Lutz, Anne-Kathrin; Putz, Stefan; Achberger, Kevin; Linta, Leonhard; Liebau, Stefan; Boeckers, Tobias M; Grabrucker, Andreas M

    2016-01-01

    Disturbances in neuronal differentiation and function are an underlying factor of many brain disorders. Zinc homeostasis and signaling are important mediators for a normal brain development and function, given that zinc deficiency was shown to result in cognitive and emotional deficits in animal models that might be associated with neurodevelopmental disorders. One underlying mechanism of the observed detrimental effects of zinc deficiency on the brain might be impaired proliferation and differentiation of stem cells participating in neurogenesis. Thus, to examine the molecular mechanisms regulating zinc metabolism and signaling in differentiating neurons, using a protocol for motor neuron differentiation, we characterized the expression of zinc homeostasis genes during neurogenesis using human induced pluripotent stem cells (hiPSCs) and evaluated the influence of altered zinc levels on the expression of zinc homeostasis genes, cell survival, cell fate, and neuronal function. Our results show that zinc transporters are highly regulated genes during neuronal differentiation and that low zinc levels are associated with decreased cell survival, altered neuronal differentiation, and, in particular, synaptic function. We conclude that zinc deficiency in a critical time window during brain development might influence brain function by modulating neuronal differentiation. PMID:27247802

  20. Cellular Zinc Homeostasis Contributes to Neuronal Differentiation in Human Induced Pluripotent Stem Cells

    PubMed Central

    Pfaender, Stefanie; Föhr, Karl; Lutz, Anne-Kathrin; Putz, Stefan; Achberger, Kevin; Linta, Leonhard; Liebau, Stefan; Boeckers, Tobias M.; Grabrucker, Andreas M.

    2016-01-01

    Disturbances in neuronal differentiation and function are an underlying factor of many brain disorders. Zinc homeostasis and signaling are important mediators for a normal brain development and function, given that zinc deficiency was shown to result in cognitive and emotional deficits in animal models that might be associated with neurodevelopmental disorders. One underlying mechanism of the observed detrimental effects of zinc deficiency on the brain might be impaired proliferation and differentiation of stem cells participating in neurogenesis. Thus, to examine the molecular mechanisms regulating zinc metabolism and signaling in differentiating neurons, using a protocol for motor neuron differentiation, we characterized the expression of zinc homeostasis genes during neurogenesis using human induced pluripotent stem cells (hiPSCs) and evaluated the influence of altered zinc levels on the expression of zinc homeostasis genes, cell survival, cell fate, and neuronal function. Our results show that zinc transporters are highly regulated genes during neuronal differentiation and that low zinc levels are associated with decreased cell survival, altered neuronal differentiation, and, in particular, synaptic function. We conclude that zinc deficiency in a critical time window during brain development might influence brain function by modulating neuronal differentiation. PMID:27247802

  1. Human stem cell neuronal differentiation on silk-carbon nanotube composite

    NASA Astrophysics Data System (ADS)

    Chen, Chi-Shuo; Soni, Sushant; Le, Catherine; Biasca, Matthew; Farr, Erik; Chen, Eric Y.-T.; Chin, Wei-Chun

    2012-02-01

    Human embryonic stem cells [hESCs] are able to differentiate into specific lineages corresponding to regulated spatial and temporal signals. This unique attribute holds great promise for regenerative medicine and cell-based therapy for many human diseases such as spinal cord injury [SCI] and multiple sclerosis [MS]. Carbon nanotubes [CNTs] have been successfully used to promote neuronal differentiation, and silk has been widely applied in tissue engineering. This study aims to build silk-CNT composite scaffolds for improved neuron differentiation efficiency from hESCs. Two neuronal markers (β-III tubulin and nestin) were utilized to determine the hESC neuronal lineage differentiation. In addition, axonal lengths were measured to evaluate the progress of neuronal development. The results demonstrated that cells on silk-CNT scaffolds have a higher β-III tubulin and nestin expression, suggesting augmented neuronal differentiation. In addition, longer axons with higher density were found to associate with silk-CNT scaffolds. Our silk-CNT-based composite scaffolds can promote neuronal differentiation of hESCs. The silk-CNT composite scaffolds developed here can serve as efficient supporting matrices for stem cell-derived neuronal transplants, offering a promising opportunity for nerve repair treatments for SCI and MS patients.

  2. Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture.

    PubMed

    Schulz, Thomas C; Noggle, Scott A; Palmarini, Gail M; Weiler, Deb A; Lyons, Ian G; Pensa, Kate A; Meedeniya, Adrian C B; Davidson, Bruce P; Lambert, Nevin A; Condie, Brian G

    2004-01-01

    The use of human embryonic stem cells (hESCs) as a source of dopaminergic neurons for Parkinson's disease cell therapy will require the development of simple and reliable cell differentiation protocols. The use of cell cocultures, added extracellular signaling factors, or transgenic approaches to drive hESC differentiation could lead to additional regulatory as well as cell production delays for these therapies. Because the neuronal cell lineage seems to require limited or no signaling for its formation, we tested the ability of hESCs to differentiate to form dopamine-producing neurons in a simple serum-free suspension culture system. BG01 and BG03 hESCs were differentiated as suspension aggregates, and neural progenitors and neurons were detectable after 2-4 weeks. Plated neurons responded appropriately to electrophysiological cues. This differentiation was inhibited by early exposure to bone morphogenic protein (BMP)-4, but a pulse of BMP-4 from days 5 to 9 caused induction of peripheral neuronal differentiation. Real-time polymerase chain reaction and whole-mount immunocytochemistry demonstrated the expression of multiple markers of the midbrain dopaminergic phenotype in serum-free differentiations. Neurons expressing tyrosine hydroxylase (TH) were killed by 6-hydroxydopamine (6-OHDA), a neurotoxic catecholamine. Upon plating, these cells released dopamine and other catecholamines in response to K+ depolarization. Surviving TH+ neurons, derived from the cells differentiated in serum-free suspension cultures, were detected 8 weeks after transplantation into 6-OHDA-lesioned rat brains. This work suggests that hESCs can differentiate in simple serum-free suspension cultures to produce the large number of cells required for transplantation studies. PMID:15579641

  3. Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro.

    PubMed

    Chun, So Young; Soker, Shay; Jang, Yu-Jin; Kwon, Tae Gyun; Yoo, Eun Sang

    2016-02-01

    We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

  4. Human periapical cyst-mesenchymal stem cells differentiate into neuronal cells.

    PubMed

    Marrelli, M; Paduano, F; Tatullo, M

    2015-06-01

    It was recently reported that human periapical cysts (hPCys), a commonly occurring odontogenic cystic lesion of inflammatory origin, contain mesenchymal stem cells (MSCs) with the capacity for self-renewal and multilineage differentiation. In this study, periapical inflammatory cysts were compared with dental pulp to determine whether this tissue may be an alternative accessible tissue source of MSCs that retain the potential for neurogenic differentiation. Flow cytometry and immunofluorescence analysis indicated that hPCy-MSCs and dental pulp stem cells spontaneously expressed the neuron-specific protein β-III tubulin and the neural stem-/astrocyte-specific protein glial fibrillary acidic protein (GFAP) in their basal state before differentiation occurs. Furthermore, undifferentiated hPCy-MSCs showed a higher expression of transcripts for neuronal markers (β-III tubulin, NF-M, MAP2) and neural-related transcription factors (MSX-1, Foxa2, En-1) as compared with dental pulp stem cells. After exposure to neurogenic differentiation conditions (neural media containing epidermal growth factor [EGF], basic fibroblast growth factor [bFGF], and retinoic acid), the hPCy-MSCs showed enhanced expression of β-III tubulin and GFAP proteins, as well as increased expression of neurofilaments medium, neurofilaments heavy, and neuron-specific enolase at the transcript level. In addition, neurally differentiated hPCy-MSCs showed upregulated expression of the neural transcription factors Pitx3, Foxa2, Nurr1, and the dopamine-related genes tyrosine hydroxylase and dopamine transporter. The present study demonstrated for the first time that hPCy-MSCs have a predisposition toward the neural phenotype that is increased when exposed to neural differentiation cues, based on upregulation of a comprehensive set of proteins and genes that define neuronal cells. In conclusion, these results provide evidence that hPCy-MSCs might be another optimal source of neural/glial cells for cell

  5. Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia.

    PubMed Central

    Lois, C; Alvarez-Buylla, A

    1993-01-01

    Subventricular zone (SVZ) cells proliferate spontaneously in vivo in the telencephalon of adult mammals. Several studies suggest that SVZ cells do not differentiate after mitosis into neurons or glia but die. In the present work, we show that SVZ cells labeled in the brains of adult mice with [3H]thymidine differentiate directly into neurons and glia in explant cultures. In vitro labeling with [3H]thymidine shows that 98% of the neurons that differentiate from the SVZ explants are derived from precursor cells that underwent their last division in vivo. This report identifies the SVZ cells as neuronal precursors in an adult mammalian brain. Images Fig. 1 Fig. 2 Fig. 3 PMID:8446631

  6. Acid Sensing Ion Channels (ASICs) in NS20Y cells - potential role in neuronal differentiation.

    PubMed

    O'Bryant, Zaven; Leng, Tiandong; Liu, Mingli; Inoue, Koichi; Vann, Kiara T; Xiong, Zhi-Gang

    2016-01-01

    Cultured neuronal cell lines can express properties of mature neurons if properly differentiated. Although the precise mechanisms underlying neuronal differentiation are not fully understood, the expression and activation of ion channels, particularly those of Ca(2+)-permeable channels, have been suggested to play a role. In this study, we explored the presence and characterized the properties of acid-sensing ion channels (ASICs) in NS20Y cells, a neuronal cell line previously used for the study of neuronal differentiation. In addition, the potential role of ASICs in cell differentiation was explored. Reverse Transcription Polymerase Chain Reaction and Western blot revealed the presence of ASIC1 subunits in these cells. Fast drops of extracellular pH activated transient inward currents which were blocked, in a dose dependent manner, by amiloride, a non-selective ASIC blocker, and by Psalmotoxin-1 (PcTX1), a specific inhibitor for homomeric ASIC1a and heteromeric ASIC1a/2b channels. Incubation of cells with PcTX1 significantly reduced the differentiation of NS20Y cells induced by cpt-cAMP, as evidenced by decreased neurite length, dendritic complexity, decreased expression of functional voltage gated Na(+) channels. Consistent with ASIC1a inhibition, ASIC1a knockdown with small interference RNA significantly attenuates cpt-cAMP-induced increase of neurite outgrowth. In summary, we described the presence of functional ASICs in NS20Y cells and demonstrate that ASIC1a plays a role in the differentiation of these cells. PMID:27342076

  7. Cannabidiol Exposure During Neuronal Differentiation Sensitizes Cells Against Redox-Active Neurotoxins.

    PubMed

    Schönhofen, Patrícia; de Medeiros, Liana M; Bristot, Ivi Juliana; Lopes, Fernanda M; De Bastiani, Marco A; Kapczinski, Flávio; Crippa, José Alexandre S; Castro, Mauro Antônio A; Parsons, Richard B; Klamt, Fábio

    2015-08-01

    Cannabidiol (CBD), one of the most abundant Cannabis sativa-derived compounds, has been implicated with neuroprotective effect in several human pathologies. Until now, no undesired side effects have been associated with CBD. In this study, we evaluated CBD's neuroprotective effect in terminal differentiation (mature) and during neuronal differentiation (neuronal developmental toxicity model) of the human neuroblastoma SH-SY5Y cell line. A dose-response curve was performed to establish a sublethal dose of CBD with antioxidant activity (2.5 μM). In terminally differentiated SH-SY5Y cells, incubation with 2.5 μM CBD was unable to protect cells against the neurotoxic effect of glycolaldehyde, methylglyoxal, 6-hydroxydopamine, and hydrogen peroxide (H2O2). Moreover, no difference in antioxidant potential and neurite density was observed. When SH-SY5Y cells undergoing neuronal differentiation were exposed to CBD, no differences in antioxidant potential and neurite density were observed. However, CBD potentiated the neurotoxicity induced by all redox-active drugs tested. Our data indicate that 2.5 μM of CBD, the higher dose tolerated by differentiated SH-SY5Y neuronal cells, does not provide neuroprotection for terminally differentiated cells and shows, for the first time, that exposure of CBD during neuronal differentiation could sensitize immature cells to future challenges with neurotoxins.

  8. Phosphatidylcholine biosynthesis during neuronal differentiation and its role in cell fate determination.

    PubMed

    Marcucci, Hebe; Paoletti, Luciana; Jackowski, Suzanne; Banchio, Claudia

    2010-08-13

    Neuronal differentiation is characterized by neuritogenesis and neurite outgrowth, processes that are dependent on membrane biosynthesis. Thus, the production of phosphatidylcholine (PtdCho), the major membrane phospholipid, should be stimulated during neuronal differentiation. We demonstrate that during retinoic acid (RA)-induced differentiation of Neuro-2a cells, PtdCho synthesis was promoted by an ordered and sequential activation of choline kinase alpha (CK(alpha)) and choline cytidylyltransferase alpha (CCT(alpha)). Early after RA stimulation, the increase in PtdCho synthesis is mainly governed by the biochemical activation of CCT(alpha). Later, the transcription of CK(alpha)- and CCT(alpha)-encoding genes was induced. Both PtdCho biosynthesis and neuronal differentiation are dependent on ERK activation. A novel mechanism is proposed by which PtdCho biosynthesis is coordinated during neuronal differentiation. Enforced expression of either CK(alpha) or CCTalpha increased the rate of synthesis and the amount of PtdCho, and these cells initiated differentiation without RA stimulation, as evidenced by cell morphology and the expression of genes associated with neuritogenesis. The differentiation resulting from enforced expression of CCT(alpha) or CK(alpha) was dependent on persistent ERK activation. These results indicate that elevated PtdCho synthesis could mimic the RA signals and thus determine neuronal cell fate. Moreover, they could explain the key role that PtdCho plays during neuronal regeneration. PMID:20525991

  9. miR-124 promotes the neuronal differentiation of mouse inner ear neural stem cells

    PubMed Central

    Jiang, Di; Du, Jintao; Zhang, Xuemei; Zhou, Wei; Zong, Lin; Dong, Chang; Chen, Kaitian; Chen, Yu; Chen, Xihui; Jiang, Hongyan

    2016-01-01

    MicroRNAs (miRNAs or miRs) act as key regulators in neuronal development, synaptic morphogenesis and plasticity. However, their role in the neuronal differentiation of inner ear neural stem cells (NSCs) remains unclear. In this study, 6 miRNAs were selected and their expression patterns during the neuronal differentiation of inner ear NSCs were examined by RT-qPCR. We demonstrated that the culture of spiral ganglion stem cells present in the inner ears of newborn mice gave rise to neurons in vitro. The expression patterns of miR-124, miR-132, miR-134, miR-20a, miR-17-5p and miR-30a-5p were examined during a 14-day neuronal differentiation period. We found that miR-124 promoted the neuronal differentiation of and neurite outgrowth in mouse inner ear NSCs, and that the changes in the expression of tropomyosin receptor kinase B (TrkB) and cell division control protein 42 homolog (Cdc42) during inner ear NSC differentiation were associated with miR-124 expression. Our findings indicate that miR-124 plays a role in the neuronal differentiation of inner ear NSCs. This finding may lead to the development of novel strategies for restoring hearing in neurodegenerative diseases.

  10. A not cytotoxic nickel concentration alters the expression of neuronal differentiation markers in NT2 cells.

    PubMed

    Ceci, Claudia; Barbaccia, Maria Luisa; Pistritto, Giuseppa

    2015-03-01

    Nickel, a known occupational/environmental hazard, may cross the placenta and reach appreciable concentrations in various fetal organs, including the brain. The aim of this study was to investigate whether nickel interferes with the process of neuronal differentiation. Following a 4 week treatment with retinoic acid (10μM), the human teratocarcinoma-derived NTera2/D1 cell line (NT2 cells) terminally differentiate into neurons which recapitulate many features of human fetal neurons. The continuous exposure of the differentiating NT2 cells to a not cytotoxic nickel concentration (10μM) increased the expression of specific neuronal differentiation markers such as neural cell adhesion molecule (NCAM) and microtubule associated protein 2 (MAP2). Furthermore, nickel exposure increased the expression of hypoxia-inducible-factor-1α (HIF-1α) and induced the activation of the AKT/PKB kinase pathway, as shown by the increase of P(Ser-9)-GSK-3β, the inactive form of glycogen synthase kinase-3β (GSK-3β). Intriguingly, by the end of the fourth week the expression of tyrosine hydroxylase (TH) protein, a marker of dopaminergic neurons, was lower in nickel-treated than in control cultures. Thus, likely by partially mimicking hypoxic conditions, a not-cytotoxic nickel concentration appears to alter the process of neuronal differentiation and hinder the expression of the dopaminergic neuronal phenotype. Taken together, these results suggest that nickel, by altering normal brain development, may increase susceptibility to neuro-psychopathology later in life.

  11. Human GPM6A is associated with differentiation and neuronal migration of neurons derived from human embryonic stem cells.

    PubMed

    Michibata, Hideo; Okuno, Tsuyoshi; Konishi, Nae; Kyono, Kiyoshi; Wakimoto, Koji; Aoki, Kan; Kondo, Yasushi; Takata, Kazuyuki; Kitamura, Yoshihisa; Taniguchi, Takashi

    2009-05-01

    Glycoprotein M6A (GPM6A) is known as a transmembrane protein and an abundant cell surface protein on neurons in the central nervous system (CNS). However, the function of GPM6A in the differentiation of neurons derived from human embryonic stem (ES) cells is unknown. To investigate the function of GPM6A in neural differentiation, we generated human ES cell lines with overexpressed (B2h-oeM6A) or suppressed (B2h-shM6A) human GPM6A. Real-time polymerase chain reaction (PCR) showed that overexpression of GPM6A markedly increased the expression of neuroectodermal-associated genes (OTX1, Lmx1b, En1, Pax2, Sox2, and Wnt1), and the number of neural stem cells (NSCs) derived from B2h-oeM6A cells compared to control vector transfected human ES cells (B2h-Mock1). Our results show an increase in the number of differentiated neuronal cells (cholinergic, catecholaminergic, and GABAergic neurons) from NSCs derived from B2h-oeM6A cells. On the other hand, suppression of human GPM6A expression using a short hairpin RNA (shRNA) in human ES cells led to a decrease in both the expression of neuroectodermal-associated genes and the number of NSCs derived from B2h-shM6A cells. In addition, our results show a decrease in the number of differentiated neuronal cells from NSCs in B2h-shM6A cells compared to control vector transfected human ES cells (B2h-shNSP1). Moreover, overexpression or suppression of human GPM6A in human ES cells led to an increase or decrease, respectively, of neuronal migration. Hence, our findings suggest that expression level of GPM6A is, directly or indirectly, associated with the differentiation and neuronal migration of neurons derived from undifferentiated human ES cells. PMID:19298174

  12. Bach2 is involved in neuronal differentiation of N1E-115 neuroblastoma cells

    SciTech Connect

    Shim, Ki Shuk; Rosner, Margit; Freilinger, Angelika; Lubec, Gert . E-mail: gert.lubec@meduniwien.ac.at; Hengstschlaeger, Markus

    2006-07-15

    Bach1 and Bach2 are evolutionarily related members of the BTB-basic region leucine zipper transcription factor family. We found that Bach2 downregulates cell proliferation of N1E-115 cells and negatively affects their potential to differentiate. Nuclear localization of the cyclin-dependent kinase inhibitor p21 is known to arrest cell cycle progression, and cytoplasmic p21 has been shown to promote neuronal differentiation of N1E-115 cells. We found that ectopic Bach2 causes upregulation of p21 expression in the nucleus and in the cytoplasm in undifferentiated N1E-115 cells. In differentiated cells, Bach2 specifically triggers upregulation of cytoplasmic p21. Our data suggest that Bach2 expression could represent a switch during the process of neuronal differentiation. Bach2 is not expressed in neuronal precursor cells. It would have negative effects on proliferation and differentiation of these cells. In differentiated neuronal cells Bach2 expression is upregulated, which could allow Bach2 to function as a gatekeeper of the differentiated status.

  13. Immunomodulation in stem cell differentiation into neurons and brain repair.

    PubMed

    Ulrich, Henning; do Nascimento, Isis Cristina; Bocsi, Jozsef; Tárnok, Attila

    2015-06-01

    Immunomodulators regulate stem cell activity at all stages of development as well as during adulthood. Embryonic stem cell (ESC) proliferation as well as neurogenic processes during embryonic development are controlled by factors of the immune system. We review here immunophenotypic expression patterns of  different stem cell types, including ESC, neural (NSC) and tissue-specific mesenchymal stem cells (MSC), and focus on immunodulatory properties of these cells. Immune and inflammatory responses, involving actions of cytokines as well as toll-like receptor (TLR) signaling are known to affect the differentiation capacity of NSC and MSC. Secretion of pro- and anti-inflammatory messengers by MSC have been observed as the consequence of TLR and cytokine activation and promotion of differentiation into specified phenotypes. As result of augmented differentiation capacity, stem cells secrete angiogenic factors including vascular endothelial growth factor, resulting in multifactorial actions in tissue repair. Immunoregulatory properties of tissue specific adult stem cells are put into the context of possible clinical applications.

  14. Differentiation of human adipose-derived stem cells into neuron-like cells by Radix Angelicae Sinensis

    PubMed Central

    Wang, Qiaozhi; Zhou, Lile; Guo, Yong; Liu, Guangyi; Cheng, Jiyan; Yu, Hong

    2013-01-01

    Human adipose tissues are an ideal source of stem cells. It is important to find inducers that can safely and effectively differentiate stem cells into functional neurons for clinical use. In this study, we investigate the use of Radix Angelicae Sinensis as an inducer of neuronal differentiation. Primary human adipose-derived stem cells were obtained from adult subcutaneous fatty tissue, then pre-induced with 10% Radix Angelicae Sinensis injection for 24 hours, and incubated in serum-free Dulbecco's modified Eagle's medium/Nutrient Mixture F-12 containing 40% Radix Angelicae Sinensis to induce its differentiation into neuron-like cells. Butylated hydroxyanisole, a common inducer for neuronal differentiation, was used as the control. After human adipose-derived stem cells differentiated into neuron-like cells under the induction of Radix Angelicae Sinensis for 24 hours, the positive expression of neuron-specific enolase was lower than that of the butylated hydroxyanisole-induced group, and the expression of glial fibrillary acidic protein was negative. After they were induced for 48 hours, the positive expression of neuron specific enolase in human adipose-derived stem cells was significantly higher than that of the butylated hydroxyanisole-induced group. Our experimental findings indicate that Radix Angelicae Sinensis can induce human adipose-derived stem cell differentiation into neuron-like cells and produce less cytotoxicity. PMID:25206657

  15. Cholinergic properties of neurons differentiated from an embryonal carcinoma cell-line (P19).

    PubMed

    Parnas, D; Linial, M

    1995-11-01

    P19 is a mouse-derived embryonal carcinoma cell-line capable of differentiation toward ectodermal, mesodermal and endodermal lineages. Following treatment with retinoic acid these cells differentiate into neurons, astrocytes and fibroblast-like cells. We induced P19 differentiation under conditions which lead to a homogeneous neuronal culture (> 95% neurons). Under these conditions, most cells (approximately 85%) express high levels of the cholinergic markers acetyl cholinesterase and choline acetyltransferase while approximately 10% of cells express the GABAergic marker glutamic acid decarboxylase. While the proportion of the GABAergic neurons is constant at different culture conditions, the cholinergic phenotype is suppressed at high cell densities. The cholinergic nature of P19 neurons is also evident in their ability to form contacts with a muscle cell-line--C2. At day 10 of differentiation cells are capable of depolarization-dependent acetylcholine release. The release is Ca2+ dependent, and drops to baseline levels at 0.5 mM Ca2+. The cells also respond to sub-nM levels of alpha-latrotoxin by acetylcholine release. All major proteins implicated in synapse functionality are expressed prior to day 10 at both at RNA and protein levels. However, the expression pattern of each gene is unique. The genes include cytoskeletal proteins, synaptic vesicle proteins and terminal specific proteins. We suggest that this cell-line can serve as an in-vitro model system for the study of neuronal phenotype acquisition. Under our conditions, the P19 cells can also provide a system in which to study the differentiation of functional cholinergic neurons. PMID:8787867

  16. Clonal Heterogeneity in the Neuronal and Glial Differentiation of Dental Pulp Stem/Progenitor Cells

    PubMed Central

    Young, Fraser I.; Telezhkin, Vsevolod; Youde, Sarah J.; Langley, Martin S.; Stack, Maria; Kemp, Paul J.; Waddington, Rachel J.; Sloan, Alastair J.; Song, Bing

    2016-01-01

    Cellular heterogeneity presents an important challenge to the development of cell-based therapies where there is a fundamental requirement for predictable and reproducible outcomes. Transplanted Dental Pulp Stem/Progenitor Cells (DPSCs) have demonstrated early promise in experimental models of spinal cord injury and stroke, despite limited evidence of neuronal and glial-like differentiation after transplantation. Here, we report, for the first time, on the ability of single cell-derived clonal cultures of murine DPSCs to differentiate in vitro into immature neuronal-like and oligodendrocyte-like cells. Importantly, only DPSC clones with high nestin mRNA expression levels were found to successfully differentiate into Map2 and NF-positive neuronal-like cells. Neuronally differentiated DPSCs possessed a membrane capacitance comparable with primary cultured striatal neurons and small inward voltage-activated K+ but not outward Na+ currents were recorded suggesting a functionally immature phenotype. Similarly, only high nestin-expressing clones demonstrated the ability to adopt Olig1, Olig2, and MBP-positive immature oligodendrocyte-like phenotype. Together, these results demonstrate that appropriate markers may be used to provide an early indication of the suitability of a cell population for purposes where differentiation into a specific lineage may be beneficial and highlight that further understanding of heterogeneity within mixed cellular populations is required. PMID:27313623

  17. Differentiation of neuronal stem cells into motor neurons using electrospun poly-L-lactic acid/gelatin scaffold.

    PubMed

    Binan, Loïc; Tendey, Charlène; De Crescenzo, Gregory; El Ayoubi, Rouwayda; Ajji, Abdellah; Jolicoeur, Mario

    2014-01-01

    Neural stem cells (NSCs) provide promising therapeutic potential for cell replacement therapy in spinal cord injury (SCI). However, high increases of cell viability and poor control of cell differentiation remain major obstacles. In this study, we have developed a non-woven material made of co-electrospun fibers of poly L-lactic acid and gelatin with a degradation rate and mechanical properties similar to peripheral nerve tissue and investigated their effect on cell survival and differentiation into motor neuronal lineages through the controlled release of retinoic acid (RA) and purmorphamine. Engineered Neural Stem-Like Cells (NSLCs) seeded on these fibers, with and without the instructive cues, differentiated into β-III-tubulin, HB-9, Islet-1, and choactase-positive motor neurons by immunostaining, in response to the release of the biomolecules. In addition, the bioactive material not only enhanced the differentiation into motor neuronal lineages but also promoted neurite outgrowth. This study elucidated that a combination of electrospun fiber scaffolds, neural stem cells, and controlled delivery of instructive cues could lead to the development of a better strategy for peripheral nerve injury repair. PMID:24161168

  18. Differentiation-specific effects of LHON mutations introduced into neuronal NT2 cells.

    PubMed

    Wong, Alice; Cavelier, Lucia; Collins-Schramm, Heather E; Seldin, Michael F; McGrogan, Michael; Savontaus, Marja-Liisa; Cortopassi, Gino A

    2002-02-15

    Inheritance of one of three primary mutations at positions 11778, 3460 or 14484 of the mitochondrial genome in subunits of Complex I causes Leber's Hereditary Optic Neuropathy (LHON), a specific degeneration of the optic nerve, resulting in bilateral blindness. It has been unclear why inheritance of a systemic mitochondrial mutation would result in a specific neurodegeneration. To address the neuron-specific degenerative phenotype of the LHON genotype, we have created cybrids using a neuronal precursor cell line, Ntera 2/D1 (NT2), containing mitochondria from patient lymphoblasts bearing the most common LHON mutation (11778) and the most severe LHON mutation (3460). The undifferentiated LHON-NT2 mutant cells were not significantly different from the parental cell control in terms of mtDNA/nDNA ratio, mitochondrial membrane potential, reactive oxygen species (ROS) production or the ability to reduce Alamar Blue. Differentiation of NT2s resulted in a neuronal morphology and neuron-specific pattern of gene expression, and a 3-fold reduction in mtDNA/nDNA ratio in both mutant and control cells; however, the differentiation protocol yielded significantly less LHON cells than controls, by 30%, indicating either a decreased proliferative potential or increased cell death of the LHON-NT2 cells. Differentiation of the cells to the neuronal form also resulted in significant increases in ROS production in the LHON-NT2 neurons versus controls, which is abolished by rotenone, a specific inhibitor of Complex I. We infer that the LHON genotype requires a differentiated neuronal environment in order to induce increased mitochondrial ROS, which may be the cause of the reduced NT2 yield; and suggest that the LHON degenerative phenotype may be the result of an increase in mitochondrial superoxide which is caused by the LHON mutations, possibly mediated through neuron-specific alterations in Complex I structure.

  19. Differentiation of mesenchymal stem cells into neuronal cells on fetal bovine acellular dermal matrix as a tissue engineered nerve scaffold.

    PubMed

    Feng, Yuping; Wang, Jiao; Ling, Shixin; Li, Zhuo; Li, Mingsheng; Li, Qiongyi; Ma, Zongren; Yu, Sijiu

    2014-11-15

    The purpose of this study was to assess fetal bovine acellular dermal matrix as a scaffold for supporting the differentiation of bone marrow mesenchymal stem cells into neural cells following induction with neural differentiation medium. We performed long-term, continuous observation of cell morphology, growth, differentiation, and neuronal development using several microscopy techniques in conjunction with immunohistochemistry. We examined specific neuronal proteins and Nissl bodies involved in the differentiation process in order to determine the neuronal differentiation of bone marrow mesenchymal stem cells. The results show that bone marrow mesenchymal stem cells that differentiate on fetal bovine acellular dermal matrix display neuronal morphology with unipolar and bi/multipolar neurite elongations that express neuronal-specific proteins, including βIII tubulin. The bone marrow mesenchymal stem cells grown on fetal bovine acellular dermal matrix and induced for long periods of time with neural differentiation medium differentiated into a multilayered neural network-like structure with long nerve fibers that was composed of several parallel microfibers and neuronal cells, forming a complete neural circuit with dendrite-dendrite to axon-dendrite to dendrite-axon synapses. In addition, growth cones with filopodia were observed using scanning electron microscopy. Paraffin sectioning showed differentiated bone marrow mesenchymal stem cells with the typical features of neuronal phenotype, such as a large, round nucleus and a cytoplasm full of Nissl bodies. The data suggest that the biological scaffold fetal bovine acellular dermal matrix is capable of supporting human bone marrow mesenchymal stem cell differentiation into functional neurons and the subsequent formation of tissue engineered nerve.

  20. Neurons Differentiated from Transplanted Stem Cells Respond Functionally to Acoustic Stimuli in the Awake Monkey Brain.

    PubMed

    Wei, Jing-Kuan; Wang, Wen-Chao; Zhai, Rong-Wei; Zhang, Yu-Hua; Yang, Shang-Chuan; Rizak, Joshua; Li, Ling; Xu, Li-Qi; Liu, Li; Pan, Ming-Ke; Hu, Ying-Zhou; Ghanemi, Abdelaziz; Wu, Jing; Yang, Li-Chuan; Li, Hao; Lv, Long-Bao; Li, Jia-Li; Yao, Yong-Gang; Xu, Lin; Feng, Xiao-Li; Yin, Yong; Qin, Dong-Dong; Hu, Xin-Tian; Wang, Zheng-Bo

    2016-07-26

    Here, we examine whether neurons differentiated from transplanted stem cells can integrate into the host neural network and function in awake animals, a goal of transplanted stem cell therapy in the brain. We have developed a technique in which a small "hole" is created in the inferior colliculus (IC) of rhesus monkeys, then stem cells are transplanted in situ to allow for investigation of their integration into the auditory neural network. We found that some transplanted cells differentiated into mature neurons and formed synaptic input/output connections with the host neurons. In addition, c-Fos expression increased significantly in the cells after acoustic stimulation, and multichannel recordings indicated IC specific tuning activities in response to auditory stimulation. These results suggest that the transplanted cells have the potential to functionally integrate into the host neural network.

  1. Neurons Differentiated from Transplanted Stem Cells Respond Functionally to Acoustic Stimuli in the Awake Monkey Brain.

    PubMed

    Wei, Jing-Kuan; Wang, Wen-Chao; Zhai, Rong-Wei; Zhang, Yu-Hua; Yang, Shang-Chuan; Rizak, Joshua; Li, Ling; Xu, Li-Qi; Liu, Li; Pan, Ming-Ke; Hu, Ying-Zhou; Ghanemi, Abdelaziz; Wu, Jing; Yang, Li-Chuan; Li, Hao; Lv, Long-Bao; Li, Jia-Li; Yao, Yong-Gang; Xu, Lin; Feng, Xiao-Li; Yin, Yong; Qin, Dong-Dong; Hu, Xin-Tian; Wang, Zheng-Bo

    2016-07-26

    Here, we examine whether neurons differentiated from transplanted stem cells can integrate into the host neural network and function in awake animals, a goal of transplanted stem cell therapy in the brain. We have developed a technique in which a small "hole" is created in the inferior colliculus (IC) of rhesus monkeys, then stem cells are transplanted in situ to allow for investigation of their integration into the auditory neural network. We found that some transplanted cells differentiated into mature neurons and formed synaptic input/output connections with the host neurons. In addition, c-Fos expression increased significantly in the cells after acoustic stimulation, and multichannel recordings indicated IC specific tuning activities in response to auditory stimulation. These results suggest that the transplanted cells have the potential to functionally integrate into the host neural network. PMID:27425612

  2. REST Regulates Non–Cell-Autonomous Neuronal Differentiation and Maturation of Neural Progenitor Cells via Secretogranin II

    PubMed Central

    Kim, Hyung Joon; Denli, Ahmet M.; Wright, Rebecca; Baul, Tithi D.; Clemenson, Gregory D.; Morcos, Ari S.; Zhao, Chunmei; Schafer, Simon T.

    2015-01-01

    RE-1 silencing transcription factor (REST), a master negative regulator of neuronal differentiation, controls neurogenesis by preventing the differentiation of neural stem cells. Here we focused on the role of REST in the early steps of differentiation and maturation of adult hippocampal progenitors (AHPs). REST knockdown promoted differentiation and affected the maturation of rat AHPs. Surprisingly, REST knockdown cells enhanced the differentiation of neighboring wild-type AHPs, suggesting that REST may play a non–cell-autonomous role. Gene expression analysis identified Secretogranin II (Scg2) as the major secreted REST target responsible for the non–cell-autonomous phenotype. Loss-of-function of Scg2 inhibited differentiation in vitro, and exogenous SCG2 partially rescued this phenotype. Knockdown of REST in neural progenitors in mice led to precocious maturation into neurons at the expense of mushroom spines in vivo. In summary, we found that, in addition to its cell-autonomous function, REST regulates differentiation and maturation of AHPs non–cell-autonomously via SCG2. SIGNIFICANCE STATEMENT Our results reveal that REST regulates differentiation and maturation of neural progenitor cells in vitro by orchestrating both cell-intrinsic and non–cell-autonomous factors and that Scg2 is a major secretory target of REST with a differentiation-enhancing activity in a paracrine manner. In vivo, REST depletion causes accelerated differentiation of newborn neurons at the expense of spine defects, suggesting a potential role for REST in the timing of the maturation of granule neurons. PMID:26538656

  3. In vitro differentiation of bone marrow stromal cells into neurons and glial cells and differential protein expression in a two-compartment bone marrow stromal cell/neuron co-culture system.

    PubMed

    Qi, Xu; Shao, Ming; Peng, Haisheng; Bi, Zhenggang; Su, Zhiqiang; Li, Hulun

    2010-07-01

    This study was performed to establish a bone marrow stromal cell (BMSC)/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC/neuron co-culture and BMSC/neuron two-compartment co-culture. Cells were examined for neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression. The electrophysiological behavior of the BMSCs was examined using patch clamping. Proteins that had significantly different expression levels in BMSCs cultured alone and co-cultured with neurons were studied using a protein chip-mass spectroscopy technique. Expression of NSE and GFAP were significantly higher in co-culture cells than in two-compartment co-culture cells, and significantly higher in both co-culture groups than in BMSCs cultured alone. Five proteins showed significant changes in expression during differentiation: TIP39_RAT and CALC_RAT underwent increases, and INSL6_RAT, PNOC_RAT and PCSK1_RAT underwent decreases in expression. We conclude that BMSCs can differentiate into neurons during both contact co-culture with neurons and two-compartment co-culture with neurons. The rate at which BMSCs differentiated into neurons was higher in contact co-culture than in non-contact co-culture.

  4. Role of lipid rafts in neuronal differentiation of dental pulp-derived stem cells.

    PubMed

    Mattei, Vincenzo; Santacroce, Costantino; Tasciotti, Vincenzo; Martellucci, Stefano; Santilli, Francesca; Manganelli, Valeria; Piccoli, Luca; Misasi, Roberta; Sorice, Maurizio; Garofalo, Tina

    2015-12-10

    Human dental pulp-derived stem cells (hDPSCs) are characterized by a typical fibroblast-like morphology. They express specific markers for mesenchymal stem cells and are capable of differentiation into osteoblasts, adipoblasts and neurons in vitro. Previous studies showed that gangliosides are involved in the induction of early neuronal differentiation of hDPSCs. This study was undertaken to investigate the role of lipid rafts in this process. Lipid rafts are signaling microdomains enriched in glycosphingolipids, cholesterol, tyrosine kinase receptors, mono- or heterotrimeric G proteins and GPI-anchored proteins. We preliminary showed that established cells expressed multipotent mesenchymal stromal-specific surface antigens. Then, we analyzed the distribution of lipid rafts, revealing plasma membrane microdomains with GM2 and EGF-R enrichment. Following stimulation with EGF/bFGF, neuronal differentiation was observed. To analyze the functional role of lipid rafts in EGF/bFGF-induced hDPSCs differentiation, cells were preincubated with lipid raft affecting agents, i.e. [D]-PDMP or methyl-β-cyclodextrin. These compounds significantly prevented neuronal-specific antigen expression, as well as Akt and ERK 1/2 phosphorylation, induced by EGF/bFGF, indicating that lipid raft integrity is essential for EGF/bFGF-induced hDPSCs differentiation. These results suggest that lipid rafts may represent specific chambers, where multimolecular signaling complexes, including lipids (gangliosides, cholesterol) and proteins (EGF-R), play a role in hDPSCs differentiation.

  5. In vitro differentiation of human amniotic fluid-derived cells: augmentation towards a neuronal dopaminergic phenotype.

    PubMed

    Pfeiffer, Shona; McLaughlin, David

    2010-09-01

    Amniotic fluid is known to yield a number of cell types which are multipotent, ethically derived, genetically stable, easily grown, expanded and possess favourable immunogenicity, which has resulted in an increasing interest for use in various neuronal disorders such as Parkinson's disease. The neuronal potential of cells derived from the adherent fraction of amniotic fluid, routinely taken by amniocentesis, are least explored. The aim of the present study was to investigate the capacity of these cells for neuronal and dopaminergic differentiation using in vitro differentiation protocols with canonical inductive factors not previously tested. To do this, samples derived from multiple donors were grown under four conditions: standard serum-containing media, NB (neurobasal) media designed specifically for propagation and maintenance of neuronal cells, NB media with addition of retinoic acid and BDNF (brain-derived neurotrophic factor) for NI (neuronal induction), and NB media with addition of FGF8 (fibroblast growth factor-8) and Shh (sonic hedgehog) after NI. Our results showed the presence of multiple neuronal markers after growth in serum-containing medium [TUJ1, MAP2, NF-M, TH (tyrosine hydroxylase)], which was significantly up-regulated after serum withdrawal in NB medium alone with induction of NeuN (neuronal nuclei) and NSE (neuron-specific enolase). NI and DA.I (dopaminergic induction) was accompanied by further increases in expression and a distinct transition to a sustained neuronal morphology. Western blot analysis confirmed increasing TH expression and NURR1, expressed in base serum-containing media, found to be down-regulated after induction. In conclusion, these cells possess a highly favourable base neuronal and dopaminergic prepotential, which may easily be accentuated by standard induction protocols. PMID:20388119

  6. Interaction of SH-SY5Y cells with nanogratings during neuronal differentiation: comparison with primary neurons.

    PubMed

    Tonazzini, Ilaria; Cecchini, Alessandra; Elgersma, Ype; Cecchini, Marco

    2014-04-01

    Controlling neuronal cell adhesion, migration, and axonal outgrowth via contact interactions with biomaterials is a critical element for tissue engineering applications and for developing artificial neuronal interfaces. One promising approach relies on the exploitation of nanostructured surfaces. Here, the human neuroblastoma cell line SH-SY5Y is interfaced with plastic nanogratings (NGs; anisotropic topographies composed by alternating lines of grooves and ridges with sub-micrometer lateral dimension). The SH-SY5Y cells' (SHs) contact guidance is investigated under proliferating conditions and upon differentiation after treatment with retinoic acid (RA) and brain-derived neurotrophin factor (BDNF), and compared with mouse primary hippocampal neurons (HNs). Quantitative readouts are obtained by measuring changes in tubulin cytoskeleton organization and cell morphology induced by mechanotransduction. Results demonstrate that SHs effectively retrieve substrate topographical signals, in particular during differentiation. Remarkably, RA/BDNF improves SH responsiveness to NG directional cues, and significantly enhances the alignment to the NG lines. HNs behave similarly, showing a marked change in network organization if cultured on NGs. These results might help the rational engineering of neuro-regenerative scaffolds to improve peripheral nerve wound healing, as well as to investigate the basic mechanisms of neuronal wiring.

  7. PDE4 Inhibition by Rolipram Promotes Neuronal Differentiation in Human Bone Marrow Mesenchymal Stem Cells.

    PubMed

    Joe, I-Seul; Cho, Goang-Won

    2016-08-01

    Increased intracellular cyclic adenosine monophosphate (cAMP) can promote axonal elongation and facilitate neuronal repair, while decreased cAMP is associated with losses in neuronal regenerative capacity. Rolipram, which upregulates intracellular cAMP by blocking phosphodiesterase-4 (PDE4) enzyme activity, can mitigate diverse neurological disorders. In this study, we investigated whether rolipram induces neuronal differentiation of human bone marrow-mesenchymal stem cells (hBM-MSCs). Rolipram-treated MSCs (Roli-MSCs) had significantly increased expression of the neuroprogenitor proteins Nestin, Musashi, GFAP, and Sox-2. When Roli-MSCs were differentiated with neuronal induction media (Roli-dMSCs), they exhibited cell body and dendritic morphologies similar to those of neurons. The neurite number and length of Roli-dMSCs were significantly increased compared to those of differentiated MSCs (dMSCs). Compared with undifferentiated hBM-MSCs, the Roli-dMSCs and dMSCs showed significantly increased expression of the neuronal-specific marker genes Nestin, Musashi, CD133, GFAP, NF-M, MAP-2, KCNH1, KCNH5, SCN3A, and CACNA1A, and decreased expression of other lineage-specific markers Adiponectin, ALP, FABP4, and MMP13. The Roli-dMSCs also showed a higher expression of the neuronal markers Nestin, Musashi, Sox-2, NF-M, and Tuj-1 compared to those of the undifferentiated hBM-MSCs, measured by immunocytochemistry and immunoblotting assay. Thus, we have shown that rolipram ameliorates neuronal differentiation by the regulation of neuroprogenitor expression in hBM-MSCs, and rolipram treatment of MSCs may improve the therapeutic efficacy of stem cell therapy for neurodegenerative disorders. PMID:27459581

  8. Down regulation of ribosomal protein mRNAs during neuronal differentiation of human NTERA2 cells.

    PubMed

    Bévort, M; Leffers, H

    2000-10-01

    We have analysed the expression of 32 ribosomal protein (RP) mRNAs during retinoic acid induced neuronal differentiation of human NTERA2 cells. Except for a new S27 variant (S27v), all were down regulated both in selectively replated differentiated neurons and the most differentiated continuous cultures, i.e., non-replated cultures. However, the expression profiles of the individual RP mRNAs were different, most (L3, L7, L8, L10, L13, L23a, L27a, L36a, L39, P0, S2, S3, S3a, S4X, S6, S9, S12, S13, S16, S19, S20, S23, and S27a) exhibited a constant down regulation, whereas a few were either initially constant (L11, L32, S8, and S11) or up regulated (L6, L15, L17, L31, and S27y) and then down regulated. The expression of S27v remained elevated in the most differentiated continuous cultures but was down regulated in replated differentiated neurons. The down regulation of RP mRNAs was variable: the expression levels in differentiated replated neurons were between 10% (S3) and 90% (S11) of the levels in undifferentiated cells. The ratio between rRNA and RP mRNA changed during the differentiation; in differentiated neurons there were, on average, about half the number of RP mRNAs per rRNA as compared to undifferentiated cells. The expression profiles of a few translation-related proteins were also determined. EF1alpha1, EF1beta1, and EF1delta were down regulated, whereas the expression of the neuron and muscle specific EF1alpha2 increased. The reduction in the expression of RP mRNAs was coordinated with a reduction in the expression level of the proliferation marker PCNA. The expression levels of most RP mRNAs were lower in purified differentiated post-mitotic neurons than in the most differentiated continuous cultures, despite similar levels of PCNA, suggesting that both the differentiation state and the proliferative status of the cells affect the expression of RP mRNAs.

  9. Crocin suppresses tumor necrosis factor-alpha-induced cell death of neuronally differentiated PC-12 cells.

    PubMed

    Soeda, S; Ochiai, T; Paopong, L; Tanaka, H; Shoyama, Y; Shimeno, H

    2001-11-01

    Crocus sativus L. is used in Chinese traditional medicine to treat some disorders of the central nervous system. Crocin is an ethanol-extractable component of Crocus sativus L.; it is reported to prevent ethanol-induced impairment of learning and memory in mice. In this study, we demonstrate that crocin suppresses the effect of tumor necrosis factor (TNF)-alpha on neuronally differentiated PC-12 cells. PC-12 cells dead from exposure to TNF-alpha show apoptotic morphological changes and DNA fragmentation. These hallmark features of cell death did not appear in cells treated in the co-presence of 10 microM crocin. Moreover, crocin suppressed the TNF-alpha-induced expression of Bcl-Xs and LICE mRNAs and simultaneously restored the cytokine-induced reduction of Bcl-X(L) mRNA expression. The modulating effects of crocin on the expression of Bcl-2 family proteins led to a marked reduction of a TNF-alpha-induced release of cytochrome c from the mitochondria. Crocin also blocked the cytochrome c-induced activation of caspase-3. To learn how crocin exhibits these anti-apoptotic actions in PC-12 cells, we tested the effect of crocin on PC-12 cell death induced by daunorubicin. We found that crocin inhibited the effect of daunorubicin as well. Our findings suggest that crocin inhibits neuronal cell death induced by both internal and external apoptotic stimuli.

  10. Overexpression of NTRK1 Promotes Differentiation of Neural Stem Cells into Cholinergic Neurons

    PubMed Central

    Wang, Limin; He, Feng; Zhong, Zhuoyuan; Lv, Ruiyan; Xiao, Songhua; Liu, Zhonglin

    2015-01-01

    Neurotrophic tyrosine kinase type 1 (NTRK1) plays critical roles in proliferation, differentiation, and survival of cholinergic neurons; however, it remains unknown whether enhanced expression of NTRK1 in neural stem cells (NSCs) can promote their differentiation into mature neurons. In this study, a plasmid encoding the rat NTRK1 gene was constructed and transfected into C17.2 mouse neural stem cells (NSCs). NTRK1 overexpression in C17.2 cells was confirmed by western blot. The NSCs overexpressing NTRK1 and the C17.2 NSCs transfected by an empty plasmid vector were treated with or without 100 ng/mL nerve growth factor (NGF) for 7 days. Expression of the cholinergic cell marker, choline acetyltransferase (ChAT), was detected by florescent immunocytochemistry (ICC). In the presence of NGF induction, the NSCs overexpressing NTRK1 differentiated into ChAT-immunopositive cells at 3-fold higher than the NSCs transfected by the plasmid vector (26% versus 9%, P < 0.05). The data suggest that elevated NTRK1 expression increases differentiation of NSCs into cholinergic neurons under stimulation of NGF. The approach also represents an efficient strategy for generation of cholinergic neurons. PMID:26509167

  11. Differential Neuronal Plasticity of Dental Pulp Stem Cells From Exfoliated Deciduous and Permanent Teeth Towards Dopaminergic Neurons.

    PubMed

    Majumdar, Debanjana; Kanafi, Mohammad; Bhonde, Ramesh; Gupta, Pawan; Datta, Indrani

    2016-09-01

    Based on early occurrence in chronological age, stem-cells from human exfoliated deciduous teeth (SHED) has been reported to possess better differentiation-potential toward certain cell-lineage in comparison to stem-cells from adult teeth (DPSCs). Whether this same property between them extends for the yield of functional central nervous system neurons is still not evaluated. Hence, we aim to assess the neuronal plasticity of SHED in comparison to DPSCs toward dopaminergic-neurons and further, if the difference is reflected in a differential expression of sonic-hedgehog (SHH)-receptors and basal-expressions of tyrosine-hydroxylase [TH; through cAMP levels]. Human SHED and DPSCs were exposed to midbrain-cues [SHH, fibroblast growth-factor8, and basic fibroblast growth-factor], and their molecular, immunophenotypical, and functional characterization was performed at different time-points of induction. Though SHED and DPSCs spontaneously expressed early-neuronal and neural-crest marker in their naïve state, only SHED expressed a high basal-expression of TH. The upregulation of dopaminergic transcription-factors Nurr1, Engrailed1, and Pitx3 was more pronounced in DPSCs. The yield of TH-expressing cells decreased from 49.8% to 32.16% in SHED while it increased from 8.09% to 77.47% in DPSCs. Dopamine release and intracellular-Ca(2+) influx upon stimulation (KCl and ATP) was higher in induced DPSCs. Significantly lower-expression of SHH-receptors was noted in naïve SHED than DPSCs, which may explain the differential neuronal plasticity. In addition, unlike DPSCs, SHED showed a down-regulation of cyclic adenosine-monophosphate (cAMP) upon exposure to SHH; possibly another contributor to the lesser differentiation-potential. Our data clearly demonstrates for the first time that DPSCs possess superior neuronal plasticity toward dopaminergic-neurons than SHED; influenced by higher SHH-receptor and lower basal TH expression. J. Cell. Physiol. 231: 2048-2063, 2016. © 2016

  12. Differential Neuronal Plasticity of Dental Pulp Stem Cells From Exfoliated Deciduous and Permanent Teeth Towards Dopaminergic Neurons.

    PubMed

    Majumdar, Debanjana; Kanafi, Mohammad; Bhonde, Ramesh; Gupta, Pawan; Datta, Indrani

    2016-09-01

    Based on early occurrence in chronological age, stem-cells from human exfoliated deciduous teeth (SHED) has been reported to possess better differentiation-potential toward certain cell-lineage in comparison to stem-cells from adult teeth (DPSCs). Whether this same property between them extends for the yield of functional central nervous system neurons is still not evaluated. Hence, we aim to assess the neuronal plasticity of SHED in comparison to DPSCs toward dopaminergic-neurons and further, if the difference is reflected in a differential expression of sonic-hedgehog (SHH)-receptors and basal-expressions of tyrosine-hydroxylase [TH; through cAMP levels]. Human SHED and DPSCs were exposed to midbrain-cues [SHH, fibroblast growth-factor8, and basic fibroblast growth-factor], and their molecular, immunophenotypical, and functional characterization was performed at different time-points of induction. Though SHED and DPSCs spontaneously expressed early-neuronal and neural-crest marker in their naïve state, only SHED expressed a high basal-expression of TH. The upregulation of dopaminergic transcription-factors Nurr1, Engrailed1, and Pitx3 was more pronounced in DPSCs. The yield of TH-expressing cells decreased from 49.8% to 32.16% in SHED while it increased from 8.09% to 77.47% in DPSCs. Dopamine release and intracellular-Ca(2+) influx upon stimulation (KCl and ATP) was higher in induced DPSCs. Significantly lower-expression of SHH-receptors was noted in naïve SHED than DPSCs, which may explain the differential neuronal plasticity. In addition, unlike DPSCs, SHED showed a down-regulation of cyclic adenosine-monophosphate (cAMP) upon exposure to SHH; possibly another contributor to the lesser differentiation-potential. Our data clearly demonstrates for the first time that DPSCs possess superior neuronal plasticity toward dopaminergic-neurons than SHED; influenced by higher SHH-receptor and lower basal TH expression. J. Cell. Physiol. 231: 2048-2063, 2016. © 2016

  13. Plasticity of marrow mesenchymal stem cells from human first-trimester fetus: from single-cell clone to neuronal differentiation.

    PubMed

    Zhang, Yihua; Shen, Wenzheng; Sun, Bingjie; Lv, Changrong; Dou, Zhongying

    2011-02-01

    Recent results have shown that bone marrow mesenchymal stem cells (BMSCs) from human first-trimester abortus (hfBMSCs) are closer to embryonic stem cells and perform greater telomerase activity and faster propagation than mid- and late-prophase fetal and adult BMSCs. However, no research has been done on the plasticity of hfBMSCs into neuronal cells using single-cell cloned strains without cell contamination. In this study, we isolated five single cells from hfBMSCs and obtained five single-cell cloned strains, and investigated their biological property and neuronal differentiation potential. We found that four of the five strains showed similar expression profile of surface antigen markers to hfBMSCs, and most of them differentiated into neuron-like cells expressing Nestin, Pax6, Sox1, β-III Tubulin, NF-L, and NSE under induction. One strain showed different expression profile of surface antigen markers from the four strains and hfBMSCs, and did not differentiate toward neuronal cells. We demonstrated for the first time that some of single-cell cloned strains from hfBMSCs can differentiate into nerve tissue-like cell clusters under induction in vitro, and that the plasticity of each single-cell cloned strain into neuronal cells is different.

  14. Investigating DNA methylation dynamics and safety of human embryonic stem cell differentiation toward striatal neurons.

    PubMed

    Baronchelli, Simona; La Spada, Alberto; Conforti, Paola; Redaelli, Serena; Dalprà, Leda; De Blasio, Pasquale; Cattaneo, Elena; Biunno, Ida

    2015-10-15

    The potential use of human embryonic stem cells (hESCs) in cell-based therapies points out the critical importance of epigenomic evaluation for cell-based therapies. Specifically, DNA methylation appears to be a crucial player in establishing cell fate commitment and lineage choices. In this study, we report the global changes observed on the CpG islands distributed in promoters, gene bodies, and intergenic regions and the major biochemical pathways and genes involved in methylation changes as H9-hESCs turn into a neuronal culture containing medium-sized spiny striatal neurons (MSNs). Using an ontogeny-recapitulating protocol of striatal neuron differentiation, we analyzed DNA methylation profiles during the conversion from pluripotency to neuropotency up to the acquisition of a mature neuronal phenotype. H9-hESCs changed the methylation pattern both through de novo methylation and hypomethylation of specific gene promoters. Bioinformatic analysis allowed us to identify a panel of striatal-associated genes, which were regulated by DNA methylation and differentially expressed during striatal commitment. Importantly, DNA methylation analysis revealed that H9-hESCs did not acquire methylation-based oncogenic properties after differentiation. Indeed, hypermethylation of cancer-associated genes that characterize transformed cells, such as Polycomb repressive complex-associated genes, was not detected in the neuronal cultures. However, the oncosuppressor gene, BCL2L11, became hypermethylated in H9-hESC-derived mature neurons. Whole-genome DNA methylation profiling could become a technological platform to predict the differentiative potential of hESC-derived cultures and establish further biosafety assessment quality control tools of the cell-based products.

  15. Metabolic reprogramming during neuronal differentiation.

    PubMed

    Agostini, M; Romeo, F; Inoue, S; Niklison-Chirou, M V; Elia, A J; Dinsdale, D; Morone, N; Knight, R A; Mak, T W; Melino, G

    2016-09-01

    Newly generated neurons pass through a series of well-defined developmental stages, which allow them to integrate into existing neuronal circuits. After exit from the cell cycle, postmitotic neurons undergo neuronal migration, axonal elongation, axon pruning, dendrite morphogenesis and synaptic maturation and plasticity. Lack of a global metabolic analysis during early cortical neuronal development led us to explore the role of cellular metabolism and mitochondrial biology during ex vivo differentiation of primary cortical neurons. Unexpectedly, we observed a huge increase in mitochondrial biogenesis. Changes in mitochondrial mass, morphology and function were correlated with the upregulation of the master regulators of mitochondrial biogenesis, TFAM and PGC-1α. Concomitant with mitochondrial biogenesis, we observed an increase in glucose metabolism during neuronal differentiation, which was linked to an increase in glucose uptake and enhanced GLUT3 mRNA expression and platelet isoform of phosphofructokinase 1 (PFKp) protein expression. In addition, glutamate-glutamine metabolism was also increased during the differentiation of cortical neurons. We identified PI3K-Akt-mTOR signalling as a critical regulator role of energy metabolism in neurons. Selective pharmacological inhibition of these metabolic pathways indicate existence of metabolic checkpoint that need to be satisfied in order to allow neuronal differentiation. PMID:27058317

  16. Metabolic reprogramming during neuronal differentiation

    PubMed Central

    Agostini, M; Romeo, F; Inoue, S; Niklison-Chirou, M V; Elia, A J; Dinsdale, D; Morone, N; Knight, R A; Mak, T W; Melino, G

    2016-01-01

    Newly generated neurons pass through a series of well-defined developmental stages, which allow them to integrate into existing neuronal circuits. After exit from the cell cycle, postmitotic neurons undergo neuronal migration, axonal elongation, axon pruning, dendrite morphogenesis and synaptic maturation and plasticity. Lack of a global metabolic analysis during early cortical neuronal development led us to explore the role of cellular metabolism and mitochondrial biology during ex vivo differentiation of primary cortical neurons. Unexpectedly, we observed a huge increase in mitochondrial biogenesis. Changes in mitochondrial mass, morphology and function were correlated with the upregulation of the master regulators of mitochondrial biogenesis, TFAM and PGC-1α. Concomitant with mitochondrial biogenesis, we observed an increase in glucose metabolism during neuronal differentiation, which was linked to an increase in glucose uptake and enhanced GLUT3 mRNA expression and platelet isoform of phosphofructokinase 1 (PFKp) protein expression. In addition, glutamate–glutamine metabolism was also increased during the differentiation of cortical neurons. We identified PI3K–Akt–mTOR signalling as a critical regulator role of energy metabolism in neurons. Selective pharmacological inhibition of these metabolic pathways indicate existence of metabolic checkpoint that need to be satisfied in order to allow neuronal differentiation. PMID:27058317

  17. Strategies to promote differentiation of newborn neurons into mature functional cells in Alzheimer brain.

    PubMed

    Schaeffer, Evelin L; Novaes, Barbara A; da Silva, Emanuelle R; Skaf, Heni D; Mendes-Neto, Alvaro G

    2009-10-01

    Adult neurogenesis occurs in the subgranular zone (SGZ) and subventricular zone (SVZ). New SGZ neurons migrate into the granule cell layer of the dentate gyrus (DG). New SVZ neurons seem to enter the association neocortex and entorhinal cortex besides the olfactory bulb. Alzheimer disease (AD) is characterized by neuron loss in the hippocampus (DG and CA1 field), entorhinal cortex, and association neocortex, which underlies the learning and memory deficits. We hypothesized that, if the AD brain can support neurogenesis, strategies to stimulate the neurogenesis process could have therapeutic value in AD. We reviewed the literature on: (a) the functional significance of adult-born neurons; (b) the occurrence of endogenous neurogenesis in AD; and (c) strategies to stimulate the adult neurogenesis process. We found that: (a) new neurons in the adult DG contribute to memory function; (b) new neurons are generated in the SGZ and SVZ of AD brains, but they fail to differentiate into mature neurons in the target regions; and (c) numerous strategies (Lithium, Glatiramer Acetate, nerve growth factor, environmental enrichment) can enhance adult neurogenesis and promote maturation of newly generated neurons. Such strategies might help to compensate for the loss of neurons and improve the memory function in AD.

  18. Thyroid Hormone-Otx2 Signaling Is Required for Embryonic Ventral Midbrain Neural Stem Cells Differentiated into Dopamine Neurons.

    PubMed

    Chen, Chunhai; Ma, Qinglong; Chen, Xiaowei; Zhong, Min; Deng, Ping; Zhu, Gang; Zhang, Yanwen; Zhang, Lei; Yang, Zhiqi; Zhang, Kuan; Guo, Lu; Wang, Liting; Yu, Zhengping; Zhou, Zhou

    2015-08-01

    Midbrain dopamine (DA) neurons are essential for maintaining multiple brain functions. These neurons have also been implicated in relation with diverse neurological disorders. However, how these neurons are developed from neuronal stem cells (NSCs) remains largely unknown. In this study, we provide both in vivo and in vitro evidence that the thyroid hormone, an important physiological factor for brain development, promotes DA neuron differentiation from embryonic ventral midbrain (VM) NSCs. We find that thyroid hormone deficiency during development reduces the midbrain DA neuron number, downregulates the expression of tyrosine hydroxylase (TH) and the dopamine transporter (DAT), and impairs the DA neuron-dependent motor behavior. In addition, thyroid hormone treatment during VM NSC differentiation in vitro increases the production of DA neurons and upregulates the expression of TH and DAT. We also found that the thyroid hormone enhances the expression of Otx2, an important determinant of DA neurogenesis, during DA neuron differentiation. Our in vitro gene silencing experiments indicate that Otx2 is required for thyroid hormone-dependent DA neuron differentiation from embryonic VM NSCs. Finally, we revealed both in vivo and in vitro that the thyroid hormone receptor alpha 1 is expressed in embryonic VM NSCs. Furthermore, it participates in the effects of thyroid hormone-induced Otx2 upregulation and DA neuron differentiation. These data demonstrate the role and molecular mechanisms of how the thyroid hormone regulates DA neuron differentiation from embryonic VM NSCs, particularly providing new mechanisms and a potential strategy for generating dopamine neurons from NSCs.

  19. Knocking down of heat-shock protein 27 directs differentiation of functional glutamatergic neurons from placenta-derived multipotent cells

    PubMed Central

    Cheng, Yu-Che; Huang, Chi-Jung; Lee, Yih-Jing; Tien, Lu-Tai; Ku, Wei-Chi; Chien, Raymond; Lee, Fa-Kung; Chien, Chih-Cheng

    2016-01-01

    This study presents human placenta-derived multipotent cells (PDMCs) as a source from which functional glutamatergic neurons can be derived. We found that the small heat-shock protein 27 (HSP27) was downregulated during the neuronal differentiation process. The in vivo temporal and spatial profiles of HSP27 expression were determined and showed inverted distributions with neuronal proteins during mouse embryonic development. Overexpression of HSP27 in stem cells led to the arrest of neuronal differentiation; however, the knockdown of HSP27 yielded a substantially enhanced ability of PDMCs to differentiate into neurons. These neurons formed synaptic networks and showed positive staining for multiple neuronal markers. Additionally, cellular phenomena including the absence of apoptosis and rare proliferation in HSP27-silenced PDMCs, combined with molecular events such as cleaved caspase-3 and the loss of stemness with cleaved Nanog, indicated that HSP27 is located upstream of neuronal differentiation and constrains that process. Furthermore, the induced neurons showed increasing intracellular calcium concentrations upon glutamate treatment. These differentiated cells co-expressed the N-methyl-D-aspartate receptor, vesicular glutamate transporter, and synaptosomal-associated protein 25 but did not show expression of tyrosine hydroxylase, choline acetyltransferase or glutamate decarboxylase 67. Therefore, we concluded that HSP27-silenced PDMCs differentiated into neurons possessing the characteristics of functional glutamatergic neurons. PMID:27444754

  20. Directed neuronal differentiation of mouse embryonic and induced pluripotent stem cells and their gene expression profiles.

    PubMed

    Chen, Xuesong; Gu, Qi; Wang, Xiang; Ma, Qingwen; Tang, Huixiang; Yan, Xiaoshuang; Guo, Xinbing; Yan, Hao; Hao, Jie; Zeng, Fanyi

    2013-07-01

    Embryonic stem cells (ESCs) may be useful as a therapeutic source of cells for the production of healthy tissue; however, they are associated with certain challenges including immunorejection as well as ethical issues. Induced pluripotent stem cells (iPSCs) are a promising substitute since a patient's own adult cells would serve as tissue precursors. Ethical concerns prevent a full evaluation of the developmental potency of human ESCs and iPSCs, therefore, mouse iPSC models are required for protocol development and safety assessments. We used a modified culturing protocol to differentiate pluripotent cells from a mouse iPS cell line and two mouse ES cell lines into neurons. Our results indicated that all three pluripotent stem cell lines underwent nearly the same differentiation process when induced to form neurons in vitro. Genomic expression microarray profiling and single-cell RT-qPCR were used to analyze the neural lineage differentiation process, and more than one thousand differentially expressed genes involved in multiple molecular processes relevant to neural development were identified.

  1. Differential vulnerability of neurons in Huntington's disease: The role of cell type-specific features

    PubMed Central

    Han, Ina; You, YiMei; Kordower, Jeffrey H.; Brady, Scott T.; Morfini, Gerardo A.

    2010-01-01

    Abnormal expansion of a polyglutamine tract in huntingtin (Htt) protein results in Huntington's disease (HD), an autosomal dominant neurodegenerative disorder involving progressive loss of motor and cognitive function. Contrasting with the ubiquitous tissue expression of polyglutamine-expanded Htt (polyQ-Htt), HD pathology is characterized by the increased vulnerability of specific neuronal populations within the striatum and the cerebral cortex. Morphological, biochemical, and functional characteristics of neurons affected in HD that might render these cells more vulnerable to the toxic effects of polyQ-Htt are covered in this review. The differential vulnerability of neurons observed in HD is discussed in the context of various major pathogenic mechanisms proposed to date, and in line with evidence showing a “dying-back” pattern of degeneration in affected neuronal populations. PMID:20236390

  2. Inner ear stem cells derived feeder layer promote directional differentiation of amniotic fluid stem cells into functional neurons.

    PubMed

    Zong, Ling; Chen, Kaitian; Zhou, Wei; Jiang, Di; Sun, Liang; Zhang, Xuemei; Jiang, Hongyan

    2014-10-01

    Intact spiral ganglion neurons are required for cochlear implantation or conventional hearing amplification as an intervention for sensorineural hearing loss. Treatment strategies to replace the loss of spiral ganglion neurons are needed. Recent reports have suggested that amniotic fluid-derived stem cells are capable of differentiating into neuron-like cells in response to cytokines and are not tumorigenic. Amniotic fluid stem cells represent a potential resource for cellular therapy of neural deafness due to spiral ganglion pathology. However, the directional differentiation of amniotic fluid stem cells is undetermined in the absence of cytokines and the consequence of inner ear supporting cells from the mouse cochlea organ of Corti on the differentiation of amniotic fluid stem cells remains to be defined. In an effort to circumvent these limitations, we investigated the effect of inner ear stem cells derived feeder layer on amniotic fluid stem cells differentiation in vitro. An inner ear stem cells derived feeder layer direct contact system was established to induce differentiation of amniotic fluid stem cells. Our results showed that inner ear stem cells derived feeder layer successfully promoted directional differentiation of amniotic fluid stem cells into neurons with characteristics of functionality. Furthermore, we showed that Wnt signaling may play an essential role in triggering neurogenesis. These findings indicate the potential use of inner ear stem cells derived feeder layer as a nerve-regenerative scaffold. A reliable and effective amniotic fluid stem cell differentiation support structure provided by inner ear stem cells derived feeder layer should contribute to efforts to translate cell-based strategies to the clinic.

  3. ALS/FTLD-linked TDP-43 regulates neurite morphology and cell survival in differentiated neurons

    SciTech Connect

    Han, Jeong-Ho; Yu, Tae-Hoon; Ryu, Hyun-Hee; Jun, Mi-Hee; Ban, Byung-Kwan; Jang, Deok-Jin; Lee, Jin-A

    2013-08-01

    Tar-DNA binding protein of 43 kDa (TDP-43) has been characterized as a major component of protein aggregates in brains with neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). However, physiological roles of TDP-43 and early cellular pathogenic effects caused by disease associated mutations in differentiated neurons are still largely unknown. Here, we investigated the physiological roles of TDP-43 and the effects of missense mutations associated with diseases in differentiated cortical neurons. The reduction of TDP-43 by siRNA increased abnormal neurites and decreased cell viability. ALS/FTLD-associated missense mutant proteins (A315T, Q331K, and M337V) were partially mislocalized to the cytosol and neurites when compared to wild-type and showed abnormal neurites similar to those observed in cases of loss of TDP-43. Interestingly, cytosolic expression of wild-type TDP-43 with mutated nuclear localization signals also induced abnormal neurtie morphology and reduction of cell viability. However, there was no significant difference in the effects of cytosolic expression in neuronal morphology and cell toxicity between wild-type and missense mutant proteins. Thus, our results suggest that mislocalization of missense mutant TDP-43 may contribute to loss of TDP-43 function and affect neuronal morphology, probably via dominant negative action before severe neurodegeneration in differentiated cortical neurons. Highlights: • The function of nuclear TDP-43 in neurite morphology in mature neurons. • Partial mislocalization of TDP-43 missense mutants into cytosol from nucleus. • Abnormal neurite morphology caused by missense mutants of TDP-43. • The effect of cytosolic expression of TDP-43 in neurite morphology and in cell survival.

  4. Denervated hippocampus provides a favorable microenvironment for neuronal differentiation of endogenous neural stem cells

    PubMed Central

    Zhang, Lei; Han, Xiao; Cheng, Xiang; Tan, Xue-feng; Zhao, He-yan; Zhang, Xin-hua

    2016-01-01

    Fimbria-fornix transection induces both exogenous and endogenous neural stem cells to differentiate into neurons in the hippocampus. This indicates that the denervated hippocampus provides an environment for neuronal differentiation of neural stem cells. However, the pathways and mechanisms in this process are still unclear. Seven days after fimbria fornix transection, our reverse transcription polymerase chain reaction, western blot assay, and enzyme linked immunosorbent assay results show a significant increase in ciliary neurotrophic factor mRNA and protein expression in the denervated hippocampus. Moreover, neural stem cells derived from hippocampi of fetal (embryonic day 17) Sprague-Dawley rats were treated with ciliary neurotrophic factor for 7 days, with an increased number of microtubule associated protein-2-positive cells and decreased number of glial fibrillary acidic protein-positive cells detected. Our results show that ciliary neurotrophic factor expression is up-regulated in the denervated hippocampus, which may promote neuronal differentiation of neural stem cells in the denervated hippocampus. PMID:27212920

  5. Expression of a plasma membrane proteolipid during differentiation of neuronal and glial cells in primary culture.

    PubMed

    Shea, T B; Fischer, I; Sapirstein, V

    1986-09-01

    Plasma membrane proteolipid protein (PM-PLP) synthesis was examined in embryonic rat neurons and neonatal rat glial cells during differentiation in culture. Glial cultures were treated with 1 mM N6, O2, dibutyryl cyclic adenosine monophosphate (dbcAMP) following confluency to induce differentiation, which resulted in the elaboration of long cellular processes. However, no changes in the biosynthetic level of PM-PLP was observed during the differentiation of these cells. Neurons differentiated spontaneously in culture, forming cellular aggregates immediately following plating and elaborating a network of neurites over 7 days. The differentiation of neurons was accompanied by a seven-fold increase in PM-PLP synthesis with increases in biosynthetic increase in PM-PLP synthesis with increases in biosynthetic rate observed between days 1 and 3 and between days 3 and 7 in culture. Ultrastructural examination of neurons indicated that the Golgi apparatus was also developing during this period of time, with an increase in both the number of lamellae and generation of vesicles. The transport of PM-PLP to the plasma membrane was therefore examined in neurons at day 7 in culture by pulse labeling experiments with monensin and colchicine. Monensin (1 microM) was found to inhibit the appearance of radiolabeled PM-PLP in the plasma membrane by 63%, indicating that a functional Golgi apparatus is required for transport of PM-PLP to its target membrane. Colchicine (125 microM) also inhibited the appearance of newly synthesized PM-PLP in the plasma membrane by greater than 40%, suggesting that microtubules may also be required for PM-PLP transport to the plasma membrane. PMID:3016181

  6. Multiparametric High Content Analysis for assessment of neurotoxicity in differentiated neuronal cell lines and human embryonic stem cell-derived neurons.

    PubMed

    Wilson, Melinda S; Graham, James R; Ball, Andrew J

    2014-05-01

    The potential for adverse neurotoxic reactions in response to therapeutics and environmental hazards continues to prompt development of novel cell-based assays to determine neurotoxic risk. A challenge remains to characterize and understand differences between assays and between neuronal cellular models in their responses to neurotoxicants if scientists are to determine the optimal model, or combination of models, for neurotoxicity screening. Most studies to date have focused on developmental neurotoxicity applications. This study reports the development of a robust multiparameter High Content Analysis (HCA) assay for neurotoxicity screening in three differentiated neuronal cell models - SH-SY5Y, PC12 and human embryonic stem cell-derived hN2™ cells. Using a multiplexed detection reagent panel (Hoechst nuclear stain; antibodies against βIII-Tubulin and phosphorylated neurofilament subunit H, and Mitotracker(®) Red CMXRos), a multiparametric HCA assay was developed and used to characterize a test set of 36 chemicals. HCA data generated were compared to data generated using MTT and LDH assays under the same assay conditions. Data showed that multiparametric High Content Analysis of differentiated neuronal cells is feasible, and represents a highly effective method for obtaining large quantities of robust data on the neurotoxic effects of compounds compared with cytotoxicity assays like MTT and LDH. Significant differences were observed between the responses to compounds across the three cellular models tested, illustrating the heterogeneity in responses to neurotoxicants across different cell types. This study provides data strongly supporting the use of cellular imaging as a tool for neurotoxicity assessment in differentiated neuronal cells, and provides novel insights into the neurotoxic effects of a test set of compounds upon differentiated neuronal cell lines and human embryonic stem cell-derived neurons.

  7. Enhanced differentiation of neural progenitor cells into neurons of the mesencephalic dopaminergic subtype on topographical patterns.

    PubMed

    Tan, Kenneth K B; Tann, Jason Y; Sathe, Sharvari R; Goh, Seok Hong; Ma, Dongliang; Goh, Eyleen L K; Yim, Evelyn K F

    2015-03-01

    Parkinson's disease (PD) is a neurodegenerative disease attributed to the loss of midbrain dopaminergic (DA) neurons. The current lack of predictive models for this disease has been hampered by the acquirement of robust cells, posing a major barrier to drug development. Differentiation of stem cells into subtype specific cells may be guided by appropriate topographical cues but the role of topography has hitherto not been well understood. We used a Multi-Architecture (MARC) chip with various topographical structures and identified three topographies, which generate DA neurons from murine hippocampal neural progenitor cells with the highest percentage of neuronal (β-III-tubulin positive) and dopaminergic (tyrosine hydroxylase positive) populations. Analysis on single pattern structures showed that 2 μm gratings with 2 μm spacing and 2 μm height (2 μm gratings) and 2 μm gratings with hierarchical structure produced cells with the highest gene expression of TH and PITX3, with the longest neurite and highest percentage of alignment. Quantitative image analysis showed the 2 μm gratings produced cells with the highest expression of pituitary homeobox 3 (PITX3), LIM homeobox transcription factor 1 alpha (LMX1a), aldehyde dehydrogenase 1 family member A1 (ALDH1a1) and microtubule associated protein 2 (MAP2), as compared to nano-gratings and unpatterned controls. These patterns also enhance DA neuron differentiation on different substrate rigidities, as seen on both poly-dimethylsiloxane (PDMS) and tissue culture polystyrene (TCPS) substrates. These results show the use of topographical influence for neuronal subtype specification, which could be translated into a wide range of clinical applications for PD. PMID:25591959

  8. Early phosphorylation of MARCKS at Ser25 in migrating precursor cells and differentiating peripheral neurons.

    PubMed

    Ruiz-Perera, Lucía M; Arruti, Cristina; Zolessi, Flavio R

    2013-06-01

    MARCKS is a ubiquitous actin-binding protein, with special functions in the development of the central nervous system. We have previously described a neuronal-specific isoform, phosphorylated at serine 25 (S25p-MARCKS), which is present very early during neuronal differentiation in the chick retina. However, very little is known about MARCKS expression or functions in the peripheral nervous system (PNS). In the present work, we analyzed migrating PNS precursor cells in the chick embryo, particularly those originating from the neural crest, and found that they all express a high amount of MARCKS and that a subpopulation of them also contained S25p-MARCKS from early developmental stages. MARCKS protein was also found in dorsal root and trigeminal ganglia during embryo development. Not only is the protein present in these structures but it is also phosphorylated in differentiating neurons with a maximal signal on the ganglion periphery, where neurogenesis is occurring. In conclusion, MARCKS is present and phosphorylated at early stages during the differentiation of PNS cells and precursors, indicating that it might also be important for the differentiation of these tissues.

  9. Early phosphorylation of MARCKS at Ser25 in migrating precursor cells and differentiating peripheral neurons.

    PubMed

    Ruiz-Perera, Lucía M; Arruti, Cristina; Zolessi, Flavio R

    2013-06-01

    MARCKS is a ubiquitous actin-binding protein, with special functions in the development of the central nervous system. We have previously described a neuronal-specific isoform, phosphorylated at serine 25 (S25p-MARCKS), which is present very early during neuronal differentiation in the chick retina. However, very little is known about MARCKS expression or functions in the peripheral nervous system (PNS). In the present work, we analyzed migrating PNS precursor cells in the chick embryo, particularly those originating from the neural crest, and found that they all express a high amount of MARCKS and that a subpopulation of them also contained S25p-MARCKS from early developmental stages. MARCKS protein was also found in dorsal root and trigeminal ganglia during embryo development. Not only is the protein present in these structures but it is also phosphorylated in differentiating neurons with a maximal signal on the ganglion periphery, where neurogenesis is occurring. In conclusion, MARCKS is present and phosphorylated at early stages during the differentiation of PNS cells and precursors, indicating that it might also be important for the differentiation of these tissues. PMID:23470634

  10. Phospholipase D1 increases Bcl-2 expression during neuronal differentiation of rat neural stem cells.

    PubMed

    Park, Shin-Young; Ma, Weina; Yoon, Sung Nyo; Kang, Min Jeong; Han, Joong-Soo

    2015-01-01

    We studied the possible role of phospholipase D1 (PLD1) in the neuronal differentiation, including neurite formation of neural stem cells. PLD1 protein and PLD activity increased during neuronal differentiation. Bcl-2 also increased. Downregulation of PLD1 by transfection with PLD1 siRNA or a dominant-negative form of PLD1 (DN-PLD1) inhibited both neurite outgrowth and Bcl-2 expression. PLD activity was dramatically reduced by a PLCγ (phospholipase Cγ) inhibitor (U73122), a Ca(2+)chelator (BAPTA-AM), and a PKCα (protein kinase Cα) inhibitor (RO320432). Furthermore, treatment with arachidonic acid (AA) which is generated by the action of PLA2 (phospholipase A2) on phosphatidic acid (a PLD1 product), increased the phosphorylation of p38 MAPK and CREB, as well as Bcl-2 expression, indicating that PLA2 is involved in the differentiation process resulting from PLD1 activation. PGE2 (prostaglandin E2), a cyclooxygenase product of AA, also increased during neuronal differentiation. Moreover, treatment with PGE2 increased the phosphorylation of p38 MAPK and CREB, as well as Bcl-2 expression, and this effect was inhibited by a PKA inhibitor (Rp-cAMP). As expected, inhibition of p38 MAPK resulted in loss of CREB activity, and when CREB activity was blocked with CREB siRNA, Bcl-2 production also decreased. We also showed that the EP4 receptor was required for the PKA/p38MAPK/CREB/Bcl-2 pathway. Taken together, these observations indicate that PLD1 is activated by PLCγ/PKCα signaling and stimulate Bcl-2 expression through PLA2/Cox2/EP4/PKA/p38MAPK/CREB during neuronal differentiation of rat neural stem cells.

  11. MicroRNA-125b promotes neuronal differentiation in human cells by repressing multiple targets.

    PubMed

    Le, Minh T N; Xie, Huangming; Zhou, Beiyan; Chia, Poh Hui; Rizk, Pamela; Um, Moonkyoung; Udolph, Gerald; Yang, Henry; Lim, Bing; Lodish, Harvey F

    2009-10-01

    MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression at the posttranscriptional level. Research on miRNAs has highlighted their importance in neural development, but the specific functions of neurally enriched miRNAs remain poorly understood. We report here the expression profile of miRNAs during neuronal differentiation in the human neuroblastoma cell line SH-SY5Y. Six miRNAs were significantly upregulated during differentiation induced by all-trans-retinoic acid and brain-derived neurotrophic factor. We demonstrated that the ectopic expression of either miR-124a or miR-125b increases the percentage of differentiated SH-SY5Y cells with neurite outgrowth. Subsequently, we focused our functional analysis on miR-125b and demonstrated the important role of this miRNA in both the spontaneous and induced differentiations of SH-SH5Y cells. miR-125b is also upregulated during the differentiation of human neural progenitor ReNcell VM cells, and miR-125b ectopic expression significantly promotes the neurite outgrowth of these cells. To identify the targets of miR-125b regulation, we profiled the global changes in gene expression following miR-125b ectopic expression in SH-SY5Y cells. miR-125b represses 164 genes that contain the seed match sequence of the miRNA and/or that are predicted to be direct targets of miR-125b by conventional methods. Pathway analysis suggests that a subset of miR-125b-repressed targets antagonizes neuronal genes in several neurogenic pathways, thereby mediating the positive effect of miR-125b on neuronal differentiation. We have further validated the binding of miR-125b to the miRNA response elements of 10 selected mRNA targets. Together, we report here for the first time the important role of miR-125b in human neuronal differentiation.

  12. Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons.

    PubMed

    Wang, Shuyan; Wang, Bin; Pan, Na; Fu, Linlin; Wang, Chaodong; Song, Gongru; An, Jing; Liu, Zhongfeng; Zhu, Wanwan; Guan, Yunqian; Xu, Zhi-Qing David; Chan, Piu; Chen, Zhiguo; Zhang, Y Alex

    2015-01-01

    It remains a challenge to differentiate human induced pluripotent stem cells (iPSCs) or embryonic stem (ES) cells to Purkinje cells. In this study, we derived iPSCs from human fibroblasts and directed the specification of iPSCs first to Purkinje progenitors, by adding Fgf2 and insulin to the embryoid bodies (EBs) in a time-sensitive manner, which activates the endogenous production of Wnt1 and Fgf8 from EBs that further patterned the cells towards a midbrain-hindbrain-boundary tissue identity. Neph3-positive human Purkinje progenitors were sorted out by using flow cytometry and cultured either alone or with granule cell precursors, in a 2-dimensional or 3-dimensional environment. However, Purkinje progenitors failed to mature further under above conditions. By co-culturing human Purkinje progenitors with rat cerebellar slices, we observed mature Purkinje-like cells with right morphology and marker expression patterns, which yet showed no appropriate membrane properties. Co-culture with human fetal cerebellar slices drove the progenitors to not only morphologically correct but also electrophysiologically functional Purkinje neurons. Neph3-posotive human cells could also survive transplantation into the cerebellum of newborn immunodeficient mice and differentiate to L7- and Calbindin-positive neurons. Obtaining mature human Purkinje cells in vitro has significant implications in studying the mechanisms of spinocerebellar ataxias and other cerebellar diseases. PMID:25782665

  13. Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons

    PubMed Central

    Wang, Shuyan; Wang, Bin; Pan, Na; Fu, Linlin; Wang, Chaodong; Song, Gongru; An, Jing; Liu, Zhongfeng; Zhu, Wanwan; Guan, Yunqian; Xu, Zhi-Qing David; Chan, Piu; Chen, Zhiguo; Zhang, Y. Alex

    2015-01-01

    It remains a challenge to differentiate human induced pluripotent stem cells (iPSCs) or embryonic stem (ES) cells to Purkinje cells. In this study, we derived iPSCs from human fibroblasts and directed the specification of iPSCs first to Purkinje progenitors, by adding Fgf2 and insulin to the embryoid bodies (EBs) in a time-sensitive manner, which activates the endogenous production of Wnt1 and Fgf8 from EBs that further patterned the cells towards a midbrain-hindbrain-boundary tissue identity. Neph3-positive human Purkinje progenitors were sorted out by using flow cytometry and cultured either alone or with granule cell precursors, in a 2-dimensional or 3-dimensional environment. However, Purkinje progenitors failed to mature further under above conditions. By co-culturing human Purkinje progenitors with rat cerebellar slices, we observed mature Purkinje-like cells with right morphology and marker expression patterns, which yet showed no appropriate membrane properties. Co-culture with human fetal cerebellar slices drove the progenitors to not only morphologically correct but also electrophysiologically functional Purkinje neurons. Neph3-posotive human cells could also survive transplantation into the cerebellum of newborn immunodeficient mice and differentiate to L7- and Calbindin-positive neurons. Obtaining mature human Purkinje cells in vitro has significant implications in studying the mechanisms of spinocerebellar ataxias and other cerebellar diseases. PMID:25782665

  14. Safrole oxide induced neuronal differentiation of rat bone-marrow mesenchymal stem cells by elevating Hsp70.

    PubMed

    Zhao, YanChun; Xin, Jie; Sun, ChunHui; Zhao, BaoXiang; Zhao, Jing; Su, Le

    2012-11-01

    In a previous study, we found that at low concentrations, safrole oxide (SFO) could induce vascular endothelial cell (VEC) transdifferentiation into neuron-like cells; however, whether SFO could induce bone-marrow mesenchymal stem cell (BMSC) neural differentiation was unknown. Here, we found that SFO could effectively induce BMSC neural differentiation in the presence of serum and fibroblast growth factor 2 and did not affect cell viability at low concentrations. The levels of neuron-specific enolase and neurofilament-L were increased greatly, but that of glial fibrillary acidic protein was absent with SFO treatment for 48h. Furthermore, SFO could increase the level of heat shock protein 70 (Hsp70), an important factor in neuronal differentiation. Knockdown of Hsp70 by its small interfering RNA blocked SFO-induced BMSC differentiation. Thus, SFO is a novel inducer of BMSC differentiation to neuron-like cells and Hsp70 is implicated in the differentiation process. We provide a new tool for obtaining neuron-like cells from BMSCs and for further investigating the new effect of Hsp70 on BMSC neuronal differentiation.

  15. Regional Differentiation of Retinoic Acid-Induced Human Pluripotent Embryonic Carcinoma Stem Cell Neurons

    PubMed Central

    Coyle, Dennis E.; Li, Jie; Baccei, Mark

    2011-01-01

    The NTERA2 cl D1 (NT2) cell line, derived from human teratocarcinoma, exhibits similar properties as embryonic stem (ES) cells or very early neuroepitheial progenitors. NT2 cells can be induced to become postmitotic central nervous system neurons (NT2N) with retinoic acid. Although neurons derived from pluripotent cells, such as NT2N, have been characterized for their neurotransmitter phenotypes, their potential suitability as a donor source for neural transplantation also depends on their ability to respond to localized environmental cues from a specific region of the CNS. Therefore, our study aimed to characterize the regional transcription factors that define the rostocaudal and dorsoventral identity of NT2N derived from a monolayer differentiation paradigm using quantitative PCR (qPCR). Purified NT2N mainly expressed both GABAergic and glutamatergic phenotypes and were electrically active but did not form functional synapses. The presence of immature astrocytes and possible radial glial cells was noted. The NT2N expressed a regional transcription factor code consistent with forebrain, hindbrain and spinal cord neural progenitors but showed minimal expression of midbrain phenotypes. In the dorsoventral plane NT2N expressed both dorsal and ventral neural progenitors. Of major interest was that even under the influence of retinoic acid, a known caudalization factor, the NT2N population maintained a rostral phenotype subpopulation which expressed cortical regional transcription factors. It is proposed that understanding the regional differentiation bias of neurons derived from pluripotent stem cells will facilitate their successful integration into existing neuronal networks within the CNS. PMID:21283767

  16. Synergistic effects of CoCl(2) and ROCK inhibition on mesenchymal stem cell differentiation into neuron-like cells.

    PubMed

    Pacary, Emilie; Legros, Hélène; Valable, Samuel; Duchatelle, Pascal; Lecocq, Myriam; Petit, Edwige; Nicole, Olivier; Bernaudin, Myriam

    2006-07-01

    Bone-marrow-derived mesenchymal stem cells (MSCs) constitute an interesting cellular source to promote brain regeneration after neurodegenerative diseases. Recently, several studies suggested that oxygen-dependent gene expression is of crucial importance in governing the essential steps of neurogenesis such as cell proliferation, survival and differentiation. In this context, we analysed the effect of the HIF-1 (hypoxia inducible factor-1) activation-mimicking agent CoCl(2) on MSCs. CoCl(2) treatment increased the expression of the anti-proliferative gene BTG2/PC3 and decreased cyclin D1 expression. Expression of HIF-1alpha and its target genes EPO, VEGF and p21 was also upregulated. These changes were followed by inhibition of cell proliferation and morphological changes resulting in neuron-like cells, which had increased neuronal marker expression and responded to neurotransmitters. Echinomycin, a molecule inhibiting HIF-1 DNA-binding activity, blocked the CoCl(2) effect on MSCs. Additionally, by using Y-27632, we demonstrated that Rho kinase (ROCK) inhibition potentiated CoCl(2)-induced MSC differentiation in particular into dopaminergic neuron-like cells as attested by its effect on tyrosine hydroxylase expression. Altogether, these results support the ability of MSCs to differentiate into neuron-like cells in response to CoCl(2), an effect that might act, in part, through HIF-1 activation and cell-cycle arrest, and which is potentiated by inhibition of ROCK.

  17. Neuron-like differentiation of mesenchymal stem cells on silicon nanowires

    NASA Astrophysics Data System (ADS)

    Kim, Hyunju; Kim, Ilsoo; Choi, Heon-Jin; Kim, So Yeon; Yang, Eun Gyeong

    2015-10-01

    The behavior of mammalian cells on vertical nanowire (NW) arrays, including cell spreading and the dynamic distribution of focal adhesions and cytoskeletal proteins, has been intensively studied to extend the implications for cellular manipulations in vitro. Prompted by the result that cells on silicon (Si) NWs showed morphological changes and reduced migration rates, we have explored the transition of mesenchymal stem cells into a neuronal lineage by using SiNWs with varying lengths. When human mesenchymal stem cells (hMSCs) were cultured on the longest SiNWs for 3 days, most of the cells exhibited elongated shapes with neurite-like extensions and dot-like focal adhesions that were prominently observed along with actin filaments. Under these circumstances, the cell motility analyzed by live cell imaging was found to decrease due to the presence of SiNWs. In addition, the slowed growth rate, as well as the reduced population of S phase cells, suggested that the cell cycle was likely arrested in response to the differentiation process. Furthermore, we measured the mRNA levels of several lineage-specific markers to confirm that the SiNWs actually induced neuron-like differentiation of the hMSCs while hampering their osteogenic differentiation. Taken together, our results implied that SiNWs were capable of inducing active reorganization of cellular behaviors, collectively guiding the fate of hMSCs into the neural lineage even in the absence of any inducing reagent.The behavior of mammalian cells on vertical nanowire (NW) arrays, including cell spreading and the dynamic distribution of focal adhesions and cytoskeletal proteins, has been intensively studied to extend the implications for cellular manipulations in vitro. Prompted by the result that cells on silicon (Si) NWs showed morphological changes and reduced migration rates, we have explored the transition of mesenchymal stem cells into a neuronal lineage by using SiNWs with varying lengths. When human mesenchymal

  18. Tracking neuronal marker expression inside living differentiating cells using molecular beacons.

    PubMed

    Ilieva, Mirolyuba; Della Vedova, Paolo; Hansen, Ole; Dufva, Martin

    2013-12-19

    Monitoring gene expression is an important tool for elucidating mechanisms of cellular function. In order to monitor gene expression during nerve cell development, molecular beacon (MB) probes targeting markers representing different stages of neuronal differentiation were designed and synthesized as 2'-O-methyl RNA backbone oligonucleotides. MBs were transfected into human mesencephalic cells (LUHMES) using streptolysin-O-based membrane permeabilization. Mathematical modeling, simulations and experiments indicated that MB concentration was equal to the MB in the transfection medium after 10 min transfection. The cells will then each contain about 60,000 MBs. Gene expression was detected at different time points using fluorescence microscopy. Nestin and NeuN mRNA were expressed in approximately 35% of the LUHMES cells grown in growth medium, and in 80-90% of cells after differentiation. MAP2 and tyrosine hydroxylase mRNAs were expressed 2 and 3 days post induction of differentiation, respectively. Oct 4 was not detected with MB in these cells and signal was not increased over time suggesting that MB are generally stable inside the cells. The gene expression changes measured using MBs were confirmed using qRT-PCR. These results suggest that MBs are simple to use sensors inside living cell, and particularly useful for studying dynamic gene expression in heterogeneous cell populations.

  19. Tetramethylpyrazine promotes SH-SY5Y cell differentiation into neurons through epigenetic regulation of Topoisomerase IIβ.

    PubMed

    Yan, Y; Zhao, J; Cao, C; Jia, Z; Zhou, N; Han, S; Wang, Y; Xu, Y; Zhao, J; Yan, Y; Cui, H

    2014-10-10

    Tetramethylpyrazine (TMP) is an active compound extracted from the traditional Chinese medicinal herb Chuanxiong. Recently, it has been reported that TMP enhances neurogenesis, and promotes neural stem cell differentiation toward neurons. However, its molecular basis remains unknown. Topoisomerase IIβ (TopoIIβ) is a nuclear enzyme with an essential role in neuronal development. This study aimed to investigate whether TopoIIβ is involved in TMP-induced neuronal differentiation. We examined the effect of TMP on neuronal differentiation of SH-SY5Y cells. It was found that TMP inhibited cell proliferation and induced G0/G1 cell cycle arrest. TMP promoted SH-SY5Y cells to differentiate toward post-mitotic neurons characterized by long, out-branched neurites and up-regulated neuronal markers, microtubule-associated protein 2 (MAP2) and tau. Meanwhile, we demonstrated that TopoIIβ was highly expressed following TMP treatment. To unravel how TMP affects TopoIIβ expression, two chromatin active markers, acetylated histone H3 (Ac-H3) and acetylated histone H4 (Ac-H4) were examined in this study. Our data showed that the levels of Ac-H3 and Ac-H4 were positively correlated with TopoIIβ expression in the processes of neuronal differentiation. We further performed chromatin immunoprecipitation (ChIP) analysis and identified that TMP enhanced the recruitment of Ac-H3 and Ac-H4 to the TopoIIβ gene promoter region. Therefore, we concluded that TMP may stimulate neuronal differentiation of SH-SY5Y cells through epigenetic regulation of TopoIIβ. These results suggest a novel molecular mechanism underlying TMP-promoted neuronal differentiation.

  20. Microsphere-Incorporated Hybrid Thermogel for Neuronal Differentiation of Tonsil Derived Mesenchymal Stem Cells.

    PubMed

    Patel, Madhumita; Moon, Hyo Jung; Jung, Bo Kyung; Jeong, Byeongmoon

    2015-07-15

    Neuronal differentiation of tonsil-derived mesenchymal stem cells (TMSCs) is investigated in a 3D hybrid system. The hybrid system is prepared by increasing the temperature of poly(ethylene glycol)-poly(l-alanine) aqueous solution to 37 °C through the heat-induced sol-to-gel transition, in which TMSCs and growth factor releasing microspheres are suspended. The in situ formed gel exhibits a modulus of 800 Pa at 37 °C, similar to that of brain tissue, and it is robust enough to hold the microspheres and cells during the 3D culture of TMSCs. The neuronal growth factors are released over 12-18 d, and the TMSCs in a spherical shape initially undergo multipolar elongation during the 3D culture. Significantly higher expressions of the neuronal biomarkers such as nuclear receptor related protein (Nurr-1), neuron specific enolase, microtubule associated protein-2, neurofilament-M, and glial fibrillary acidic protein are observed in both mRNA level and protein level in the hybrid systems than in the control experiments. This study proves the significance of a controlled drug delivery concept in tissue engineering or regenerative medicine, and a 3D hybrid system with controlled release of growth factors from microspheres in a thermogel can be a very promising tool. PMID:26033880

  1. Microsphere-Incorporated Hybrid Thermogel for Neuronal Differentiation of Tonsil Derived Mesenchymal Stem Cells.

    PubMed

    Patel, Madhumita; Moon, Hyo Jung; Jung, Bo Kyung; Jeong, Byeongmoon

    2015-07-15

    Neuronal differentiation of tonsil-derived mesenchymal stem cells (TMSCs) is investigated in a 3D hybrid system. The hybrid system is prepared by increasing the temperature of poly(ethylene glycol)-poly(l-alanine) aqueous solution to 37 °C through the heat-induced sol-to-gel transition, in which TMSCs and growth factor releasing microspheres are suspended. The in situ formed gel exhibits a modulus of 800 Pa at 37 °C, similar to that of brain tissue, and it is robust enough to hold the microspheres and cells during the 3D culture of TMSCs. The neuronal growth factors are released over 12-18 d, and the TMSCs in a spherical shape initially undergo multipolar elongation during the 3D culture. Significantly higher expressions of the neuronal biomarkers such as nuclear receptor related protein (Nurr-1), neuron specific enolase, microtubule associated protein-2, neurofilament-M, and glial fibrillary acidic protein are observed in both mRNA level and protein level in the hybrid systems than in the control experiments. This study proves the significance of a controlled drug delivery concept in tissue engineering or regenerative medicine, and a 3D hybrid system with controlled release of growth factors from microspheres in a thermogel can be a very promising tool.

  2. Differentiation of human gingival mesenchymal stem cells into neuronal lineages in 3D bioconjugated injectable protein hydrogel construct for the management of neuronal disorder

    PubMed Central

    Rao, Suresh Ranga; Subbarayan, Rajasekaran; Dinesh, Murugan Girija; Arumugam, Gnanamani; Raja, Selvaraj Thirupathi Kumara

    2016-01-01

    The success of regeneration attempt is based on an ideal combination of stem cells, scaffolding and growth factors. Tissue constructs help to maintain stem cells in a required area for a desired time. There is a need for easily obtainable cells, potentially autologous stem cells and a biologically acceptable scaffold for use in humans in different difficult situations. This study aims to address these issues utilizing a unique combination of stem cells from gingiva and a hydrogel scaffold, based on a natural product for regenerative application. Human gingival mesenchymal stem cells (HGMSCs) were, with due induction, differentiated to neuronal lineages to overcome the problems associated with birth tissue-related stem cells. The differentiation potential of neuronal lineages was confirmed with suitable specific markers. The properties of mesenchymal stem cells in encapsulated form were observed to be similar to free cells. The encapsulated cells (3D) were then subjected to differentiation into neuronal lineages with suitable inducers, and the morphology and gene expression of transient cells were analyzed. HGMSCs was differentiated into neuronal lineages as both free and encapsulated forms without any significant differences. The presence of Nissl bodies and the neurite outgrowth confirm the differentiation. The advantages of this new combination appear to make it a promising tissue construct for translational application. PMID:26869025

  3. Cell-Specific Fine-Tuning of Neuronal Excitability by Differential Expression of Modulator Protein Isoforms

    PubMed Central

    Jepson, James; Sheldon, Amanda; Shahidullah, Mohammad; Fei, Hong; Koh, Kyunghee

    2013-01-01

    SLOB (SLOWPOKE-binding protein) modulates the Drosophila SLOWPOKE calcium-activated potassium channel. We have shown previously that SLOB deletion or RNAi knockdown decreases excitability of neurosecretory pars intercerebralis (PI) neurons in the adult Drosophila brain. In contrast, we found that SLOB deletion/knockdown enhances neurotransmitter release from motor neurons at the fly larval neuromuscular junction, suggesting an increase in excitability. Because two prominent SLOB isoforms, SLOB57 and SLOB71, modulate SLOWPOKE channels in opposite directions in vitro, we investigated whether divergent expression patterns of these two isoforms might underlie the differential modulation of excitability in PI and motor neurons. By performing detailed in vitro and in vivo analysis, we found strikingly different modes of regulatory control by the slob57 and slob71 promoters. The slob71, but not slob57, promoter contains binding sites for the Hunchback and Mirror transcriptional repressors. Furthermore, several core promoter elements that are absent in the slob57 promoter coordinately drive robust expression of a luciferase vector by the slob71 promoter in vitro. In addition, we visualized the expression patterns of the slob57 and slob71 promoters in vivo and found clear spatiotemporal differences in promoter activity. SLOB57 is expressed prominently in adult PI neurons, whereas larval motor neurons exclusively express SLOB71. In contrast, at the larval neuromuscular junction, SLOB57 expression appears to be restricted mainly to a subset of glial cells. Our results illustrate how the use of alternative transcriptional start sites within an ion channel modulator locus coupled with functionally relevant alternative splicing can be used to fine-tune neuronal excitability in a cell-specific manner. PMID:24133277

  4. Pleiotrophin is involved in the amniotic epithelial cell-induced differentiation of human umbilical cord blood-derived mesenchymal stem cells into dopaminergic neuron-like cells.

    PubMed

    Yang, Shu; Xue, Dan-Dan; Wu, Bo; Sun, Hai-Mei; Li, Xiao-Shuang; Dong, Fang; Li, Wen-Shuai; Ji, Feng-Qing; Zhou, De-Shan

    2013-02-28

    We have reported that human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) are capable of differentiating into dopaminergic (DA) neuron-like cells upon being induced by amniotic epithelial cells (AECs). However, what factor(s) is involved in the differentiation process has not been explored out thoroughly. Because pleiotrophin (PTN) is known to exert important trophic effects on DA neurons, in the present study, we investigated whether PTN is released by AECs and whether it is involved in the differentiation of hUCB-MSCs into DA neuron-like cells. The expression and secretion of PTN by AECs were detected by immunofluorescence, RT-PCR and ELISA. The hUCB-MSCs were isolated and treated with AEC-conditioned medium (ACM) or recombinant human PTN. Compared to the controls, a higher proportion of treated cells differentiated into DA neuron-like cells, indicated by the increased expression of TH and DAT and the increased dopamine content. These results indicate that PTN released by AECs acts as a synergetic factor with other neurotrophic factors and is involved in the differentiation of hUCB-MSCs into DA neuron-like cells. We suggest that ACM, which contains PTN and other neurotrophic factors, could potentially be used as an agent to promote the differentiation of DA neuron-like cells from hUCB-MSCs for cell therapy of Parkinson's disease without creating legal or ethical issues.

  5. Conditional induction of Math1 specifies embryonic stem cells to cerebellar granule neuron lineage and promotes differentiation into mature granule neurons.

    PubMed

    Srivastava, Rupali; Kumar, Manoj; Peineau, Stéphane; Csaba, Zsolt; Mani, Shyamala; Gressens, Pierre; El Ghouzzi, Vincent

    2013-04-01

    Directing differentiation of embryonic stem cells (ESCs) to specific neuronal subtype is critical for modeling disease pathology in vitro. An attractive means of action would be to combine regulatory differentiation factors and extrinsic inductive signals added to the culture medium. In this study, we have generated mature cerebellar granule neurons by combining a temporally controlled transient expression of Math1, a master gene in granule neuron differentiation, with inductive extrinsic factors involved in cerebellar development. Using a Tetracyclin-On transactivation system, we overexpressed Math1 at various stages of ESCs differentiation and found that the yield of progenitors was considerably increased when Math1 was induced during embryonic body stage. Math1 triggered expression of Mbh1 and Mbh2, two target genes directly involved in granule neuron precursor formation and strong expression of early cerebellar territory markers En1 and NeuroD1. Three weeks after induction, we observed a decrease in the number of glial cells and an increase in that of neurons albeit still immature. Combining Math1 induction with extrinsic factors specifically increased the number of neurons that expressed Pde1c, Zic1, and GABAα6R characteristic of mature granule neurons, formed "T-shaped" axons typical of granule neurons, and generated synaptic contacts and action potentials in vitro. Finally, in vivo implantation of Math1-induced progenitors into young adult mice resulted in cell migration and settling of newly generated neurons in the cerebellum. These results show that conditional induction of Math1 drives ESCs toward the cerebellar fate and indicate that acting on both intrinsic and extrinsic factors is a powerful means to modulate ESCs differentiation and maturation into a specific neuronal lineage.

  6. EVALUATION OF PROTEIN MARKERS FOR NEURONAL DIFFERENTIATION IN PC12 CELLS.

    EPA Science Inventory

    Chemical-induced injury of the developing nervous system can be manifested as a change in the differentiation or growth of neurons. The present study evaluated the use of proteins associated with axonal growth and synaptogenesis as markers for neuronal differentiation in vitro. ...

  7. Zhichan decoction induces differentiation of dopaminergic neurons in Parkinson's disease rats after neural stem cell transplantation

    PubMed Central

    Shi, Huifen; Song, Jie; Yang, Xuming

    2014-01-01

    The goal of this study was to increase the dopamine content and reduce dopaminergic metabolites in the brain of Parkinson's disease rats. Using high-performance liquid chromatography, we found that dopamine and dopaminergic metabolite (dihydroxyphenylacetic acid and homovanillic acid) content in the midbrain of Parkinson's disease rats was increased after neural stem cell transplantation + Zhichan decoction, compared with neural stem cell transplantation alone. Our genetic algorithm results show that dihydroxyphenylacetic acid and homovanillic acid levels achieve global optimization. Neural stem cell transplantation + Zhichan decoction increased dihydroxyphenylacetic acid levels up to 10-fold, while transplantation alone resulted in a 3-fold increment. Homovanillic acid levels showed no apparent change. Our experimental findings show that after neural stem cell transplantation in Parkinson's disease rats, Zhichan decoction can promote differentiation of neural stem cells into dopaminergic neurons. PMID:25206914

  8. BM88 is an early marker of proliferating precursor cells that will differentiate into the neuronal lineage.

    PubMed

    Koutmani, Yassemi; Hurel, Catherine; Patsavoudi, Evangelia; Hack, Michael; Gotz, Magdalena; Thomaidou, Dimitra; Matsas, Rebecca

    2004-11-01

    Progression of progenitor cells towards neuronal differentiation is tightly linked with cell cycle control and the switch from proliferative to neuron-generating divisions. We have previously shown that the neuronal protein BM88 drives neuroblastoma cells towards exit from the cell cycle and differentiation into a neuronal phenotype in vitro. Here, we explored the role of BM88 during neuronal birth, cell cycle exit and the initiation of differentiation in vivo. By double- and triple-labelling with the S-phase marker BrdU or the late G2 and M-phase marker cyclin B1, antibodies to BM88 and markers of the neuronal or glial cell lineages, we demonstrate that in the rodent forebrain, BM88 is expressed in multipotential progenitor cells before terminal mitosis and in their neuronal progeny during the neurogenic interval, as well as in the adult. Further, we defined at E16 a cohort of proliferative progenitors that exit S phase in synchrony, and by following their fate for 24 h we show that BM88 is associated with the dynamics of neuron-generating divisions. Expression of BM88 was also evident in cycling cortical radial glial cells, which constitute the main neurogenic population in the cerebral cortex. In agreement, BM88 expression was markedly reduced and restricted to a smaller percentage of cells in the cerebral cortex of the Small eye mutant mice, which lack functional Pax6 and exhibit severe neurogenesis defects. Our data show an interesting correlation between BM88 expression and the progression of progenitor cells towards neuronal differentiation during the neurogenic interval.

  9. Overexpression of glial cell line-derived neurotrophic factor induces genes regulating migration and differentiation of neuronal progenitor cells.

    PubMed

    Pahnke, Jens; Mix, Eilhard; Knoblich, Rupert; Müller, Jana; Zschiesche, Marlies; Schubert, Beke; Koczan, Dirk; Bauer, Peter; Böttcher, Tobias; Thiesen, Hans-Jürgen; Lazarov, Ludmil; Wree, Andreas; Rolfs, Arndt

    2004-07-15

    The glial cell line-derived neurotrophic factor (GDNF) is involved in the development and maintenance of neural tissues. Mutations in components of its signaling pathway lead to severe migration deficits of neuronal crest stem cells, tumor formation, or ablation of the urinary system. In animal models of Parkinson's disease, GDNF has been recognized to be neuroprotective and to improve motor function when delivered into the cerebral ventricles or into the substantia nigra. Here, we characterize the network of 43 genes induced by GDNF overproduction of neuronal progenitor cells (ST14A), which mainly regulate migration and differentiation of neuronal progenitor cells. GDNF down-regulates doublecortin, Paf-ah1b (Lis1), dynamin, and alpha-tubulin, which are involved in neocortical lamination and cytoskeletal reorganization. Axonal guidance depends on cell-surface molecules and extracellular matrix proteins. Laminin, Mpl3, Alcam, Bin1, Id1, Id2, Id3, neuregulin1, the ephrinB2-receptor, neuritin, focal adhesion kinase (FAK), Tc10, Pdpk1, clusterin, GTP-cyclooxygenase1, and follistatin are genes up-regulated by GDNF overexpression. Moreover, we found four key enzymes of the cholesterol-synthesis pathway to be down-regulated leading to decreased farnesyl-pyrophospate production. Many proteins are anchored by farnesyl-derivates at the cell membrane. The identification of these GDNF-regulated genes may open new opportunities for directly influencing differentiation and developmental processes of neurons. PMID:15212950

  10. Inhibition of mouse GPM6A expression leads to decreased differentiation of neurons derived from mouse embryonic stem cells.

    PubMed

    Michibata, Hideo; Okuno, Tsuyoshi; Konishi, Nae; Wakimoto, Koji; Kyono, Kiyoshi; Aoki, Kan; Kondo, Yasushi; Takata, Kazuyuki; Kitamura, Yoshihisa; Taniguchi, Takashi

    2008-08-01

    Glycoprotein M6A (GPM6A) is known as a transmembrane protein and an abundant cell surface protein on neurons in the central nervous system (CNS). However, the function of GPM6A is still unknown in the differentiation of neurons derived from embryonic stem (ES) cells. To investigate the function of GPM6A, we generated knockdown mouse ES cell lines (D3m-shM6A) using a short hairpin RNA (shRNA) expression vector driven by the U6 small nuclear RNA promoter, which can significantly suppress the expression of mouse GPM6A mRNA. Real-time polymerase chain reaction (real-time PCR) and immunocytochemical analysis showed that expression of shRNA against GPM6A markedly reduced the expression of neuroectodermal-associated genes (OTX1, Lmx1b, En1, Pax2, Pax5, Sox1, Sox2, and Wnt1), and also the number of neural stem cells (NSC) derived from D3mshM6A cells compared to control vector-transfected mouse ES cells (D3m-Mock). Moreover, our results show a decrease in both the number of neuronal markers and the number of differentiating neuronal cells (cholinergic, catecholaminergic, and GABAergic neurons) from NSC in D3m-shM6A cells. Hence, our findings suggest that expression level of GPM6A is directly or indirectly associated with the differentiation of neurons derived from undifferentiated ES cells. PMID:18522499

  11. Taurine Protected Against the Impairments of Neural Stem Cell Differentiated Neurons Induced by Oxygen-Glucose Deprivation.

    PubMed

    Xiao, Bo; Liu, Huazhen; Gu, Zeyun; Liu, Sining; Ji, Cheng

    2015-11-01

    Cell transplantation of neural stem cells (NSCs) is a promising approach for neurological recovery both structurally and functionally. However, one big obstacle is to promote differentiation of NSCs into neurons and the followed maturation. In the present study, we aimed to investigate the protective effect of taurine on the differentiation of NSCs and subsequent maturation of their neuronal lineage, when exposed to oxygen-glucose deprivation (OGD). The results suggested that taurine (5-20 mM) promoted the viability and proliferation of NSCs, and it protected against 8 h of OGD induced impairments. Furthermore, 20 mM taurine promoted NSCs to differentiate into neurons after 7 days of culture, and it also protected against the suppressive impairments of 8 h of OGD. Consistently, taurine (20 mM) promoted the neurite sprouting and outgrowth of the NSC differentiated neurons after 14 days of differentiation, which were significantly inhibited by OGD (8 h). At D21, the mushroom spines and spine density were promoted or restored by 20 mM taurine. Taken together, the enhanced viability and proliferation of NSCs, more differentiated neurons and the promoted maturation of neurons by 20 mM taurine support its therapeutic application during stem cell therapy to enhance neurological recovery. Moreover, it protected against the impairments induced by OGD, which may highlight its role for a more direct therapeutic application especially in an ischemic stroke environment. PMID:26415593

  12. Prenatal exposure of ethanol induces increased glutamatergic neuronal differentiation of neural progenitor cells

    PubMed Central

    2010-01-01

    Background Prenatal ethanol exposure during pregnancy induces a spectrum of mental and physical disorders called fetal alcohol spectrum disorder (FASD). The central nervous system is the main organ influenced by FASD, and neurological symptoms include mental retardation, learning abnormalities, hyperactivity and seizure susceptibility in childhood along with the microcephaly. In this study, we examined whether ethanol exposure adversely affects the proliferation of NPC and de-regulates the normal ratio between glutamatergic and GABAergic neuronal differentiation using primary neural progenitor culture (NPC) and in vivo FASD models. Methods Neural progenitor cells were cultured from E14 embryo brain of Sprague-Dawley rat. Pregnant mice and rats were treated with ethanol (2 or 4 g/kg/day) diluted with normal saline from E7 to E16 for in vivo FASD animal models. Expression level of proteins was investigated by western blot analysis and immunocytochemical assays. MTT was used for cell viability. Proliferative activity of NPCs was identified by BrdU incorporation, immunocytochemistry and FACS analysis. Results Reduced proliferation of NPCs by ethanol was demonstrated using BrdU incorporation, immunocytochemistry and FACS analysis. In addition, ethanol induced the imbalance between glutamatergic and GABAergic neuronal differentiation via transient increase in the expression of Pax6, Ngn2 and NeuroD with concomitant decrease in the expression of Mash1. Similar pattern of expression of those transcription factors was observed using an in vivo model of FASD as well as the increased expression of PSD-95 and decreased expression of GAD67. Conclusions These results suggest that ethanol induces hyper-differentiation of glutamatergic neuron through Pax6 pathway, which may underlie the hyper-excitability phenotype such as hyperactivity or seizure susceptibility in FASD patients. PMID:21073715

  13. Hbp1 regulates the timing of neuronal differentiation during cortical development by controlling cell cycle progression.

    PubMed

    Watanabe, Naoki; Kageyama, Ryoichiro; Ohtsuka, Toshiyuki

    2015-07-01

    In the developing mammalian brain, neural stem cells (NSCs) initially expand the progenitor pool by symmetric divisions. NSCs then shift from symmetric to asymmetric division and commence neurogenesis. Although the precise mechanisms regulating the developmental timing of this transition have not been fully elucidated, gradual elongation in the length of the cell cycle and coinciding accumulation of determinants that promote neuronal differentiation might function as a biological clock that regulates the onset of asymmetric division and neurogenesis. We conducted gene expression profiling of embryonic NSCs in the cortical regions and found that expression of high mobility group box transcription factor 1 (Hbp1) was upregulated during neurogenic stages. Induced conditional knockout mice of Hbp1, generated by crossing with Nestin-CreER(T2) mice, exhibited a remarkable dilatation of the telencephalic vesicles with a tangentially expanded ventricular zone and a thinner cortical plate containing reduced numbers of neurons. In these Hbp1-deficient mouse embryos, neural stem/progenitor cells continued to divide with a shorter cell cycle length. Moreover, downstream target genes of the Wnt signaling, such as cyclin D1 (Ccnd1) and c-jun (Jun), were upregulated in the germinal zone of the cortical regions. These results indicate that Hbp1 plays a crucial role in regulating the timing of cortical neurogenesis by elongating the cell cycle and that it is essential for normal cortical development.

  14. A Flavonoid Compound Promotes Neuronal Differentiation of Embryonic Stem Cells via PPAR-β Modulating Mitochondrial Energy Metabolism.

    PubMed

    Mei, Yu-Qin; Pan, Zong-Fu; Chen, Wen-Teng; Xu, Min-Hua; Zhu, Dan-Yan; Yu, Yong-Ping; Lou, Yi-Jia

    2016-01-01

    Relatively little is known regarding mitochondrial metabolism in neuronal differentiation of embryonic stem (ES) cells. By using a small molecule, present research has investigated the pattern of cellular energy metabolism in neural progenitor cells derived from mouse ES cells. Flavonoid compound 4a faithfully facilitated ES cells to differentiate into neurons morphologically and functionally. The expression and localization of peroxisome proliferator-activated receptors (PPARs) were examined in neural progenitor cells. PPAR-β expression showed robust upregulation compared to solvent control. Treatment with PPAR-β agonist L165041 alone or together with compound 4a significantly promoted neuronal differentiation, while antagonist GSK0660 blocked the neurogenesis-promoting effect of compound 4a. Consistently, knockdown of PPAR-β in ES cells abolished compound 4a-induced neuronal differentiation. Interestingly, we found that mitochondrial fusion protein Mfn2 was also abolished by sh-PPAR-β, resulting in abnormal mitochondrial Ca2+ ([Ca2+]M) transients as well as impaired mitochondrial bioenergetics. In conclusion, we demonstrated that by modulating mitochondrial energy metabolism through Mfn2 and mitochondrial Ca2+, PPAR-β took an important role in neuronal differentiation induced by flavonoid compound 4a. PMID:27315062

  15. A Flavonoid Compound Promotes Neuronal Differentiation of Embryonic Stem Cells via PPAR-β Modulating Mitochondrial Energy Metabolism

    PubMed Central

    Mei, Yu-qin; Pan, Zong-fu; Chen, Wen-teng; Xu, Min-hua; Zhu, Dan-yan; Yu, Yong-ping; Lou, Yi-jia

    2016-01-01

    Relatively little is known regarding mitochondrial metabolism in neuronal differentiation of embryonic stem (ES) cells. By using a small molecule, present research has investigated the pattern of cellular energy metabolism in neural progenitor cells derived from mouse ES cells. Flavonoid compound 4a faithfully facilitated ES cells to differentiate into neurons morphologically and functionally. The expression and localization of peroxisome proliferator-activated receptors (PPARs) were examined in neural progenitor cells. PPAR-β expression showed robust upregulation compared to solvent control. Treatment with PPAR-β agonist L165041 alone or together with compound 4a significantly promoted neuronal differentiation, while antagonist GSK0660 blocked the neurogenesis-promoting effect of compound 4a. Consistently, knockdown of PPAR-β in ES cells abolished compound 4a-induced neuronal differentiation. Interestingly, we found that mitochondrial fusion protein Mfn2 was also abolished by sh-PPAR-β, resulting in abnormal mitochondrial Ca2+ ([Ca2+]M) transients as well as impaired mitochondrial bioenergetics. In conclusion, we demonstrated that by modulating mitochondrial energy metabolism through Mfn2 and mitochondrial Ca2+, PPAR-β took an important role in neuronal differentiation induced by flavonoid compound 4a. PMID:27315062

  16. A potent and selective small molecule inhibitor of sirtuin 1 promotes differentiation of pluripotent P19 cells into functional neurons

    PubMed Central

    Kim, Beom Seok; Lee, Chang-Hee; Chang, Gyeong-Eon; Cheong, Eunji; Shin, Injae

    2016-01-01

    Sirtuin 1 (SIRT1) is known to suppress differentiation of pluripotent/multipotent cells and neural progenitor cells into neurons by blocking activation of transcription factors critical for neurogenesis. EX-527 is a highly selective and potent inhibitor against SIRT1 and has been used as a chemical probe that modulates SIRT1-associated biological processes. However, the effect of EX-527 on neuronal differentiation in pluripotent cells has not been well elucidated. Here, we report an examination of EX-527 effects on neurogenesis of pluripotent P19 cells. The results showed that EX-527 greatly accelerated differentiation of P19 cells into neurons without generation of cardiac cells and astrocytes. Importantly, neurons derived from P19 cells treated with EX-527 generated voltage-dependent sodium currents and depolarization-induced action potentials. The findings indicate that the differentiated cells have electrophysiological properties. The present study suggests that the selective SIRT1 inhibitor could have the potential of being employed as a chemical inducer to generate functionally active neurons. PMID:27680533

  17. The Ubiquitin Ligase Praja1 Reduces NRAGE Expression and Inhibits Neuronal Differentiation of PC12 Cells

    PubMed Central

    Teuber, Jan; Mueller, Bettina; Fukabori, Ryoji; Lang, Daniel; Albrecht, Anne; Stork, Oliver

    2013-01-01

    Evidence suggests that regulated ubiquitination of proteins plays a critical role in the development and plasticity of the central nervous system. We have previously identified the ubiquitin ligase Praja1 as a gene product induced during fear memory consolidation. However, the neuronal function of this enzyme still needs to be clarified. Here, we investigate its involvement in the nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma (PC12) cells. Praja1 co-localizes with cytoskeleton components and the neurotrophin receptor interacting MAGE homologue (NRAGE). We observed an enhanced expression of Praja1 after 3 days of NGF treatment and a suppression of neurite formation upon Praja1 overexpression in stably transfected PC12 cell lines, which was associated with a proteasome-dependent reduction of NRAGE levels. Our data suggest that Praja1, through ubiquitination and degradation of NRAGE, inhibits neuronal differentiation. The two murine isoforms, Praja1.1 and Praja1.2, appear to be functionally homologous in this respect. PMID:23717400

  18. A High-content screen identifies compounds promoting the neuronal differentiation and the midbrain dopamine neuron specification of human neural progenitor cells

    PubMed Central

    Rhim, Ji heon; Luo, Xiangjian; Xu, Xiaoyun; Gao, Dongbing; Zhou, Tieling; Li, Fuhai; Qin, Lidong; Wang, Ping; Xia, Xiaofeng; Wong, Stephen T. C.

    2015-01-01

    Small molecule compounds promoting the neuronal differentiation of stem/progenitor cells are of pivotal importance to regenerative medicine. We carried out a high-content screen to systematically characterize known bioactive compounds, on their effects on the neuronal differentiation and the midbrain dopamine (mDA) neuron specification of neural progenitor cells (NPCs) derived from the ventral mesencephalon of human fetal brain. Among the promoting compounds three major pharmacological classes were identified including the statins, TGF-βRI inhibitors, and GSK-3 inhibitors. The function of each class was also shown to be distinct, either to promote both the neuronal differentiation and mDA neuron specification, or selectively the latter, or promote the former but suppress the latter. We then carried out initial investigation on the possible mechanisms underlying, and demonstrated their applications on NPCs derived from human pluripotent stem cells (PSCs). Our study revealed the potential of several small molecule compounds for use in the directed differentiation of human NPCs. The screening result also provided insight into the signaling network regulating the differentiation of human NPCs. PMID:26542303

  19. Expression of PKC iota affects neuronal differentiation of PC12 cells at least partly independent of kinase function

    PubMed Central

    Doonachar, Alana; Schoenfeld, Alan R.

    2014-01-01

    Atypical PKC (aPKC) plays a role in establishing cell polarity and has been indicated in neuronal differentiation and polarization, including neurite formation in rat pheochromocytoma PC12 cells, albeit by unclear mechanisms. Here, the role of the aPKC isoform, PKC iota (PKCι), in the early neuronal differentiation of PC12 cells was investigated. NGF-treated PC12 cells with stably expressed exogenous wild-type PKCι showed decreased expression of a neuroendocrine marker, increased expression of a neuronal marker, and increased neurite formation. Stable expression of a kinase- inactive PKCι, but not constitutively active PKCι lacking a regulatory domain, had similar although less potent effects. Pharmacological inhibition of endogenous aPKC kinase activity in parental PC12 cells did not inhibit neurite formation, suggesting that some of the observed effects of PKCι expression on neuronal differentiation are kinase- independent. Interestingly, exogenous expression of wild-type and kinase-inactive PKCι had little effect on overall PKCι activity, but caused a decrease in PKC zeta (PKCζ) kinase activity, suggesting an interplay between the two isoforms that may underlie the observed results. Overall, these findings suggest that in PC12 and perhaps other neuroendocrine precursor cells, PKCι influences an early differentiation decision between the neuroendocrine (chromaffin) and sympathetic neuron cell lineages, potentially by affecting PKCζ function. PMID:24910851

  20. Differentiation-Dependent Energy Production and Metabolite Utilization: A Comparative Study on Neural Stem Cells, Neurons, and Astrocytes.

    PubMed

    Jády, Attila Gy; Nagy, Ádám M; Kőhidi, Tímea; Ferenczi, Szilamér; Tretter, László; Madarász, Emília

    2016-07-01

    While it is evident that the metabolic machinery of stem cells should be fairly different from that of differentiated neurons, the basic energy production pathways in neural stem cells (NSCs) or in neurons are far from clear. Using the model of in vitro neuron production by NE-4C NSCs, this study focused on the metabolic changes taking place during the in vitro neuronal differentiation. O2 consumption, H(+) production, and metabolic responses to single metabolites were measured in cultures of NSCs and in their neuronal derivatives, as well as in primary neuronal and astroglial cultures. In metabolite-free solutions, NSCs consumed little O2 and displayed a higher level of mitochondrial proton leak than neurons. In stem cells, glycolysis was the main source of energy for the survival of a 2.5-h period of metabolite deprivation. In contrast, stem cell-derived or primary neurons sustained a high-level oxidative phosphorylation during metabolite deprivation, indicating the consumption of own cellular material for energy production. The stem cells increased O2 consumption and mitochondrial ATP production in response to single metabolites (with the exception of glucose), showing rapid adaptation of the metabolic machinery to the available resources. In contrast, single metabolites did not increase the O2 consumption of neurons or astrocytes. In "starving" neurons, neither lactate nor pyruvate was utilized for mitochondrial ATP production. Gene expression studies also suggested that aerobic glycolysis and rapid metabolic adaptation characterize the NE-4C NSCs, while autophagy and alternative glucose utilization play important roles in the metabolism of stem cell-derived neurons.

  1. Differentiation-Dependent Energy Production and Metabolite Utilization: A Comparative Study on Neural Stem Cells, Neurons, and Astrocytes

    PubMed Central

    Jády, Attila Gy.; Nagy, Ádám M.; Kőhidi, Tímea; Ferenczi, Szilamér; Tretter, László

    2016-01-01

    While it is evident that the metabolic machinery of stem cells should be fairly different from that of differentiated neurons, the basic energy production pathways in neural stem cells (NSCs) or in neurons are far from clear. Using the model of in vitro neuron production by NE-4C NSCs, this study focused on the metabolic changes taking place during the in vitro neuronal differentiation. O2 consumption, H+ production, and metabolic responses to single metabolites were measured in cultures of NSCs and in their neuronal derivatives, as well as in primary neuronal and astroglial cultures. In metabolite-free solutions, NSCs consumed little O2 and displayed a higher level of mitochondrial proton leak than neurons. In stem cells, glycolysis was the main source of energy for the survival of a 2.5-h period of metabolite deprivation. In contrast, stem cell-derived or primary neurons sustained a high-level oxidative phosphorylation during metabolite deprivation, indicating the consumption of own cellular material for energy production. The stem cells increased O2 consumption and mitochondrial ATP production in response to single metabolites (with the exception of glucose), showing rapid adaptation of the metabolic machinery to the available resources. In contrast, single metabolites did not increase the O2 consumption of neurons or astrocytes. In “starving” neurons, neither lactate nor pyruvate was utilized for mitochondrial ATP production. Gene expression studies also suggested that aerobic glycolysis and rapid metabolic adaptation characterize the NE-4C NSCs, while autophagy and alternative glucose utilization play important roles in the metabolism of stem cell-derived neurons. PMID:27116891

  2. Concomitant inhibition of prolyl hydroxylases and ROCK initiates differentiation of mesenchymal stem cells and PC12 towards the neuronal lineage.

    PubMed

    Pacary, Emilie; Petit, Edwige; Bernaudin, Myriam

    2008-12-12

    This study demonstrates that a prolyl hydroxylase inhibitor, FG-0041, is able, in combination with the ROCK inhibitor, Y-27632, to initiate differentiation of mesenchymal stem cells (MSCs) into neuron-like cells. FG-0041/Y-27632 co-treatment provokes morphological changes into neuron-like cells, increases neuronal marker expression and provokes modifications of cell cycle-related gene expression consistent with a cell cycle arrest of MSC, three events showing the engagement of MSC towards the neuronal lineage. Moreover, as we observed in our previous studies with cobalt chloride and desferroxamine, the activation of HIF-1 by this prolyl hydroxylase inhibitor is potentiated by Y-27632 which could explain at least in part the effect of this co-treatment on MSC neuronal differentiation. In addition, we show that this co-treatment enhances neurite outgrowth and tyrosine hydroxylase expression in PC12 cells. Altogether, these results evidence that concomitant inhibition of prolyl hydroxylases and ROCK represents a relevant protocol to initiate neuronal differentiation.

  3. Multiple Sclerosis Patient-Specific Primary Neurons Differentiated from Urinary Renal Epithelial Cells via Induced Pluripotent Stem Cells

    PubMed Central

    Massa, Megan G.; Gisevius, Barbara; Hirschberg, Sarah; Hinz, Lisa; Schmidt, Matthias; Gold, Ralf; Prochnow, Nora; Haghikia, Aiden

    2016-01-01

    As multiple sclerosis research progresses, it is pertinent to continue to develop suitable paradigms to allow for ever more sophisticated investigations. Animal models of multiple sclerosis, despite their continuing contributions to the field, may not be the most prudent for every experiment. Indeed, such may be either insufficient to reflect the functional impact of human genetic variations or unsuitable for drug screenings. Thus, we have established a cell- and patient-specific paradigm to provide an in vitro model within which to perform future genetic investigations. Renal proximal tubule epithelial cells were isolated from multiple sclerosis patients’ urine and transfected with pluripotency-inducing episomal factors. Subsequent induced pluripotent stem cells were formed into embryoid bodies selective for ectodermal lineage, resulting in neural tube-like rosettes and eventually neural progenitor cells. Differentiation of these precursors into primary neurons was achieved through a regimen of neurotrophic and other factors. These patient-specific primary neurons displayed typical morphology and functionality, also staining positive for mature neuronal markers. The development of such a non-invasive procedure devoid of permanent genetic manipulation during the course of differentiation, in the context of multiple sclerosis, provides an avenue for studies with a greater cell- and human-specific focus, specifically in the context of genetic contributions to neurodegeneration and drug discovery. PMID:27158987

  4. [NEURONAL DIFFERENTIATION OF PC12 CELL LINE AND MURINE NEURAL STEM CELLS ON THE CARBON NANOTUBES FILMS].

    PubMed

    Posypanova, G A; Gaiduchenko, A I; Moskaleva, E Yu; Fedorov, G E

    2016-01-01

    The study of the interaction of nerve cells with specially designed substrates (scaffolds) with different surface characteristics at the nanoscale is a necessary step in the development of methods of stimulation of regeneration of nervous tissues, as well as to create next generation of bioelectronic devices. A promising material for such scaffolds may be carbon nanotubes (CNT) that are flexible films of graphene rolled into nano-sized cylindrical tubes. CNT were produced by chemical deposition from the gas phase. The analysis of the PC12 cells cultivated on quartz glass coated by carbon nanotubes films using electron and light microscopy has shown that CNT stimulate the proliferation and do not inhibit neuronal differentiation of PC12 cells. We have found that it is possible to obtain differentiated neurons from murine neural stem cells on the quartz glasses covered with CNT films. The data obtained indicate that the CNT films produced by chemical deposition from the gas phase onto quartz glass may be used as the electro conductive scaffold to obtain and study the functions of neural cells and possibly of mature neurons.

  5. Label-Free Detection of Neuronal Differentiation in Cell Populations Using High-Throughput Live-Cell Imaging of PC12 Cells

    PubMed Central

    Nascimento, Juliana M.; Knauer, Steffen; Offermann, Barbara; Murphy, Robert F.

    2013-01-01

    Detection of neuronal cell differentiation is essential to study cell fate decisions under various stimuli and/or environmental conditions. Many tools exist that quantify differentiation by neurite length measurements of single cells. However, quantification of differentiation in whole cell populations remains elusive so far. Because such populations can consist of both proliferating and differentiating cells, the task to assess the overall differentiation status is not trivial and requires a high-throughput, fully automated approach to analyze sufficient data for a statistically significant discrimination to determine cell differentiation. We address the problem of detecting differentiation in a mixed population of proliferating and differentiating cells over time by supervised classification. Using nerve growth factor induced differentiation of PC12 cells, we monitor the changes in cell morphology over days by phase-contrast live-cell imaging. For general applicability, the classification procedure starts out with many features to identify those that maximize discrimination of differentiated and undifferentiated cells and to eliminate features sensitive to systematic measurement artifacts. The resulting image analysis determines the optimal post treatment day for training and achieves a near perfect classification of differentiation, which we confirmed in technically and biologically independent as well as differently designed experiments. Our approach allows to monitor neuronal cell populations repeatedly over days without any interference. It requires only an initial calibration and training step and is thereafter capable to discriminate further experiments. In conclusion, this enables long-term, large-scale studies of cell populations with minimized costs and efforts for detecting effects of external manipulation of neuronal cell differentiation. PMID:23451069

  6. Some Rat Sensory Neurons in Culture Express Characteristics of Differentiated Pain Sensory Cells

    NASA Astrophysics Data System (ADS)

    Baccaglini, Paola I.; Hogan, Patrick G.

    1983-01-01

    Sensory neurons were dissociated from trigeminal ganglia or from dorsal root ganglia of rats, grown in culture, and examined for expression of properties of pain sensory cells. Many sensory neurons in culture are excited by low concentrations of capsaicin, reportedly a selective stimulus for pain sensory neurons. Many are excited by bradykinin, sensitized by prostaglandin E2, or specifically stained by an antiserum against substance P. These experiments provide a basis for the study of pain mechanisms in cell culture.

  7. Pleiotrophin antagonizes Brd2 during neuronal differentiation.

    PubMed

    Garcia-Gutierrez, Pablo; Juarez-Vicente, Francisco; Wolgemuth, Debra J; Garcia-Dominguez, Mario

    2014-06-01

    Bromodomain-containing protein 2 (Brd2) is a BET family chromatin adaptor required for expression of cell-cycle-associated genes and therefore involved in cell cycle progression. Brd2 is expressed in proliferating neuronal progenitors, displays cell-cycle-stimulating activity and, when overexpressed, impairs neuronal differentiation. Paradoxically, Brd2 is also detected in differentiating neurons. To shed light on the role of Brd2 in the transition from cell proliferation to differentiation, we had previously looked for proteins that interacted with Brd2 upon induction of neuronal differentiation. Surprisingly, we identified the growth factor pleiotrophin (Ptn). Here, we show that Ptn antagonized the cell-cycle-stimulating activity associated with Brd2, thus enhancing induced neuronal differentiation. Moreover, Ptn knockdown reduced neuronal differentiation. We analyzed Ptn-mediated antagonism of Brd2 in a cell differentiation model and in two embryonic processes associated with the neural tube: spinal cord neurogenesis and neural crest migration. Finally, we investigated the mechanisms of Ptn-mediated antagonism and determined that Ptn destabilizes the association of Brd2 with chromatin. Thus, Ptn-mediated Brd2 antagonism emerges as a modulation system accounting for the balance between cell proliferation and differentiation in the vertebrate nervous system.

  8. Bilobalide induces neuronal differentiation of P19 embryonic carcinoma cells via activating Wnt/β-catenin pathway.

    PubMed

    Liu, Mei; Guo, Jingjing; Wang, Juan; Zhang, Luyong; Pang, Tao; Liao, Hong

    2014-08-01

    Bilobalide, a natural product extracted from Ginkgo biloba leaf, is known to exhibit a number of pharmacological activities. So far, whether it could affect embryonic stem cell differentiation is still unknown. The main aim of this study was to investigate the effect of bilobalide on P19 embryonic carcinoma cells differentiation and the underlying mechanisms. Our results showed that bilobalide induced P19 cells differentiation into neurons in a concentration- and time-dependent manner. We also found that bilobalide promoted neuronal differentiation through activation of Wnt/β-catenin signaling pathway. Exposure to bilobalide increased inactive GSK-3β phosphorylation, further induced the nuclear accumulation of β-catenin, and also up-regulated the expression of Wnt ligands Wnt1 and Wnt7a. Neuronal differentiation induced by bilobalide was totally abolished by XAV939, an inhibitor of Wnt/β-catenin pathway. These results revealed a novel role of bilobalide in neuronal differentiation from P19 embryonic cells acting through Wnt/β-catenin signaling pathway, which would provide a better insight into the beneficial effects of bilobalide in brain diseases.

  9. Conditioned medium from human amniotic epithelial cells may induce the differentiation of human umbilical cord blood mesenchymal stem cells into dopaminergic neuron-like cells.

    PubMed

    Yang, Shu; Sun, Hai-Mei; Yan, Ji-Hong; Xue, Hong; Wu, Bo; Dong, Fang; Li, Wen-Shuai; Ji, Feng-Qing; Zhou, De-Shan

    2013-07-01

    Dopaminergic (DA) neuron therapy has been established as a new clinical tool for treating Parkinson's disease (PD). Prior to cell transplantation, there are two primary issues that must be resolved: one is the appropriate seed cell origin, and the other is the efficient inducing technique. In the present study, human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) were used as the available seed cells, and conditioned medium from human amniotic epithelial cells (ACM) was used as the inducing reagent. Results showed that the proportion of DA neuron-like cells from hUCB-MSCs was significantly increased after cultured in ACM, suggested by the upregulation of DAT, TH, Nurr1, and Pitx3. To identify the process by which ACM induces DA neuron differentiation, we pretreated hUCB-MSCs with k252a, the Trk receptor inhibitor of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and found that the proportion of DA neuron-like cells was significantly decreased compared with ACM-treated hUCB-MSCs, suggesting that NGF and BDNF in ACM were involved in the differentiation process. However, we could not rule out the involvement of other unidentified factors in the ACM, because ACM + k252a treatment does not fully block DA neuron-like cell differentiation compared with control. The transplantation of ACM-induced hUCB-MSCs could ameliorate behavioral deficits in PD rats, which may be associated with the survival of engrafted DA neuron-like cells. In conclusion, we propose that hUCB-MSCs are a good source of DA neuron-like cells and that ACM is a potential inducer to obtain DA neuron-like cells from hUCB-MSCs in vitro for an ethical and legal cell therapy for PD.

  10. Genome-wide characterisation of Foxa1 binding sites reveals several mechanisms for regulating neuronal differentiation in midbrain dopamine cells.

    PubMed

    Metzakopian, Emmanouil; Bouhali, Kamal; Alvarez-Saavedra, Matías; Whitsett, Jeffrey A; Picketts, David J; Ang, Siew-Lan

    2015-04-01

    Midbrain dopamine neuronal progenitors develop into heterogeneous subgroups of neurons, such as substantia nigra pars compacta, ventral tegmental area and retrorubal field, that regulate motor control, motivated and addictive behaviours. The development of midbrain dopamine neurons has been extensively studied, and these studies indicate that complex cross-regulatory interactions between extrinsic and intrinsic molecules regulate a precise temporal and spatial programme of neurogenesis in midbrain dopamine progenitors. To elucidate direct molecular interactions between multiple regulatory factors during neuronal differentiation in mice, we characterised genome-wide binding sites of the forkhead/winged helix transcription factor Foxa1, which functions redundantly with Foxa2 to regulate the differentiation of mDA neurons. Interestingly, our studies identified a rostral brain floor plate Neurog2 enhancer that requires direct input from Otx2, Foxa1, Foxa2 and an E-box transcription factor for its transcriptional activity. Furthermore, the chromatin remodelling factor Smarca1 was shown to function downstream of Foxa1 and Foxa2 to regulate differentiation from immature to mature midbrain dopaminergic neurons. Our genome-wide Foxa1-bound cis-regulatory sequences from ChIP-Seq and Foxa1/2 candidate target genes from RNA-Seq analyses of embryonic midbrain dopamine cells also provide an excellent resource for probing mechanistic insights into gene regulatory networks involved in the differentiation of midbrain dopamine neurons.

  11. The effects of diazinon and cypermethrin on the differentiation of neuronal and glial cell lines

    SciTech Connect

    Flaskos, J.; Harris, W.; Sachana, M.; Munoz, D.; Tack, J.; Hargreaves, A.J. . E-mail: alan.hargreaves@ntu.ac.uk

    2007-03-15

    Diazinon and cypermethrin are pesticides extensively used in sheep dipping. Diazinon is a known anti-cholinesterase, but there is limited information regarding its molecular mechanism of action. This paper describes the effects of diazinon and cypermethrin at a morphological and molecular level on differentiating mouse N2a neuroblastoma and rat C6 glioma cell lines. Concentrations up to 10 {mu}M of both compounds and their mixture had no effect on the viability of either cell line, as determined by methyl blue tetrazolium reduction and total protein assays. Microscopic analysis revealed that 1 {mu}M and 10 {mu}M diazinon but not cypermethrin inhibited the outgrowth of axon-like processes in N2a cells after a 24-h exposure but neither compound affected process outgrowth by differentiating C6 cells at these concentrations. Under these conditions, 10 {mu}M diazinon inhibited AChE slightly compared to the control after a 4-h exposure but not after 24 h. Western blotting analysis showed that morphological changes were associated with reduced cross-reactivity with antibodies that recognize the neurofilament heavy chain (NFH), microtubule associated protein MAP 1B and HSP-70 compared to control cell extracts, whereas reactivity with anti-{alpha}-tubulin antibodies was unchanged. Aggregation of NFH was observed in cell bodies of diazinon-treated N2a cells, as determined by indirect immunofluorescence staining. These data demonstrate that diazinon specifically targets neurite outgrowth in neuronal cells and that this effect is associated with disruption of axonal cytoskeleton proteins, whereas cypermethrin has no effect on the same parameters.

  12. HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation

    NASA Astrophysics Data System (ADS)

    Rouhiainen, Ari; Zhao, Xiang; Vanttola, Päivi; Qian, Kui; Kulesskiy, Evgeny; Kuja-Panula, Juha; Gransalke, Kathleen; Grönholm, Mikaela; Unni, Emmanual; Meistrich, Marvin; Tian, Li; Auvinen, Petri; Rauvala, Heikki

    2016-09-01

    HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A -processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers.

  13. HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation

    PubMed Central

    Rouhiainen, Ari; Zhao, Xiang; Vanttola, Päivi; Qian, Kui; Kulesskiy, Evgeny; Kuja-Panula, Juha; Gransalke, Kathleen; Grönholm, Mikaela; Unni, Emmanual; Meistrich, Marvin; Tian, Li; Auvinen, Petri; Rauvala, Heikki

    2016-01-01

    HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A –processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers. PMID:27608812

  14. HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation

    NASA Astrophysics Data System (ADS)

    Rouhiainen, Ari; Zhao, Xiang; Vanttola, Päivi; Qian, Kui; Kulesskiy, Evgeny; Kuja-Panula, Juha; Gransalke, Kathleen; Grönholm, Mikaela; Unni, Emmanual; Meistrich, Marvin; Tian, Li; Auvinen, Petri; Rauvala, Heikki

    2016-09-01

    HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A –processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers.

  15. HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation.

    PubMed

    Rouhiainen, Ari; Zhao, Xiang; Vanttola, Päivi; Qian, Kui; Kulesskiy, Evgeny; Kuja-Panula, Juha; Gransalke, Kathleen; Grönholm, Mikaela; Unni, Emmanual; Meistrich, Marvin; Tian, Li; Auvinen, Petri; Rauvala, Heikki

    2016-01-01

    HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A -processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers. PMID:27608812

  16. New methods for inducing the differentiation of amniotic-derived mesenchymal stem cells into motor neuron precursor cells.

    PubMed

    Hu, Wei; Guan, Fang-xia; Li, Yuan; Tang, You-jia; Yang, Feng; Yang, Bo

    2013-10-01

    Our study investigates the differentiation of amniotic-derived mesenchymal stem cells (ADMSCs) into motor neuron (MN) precursor cells induced by a combination of extracellular matrix (ECM) and multi-cell factors. Membrane-like ECM was made by an enzymatic and chemical extraction method and exhibited good biological compatibility. Cells in the experimental group (EG) were treated with ECM and multi-cell factors in a multi-step induction process, while the control group (CG) was treated similarly, except without ECM. In the EG, after induction, the cells formed processes that connected with neighboring cells to form a net that had directionality. In these cells, neuron-specific enolase (NSE) and synaptophysin (SYN) expression levels increased and glial fibrillary acidic protein (GFAP) expression decreased. The SYN expression in the EG cells was higher compared with those in the CG. In the CG, NSE expression increased, while the expression of Nestin and SYN did not change. These were several changes in the levels of other genes: ADMSCs at passage 1 expressed Nanog, SOX2, octamer-binding transcription factor 4 (OCT4) and Nestin. In the EG, at the beginning of induction, the expression of Nanog decreased and that of SOX2 and Nestin increased. After 2 days, the cells expressed Nestin, OCT4 and SYNIII, and after 3 days, they expressed Olig2, OCT4, Nestin, SYNII and Islet1 (ISL1). Finally, at day 6, the cells expressed Nestin, SYNI, SYNIII, ISL-1, homeobox 9 (Hb9) and oligodendrocyte lineage transcription factor 2 (Olig2). In the CG, the cells never expressed SYNI, SYNII or Hb9. Our studies therefore demonstrate that the extracted ECM was capable of promoting the maturation of synapses. Human ADMSCs are composed of multiple cell subsets, including neural progenitor cells. The multi-step induction method used in this study causes human ADMSCs to differentiate into MN precursor cells.

  17. Nanotopography induced contact guidance of the F11 cell line during neuronal differentiation: a neuronal model cell line for tissue scaffold development

    NASA Astrophysics Data System (ADS)

    Wieringa, Paul; Tonazzini, Ilaria; Micera, Silvestro; Cecchini, Marco

    2012-07-01

    The F11 hybridoma, a dorsal root ganglion-derived cell line, was used to investigate the response of nociceptive sensory neurons to nanotopographical guidance cues. This established this cell line as a model of peripheral sensory neuron growth for tissue scaffold design. Cells were seeded on substrates of cyclic olefin copolymer (COC) films imprinted via nanoimprint lithography (NIL) with a grating pattern of nano-scale grooves and ridges. Different ridge widths were employed to alter the focal adhesion formation, thereby changing the cell/substrate interaction. Differentiation was stimulated with forskolin in culture medium consisting of either 1 or 10% fetal bovine serum (FBS). Per medium condition, similar neurite alignment was achieved over the four day period, with the 1% serum condition exhibiting longer, more aligned neurites. Immunostaining for focal adhesions found the 1% FBS condition to also have fewer, less developed focal adhesions. The robust response of the F11 to guidance cues further builds on the utility of this cell line as a sensory neuron model, representing a useful tool to explore the design of regenerative guidance tissue scaffolds.

  18. Differentiation of rat adipose tissue-derived stem cells into neuron-like cells by valproic acid, a histone deacetylase inhibitor.

    PubMed

    Okubo, Takumi; Hayashi, Daiki; Yaguchi, Takayuki; Fujita, Yudai; Sakaue, Motoharu; Suzuki, Takehito; Tsukamoto, Atsushi; Murayama, Ohoshi; Lynch, Jonathan; Miyazaki, Yoko; Tanaka, Kazuaki; Takizawa, Tatsuya

    2016-01-01

    Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to modulate the neuronal differentiation of adipose tissue-derived stem cells (ASCs) in humans and dogs. However, controversy exists as to whether VPA really acts as an inducer of neuronal differentiation of ASCs. The present study aimed to elucidate the effect of VPA in neuronal differentiation of rat ASCs. One or three days of pretreatment with VPA (2 mM) followed by neuronal induction enhanced the ratio of immature neuron marker βIII-tubulin-positive cells in a time-dependent manner, where the majority of cells also had a positive signal for neurofilament medium polypeptide (NEFM), a mature neuron marker. RT-PCR analysis revealed increases in the mRNA expression of microtubule-associated protein 2 (MAP2) and NEFM mature neuron markers, even without neuronal induction. Three-days pretreatment of VPA increased acetylation of histone H3 of ASCs as revealed by immunofluorescence staining. Chromatin immunoprecipitation assay also showed that the status of histone acetylation at H3K9 correlated with the gene expression of TUBB3 in ASCs by VPA. These results indicate that VPA significantly promotes the differentiation of rat ASCs into neuron-like cells through acetylation of histone H3, which suggests that VPA may serve as a useful tool for producing transplantable cells for future applications in clinical treatments. PMID:26411320

  19. Development of ion channels and neurofilaments during neuronal differentiation of mouse embryonal carcinoma cell lines.

    PubMed Central

    Kubo, Y

    1989-01-01

    1. an embryonal carcinoma cell line, PCC4-Aza1-ECA2, was induced to differentiate to neurones by two different procedures: an addition of retinoic acid to the culture medium or a reduction of serum concentration. The changes in membrane currents during differentiation were studied by the whole-cell variation of the patch-clamp technique and the change in neurofilament expression was studied immunohistochemically. 2. Stem cells showed the outward K+ current which inactivated slightly, but no inward currents were observed. These cells did not express neurofilament. 3. Three days after an addition of 10(-7) M-retinoic acid, neurofilament-positive round cells without processes began to appear. The inward currents observed in these cells were the Na+ current and fast-inactivating Ca2+-channel current. Four days after an addition of 10(-7) M-retinoic acid, the cells began to extend processes and showed an intense neurofilament expression. The inward currents were the Na+ current and slow-inactivating Ca2+-channel current, while the fast-inactivating Ca2+-channel current observed previously had almost disappeared. The amplitude of the outward K+ current was larger than that in the stem cell and it did not show clear inactivation. 4. By reducing the serum concentration in the medium from 10 to 0.1%, cells with processes were observed after 6 days. They were neurofilament-positive and had the Na+ current, both fast- and slow-inactivating Ca2+-channel currents, and the outward K+ current which inactivated slightly. 5. The properties of these ionic currents observed in induced neurones were studied. The Na+ current was blocked by 0.1 microM-tetrodotoxin at any stage. The Na+ current was evoked by a depolarization pulse to a level above -40 mV with a maximum amplitude at around -10 mV. The fast-inactivating Ca2+-channel current was evoked by a depolarization to a level above -50 mV with a maximum amplitude at around -15 mV. It was resistant to 50 microM-Cd2+. The slow

  20. Transgenic GDNF Positively Influences Proliferation, Differentiation, Maturation and Survival of Motor Neurons Produced from Mouse Embryonic Stem Cells

    PubMed Central

    Cortés, Daniel; Robledo-Arratia, Yolanda; Hernández-Martínez, Ricardo; Escobedo-Ávila, Itzel; Bargas, José; Velasco, Iván

    2016-01-01

    Embryonic stem cells (ESC) are pluripotent and thus can differentiate into every cell type present in the body. Directed differentiation into motor neurons (MNs) has been described for pluripotent cells. Although neurotrophic factors promote neuronal survival, their role in neuronal commitment is elusive. Here, we developed double-transgenic lines of mouse ESC (mESC) that constitutively produce glial cell line-derived neurotrophic factor (GDNF) and also contain a GFP reporter, driven by HB9, which is expressed only by postmitotic MNs. After lentiviral transduction, ESC lines integrated and expressed the human GDNF (hGDNF) gene without altering pluripotency markers before differentiation. Further, GDNF-ESC showed significantly higher spontaneous release of this neurotrophin to the medium, when compared to controls. To study MN induction, control and GDNF cell lines were grown as embryoid bodies and stimulated with retinoic acid and Sonic Hedgehog. In GDNF-overexpressing cells, a significant increase of proliferative Olig2+ precursors, which are specified as spinal MNs, was found. Accordingly, GDNF increases the yield of cells with the pan motor neuronal markers HB9, monitored by GFP expression, and Isl1. At terminal differentiation, almost all differentiated neurons express phenotypic markers of MNs in GDNF cultures, with lower proportions in control cells. To test if the effects of GDNF were present at early differentiation stages, exogenous recombinant hGDNF was added to control ESC, also resulting in enhanced MN differentiation. This effect was abolished by the co-addition of neutralizing anti-GDNF antibodies, strongly suggesting that differentiating ESC are responsive to GDNF. Using the HB9::GFP reporter, MNs were selected for electrophysiological recordings. MNs differentiated from GDNF-ESC, compared to control MNs, showed greater electrophysiological maturation, characterized by increased numbers of evoked action potentials (APs), as well as by the appearance

  1. Transgenic GDNF Positively Influences Proliferation, Differentiation, Maturation and Survival of Motor Neurons Produced from Mouse Embryonic Stem Cells

    PubMed Central

    Cortés, Daniel; Robledo-Arratia, Yolanda; Hernández-Martínez, Ricardo; Escobedo-Ávila, Itzel; Bargas, José; Velasco, Iván

    2016-01-01

    Embryonic stem cells (ESC) are pluripotent and thus can differentiate into every cell type present in the body. Directed differentiation into motor neurons (MNs) has been described for pluripotent cells. Although neurotrophic factors promote neuronal survival, their role in neuronal commitment is elusive. Here, we developed double-transgenic lines of mouse ESC (mESC) that constitutively produce glial cell line-derived neurotrophic factor (GDNF) and also contain a GFP reporter, driven by HB9, which is expressed only by postmitotic MNs. After lentiviral transduction, ESC lines integrated and expressed the human GDNF (hGDNF) gene without altering pluripotency markers before differentiation. Further, GDNF-ESC showed significantly higher spontaneous release of this neurotrophin to the medium, when compared to controls. To study MN induction, control and GDNF cell lines were grown as embryoid bodies and stimulated with retinoic acid and Sonic Hedgehog. In GDNF-overexpressing cells, a significant increase of proliferative Olig2+ precursors, which are specified as spinal MNs, was found. Accordingly, GDNF increases the yield of cells with the pan motor neuronal markers HB9, monitored by GFP expression, and Isl1. At terminal differentiation, almost all differentiated neurons express phenotypic markers of MNs in GDNF cultures, with lower proportions in control cells. To test if the effects of GDNF were present at early differentiation stages, exogenous recombinant hGDNF was added to control ESC, also resulting in enhanced MN differentiation. This effect was abolished by the co-addition of neutralizing anti-GDNF antibodies, strongly suggesting that differentiating ESC are responsive to GDNF. Using the HB9::GFP reporter, MNs were selected for electrophysiological recordings. MNs differentiated from GDNF-ESC, compared to control MNs, showed greater electrophysiological maturation, characterized by increased numbers of evoked action potentials (APs), as well as by the appearance

  2. Transgenic GDNF Positively Influences Proliferation, Differentiation, Maturation and Survival of Motor Neurons Produced from Mouse Embryonic Stem Cells.

    PubMed

    Cortés, Daniel; Robledo-Arratia, Yolanda; Hernández-Martínez, Ricardo; Escobedo-Ávila, Itzel; Bargas, José; Velasco, Iván

    2016-01-01

    Embryonic stem cells (ESC) are pluripotent and thus can differentiate into every cell type present in the body. Directed differentiation into motor neurons (MNs) has been described for pluripotent cells. Although neurotrophic factors promote neuronal survival, their role in neuronal commitment is elusive. Here, we developed double-transgenic lines of mouse ESC (mESC) that constitutively produce glial cell line-derived neurotrophic factor (GDNF) and also contain a GFP reporter, driven by HB9, which is expressed only by postmitotic MNs. After lentiviral transduction, ESC lines integrated and expressed the human GDNF (hGDNF) gene without altering pluripotency markers before differentiation. Further, GDNF-ESC showed significantly higher spontaneous release of this neurotrophin to the medium, when compared to controls. To study MN induction, control and GDNF cell lines were grown as embryoid bodies and stimulated with retinoic acid and Sonic Hedgehog. In GDNF-overexpressing cells, a significant increase of proliferative Olig2+ precursors, which are specified as spinal MNs, was found. Accordingly, GDNF increases the yield of cells with the pan motor neuronal markers HB9, monitored by GFP expression, and Isl1. At terminal differentiation, almost all differentiated neurons express phenotypic markers of MNs in GDNF cultures, with lower proportions in control cells. To test if the effects of GDNF were present at early differentiation stages, exogenous recombinant hGDNF was added to control ESC, also resulting in enhanced MN differentiation. This effect was abolished by the co-addition of neutralizing anti-GDNF antibodies, strongly suggesting that differentiating ESC are responsive to GDNF. Using the HB9::GFP reporter, MNs were selected for electrophysiological recordings. MNs differentiated from GDNF-ESC, compared to control MNs, showed greater electrophysiological maturation, characterized by increased numbers of evoked action potentials (APs), as well as by the appearance

  3. Minocycline inhibited the pro-apoptotic effect of microglia on neural progenitor cells and protected their neuronal differentiation in vitro.

    PubMed

    Liu, Xuqing; Su, Huanxing; Chu, Tak-Ho; Guo, Anchen; Wu, Wutian

    2013-05-10

    Neural progenitor cell (NPC) transplantation offers great potential to treat spinal cord injury (SCI), but their efficiency is limited by poor survival and neuronal differentiation after transplantation. In the injury site, microglia may become activated and participate in the inflammation reaction. In vitro studies indicated that activated microglia might impair NPC survival and neuronal differentiation, but resting microglia did not. This study investigated the potential of minocycline to modify the negative effects of activated microglia on NPCs in vitro. First, the direct effects of minocycline on NPCs were tested. The results showed that at the concentration of 10μg/ml or lower, minocycline did not affect NPC survival and proliferation, but impaired neuronal differentiation. Then microglia were activated with lipopolysaccharide (LPS) or treated with LPS plus minocycline (LPSMC), and the effects of conditioned media on NPC apoptosis and differentiation were studied. The results showed that, compared with LPS treatment group, the microglia conditioned media of LPSMC treatment group resulted in a significantly lower apoptotic rate of NPCs, and increased the neuronal differentiation of NPCs. This suggested that minocycline might inhibit the negative effects of microglia on NPCs, and have the potential to support the survival and neuronal differentiation of transplanted NPCs for SCI.

  4. Differentiation of human epidermal neural crest stem cells (hEPI-NCSC) into virtually homogenous populations of dopaminergic neurons.

    PubMed

    Narytnyk, Alla; Verdon, Bernard; Loughney, Andrew; Sweeney, Michele; Clewes, Oliver; Taggart, Michael J; Sieber-Blum, Maya

    2014-04-01

    Here we provide a protocol for the directed differentiation of hEPI-NCSC into midbrain dopaminergic neurons, which degenerate in Parkinson's disease. hEPI-NCSC are neural crest-derived multipotent stem cells that persist into adulthood in the bulge of hair follicles. The experimental design is distinctly different from conventional protocols for embryonic stem cells and induced pluripotent stem (iPS) cells. It includes pre-differentiation of the multipotent hEPI-NCSC into neural stem cell-like cells, followed by ventralizing, patterning, continued exposure to the TGFβ receptor inhibitor, SB431542, and at later stages of differentiation the presence of the WNT inhibitor, IWP-4. All cells expressed A9 midbrain dopaminergic neuron progenitor markers with gene expression levels comparable to those in normal human substantia nigra. The current study shows for the first time that virtually homogeneous populations of dopaminergic neurons can be derived ex vivo from somatic stem cells without the need for purification, with useful timeliness and high efficacy. This novel development is an important first step towards the establishment of fully functional dopaminergic neurons from an ontologically relevant stem cell type, hEPI-NCSC. PMID:24399192

  5. Non-Faradaic electrical impedimetric investigation of the interfacial effects of neuronal cell growth and differentiation on silicon nanowire transistors.

    PubMed

    Lin, Shu-Ping; Vinzons, Lester U; Kang, Yu-Shan; Lai, Tung-Yen

    2015-05-13

    Silicon nanowire field-effect transistor (SiNW FET) devices have been interfaced with cells; however, their application for noninvasive, real-time monitoring of interfacial effects during cell growth and differentiation on SiNW has not been fully explored. Here, we cultured rat adrenal pheochromocytoma (PC12) cells, a type of neural progenitor cell, directly on SiNW FET devices to monitor cell adhesion during growth and morphological changes during neuronal differentiation for a period of 5-7 d. Monitoring was performed by measuring the non-Faradaic electrical impedance of the cell-SiNW FET system using a precision LCR meter. Our SiNW FET devices exhibited changes in impedance parameters during cell growth and differentiation because of the negatively charged cell membrane, seal resistance, and membrane capacitance at the cell/SiNW interface. It was observed that during both PC12 cell growth and neuronal differentiation, the impedance magnitude increased and the phase shifted to more negative values. However, impedance changes during cell growth already plateaued 3 d after seeding, while impedance changes continued until the last observation day during differentiation. Our results also indicate that the frequency shift to above 40 kHz after growth factor induction resulted from a larger coverage of cell membrane on the SiNWs due to distinctive morphological changes according to vinculin staining. Encapsulation of PC12 cells in a hydrogel scaffold resulted in a lack of trend in impedance parameters and confirmed that impedance changes were due to the cells. Moreover, cytolysis of the differentiated PC12 cells led to significant changes in impedance parameters. Equivalent electrical circuits were used to analyze the changes in impedance values during cell growth and differentiation. The technique employed in this study can provide a platform for performing investigations of growth-factor-induced progenitor cell differentiation.

  6. Why are enteric ganglia so small? Role of differential adhesion of enteric neurons and enteric neural crest cells.

    PubMed Central

    Rollo, Benjamin N.; Zhang, Dongcheng; Simkin, Johanna E.; Menheniott, Trevelyan R.; Newgreen, Donald F.

    2015-01-01

    The avian enteric nervous system (ENS) consists of a vast number of unusually small ganglia compared to other peripheral ganglia. Each ENS ganglion at mid-gestation has a core of neurons and a shell of mesenchymal precursor/glia-like enteric neural crest (ENC) cells. To study ENS cell ganglionation we isolated midgut ENS cells by HNK-1 fluorescence-activated cell sorting (FACS) from E5 and E8 quail embryos, and from E9 chick embryos. We performed cell-cell aggregation assays which revealed a developmentally regulated functional increase in ENS cell adhesive function, requiring both Ca 2+ -dependent and independent adhesion. This was consistent with N-cadherin and NCAM labelling. Neurons sorted to the core of aggregates, surrounded by outer ENC cells, showing that neurons had higher adhesion than ENC cells. The outer surface of aggregates became relatively non-adhesive, correlating with low levels of NCAM and N-cadherin on this surface of the outer non-neuronal ENC cells. Aggregation assays showed that ENS cells FACS selected for NCAM-high and enriched for enteric neurons formed larger and more coherent aggregates than unsorted ENS cells. In contrast, ENS cells of the NCAM-low FACS fraction formed small, disorganised aggregates.  This suggests a novel mechanism for control of ENS ganglion morphogenesis where i) differential adhesion of ENS neurons and ENC cells controls the core/shell ganglionic structure and ii) the ratio of neurons to ENC cells dictates the equilibrium ganglion size by generation of an outer non-adhesive surface. PMID:26064478

  7. Why are enteric ganglia so small? Role of differential adhesion of enteric neurons and enteric neural crest cells.

    PubMed

    Rollo, Benjamin N; Zhang, Dongcheng; Simkin, Johanna E; Menheniott, Trevelyan R; Newgreen, Donald F

    2015-01-01

    The avian enteric nervous system (ENS) consists of a vast number of unusually small ganglia compared to other peripheral ganglia. Each ENS ganglion at mid-gestation has a core of neurons and a shell of mesenchymal precursor/glia-like enteric neural crest (ENC) cells. To study ENS cell ganglionation we isolated midgut ENS cells by HNK-1 fluorescence-activated cell sorting (FACS) from E5 and E8 quail embryos, and from E9 chick embryos. We performed cell-cell aggregation assays which revealed a developmentally regulated functional increase in ENS cell adhesive function, requiring both Ca (2+) -dependent and independent adhesion. This was consistent with N-cadherin and NCAM labelling. Neurons sorted to the core of aggregates, surrounded by outer ENC cells, showing that neurons had higher adhesion than ENC cells. The outer surface of aggregates became relatively non-adhesive, correlating with low levels of NCAM and N-cadherin on this surface of the outer non-neuronal ENC cells. Aggregation assays showed that ENS cells FACS selected for NCAM-high and enriched for enteric neurons formed larger and more coherent aggregates than unsorted ENS cells. In contrast, ENS cells of the NCAM-low FACS fraction formed small, disorganised aggregates.  This suggests a novel mechanism for control of ENS ganglion morphogenesis where i) differential adhesion of ENS neurons and ENC cells controls the core/shell ganglionic structure and ii) the ratio of neurons to ENC cells dictates the equilibrium ganglion size by generation of an outer non-adhesive surface.

  8. CDKL5, a novel MYCN-repressed gene, blocks cell cycle and promotes differentiation of neuronal cells

    PubMed Central

    Valli, Emanuele; Trazzi, Stefania; Fuchs, Claudia; Erriquez, Daniela; Bartesaghi, Renata; Perini, Giovanni; Ciani, Elisabetta

    2012-01-01

    Mutations in the CDKL5 (cyclin-dependent kinase-like 5) gene are associated with a severe epileptic encephalopathy (early infantile epileptic encephalopathy type 2, EIEE2) characterized by early-onset intractable seizures, infantile spasms, severe developmental delay, intellectual disability, and Rett syndrome (RTT)-like features. Despite the clear involvement of CDKL5 mutations in intellectual disability, the function of this protein during brain development and the molecular mechanisms involved in its regulation are still unknown. Using human neuroblastoma cells as a model system we found that an increase in CDKL5 expression caused an arrest of the cell cycle in the G0/G1 phases and induced cellular differentiation. Interestingly, CDKL5 expression was inhibited by MYCN, a transcription factor that promotes cell proliferation during brain development and plays a relevant role in neuroblastoma biology. Through a combination of different and complementary molecular and cellular approaches we could show that MYCN acts as a direct repressor of the CDKL5 promoter. Overall our findings unveil a functional axis between MYCN and CDKL5 governing both neuron proliferation rate and differentiation. The fact that CDKL5 is involved in the control of both neuron proliferation and differentiation may help understand the early appearance of neurological symptoms in patients with mutations in CDKL5. PMID:22921766

  9. Markers of Pluripotency in Human Amniotic Epithelial Cells and Their Differentiation to Progenitor of Cortical Neurons.

    PubMed

    García-Castro, Irma Lydia; García-López, Guadalupe; Ávila-González, Daniela; Flores-Herrera, Héctor; Molina-Hernández, Anayansi; Portillo, Wendy; Ramón-Gallegos, Eva; Díaz, Néstor Fabián

    2015-01-01

    Human pluripotent stem cells (hPSC) have promise for regenerative medicine due to their auto-renovation and differentiation capacities. Nevertheless, there are several ethical and methodological issues about these cells that have not been resolved. Human amniotic epithelial cells (hAEC) have been proposed as source of pluripotent stem cells. Several groups have studied hAEC but have reported inconsistencies about their pluripotency properties. The aim of the present study was the in vitro characterization of hAEC collected from a Mexican population in order to identify transcription factors involved in the pluripotency circuitry and to determine their epigenetic state. Finally, we evaluated if these cells differentiate to cortical progenitors. We analyzed qualitatively and quantitatively the expression of the transcription factors of pluripotency (OCT4, SOX2, NANOG, KLF4 and REX1) by RT-PCR and RT-qPCR in hAEC. Also, we determined the presence of OCT4, SOX2, NANOG, SSEA3, SSEA4, TRA-1-60, E-cadherin, KLF4, TFE3 as well as the proliferation and epigenetic state by immunocytochemistry of the cells. Finally, hAEC were differentiated towards cortical progenitors using a protocol of two stages. Here we show that hAEC, obtained from a Mexican population and cultured in vitro (P0-P3), maintained the expression of several markers strongly involved in pluripotency maintenance (OCT4, SOX2, NANOG, TFE3, KLF4, SSEA3, SSEA4, TRA-1-60 and E-cadherin). Finally, when hAEC were treated with growth factors and small molecules, they expressed markers characteristic of cortical progenitors (TBR2, OTX2, NeuN and β-III-tubulin). Our results demonstrated that hAEC express naïve pluripotent markers (KLF4, REX1 and TFE3) as well as the cortical neuron phenotype after differentiation. This highlights the need for further investigation of hAEC as a possible source of hPSC.

  10. Markers of Pluripotency in Human Amniotic Epithelial Cells and Their Differentiation to Progenitor of Cortical Neurons

    PubMed Central

    García-Castro, Irma Lydia; García-López, Guadalupe; Ávila-González, Daniela; Flores-Herrera, Héctor; Molina-Hernández, Anayansi; Portillo, Wendy; Ramón-Gallegos, Eva; Díaz, Néstor Fabián

    2015-01-01

    Human pluripotent stem cells (hPSC) have promise for regenerative medicine due to their auto-renovation and differentiation capacities. Nevertheless, there are several ethical and methodological issues about these cells that have not been resolved. Human amniotic epithelial cells (hAEC) have been proposed as source of pluripotent stem cells. Several groups have studied hAEC but have reported inconsistencies about their pluripotency properties. The aim of the present study was the in vitro characterization of hAEC collected from a Mexican population in order to identify transcription factors involved in the pluripotency circuitry and to determine their epigenetic state. Finally, we evaluated if these cells differentiate to cortical progenitors. We analyzed qualitatively and quantitatively the expression of the transcription factors of pluripotency (OCT4, SOX2, NANOG, KLF4 and REX1) by RT-PCR and RT-qPCR in hAEC. Also, we determined the presence of OCT4, SOX2, NANOG, SSEA3, SSEA4, TRA-1-60, E-cadherin, KLF4, TFE3 as well as the proliferation and epigenetic state by immunocytochemistry of the cells. Finally, hAEC were differentiated towards cortical progenitors using a protocol of two stages. Here we show that hAEC, obtained from a Mexican population and cultured in vitro (P0-P3), maintained the expression of several markers strongly involved in pluripotency maintenance (OCT4, SOX2, NANOG, TFE3, KLF4, SSEA3, SSEA4, TRA-1-60 and E-cadherin). Finally, when hAEC were treated with growth factors and small molecules, they expressed markers characteristic of cortical progenitors (TBR2, OTX2, NeuN and β-III-tubulin). Our results demonstrated that hAEC express naïve pluripotent markers (KLF4, REX1 and TFE3) as well as the cortical neuron phenotype after differentiation. This highlights the need for further investigation of hAEC as a possible source of hPSC. PMID:26720151

  11. 14-3-3ε and ζ Regulate Neurogenesis and Differentiation of Neuronal Progenitor Cells in the Developing Brain

    PubMed Central

    Wachi, Tomoka; Hunt, Robert F.; Baraban, Scott C.; Taya, Shinichiro; Ramshaw, Hayley; Kaibuchi, Kozo; Schwarz, Quenten P.; Lopez, Angel F.

    2014-01-01

    During brain development, neural progenitor cells proliferate and differentiate into neural precursors. These neural precursors migrate along the radial glial processes and localize at their final destination in the cortex. Numerous reports have revealed that 14-3-3 proteins are involved in many neuronal activities, although their functions in neurogenesis remain unclear. Here, using 14-3-3ε/ζ double knock-out mice, we found that 14-3-3 proteins are important for proliferation and differentiation of neural progenitor cells in the cortex, resulting in neuronal migration defects and seizures. 14-3-3 deficiency resulted in the increase of δ-catenin and the decrease of β-catenin and αN-catenin. 14-3-3 proteins regulated neuronal differentiation into neurons via direct interactions with phosphorylated δ-catenin to promote F-actin formation through a catenin/Rho GTPase/Limk1/cofilin signaling pathway. Conversely, neuronal migration defects seen in the double knock-out mice were restored by phosphomimic Ndel1 mutants, but not δ-catenin. Our findings provide new evidence that 14-3-3 proteins play important roles in neurogenesis and neuronal migration via the regulation of distinct signaling cascades. PMID:25186760

  12. TRF2 dysfunction elicits DNA damage responses associated with senescence in proliferating neural cells and differentiation of neurons.

    PubMed

    Zhang, Peisu; Furukawa, Katsutoshi; Opresko, Patricia L; Xu, Xiangru; Bohr, Vilhelm A; Mattson, Mark P

    2006-04-01

    Telomeres are specialized structures at the ends of chromosomes that consist of tandem repeats of the DNA sequence TTAGGG and several proteins that protect the DNA and regulate the plasticity of the telomeres. The telomere-associated protein TRF2 (telomeric repeat binding factor 2) is critical for the control of telomere structure and function; TRF2 dysfunction results in the exposure of the telomere ends and activation of ATM (ataxia telangiectasin mutated)-mediated DNA damage response. Recent findings suggest that telomere attrition can cause senescence or apoptosis of mitotic cells, but the function of telomeres in differentiated neurons is unknown. Here, we examined the impact of telomere dysfunction via TRF2 inhibition in neurons (primary embryonic hippocampal neurons) and mitotic neural cells (astrocytes and neuroblastoma cells). We demonstrate that telomere dysfunction induced by adenovirus-mediated expression of dominant-negative TRF2 (DN-TRF2) triggers a DNA damage response involving the formation of nuclear foci containing phosphorylated histone H2AX and activated ATM in each cell type. In mitotic neural cells DN-TRF2 induced activation of both p53 and p21 and senescence (as indicated by an up-regulation of beta-galactosidase). In contrast, in neurons DN-TRF2 increased p21, but neither p53 nor beta-galactosidase was induced. In addition, TRF2 inhibition enhanced the morphological, molecular and biophysical differentiation of hippocampal neurons. These findings demonstrate divergent molecular and physiological responses to telomere dysfunction in mitotic neural cells and neurons, indicate a role for TRF2 in regulating neuronal differentiation, and suggest a potential therapeutic application of inhibition of TRF2 function in the treatment of neural tumors. PMID:16539655

  13. Possible promotion of neuronal differentiation in fetal rat brain neural progenitor cells after sustained exposure to static magnetism.

    PubMed

    Nakamichi, Noritaka; Ishioka, Yukichi; Hirai, Takao; Ozawa, Shusuke; Tachibana, Masaki; Nakamura, Nobuhiro; Takarada, Takeshi; Yoneda, Yukio

    2009-08-15

    We have previously shown significant potentiation of Ca(2+) influx mediated by N-methyl-D-aspartate receptors, along with decreased microtubules-associated protein-2 (MAP2) expression, in hippocampal neurons cultured under static magnetism without cell death. In this study, we investigated the effects of static magnetism on the functionality of neural progenitor cells endowed to proliferate for self-replication and differentiate into neuronal, astroglial, and oligodendroglial lineages. Neural progenitor cells were isolated from embryonic rat neocortex and hippocampus, followed by culture under static magnetism at 100 mT and subsequent determination of the number of cells immunoreactive for a marker protein of particular progeny lineages. Static magnetism not only significantly decreased proliferation of neural progenitor cells without affecting cell viability, but also promoted differentiation into cells immunoreactive for MAP2 with a concomitant decrease in that for an astroglial marker, irrespective of the presence of differentiation inducers. In neural progenitors cultured under static magnetism, a significant increase was seen in mRNA expression of several activator-type proneural genes, such as Mash1, Math1, and Math3, together with decreased mRNA expression of the repressor type Hes5. These results suggest that sustained static magnetism could suppress proliferation for self-renewal and facilitate differentiation into neurons through promoted expression of activator-type proneural genes by progenitor cells in fetal rat brain.

  14. The parkinson's disease-associated LRRK2 mutation R1441G inhibits neuronal differentiation of neural stem cells.

    PubMed

    Bahnassawy, Lamia'a; Nicklas, Sarah; Palm, Thomas; Menzl, Ingeborg; Birzele, Fabian; Gillardon, Frank; Schwamborn, Jens C

    2013-09-15

    Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause familial as well as sporadic Parkinson's disease (PD) that is characterized by an age-dependent degeneration of dopaminergic neurons. LRRK2 is strongly expressed in neural stem cells (NSCs), but still the exact molecular function of LRRK2 in these cells remains unknown. By performing a systemic analysis of the gene expression profile of LRRK2-deficient NSCs, we found that the expression of several PD-associated genes, such as oxidation and reduction in mitochondria, are deregulated on LRRK2 absence. Our data, indeed, indicate that LRRK2 regulates the level of cellular oxidative stress and thereby influences the survival of NSCs. Furthermore, the lack of LRRK2 leads to an up-regulation of neuronal differentiation-inducing processes, including the Let-7a pathway. On the other hand, the constitutive mutant of LRRK2(R1441G), known to cause PD, leads to down-regulation of the same pathway. In agreement with the function of Let-7a during neuronal differentiation, LRRK2-deficient NSCs differentiate faster than wild-type cells, while LRRK2(R1441G)-expressing NSCs show impaired neuronal differentiation. These results might help better characterize the molecular mechanisms underlying the role of LRRK2 in NSCs and would further improve potential cell-replacement strategies as well as drug discovery approaches.

  15. Tryptophan hydroxylase 2 (TPH2) in a neuronal cell line: modulation by cell differentiation and NRSF/rest activity.

    PubMed

    Gentile, Maria Teresa; Nawa, Yukino; Lunardi, Gianluigi; Florio, Tullio; Matsui, Hiroaki; Colucci-D'Amato, Luca

    2012-12-01

    Serotonin (5-HT) is a neurotransmitter involved in many aspects of the neuronal function. The synthesis of 5-HT is initiated by the hydroxylation of tryptophan, catalyzed by tryptophan hydroxylase (TPH). Two isoforms of TPH (TPH1 and TPH2) have been identified, with TPH2 almost exclusively expressed in the brain. Following TPH2 discovery, it was reported that polymorphisms of both gene and non-coding regions are associated with a spectrum of psychiatric disorders. Thus, insights into the mechanisms that specifically regulate TPH2 expression and its modulation by exogenous stimuli may represent a new therapeutic approach to modify serotonergic neurotransmission. To this aim, a CNS-originated cell line expressing TPH2 endogenously represents a valid model system. In this study, we report that TPH2 transcript and protein are modulated by neuronal differentiation in the cell line A1 mes-c-myc (A1). Moreover, we show luciferase activity driven by the human TPH2 promoter region and demonstrate that upon mutation of the NRSF/REST responsive element, the promoter activity strongly increases with cell differentiation. Our data suggest that A1 cells could represent a model system, allowing an insight into the mechanisms of regulation of TPH2 and to identify novel therapeutic targets in the development of drugs for the management of psychiatric disorders. PMID:22958208

  16. Modulation of the Mesenchymal Stem Cell Secretome Using Computer-Controlled Bioreactors: Impact on Neuronal Cell Proliferation, Survival and Differentiation

    PubMed Central

    Teixeira, Fábio G.; Panchalingam, Krishna M.; Assunção-Silva, Rita; Serra, Sofia C.; Mendes-Pinheiro, Bárbara; Patrício, Patrícia; Jung, Sunghoon; Anjo, Sandra I.; Manadas, Bruno; Pinto, Luísa; Sousa, Nuno; Behie, Leo A.; Salgado, António J.

    2016-01-01

    In recent years it has been shown that the therapeutic benefits of human mesenchymal stem/stromal cells (hMSCs) in the Central Nervous System (CNS) are mainly attributed to their secretome. The implementation of computer-controlled suspension bioreactors has shown to be a viable route for the expansion of these cells to large numbers. As hMSCs actively respond to their culture environment, there is the hypothesis that one can modulate its secretome through their use. Herein, we present data indicating that the use of computer-controlled suspension bioreactors enhanced the neuroregulatory profile of hMSCs secretome. Indeed, higher levels of in vitro neuronal differentiation and NOTCH1 expression in human neural progenitor cells (hNPCs) were observed when these cells were incubated with the secretome of dynamically cultured hMSCs. A similar trend was also observed in the hippocampal dentate gyrus (DG) of rat brains where, upon injection, an enhanced neuronal and astrocytic survival and differentiation, was observed. Proteomic analysis also revealed that the dynamic culturing of hMSCs increased the secretion of several neuroregulatory molecules and miRNAs present in hMSCs secretome. In summary, the appropriate use of dynamic culture conditions can represent an important asset for the development of future neuro-regenerative strategies involving the use of hMSCs secretome. PMID:27301770

  17. Modulation of the Mesenchymal Stem Cell Secretome Using Computer-Controlled Bioreactors: Impact on Neuronal Cell Proliferation, Survival and Differentiation.

    PubMed

    Teixeira, Fábio G; Panchalingam, Krishna M; Assunção-Silva, Rita; Serra, Sofia C; Mendes-Pinheiro, Bárbara; Patrício, Patrícia; Jung, Sunghoon; Anjo, Sandra I; Manadas, Bruno; Pinto, Luísa; Sousa, Nuno; Behie, Leo A; Salgado, António J

    2016-01-01

    In recent years it has been shown that the therapeutic benefits of human mesenchymal stem/stromal cells (hMSCs) in the Central Nervous System (CNS) are mainly attributed to their secretome. The implementation of computer-controlled suspension bioreactors has shown to be a viable route for the expansion of these cells to large numbers. As hMSCs actively respond to their culture environment, there is the hypothesis that one can modulate its secretome through their use. Herein, we present data indicating that the use of computer-controlled suspension bioreactors enhanced the neuroregulatory profile of hMSCs secretome. Indeed, higher levels of in vitro neuronal differentiation and NOTCH1 expression in human neural progenitor cells (hNPCs) were observed when these cells were incubated with the secretome of dynamically cultured hMSCs. A similar trend was also observed in the hippocampal dentate gyrus (DG) of rat brains where, upon injection, an enhanced neuronal and astrocytic survival and differentiation, was observed. Proteomic analysis also revealed that the dynamic culturing of hMSCs increased the secretion of several neuroregulatory molecules and miRNAs present in hMSCs secretome. In summary, the appropriate use of dynamic culture conditions can represent an important asset for the development of future neuro-regenerative strategies involving the use of hMSCs secretome. PMID:27301770

  18. Forcing neural progenitor cells to cycle is insufficient to alter cell-fate decision and timing of neuronal differentiation in the spinal cord

    PubMed Central

    Lobjois, Valérie; Bel-Vialar, Sophie; Trousse, Françoise; Pituello, Fabienne

    2008-01-01

    Background During the development of the nervous system, neural progenitor cells can either stay in the pool of proliferating undifferentiated cells or exit the cell cycle and differentiate. Two main factors will determine the fate of a neural progenitor cell: its position within the neuroepithelium and the time at which the cell initiates differentiation. In this paper we investigated the importance of the timing of cell cycle exit on cell-fate decision by forcing neural progenitors to cycle and studying the consequences on specification and differentiation programs. Results As a model, we chose the spinal progenitors of motor neurons (pMNs), which switch cell-fate from motor neurons to oligodendrocytes with time. To keep pMNs in the cell cycle, we forced the expression of G1-phase regulators, the D-type cyclins. We observed that keeping neural progenitor cells cycling is not sufficient to retain them in the progenitor domain (ventricular zone); transgenic cells instead migrate to the differentiating field (mantle zone) regardless of cell cycle exit. Cycling cells located in the mantle zone do not retain markers of neural progenitor cells such as Sox2 or Olig2 but upregulate transcription factors involved in motor neuron specification, including MNR2 and Islet1/2. These cycling cells also progress through neuronal differentiation to axonal extension. We also observed mitotic cells displaying all the features of differentiating motor neurons, including axonal projection via the ventral root. However, the rapid decrease observed in the proliferation rate of the transgenic motor neuron population suggests that they undergo only a limited number of divisions. Finally, quantification of the incidence of the phenotype in young and more mature neuroepithelium has allowed us to propose that once the transcriptional program assigning neural progenitor cells to a subtype of neurons is set up, transgenic cells progress in their program of differentiation regardless of cell

  19. Dynamin-related protein 1 controls the migration and neuronal differentiation of subventricular zone-derived neural progenitor cells

    PubMed Central

    Kim, Hyun Jung; Shaker, Mohammed R.; Cho, Bongki; Cho, Hyo Min; Kim, Hyun; Kim, Joo Yeon; Sun, Woong

    2015-01-01

    Mitochondria are important in many essential cellular functions, including energy production, calcium homeostasis, and apoptosis. The organelles are scattered throughout the cytoplasm, but their distribution can be altered in response to local energy demands, such as cell division and neuronal maturation. Mitochondrial distribution is closely associated with mitochondrial fission, and blocking the fission-promoting protein dynamin-related protein 1 (Drp1) activity often results in mitochondrial elongation and clustering. In this study, we observed that mitochondria were preferentially localized at the leading process of migratory adult neural stem cells (aNSCs), whereas neuronal differentiating cells transiently exhibited perinuclear condensation of mitochondria. Inhibiting Drp1 activity altered the typical migratory cell morphology into round shapes while the polarized mitochondrial distribution was maintained. With these changes, aNSCs failed to migrate, and neuronal differentiation was prevented. Because Drp1 blocking also impaired the mitochondrial membrane potential, we tested whether supplementing with L-carnitine, a compound that restores mitochondrial membrane potential and ATP synthesis, could revert the defects induced by Drp1 inhibition. Interestingly, L-carnitine fully restored the aNSC defects, including cell shrinkage, migration, and impaired neuronal differentiation. These results suggest that Drp1 is required for functionally active mitochondria, and supplementing with ATP can restore the defects induced by Drp1 suppression. PMID:26514444

  20. Tumour necrosis factor-alpha impairs neuronal differentiation but not proliferation of hippocampal neural precursor cells: Role of Hes1.

    PubMed

    Keohane, Aoife; Ryan, Sinead; Maloney, Eimer; Sullivan, Aideen M; Nolan, Yvonne M

    2010-01-01

    Tumour necrosis factor-alpha (TNFalpha) is a pro-inflammatory cytokine, which influences neuronal survival and function yet there is limited information available on its effects on hippocampal neural precursor cells (NPCs). We show that TNFalpha treatment during proliferation had no effect on the percentage of proliferating cells prepared from embryonic rat hippocampal neurosphere cultures, nor did it affect cell fate towards either an astrocytic or neuronal lineage when cells were then allowed to differentiate. However, when cells were differentiated in the presence of TNFalpha, significantly reduced percentages of newly born and post-mitotic neurons, significantly increased percentages of astrocytes and increased expression of TNFalpha receptors, TNF-R1 and TNF-R2, as well as expression of the anti-neurogenic Hes1 gene, were observed. These data indicate that exposure of hippocampal NPCs to TNFalpha when they are undergoing differentiation but not proliferation has a detrimental effect on their neuronal lineage fate, which may be mediated through increased expression of Hes1.

  1. Neural stem cells transplanted in a mouse model of Parkinson's disease differentiate to neuronal phenotypes and reduce rotational deficit.

    PubMed

    Ziavra, Despina; Makri, Georgia; Giompres, Panagiotis; Taraviras, Stavros; Thomaidou, Dimitra; Matsas, Rebecca; Mitsacos, Ada; Kouvelas, Elias D

    2012-11-01

    The most prominent pathological feature in Parkinson's disease (PD) is the progressive and selective loss of mesencephalic dopaminergic neurons of the nigrostriatal tract. The present study was conducted in order to investigate whether naive and or genetically modified neural stem/precursor cells (NPCs) can survive, differentiate and functionally integrate in the lesioned striatum. To this end, stereotaxic injections of 6-OHDA in the right ascending nigrostriatal dopaminergic pathway of mice and subsequent NPC transplantations were performed, followed by apomorphine-induced rotations and double-immunofluorescence experiments. Our results demonstrate that transplanted embryonic NPCs derived from the cortical ventricular zone of E14.5 transgenic mouse embryos expressing the green fluorescent protein (GFP) under control of the beta-actin promoter and cultured as neurospheres can survive in the host striatum for at least three weeks after transplantation. The percentage of surviving GFP-positive cells in the host striatum ranges from 0.2% to 0.6% of the total transplanted NPCs. Grafted cells functionally integrate in the striatum, as indicated by the statistically significant decrease of contralateral rotations after apomorphine treatment. Furthermore, we show that within the striatal environment GFP-positive cells differentiate into beta-III tubulin-expressing neurons, but not glial cells. Most importantly, GFP-positive cells further differentiate to dopaminergic (TH-positive) and medium size spiny (DARPP-32- positive) neuronal phenotypes. Over-expression of the cell cycle exit and neuronal differentiation protein Cend1 in NPCs enhances the generation of GABAergic, but not dopaminergic, neuronal phenotypes after grafting in the lesioned striatum. Our results encourage the development of strategies involving NPC transplantation for the treatment of neurodegenerative diseases.

  2. Surface-Functionalized Silk Fibroin Films as a Platform To Guide Neuron-like Differentiation of Human Mesenchymal Stem Cells.

    PubMed

    Manchineella, Shivaprasad; Thrivikraman, Greeshma; Basu, Bikramjit; Govindaraju, T

    2016-09-01

    Surface interactions at the biomaterial-cellular interface determine the proliferation and differentiation of stem cells. Manipulating such interactions through the surface chemistry of scaffolds renders control over directed stem cell differentiation into the cell lineage of interest. This approach is of central importance for stem cell-based tissue engineering and regenerative therapy applications. In the present study, silk fibroin films (SFFs) decorated with integrin-binding laminin peptide motifs (YIGSR and GYIGSR) were prepared and employed for in vitro adult stem cell-based neural tissue engineering applications. Functionalization of SFFs with short peptides showcased the peptide sequence and nature of functionalization-dependent differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs). Intriguingly, covalently functionalized SFFs with GYIGSR hexapeptide (CL2-SFF) supported hMSC proliferation and maintenance in an undifferentiated pluripotent state and directed the differentiation of hMSCs into neuron-like cells in the presence of a biochemical cue, on-demand. The observed morphological changes were further corroborated by the up-regulation of neuronal-specific marker gene expression (MAP2, TUBB3, NEFL), confirmed through semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. The enhanced proliferation and on-demand directed differentiation of adult stem cells (hMSCs) by the use of an economically viable short recognition peptide (GYIGSR), as opposed to the integrin recognition protein laminin, establishes the potential of SFFs for neural tissue engineering and regenerative therapy applications. PMID:27518901

  3. Carbon Monoxide Releasing Molecule-A1 (CORM-A1) Improves Neurogenesis: Increase of Neuronal Differentiation Yield by Preventing Cell Death.

    PubMed

    Almeida, Ana S; Soares, Nuno L; Vieira, Melissa; Gramsbergen, Jan Bert; Vieira, Helena L A

    2016-01-01

    Cerebral ischemia and neurodegenerative diseases lead to impairment or death of neurons in the central nervous system. Stem cell based therapies are promising strategies currently under investigation. Carbon monoxide (CO) is an endogenous product of heme degradation by heme oxygenase (HO) activity. Administration of CO at low concentrations produces several beneficial effects in distinct tissues, namely anti-apoptotic and anti-inflammatory. Herein the CO role on modulation of neuronal differentiation was assessed. Three different models with increasing complexity were used: human neuroblastoma SH-S5Y5 cell line, human teratocarcinoma NT2 cell line and organotypic hippocampal slice cultures (OHSC). Cell lines were differentiated into post-mitotic neurons by treatment with retinoic acid (RA) supplemented with CO-releasing molecule A1 (CORM-A1). CORM-A1 positively modulated neuronal differentiation, since it increased final neuronal production and enhanced the expression of specific neuronal genes: Nestin, Tuj1 and MAP2. Furthermore, during neuronal differentiation process, there was an increase in proliferative cell number (ki67 mRNA expressing cells) and a decrease in cell death (lower propidium iodide (PI) uptake, limitation of caspase-3 activation and higher Bcl-2 expressing cells). CO supplementation did not increase the expression of RA receptors. In the case of SH-S5Y5 model, small amounts of reactive oxygen species (ROS) generation emerges as important signaling molecules during CO-promoted neuronal differentiation. CO's improvement of neuronal differentiation yield was validated using OHSC as ex vivo model. CORM-A1 treatment of OHSC promoted higher levels of cells expressing the neuronal marker Tuj1. Still, CORM-A1 increased cell proliferation assessed by ki67 expression and also prevented cell death, which was followed by increased Bcl-2 expression, decreased levels of active caspase-3 and PI uptake. Likewise, ROS signaling emerged as key factors in CO

  4. Induction of apoptotic death and retardation of neuronal differentiation of human neural stem cells by sodium arsenite treatment

    SciTech Connect

    Ivanov, Vladimir N.; Hei, Tom K.

    2013-04-01

    Chronic arsenic toxicity is a global health problem that affects more than 100 million people worldwide. Long-term health effects of inorganic sodium arsenite in drinking water may result in skin, lung and liver cancers and in severe neurological abnormalities. We investigated in the present study whether sodium arsenite affects signaling pathways that control cell survival, proliferation and neuronal differentiation of human neural stem cells (NSC). We demonstrated that the critical signaling pathway, which was suppressed by sodium arsenite in NSC, was the protective PI3K–AKT pathway. Sodium arsenite (2–4 μM) also caused down-regulation of Nanog, one of the key transcription factors that control pluripotency and self-renewal of stem cells. Mitochondrial damage and cytochrome-c release induced by sodium arsenite exposure was followed by initiation of the mitochondrial apoptotic pathway in NSC. Beside caspase-9 and caspase-3 inhibitors, suppression of JNK activity decreased levels of arsenite-induced apoptosis in NSC. Neuronal differentiation of NSC was substantially inhibited by sodium arsenite exposure. Overactivation of JNK1 and ERK1/2 and down-regulation of PI3K–AKT activity induced by sodium arsenite were critical factors that strongly affected neuronal differentiation. In conclusion, sodium arsenite exposure of human NSC induces the mitochondrial apoptotic pathway, which is substantially accelerated due to the simultaneous suppression of PI3K–AKT. Sodium arsenite also negatively affects neuronal differentiation of NSC through overactivation of MEK–ERK and suppression of PI3K–AKT. - Highlights: ► Arsenite induces the mitochondrial apoptotic pathway in human neural stem cells. ► Arsenite-induced apoptosis is strongly upregulated by suppression of PI3K–AKT. ► Arsenite-induced apoptosis is strongly down-regulated by inhibition of JNK–cJun. ► Arsenite negatively affects neuronal differentiation by inhibition of PI3K–AKT.

  5. Nanofiber Matrices Promote the Neuronal Differentiation of Human Embryonic Stem Cell-Derived Neural Precursors In Vitro

    PubMed Central

    Lim, Shawn H.; Christopherson, Gregory T.; Xu, Leyan; Nasonkin, Igor; Yu, Christopher; Mao, Hai-Quan; Koliatsos, Vassilis E.

    2011-01-01

    The potential of human embryonic stem (ES) cells as experimental therapies for neuronal replacement has recently received considerable attention. In view of the organization of the mature nervous system into distinct neural circuits, key challenges of such therapies are the directed differentiation of human ES cell-derived neural precursors (NPs) into specific neuronal types and the directional growth of axons along specified trajectories. In the present study, we cultured human NPs derived from the NIH-approved ES line BGO1 on polycaprolactone fiber matrices of different diameter (i.e., nanofibers and microfibers) and orientation (i.e., aligned and random); fibers were coated with poly-L-ornithine/laminin to mimic the extracellular matrix and support the adhesion, viability, and differentiation of NPs. On aligned fibrous meshes, human NPs adopt polarized cell morphology with processes extending along the axis of the fiber, whereas NPs on plain tissue culture surfaces or random fiber substrates form nonpolarized neurite networks. Under differentiation conditions, human NPs cultured on aligned fibrous substrates show a higher rate of neuronal differentiation than other matrices; 62% and 86% of NPs become TUJ1 (+) early neurons on aligned micro- and nanofibers, respectively, whereas only 32% and 27% of NPs acquire the same fate on random micro- and nanofibers. Metabolic cell activity/viability studies reveal that fiber alignment and diameter also have an effect on NP viability, but only in the presence of mitogens. Our findings demonstrate that fibrous substrates serve as an artificial extracellular matrix and provide a microenviroment that influences key aspects of the neuronal differentiation of ES-derived NPs. PMID:20973749

  6. Neuronal growth and differentiation on biodegradable membranes.

    PubMed

    Morelli, Sabrina; Piscioneri, Antonella; Messina, Antonietta; Salerno, Simona; Al-Fageeh, Mohamed B; Drioli, Enrico; De Bartolo, Loredana

    2015-02-01

    Semipermeable polymeric membranes with appropriate morphological, physicochemical and transport properties are relevant to inducing neural regeneration. We developed novel biodegradable membranes to support neuronal differentiation. In particular, we developed chitosan, polycaprolactone and polyurethane flat membranes and a biosynthetic blend between polycaprolactone and polyurethane by phase-inversion techniques. The biodegradable membranes were characterized in order to evaluate their morphological, physicochemical, mechanical and degradation properties. We investigated the efficacy of these different membranes to promote the adhesion and differentiation of neuronal cells. We employed as model cell system the human neuroblastoma cell line SHSY5Y, which is a well-established system for studying neuronal differentiation. The investigation of viability and specific neuronal marker expression allowed assessment that the correct neuronal differentiation and the formation of neuronal network had taken place in vitro in the cells seeded on different biodegradable membranes. Overall, this study provides evidence that neural cell responses depend on the nature of the biodegradable polymer used to form the membranes, as well as on the dissolution, hydrophilic and, above all, mechanical membrane properties. PCL-PU membranes exhibit mechanical properties that improve neurite outgrowth and the expression of specific neuronal markers.

  7. Metabolic Differentiation of Neuronal Phenotypes by Single Cell CE-ESI-MS

    PubMed Central

    Nemes, Peter; Knolhoff, Ann M.; Rubakhin, Stanislav S.; Sweedler, Jonathan V.

    2011-01-01

    Single cell mass spectrometry (MS) is a rapidly emerging field in metabolic investigations. The inherent chemical complexity of most biological samples poses analytical challenges when using MS platforms to measure sample content without prior chemical separation. Here, a single-cell capillary electrophoresis (CE) system was coupled with electrospray ionization (ESI) MS to enable the simultaneous measurement of a vast array of endogenous compounds in over 50 identified and isolated large neurons from the Aplysia californica central nervous system. More than 300 distinct ion signals (m/z values) were detected from a single neuron in positive ion mode, 140 of which were selected for chemometric data analysis. Metabolic features were evaluated among six different neuron types (B1, B2, left pleural 1 (LPl1), metacerebral cell (MCC), R2, and R15), chosen for their various physiological functions. The results indicated chemical similarities among some neuron types (B1 to B2 and LPl1 to R2) and distinctive features for others (MCC and R15 cells). The quantitative nature of the MS platform allowed the comparison of metabolite levels for specific neurons. The CE-ESI-MS approach for examination of individual nanoliter-volume cells as described herein is readily adaptable to other volume-limited samples. PMID:21809850

  8. Morphological Differentiation Towards Neuronal Phenotype of SH-SY5Y Neuroblastoma Cells by Estradiol, Retinoic Acid and Cholesterol.

    PubMed

    Teppola, Heidi; Sarkanen, Jertta-Riina; Jalonen, Tuula O; Linne, Marja-Leena

    2016-04-01

    Human SH-SY5Y neuroblastoma cells maintain their potential for differentiation and regression in culture conditions. The induction of differentiation could serve as a strategy to inhibit cell proliferation and tumor growth. Previous studies have shown that differentiation of SH-SY5Y cells can be induced by all-trans-retinoic-acid (RA) and cholesterol (CHOL). However, signaling pathways that lead to terminal differentiation of SH-SY5Y cells are still largely unknown. The goal of this study was to examine in the RA and CHOL treated SH-SY5Y cells the additive impacts of estradiol (E2) and brain-derived neurotrophic factor (BDNF) on cell morphology, cell population growth, synaptic vesicle recycling and presence of neurofilaments. The above features indicate a higher level of neuronal differentiation. Our data show that treatment for 10 days in vitro (DIV) with RA alone or when combined with E2 (RE) or CHOL (RC), but not when combined with BDNF (RB), significantly (p < 0.01) inhibited the cell population growth. Synaptic vesicle recycling, induced by high-K(+) depolarization, was significantly increased in all treatments where RA was included (RE, RC, RB, RCB), and when all agents were added together (RCBE). Specifically, our results show for the first time that E2 treatment can alone increase synaptic vesicle recycling in SH-SY5Y cells. This work contributes to the understanding of the ways to improve suppression of neuroblastoma cells' population growth by inducing maturation and differentiation.

  9. Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells.

    PubMed

    Sun, Yubing; Yong, Koh Meng Aw; Villa-Diaz, Luis G; Zhang, Xiaoli; Chen, Weiqiang; Philson, Renee; Weng, Shinuo; Xu, Haoxing; Krebsbach, Paul H; Fu, Jianping

    2014-06-01

    Our understanding of the intrinsic mechanosensitive properties of human pluripotent stem cells (hPSCs), in particular the effects that the physical microenvironment has on their differentiation, remains elusive. Here, we show that neural induction and caudalization of hPSCs can be accelerated by using a synthetic microengineered substrate system consisting of poly(dimethylsiloxane) micropost arrays (PMAs) with tunable mechanical rigidities. The purity and yield of functional motor neurons derived from hPSCs within 23 days of culture using soft PMAs were improved more than fourfold and tenfold, respectively, compared with coverslips or rigid PMAs. Mechanistic studies revealed a multi-targeted mechanotransductive process involving Smad phosphorylation and nucleocytoplasmic shuttling, regulated by rigidity-dependent Hippo/YAP activities and actomyosin cytoskeleton integrity and contractility. Our findings suggest that substrate rigidity is an important biophysical cue influencing neural induction and subtype specification, and that microengineered substrates can thus serve as a promising platform for large-scale culture of hPSCs. PMID:24728461

  10. Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells

    NASA Astrophysics Data System (ADS)

    Sun, Yubing; Yong, Koh Meng Aw; Villa-Diaz, Luis G.; Zhang, Xiaoli; Chen, Weiqiang; Philson, Renee; Weng, Shinuo; Xu, Haoxing; Krebsbach, Paul H.; Fu, Jianping

    2014-06-01

    Our understanding of the intrinsic mechanosensitive properties of human pluripotent stem cells (hPSCs), in particular the effects that the physical microenvironment has on their differentiation, remains elusive. Here, we show that neural induction and caudalization of hPSCs can be accelerated by using a synthetic microengineered substrate system consisting of poly(dimethylsiloxane) micropost arrays (PMAs) with tunable mechanical rigidities. The purity and yield of functional motor neurons derived from hPSCs within 23 days of culture using soft PMAs were improved more than fourfold and tenfold, respectively, compared with coverslips or rigid PMAs. Mechanistic studies revealed a multi-targeted mechanotransductive process involving Smad phosphorylation and nucleocytoplasmic shuttling, regulated by rigidity-dependent Hippo/YAP activities and actomyosin cytoskeleton integrity and contractility. Our findings suggest that substrate rigidity is an important biophysical cue influencing neural induction and subtype specification, and that microengineered substrates can thus serve as a promising platform for large-scale culture of hPSCs.

  11. Interferon-gamma but not TNF alpha promotes neuronal differentiation and neurite outgrowth of murine adult neural stem cells.

    PubMed

    Wong, Galaxy; Goldshmit, Yona; Turnley, Ann M

    2004-05-01

    Neural trauma, such as traumatic brain injury or stroke, results in a vigorous inflammatory response at and near the site of injury, with cytokine production by endogenous glial cells and invading immune cells. Little is known of the effect that these cytokines have on neural stem cell function. Here we examine the effects of two inflammatory cytokines, interferon-gamma (IFN gamma) and tumour necrosis factor-alpha (TNFalpha), on adult neural stem cells. Neural stem cells grown in the presence of either cytokine failed to generate neurospheres. Cytotoxicity assays showed that TNF alpha but not IFN gamma was toxic to the neural stem cells under proliferative conditions. Under differentiating conditions, neither cytokine was toxic; however, IFN gamma enhanced neuronal differentiation, rapidly increasing beta III-tubulin positive cell numbers 3-4 fold and inhibiting astrocyte generation. Furthermore, neurite outgrowth and the number of neurites per neuron was enhanced in cells differentiated in the presence of IFN gamma. Therefore, both inflammatory cytokines examined have substantial, but different effects on neural stem cell function and suggests that regulation of the inflammatory environment following brain injury may influence the ability of neural stem cells to repair the damage. PMID:15081598

  12. Flash photo stimulation of human neural stem cells on graphene/TiO2 heterojunction for differentiation into neurons

    NASA Astrophysics Data System (ADS)

    Akhavan, Omid; Ghaderi, Elham

    2013-10-01

    For the application of human neural stem cells (hNSCs) in neural regeneration and brain repair, it is necessary to stimulate hNSC differentiation towards neurons rather than glia. Due to the unique properties of graphene in stem cell differentiation, here we introduce reduced graphene oxide (rGO)/TiO2 heterojunction film as a biocompatible flash photo stimulator for effective differentiation of hNSCs into neurons. Using the stimulation, the number of cell nuclei on rGO/TiO2 increased by a factor of ~1.5, while on GO/TiO2 and TiO2 it increased only ~48 and 24%, respectively. Moreover, under optimum conditions of flash photo stimulation (10 mW cm-2 flash intensity and 15.0 mM ascorbic acid in cell culture medium) not only did the number of cell nuclei and neurons differentiated on rGO/TiO2 significantly increase (by factors of ~2.5 and 3.6), but also the number of glial cells decreased (by a factor of ~0.28). This resulted in a ~23-fold increase in the neural to glial cell ratio. Such highly accelerated differentiation was assigned to electron injection from the photoexcited TiO2 into the cells on the rGO through Ti-C and Ti-O-C bonds. The role of ascorbic acid, as a scavenger of the photoexcited holes, in flash photo stimulation was studied at various concentrations and flash intensities.

  13. The BM88 antigen, a novel neuron-specific molecule, enhances the differentiation of mouse neuroblastoma cells.

    PubMed

    Mamalaki, A; Boutou, E; Hurel, C; Patsavoudi, E; Tzartos, S; Matsas, R

    1995-06-01

    The BM88 antigen is a neuron-specific molecule widely distributed in the mammalian nervous system. It is a 22-kDa, apparently not glycosylated, integral membrane protein, which appears early during brain development and remains at high levels in the mature animal. Here, we describe the cDNA cloning of the porcine BM88 antigen and present evidence that this protein is involved in neuroblastoma cell differentiation. The deduced protein is a novel molecule consisting of 140 amino acids and bears a putative transmembrane domain at the COOH-terminal region. The mRNA of this protein is expressed only in neural tissues, where it is restricted to neurons. Stably transfected Neuro-2a cells overexpressing the BM88 antigen exhibited a significant change in morphology, reflected by enhanced process outgrowth, and a slower rate of division. Moreover, in the presence of differentiation agents, such as sucrose and retinoic acid, an accelerated differentiation of the transfected Neuro-2a cells was observed. Especially in the presence of sucrose, the consequent overexpression of the BM88 antigen in the transfected cells resulted in their enhanced morphological differentiation accompanied by the induction of neurofilament protein expression. Our results suggest that the BM88 antigen plays a role in the differentiation of neuroblastoma cells.

  14. Elevated IKKα accelerates the differentiation of human neuronal progenitor cells and induces MeCP2-dependent BDNF expression.

    PubMed

    Khoshnan, Ali; Patterson, Paul H

    2012-01-01

    The IκB kinase α (IKKα) is implicated in the differentiation of epithelial and immune cells. We examined whether IKKα also plays a role in the differentiation and maturation of embryonic human neuronal progenitor cells (NPCs). We find that expression of an extra copy of IKKα (IKKα+) blocks self-renewal and accelerates the differentiation of NPCs. This coincides with reduced expression of the Repressor Element Silencing Transcription Factor/Neuron-Restrictive Silencing Factor (REST/NRSF), which is a prominent inhibitor of neurogenesis, and subsequent induction of the pro-differentiation non-coding RNA, miR-124a. However, the effects of IKKα on REST/NRSF and miR-124a expression are likely to be indirect. IKKα+ neurons display extensive neurite outgrowth and accumulate protein markers of neuronal maturation such as SCG10/stathmin-2, postsynaptic density 95 (PSD95), syntaxin, and methyl-CpG binding protein 2 (MeCP2). Interestingly, IKKα associates with MeCP2 in the nuclei of human neurons and can phosphorylate MeCP2 in vitro. Using chromatin immunoprecipitation assays, we find that IKKα is recruited to the exon-IV brain-derived neurotrophic factor (BDNF) promoter, which is a well-characterized target of MeCP2 activity. Moreover, IKKα induces the transcription of BDNF and knockdown expression of MeCP2 interferes with this event. These studies highlight a role for IKKα in accelerating the differentiation of human NPCs and identify IKKα as a potential regulator of MeCP2 function and BDNF expression. PMID:22848609

  15. RAF Kinase Activity Regulates Neuroepithelial Cell Proliferation and Neuronal Progenitor Cell Differentiation during Early Inner Ear Development

    PubMed Central

    Magariños, Marta; Aburto, María R.; Sánchez-Calderón, Hortensia; Muñoz-Agudo, Carmen; Rapp, Ulf R.; Varela-Nieto, Isabel

    2010-01-01

    Background Early inner ear development requires the strict regulation of cell proliferation, survival, migration and differentiation, coordinated by the concerted action of extrinsic and intrinsic factors. Deregulation of these processes is associated with embryonic malformations and deafness. We have shown that insulin-like growth factor I (IGF-I) plays a key role in embryonic and postnatal otic development by triggering the activation of intracellular lipid and protein kinases. RAF kinases are serine/threonine kinases that regulate the highly conserved RAS-RAF-MEK-ERK signaling cascade involved in transducing the signals from extracellular growth factors to the nucleus. However, the regulation of RAF kinase activity by growth factors during development is complex and still not fully understood. Methodology/Principal Findings By using a combination of qRT-PCR, Western blotting, immunohistochemistry and in situ hybridization, we show that C-RAF and B-RAF are expressed during the early development of the chicken inner ear in specific spatiotemporal patterns. Moreover, later in development B-RAF expression is associated to hair cells in the sensory patches. Experiments in ex vivo cultures of otic vesicle explants demonstrate that the influence of IGF-I on proliferation but not survival depends on RAF kinase activating the MEK-ERK phosphorylation cascade. With the specific RAF inhibitor Sorafenib, we show that blocking RAF activity in organotypic cultures increases apoptosis and diminishes the rate of cell proliferation in the otic epithelia, as well as severely impairing neurogenesis of the acoustic-vestibular ganglion (AVG) and neuron maturation. Conclusions/Significance We conclude that RAF kinase activity is essential to establish the balance between cell proliferation and death in neuroepithelial otic precursors, and for otic neuron differentiation and axonal growth at the AVG. PMID:21203386

  16. A Novel Protocol to Differentiate Induced Pluripotent Stem Cells by Neuronal microRNAs to Provide a Suitable Cellular Model.

    PubMed

    Zare, Mehrak; Soleimani, Masoud; Akbarzadeh, Abolfazl; Bakhshandeh, Behnaz; Aghaee-Bakhtiari, Seyed Hamid; Zarghami, Nosratollah

    2015-08-01

    Neurodegenerative diseases are one of the most challenging subjects in medicine. Investigation of their underlying genetic or epigenetic factors is hampered by lack of suitable models. Patient-specific induced pluripotent stem cells (iPS cells) represent a valuable approach to provide a proper model for poorly understood mechanisms of neuronal diseases and the related drug screenings. miR-124 and miR-128 are the two brain-enriched miRNAs with different time-points of expression during neuronal development. Herein, we transduced human iPS cells with miR-124 and miR-128 harboring lentiviruses sequentially. The transduced plasmids contained GFP and puromycin antibiotic-resistant genes for easier selection and identification. Morphological assessment and immunocytochemistry (overexpressions of beta-tubulin and neuron-specific enolase) confirmed that induced hiPS cells by miR-124 and miR-128 represent similar characteristics to those of mature neurons. In addition, the upregulation of neuron-specific enolase, beta-tubulin, Map2, GFAP, and BDNF was detected by quantitative real-time PCR. In conclusion, it seems that our novel protocol remarks the combinatorial effect of miR-124 and miR-128 on neural differentiation in the absence of any extrinsic factor. Moreover, such cellular models could be used in personalized drug screening and applied for more effective therapies.

  17. Homocysteinylation of neuronal proteins contributes to folate deficiency-associated alterations of differentiation, vesicular transport, and plasticity in hippocampal neuronal cells.

    PubMed

    Akchiche, Nassila; Bossenmeyer-Pourié, Carine; Kerek, Racha; Martin, Nicolas; Pourié, Grégory; Koziel, Violette; Helle, Déborah; Alberto, Jean-Marc; Ortiou, Sandrine; Camadro, Jean-Michel; Léger, Thibaut; Guéant, Jean-Louis; Daval, Jean-Luc

    2012-10-01

    Despite the key role in neuronal development of a deficit in the methyl donor folate, little is known on the underlying mechanisms. We therefore studied the consequences of folate deficiency on proliferation, differentiation, and plasticity of the rat H19-7 hippocampal cell line. Folate deficit reduced proliferation (17%) and sensitized cells to differentiation-associated apoptosis (+16%). Decreased production (-58%) of S-adenosylmethionine (the universal substrate for transmethylation reactions) and increased expression of histone deacetylases (HDAC4,6,7) would lead to epigenomic changes that may impair the differentiation process. Cell polarity, vesicular transport, and synaptic plasticity were dramatically affected, with poor neurite outgrowth (-57%). Cell treatment by an HDAC inhibitor (SAHA) led to a noticeable improvement of cell polarity and morphology, with longer processes. Increased homocysteine levels (+55%) consecutive to folate shortage produced homocysteinylation, evidenced by coimmunoprecipitations and mass spectrometry, and aggregation of motor proteins dynein and kinesin, along with functional alterations, as reflected by reduced interactions with partner proteins. Prominent homocysteinylation of key neuronal proteins and subsequent aggregation certainly constitute major adverse effects of folate deficiency, affecting normal development with possible long-lasting consequences.

  18. Dopaminergic neurons differentiating from LRRK2 G2019S induced pluripotent stem cells show early neuritic branching defects.

    PubMed

    Borgs, Laurence; Peyre, Elise; Alix, Philippe; Hanon, Kevin; Grobarczyk, Benjamin; Godin, Juliette D; Purnelle, Audrey; Krusy, Nathalie; Maquet, Pierre; Lefebvre, Philippe; Seutin, Vincent; Malgrange, Brigitte; Nguyen, Laurent

    2016-01-01

    Some mutations of the LRRK2 gene underlie autosomal dominant form of Parkinson's disease (PD). The G2019S is a common mutation that accounts for about 2% of PD cases. To understand the pathophysiology of this mutation and its possible developmental implications, we developed an in vitro assay to model PD with human induced pluripotent stem cells (hiPSCs) reprogrammed from skin fibroblasts of PD patients suffering from the LRKK2 G2019S mutation. We differentiated the hiPSCs into neural stem cells (NSCs) and further into dopaminergic neurons. Here we show that NSCs bearing the mutation tend to differentiate less efficiently into dopaminergic neurons and that the latter exhibit significant branching defects as compared to their controls. PMID:27640816

  19. Dopaminergic neurons differentiating from LRRK2 G2019S induced pluripotent stem cells show early neuritic branching defects

    PubMed Central

    Borgs, Laurence; Peyre, Elise; Alix, Philippe; Hanon, Kevin; Grobarczyk, Benjamin; Godin, Juliette D.; Purnelle, Audrey; Krusy, Nathalie; Maquet, Pierre; Lefebvre, Philippe; Seutin, Vincent; Malgrange, Brigitte; Nguyen, Laurent

    2016-01-01

    Some mutations of the LRRK2 gene underlie autosomal dominant form of Parkinson’s disease (PD). The G2019S is a common mutation that accounts for about 2% of PD cases. To understand the pathophysiology of this mutation and its possible developmental implications, we developed an in vitro assay to model PD with human induced pluripotent stem cells (hiPSCs) reprogrammed from skin fibroblasts of PD patients suffering from the LRKK2 G2019S mutation. We differentiated the hiPSCs into neural stem cells (NSCs) and further into dopaminergic neurons. Here we show that NSCs bearing the mutation tend to differentiate less efficiently into dopaminergic neurons and that the latter exhibit significant branching defects as compared to their controls. PMID:27640816

  20. Dual Inhibition of Activin/Nodal/TGF-β and BMP Signaling Pathways by SB431542 and Dorsomorphin Induces Neuronal Differentiation of Human Adipose Derived Stem Cells

    PubMed Central

    Madhu, Vedavathi; Dighe, Abhijit S.; Cui, Quanjun; Deal, D. Nicole

    2016-01-01

    Damage to the nervous system can cause devastating diseases or musculoskeletal dysfunctions and transplantation of progenitor stem cells can be an excellent treatment option in this regard. Preclinical studies demonstrate that untreated stem cells, unlike stem cells activated to differentiate into neuronal lineage, do not survive in the neuronal tissues. Conventional methods of inducing neuronal differentiation of stem cells are complex and expensive. We therefore sought to determine if a simple, one-step, and cost effective method, previously reported to induce neuronal differentiation of embryonic stem cells and induced-pluripotent stem cells, can be applied to adult stem cells. Indeed, dual inhibition of activin/nodal/TGF-β and BMP pathways using SB431542 and dorsomorphin, respectively, induced neuronal differentiation of human adipose derived stem cells (hADSCs) as evidenced by formation of neurite extensions, protein expression of neuron-specific gamma enolase, and mRNA expression of neuron-specific transcription factors Sox1 and Pax6 and matured neuronal marker NF200. This process correlated with enhanced phosphorylation of p38, Erk1/2, PI3K, and Akt1/3. Additionally, in vitro subcutaneous implants of SB431542 and dorsomorphin treated hADSCs displayed significantly higher expression of active-axonal-growth-specific marker GAP43. Our data offers novel insights into cell-based therapies for the nervous system repair. PMID:26798350

  1. Stress Conditions Increase Vimentin Cleavage by Omi/HtrA2 Protease in Human Primary Neurons and Differentiated Neuroblastoma Cells.

    PubMed

    Lucotte, Bérangère; Tajhizi, Mehdi; Alkhatib, Dareen; Samuelsson, Eva-Britt; Wiehager, Birgitta; Schedin-Weiss, Sophia; Sundström, Erik; Winblad, Bengt; Tjernberg, Lars O; Behbahani, Homira

    2015-12-01

    Dysfunctional Omi/HtrA2, a mitochondrial serine protease, has been implicated in various neurodegenerative disorders. Despite the wealth of evidence on the roles of Omi/HtrA2 in apoptosis, little is known about its cytosolic targets, the cleavage of which could account for the observed morphological changes such as cytoskeletal reorganizations in axons. By proteomic analysis, vimentin was identified as a substrate for Omi/HtrA2 and we have reported increased Omi/HtrA2 protease activity in Alzheimer disease (AD) brain. Here, we investigated a possible link between Omi/HtrA2 and vimentin cleavage, and consequence of this cleavage on mitochondrial distribution in neurons. In vitro protease assays showed vimentin to be cleaved by Omi/HtrA2 protease, and proximity ligation assay demonstrated an increased interaction between Omi/HtrA2 and vimentin in human primary neurons upon stress stimuli. Using differentiated neuroblastoma SH-SY5Y cells, we showed that Omi/HtrA2 under several different stress conditions induces cleavage of vimentin in wild-type as well as SH-SY5Y cells transfected with amyloid precursor protein with the Alzheimer disease-associated Swedish mutation. After stress treatment, inhibition of Omi/HtrA2 protease activity by the Omi/HtrA2 specific inhibitor, Ucf-101, reduced the cleavage of vimentin in wild-type cells. Following altered vimentin filaments integrity by stress stimuli, mitochondria was redistributed in differentiated SH-SY5Y cells and human primary neurons. In summary, the findings outlined in this paper suggest a role of Omi/HtrA2 in modulation of vimentin filamentous structure in neurons. Our results provide important findings for understanding the biological role of Omi/HtrA2 activity during stress conditions, and give knowledge of interplay between Omi/HtrA2 and vimentin which might affect mitochondrial distribution in neurons.

  2. BDNF Increases Survival and Neuronal Differentiation of Human Neural Precursor Cells Cotransplanted with a Nanofiber Gel to the Auditory Nerve in a Rat Model of Neuronal Damage

    PubMed Central

    Jiao, Yu; Palmgren, Björn; Novozhilova, Ekaterina; Englund Johansson, Ulrica; Spieles-Engemann, Anne L.; Kale, Ajay; Stupp, Samuel I.; Olivius, Petri

    2014-01-01

    Objectives. To study possible nerve regeneration of a damaged auditory nerve by the use of stem cell transplantation. Methods. We transplanted HNPCs to the rat AN trunk by the internal auditory meatus (IAM). Furthermore, we studied if addition of BDNF affects survival and phenotypic differentiation of the grafted HNPCs. A bioactive nanofiber gel (PA gel), in selected groups mixed with BDNF, was applied close to the implanted cells. Before transplantation, all rats had been deafened by a round window niche application of β-bungarotoxin. This neurotoxin causes a selective toxic destruction of the AN while keeping the hair cells intact. Results. Overall, HNPCs survived well for up to six weeks in all groups. However, transplants receiving the BDNF-containing PA gel demonstrated significantly higher numbers of HNPCs and neuronal differentiation. At six weeks, a majority of the HNPCs had migrated into the brain stem and differentiated. Differentiated human cells as well as neurites were observed in the vicinity of the cochlear nucleus. Conclusion. Our results indicate that human neural precursor cells (HNPC) integration with host tissue benefits from additional brain derived neurotrophic factor (BDNF) treatment and that these cells appear to be good candidates for further regenerative studies on the auditory nerve (AN). PMID:25243135

  3. Interaction between Oc-1 and Lmx1a promotes ventral midbrain dopamine neural stem cells differentiation into dopamine neurons.

    PubMed

    Yuan, Jian; Lei, Zhi-nian; Wang, Xi; Deng, Yong-Jian; Chen, Dong-Bo

    2015-05-22

    Recent studies have shown that Onecut (Oc) transcription factors may be involved in the early development of midbrain dopaminergic neurons (mdDA). The expression profile of Oc factors matches that of Lmx1a, an important intrinsic transcription factor in the development of mDA neuron. Moreover, the Wnt1-Lmx1a pathway controls the mdDA differentiation. However, their expression dynamics and molecular mechanisms remain to be determined. To address these issues, we hypothesize that cross-talk between Oc-1 and Lmx1a regulates the mdDA specification and differentiation through the canonical Wnt-β-catenin pathway. We found that Oc-1 and Lmx1a displayed a very similar expression profile from embryonic to adult ventral midbrain (VM) tissues. Oc-1 regulated the proliferation and differentiation of ventral midbrain neural stem cells (vmNSCs). Downregulation of Oc-1 decreased both transcript and protein level of Lmx1a. Oc-1 interacted with lmx1a in vmNSCs in vitro and in VM tissues in vivo. Knockdown of Lmx1a reduced the expression of Oc-1 and Wnt1 in vmNSCs. Inhibiting Wnt1 signaling in vmNSCs provoked similar responses. Our data suggested that Oc-1 interacts with Lmx1a to promote vmNSCs differentiation into dopamine neuron through Wnt1-Lmx1a pathway.

  4. The effects of functional magnetic nanotubes with incorporated nerve growth factor in neuronal differentiation of PC12 cells

    NASA Astrophysics Data System (ADS)

    Xie, Jining; Chen, Linfeng; Varadan, Vijay K.; Yancey, Justin; Srivatsan, Malathi

    2008-03-01

    In this in vitro study the efficiency of magnetic nanotubes to bind with nerve growth factor (NGF) and the ability of NGF-incorporated magnetic nanotubes to release the bound NGF are investigated using rat pheochromocytoma cells (PC12 cells). It is found that functional magnetic nanotubes with NGF incorporation enabled the differentiation of PC12 cells into neurons exhibiting growth cones and neurite outgrowth. Microscope observations show that filopodia extending from neuron growth cones were in close proximity to the NGF-incorporated magnetic nanotubes, at times appearing to extend towards or into them. These results show that magnetic nanotubes can be used as a delivery vehicle for NGF and thus may be exploited in attempts to treat neurodegenerative disorders such as Parkinson's disease with neurotrophins. Further neurite outgrowth can be controlled by manipulating magnetic nanotubes with external magnetic fields, thus helping in directed regeneration.

  5. YAP regulates neuronal differentiation through Sonic hedgehog signaling pathway

    SciTech Connect

    Lin, Yi-Ting; Ding, Jing-Ya; Li, Ming-Yang; Yeh, Tien-Shun; Wang, Tsu-Wei; Yu, Jenn-Yah

    2012-09-10

    Tight regulation of cell numbers by controlling cell proliferation and apoptosis is important during development. Recently, the Hippo pathway has been shown to regulate tissue growth and organ size in Drosophila. In mammalian cells, it also affects cell proliferation and differentiation in various tissues, including the nervous system. Interplay of several signaling cascades, such as Notch, Wnt, and Sonic Hedgehog (Shh) pathways, control cell proliferation during neuronal differentiation. However, it remains unclear whether the Hippo pathway coordinates with other signaling cascades in regulating neuronal differentiation. Here, we used P19 cells, a mouse embryonic carcinoma cell line, as a model to study roles of YAP, a core component of the Hippo pathway, in neuronal differentiation. P19 cells can be induced to differentiate into neurons by expressing a neural bHLH transcription factor gene Ascl1. Our results showed that YAP promoted cell proliferation and inhibited neuronal differentiation. Expression of Yap activated Shh but not Wnt or Notch signaling activity during neuronal differentiation. Furthermore, expression of Yap increased the expression of Patched homolog 1 (Ptch1), a downstream target of the Shh signaling. Knockdown of Gli2, a transcription factor of the Shh pathway, promoted neuronal differentiation even when Yap was over-expressed. We further demonstrated that over-expression of Yap inhibited neuronal differentiation in primary mouse cortical progenitors and Gli2 knockdown rescued the differentiation defect in Yap over-expressing cells. In conclusion, our study reveals that Shh signaling acts downstream of YAP in regulating neuronal differentiation. -- Highlights: Black-Right-Pointing-Pointer YAP promotes cell proliferation and inhibits neuronal differentiation in P19 cells. Black-Right-Pointing-Pointer YAP promotes Sonic hedgehog signaling activity during neuronal differentiation. Black-Right-Pointing-Pointer Knockdown of Gli2 rescues the Yap

  6. The matrix metalloproteinase inhibitor marimastat promotes neural progenitor cell differentiation into neurons by gelatinase-independent TIMP-2-dependent mechanisms.

    PubMed

    Sinno, Maddalena; Biagioni, Stefano; Ajmone-Cat, Maria Antonietta; Pafumi, Irene; Caramanica, Pasquale; Medda, Virginia; Tonti, Gaetana; Minghetti, Luisa; Mannello, Ferdinando; Cacci, Emanuele

    2013-02-01

    Metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs), produced in the brain by cells of non-neural and neural origin, including neural progenitors (NPs), are emerging as regulators of nervous system development and adult brain functions. In the present study, we explored whether MMP-2, MMP-9, and TIMP-2, abundantly produced in the brain, modulate NP developmental properties. We found that treatment of NPs, isolated from the murine fetal cerebral cortex or adult subventricular zone, with the clinically tested broad-spectrum MMP inhibitor Marimastat profoundly affected the NP differentiation fate. Marimastat treatment allowed for an enrichment of our cultures in neuronal cells, inducing NPs to generate higher percentage of neurons and a lower percentage of astrocytes, possibly affecting NP commitment. Consistently with its proneurogenic effect, Marimastat early downregulated the expression of Notch target genes, such as Hes1 and Hes5. MMP-2 and MMP-9 profiling on proliferating and differentiating NPs revealed that MMP-9 was not expressed under these conditions, whereas MMP-2 increased in the medium as pro-MMP-2 (72 kDa) during differentiation; its active form (62 kDa) was not detectable by gel zymography. MMP-2 silencing or administration of recombinant active MMP-2 demonstrated that MMP-2 does not affect NP neuronal differentiation, nor it is involved in the Marimastat proneurogenic effect. We also found that TIMP-2 is expressed in NPs and increases during late differentiation, mainly as a consequence of astrocyte generation. Endogenous TIMP-2 did not modulate NP neurogenic potential; however, the proneurogenic action of Marimastat was mediated by TIMP-2, as demonstrated by silencing experiments. In conclusion, our data exclude a major involvement of MMP-2 and MMP-9 in the regulation of basal NP differentiation, but highlight the ability of TIMP-2 to act as key effector of the proneurogenic response to an inducing stimulus such as Marimastat. PMID

  7. The matrix metalloproteinase inhibitor marimastat promotes neural progenitor cell differentiation into neurons by gelatinase-independent TIMP-2-dependent mechanisms.

    PubMed

    Sinno, Maddalena; Biagioni, Stefano; Ajmone-Cat, Maria Antonietta; Pafumi, Irene; Caramanica, Pasquale; Medda, Virginia; Tonti, Gaetana; Minghetti, Luisa; Mannello, Ferdinando; Cacci, Emanuele

    2013-02-01

    Metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs), produced in the brain by cells of non-neural and neural origin, including neural progenitors (NPs), are emerging as regulators of nervous system development and adult brain functions. In the present study, we explored whether MMP-2, MMP-9, and TIMP-2, abundantly produced in the brain, modulate NP developmental properties. We found that treatment of NPs, isolated from the murine fetal cerebral cortex or adult subventricular zone, with the clinically tested broad-spectrum MMP inhibitor Marimastat profoundly affected the NP differentiation fate. Marimastat treatment allowed for an enrichment of our cultures in neuronal cells, inducing NPs to generate higher percentage of neurons and a lower percentage of astrocytes, possibly affecting NP commitment. Consistently with its proneurogenic effect, Marimastat early downregulated the expression of Notch target genes, such as Hes1 and Hes5. MMP-2 and MMP-9 profiling on proliferating and differentiating NPs revealed that MMP-9 was not expressed under these conditions, whereas MMP-2 increased in the medium as pro-MMP-2 (72 kDa) during differentiation; its active form (62 kDa) was not detectable by gel zymography. MMP-2 silencing or administration of recombinant active MMP-2 demonstrated that MMP-2 does not affect NP neuronal differentiation, nor it is involved in the Marimastat proneurogenic effect. We also found that TIMP-2 is expressed in NPs and increases during late differentiation, mainly as a consequence of astrocyte generation. Endogenous TIMP-2 did not modulate NP neurogenic potential; however, the proneurogenic action of Marimastat was mediated by TIMP-2, as demonstrated by silencing experiments. In conclusion, our data exclude a major involvement of MMP-2 and MMP-9 in the regulation of basal NP differentiation, but highlight the ability of TIMP-2 to act as key effector of the proneurogenic response to an inducing stimulus such as Marimastat.

  8. Factor-Reduced Human Induced Pluripotent Stem Cells Efficiently Differentiate into Neurons Independent of the Number of Reprogramming Factors.

    PubMed

    Hermann, Andreas; Kim, Jeong Beom; Srimasorn, Sumitra; Zaehres, Holm; Reinhardt, Peter; Schöler, Hans R; Storch, Alexander

    2016-01-01

    Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by overexpression of the transcription factors OCT4, SOX2, KLF4, and c-Myc holds great promise for the development of personalized cell replacement therapies. In an attempt to minimize the risk of chromosomal disruption and to simplify reprogramming, several studies demonstrated that a reduced set of reprogramming factors is sufficient to generate iPSC. We recently showed that a reduction of reprogramming factors in murine cells not only reduces reprogramming efficiency but also may worsen subsequent differentiation. To prove whether this is also true for human cells, we compared the efficiency of neuronal differentiation of iPSC generated from fetal human neural stem cells with either one (OCT4; hiPSC1F-NSC) or two (OCT4, KLF4; hiPSC2F-NSC) reprogramming factors with iPSC produced from human fibroblasts using three (hiPSC3F-FIB) or four reprogramming factors (hiPSC4F-FIB). After four weeks of coculture with PA6 stromal cells, neuronal differentiation of hiPSC1F-NSC and hiPSC2F-NSC was as efficient as iPSC3F-FIB or iPSC4F-FIB. We conclude that a reduction of reprogramming factors in human cells does reduce reprogramming efficiency but does not alter subsequent differentiation into neural lineages. This is of importance for the development of future application of iPSC in cell replacement therapies.

  9. Established Monolayer Differentiation of Mouse Embryonic Stem Cells Generates Heterogeneous Neocortical-Like Neurons Stalled at a Stage Equivalent to Midcorticogenesis

    PubMed Central

    Sadegh, Cameron; Macklis, Jeffrey D.

    2014-01-01

    Two existing and widely applied protocols of embryonic stem (ES) cell differentiation have been developed to enable in vitro generation of neurons resembling neocortical projection neurons in monolayer culture and from embryoid bodies. The monolayer approach offers advantages for detailed in vitro characterizations and potential mechanistic and therapeutic screening. We investigated whether mouse ES cells undergoing largely undirected neocortical differentiation in monolayer culture recapitulate progressive developmental programs of in vivo progenitor and postmitotic differentiation and whether they develop into specific neocortical subtypes. We find that ES-derived mitotic cells that have been dorsalized by the sonic hedgehog antagonist cyclopamine, and that express, as a total population, cardinal markers of telencephalic progenitors, are, in fact, molecularly heterogeneous. We next show that these progenitors subsequently generate small numbers of heterogeneous neocortical-like neurons that are “stalled” at an immature stage of differentiation, based on multiple developmental criteria. Although some aspects of neocortical development are recapitulated by existing protocols of ES cell differentiation, these data indicate that mouse ES-derived neocortical progenitors both are more heterogeneous than their in vivo counterparts and seemingly include many incorrectly specified progenitors. Furthermore, these ES-derived progenitors spontaneously differentiate into sparse, and incompletely and largely imprecisely differentiated, neocortical-like neurons that fail to adopt specific neuronal identities in vitro. These results provide both foundation and motivation for refining and enhancing directed differentiation of clinically important neocortical projection neuron subtypes. PMID:24610556

  10. Increased dosage of DYRK1A and DSCR1 delays neuronal differentiation in neocortical progenitor cells.

    PubMed

    Kurabayashi, Nobuhiro; Sanada, Kamon

    2013-12-15

    Down's syndrome (DS), a major genetic cause of mental retardation, arises from triplication of genes on human chromosome 21. Here we show that DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) and DSCR1 (DS critical region 1), two genes lying within human chromosome 21 and encoding for a serine/threonine kinase and calcineurin regulator, respectively, are expressed in neural progenitors in the mouse developing neocortex. Increasing the dosage of both proteins in neural progenitors leads to a delay in neuronal differentiation, resulting ultimately in alteration of their laminar fate. This defect is mediated by the cooperative actions of DYRK1A and DSCR1 in suppressing the activity of the transcription factor NFATc. In Ts1Cje mice, a DS mouse model, dysregulation of NFATc in conjunction with increased levels of DYRK1A and DSCR1 was observed. Furthermore, counteracting the dysregulated pathway ameliorates the delayed neuronal differentiation observed in Ts1Cje mice. In sum, our findings suggest that dosage of DYRK1A and DSCR1 is critical for proper neurogenesis through NFATc and provide a potential mechanism to explain the neurodevelopmental defects in DS. PMID:24352425

  11. Dopaminergic Neuronal Differentiation from the Forebrain-Derived Human Neural Stem Cells Induced in Cultures by Using a Combination of BMP-7 and Pramipexole with Growth Factors

    PubMed Central

    Yang, HongNa; Wang, Jing; Wang, Feng; Liu, XiaoDun; Chen, Heng; Duan, WeiMing; Qu, TingYu

    2016-01-01

    Transplantation of dopaminergic (DA) neurons is considered to be the most promising therapeutic strategy for replacing degenerated dopamine cells in the midbrain of Parkinson's disease (PD), thereby restoring normal neural circuit function and slow clinical progression of the disease. Human neural stem cells (hNSCs) derived from fetal forebrain are thought to be the important cell sources for producing DA neurons because of their multipotency for differentiation and long-term expansion property in cultures. However, low DA differentiation of the forebrain-derived hNSCs limited their therapeutic potential in PD. In the current study, we explored a combined application of Pramipexole (PRX), bone morphogenetic proteins 7 (BMP-7), and growth factors, including acidic fibroblast factor (aFGF), forskolin, and phorbol-12-myristae-13-acetate (TPA), to induce differentiation of forebrain-derived hNSCs toward DA neurons in cultures. We found that DA neuron-associated genes, including Nurr1, Neurogenin2 (Ngn2), and tyrosine hydroxylase (TH) were significantly increased after 24 h of differentiation by RT-PCR analysis (p < 0.01). Fluorescent examination showed that about 25% of cells became TH-positive neurons at 24 h, about 5% of cells became VMAT2 (vascular monoamine transporter 2)-positive neurons, and less than 5% of cells became DAT (dopamine transporter)-positive neurons at 72 h following differentiation in cultures. Importantly, these TH-, VMAT2-, and DAT-expressing neurons were able to release dopamine into cultures under both of the basal and evoked conditions. Dopamine levels released by DA neurons produced using our protocol were significantly higher compared to the control groups (P < 0.01), as examined by ELISA. Our results demonstrated that the combination of PRX, BMP-7, and growth factors was able to greatly promote differentiation of the forebrain-derived hNSCs into DA-releasing neurons. PMID:27147976

  12. Neuronal adhesion, proliferation and differentiation of embryonic stem cells on hybrid scaffolds made of xanthan and magnetite nanoparticles.

    PubMed

    Glaser, Talita; Bueno, Vânia B; Cornejo, Daniel R; Petri, Denise F S; Ulrich, Henning

    2015-08-01

    Hybrid scaffolds made of xanthan and magnetite nanoparticles (XCA/mag) were prepared by dipping xanthan membranes (XCA) into dispersions of magnetic nanoparticles for different periods of time. The resulting hybrid scaffolds presented magnetization values ranging from 0.25 emu g(-1) to 1.80 emu g(-1) at 70 kOe and corresponding iron contents ranging from 0.25% to 2.3%, respectively. They were applied as matrices for in vitro embryoid body adhesion and neuronal differentiation of embryonic stem cells; for comparison, neat XCA and commercial plastic plates were also used. Adhesion rates were more pronounced when cells were seeded on XCA/mag than on neat XCA or plastic dishes; however, proliferation levels were independent from those of the scaffold type. Embryonic stem cells showed similar differentiation rates on XCA/mag scaffolds with magnetization of 0.25 and 0.60 emu g(-1), but did not survive on scaffolds with 1.80 emu g(-1). Differentiation rates, expressed as the number of neurons obtained on the chosen scaffolds, were the largest on neat XCA, which has a high density of negative charge, and were smallest on the commercial plastic dishes. The local magnetic field inherent of magnetite particles present on the surface of XCA/mag facilitates synapse formation, because synaptophysin expression and electrical transmission were increased when compared to the other scaffolds used. We conclude that XCA/mag and XCA hydrogels are scaffolds with distinguishable performance for adhesion and differentiation of ESCs into neurons. PMID:26154495

  13. Differential distribution of glycine transporters in Müller cells and neurons in amphibian retinas.

    PubMed

    Jiang, Zheng; Li, Baoqin; Jursky, Frantisek; Shen, Wen

    2007-01-01

    Amphibian retinas are commonly used for electrophysiological studies on neural function and transduction because they share the same general properties as higher vertebrate retinas. Glycinergic synapses have been well described in amphibian retinas. However, the role of glycine transporters in the synapses is largely unknown. We studied the distribution and function of glycine transporters in the retinas from tiger salamanders, mudpuppies, and leopard frogs by immunofluorescence labeling and whole-cell recording methods. Our results indicated that GlyT1- and GlyT2-like transporters were present in Müller cells and neurons, respectively. GlyT1 labeling was present in Müller glial cells and co-localized with Glial fibrillary acidic protein (GFAP), a Müller cell marker, whereas the GlyT2 immunoreactivity was present in the somas of amacrine cells (ACs) and processes in the inner plexiform layer (IPL) and the outer plexiform layer (OPL). Because the axon processes of glycinergic interplexiform cells (IPCs) are the only source of glycine input in the OPL, GlyT2 staining revealed a spatial pattern of the axon processes of IPCs in the OPL. The function of GlyT2 in the IPCs was studied in tiger salamander retinal horizontal cells (HCs) by whole-cell gramicidin perforated recording. The results demonstrated that inhibition of GlyT2 by a specific inhibitor, amoxapine, increased a tonic glycine input to HCs. Thus, the GlyT2 transporter is responsible for uptake of synaptic glycine in the outer retina. We also compared the distribution of glycine transporters in other amphibian species: salamander, mudpuppy, and frog. The results are consistent with the general pattern that GlyT1-like transporters are present in Müller cells and GlyT2-like transporters in neurons in amphibian retinas. PMID:17640406

  14. Prenatal Hypoxia in Different Periods of Embryogenesis Differentially Affects Cell Migration, Neuronal Plasticity, and Rat Behavior in Postnatal Ontogenesis

    PubMed Central

    Vasilev, Dmitrii S.; Dubrovskaya, Nadezhda M.; Tumanova, Natalia L.; Zhuravin, Igor A.

    2016-01-01

    Long-term effects of prenatal hypoxia on embryonic days E14 or E18 on the number, type and localization of cortical neurons, density of labile synaptopodin-positive dendritic spines, and parietal cortex-dependent behavioral tasks were examined in the postnatal ontogenesis of rats. An injection of 5′ethynyl-2′deoxyuridine to pregnant rats was used to label neurons generated on E14 or E18 in the fetuses. In control rat pups a majority of cells labeled on E14 were localized in the lower cortical layers V-VI while the cells labeled on E18 were mainly found in the superficial cortical layers II-III. It was shown that hypoxia both on E14 and E18 results in disruption of neuroblast generation and migration but affects different cell populations. In rat pups subjected to hypoxia on E14, the total number of labeled cells in the parietal cortex was decreased while the number of labeled neurons scattered within the superficial cortical layers was increased. In rat pups subjected to hypoxia on E18, the total number of labeled cells in the parietal cortex was also decreased but the number of scattered labeled neurons was higher in the lower cortical layers. It can be suggested that prenatal hypoxia both on E14 and E18 causes a disruption in neuroblast migration but with a different outcome. Only in rats subjected to hypoxia on E14 did we observe a reduction in the total number of pyramidal cortical neurons and the density of labile synaptopodin-positive dendritic spines in the molecular cortical layer during the first month after birth which affected development of the cortical functions. As a result, rats subjected to hypoxia on E14, but not on E18, had impaired development of the whisker-placing reaction and reduced ability to learn reaching by a forepaw. The data obtained suggest that hypoxia on E14 in the period of generation of the cells, which later differentiate into the pyramidal cortical neurons of the V-VI layers and form cortical minicolumns, affects formation of

  15. Oleanolic Acid Induces Differentiation of Neural Stem Cells to Neurons: An Involvement of Transcription Factor Nkx-2.5

    PubMed Central

    Ning, You; Huang, Jianhua; Kalionis, Bill; Bian, Qin; Dong, Jingcheng; Wu, Junzhen; Tai, Xiantao; Xia, Shijin; Shen, Ziyin

    2015-01-01

    Neural stem cells (NSCs) harbor the potential to differentiate into neurons, astrocytes, and oligodendrocytes under normal conditions and/or in response to tissue damage. NSCs open a new way of treatment of the injured central nervous system and neurodegenerative disorders. Thus far, few drugs have been developed for controlling NSC functions. Here, the effect as well as mechanism of oleanolic acid (OA), a pentacyclic triterpenoid, on NSC function was investigated. We found OA significantly inhibited neurosphere formation in a dose-dependent manner and achieved a maximum effect at 10 nM. OA also reduced 5-ethynyl-2′-deoxyuridine (EdU) incorporation into NSCs, which was indicative of inhibited NSC proliferation. Western blotting analysis revealed the protein levels of neuron-specific marker tubulin-βIII (TuJ1) and Mash1 were increased whilst the astrocyte-specific marker glial fibrillary acidic protein (GFAP) decreased. Immunofluorescence analysis showed OA significantly elevated the percentage of TuJ1-positive cells and reduced GFAP-positive cells. Using DNA microarray analysis, 183 genes were differentially regulated by OA. Through transcription factor binding site analyses of the upstream regulatory sequences of these genes, 87 genes were predicted to share a common motif for Nkx-2.5 binding. Finally, small interfering RNA (siRNA) methodology was used to silence Nkx-2.5 expression and found silence of Nkx-2.5 alone did not change the expression of TuJ-1 and the percentage of TuJ-1-positive cells. But in combination of OA treatment and silence of Nkx-2.5, most effects of OA on NSCs were abolished. These results indicated that OA is an effective inducer for NSCs differentiation into neurons at least partially by Nkx-2.5-dependent mechanism. PMID:26240574

  16. Differentiation from human pluripotent stem cells of cortical neurons of the superficial layers amenable to psychiatric disease modeling and high-throughput drug screening.

    PubMed

    Boissart, C; Poulet, A; Georges, P; Darville, H; Julita, E; Delorme, R; Bourgeron, T; Peschanski, M; Benchoua, A

    2013-01-01

    Cortical neurons of the superficial layers (II-IV) represent a pivotal neuronal population involved in the higher cognitive functions of the human and are particularly affected by psychiatric diseases with developmental manifestations such as schizophrenia and autism. Differentiation protocols of human pluripotent stem cells (PSC) into cortical neurons have been achieved, opening the way to in vitro modeling of neuropsychiatric diseases. However, these protocols commonly result in the asynchronous production of neurons typical for the different layers of the cortex within an extended period of culture, thus precluding the analysis of specific subtypes of neurons in a standardized manner. Addressing this issue, we have successfully captured a stable population of self-renewing late cortical progenitors (LCPs) that synchronously and massively differentiate into glutamatergic cortical neurons of the upper layers. The short time course of differentiation into neurons of these progenitors has made them amenable to high-throughput assays. This has allowed us to analyze the capability of LCPs at differentiating into post mitotic neurons as well as extending and branching neurites in response to a collection of selected bioactive molecules. LCPs and cortical neurons of the upper layers were successfully produced from patient-derived-induced PSC, indicating that this system enables functional studies of individual-specific cortical neurons ex vivo for disease modeling and therapeutic purposes. PMID:23962924

  17. Biologically synthesized silver nanoparticles induce neuronal differentiation of SH-SY5Y cells via modulation of reactive oxygen species, phosphatases, and kinase signaling pathwayss.

    PubMed

    Dayem, Ahmed Abdal; Kim, Bongwoo; Gurunathan, Sangiliyandi; Choi, Hye Yeon; Yang, Gwangmo; Saha, Subbroto Kumar; Han, Dawoon; Han, Jihae; Kim, Kyeongseok; Kim, Jin-Hoi; Cho, Ssang-Goo

    2014-04-22

    The relevant in vitro cellular model resembling functional neurons is important for the mechanistic research on various neuronal diseases. Human neuroblastoma SH-SY5Y cells may be considered one of the ideal in vitro models for studying neuroscience, as they can be maintained in an undifferentiated state or be induced to differentiate into neuron-like phenotypes in vitro by several differentiation-inducing agents. In this study, we prepared spherical silver nanoparticles (AgNPs) with an average size of about 30 nm and tested their potency to induce neuronal differentiation of SH-SY5Y cells. Treatment of SH-SY5Y cells by biologically synthesized AgNPs led to cell morphological changes and significant increase in neurite length and enhanced the expression of neuronal differentiation markers such as Map-2, β-tubulin III, synaptophysin, neurogenin-1, Gap-43, and Drd-2. Furthermore, we observed an increase in generation of intracellular reactive oxygen species (ROS), activation of several kinases such as ERK and AKT, and down-regulation of expression of dual-specificity phosphatases (DUSPs) in AgNPs-exposed SH-SY5Y cells. Our results suggest that AgNPs could modulate the intracellular signaling pathways to lead to neuronal differentiation, and could be applied as promising nanomaterials for stem cell research and therapy.

  18. Levetiracetam Differentially Alters CD95 Expression of Neuronal Cells and the Mitochondrial Membrane Potential of Immune and Neuronal Cells in vitro

    PubMed Central

    Rogers, Susannah K.; Shapiro, Lee A.; Tobin, Richard P.; Tow, Benjamin; Zuzek, Aleksej; Mukherjee, Sanjib; Newell-Rogers, M. Karen

    2014-01-01

    Epilepsy is a neurological seizure disorder that affects over 100 million people worldwide. Levetiracetam, either alone, as monotherapy, or as adjunctive treatment, is widely used to control certain types of seizures. Despite its increasing popularity as a relatively safe and effective anti-convulsive treatment option, its mechanism(s) of action are poorly understood. Studies have suggested neuronal, glial, and immune mechanisms of action. Understanding the precise mechanisms of action of levetiracetam would be extremely beneficial in helping to understand the processes involved in seizure generation and epilepsy. Moreover, a full understanding of these mechanisms would help to create more efficacious treatments while minimizing side-effects. The current study examined the effects of levetiracetam on the mitochondrial membrane potential of neuronal and non-neuronal cells, in vitro, in order to determine if levetiracetam influences metabolic processes in these cell types. In addition, this study sought to address possible immune-mediated mechanisms by determining if levetiracetam alters the expression of immune receptor–ligand pairs. The results show that levetiracetam induces expression of CD95 and CD178 on NGF-treated C17.2 neuronal cells. The results also show that levetiracetam increases mitochondrial membrane potential on C17.2 neuronal cells in the presence of nerve growth factor. In contrast, levetiracetam decreases the mitochondrial membrane potential of splenocytes and this effect was dependent on intact invariant chain, thus implicating immune cell interactions. These results suggest that both neuronal and non-neuronal anti-epileptic activities of levetiracetam involve control over energy metabolism, more specifically, mΔΨ. Future studies are needed to further investigate this potential mechanism of action. PMID:24600432

  19. Safrole oxide induced human umbilical vein vascular endothelial cell differentiation into neuron-like cells by depressing the reactive oxygen species level at the low concentration.

    PubMed

    Su, Le; Zhao, Jing; Zhao, Bao Xiang; Miao, Jun Ying; Yin, De Ling; Zhang, Shang Li

    2006-02-01

    Previously, we found that 5-25 microg/ml safrole oxide could inhibit apoptosis and dramatically make a morphological change in human umbilical vein vascular endothelial cells (HUVECs). But the possible mechanism by which safrole oxide function is unknown. To answer this question, in this study, we first investigated the effects of it on the activity of nitric oxide synthetase (NOS), the expressions of Fas and integrin beta4, which play important roles in HUVEC growth and apoptosis, respectively. The results showed that, at the low concentration (10 microg/ml), safrole oxide had no effects on NOS activity and the expressions of Fas and integrin beta4. Then, we investigated whether HUVECs underwent differentiation. We examined the expressions of neuron-specific enolase (NSE) and neurofilament-L (NF-L). Furthermore, we analyzed the changes of intracellular reactive oxygen species (ROS). After 10 h of treatment with 10 microg/ml safrole oxide, some HUVECs became neuron-like cells in morphology, and intensively displayed positive NSE and NF-L. Simultaneously, ROS levels dramatically decreased during HUVECs differentiation towards neuron-like cells. At the low concentration, safrole oxide induced HUVECs differentiation into neuron-like cells. Furthermore, our data suggested that safrole oxide might perform this function by depressing intracellular ROS levels instead of by affecting cell growth or apoptosis signal pathways.

  20. The neuronal differentiation factor NeuroD1 downregulates the neuronal repellent factor Slit2 expression and promotes cell motility and tumor formation of neuroblastoma.

    PubMed

    Huang, Peng; Kishida, Satoshi; Cao, Dongliang; Murakami-Tonami, Yuko; Mu, Ping; Nakaguro, Masato; Koide, Naoshi; Takeuchi, Ichiro; Onishi, Akira; Kadomatsu, Kenji

    2011-04-15

    The basic helix-loop-helix transcription factor NeuroD1 has been implicated in the neurogenesis and early differentiation of pancreatic endocrine cells. However, its function in relation to cancer has been poorly examined. In this study, we found that NeuroD1 is involved in the tumorigenesis of neuroblastoma. NeuroD1 was strongly expressed in a hyperplastic region comprising neuroblasts in the celiac sympathetic ganglion of 2-week-old MYCN transgenic (Tg) mice and was consistently expressed in the subsequently generated neuroblastoma tissue. NeuroD1 knockdown by short hairpin RNA (shRNA) resulted in motility inhibition of the human neuroblastoma cell lines, and this effect was reversed by shRNA-resistant NeuroD1. The motility inhibition by NeuroD1 knockdown was associated with induction of Slit2 expression, and knockdown of Slit2 could restore cell motility. Consistent with this finding, shRNA-resistant NeuroD1 suppressed Slit2 expression. NeuroD1 directly bound to the first and second E-box of the Slit2 promoter region. Moreover, we found that the growth of tumor spheres, established from neuroblastoma cell lines in MYCN Tg mice, was suppressed by NeuroD1 suppression. The functions identified for NeuroD1 in cell motility and tumor sphere growth may suggest a link between NeuroD1 and the tumorigenesis of neuroblastoma. Indeed, tumor formation of tumor sphere-derived cells was significantly suppressed by NeuroD1 knockdown. These data are relevant to the clinical features of human neuroblastoma: high NeuroD1 expression was closely associated with poor prognosis. Our findings establish the critical role of the neuronal differentiation factor NeuroD1 in neuroblastoma as well as its functional relationship with the neuronal repellent factor Slit2.

  1. Neuritin (cpg15) enhances the differentiating effect of NGF on neuronal PC12 cells.

    PubMed

    Cappelletti, Graziella; Galbiati, Mariarita; Ronchi, Cristina; Maggioni, Maria Grazia; Onesto, Elisa; Poletti, Angelo

    2007-09-01

    Neuritin is a small, highly conserved GPI-anchored protein involved in neurite outgrowth. We have analyzed the involvement of neuritin in NGF-induced differentiation of PC12 cells by investigating the time-course of neuritin expression, the effects of its overexpression or silencing, and the possible mechanisms of its regulation and action. Real-time PCR analysis has shown that neuritin gene is upregulated by NGF in PC12 cells hours before neurite outgrowth becomes appreciable. PC12 cells transfected with a plasmid expressing neuritin display a significant increase in the response to NGF: 1) in the levels of SMI312 positive phosphorylated neurofilament proteins (markers for axonal processes) and tyrosine hydroxylase; 2) in the percentage of cells bearing neurites; as well as 3) in the average length of neurites when compared to control cells. On the contrary, neuritin silencing significantly reduces neurite outgrowth. These data suggest that neuritin is a modulator of NGF-induced neurite extension in PC12 cells. We also showed that neuritin potentiated the NGF-induced differentiation of PC12 cells without affecting TrkA or EGF receptor mRNAs expression. Moreover, the S-methylisothiourea (MIU), a potent inhibitor of inducible nitric oxide synthases, partially counteracts the NGF-mediated neuritin induction. These data suggest that NGF regulates neuritin expression in PC12 cells via the signaling pathway triggered by NO. This study reports the first evidence that neuritin plays a role in modulating neurite outgrowth during the progression of NGF-induced differentiation of PC12 cells. PC12 cells could be considered a valuable model to unravel the mechanism of action of neuritin on neurite outgrowth. (c) 2007 Wiley-Liss, Inc. PMID:17335086

  2. Claulansine F promoted the neuronal differentiation of neural stem and progenitor cells through Akt/GSK-3β/β-catenin pathway.

    PubMed

    Huang, Ju-Yang; Ma, Yin-Zhong; Yuan, Yu-He; Zuo, Wei; Chu, Shi-Feng; Liu, Hang; Du, Guan-Hua; Zhang, Dong-Ming; Chen, Nai-Hong

    2016-09-01

    The persistence of neurogenesis raises the idea that neurons produced by the resident or transplanted neural stem cells could replace the neurons lost from brain injury or neurodegenerative disease. Therefore, compounds or methods for promoting neuronal differentiation become the focus of neurodegenerative disease therapy research. Claulansine F (Clau F), a newly discovered carbazole alkaloid, has been showed to induce neuritogenesis in PC12 cells. Herein, we studied the effect of Clau F on neuronal differentiation of neural stem/progenitor cells (NS/PCs). The current study demonstrated that Clau F initiated neuronal differentiation with a significant increase of TuJ1-positive cells and TuJ1 protein levels. We also found that Clau F promoted the maturity and sustainability of neurons by increasing MAP2-positive cells and MAP2 protein levels. At the same time, Clau F significantly inhibited the proliferation of NS/PCs. The underlying mechanism of Clau F was preliminary explored. Clau F treatment resulted in a profound increase of phosphorylation of Akt and GSK-3β, which led to GSK-3β inhibition and subsequently the nuclear accumulation of β-catenin. Further, the interaction between β-catenin and p300 in the nucleus was enhanced and the transcription of p300/β-catenin responsive genes were increased significantly (c-jun, fra-1) by Clau F. Importantly, the positive effect of Clau F on neuronal differentiation was abolished by Akti-1/2, a specific inhibitor of Akt-1/2 kinase, which indicated the involvement of Akt/GSK-3β in Clau F-mediated neuronal differentiation. In conclusion, these data suggested that Clau F promoted neuronal differentiation through Akt/GSK-3β/β-catenin signaling pathway in NS/PCs.

  3. Tetramethylpyrazine induces SH-SY5Y cell differentiation toward the neuronal phenotype through activation of the PI3K/Akt/Sp1/TopoIIβ pathway.

    PubMed

    Yan, Yong-Xin; Zhao, Jun-Xia; Han, Shuo; Zhou, Na-Jing; Jia, Zhi-Qiang; Yao, Sheng-Jie; Cao, Cui-Li; Wang, Yan-Ling; Xu, Yan-Nan; Zhao, Juan; Yan, Yun-Li; Cui, Hui-Xian

    2015-12-01

    Tetramethylpyrazine (TMP) is an active compound extracted from the traditional Chinese medicinal herb Chuanxiong. Previously, we have shown that TMP induces human SH-SY5Y neuroblastoma cell differentiation toward the neuronal phenotype by targeting topoisomeraseIIβ (TopoIIβ), a protein implicated in neural development. In the present study, we aimed to elucidate whether the transcriptional factors specificity protein 1 (Sp1) and nuclear factor Y (NF-Y), in addition to the upstream signaling pathways ERK1/2 and PI3K/Akt, are involved in modulating TopoIIβ expression in the neuronal differentiation process. We demonstrated that SH-SY5Y cells treated with TMP (80μM) terminally differentiated into neurons, characterized by increased neuronal markers, tubulin βIII and microtubule associated protein 2 (MAP2), and increased neurite outgrowth, with no negative effect on cell survival. TMP also increased the expression of TopoIIβ, which was accompanied by increased expression of Sp1 in the differentiated neuron-like cells, whereas NF-Y protein levels remained unchanged following the differentiation progression. We also found that the phosphorylation level of Akt, but not ERK1/2, was significantly increased as a result of TMP stimulation. Furthermore, as established by chromatin immunoprecipitation (ChIP) assay, activation of the PI3K/Akt pathway increased Sp1 binding to the promoter of the TopoIIβ gene. Blockage of PI3K/Akt was shown to lead to subsequent inhibition of TopoIIβ expression and neuronal differentiation. Collectively, the results indicate that the PI3K/Akt/Sp1/TopoIIβ signaling pathway is necessary for TMP-induced neuronal differentiation. Our findings offer mechanistic insights into understanding the upstream regulation of TopoIIβ in neuronal differentiation, and suggest potential applications of TMP both in neuroscience research and clinical practice to treat relevant diseases of the nervous system. PMID:26518113

  4. Requirement for neurogenesis to proceed through the division of neuronal progenitors following differentiation of epidermal growth factor and fibroblast growth factor-2-responsive human neural stem cells.

    PubMed

    Ostenfeld, Thor; Svendsen, Clive N

    2004-01-01

    Epidermal growth factor (EGF)- and fibroblast growth factor-2 (FGF-2)-responsive human neural stem cells may provide insight into mechanisms of neural development and have applications in cell-based therapeutics for neurological disease. However, their biology after expansion in vitro is currently poorly understood. Cells grown in either EGF or FGF-2 or a combination of both mitogens displayed characteristically similar levels of transcriptional activation and comparable proliferative profiles with linear cell-cycle kinetics and possessed similar neuronal differentiation capabilities. These data support the view that human neurospheres at later stages of expansion (>10 weeks) are comprised overwhelmingly of a single type of stem cell responsive to both EGF and FGF-2. After mitogen withdrawal and neurosphere plating, bromodeoxyuridine pulse-chase experiments revealed that the stem cells did not undergo differentiation directly into neurons. Instead, most immature neurons arose via the division of emerging progenitor cells in the absence of exogenous EGF or FGF-2. Neurogenesis was abolished by application of high concentrations of either EGF/FGF-2 or the mitotic inhibitor cytosine-b-arabinofuranoside, suggesting that there is an obligatory requirement for at least one round of cell division in the absence of mitogens as a prelude to terminal neuronal differentiation. The differentiation of human neurospheres provides a useful model of human neurogenesis, and the data presented indicate that it proceeds through the division of committed neuronal progenitor cells rather than directly from the neural stem cell. PMID:15342944

  5. Nerve growth factor (NGF) induces neuronal differentiation in neuroblastoma cells transfected with the NGF receptor cDNA

    SciTech Connect

    Matsushima, H.; Bogenmann, E. )

    1990-09-01

    Human nerve growth factor (NGF) receptor (NGFR) cDNA was transfected into a neuroblastoma cell line (HTLA 230) which does not express a functional NGF-NGFR signal transduction cascade. Short-term treatment of stably transfected cells (98-3) expressing membrane-bound NGF receptor molecules resulted in a cell cycle-dependent, transient expression of the c-fos gene upon treatment with NGF, suggesting the presence of functional high-affinity NGFR. Extensive outgrowth of neurites and cessation of DNA synthesis occurred in transfectants grown on an extracellular matrix after long-term treatment with NGF, suggesting terminal differentiation. Our data support the idea that introduction of a constitutively expressed NGFR cDNA into cells with neuronal background results in the assembly of a functional NGF-NGFR signal cascade in a permissive extracellular environment.

  6. The Down syndrome-related protein kinase DYRK1A phosphorylates p27Kip1 and Cyclin D1 and induces cell cycle exit and neuronal differentiation

    PubMed Central

    Soppa, Ulf; Schumacher, Julian; Florencio Ortiz, Victoria; Pasqualon, Tobias; Tejedor, Francisco J; Becker, Walter

    2014-01-01

    A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G1 phase. Sustained overexpression of DYRK1A induced G0 cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27Kip1 on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27Kip1 Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome. PMID:24806449

  7. The Down syndrome-related protein kinase DYRK1A phosphorylates p27(Kip1) and Cyclin D1 and induces cell cycle exit and neuronal differentiation.

    PubMed

    Soppa, Ulf; Schumacher, Julian; Florencio Ortiz, Victoria; Pasqualon, Tobias; Tejedor, Francisco J; Becker, Walter

    2014-01-01

    A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G₁ phase. Sustained overexpression of DYRK1A induced G₀ cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27(Kip1) on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27(Kip1) Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome.

  8. The Down syndrome-related protein kinase DYRK1A phosphorylates p27(Kip1) and Cyclin D1 and induces cell cycle exit and neuronal differentiation.

    PubMed

    Soppa, Ulf; Schumacher, Julian; Florencio Ortiz, Victoria; Pasqualon, Tobias; Tejedor, Francisco J; Becker, Walter

    2014-01-01

    A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G₁ phase. Sustained overexpression of DYRK1A induced G₀ cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27(Kip1) on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27(Kip1) Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome. PMID:24806449

  9. Stepwise, non-adherent differentiation of human pluripotent stem cells to generate basal forebrain cholinergic neurons via hedgehog signaling.

    PubMed

    Crompton, Lucy A; Byrne, Meg L; Taylor, Hannah; Kerrigan, Talitha L; Bru-Mercier, Gilles; Badger, Jennifer L; Barbuti, Peter A; Jo, Jihoon; Tyler, Sue J; Allen, Shelley J; Kunath, Tilo; Cho, Kwangwook; Caldwell, Maeve A

    2013-11-01

    Basal forebrain cholinergic neurons (bfCNs) which provide innervation to the hippocampus and cortex, are required for memory and learning, and are primarily affected in Alzheimer's Disease (AD), resulting in related cognitive decline. Therefore generation of a source of bfCNs from human pluripotent stem cells (hPSCs) is crucial for in vitro disease modeling and development of novel AD therapies. In addition, for the advancement of regenerative approaches there is a requirement for an accurate developmental model to study the neurogenesis and survival of this population. Here we demonstrate the efficient production of bfCNs, using a novel embryoid body (EB) based non-adherent differentiation (NAdD) protocol. We establish a specific basal forebrain neural stem cell (NSC) phenotype via expression of the basal forebrain transcription factors NKX2.1 and LHX8, as well as the general forebrain marker FOXG1. We present evidence that this lineage is achieved via recapitulation of embryonic events, with induction of intrinsic hedgehog signaling, through the use of a 3D non-adherent differentiation system. This is the first example of hPSC-derived basal forebrain-like NSCs, which are scalable via self-renewal in prolonged culture. Furthermore upon terminal differentiation these basal forebrain-like NSCs generate high numbers of cholinergic neurons expressing the specific markers ChAT, VACht and ISL1. These hPSC-derived bfCNs possess characteristics that are crucial in a model to study AD related cholinergic neuronal loss in the basal forebrain. Examples are expression of the therapeutic target p75(NTR), the release of acetylcholine, and demonstration of a mature, and functional electrophysiological profile. In conclusion, this work provides a renewable source of human functional bfCNs applicable for studying AD specifically in the cholinergic system, and also provides a model of the key embryonic events in human bfCN development. PMID:24013066

  10. Tamoxifen and raloxifene modulate gap junction coupling during early phases of retinoic acid-dependent neuronal differentiation of NTera2/D1 cells

    PubMed Central

    Dahm, Liane; Klugmann, Fanny; Gonzalez-Algaba, Angeles

    2010-01-01

    Gap junctions (GJ) represent a cellular communication system known to influence neuronal differentiation and survival. To assess a putative role of this system for neural effects of tamoxifen (TAM) and raloxifene (RAL), we used the human teratocarcinoma cell line NTera2/D1, retinoic acid (RA)-dependent neuronal differentiation of which is regulated by gap junctions formed of connexin43 (Cx43). As demonstrated by Western blot analysis, concentrations above 1 µmol/l for TAM, and 0.1 µmol/l for RAL lead to a temporary time- and concentration-dependent increase in Cx43 immunoreactivity, which reached a peak for TAM after 1 day and for RAL after 2 days. Immunocytochemical stainings revealed the increase in Cx43 immunoreactivity to result from an accumulation in intracellular compartments such as the Golgi apparatus or lysosomes. In addition, TAM and RAL were able to prevent the RA-dependent decrease of Cx43 immunoreactivity in NTera2/D1 cells, normally observed during neuronal differentiation. This suggested a suppression of neuronal differentiation to result from these substances. According to this, treatment of NTera2/D1 cells with 10 µmol/l TAM or RAL during weeks 1 and 2 of a 6 weeks RA-driven differentiation schedule impaired, whereas treatment during weeks 5 and 6 did not impair, neuronal differentiation of these cells. Modulation of GJ coupling between NTera2/D1 cells by TAM and RAL seems therefore to perturb early neuronal differentiation, whereas differentiated neurons in the mature brain seem to be not affected. These effects could be of importance for actions of TAM and RAL on early embryonic steps of nervous system formation. PMID:20437090

  11. Mesenchymal Stem Cells Increase Hippocampal Neurogenesis and Neuronal Differentiation by Enhancing the Wnt Signaling Pathway in an Alzheimer's Disease Model.

    PubMed

    Oh, Se Hee; Kim, Ha Na; Park, Hyun-Jung; Shin, Jin Young; Lee, Phil Hyu

    2015-01-01

    Neurogenesis in the subgranular zone of the hippocampal dentate gyrus may act as an endogenous repair mechanism in Alzheimer's disease (AD), and the Wnt signaling pathway has been suggested to closely modulate neurogenesis in amyloid-β (Aβ)-related AD models. The present study investigated whether mesenchymal stem cells (MSCs) would modulate hippocampal neurogenesis via modulation of the Wnt signaling pathway in a model of AD. In Aβ-treated neuronal progenitor cells (NPCs), the coculture with MSCs increased significantly the expression of Ki-67, GFAP, SOX2, nestin, and HuD compared to Aβ treatment alone. In addition, MSC treatment in Aβ-treated NPCs enhanced the expression of β-catenin and Ngn1 compared to Aβ treatment alone. MSC treatment in Aβ-treated animals significantly increased the number of BrdU-ir cells in the hippocampus at 2 and 4 weeks compared to Aβ treatment alone. In addition, quantitative analysis showed that the number of BrdU and HuD double-positive cells in the dentate gyrus was significantly higher in the MSC-treated group than in controls or after Aβ treatment alone. These results demonstrate that MSC administration significantly augments hippocampal neurogenesis and enhances the differentiation of NPCs into mature neurons in AD models by augmenting the Wnt signaling pathway. The use of MSCs to modulate endogenous adult neurogenesis may have a significant impact on future strategies for AD treatment.

  12. Specific Intensity Direct Current (DC) Electric Field Improves Neural Stem Cell Migration and Enhances Differentiation towards βIII-Tubulin+ Neurons

    PubMed Central

    Zhao, Huiping; Steiger, Amanda; Nohner, Mitch; Ye, Hui

    2015-01-01

    Control of stem cell migration and differentiation is vital for efficient stem cell therapy. Literature reporting electric field–guided migration and differentiation is emerging. However, it is unknown if a field that causes cell migration is also capable of guiding cell differentiation—and the mechanisms for these processes remain unclear. Here, we report that a 115 V/m direct current (DC) electric field can induce directional migration of neural precursor cells (NPCs). Whole cell patching revealed that the cell membrane depolarized in the electric field, and buffering of extracellular calcium via EGTA prevented cell migration under these conditions. Immunocytochemical staining indicated that the same electric intensity could also be used to enhance differentiation and increase the percentage of cell differentiation into neurons, but not astrocytes and oligodendrocytes. The results indicate that DC electric field of this specific intensity is capable of promoting cell directional migration and orchestrating functional differentiation, suggestively mediated by calcium influx during DC field exposure. PMID:26068466

  13. Effects of nerve growth factor and basic fibroblast growth factor dual gene modification on rat bone marrow mesenchymal stem cell differentiation into neuron-like cells in vitro

    PubMed Central

    HU, YANG; ZHANG, YAN; TIAN, KANG; XUN, CHONG; WANG, SHOUYU; LV, DECHENG

    2016-01-01

    Recent studies regarding regenerative medicine have focused on bone marrow mesenchymal stem cells (BMSCs), which have the potential to undergo neural differentiation, and may be transfected with specific genes. BMSCs can differentiate into neuron-like cells in certain neurotropic circumstances in vitro. Basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) are often used to induce neural differentiation in BMSCs in vitro. However, previous studies regarding their combined actions are insufficient. The present study is the first, to the best of our knowledge, to thoroughly assess the enhancement of neural differentiation of BMSCs following transfection with bFGF and NGF. Sprague-Dawley (SD) rat BMSCs were separated through whole bone marrow adherence, and were then passaged to the third generation. The cells were subsequently divided into five groups: The control group, which consisted of untransfected BMSCs; the plv-blank-transfected BMSCs group; the plv-bFGF-trans-fected BMSCs group; the plv-NGF-transfected BMSCs group; and the plv-NGF-bFGF co-transfected BMSCs group. Cell neural differentiation was characterized in terms of stem cell molecular expression, and the neuronal morphology and expression of neural-like molecules was detected in each of the groups. A total of 72 h post-transfection, the expression levels of neuron-specific enolase, glial fibrillary acidic protein, and nestin protein, were higher in the co-transfected group, as compared with the other groups, the expression levels of β-tubulin III were also increased in the co-transfected cells, thus suggesting the maturation of differentiated neuron-like cells. Furthermore, higher neuronal proliferation was observed in the co-transfected group, as compared with the other groups at passages 2, 4, 6 and 8. Western blotting demonstrated that the transfected groups exhibited a simultaneous increase in phosphorylation of the AKT and extracellular signal-regulated kinases (ERK) signaling pathway

  14. High neuronal/astroglial differentiation plasticity of adult rat hippocampal neural stem/progenitor cells in response to the effects of embryonic and adult cerebrospinal fluids

    PubMed Central

    Peirouvi, T.; Yekani, F.; Azarnia, M.; Massumi, M.

    2015-01-01

    Hippocampal neural stem/progenitor cells (hipp-NS/PCs) of the adult mammalian brain are important sources of neuronal and gial cell production. In this study, the main goal is to investigate the plasticity of these cells in neuronal/astroglial differentiations. To this end, the differentiation of the hipp-NS/PCs isolated from 3-month-old Wistar rats was investigated in response to the embryonic cerebrospinal fluid (E-CSF) including E13.5, E17-CSF and the adult cerebrospinal fluid (A-CSF), all extracted from rats. CSF samples were selected based on their effects on cell behavioral parameters. Primary cell culture was performed in the presence of either normal or high levels of KCL in a culture medium. High levels of KCL cause cell depolarization, and thus the activation of quiescent NSCs. Results from immunocytochemistry (ICC) and semi-quantitative RT-PCR (sRT-PCR) techniques showed that in E-CSF-treated groups, neuronal differentiation increased (E17>E13.5). In contrast, A-CSF decreased and increased neuronal and astroglial differentiations, respectively. Cell survivability and/or proliferation (S/P), evaluated by an MTT assay, increased by E13.5 CSF, but decreased by both E17 CSF and A-CSF. Based on the results, it is finally concluded that adult rat hippocampal proliferative cells are not restricted progenitors but rather show high plasticity in neuronal/astroglial differentiation according to the effects of CSF samples. In addition, using high concentrations of KCL in the primary cell culture led to an increase in the number of NSCs, which in turn resulted in the increase in neuronal or astroglial differentiations after CSF treatment. PMID:27175157

  15. Healthy human CSF promotes glial differentiation of hESC-derived neural cells while retaining spontaneous activity in existing neuronal networks.

    PubMed

    Kiiski, Heikki; Aänismaa, Riikka; Tenhunen, Jyrki; Hagman, Sanna; Ylä-Outinen, Laura; Aho, Antti; Yli-Hankala, Arvi; Bendel, Stepani; Skottman, Heli; Narkilahti, Susanna

    2013-06-15

    The possibilities of human pluripotent stem cell-derived neural cells from the basic research tool to a treatment option in regenerative medicine have been well recognized. These cells also offer an interesting tool for in vitro models of neuronal networks to be used for drug screening and neurotoxicological studies and for patient/disease specific in vitro models. Here, as aiming to develop a reductionistic in vitro human neuronal network model, we tested whether human embryonic stem cell (hESC)-derived neural cells could be cultured in human cerebrospinal fluid (CSF) in order to better mimic the in vivo conditions. Our results showed that CSF altered the differentiation of hESC-derived neural cells towards glial cells at the expense of neuronal differentiation. The proliferation rate was reduced in CSF cultures. However, even though the use of CSF as the culture medium altered the glial vs. neuronal differentiation rate, the pre-existing spontaneous activity of the neuronal networks persisted throughout the study. These results suggest that it is possible to develop fully human cell and culture-based environments that can further be modified for various in vitro modeling purposes.

  16. Histone methylation, alternative splicing and neuronal differentiation.

    PubMed

    Fiszbein, Ana; Kornblihtt, Alberto R

    2016-01-01

    Alternative splicing, as well as chromatin structure, greatly contributes to specific transcriptional programs that promote neuronal differentiation. The activity of G9a, the enzyme responsible for mono- and di-methylation of lysine 9 on histone H3 (H3K9me1 and H3K9me2) in mammalian euchromatin, has been widely implicated in the differentiation of a variety of cell types and tissues. In a recent work from our group (Fiszbein et al., 2016) we have shown that alternative splicing of G9a regulates its nuclear localization and, therefore, the efficiency of H3K9 methylation, which promotes neuronal differentiation. We discuss here our results in the light of a report from other group (Laurent et al. 2015) demonstrating a key role for the alternative splicing of the histone demethylase LSD1 in controlling specific gene expression in neurons. All together, these results illustrate the importance of alternative splicing in the generation of a proper equilibrium between methylation and demethylation of histones for the regulation of neuron-specific transcriptional programs. PMID:27606339

  17. Histone methylation, alternative splicing and neuronal differentiation.

    PubMed

    Fiszbein, Ana; Kornblihtt, Alberto R

    2016-01-01

    Alternative splicing, as well as chromatin structure, greatly contributes to specific transcriptional programs that promote neuronal differentiation. The activity of G9a, the enzyme responsible for mono- and di-methylation of lysine 9 on histone H3 (H3K9me1 and H3K9me2) in mammalian euchromatin, has been widely implicated in the differentiation of a variety of cell types and tissues. In a recent work from our group (Fiszbein et al., 2016) we have shown that alternative splicing of G9a regulates its nuclear localization and, therefore, the efficiency of H3K9 methylation, which promotes neuronal differentiation. We discuss here our results in the light of a report from other group (Laurent et al. 2015) demonstrating a key role for the alternative splicing of the histone demethylase LSD1 in controlling specific gene expression in neurons. All together, these results illustrate the importance of alternative splicing in the generation of a proper equilibrium between methylation and demethylation of histones for the regulation of neuron-specific transcriptional programs.

  18. Foxp2 regulates neuronal differentiation and neuronal subtype specification.

    PubMed

    Chiu, Yi-Chi; Li, Ming-Yang; Liu, Yuan-Hsuan; Ding, Jing-Ya; Yu, Jenn-Yah; Wang, Tsu-Wei

    2014-07-01

    Mutations of the transcription factor FOXP2 in humans cause a severe speech and language disorder. Disruption of Foxp2 in songbirds or mice also leads to deficits in song learning or ultrasonic vocalization, respectively. These data suggest that Foxp2 plays important roles in the developing nervous system. However, the mechanism of Foxp2 in regulating neural development remains elusive. In the current study, we found that Foxp2 increased neuronal differentiation without affecting cell proliferation and cell survival in primary neural progenitors from embryonic forebrains. Foxp2 induced the expression of platelet-derived growth factor receptor α, which mediated the neurognic effect of Foxp2. In addition, Foxp2 positively regulated the differentiation of medium spiny neurons derived from the lateral ganglionic eminence and negatively regulated the formation of interneurons derived from dorsal medial ganglionic eminence by interacting with the Sonic hedgehog pathway. Taken together, our results suggest that Foxp2 regulates multiple aspects of neuronal development in the embryonic forebrain.

  19. The risk-associated long noncoding RNA NBAT-1 controls neuroblastoma progression by regulating cell proliferation and neuronal differentiation.

    PubMed

    Pandey, Gaurav Kumar; Mitra, Sanhita; Subhash, Santhilal; Hertwig, Falk; Kanduri, Meena; Mishra, Kankadeb; Fransson, Susanne; Ganeshram, Abiarchana; Mondal, Tanmoy; Bandaru, Sashidhar; Ostensson, Malin; Akyürek, Levent M; Abrahamsson, Jonas; Pfeifer, Susan; Larsson, Erik; Shi, Leming; Peng, Zhiyu; Fischer, Matthias; Martinsson, Tommy; Hedborg, Fredrik; Kogner, Per; Kanduri, Chandrasekhar

    2014-11-10

    Neuroblastoma is an embryonal tumor of the sympathetic nervous system and the most common extracranial tumor of childhood. By sequencing transcriptomes of low- and high-risk neuroblastomas, we detected differentially expressed annotated and nonannotated long noncoding RNAs (lncRNAs). We identified a lncRNA neuroblastoma associated transcript-1 (NBAT-1) as a biomarker significantly predicting clinical outcome of neuroblastoma. CpG methylation and a high-risk neuroblastoma associated SNP on chromosome 6p22 functionally contribute to NBAT-1 differential expression. Loss of NBAT-1 increases cellular proliferation and invasion. It controls these processes via epigenetic silencing of target genes. NBAT-1 loss affects neuronal differentiation through activation of the neuronal-specific transcription factor NRSF/REST. Thus, loss of NBAT-1 contributes to aggressive neuroblastoma by increasing proliferation and impairing differentiation of neuronal precursors. PMID:25517750

  20. Cell-type-specific and differentiation-status-dependent variations in cytotoxicity of tributyltin in cultured rat cerebral neurons and astrocytes.

    PubMed

    Oyanagi, Koshi; Tashiro, Tomoko; Negishi, Takayuki

    2015-08-01

    Tributyltin (TBT) is an organotin used as an anti-fouling agent for fishing nets and ships and it is a widespread environmental contaminant at present. There is an increasing concern about imperceptible but serious adverse effect(s) of exposure to chemicals existing in the environment on various organs and their physiological functions, e.g. brain and mental function. Here, so as to contribute to improvement of and/or advances in in vitro cell-based assay systems for evaluating brain-targeted adverse effect of chemicals, we tried to evaluate cell-type-specific and differentiation-status-dependent variations in the cytotoxicity of TBT towards neurons and astrocytes using the four culture systems differing in the relative abundance of these two types of cells; primary neuron culture (> 95% neurons), primary neuron-astrocyte (2 : 1) mix culture, primary astrocyte culture (> 95% astrocytes), and passaged astrocyte culture (100% proliferative astrocytes). Cell viability was measured at 48 hr after exposure to TBT in serum-free medium. IC50's of TBT were 198 nM in primary neuron culture, 288 nM in primary neuron-astrocyte mix culture, 2001 nM in primary astrocyte culture, and 1989 nM in passaged astrocyte culture. Furthermore, in primary neuron-astrocyte mix culture, vulnerability of neurons cultured along with astrocytes to TBT toxicity was lower than that of neurons cultured purely in primary neuron culture. On the other hand, astrocytes in primary neuron-astrocyte mix culture were considered to be more vulnerable to TBT than those in primary or passaged astrocyte culture. The present study demonstrated variable cytotoxicity of TBT in neural cells depending on the culture condition.

  1. Cochlear nucleus whole mount explants promote the differentiation of neuronal stem cells from the cochlear nucleus in co-culture experiments.

    PubMed

    Rak, Kristen; Völker, Johannes; Jürgens, Lukas; Völker, Christine; Frenz, Silke; Scherzad, Agmal; Schendzielorz, Philipp; Jablonka, Sibylle; Mlynski, Robert; Radeloff, Andreas; Hagen, Rudolf

    2015-08-01

    The cochlear nucleus is the first brainstem nucleus to receive sensory input from the cochlea. Depriving this nucleus of auditory input leads to cellular and molecular disorganization which may potentially be counteracted by the activation or application of stem cells. Neuronal stem cells (NSCs) have recently been identified in the neonatal cochlear nucleus and a persistent neurogenic niche was demonstrated in this brainstem nucleus until adulthood. The present work investigates whether the neurogenic environment of the cochlear nucleus can promote the survival of engrafted NSCs and whether cochlear nucleus-derived NSCs can differentiate into neurons and glia in brain tissue. Therefore, cochlear nucleus whole-mount explants were co-cultured with NSCs extracted from either the cochlear nucleus or the hippocampus and compared to a second environment using whole-mount explants from the hippocampus. Factors that are known to induce neuronal differentiation were also investigated in these NSC-explant experiments. NSCs derived from the cochlear nucleus engrafted in the brain tissue and differentiated into all cells of the neuronal lineage. Hippocampal NSCs also immigrated in cochlear nucleus explants and differentiated into neurons, astrocytes and oligodendrocytes. Laminin expression was up-regulated in the cochlear nucleus whole-mounts and regulated the in vitro differentiation of NSCs from the cochlear nucleus. These experiments confirm a neurogenic environment in the cochlear nucleus and the capacity of cochlear nucleus-derived NSCs to differentiate into neurons and glia. Consequently, the presented results provide a first step for the possible application of stem cells to repair the disorganization of the cochlear nucleus, which occurs after hearing loss. PMID:25960344

  2. Crosstalk between HIF-1 and ROCK pathways in neuronal differentiation of mesenchymal stem cells, neurospheres and in PC12 neurite outgrowth.

    PubMed

    Pacary, Emilie; Tixier, Emmanuelle; Coulet, Florence; Roussel, Simon; Petit, Edwige; Bernaudin, Myriam

    2007-07-01

    This study demonstrates that the Rho-kinase (ROCK) inhibitor, Y-27632, potentiates not only the effect of cobalt chloride (CoCl(2)) but also that of deferoxamine, another HIF-1 inducer, on mesenchymal stem cell (MSC) neuronal differentiation. HIF-1 is essential for CoCl(2)+/-Y-27632-induced MSC neuronal differentiation, since agents inhibiting HIF-1 abolish the changes of morphology and cell cycle arrest-related gene or protein expressions (p21, cyclin D1) and the increase of neuronal marker expressions (Tuj1, NSE). Y-27632 potentiates the CoCl(2)-induced decrease of cyclin D1 and nestin expressions, the increase of HIF-1 activation and EPO expression, and decreases pVHL expression. Interestingly, CoCl(2) decreases RhoA expression, an effect potentiated by Y-27632, revealing crosstalk between HIF-1 and RhoA/ROCK pathways. Moreover, we demonstrate a synergistic effect of CoCl(2) and Y-27632 on neurosphere differentiation into neurons and PC12 neurite outgrowth underlining that a co-treatment targeting both HIF-1 and ROCK pathways might be relevant to differentiate stem cells into neurons.

  3. Quinazoline-based tricyclic compounds that regulate programmed cell death, induce neuronal differentiation, and are curative in animal models for excitotoxicity and hereditary brain disease

    PubMed Central

    Vainshtein, A; Veenman, L; Shterenberg, A; Singh, S; Masarwa, A; Dutta, B; Island, B; Tsoglin, E; Levin, E; Leschiner, S; Maniv, I; Pe’er, L; Otradnov, I; Zubedat, S; Aga-Mizrachi, S; Weizman, A; Avital, A; Marek, I; Gavish, M

    2015-01-01

    Expanding on a quinazoline scaffold, we developed tricyclic compounds with biological activity. These compounds bind to the 18 kDa translocator protein (TSPO) and protect U118MG (glioblastoma cell line of glial origin) cells from glutamate-induced cell death. Fascinating, they can induce neuronal differentiation of PC12 cells (cell line of pheochromocytoma origin with neuronal characteristics) known to display neuronal characteristics, including outgrowth of neurites, tubulin expression, and NeuN (antigen known as ‘neuronal nuclei’, also known as Rbfox3) expression. As part of the neurodifferentiation process, they can amplify cell death induced by glutamate. Interestingly, the compound 2-phenylquinazolin-4-yl dimethylcarbamate (MGV-1) can induce expansive neurite sprouting on its own and also in synergy with nerve growth factor and with glutamate. Glycine is not required, indicating that N-methyl-D-aspartate receptors are not involved in this activity. These diverse effects on cells of glial origin and on cells with neuronal characteristics induced in culture by this one compound, MGV-1, as reported in this article, mimic the diverse events that take place during embryonic development of the brain (maintenance of glial integrity, differentiation of progenitor cells to mature neurons, and weeding out of non-differentiating progenitor cells). Such mechanisms are also important for protective, curative, and restorative processes that occur during and after brain injury and brain disease. Indeed, we found in a rat model of systemic kainic acid injection that MGV-1 can prevent seizures, counteract the process of ongoing brain damage, including edema, and restore behavior defects to normal patterns. Furthermore, in the R6-2 (transgenic mouse model for Huntington disease; Strain name: B6CBA-Tg(HDexon1)62Gpb/3J) transgenic mouse model for Huntington disease, derivatives of MGV-1 can increase lifespan by >20% and reduce incidence of abnormal movements. Also in vitro

  4. BMP2 promotes the differentiation of neural stem cells into dopaminergic neurons in vitro via miR-145-mediated upregulation of Nurr1 expression

    PubMed Central

    Yan, Wang; Chen, Zhao-Ying; Chen, Jia-Qi; Chen, Hui-Min

    2016-01-01

    Background: Neural stem cells (NSCs) are pluripotent and self-renewing cells which could differentiate into diverse types of neural cells, such as dopaminergic (DA) neurons, the loss of which is the typical characteristic of Parkinson’s disease (PD). This study aimed to examine the molecular mechanisms of BMP2-mediating NSCs differentiation into DA neurons. Methods: Different concentrations of BMP2 were used to induce the differentiation of NSCs into DA neurons, which were characterized by the number and the neurite lengths of tyrosine hydroxylase (TH)+ and dopamine transporter (DAT)+ neurons by immunocytochemistry. qRT-PCR and Western blot were performed to explore the expression of miR-145 and Nurr1. The methylation level of miR-145 promoter was examined by DNA methylation analyses. The regulation of miR-145 on Nurr1 was detected by Dual-Luciferase reporter assay. Results: The number of TH+ and DAT+ neurons were significantly increased in NSCs treated with 20 and 100 ng/ml of BMP2, as well as the neurite lengths of TH+ and DAT+ neurons. The reduced level of miR-145 and up-regulated Nurr1 were observed in NSCs induced by BMP2. The hypermethylation level of miR-145 promoter down-regulated the expression of miR-145 in NSCs pretreated with BMP2, which was regulated by DNMT3b. Luciferase reporter assay showed that Nurr1 was a direct target of miR-145. miR-145 overexpression restrained the differentiating effect of BMP2. Moreover, overexpression of Nurr1 abrogated this effect of miR-145 overexpression. Conclusion: Our results showed that BMP2 promoted the differentiation of NSCs into DA neurons in vitro and miR-145 and Nurr1 were involved in the neurotrophic effects of BMP2. PMID:27725851

  5. Epilepsy-induced motility of differentiated neurons.

    PubMed

    Chai, Xuejun; Münzner, Gert; Zhao, Shanting; Tinnes, Stefanie; Kowalski, Janina; Häussler, Ute; Young, Christina; Haas, Carola A; Frotscher, Michael

    2014-08-01

    Neuronal ectopia, such as granule cell dispersion (GCD) in temporal lobe epilepsy (TLE), has been assumed to result from a migration defect during development. Indeed, recent studies reported that aberrant migration of neonatal-generated dentate granule cells (GCs) increased the risk to develop epilepsy later in life. On the contrary, in the present study, we show that fully differentiated GCs become motile following the induction of epileptiform activity, resulting in GCD. Hippocampal slice cultures from transgenic mice expressing green fluorescent protein in differentiated, but not in newly generated GCs, were incubated with the glutamate receptor agonist kainate (KA), which induced GC burst activity and GCD. Using real-time microscopy, we observed that KA-exposed, differentiated GCs translocated their cell bodies and changed their dendritic organization. As found in human TLE, KA application was associated with decreased expression of the extracellular matrix protein Reelin, particularly in hilar interneurons. Together these findings suggest that KA-induced motility of differentiated GCs contributes to the development of GCD and establish slice cultures as a model to study neuronal changes induced by epileptiform activity.

  6. Corexit-EC9527A Disrupts Retinol Signaling and Neuronal Differentiation in P19 Embryonal Pluripotent Cells

    PubMed Central

    Chen, Yanling; Reese, David H.

    2016-01-01

    Corexit-EC9500A and Corexit-EC9527A are two chemical dispersants that have been used to remediate the impact of the 2010 Deepwater Horizon oil spill. Both dispersants are composed primarily of organic solvents and surfactants and act by emulsifying the crude oil to facilitate biodegradation. The potential adverse effect of the Corexit chemicals on mammalian embryonic development remains largely unknown. Retinol (vitamin A) signaling, mediated by all-trans retinoic acid (RA), is essential for neural tube formation and the development of many organs in the embryo. The physiological levels of RA in cells and tissues are maintained by the retinol signaling pathway (RSP), which controls the biosynthesis of RA from dietary retinol and the catabolism of RA to polar metabolites for removal. RA is a potent activating ligand for the RAR/RXR nuclear receptors. Through RA and the receptors, the RSP modulates the expression of many developmental genes; interference with the RSP is potentially teratogenic. In this study the mouse P19 embryonal pluripotent cell, which contains a functional RSP, was used to evaluate the effects of the Corexit dispersants on retinol signaling and associated neuronal differentiation. The results showed that Corexit-EC9500A was more cytotoxic than Corexit-EC9527A to P19 cells. At non-cytotoxic doses, Corexit-EC9527A inhibited retinol-induced expression of the Hoxa1 gene, which encodes a transcription factor for the regulation of body patterning in the embryo. Such inhibition was seen in the retinol- and retinal- induced, but not RA-induced, Hoxa1 up-regulation, indicating that the Corexit chemicals primarily inhibit RA biosynthesis from retinal. In addition, Corexit-EC9527A suppressed retinol-induced P19 cell differentiation into neuronal cells, indicating potential neurotoxic effect of the chemicals under the tested conditions. The surfactant ingredient, dioctyl sodium sulfosuccinate (DOSS), may be a major contributor to the observed effect of

  7. ZNF804A Transcriptional Networks in Differentiating Neurons Derived from Induced Pluripotent Stem Cells of Human Origin

    PubMed Central

    Hrabovsky, Anastasia; Pedrosa, Erika; Dean, Jason; Jain, Swati; Zheng, Deyou; Lachman, Herbert M.

    2015-01-01

    ZNF804A (Zinc Finger Protein 804A) has been identified as a candidate gene for schizophrenia (SZ), autism spectrum disorders (ASD), and bipolar disorder (BD) in replicated genome wide association studies (GWAS) and by copy number variation (CNV) analysis. Although its function has not been well-characterized, ZNF804A contains a C2H2-type zinc-finger domain, suggesting that it has DNA binding properties, and consequently, a role in regulating gene expression. To further explore the role of ZNF804A on gene expression and its downstream targets, we used a gene knockdown (KD) approach to reduce its expression in neural progenitor cells (NPCs) derived from induced pluripotent stem cells (iPSCs). KD was accomplished by RNA interference (RNAi) using lentiviral particles containing shRNAs that target ZNF804A mRNA. Stable transduced NPC lines were generated after puromycin selection. A control cell line expressing a random (scrambled) shRNA was also generated. Neuronal differentiation was induced, RNA was harvested after 14 days and transcriptome analysis was carried out using RNA-seq. 1815 genes were found to be differentially expressed at a nominally significant level (p<0.05); 809 decreased in expression in the KD samples, while 1106 increased. Of these, 370 achieved genome wide significance (FDR<0.05); 125 were lower in the KD samples, 245 were higher. Pathway analysis showed that genes involved in interferon-signaling were enriched among those that were down-regulated in the KD samples. Correspondingly, ZNF804A KD was found to affect interferon-alpha 2 (IFNA2)-mediated gene expression. The findings suggest that ZNF804A may affect a differentiating neuron’s response to inflammatory cytokines, which is consistent with models of SZ and ASD that support a role for infectious disease, and/or autoimmunity in a subgroup of patients. PMID:25905630

  8. The cellular form of the prion protein guides the differentiation of human embryonic stem cells into neuron-, oligodendrocyte-, and astrocyte-committed lineages.

    PubMed

    Lee, Young Jin; Baskakov, Ilia V

    2014-01-01

    Prion protein, PrP(C), is a glycoprotein that is expressed on the cell surface beginning with the early stages of embryonic stem cell differentiation. Previously, we showed that ectopic expression of PrP(C) in human embryonic stem cells (hESCs) triggered differentiation toward endodermal, mesodermal, and ectodermal lineages, whereas silencing of PrP(C) suppressed differentiation toward ectodermal but not endodermal or mesodermal lineages. Considering that PrP(C) might be involved in controlling the balance between cells of different lineages, the current study was designed to test whether PrP(C) controls differentiation of hESCs into cells of neuron-, oligodendrocyte-, and astrocyte-committed lineages. PrP(C) was silenced in hESCs cultured under three sets of conditions that were previously shown to induce hESCs differentiation into predominantly neuron-, oligodendrocyte-, and astrocyte-committed lineages. We found that silencing of PrP(C) suppressed differentiation toward all three lineages. Similar results were observed in all three protocols, arguing that the effect of PrP(C) was independent of differentiation conditions employed. Moreover, switching PrP(C) expression during a differentiation time course revealed that silencing PrP(C) expression during the very initial stage that corresponds to embryonic bodies has a more significant impact than silencing at later stages of differentiation. The current work illustrates that PrP(C) controls differentiation of hESCs toward neuron-, oligodendrocyte-, and astrocyte-committed lineages and is likely involved at the stage of uncommitted neural progenitor cells rather than lineage-committed neural progenitors.

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

    PubMed Central

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

    2015-01-01

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

  10. Overexpression of β-NGF promotes differentiation of bone marrow mesenchymal stem cells into neurons through regulation of AKT and MAPK pathway.

    PubMed

    Yuan, Jun; Huang, Guorong; Xiao, Zhe; Lin, Lvbiao; Han, Tianwang

    2013-11-01

    Bone marrow stromal stem cells (BMSCs) are fibroblastic in shape and capable of self-renewal and have the potential for multi-directional differentiation. Nerve growth factor (NGF), a homodimeric polypeptide, plays an important role in the nervous system by supporting the survival and growth of neural cells, regulating cell growth, promoting differentiation into neuron, and neuron migration. Adenoviral vectors are DNA viruses that contain 36 kb of double-stranded DNA allowing for transmission of the genes to the host nucleus but not inserting them into the host chromosome. The present study aimed to investigate the induction efficiency and differentiation of neural cells from BMSCs by β-NGF gene transfection with recombinant adenoviral vector (Ad-β-NGF) in vitro. The results of immunochemical assay confirmed the induced cells as neuron cells. Moreover, flow cytometric analysis, Annexin-V-FITC/PI, and BrdU assay revealed that chemical inducer β-mercaptoethanol (β-met) triggered apoptosis of BMSCs, as evidenced by inhibition of DNA fragmentation, nuclear condensation, translocation of phospholipid phosphatidylserine, and activation of caspase-3. Furthermore, the results of western blotting showed that β-met suppressed AKT signaling pathway and regulated the MAPKs during differentiation of BMSCs. In contrast, Ad-β-NGF effectively induced the differentiation of BMSCs without causing any cytopathic phenomenon and apoptotic cell death. Moreover, Ad-β-NGF recovered the expression level of phosphorylated AKT and MAPKs in cells exposed to chemical reagents. Taken together, these results suggest that β-NGF gene transfection promotes the differentiation of BMSCs into neurons through regulation of AKT and MAPKs signaling pathways. PMID:23934089

  11. Effects of brain-derived neurotrophic factor and neurotrophin-3 on the neuronal differentiation of rat adipose-derived stem cells

    PubMed Central

    JI, WENCHEN; ZHANG, XIAOWEI; JI, LE; WANG, KUNZHENG; QIU, YUSHENG

    2015-01-01

    Tissue engineering is a promising method that may be used to treat spinal cord injury (SCI). The underlying repair mechanism of tissue engineering involves the stable secretion of neurotrophins from seed cells, which eventually differentiate into neurons; therefore, the selection of appropriate seed cells, which stably secrete neurotrophins that easily differentiate into neurons requires investigation. Adipose-derived stem cells (ADSCs), which are adult SCs, are advantageous due to convenience sampling and easy expansion; therefore, ADSCs are currently the most popular type of seed cell. Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) possess superior properties, when compared with other neurotrophic factors, in the maintenance of neuronal survival and promotion of SC differentiation into neurons. The present study used two lentiviruses, which specifically express BDNF and NT-3 [Lenti-BDNF-green fluorescent protein (GFP), Lenti-NT-3-red fluorescent protein (RFP)], to transfect third-generation ADSCs. Three types of seed cell were obtained: i) Seed cells overexpressing BDNF (ADSC/Lenti-BDNF-GFP); ii) seed cells overexpressing NT-3 (ADSC/Lenti-NT-3-RFP); and iii) seed cells overexpressing BDNF and NT-3 (ADSC/Lenti-BDNF-GFP and NT-3-RFP). The transfected cells were then induced to differentiate into neurons and were divided into a further four groups: i) The BDNF and NT-3 co-overexpression group; ii) the BDNF overexpression group; iii) the NT-3 overexpression group; and iv) the control group, which consisted of untransfected ADSCs. The results of the present study demonstrate that BDNF and NT-3 expression was higher 10 days after induction, as detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. Neuron-specific enolase is a neuronal marker, the expression of which was highest in the BDNF and NT-3 co-overexpression group, followed by the BDNF overexpression group and then by the NT-3 overexpression

  12. HLXB9 Gene Expression, and Nuclear Location during In Vitro Neuronal Differentiation in the SK-N-BE Neuroblastoma Cell Line

    PubMed Central

    Leotta, Claudia Giovanna; Federico, Concetta; Brundo, Maria Violetta; Tosi, Sabrina; Saccone, Salvatore

    2014-01-01

    Different parts of the genome occupy specific compartments of the cell nucleus based on the gene content and the transcriptional activity. An example of this is the altered nuclear positioning of the HLXB9 gene in leukaemia cells observed in association with its over-expression. This phenomenon was attributed to the presence of a chromosomal translocation with breakpoint proximal to the HLXB9 gene. Before becoming an interesting gene in cancer biology, HLXB9 was studied as a developmental gene. This homeobox gene is also known as MNX1 (motor neuron and pancreas homeobox 1) and it is relevant for both motor neuronal and pancreatic beta cells development. A spectrum of mutations in this gene are causative of sacral agenesis and more broadly, of what is known as the Currarino Syndrome, a constitutional autosomal dominant disorder. Experimental work on animal models has shown that HLXB9 has an essential role in motor neuronal differentiation. Here we present data to show that, upon treatment with retinoic acid, the HLXB9 gene becomes over-expressed during the early stages of neuronal differentiation and that this corresponds to a reposition of the gene in the nucleus. More precisely, we used the SK-N-BE human neuroblastoma cell line as an in vitro model and we demonstrated a transient transcription of HLXB9 at the 4th and 5th days of differentiation that corresponded to the presence, predominantly in the cell nuclei, of the encoded protein HB9. The nuclear positioning of the HLXB9 gene was monitored at different stages: a peripheral location was noted in the proliferating cells whereas a more internal position was noted during differentiation, that is while HLXB9 was transcriptionally active. Our findings suggest that HLXB9 can be considered a marker of early neuronal differentiation, possibly involving chromatin remodeling pathways. PMID:25136833

  13. HLXB9 gene expression, and nuclear location during in vitro neuronal differentiation in the SK-N-BE neuroblastoma cell line.

    PubMed

    Leotta, Claudia Giovanna; Federico, Concetta; Brundo, Maria Violetta; Tosi, Sabrina; Saccone, Salvatore

    2014-01-01

    Different parts of the genome occupy specific compartments of the cell nucleus based on the gene content and the transcriptional activity. An example of this is the altered nuclear positioning of the HLXB9 gene in leukaemia cells observed in association with its over-expression. This phenomenon was attributed to the presence of a chromosomal translocation with breakpoint proximal to the HLXB9 gene. Before becoming an interesting gene in cancer biology, HLXB9 was studied as a developmental gene. This homeobox gene is also known as MNX1 (motor neuron and pancreas homeobox 1) and it is relevant for both motor neuronal and pancreatic beta cells development. A spectrum of mutations in this gene are causative of sacral agenesis and more broadly, of what is known as the Currarino Syndrome, a constitutional autosomal dominant disorder. Experimental work on animal models has shown that HLXB9 has an essential role in motor neuronal differentiation. Here we present data to show that, upon treatment with retinoic acid, the HLXB9 gene becomes over-expressed during the early stages of neuronal differentiation and that this corresponds to a reposition of the gene in the nucleus. More precisely, we used the SK-N-BE human neuroblastoma cell line as an in vitro model and we demonstrated a transient transcription of HLXB9 at the 4th and 5th days of differentiation that corresponded to the presence, predominantly in the cell nuclei, of the encoded protein HB9. The nuclear positioning of the HLXB9 gene was monitored at different stages: a peripheral location was noted in the proliferating cells whereas a more internal position was noted during differentiation, that is while HLXB9 was transcriptionally active. Our findings suggest that HLXB9 can be considered a marker of early neuronal differentiation, possibly involving chromatin remodeling pathways. PMID:25136833

  14. Dickkopf 3 Promotes the Differentiation of a Rostrolateral Midbrain Dopaminergic Neuronal Subset In Vivo and from Pluripotent Stem Cells In Vitro in the Mouse.

    PubMed

    Fukusumi, Yoshiyasu; Meier, Florian; Götz, Sebastian; Matheus, Friederike; Irmler, Martin; Beckervordersandforth, Ruth; Faus-Kessler, Theresa; Minina, Eleonora; Rauser, Benedict; Zhang, Jingzhong; Arenas, Ernest; Andersson, Elisabet; Niehrs, Christof; Beckers, Johannes; Simeone, Antonio; Wurst, Wolfgang; Prakash, Nilima

    2015-09-30

    Wingless-related MMTV integration site 1 (WNT1)/β-catenin signaling plays a crucial role in the generation of mesodiencephalic dopaminergic (mdDA) neurons, including the substantia nigra pars compacta (SNc) subpopulation that preferentially degenerates in Parkinson's disease (PD). However, the precise functions of WNT1/β-catenin signaling in this context remain unknown. Stem cell-based regenerative (transplantation) therapies for PD have not been implemented widely in the clinical context, among other reasons because of the heterogeneity and incomplete differentiation of the transplanted cells. This might result in tumor formation and poor integration of the transplanted cells into the dopaminergic circuitry of the brain. Dickkopf 3 (DKK3) is a secreted glycoprotein implicated in the modulation of WNT/β-catenin signaling. Using mutant mice, primary ventral midbrain cells, and pluripotent stem cells, we show that DKK3 is necessary and sufficient for the correct differentiation of a rostrolateral mdDA neuron subset. Dkk3 transcription in the murine ventral midbrain coincides with the onset of mdDA neurogenesis and is required for the activation and/or maintenance of LMX1A (LIM homeobox transcription factor 1α) and PITX3 (paired-like homeodomain transcription factor 3) expression in the corresponding mdDA precursor subset, without affecting the proliferation or specification of their progenitors. Notably, the treatment of differentiating pluripotent stem cells with recombinant DKK3 and WNT1 proteins also increases the proportion of mdDA neurons with molecular SNc DA cell characteristics in these cultures. The specific effects of DKK3 on the differentiation of rostrolateral mdDA neurons in the murine ventral midbrain, together with its known prosurvival and anti-tumorigenic properties, make it a good candidate for the improvement of regenerative and neuroprotective strategies in the treatment of PD. Significance statement: We show here that Dickkopf 3 (DKK3), a

  15. Pax6 Is Essential for the Maintenance and Multi-Lineage Differentiation of Neural Stem Cells, and for Neuronal Incorporation into the Adult Olfactory Bulb

    PubMed Central

    Curto, Gloria G.; Nieto-Estévez, Vanesa; Hurtado-Chong, Anahí; Valero, Jorge; Gómez, Carmela; Alonso, José R.; Weruaga, Eduardo

    2014-01-01

    The paired type homeobox 6 (Pax6) transcription factor (TF) regulates multiple aspects of neural stem cell (NSC) and neuron development in the embryonic central nervous system. However, less is known about the role of Pax6 in the maintenance and differentiation of adult NSCs and in adult neurogenesis. Using the +/SeyDey mouse, we have analyzed how Pax6 heterozygosis influences the self-renewal and proliferation of adult olfactory bulb stem cells (aOBSCs). In addition, we assessed its influence on neural differentiation, neuronal incorporation, and cell death in the adult OB, both in vivo and in vitro. Our results indicate that the Pax6 mutation alters Nestin+-cell proliferation in vivo, as well as self-renewal, proliferation, and survival of aOBSCs in vitro although a subpopulation of +/SeyDey progenitors is able to expand partially similar to wild-type progenitors. This mutation also impairs aOBSC differentiation into neurons and oligodendrocytes, whereas it increases cell death while preserving astrocyte survival and differentiation. Furthermore, Pax6 heterozygosis causes a reduction in the variety of neurochemical interneuron subtypes generated from aOBSCs in vitro and in the incorporation of newly generated neurons into the OB in vivo. Our findings support an important role of Pax6 in the maintenance of aOBSCs by regulating cell death, self-renewal, and cell fate, as well as in neuronal incorporation into the adult OB. They also suggest that deregulation of the cell cycle machinery and TF expression in aOBSCs which are deficient in Pax6 may be at the origin of the phenotypes observed in this adult NSC population. PMID:25117830

  16. Protective role of olesoxime against wild-type α-synuclein-induced toxicity in human neuronally differentiated SHSY-5Y cells

    PubMed Central

    Gouarné, C; Tracz, J; Paoli, M Giraudon; Deluca, V; Seimandi, M; Tardif, G; Xilouri, M; Stefanis, L; Bordet, T; Pruss, R M

    2015-01-01

    BACKGROUND AND PURPOSE Parkinson's disease (PD) is usually diagnosed clinically from classical motor symptoms, while definitive diagnosis is made postmortem, based on the presence of Lewy bodies and nigral neuron cell loss. α-Synuclein (ASYN), the main protein component of Lewy bodies, clearly plays a role in the neurodegeneration that characterizes PD. Additionally, mutation in the SNCA gene or copy number variations are associated with some forms of familial PD. Here, the objective of the study was to evaluate whether olesoxime, a promising neuroprotective drug can prevent ASYN-mediated neurotoxicity. EXPERIMENTAL APPROACH We used here a novel, mechanistically approachable and attractive cellular model based on the inducible overexpression of human wild-type ASYN in neuronally differentiated human neuroblastoma (SHSY-5Y) cells. This model demonstrates gradual cellular degeneration, coinciding temporally with the appearance of soluble and membrane-bound ASYN oligomers and cell death combining both apoptotic and non-apoptotic pathways. KEY RESULTS Olesoxime fully protected differentiated SHSY-5Y cells from cell death, neurite retraction and cytoplasmic shrinkage induced by moderate ASYN overexpression. This protection was associated with a reduction in cytochrome c release from mitochondria and caspase-9 activation suggesting that olesoxime prevented ASYN toxicity by preserving mitochondrial integrity and function. In addition, olesoxime displayed neurotrophic effects on neuronally differentiated SHSY-5Y cells, independent of ASYN expression, by promoting their differentiation. CONCLUSIONS AND IMPLICATIONS Because ASYN is a common underlying factor in many cases of PD, olesoxime could be a promising therapy to slow neurodegeneration in PD. PMID:25220617

  17. Effects of brain‑derived neurotrophic factor and neurotrophin‑3 on the neuronal differentiation of rat adipose‑derived stem cells.

    PubMed

    Ji, Wenchen; Zhang, Xiaowei; Ji, Le; Wang, Kunzheng; Qiu, Yusheng

    2015-10-01

    Tissue engineering is a promising method that may be used to treat spinal cord injury (SCI). The underlying repair mechanism of tissue engineering involves the stable secretion of neurotrophins from seed cells, which eventually differentiate into neurons; therefore, the selection of appropriate seed cells, which stably secrete neurotrophins that easily differentiate into neurons requires investigation. Adipose‑derived stem cells (ADSCs), which are adult SCs, are advantageous due to convenience sampling and easy expansion; therefore, ADSCs are currently the most popular type of seed cell. Brain‑derived neurotrophic factor (BDNF) and neurotrophin‑3 (NT‑3) possess superior properties, when compared with other neurotrophic factors, in the maintenance of neuronal survival and promotion of SC differentiation into neurons. The present study used two lentiviruses, which specifically express BDNF and NT‑3 [Lenti‑BDNF‑green fluorescent protein (GFP), Lenti‑NT‑3‑red fluorescent protein (RFP)], to transfect third‑generation ADSCs. Three types of seed cell were obtained: i) Seed cells overexpressing BDNF (ADSC/Lenti‑BDNF‑GFP); ii) seed cells overexpressing NT‑3 (ADSC/Lenti‑NT‑3‑RFP); and iii) seed cells overexpressing BDNF and NT‑3 (ADSC/Lenti‑BDNF‑GFP and NT‑3‑RFP). The transfected cells were then induced to differentiate into neurons and were divided into a further four groups: i) The BDNF and NT‑3 co‑overexpression group; ii) the BDNF overexpression group; iii) the NT‑3 overexpression group; and iv) the control group, which consisted of untransfected ADSCs. The results of the present study demonstrate that BDNF and NT‑3 expression was higher 10 days after induction, as detected by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blotting. Neuron‑specific enolase is a neuronal marker, the expression of which was highest in the BDNF and NT‑3 co‑overexpression group, followed by the

  18. Regulation of Shootin1 Gene Expression Involves NGF-induced Alternative Splicing during Neuronal Differentiation of PC12 Cells

    PubMed Central

    Ergin, Volkan; Erdogan, Mutlu; Menevse, Adnan

    2015-01-01

    Shootin1 is a protein involved in neuronal polarization, and has been shown to be a key molecule for the positive/negative feedback loop for axon induction required during neuronal symmetry breaking. To better understand the molecular basis of shootin1 dynamics, we analysed the regulatory pathways and the expressional status of shootin1 gene during NGF-induced neuronal differentiation. We demonstrated that the isoform-1 and isoform-2 of shootin1 is differentially expressed during neuronal differentiation. By blocking individual downstream pathways of NGF signalling, we found that PI3K/Akt pathway plays a major role in the expression of shootin1 isoform-2. Western blot and RT-PCR results showed that the isoform-1 of shootin1 is constitutively expressed, while the isoform-2 is expressed in a manner that is strictly dependent on NGF-stimulation. Isoform-specific RT-PCR results demonstrated that the differential expression of the isoform-1 and isoform-2 of shootin1 is a consequence of alternative splicing of shootin1 pre-mRNA, in response to NGF-signalling. Collectively these findings provide the first information on the molecular mechanisms regulating the expression of shootin1 gene and represent the first example of NGF-induced alternative splicing process that has a regulatory role in neuritogenesis. PMID:26648138

  19. Inhibition of BCL-2 leads to increased apoptosis and delayed neuronal differentiation in human ReNcell VM cells in vitro.

    PubMed

    Fröhlich, Michael; Jaeger, Alexandra; Weiss, Dieter G; Kriehuber, Ralf

    2016-02-01

    BCL-2 is a multifunctional protein involved in the regulation of apoptosis, cell cycle progression and neural developmental processes. Its function in the latter process is not well understood and needs further elucidation. Therefore, we characterized the protein expression kinetics of BCL-2 and associated regulatory proteins of the intrinsic apoptosis pathway during the process of neuronal differentiation in ReNcell VM cells with and without functional inhibition of BCL-2 by its competitive ligand HA14-1. Inhibition of BCL-2 caused a diminished BCL-2 expression and higher levels of cleaved BAX, activated Caspase-3 and cleaved PARP, all pro-apoptotic markers, when compared with untreated differentiating cells. In parallel, flow cytometric analysis of HA14-1-treated cells revealed a delayed differentiation into HuC/D+ neuronal cells when compared to untreated differentiating cells. In conclusion, BCL-2 possess a protective function in fully differentiated ReNcell VM cells. We propose that the pro-survival signaling of BCL-2 is closely connected with its stimulatory effects on neurogenesis of human neural progenitor cells.

  20. Different regulation of aryl hydrocarbon receptor-regulated genes in response to dioxin in undifferentiated and neuronally differentiated human neuroblastoma SH-SY5Y cells.

    PubMed

    Imran, Saima; Ferretti, Patrizia; Vrzal, Radim

    2015-01-01

    Some environmental pollutants derived from industrial processes have been suggested to be responsible for neurological impairment in children, especially in heavily polluted areas. Since these compounds are usually activators of aryl hydrocarbon receptor (AhR), it would be important to better understand the molecular pathways downstream of AhR leading to neural deficits. To this purpose, appropriate in vitro human neural model is much needed. Here we have investigated whether undifferentiated and neuronally differentiated human neuroblastoma cells, SH-SY5Y cells, can provide a suitable model for monitoring AhR activity induced by environmental pollutants, focusing on 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD), a known activator of AhR. Further characterization of differentiated SH-SY5Y showed an increase in AhRR (aryl hydrocarbon receptor repressor), no change in ARNT1 (AhR nuclear translocator 1), and a decrease in ARNT2 expression with differentiation; in contrast, AhR was undetectable in both undifferentiated and differentiated cells. Nonetheless, treatment of parental as well as differentiated SH-SY5Y cells with TCDD resulted in the induction of AhR-regulated genes, CYP1A1 and CYP1B1; AhRR expression was also affected, but to a much smaller extent. These results indicate that undifferentiated SH-SY5Y are less sensitive to TCDD than neuronally differentiated ones, suggesting a higher resistance of the undifferentiated tumor cells to toxic insults. They also suggest that TCDD in these cells may not act via direct activation of AhR that is undetectable in SH-SY5Y as well as in differentiated neurons. Hence, these cells do not provide an appropriate model for studying ligand-mediated activation of AhR.

  1. Role of PPARγ in the Differentiation and Function of Neurons

    PubMed Central

    Quintanilla, Rodrigo A.; Utreras, Elias; Cabezas-Opazo, Fabián A.

    2014-01-01

    Neuronal processes (neurites and axons) have an important role in brain cells communication and, generally, they are damaged in neurodegenerative diseases. Recent evidence has showed that the activation of PPARγ pathway promoted neuronal differentiation and axon polarity. In addition, activation of PPARγ using thiazolidinediones (TZDs) prevented neurodegeneration by reducing neuronal death, improving mitochondrial function, and decreasing neuroinflammation in neuropathic pain. In this review, we will discuss important evidence that supports a possible role of PPARγ in neuronal development, improvement of neuronal health, and pain signaling. Therefore, activation of PPARγ is a potential target with therapeutic applications against neurodegenerative disorders, brain injury, and pain regulation. PMID:25246934

  2. Cannabinoids and neuronal damage: differential effects of THC, AEA and 2-AG on activated microglial cells and degenerating neurons in excitotoxically lesioned rat organotypic hippocampal slice cultures.

    PubMed

    Kreutz, Susanne; Koch, Marco; Ghadban, Chalid; Korf, Horst-Werner; Dehghani, Faramarz

    2007-01-01

    Cannabinoids (CBs) are attributed neuroprotective effects in vivo. Here, we determined the neuroprotective potential of CBs during neuronal damage in excitotoxically lesioned organotypic hippocampal slice cultures (OHSCs). OHSCs are the best characterized in vitro model to investigate the function of microglial cells in neuronal damage since blood-borne monocytes and T-lymphocytes are absent and microglial cells represent the only immunocompetent cell type. Excitotoxic neuronal damage was induced by NMDA (50 microM) application for 4 h. Neuroprotective properties of 9-carboxy-11-nor-delta-9-tetrahydrocannabinol (THC), N-arachidonoylethanolamide (AEA) or 2-arachidonoylglycerol (2-AG) in different concentrations were determined after co-application with NMDA by counting degenerating neurons identified by propidium iodide labeling (PI(+)) and microglial cells labeled by isolectin B(4) (IB(4)(+)). All three CBs used significantly decreased the number of IB(4)(+) microglial cells in the dentate gyrus but the number of PI(+) neurons was reduced only after 2-AG treatment. Application of AM630, antagonizing CB2 receptors highly expressed by activated microglial cells, did not counteract neuroprotective effects of 2-AG, but affected THC-mediated reduction of IB(4)(+) microglial cells. Our results indicate that (1) only 2-AG exerts neuroprotective effects in OHSCs; (2) reduction of IB(4)(+) microglial cells is not a neuroprotective event per se and involves other CB receptors than the CB2 receptor; (3) the discrepancy in the neuroprotective effects of CBs observed in vivo and in our in vitro model system may underline the functional relevance of invading monocytes and T-lymphocytes that are absent in OHSCs. PMID:17010339

  3. [Astragalus induces human amniotic epithelial cells (WISH) to differentiate toward neurons, inhibits the expression of Notch1 and promotes cell survival].

    PubMed

    Chen, Xu-Dong; Wang, Jian-Guo

    2012-12-25

    The aim of the study was to investigate the effect of astragalus on differentiation of human amniotic epithelial cell line WISH into neurons, the expression of Notch1 gene and cell viability. WISH were randomly divided into astragalus group (4 subgroups), alltransretinoic acid (RA) group and control group. Astragalus group and RA group were induced to differentiate into neurocytes by using chemical inducer RA and astragalus, respectively. The expression of neuron-specific enolase (NSE), microtubule associated protein 2 (MAP-2), Nestin and GFAP of induced cells in three groups were detected using immunocytochemical method. RT- PCR was further used to detect the expression of Oct4, Notch1, Hes1, Nestin and NSE. The cell viability was measured by methyl thiazolyl tetrazolium methods. Under the convert microscope it was observed that WISH cells started to change their shape, and there were several axon or dendrite-like processes out from the cell body induced by astragalus for 24 h or RA for 12 h. The positive cell rates of NSE and MAP-2 in 100 μL/mL astragalus-induced group were less than those in RA-induced group at 48 h (P < 0.05), but higher than those in control group. Cell viability in astragalus group was higher than that of RA group (P < 0.05). While the positive cell rates of Nestin and GFAP in 100 μL/mL astragalus-induced group were higher than those in RA-induced group at 48 h (P < 0.05). The positive cell rates of Nestin in the two induced groups were lower than those in control group. RT-PCR showed that the expressions of Oct4, Notch1 and Hes1 in RA and astragalus (100 μL/mL) groups were less than those in control group, but the expression of NSE was higher than that in control group. These results suggest that astragalus (especially at 100 μL/mL, 48 h) and RA can both induce human amniotic epithelial cell line WISH cells into neuron-like cells, but astragalus induction has a higher cell survival rate than RA induction, and the expression of Notch1

  4. Survival, Differentiation, and Migration of High-Purity Mouse Embryonic Stem Cell-derived Progenitor Motor Neurons in Fibrin Scaffolds after Sub-Acute Spinal Cord Injury.

    PubMed

    McCreedy, D A; Wilems, T S; Xu, H; Butts, J C; Brown, C R; Smith, A W; Sakiyama-Elbert, S E

    2014-11-01

    Embryonic stem (ES) cells can be differentiated into many neural cell types that hold great potential as cell replacement therapies following spinal cord injury (SCI). Coupling stem cell transplantation with biomaterial scaffolds can produce a unified combination therapy with several potential advantages including enhanced cell survival, greater transplant retention, reduced scarring, and improved integration at the transplant/host interface. Undesired cell types, however, are commonly present in ES-cell derived cultures due to the limited efficiency of most ES cell induction protocols. Heterogeneous cell populations can confound the interaction between the biomaterial and specific neural populations leading to undesired outcomes. In particular, biomaterials scaffolds may enhance tumor formation by promoting survival and proliferation of undifferentiated ES cells that can persist after induction. Methods for purification of specific ES cell-derived neural populations are necessary to recognize the full potential of combination therapies involving biomaterials and ES cell-derived neural populations. We previously developed a method for enriching ES cell-derived progenitor motor neurons (pMNs) induced from mouse ES cells via antibiotic selection and showed that the enriched cell populations are depleted of pluripotent stem cells. In this study, we demonstrate the survival and differentiation of enriched pMNs within three dimensional (3D) fibrin scaffolds in vitro and when transplanted into a sub-acute dorsal hemisection model of SCI into neurons, oligodendrocytes and astrocytes. PMID:25346848

  5. Differentiation of Human Adipose-Derived Stem Cells into Neuron-Like Cells Which Are Compatible with Photocurable Three-Dimensional Scaffolds

    PubMed Central

    Gao, Shane; Zhao, Peng; Lin, Chao; Sun, Yuxi; Wang, Yilei; Zhou, Zhichong; Yang, Danjing; Wang, Xianli; Xu, Hongzhen; Zhou, Fei; Cao, Limei; Zhou, Wei; Ning, Ke

    2014-01-01

    Multipotent human adipose-derived stromal/stem cells (hADSCs) hold a great promise for cell-based therapy for many devastating human diseases, such as spinal cord injury and stroke. If exogenous hADSCs can be cultured in a three-dimensional (3D) scaffold with effective proliferation and differentiation capacity, it will better mimic the in vivo environment, which will have profound impact on the therapeutic application of hADSCs. In this study, a group of elastic-dominant, porous bioscaffolds from photocurable chitosan and gelatin were fabricated and proven to be biocompatible with both hADSCs and hADSC-derived neuron-like cells (hADSC-NLCs) in vitro. The identity of harvested hADSCs was confirmed by their positive immunostaining of mesenchymal stem cell surface markers, CD29, CD44, and CD105, and also positive expression of stem markers, Sox-2, Oct-4, c-Myc, Nanog, and Klf4. Their multipotency was further confirmed by trilineage differentiation of hADSCs toward adipocyte, osteoblast, and chondrocyte. It was found that hADSCs could be conditioned to differentiate into neurons in vitro as determined by immunostaining the markers of Tuj1, MAP2, NeuN, and Synapsin. The hADSCs and hADSC-NLCs were proven to be biocompatible with 3D scaffold, which actually facilitated the proliferation and differentiation of hADSCs in vitro, by MTT assay and their neuronal gene expression profiling. Moreover, hADSC-NLCs, which were mixed with 3D scaffold and transplanted into traumatic brain injury mouse model, survived in vivo and led to the better repair of the damaged brain area. The immunohistochemical studies revealed that 3D scaffold indeed improved the viability of transplanted cells, their ability to incorporate into the in vivo neural circuit, and their capacity for tissue repair. This study indicates that hADSCs would have great therapeutic application potential as seeding cells for in vivo transplantation to treat various neurological diseases when co-applied with porous

  6. Non ionising radiation as a non chemical strategy in regenerative medicine: Ca(2+)-ICR "In Vitro" effect on neuronal differentiation and tumorigenicity modulation in NT2 cells.

    PubMed

    Ledda, Mario; Megiorni, Francesca; Pozzi, Deleana; Giuliani, Livio; D'Emilia, Enrico; Piccirillo, Sara; Mattei, Cristiana; Grimaldi, Settimio; Lisi, Antonella

    2013-01-01

    In regenerative medicine finding a new method for cell differentiation without pharmacological treatment or gene modification and minimal cell manipulation is a challenging goal. In this work we reported a neuronal induced differentiation and consequent reduction of tumorigenicity in NT2 human pluripotent embryonal carcinoma cells exposed to an extremely low frequency electromagnetic field (ELF-EMF), matching the cyclotron frequency corresponding to the charge/mass ratio of calcium ion (Ca(2+)-ICR). These cells, capable of differentiating into post-mitotic neurons following treatment with Retinoic Acid (RA), were placed in a solenoid and exposed for 5 weeks to Ca(2+)-ICR. The solenoid was installed in a μ-metal shielded room to avoid the effect of the geomagnetic field and obtained totally controlled and reproducible conditions. Contrast microscopy analysis reveled, in the NT2 exposed cells, an important change in shape and morphology with the outgrowth of neuritic-like structures together with a lower proliferation rate and metabolic activity alike those found in the RA treated cells. A significant up-regulation of early and late neuronal differentiation markers and a significant down-regulation of the transforming growth factor-α (TGF-α) and the fibroblast growth factor-4 (FGF-4) were also observed in the exposed cells. The decreased protein expression of the transforming gene Cripto-1 and the reduced capability of the exposed NT2 cells to form colonies in soft agar supported these last results. In conclusion, our findings demonstrate that the Ca(2+)-ICR frequency is able to induce differentiation and reduction of tumorigenicity in NT2 exposed cells suggesting a new potential therapeutic use in regenerative medicine.

  7. Programming embryonic stem cells to neuronal subtypes

    PubMed Central

    Peljto, Mirza; Wichterle, Hynek

    2010-01-01

    Richness of neural circuits and specificity of neuronal connectivity depends on the diversification of nerve cells into functionally and molecularly distinct subtypes. While efficient methods for directed differentiation of embryonic stem cells (ESCs) into multiple principal neuronal classes have been established, only a few studies systematically examined the subtype diversity of in vitro derived nerve cells. Here we review evidence based on molecular and in vivo transplantation studies that ESC-derived spinal motor neurons and cortical layer V pyramidal neurons acquire subtype specific functional properties. We discuss similarities and differences in the role of cell intrinsic transcriptional programs, extrinsic signals and cell-cell interactions during subtype diversification of the two classes of nerve cells. We conclude that the high degree of fidelity with which differentiating ESCs recapitulate normal embryonic development provides a unique opportunity to explore developmental processes underlying specification of mammalian neuronal diversity in a simplified and experimentally accessible system. PMID:20970319

  8. Lead decreases cell survival, proliferation, and neuronal differentiation of primary cultured adult neural precursor cells through activation of the JNK and p38 MAP kinases.

    PubMed

    Engstrom, Anna; Wang, Hao; Xia, Zhengui

    2015-08-01

    Adult hippocampal neurogenesis is the process whereby adult neural precursor cells (aNPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate adult-born, functional neurons in the hippocampus. This process is modulated by various extracellular and intracellular stimuli, and the adult-born neurons have been implicated in hippocampus-dependent learning and memory. However, studies on how neurotoxic agents affect this process and the underlying mechanisms are limited. The goal of this study was to determine whether lead, a heavy metal, directly impairs critical processes in adult neurogenesis and to characterize the underlying signaling pathways using primary cultured SGZ-aNPCs isolated from adult mice. We report here that lead significantly increases apoptosis and inhibits proliferation in SGZ-aNPCs. In addition, lead significantly impairs spontaneous neuronal differentiation and maturation. Furthermore, we found that activation of the c-Jun NH2-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase signaling pathways are important for lead cytotoxicity. Our data suggest that lead can directly act on adult neural stem cells and impair critical processes in adult hippocampal neurogenesis, which may contribute to its neurotoxicity and adverse effects on cognition in adults.

  9. Sexual Behavior Increases Cell Proliferation in the Rostral Migratory Stream and Promotes the Differentiation of the New Cells into Neurons in the Accessory Olfactory Bulb of Female Rats

    PubMed Central

    Corona, Rebeca; Retana-Márquez, Socorro; Portillo, Wendy; Paredes, Raúl G.

    2016-01-01

    We have previously demonstrated, that 15 days after female rats pace the sexual interaction, there is an increase in the number of new cells that reach the granular cell layer (GrL) of the accessory olfactory bulb (AOB). The aim of the present study was to evaluate, if the first sexual experience in the female rat increases cell proliferation in the subventricular zone (SVZ) and the rostral migratory stream (RMS). We also tested if this behavior promotes the survival of the new cells that integrate into the main olfactory bulb (MOB) and AOB 45 days after the behavioral test. Sexually, naive female rats were injected with the DNA synthesis marker 5′-bromo-2′-deoxyuridine (BrdU) on the day of the behavioral test. They were randomly divided into the following groups: Female rats placed alone in the mating cage (1); Females exposed to amyl acetate odor [banana scent, (2)]; Females that could see, hear, and smell the male but physical contact was not possible [exposed to male, (3)]; Female rats that could pace the sexual interaction (4); and females that mated without the possibility of pacing the sexual interaction (5). Animals were sacrificed 2 days after the behavioral test (proliferation) or 45 days later (survival). Our results show that 2 days after females were exposed to banana scent or to the male, they had a higher number of cells in the SVZ. Females, that mated in pace and no-paced conditions had more new cells in the RMS. At 45 days, no significant differences were found in the number of new cells that survived in the MOB or in the AOB. However, mating increased the percentage of new cells, that differentiated into neurons in the GrL of the AOB. These new cells expressed c-Fos after a second sexual encounter just before the females were sacrificed. No significant differences in plasma levels of estradiol and progesterone were observed between groups. Our results indicate that the first sexual experience increases cell proliferation in the RMS and mating

  10. Extremely Low-Frequency Electromagnetic Fields Promote In Vitro Neuronal Differentiation and Neurite Outgrowth of Embryonic Neural Stem Cells via Up-Regulating TRPC1

    PubMed Central

    Ma, Qinlong; Chen, Chunhai; Deng, Ping; Zhu, Gang; Lin, Min; Zhang, Lei; Xu, Shangcheng; He, Mindi; Lu, Yonghui; Duan, Weixia; Pi, Huifeng; Cao, Zhengwang; Pei, Liping; Li, Min; Liu, Chuan; Zhang, Yanwen; Zhong, Min; Zhou, Zhou; Yu, Zhengping

    2016-01-01

    Exposure to extremely low-frequency electromagnetic fields (ELF-EMFs) can enhance hippocampal neurogenesis in adult mice. However, little is focused on the effects of ELF-EMFs on embryonic neurogenesis. Here, we studied the potential effects of ELF-EMFs on embryonic neural stem cells (eNSCs). We exposed eNSCs to ELF-EMF (50 Hz, 1 mT) for 1, 2, and 3 days with 4 hours per day. We found that eNSC proliferation and maintenance were significantly enhanced after ELF-EMF exposure in proliferation medium. ELF-EMF exposure increased the ratio of differentiated neurons and promoted the neurite outgrowth of eNSC-derived neurons without influencing astrocyes differentiation and the cell apoptosis. In addition, the expression of the proneural genes, NeuroD and Ngn1, which are crucial for neuronal differentiation and neurite outgrowth, was increased after ELF-EMF exposure. Moreover, the expression of transient receptor potential canonical 1 (TRPC1) was significantly up-regulated accompanied by increased the peak amplitude of intracellular calcium level induced by ELF-EMF. Furthermore, silencing TRPC1 expression eliminated the up-regulation of the proneural genes and the promotion of neuronal differentiation and neurite outgrowth induced by ELF-EMF. These results suggest that ELF-EMF exposure promotes the neuronal differentiation and neurite outgrowth of eNSCs via up-regulation the expression of TRPC1 and proneural genes (NeuroD and Ngn1). These findings also provide new insights in understanding the effects of ELF-EMF exposure on embryonic brain development. PMID:26950212

  11. Extremely Low-Frequency Electromagnetic Fields Promote In Vitro Neuronal Differentiation and Neurite Outgrowth of Embryonic Neural Stem Cells via Up-Regulating TRPC1.

    PubMed

    Ma, Qinlong; Chen, Chunhai; Deng, Ping; Zhu, Gang; Lin, Min; Zhang, Lei; Xu, Shangcheng; He, Mindi; Lu, Yonghui; Duan, Weixia; Pi, Huifeng; Cao, Zhengwang; Pei, Liping; Li, Min; Liu, Chuan; Zhang, Yanwen; Zhong, Min; Zhou, Zhou; Yu, Zhengping

    2016-01-01

    Exposure to extremely low-frequency electromagnetic fields (ELF-EMFs) can enhance hippocampal neurogenesis in adult mice. However, little is focused on the effects of ELF-EMFs on embryonic neurogenesis. Here, we studied the potential effects of ELF-EMFs on embryonic neural stem cells (eNSCs). We exposed eNSCs to ELF-EMF (50 Hz, 1 mT) for 1, 2, and 3 days with 4 hours per day. We found that eNSC proliferation and maintenance were significantly enhanced after ELF-EMF exposure in proliferation medium. ELF-EMF exposure increased the ratio of differentiated neurons and promoted the neurite outgrowth of eNSC-derived neurons without influencing astrocyes differentiation and the cell apoptosis. In addition, the expression of the proneural genes, NeuroD and Ngn1, which are crucial for neuronal differentiation and neurite outgrowth, was increased after ELF-EMF exposure. Moreover, the expression of transient receptor potential canonical 1 (TRPC1) was significantly up-regulated accompanied by increased the peak amplitude of intracellular calcium level induced by ELF-EMF. Furthermore, silencing TRPC1 expression eliminated the up-regulation of the proneural genes and the promotion of neuronal differentiation and neurite outgrowth induced by ELF-EMF. These results suggest that ELF-EMF exposure promotes the neuronal differentiation and neurite outgrowth of eNSCs via up-regulation the expression of TRPC1 and proneural genes (NeuroD and Ngn1). These findings also provide new insights in understanding the effects of ELF-EMF exposure on embryonic brain development.

  12. Differentiation of SH-SY5Y cells to a neuronal phenotype changes cellular bioenergetics and the response to oxidative stress.

    PubMed

    Schneider, Lonnie; Giordano, Samantha; Zelickson, Blake R; S Johnson, Michelle; A Benavides, Gloria; Ouyang, Xiaosen; Fineberg, Naomi; Darley-Usmar, Victor M; Zhang, Jianhua

    2011-12-01

    differentiation of neuroblastoma cells to a neuron-like phenotype. PMID:21945098

  13. Differentiation of Rat Bone Marrow Mesenchymal Stem Cells Into Neuron-Like Cells In Vitro and Co-Cultured with Biological Scaffold as Transplantation Carrier.

    PubMed

    Yue, Wei; Yan, Feng; Zhang, Yue-Lin; Liu, Shu-Ling; Hou, Shu-Ping; Mao, Guo-Chao; Liu, Ning; Ji, Yong

    2016-01-01

    BACKGROUND Autograft and allograft transplantation are used to prompt the regeneration of axons after nerve injury. However, the poor self-regeneration caused by the glial scar and growth inhibitory factors after neuronal necrosis limit the efficacy of these methods. The purpose of this study was to develop a new chitosan porous scaffold for cell seeding. MATERIAL AND METHODS The bone marrow mesenchymal stem cells (BMSCs) and tissue-engineered biomaterial scaffold compound were constructed and co-cultured in vitro with the differentiated BMSCs of Wistar rats and chitosan scaffold in a 3D environment. The purity of the third-generation BMSCs culture was identified using flow cytometry and assessment of induced neuronal differentiation. The scaffolds were prepared by the freeze-drying method. The internal structure of scaffolds and the change of cells' growth and morphology were observed under a scanning electron microscope. The proliferation of cells was detected with the MTT method. RESULTS On day 5 there was a significant difference in the absorbance value of the experimental group (0.549±0.0256) and the control group (0.487±0.0357) (P>0.05); but on day 7 there was no significant difference in the proliferation of the experimental group (0.751±0.011) and the control group and (0.78±0.017) (P>0.05). CONCLUSIONS Tissue engineering technology can provide a carrier for cells seeding and is expected to become an effective method for the regeneration and repair of nerve cells. Our study showed that chitosan porous scaffolds can be used for such purposes. PMID:27225035

  14. Marked change in microRNA expression during neuronal differentiation of human teratocarcinoma NTera2D1 and mouse embryonal carcinoma P19 cells

    SciTech Connect

    Hohjoh, Hirohiko Fukushima, Tatsunobu

    2007-10-19

    MicroRNAs (miRNAs) are small noncoding RNAs, with a length of 19-23 nucleotides, which appear to be involved in the regulation of gene expression by inhibiting the translation of messenger RNAs carrying partially or nearly complementary sequences to the miRNAs in their 3' untranslated regions. Expression analysis of miRNAs is necessary to understand their complex role in the regulation of gene expression during the development, differentiation and proliferation of cells. Here we report on the expression profile analysis of miRNAs in human teratocarcinoma NTere2D1, mouse embryonic carcinoma P19, mouse neuroblastoma Neuro2a and rat pheochromocytoma PC12D cells, which can be induced into differentiated cells with long neuritic processes, i.e., after cell differentiation, such that the resultant cells look similar to neuronal cells. The data presented here indicate marked changes in the expression of miRNAs, as well as genes related to neuronal development, occurred in the differentiation of NTera2D1 and P19 cells. Significant changes in miRNA expression were not observed in Neuro2a and PC12D cells, although they showed apparent morphologic change between undifferentiated and differentiated cells. Of the miRNAs investigated, the expression of miRNAs belonging to the miR-302 cluster, which is known to be specifically expressed in embryonic stem cells, and of miR-124a specific to the brain, appeared to be markedly changed. The miR-302 cluster was potently expressed in undifferentiated NTera2D1 and P19 cells, but hardly in differentiated cells, such that miR-124a showed an opposite expression pattern to the miR-302 cluster. Based on these observations, it is suggested that the miR-302 cluster and miR-124a may be useful molecular indicators in the assessment of degree of undifferentiation and/or differentiation in the course of neuronal differentiation.

  15. Differentiation of Rat Bone Marrow Mesenchymal Stem Cells Into Neuron-Like Cells In Vitro and Co-Cultured with Biological Scaffold as Transplantation Carrier

    PubMed Central

    Yue, Wei; Yan, Feng; Zhang, Yue-Lin; Liu, Shu-Ling; Hou, Shu-Ping; Mao, Guo-Chao; Liu, Ning; Ji, Yong

    2016-01-01

    Background Autograft and allograft transplantation are used to prompt the regeneration of axons after nerve injury. However, the poor self-regeneration caused by the glial scar and growth inhibitory factors after neuronal necrosis limit the efficacy of these methods. The purpose of this study was to develop a new chitosan porous scaffold for cell seeding. Material/Methods The bone marrow mesenchymal stem cells (BMSCs) and tissue-engineered biomaterial scaffold compound were constructed and co-cultured in vitro with the differentiated BMSCs of Wistar rats and chitosan scaffold in a 3D environment. The purity of the third-generation BMSCs culture was identified using flow cytometry and assessment of induced neuronal differentiation. The scaffolds were prepared by the freeze-drying method. The internal structure of scaffolds and the change of cells’ growth and morphology were observed under a scanning electron microscope. The proliferation of cells was detected with the MTT method. Results On day 5 there was a significant difference in the absorbance value of the experimental group (0.549±0.0256) and the control group (0.487±0.0357) (P>0.05); but on day 7 there was no significant difference in the proliferation of the experimental group (0.751±0.011) and the control group and (0.78±0.017) (P>0.05). Conclusions Tissue engineering technology can provide a carrier for cells seeding and is expected to become an effective method for the regeneration and repair of nerve cells. Our study showed that chitosan porous scaffolds can be used for such purposes. PMID:27225035

  16. Dopaminergic neurons differentiating from LRRK2 G2019S induced pluripotent stem cells show early neuritic branching defects

    PubMed Central

    Borgs, Laurence; Peyre, Elise; Alix, Philippe; Hanon, Kevin; Grobarczyk, Benjamin; Godin, Juliette D.; Purnelle, Audrey; Krusy, Nathalie; Maquet, Pierre; Lefebvre, Philippe; Seutin, Vincent; Malgrange, Brigitte; Nguyen, Laurent

    2016-01-01

    Some mutations of the LRRK2 gene underlie autosomal dominant form of Parkinson’s disease (PD). The G2019S is a common mutation that accounts for about 2% of PD cases. To understand the pathophysiology of this mutation and its possible developmental implications, we developed an in vitro assay to model PD with human induced pluripotent stem cells (hiPSCs) reprogrammed from skin fibroblasts of PD patients suffering from the LRKK2 G2019S mutation. We differentiated the hiPSCs into neural stem cells (NSCs) and further into dopaminergic neurons. Here we show that NSCs bearing the mutation tend to differentiate less efficiently into dopaminergic neurons and that the latter exhibit significant branching defects as compared to their controls. PMID:27640816

  17. Bcl-2 accelerates the neuronal differentiation: new evidence approaching to the biofunction of bcl-2 in the neuronal system.

    PubMed

    Suzuki, A; Tsutomi, Y

    1998-08-10

    The proto-oncogene product Bcl-2 is unique in that it inhibits apoptosis rather than promoting cell proliferation. In the present study, we encountered a new possible role of Bcl-2 in the neuronal differentiation. Rat pheochromocytoma PC12 cells have been known as the model of neuronal differentiation by the stimulation of NGF. Bcl-2 transfected PC12 (MB2) cells showed the accelerated neuronal differentiation, as compared with control PC12 (V4) cells. In addition, chemotherapeutic agents Taxol which has been known as neurotoxic compound, induced the acute neuronal cell atrophy and suppressed neuronal differentiation. This neuronal cell atrophy and suppression of neuronal differentiation were not due to apoptotic cell death. Interestingly, Bcl-2 rescued PC12 cells from both neuronal cell atrophy and suppression of neuronal differentiation. Taxol suppressed polymerization between neurofilament light and heavy (NF-L and NF-H), and MB2 cell extract rescued it. We, therefore, suggest the acceleration of polymerization between NF-L and NF-H as the new possible role of Bcl-2.

  18. Negative regulation of Yap during neuronal differentiation

    PubMed Central

    Zhang, Huanqing; Deo, Monika; Thompson, Robert C.; Uhler, Michael D.; Turner, David L.

    2011-01-01

    Regulated proliferation and cell cycle exit are essential aspects of neurogenesis. The Yap transcriptional coactivator controls proliferation in a variety of tissues during development, and this activity is negatively regulated by kinases in the Hippo signaling pathway. We find that Yap is expressed in mitotic mouse retinal progenitors and it is downregulated during neuronal differentiation. Forced expression of Yap prolongs proliferation in the postnatal mouse retina, whereas inhibition of Yap by RNA interference (RNAi) decreases proliferation and increases differentiation. We show Yap is subject to post-translational inhibition in the retina, and also downregulated at the level of mRNA expression. Using a cell culture model, we find that expression of the proneural basic helix-loop-helix (bHLH) transcription factors Neurog2 or Ascl1 downregulates Yap mRNA levels, and simultaneously inhibits Yap protein via activation of the Lats1 and/or Lats2 kinases. Conversely, overexpression of Yap prevents proneural bHLH proteins from initiating cell cycle exit. We propose that mutual inhibition between proneural bHLH proteins and Yap is an important regulator of proliferation and cell cycle exit during mammalian neurogenesis. PMID:22037235

  19. Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation

    PubMed Central

    Bergström, Petra; Agholme, Lotta; Nazir, Faisal Hayat; Satir, Tugce Munise; Toombs, Jamie; Wellington, Henrietta; Strandberg, Joakim; Bontell, Thomas Olsson; Kvartsberg, Hlin; Holmström, Maria; Boreström, Cecilia; Simonsson, Stina; Kunath, Tilo; Lindahl, Anders; Blennow, Kaj; Hanse, Eric; Portelius, Erik; Wray, Selina; Zetterberg, Henrik

    2016-01-01

    Amyloid precursor protein (APP) and its cleavage product amyloid β (Aβ) have been thoroughly studied in Alzheimer’s disease. However, APP also appears to be important for neuronal development. Differentiation of induced pluripotent stem cells (iPSCs) towards cortical neurons enables in vitro mechanistic studies on human neuronal development. Here, we investigated expression and proteolytic processing of APP during differentiation of human iPSCs towards cortical neurons over a 100-day period. APP expression remained stable during neuronal differentiation, whereas APP processing changed. α-Cleaved soluble APP (sAPPα) was secreted early during differentiation, from neuronal progenitors, while β-cleaved soluble APP (sAPPβ) was first secreted after deep-layer neurons had formed. Short Aβ peptides, including Aβ1-15/16, peaked during the progenitor stage, while processing shifted towards longer peptides, such as Aβ1-40/42, when post-mitotic neurons appeared. This indicates that APP processing is regulated throughout differentiation of cortical neurons and that amyloidogenic APP processing, as reflected by Aβ1-40/42, is associated with mature neuronal phenotypes. PMID:27383650

  20. The Relationship between the Bcl-2/Bax Proteins and the Mitochondria-Mediated Apoptosis Pathway in the Differentiation of Adipose-Derived Stromal Cells into Neurons

    PubMed Central

    Wang, Quanquan; Zhang, Lili; Yuan, Xiaodong; Ou, Ya; Zhu, Xuhong; Cheng, Zanzan; Zhang, Pingshu; Wu, Xiaoying; Meng, Yan; Zhang, Liping

    2016-01-01

    Our objective is to study the relationship between the regulatory proteins Bcl-2/Bax and mitochondria-mediated apoptosis during the differentiation of adipose-derived stromal cells (ADSCs) into neurons. Immunocytochemistry and western blotting showed that the cells weakly expressed neuron-specific enolase (NSE) in the non-induced group and expressed NSE more strongly in the groups induced for 1 h, 3 h, 5 h and 8 h. NSE expression peaked at 5 h (P < 0.05), although there was no significant difference between 5 and 8 h (P > 0.05). Bcl-2 expression gradually decreased over time in the non-induced group (P < 0.05). However, Bax, caspase-9, Cyt-c and caspase-3 expression gradually increased and peaked at 8 h (P < 0.05). Transmission electron microscopy revealed karyopyknosis, chromatin edge setting, mitochondria swelling and cavitation in cells at 5 h, and the mitochondrial membrane potential decreased over time, as demonstrated by laser scanning confocal microscopy. After a 5 h induction, cells differentiated into typical neurons and expressed Bcl-2, which inhibited apoptosis. Bax showed a strong apoptosis-promoting capacity, leading to changes in the mitochondrial membrane potential and structure, and then triggered the caspase-independent apoptotic response through the mitochondrial pathway. At the same time, Cyt-c was directly or indirectly released from the mitochondria to the cytoplasm to trigger the caspase-dependent apoptotic response through the mitochondrial pathway. Therefore, Bcl-2/Bax play an important role in regulating caspase-dependent and caspase-independent apoptosis mediated by the mitochondrial pathway during the differentiation of ADSCs into neurons. PMID:27706181

  1. Ethanol Diverts Early Neuronal Differentiation Trajectory of Embryonic Stem Cells by Disrupting the Balance of Lineage Specifiers

    PubMed Central

    Sánchez-Alvarez, Rosa; Gayen, Saurabh; Vadigepalli, Rajanikanth; Anni, Helen

    2013-01-01

    Background Ethanol is a toxin responsible for the neurodevelopmental deficits of Fetal Alcohol Spectrum Disorders (FASD). Recent evidence suggests that ethanol modulates the protein expression of lineage specifier transcription factors Oct4 (Pou5f1) and Sox2 in early stages of mouse embryonic stem (ES) cell differentiation. We hypothesized that ethanol induced an imbalance in the expression of Oct4 and Sox2 in early differentiation, that dysregulated the expression of associated and target genes and signaling molecules and diverted cells from neuroectodermal (NE) formation. Methodology/Principal Findings We showed modulation by ethanol of 33 genes during ES cell differentiation, using high throughput microfluidic dynamic array chips measuring 2,304 real time quantitative PCR assays. Based on the overall gene expression dynamics, ethanol drove cells along a differentiation trajectory away from NE fate. These ethanol-induced gene expression changes were observed as early as within 2 days of differentiation, and were independent of cell proliferation or apoptosis. Gene expression changes were correlated with fewer βIII-tubulin positive cells of an immature neural progenitor phenotype, as well as a disrupted actin cytoskeleton were observed. Moreover, Tuba1a and Gapdh housekeeping genes were modulated by ethanol during differentiation and were replaced by a set of ribosomal genes with stable expression. Conclusions/Significance These findings provided an ethanol-response gene signature and pointed to the transcriptional dynamics underlying lineage imbalance that may be relevant to FASD phenotype. PMID:23724002

  2. RAGE Expression and ROS Generation in Neurons: Differentiation versus Damage.

    PubMed

    Piras, S; Furfaro, A L; Domenicotti, C; Traverso, N; Marinari, U M; Pronzato, M A; Nitti, M

    2016-01-01

    RAGE is a multiligand receptor able to bind advanced glycation end-products (AGEs), amphoterin, calgranulins, and amyloid-beta peptides, identified in many tissues and cells, including neurons. RAGE stimulation induces the generation of reactive oxygen species (ROS) mainly through the activity of NADPH oxidases. In neuronal cells, RAGE-induced ROS generation is able to favor cell survival and differentiation or to induce death through the imbalance of redox state. The dual nature of RAGE signaling in neurons depends not only on the intensity of RAGE activation but also on the ability of RAGE-bearing cells to adapt to ROS generation. In this review we highlight these aspects of RAGE signaling regulation in neuronal cells.

  3. RAGE Expression and ROS Generation in Neurons: Differentiation versus Damage

    PubMed Central

    Piras, S.; Furfaro, A. L.; Domenicotti, C.; Traverso, N.; Marinari, U. M.; Pronzato, M. A.; Nitti, M.

    2016-01-01

    RAGE is a multiligand receptor able to bind advanced glycation end-products (AGEs), amphoterin, calgranulins, and amyloid-beta peptides, identified in many tissues and cells, including neurons. RAGE stimulation induces the generation of reactive oxygen species (ROS) mainly through the activity of NADPH oxidases. In neuronal cells, RAGE-induced ROS generation is able to favor cell survival and differentiation or to induce death through the imbalance of redox state. The dual nature of RAGE signaling in neurons depends not only on the intensity of RAGE activation but also on the ability of RAGE-bearing cells to adapt to ROS generation. In this review we highlight these aspects of RAGE signaling regulation in neuronal cells. PMID:27313835

  4. Modulation of neuronal differentiation by CD40 isoforms

    SciTech Connect

    Hou Huayu; Obregon, Demian; Lou, Deyan; Ehrhart, Jared; Fernandez, Frank; Silver, Archie; Tan Jun

    2008-05-02

    Neuron differentiation is a complex process involving various cell-cell interactions, and multiple signaling pathways. We showed previously that CD40 is expressed and functional on mouse and human neurons. In neurons, ligation of CD40 protects against serum withdrawal-induced injury and plays a role in survival and differentiation. CD40 deficient mice display neuron dysfunction, aberrant neuron morphologic changes, and associated gross brain abnormalities. Previous studies by Tone and colleagues suggested that five isoforms of CD40 exist with two predominant isoforms expressed in humans: signal-transducible CD40 type I and a C-terminal truncated, non-signal-transducible CD40 type II. We hypothesized that differential expression of CD40 isoform type I and type II in neurons may modulate neuron differentiation. Results show that adult wild-type, and CD40{sup -/-} deficient mice predominantly express CD40 type I and II isoforms. Whereas adult wild-type mice express mostly CD40 type I in cerebral tissues at relatively high levels, in age and gender-matched CD40{sup -/-} mice CD40 type I expression was almost completely absent; suggesting a predominance of the non-signal-transducible CD40 type II isoform. Younger, 1 day old wild-type mice displayed less CD40 type I, and more CD40 type II, as well as, greater expression of soluble CD40 (CD40L/CD40 signal inhibitor), compared with 1 month old mice. Neuron-like N2a cells express CD40 type I and type II isoforms while in an undifferentiated state, however once induced to differentiate, CD40 type I predominates. Further, differentiated N2a cells treated with CD40 ligand express high levels of neuron specific nuclear protein (NeuN); an effect reduced by anti-CD40 type I siRNA, but not by control (non-targeting) siRNA. Altogether these data suggest that CD40 isoforms may act in a temporal fashion to modulate neuron differentiation during brain development. Thus, modulation of neuronal CD40 isoforms and CD40 signaling may

  5. Nucleosome assembly proteins and their interacting proteins in neuronal differentiation.

    PubMed

    Attia, Mikaël; Rachez, Christophe; Avner, Philip; Rogner, Ute Christine

    2013-06-01

    Neuronal differentiation from neural stem cells into mature neurons is guided by the concerted action of specific transcription factors that stepwise exercise their role in the context of defined chromatin states. Amongst the classes of proteins that influence chromatin compaction and modification are nucleosome assembly proteins (NAPs). Mammals possess several nucleosome assembly protein 1 like proteins (NAP1L) that show either ubiquitous or neuron-restricted expression. The latter group is presumably involved in the process of neuronal differentiation. Mammalian NAP1Ls can potentially form both homo- and hetero-dimers and octamers, in theory allowing thousands of different combinations to be formed. Detailed studies have been performed on several of the NAP1Ls that point to a range of molecular roles, including transcriptional regulation, nuclear import, and control of cell division. This article aims at summarizing current knowledge of the mammalian NAP1L family and its interactions.

  6. Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy

    PubMed Central

    2011-01-01

    Background Even in the presence of oxygen, malignant cells often highly depend on glycolysis for energy generation, a phenomenon known as the Warburg effect. One strategy targeting this metabolic phenotype is glucose restriction by administration of a high-fat, low-carbohydrate (ketogenic) diet. Under these conditions, ketone bodies are generated serving as an important energy source at least for non-transformed cells. Methods To investigate whether a ketogenic diet might selectively impair energy metabolism in tumor cells, we characterized in vitro effects of the principle ketone body 3-hydroxybutyrate in rat hippocampal neurons and five glioma cell lines. In vivo, a non-calorie-restricted ketogenic diet was examined in an orthotopic xenograft glioma mouse model. Results The ketone body metabolizing enzymes 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and 2), 3-oxoacid-CoA transferase 1 (OXCT1) and acetyl-CoA acetyltransferase 1 (ACAT1) were expressed at the mRNA and protein level in all glioma cell lines. However, no activation of the hypoxia-inducible factor-1α (HIF-1α) pathway was observed in glioma cells, consistent with the absence of substantial 3-hydroxybutyrate metabolism and subsequent accumulation of succinate. Further, 3-hydroxybutyrate rescued hippocampal neurons from glucose withdrawal-induced cell death but did not protect glioma cell lines. In hypoxia, mRNA expression of OXCT1, ACAT1, BDH1 and 2 was downregulated. In vivo, the ketogenic diet led to a robust increase of blood 3-hydroxybutyrate, but did not alter blood glucose levels or improve survival. Conclusion In summary, glioma cells are incapable of compensating for glucose restriction by metabolizing ketone bodies in vitro, suggesting a potential disadvantage of tumor cells compared to normal cells under a carbohydrate-restricted ketogenic diet. Further investigations are necessary to identify co-treatment modalities, e.g. glycolysis inhibitors or antiangiogenic agents that efficiently

  7. Neural differentiation of human embryonic stem cells as an in vitro tool for the study of the expression patterns of the neuronal cytoskeleton during neurogenesis.

    PubMed

    Liu, Chao; Zhong, Yongwang; Apostolou, Andria; Fang, Shengyun

    2013-09-13

    The neural differentiation of human embryonic stem cells (ESCs) is a potential tool for elucidating the key mechanisms involved in human neurogenesis. Nestin and β-III-tubulin, which are cytoskeleton proteins, are marker proteins of neural stem cells (NSCs) and neurons, respectively. However, the expression patterns of nestin and β-III-tubulin in neural derivatives from human ESCs remain unclear. In this study, we found that neural progenitor cells (NPCs) derived from H9 cells express high levels of nestin and musashi-1. In contrast, β-III-tubulin was weakly expressed in a few NPCs. Moreover, in these cells, nestin formed filament networks, whereas β-III-tubulin was distributed randomly as small particles. As the differentiation proceeded, the nestin filament networks and the β-III-tubulin particles were found in both the cell soma and the cellular processes. Moreover, the colocalization of nestin and β-III-tubulin was found mainly in the cell processes and neurite-like structures and not in the cell soma. These results may aid our understanding of the expression patterns of nestin and β-III-tubulin during the neural differentiation of H9 cells.

  8. Interactome analysis of the EV71 5' untranslated region in differentiated neuronal cells SH-SY5Y and regulatory role of FBP3 in viral replication.

    PubMed

    Huang, Hsing-I; Chang, Ying-Ying; Lin, Jhao-Yin; Kuo, Rei-Lin; Liu, Hao-Ping; Shih, Shin-Ru; Wu, Chih-Ching

    2016-09-01

    Enterovirus 71 (EV71), a single-stranded RNA virus, is one of the most serious neurotropic pathogens in the Asia-Pacific region. Through interactions with host proteins, the 5' untranslated region (5'UTR) of EV71 is important for viral replication. To gain a protein profile that interact with the EV71 5'UTR in neuronal cells, we performed a biotinylated RNA-protein pull-down assay in conjunction with LC-MS/MS analysis. A total of 109 proteins were detected and subjected to Database for Annotation, Visualization and Integrated Discovery (DAVID) analyses. These proteins were found to be highly correlated with biological processes including RNA processing/splicing, epidermal cell differentiation, and protein folding. A protein-protein interaction network was constructed using the STRING online database to illustrate the interactions of those proteins that are mainly involved in RNA processing/splicing or protein folding. Moreover, we confirmed that the far-upstream element binding protein 3 (FBP3) was able to bind to the EV71 5'UTR. The redistribution of FBP3 in subcellular compartments was observed after EV71 infection, and the decreased expression of FBP3 in host neuronal cells markedly inhibited viral replication. Our results reveal various host proteins that potentially interact with the EV71 5'UTR in neuronal cells, and we found that FBP3 could serve as a positive regulator in host cells. PMID:27291656

  9. Interactome analysis of the EV71 5' untranslated region in differentiated neuronal cells SH-SY5Y and regulatory role of FBP3 in viral replication.

    PubMed

    Huang, Hsing-I; Chang, Ying-Ying; Lin, Jhao-Yin; Kuo, Rei-Lin; Liu, Hao-Ping; Shih, Shin-Ru; Wu, Chih-Ching

    2016-09-01

    Enterovirus 71 (EV71), a single-stranded RNA virus, is one of the most serious neurotropic pathogens in the Asia-Pacific region. Through interactions with host proteins, the 5' untranslated region (5'UTR) of EV71 is important for viral replication. To gain a protein profile that interact with the EV71 5'UTR in neuronal cells, we performed a biotinylated RNA-protein pull-down assay in conjunction with LC-MS/MS analysis. A total of 109 proteins were detected and subjected to Database for Annotation, Visualization and Integrated Discovery (DAVID) analyses. These proteins were found to be highly correlated with biological processes including RNA processing/splicing, epidermal cell differentiation, and protein folding. A protein-protein interaction network was constructed using the STRING online database to illustrate the interactions of those proteins that are mainly involved in RNA processing/splicing or protein folding. Moreover, we confirmed that the far-upstream element binding protein 3 (FBP3) was able to bind to the EV71 5'UTR. The redistribution of FBP3 in subcellular compartments was observed after EV71 infection, and the decreased expression of FBP3 in host neuronal cells markedly inhibited viral replication. Our results reveal various host proteins that potentially interact with the EV71 5'UTR in neuronal cells, and we found that FBP3 could serve as a positive regulator in host cells.

  10. Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation

    PubMed Central

    Zheng, Xinde; Boyer, Leah; Jin, Mingji; Mertens, Jerome; Kim, Yongsung; Ma, Li; Ma, Li; Hamm, Michael; Gage, Fred H; Hunter, Tony

    2016-01-01

    How metabolism is reprogrammed during neuronal differentiation is unknown. We found that the loss of hexokinase (HK2) and lactate dehydrogenase (LDHA) expression, together with a switch in pyruvate kinase gene splicing from PKM2 to PKM1, marks the transition from aerobic glycolysis in neural progenitor cells (NPC) to neuronal oxidative phosphorylation. The protein levels of c-MYC and N-MYC, transcriptional activators of the HK2 and LDHA genes, decrease dramatically. Constitutive expression of HK2 and LDHA during differentiation leads to neuronal cell death, indicating that the shut-off aerobic glycolysis is essential for neuronal survival. The metabolic regulators PGC-1α and ERRγ increase significantly upon neuronal differentiation to sustain the transcription of metabolic and mitochondrial genes, whose levels are unchanged compared to NPCs, revealing distinct transcriptional regulation of metabolic genes in the proliferation and post-mitotic differentiation states. Mitochondrial mass increases proportionally with neuronal mass growth, indicating an unknown mechanism linking mitochondrial biogenesis to cell size. DOI: http://dx.doi.org/10.7554/eLife.13374.001 PMID:27282387

  11. Inhibitory effects of multiwall carbon nanotubes with high iron impurity on viability and neuronal differentiation in cultured PC12 cells.

    PubMed

    Meng, Li; Jiang, Aihua; Chen, Rui; Li, Chen-zhong; Wang, Liming; Qu, Ying; Wang, Peng; Zhao, Yuliang; Chen, Chunying

    2013-11-01

    The increasing use of carbon nanotubes (CNTs) in biomedical applications has garnered a great concern on their potential negative effects to human health. CNTs have been reported to potentially disrupt normal neuronal function and they were speculated to accumulate and cause brain damage, although a lot of distinct and exceptional properties and potential wide applications have been associated with this material in neurobiology. Fe impurities strapped inside the CNTs may be partially responsible for neurotoxicity generation. In the present study, we selected rat pheochromocytoma (PC12) cells to investigate and compare the effects of two kinds of multiwall carbon nanotubes (MWCNTs) with different concentrations of Fe impurities which usually come from the massive production of CNTs by chemical vapor deposition. Exposure to Fe-high MWCNTs can reduce cell viability and increase cytoskeletal disruption of undifferentiated PC12 cells, diminish the ability to form mature neurites, and then adversely influence the neuronal dopaminergic phenotype in NGF-treated PC-12 cells. The present results highlight the critical role of iron residue in the adverse response to MWCNTs exposure in neural cells. These findings provide useful information for understanding the toxicity and safe application of carbon nanotubes.

  12. Alternative Splicing of G9a Regulates Neuronal Differentiation.

    PubMed

    Fiszbein, Ana; Giono, Luciana E; Quaglino, Ana; Berardino, Bruno G; Sigaut, Lorena; von Bilderling, Catalina; Schor, Ignacio E; Steinberg, Juliana H Enriqué; Rossi, Mario; Pietrasanta, Lía I; Caramelo, Julio J; Srebrow, Anabella; Kornblihtt, Alberto R

    2016-03-29

    Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10) through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain. Although E10 inclusion greatly stimulates overall H3K9me2 levels, it does not affect G9a catalytic activity. Instead, E10 increases G9a nuclear localization. We show that the G9a E10(+) isoform is necessary for neuron differentiation and regulates the alternative splicing pattern of its own pre-mRNA, enhancing E10 inclusion. Overall, our findings indicate that by regulating its own alternative splicing, G9a promotes neuron differentiation and creates a positive feedback loop that reinforces cellular commitment to differentiation.

  13. hESC Differentiation toward an Autonomic Neuronal Cell Fate Depends on Distinct Cues from the Co-Patterning Vasculature

    PubMed Central

    Acevedo, Lisette M.; Lindquist, Jeffrey N.; Walsh, Breda M.; Sia, Peik; Cimadamore, Flavio; Chen, Connie; Denzel, Martin; Pernia, Cameron D.; Ranscht, Barbara; Terskikh, Alexey; Snyder, Evan Y.; Cheresh, David A.

    2015-01-01

    Summary To gain insight into the cellular and molecular cues that promote neurovascular co-patterning at the earliest stages of human embryogenesis, we developed a human embryonic stem cell model to mimic the developing epiblast. Contact of ectoderm-derived neural cells with mesoderm-derived vasculature is initiated via the neural crest (NC), not the neural tube (NT). Neurovascular co-patterning then ensues with specification of NC toward an autonomic fate requiring vascular endothelial cell (EC)-secreted nitric oxide (NO) and direct contact with vascular smooth muscle cells (VSMCs) via T-cadherin-mediated homotypic interactions. Once a neurovascular template has been established, NT-derived central neurons then align themselves with the vasculature. Our findings reveal that, in early human development, the autonomic nervous system forms in response to distinct molecular cues from VSMCs and ECs, providing a model for how other developing lineages might coordinate their co-patterning. PMID:26004631

  14. Human dental pulp stem cells respond to cues from the rat retina and differentiate to express the retinal neuronal marker rhodopsin.

    PubMed

    Bray, A F; Cevallos, R R; Gazarian, K; Lamas, M

    2014-11-01

    Human adult dental pulp stem cells (DPSCs) are self-renewing stem cells that originate from the neural crest during development and remain within the dental pulp niche through adulthood. Due to their multi-lineage differentiation potential and their relative ease of access they represent an exciting alternative for autologous stem cell-based therapies in neurodegenerative diseases. In animal models, DPSCs transplanted into the brain differentiate into functional neurons or astrocytes in response to local environmental cues that appear to influence the fate of the surviving cells. Here we tested the hypothesis that DPSCs might be able to respond to factors present in the retina enabling the regenerative potential of these cells. We evaluated the response of DPSCs to conditioned media from organotypic explants from control and chemically damaged rat retinas. To evaluate cell differentiation, we analyzed the expression of glial fibrillary acidic protein (GFAP), early neuronal and retinal markers (polysialic acid-neural cell adhesion molecule (PSA-NCAM); Pax6; Ascl1; NeuroD1) and the late photoreceptor marker rhodopsin, by immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR). Exposure of DPSC cultures to conditioned media from control retinas induced a 39% reduction on the number of DPSCs that expressed GFAP; the expression of Pax6, Ascl1, PSA-NCAM or NeuroD1 was undetectable or did not change significantly. Expression of rhodopsin was not detectable in control or after exposure of the cultures with retinal conditioned media. By contrast, 44% of DPSCs exposed to conditioned media from damaged retinas were immunopositive to this protein. This response could not be reproduced when conditioned media from Müller-enriched primary cultures was used. Finally, quantitative RT-PCR was performed to compare the relative expression of glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF) and brain

  15. Can the 'neuron theory' be complemented by a universal mechanism for generic neuronal differentiation.

    PubMed

    Ernsberger, Uwe

    2015-01-01

    With the establishment of the 'neuron theory' at the turn of the twentieth century, this remarkably powerful term was introduced to name a breathtaking diversity of cells unified by a characteristic structural compartmentalization and unique information processing and propagating features. At the beginning of the twenty-first century, developmental, stem cell and reprogramming studies converged to suggest a common mechanism involved in the generation of possibly all vertebrate, and at least a significant number of invertebrate, neurons. Sox and, in particular, SoxB and SoxC proteins as well as basic helix-loop-helix proteins play major roles, even though their precise contributions to progenitor programming, proliferation and differentiation are not fully resolved. In addition to neuronal development, these transcription factors also regulate sensory receptor and endocrine cell development, thus specifying a range of cells with regulatory and communicative functions. To what extent microRNAs contribute to the diversification of these cell types is an upcoming question. Understanding the transcriptional and post-transcriptional regulation of genes coding for cell type-specific cytoskeletal and motor proteins as well as synaptic and ion channel proteins, which mark differences but also similarities between the three communicator cell types, will provide a key to the comprehension of their diversification and the signature of 'generic neuronal' differentiation. Apart from the general scientific significance of a putative universal core instruction for neuronal development, the impact of this line of research for cell replacement therapy and brain tumor treatment will be of considerable interest.

  16. METHYLMERCURY IMPAIRS NEURONAL DIFFERENTIATION BY ALTERING NEUROTROPHIN SIGNALING.

    EPA Science Inventory

    In previous in vivo studies, we observed that developmental exposure to CH3Hg can alter neocortical morphology and neurotrophin signaling. Using primed PC12 cells as a model system for neuronal differentiation, we examined the hypothesis that the developmental effects of CH3Hg ma...

  17. Neogenin, an avian cell surface protein expressed during terminal neuronal differentiation, is closely related to the human tumor suppressor molecule deleted in colorectal cancer

    PubMed Central

    1994-01-01

    Using a monoclonal antibody, we have identified and characterized a previously unknown cell surface protein in chicken that we call neogenin and have determined its primary sequence. The deduced amino acid sequence and structure of neogenin characterize it as a member of the immunoglobulin (Ig) superfamily. Based on amino acid sequence similarities, neogenin is closely related to the human tumor suppressor molecule DCC (deleted in colorectal cancer). Neogenin and DCC define a subgroup of Ig superfamily proteins structurally distinct from other Ig molecules such as N-CAM, Ng-CAM, and Bravo/Nr-CAM. As revealed by antibody staining of tissue sections and Western blots, neogenin expression correlates with the onset of neuronal differentiation. Neogenin is also found on cells in the lower gastrointestinal tract of embryonic chickens. DCC has been observed in human neural tissues and has been shown to be essential for terminal differentiation of specific cell types in the adult human colon. These parallels suggest that neogenin, like DCC, is functionally involved in the transition from cell proliferation to terminal differentiation of specific cell types. Since neogenin is expressed on growing neurites and downregulated at termination of neurite growth, it may also play an important role in many of the complex functional aspects of neurite extension and intercellular signaling. PMID:7806578

  18. Perfluorooctane sulfonate induces neuronal and oligodendrocytic differentiation in neural stem cells and alters the expression of PPARγ in vitro and in vivo

    SciTech Connect

    Wan Ibrahim, Wan Norhamidah; Tofighi, Roshan; Onishchenko, Natalia; Rebellato, Paola; Bose, Raj; Uhlén, Per; Ceccatelli, Sandra

    2013-05-15

    Perfluorinated compounds are ubiquitous chemicals of major concern for their potential adverse effects on the human population. We have used primary rat embryonic neural stem cells (NSCs) to study the effects of perfluorooctane sulfonate (PFOS) on the process of NSC spontaneous differentiation. Upon removal of basic fibroblast growth factor, NSCs were exposed to nanomolar concentrations of PFOS for 48 h, and then allowed to differentiate for additional 5 days. Exposure to 25 or 50 nM concentration resulted in a lower number of proliferating cells and a higher number of neurite-bearing TuJ1-positive cells, indicating an increase in neuronal differentiation. Exposure to 50 nM also significantly increased the number of CNPase-positive cells, pointing to facilitation of oligodendrocytic differentiation. PPAR genes have been shown to be involved in PFOS toxicity. By q-PCR we detected an upregulation of PPARγ with no changes in PPARα or PPARδ genes. One of the downstream targets of PPARs, the mitochondrial uncoupling protein 2 (UCP2) was also upregulated. The number of TuJ1- and CNPase-positive cells increased after exposure to PPARγ agonist rosiglitazone (RGZ, 3 μM) and decreased after pre-incubation with the PPARγ antagonist GW9662 (5 μM). RGZ also upregulated the expression of PPARγ and UCP2 genes. Meanwhile GW9662 abolished the UCP2 upregulation and decreased Ca{sup 2+} activity induced by PFOS. Interestingly, a significantly higher expression of PPARγ and UCP3 genes was also detected in mouse neonatal brain after prenatal exposure to PFOS. These data suggest that PPARγ plays a role in the alteration of spontaneous differentiation of NSCs induced by nanomolar concentrations of PFOS. - Highlights: • PFOS decreases proliferation of neural stem cells (NSCs). • PFOS induces neuronal and oligodendrocytic differentiation in NSCs. • PFOS alters expression of PPARγ and UCP2 in vitro. • PFOS alters expression of PPARγ and UCP3 in vivo. • Block of PPAR

  19. Carbon monoxide improves neuronal differentiation and yield by increasing the functioning and number of mitochondria.

    PubMed

    Almeida, Ana S; Sonnewald, Ursula; Alves, Paula M; Vieira, Helena L A

    2016-08-01

    The process of cell differentiation goes hand-in-hand with metabolic adaptations, which are needed to provide energy and new metabolites. Carbon monoxide (CO) is an endogenous cytoprotective molecule able to inhibit cell death and improve mitochondrial metabolism. Neuronal differentiation processes were studied using the NT2 cell line, which is derived from human testicular embryonic teratocarcinoma and differentiates into post-mitotic neurons upon retinoic acid treatment. CO-releasing molecule A1 (CORM-A1) was used do deliver CO into cell culture. CO treatment improved NT2 neuronal differentiation and yield, since there were more neurons and the total cell number increased following the differentiation process. CO supplementation enhanced the mitochondrial population in post-mitotic neurons derived from NT2 cells, as indicated by an increase in mitochondrial DNA. CO treatment during neuronal differentiation increased the extent of the classical metabolic change that occurs during neuronal differentiation, from glycolytic to more oxidative metabolism, by decreasing the ratio of lactate production and glucose consumption. The expression of pyruvate and lactate dehydrogenases was higher, indicating an augmented oxidative metabolism. Moreover, these findings were corroborated by an increased percentage of (13) C incorporation from [U-(13) C]glucose into the tricarboxylic acid cycle metabolites malate and citrate, and also glutamate and aspartate in CO-treated cells. Finally, under low levels of oxygen (5%), which enhances glycolytic metabolism, some of the enhancing effects of CO on mitochondria were not observed. In conclusion, our data show that CO improves neuronal and mitochondrial yield by stimulation of tricarboxylic acid cycle activity, and thus oxidative metabolism of NT2 cells during the process of neuronal differentiation. The process of cell differentiation is coupled with metabolic adaptations. Carbon monoxide (CO) is an endogenous cytoprotective

  20. Changes in the expression of DNA-binding/differentiation protein inhibitors in neurons and glial cells of the gerbil hippocampus following transient global cerebral ischemia

    PubMed Central

    LEE, JAE-CHUL; CHEN, BAI HUI; CHO, JEONG-HWI; KIM, IN HYE; AHN, JI HYEON; PARK, JOON HA; TAE, HYUN-JIN; CHO, GEUM-SIL; YAN, BING CHUN; KIM, DAE WON; HWANG, IN KOO; PARK, JINSEU; LEE, YUN LYUL; CHOI, SOO YOUNG; WON, MOO-HO

    2015-01-01

    Inhibitors of DNA-binding/differentiation (ID) proteins bind to basic helix-loop-helix (bHLH) transcription factors, including those that regulate differentiation and cell-cycle progression during development, and regulate gene transcription. However, little is known about the role of ID proteins in the brain under transient cerebral ischemic conditions. In the present study, we examined the effects of ischemia-reperfusion (I-R) injury on the immunoreactivity and protein levels of IDs 1–4 in the gerbil hippocampus proper Cornu Ammonis regions CA1–3 following 5 min of transient cerebral ischemia. Strong ID1 immunoreactivity was detected in the nuclei of pyramidal neurons in the hippocampal CA1–3 regions; immunoreactivity was significantly changed following I-R in the CA1 region, but not in the CA2/3 region. Five days following I-R, ID1 immunoreactivity was not detected in the CA1 pyramidal neurons. ID1 immunoreactivity was detected only in GABAergic interneurons in the ischemic CA1 region. Weak ID4 immunoreactivity was detected in non-pyramidal cells, and immunoreactivity was again only changed in the ischemic CA1 region. Five days following I-R, strong ID4 immunoreactivity was detected in non-pyramidal cells, which were identified as microglia, and not astrocytes, in the ischemic CA1 region. Furthermore, changes in the protein levels of ID1 and ID4 in the ischemic CA1 region studied by western blot were consistent with patterns of immunoreactivity. In summary, these results indicate that immunoreactivity and protein levels of ID1 and ID4 are distinctively altered following transient cerebral ischemia only in the CA1 region, and that the changes in ID1 and ID4 expression may relate to the ischemia-induced delayed neuronal death. PMID:25503067

  1. Murine Mueller cells are progenitor cells for neuronal cells and fibrous tissue cells

    SciTech Connect

    Florian, Christian; Langmann, Thomas; Weber, Bernhard H.F.; Morsczeck, Christian

    2008-09-19

    Mammalian Mueller cells have been reported to possess retinal progenitor cell properties and generate new neurons after injury. This study investigates murine Mueller cells under in vitro conditions for their capability of dedifferentiation into retinal progenitor cells. Mueller cells were isolated from mouse retina, and proliferating cells were expanded in serum-containing medium. For dedifferentiation, the cultured cells were transferred to serum-replacement medium (SRM) at different points in time after their isolation. Interestingly, early cell passages produced fibrous tissue in which extracellular matrix proteins and connective tissue markers were differentially expressed. In contrast, aged Mueller cell cultures formed neurospheres in SRM that are characteristic for neuronal progenitor cells. These neurospheres differentiated into neuron-like cells after cultivation on laminin/ornithine cell culture substrate. Here, we report for the first time that murine Mueller cells can be progenitors for both, fibrous tissue cells and neuronal cells, depending on the age of the cell culture.

  2. Microarray and synchronization of neuronal differentiation with pathway changes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) databank in nerve growth factor-treated PC12 cells.

    PubMed

    Lin, Chih-Ming; Feng, Wayne

    2012-08-01

    The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database creates networks from interrelations between molecular biology and underlying chemical elements. This allows for analysis of biologic networks, genomic information, and higher-order functional information at a systems level. We performed microarray experiments and used the KEGG database, systems biology analysis, and annotation of pathway function to study nerve growth factor (NGF)-induced differentiation of PC12 cells. Cells were cultured to 70%-80% confluence, treated with NGF for 1 or 3 hours (h), and RNA was extracted. Stage 1 data analysis involved analysis of variance (ANOVA), and stage 2 involved cluster analysis and heat map generation. We identified 2020 NGF-induced PC12 genes (1038 at 1 h and 1554 at 3 h). Results showed changes in gene expression over time. We compared these genes with 6035 genes from the KEGG database. Cross-matching resulted in 830 genes. Among these, we identified 395 altered genes (155 at 1 h and 301 at 3 h; 2-fold increase from 1 h to 3 h). We identified 191 biologic pathways in the KEGG database; the top 15 showed correlations with neuronal differentiation (mitogen-activated protein kinase [MAPK] pathway: 35 genes at 1 h, 54 genes at 3 h; genes associated with axonal guidance: 12 at 1 h, 26 at 3 h; Wnt pathway: 16 at 1 h, 25 at 3 h; neurotrophin pathway: 4 at 1 h, 14 at 3 h). Thus, we identified changes in neuronal differentiation pathways with the KEGG database, which were synchronized with NGF-induced differentiation.

  3. GluD1 is a common altered player in neuronal differentiation from both MECP2-mutated and CDKL5-mutated iPS cells.

    PubMed

    Livide, Gabriella; Patriarchi, Tommaso; Amenduni, Mariangela; Amabile, Sonia; Yasui, Dag; Calcagno, Eleonora; Lo Rizzo, Caterina; De Falco, Giulia; Ulivieri, Cristina; Ariani, Francesca; Mari, Francesca; Mencarelli, Maria Antonietta; Hell, Johannes Wilhelm; Renieri, Alessandra; Meloni, Ilaria

    2015-02-01

    Rett syndrome is a monogenic disease due to de novo mutations in either MECP2 or CDKL5 genes. In spite of their involvement in the same disease, a functional interaction between the two genes has not been proven. MeCP2 is a transcriptional regulator; CDKL5 encodes for a kinase protein that might be involved in the regulation of gene expression. Therefore, we hypothesized that mutations affecting the two genes may lead to similar phenotypes by dysregulating the expression of common genes. To test this hypothesis we used induced pluripotent stem (iPS) cells derived from fibroblasts of one Rett patient with a MECP2 mutation (p.Arg306Cys) and two patients with mutations in CDKL5 (p.Gln347Ter and p.Thr288Ile). Expression profiling was performed in CDKL5-mutated cells and genes of interest were confirmed by real-time RT-PCR in both CDKL5- and MECP2-mutated cells. The only major change in gene expression common to MECP2- and CDKL5-mutated cells was for GRID1, encoding for glutamate D1 receptor (GluD1), a member of the δ-family of ionotropic glutamate receptors. GluD1 does not form AMPA or NMDA glutamate receptors. It acts like an adhesion molecule by linking the postsynaptic and presynaptic compartments, preferentially inducing the inhibitory presynaptic differentiation of cortical neurons. Our results demonstrate that GRID1 expression is downregulated in both MECP2- and CDKL5-mutated iPS cells and upregulated in neuronal precursors and mature neurons. These data provide novel insights into disease pathophysiology and identify possible new targets for therapeutic treatment of Rett syndrome.

  4. Mu-opioid receptor activation prevents apoptosis following serum withdrawal in differentiated SH-SY5Y cells and cortical neurons via phosphatidylinositol 3-kinase.

    PubMed

    Iglesias, M; Segura, M F; Comella, J X; Olmos, G

    2003-03-01

    Opioid peptides and alkaloids exert their effects via G protein-coupled receptors (GPCRs). It has been shown that, in addition to trophic factors, some GPCRs are able to activate the phosphatidylinositol 3-kinase/Akt (PI 3-K/Akt) signal transduction pathway, thus leading to cell survival. The aim of this study was to test whether activation of mu-opioid receptors has protective effects on serum withdrawal-induced cell death and to study the possible implication of PI 3-K in this process. In SH-SY5Y neuroblastoma cells fully differentiated by exposure to retinoic acid for five days, the enkephalin derivative selective mu-agonist DAMGO (0.1-2 microM) and the alkaloid morphine (0.1-10 microM) promoted cell survival after serum deprivation (MTT and trypan blue exclusion assays), without inducing cell proliferation. These effects were fully reversed by naloxone, by the selective mu-antagonist beta-funaltrexamine (beta-FNA) and also by the specific PI 3-K inhibitor LY294002. The two agonists stimulated Akt phosphorylation and the effect was also abolished by beta-FNA and by LY294002. In mouse primary cortical neurons, DAMGO reduced the percentage of apoptosis after 6, 12, 24 and 48 h of serum withdrawal; as determined by Hoechst staining. This effect was blocked by beta-FNA, by pre-treatment with pertussis toxin and by LY294002. DAMGO also stimulated Akt phosphorylation via PI 3-K in this primary neuronal culture. Together, these results indicate that stimulation of the mu-opioid receptor promotes neuronal survival in a G(i/o)-linked, PI 3-K-dependent signaling cascade and suggest that Akt may be a key downstream kinase involved in this anti-apoptotic effect. PMID:12646285

  5. GluD1 is a common altered player in neuronal differentiation from both MECP2-mutated and CDKL5-mutated iPS cells

    PubMed Central

    Livide, Gabriella; Patriarchi, Tommaso; Amenduni, Mariangela; Amabile, Sonia; Yasui, Dag; Calcagno, Eleonora; Lo Rizzo, Caterina; De Falco, Giulia; Ulivieri, Cristina; Ariani, Francesca; Mari, Francesca; Mencarelli, Maria Antonietta; Hell, Johannes Wilhelm; Renieri, Alessandra; Meloni, Ilaria

    2015-01-01

    Rett syndrome is a monogenic disease due to de novo mutations in either MECP2 or CDKL5 genes. In spite of their involvement in the same disease, a functional interaction between the two genes has not been proven. MeCP2 is a transcriptional regulator; CDKL5 encodes for a kinase protein that might be involved in the regulation of gene expression. Therefore, we hypothesized that mutations affecting the two genes may lead to similar phenotypes by dysregulating the expression of common genes. To test this hypothesis we used induced pluripotent stem (iPS) cells derived from fibroblasts of one Rett patient with a MECP2 mutation (p.Arg306Cys) and two patients with mutations in CDKL5 (p.Gln347Ter and p.Thr288Ile). Expression profiling was performed in CDKL5-mutated cells and genes of interest were confirmed by real-time RT-PCR in both CDKL5- and MECP2-mutated cells. The only major change in gene expression common to MECP2- and CDKL5-mutated cells was for GRID1, encoding for glutamate D1 receptor (GluD1), a member of the δ-family of ionotropic glutamate receptors. GluD1 does not form AMPA or NMDA glutamate receptors. It acts like an adhesion molecule by linking the postsynaptic and presynaptic compartments, preferentially inducing the inhibitory presynaptic differentiation of cortical neurons. Our results demonstrate that GRID1 expression is downregulated in both MECP2- and CDKL5-mutated iPS cells and upregulated in neuronal precursors and mature neurons. These data provide novel insights into disease pathophysiology and identify possible new targets for therapeutic treatment of Rett syndrome. PMID:24916645

  6. Transient suppression of late-stage neuronal progenitor cell differentiation in the hippocampal dentate gyrus of rat offspring after maternal exposure to nicotine.

    PubMed

    Ohishi, Takumi; Wang, Liyun; Akane, Hirotoshi; Shiraki, Ayako; Itahashi, Megu; Mitsumori, Kunitoshi; Shibutani, Makoto

    2014-02-01

    To examine the developmental exposure effect of nicotine (NIC) on hippocampal neurogenesis, pregnant Sprague-Dawley rats were treated with (-)-NIC hydrogen tartrate salt through drinking water at 2, 10 or 50 ppm from gestational day 6 to day 21 after delivery. On postnatal day (PND) 21, immunohistochemically doublecortin (Dcx)(+) cells increased at ≥10 ppm in the dentate subgranular zone (SGZ) as examined in male offspring; however, dihydropyrimidinase-like 3 (TUC4)(+) cells decreased at 2 ppm, and T box brain 2 (Tbr2)(+) cells were unchanged at any dose. Double immunohistochemistry revealed decreases in TUC4(+)/Dcx(+) and TUC4(+)/Dcx(-) cells, an increase in TUC4(-)/Dcx(+) cells at 2 and 10 ppm and an increase in Tbr2(-)/Dcx(+) cells at 50 ppm, suggesting an increase in type-3 progenitor cells at ≥2 ppm and decrease in immature granule cells at 2 and 10 ppm. The number of mature neuron-specific NeuN(-) progenitor cells expressing nicotinic acetylcholine receptor α7 in the SGZ and mRNA levels of Chrna7 and Chrnb2 in the dentate gyrus was unchanged at any dose, suggesting a lack of direct nicotinic stimulation on progenitor cells. In the dentate hilus, glutamic acid decarboxylase 67(+) interneurons increased at ≥10 ppm. All changes disappeared on PND 77. Therefore, maternal exposure to NIC reversibly affects hippocampal neurogenesis targeting late-stage differentiation in rat offspring. An increase in interneurons suggested that their activation affected granule cell differentiation. The lowest observed adverse effect level was at 2 ppm (0.091 mg/kg/day as a free base) by the affection of hippocampal neurogenesis at ≥2 ppm.

  7. Spatial training promotes short-term survival and neuron-like differentiation of newborn cells in Aβ1-42-injected rats.

    PubMed

    Zeng, Juan; Jiang, Xia; Hu, Xian-Feng; Ma, Rong-Hong; Chai, Gao-Shang; Sun, Dong-Sheng; Xu, Zhi-Peng; Li, Li; Bao, Jian; Feng, Qiong; Hu, Yu; Chu, Jiang; Chai, Da-Min; Hong, Xiao-Yue; Wang, Jian-Zhi; Liu, Gong-Ping

    2016-09-01

    Neurogenesis plays a role in hippocampus-dependent learning and impaired neurogenesis may correlate with cognitive deficits in Alzheimer's disease. Spatial training influences the production and fate of newborn cells in hippocampus of normal animals, whereas the effects on neurogenesis in Alzheimer-like animal are not reported until now. Here, for the first time, we investigated the effect of Morris water maze training on proliferation, survival, apoptosis, migration, and differentiation of newborn cells in β-amyloid-treated Alzheimer-like rats. We found that spatial training could preserve a short-term survival of newborn cells generated before training, during the early phase, and the late phase of training. However, the training had no effect on the long-term survival of mature newborn cells generated at previously mentioned 3 different phases. We also demonstrated that spatial training promoted newborn cell differentiation preferentially to the neuron direction. These findings suggest a time-independent neurogenesis induced by spatial training, which may be indicative for the cognitive stimulation in Alzheimer's disease therapy. PMID:27459927

  8. Spatial training promotes short-term survival and neuron-like differentiation of newborn cells in Aβ1-42-injected rats.

    PubMed

    Zeng, Juan; Jiang, Xia; Hu, Xian-Feng; Ma, Rong-Hong; Chai, Gao-Shang; Sun, Dong-Sheng; Xu, Zhi-Peng; Li, Li; Bao, Jian; Feng, Qiong; Hu, Yu; Chu, Jiang; Chai, Da-Min; Hong, Xiao-Yue; Wang, Jian-Zhi; Liu, Gong-Ping

    2016-09-01

    Neurogenesis plays a role in hippocampus-dependent learning and impaired neurogenesis may correlate with cognitive deficits in Alzheimer's disease. Spatial training influences the production and fate of newborn cells in hippocampus of normal animals, whereas the effects on neurogenesis in Alzheimer-like animal are not reported until now. Here, for the first time, we investigated the effect of Morris water maze training on proliferation, survival, apoptosis, migration, and differentiation of newborn cells in β-amyloid-treated Alzheimer-like rats. We found that spatial training could preserve a short-term survival of newborn cells generated before training, during the early phase, and the late phase of training. However, the training had no effect on the long-term survival of mature newborn cells generated at previously mentioned 3 different phases. We also demonstrated that spatial training promoted newborn cell differentiation preferentially to the neuron direction. These findings suggest a time-independent neurogenesis induced by spatial training, which may be indicative for the cognitive stimulation in Alzheimer's disease therapy.

  9. Interferon-gamma inhibits the neuronal differentiation of neural progenitor cells by inhibiting the expression of Neurogenin2 via the JAK/STAT1 pathway.

    PubMed

    Ahn, Jyhyun; Lee, Junsub; Kim, Sunyoung

    2015-10-01

    Interferon-gamma (IFN-γ) is one of the critical cytokines released by host immune cells upon infection. Despite the important role(s) of IFN-γ in host immune responses, there has been no in vivo study regarding the effects of IFN-γ on brain development, and the results from many in vitro studies are controversial. In this study, the effects of IFN-γ on embryonic neurogenesis were investigated. Treatment of E14.5 mouse neural progenitor cells (NPCs) with IFN-γ resulted in a decrease in the percentage of TuJ1-positive immature neurons but an increase in the percentage of Nestin-positive NPCs. Similar results were obtained in vivo. Treatment of NPCs with a JAK inhibitor or the knockdown of STAT1 expression abrogated the IFN-γ-mediated inhibition of neurogenesis. Interestingly, the expression of one of proneural genes, Neurogenin2 (Neurog2) was dramatically inhibited upon IFN-γ treatment, and cells overexpressing Neurog2 did not respond to IFN-γ. Taken together, our results demonstrate that IFN-γ inhibits neuronal differentiation of NPCs by negatively regulating the expression of Neurog2 via the JAK/STAT1 pathway. Our findings may provide an insight into the role of IFN-γ in the development of embryonic brain.

  10. Electrohydrodynamic jetting of mouse neuronal cells

    PubMed Central

    Eagles, Peter A. M.; Qureshi, Amer N.; Jayasinghe, Suwan N.

    2006-01-01

    CAD (Cath.a-differentiated) cells, a mouse neuronal cell line, were subjected to electrohydrodynamic jetting at a field strength of 0.47–0.67 kV/mm, corresponding to an applied voltage of 7–10 kV. After jetting, the cells appeared normal and continued to divide at rates similar to those shown by control samples. Jetted cells, when placed in serum-free medium, underwent differentiation that was sustained for at least 1 month. Some of the droplets produced by jetting contained only a few cells. These results indicate that the process of jetting does not significantly perturb neuronal cells and that this novel approach might in the future be a useful way to deposit small numbers of living nerve cells on to surfaces. PMID:16393140

  11. Engineered N-cadherin and L1 biomimetic substrates concertedly promote neuronal differentiation, neurite extension and neuroprotection of human neural stem cells.

    PubMed

    Cherry, Jocie F; Bennett, Neal K; Schachner, Melitta; Moghe, Prabhas V

    2014-10-01

    We investigated the design of neurotrophic biomaterial constructs for human neural stem cells, guided by neural developmental cues of N-cadherin and L1 adhesion molecules. Polymer substrates fabricated either as two-dimensional (2-D) films or three-dimensional (3-D) microfibrous scaffolds were functionalized with fusion chimeras of N-cadherin-Fc alone and in combination with L1-Fc, and the effects on differentiation, neurite extension and survival of H9 human-embryonic-stem-cell-derived neural stem cells (H9-NSCs) were quantified. Combinations of N-cadherin and L1-Fc co-operatively enhanced neuronal differentiation profiles, indicating the critical nature of the two complementary developmental cues. Notably, substrates presenting low levels of N-cadherin-Fc concentrations, combined with proportionately higher L1-Fc concentration, most enhanced neurite outgrowth and the degree of MAP2+ and neurofilament-M+ H9-NSCs. Low N-cadherin-Fc alone promoted improved cell survival following oxidative stress, compared to higher concentrations of N-cadherin-Fc alone or combinations with L1-Fc. Pharmacological and antibody blockage studies revealed that substrates presenting low levels of N-cadherin are functionally competent so long as they elicit a threshold signal mediated by homophilic N-cadherin and fibroblast growth factor signaling. Overall, these studies highlight the ability of optimal combinations of N-cadherin and L1 to recapitulate a "neurotrophic" microenvironment that enhances human neural stem cell differentiation and neurite outgrowth. Additionally, 3-D fibrous scaffolds presenting low N-cadherin-Fc further enhanced the survival of H9-NSCs compared to equivalent 2-D films. This indicates that similar biofunctionalization approaches based on N-cadherin and L1 can be translated to 3-D "transplantable" scaffolds with enhanced neurotrophic behaviors. Thus, the insights from this study have fundamental and translational impacts for neural-stem-cell-based regenerative

  12. Ionizing Radiation Induces Altered Neuronal Differentiation by mGluR1 through PI3K-STAT3 Signaling in C17.2 Mouse Neural Stem-Like Cells.

    PubMed

    Eom, Hyeon Soo; Park, Hae Ran; Jo, Sung Kee; Kim, Young Sang; Moon, Changjong; Kim, Sung-Ho; Jung, Uhee

    2016-01-01

    Most studies of IR effects on neural cells and tissues in the brain are still focused on loss of neural stem cells. On the other hand, the effects of IR on neuronal differentiation and its implication in IR-induced brain damage are not well defined. To investigate the effects of IR on C17.2 mouse neural stem-like cells and mouse primary neural stem cells, neurite outgrowth and expression of neuronal markers and neuronal function-related genes were examined. To understand this process, the signaling pathways including PI3K, STAT3, metabotrophic glutamate receptor 1 (mGluR1) and p53 were investigated. In C17.2 cells, irradiation significantly increased the neurite outgrowth, a morphological hallmark of neuronal differentiation, in a dose-dependent manner. Also, the expression levels of neuronal marker proteins, β-III tubulin were increased by IR. To investigate whether IR-induced differentiation is normal, the expression of neuronal function-related genes including synaptophysin, a synaptic vesicle forming proteins, synaptotagmin1, a calcium ion sensor, γ-aminobutyric acid (GABA) receptors, inhibitory neurotransmitter receptors and glutamate receptors, excitatory neurotransmitter receptors was examined and compared to that of neurotrophin-stimulated differentiation. IR increased the expression of synaptophysin, synaptotagmin1 and GABA receptors mRNA similarly to normal differentiation by stimulation of neurotrophin. Interestingly, the overall expression of glutamate receptors was significantly higher in irradiated group than normal differentiation group, suggesting that the IR-induced neuronal differentiation may cause altered neuronal function in C17.2 cells. Next, the molecular mechanism of the altered neuronal differentiation induced by IR was studied by investigating signaling pathways including p53, mGluR1, STAT3 and PI3K. Increases of neurite outgrowth, neuronal marker and neuronal function-related gene expressions by IR were abolished by inhibition of p53, m

  13. Ionizing Radiation Induces Altered Neuronal Differentiation by mGluR1 through PI3K-STAT3 Signaling in C17.2 Mouse Neural Stem-Like Cells

    PubMed Central

    Eom, Hyeon Soo; Park, Hae Ran; Jo, Sung Kee; Kim, Young Sang; Moon, Changjong; Kim, Sung-Ho; Jung, Uhee

    2016-01-01

    Most studies of IR effects on neural cells and tissues in the brain are still focused on loss of neural stem cells. On the other hand, the effects of IR on neuronal differentiation and its implication in IR-induced brain damage are not well defined. To investigate the effects of IR on C17.2 mouse neural stem-like cells and mouse primary neural stem cells, neurite outgrowth and expression of neuronal markers and neuronal function-related genes were examined. To understand this process, the signaling pathways including PI3K, STAT3, metabotrophic glutamate receptor 1 (mGluR1) and p53 were investigated. In C17.2 cells, irradiation significantly increased the neurite outgrowth, a morphological hallmark of neuronal differentiation, in a dose-dependent manner. Also, the expression levels of neuronal marker proteins, β-III tubulin were increased by IR. To investigate whether IR-induced differentiation is normal, the expression of neuronal function-related genes including synaptophysin, a synaptic vesicle forming proteins, synaptotagmin1, a calcium ion sensor, γ-aminobutyric acid (GABA) receptors, inhibitory neurotransmitter receptors and glutamate receptors, excitatory neurotransmitter receptors was examined and compared to that of neurotrophin-stimulated differentiation. IR increased the expression of synaptophysin, synaptotagmin1 and GABA receptors mRNA similarly to normal differentiation by stimulation of neurotrophin. Interestingly, the overall expression of glutamate receptors was significantly higher in irradiated group than normal differentiation group, suggesting that the IR-induced neuronal differentiation may cause altered neuronal function in C17.2 cells. Next, the molecular mechanism of the altered neuronal differentiation induced by IR was studied by investigating signaling pathways including p53, mGluR1, STAT3 and PI3K. Increases of neurite outgrowth, neuronal marker and neuronal function-related gene expressions by IR were abolished by inhibition of p53, m

  14. Human embryonic stem cell-derived neuronal cells form spontaneously active neuronal networks in vitro.

    PubMed

    Heikkilä, Teemu J; Ylä-Outinen, Laura; Tanskanen, Jarno M A; Lappalainen, Riikka S; Skottman, Heli; Suuronen, Riitta; Mikkonen, Jarno E; Hyttinen, Jari A K; Narkilahti, Susanna

    2009-07-01

    The production of functional human embryonic stem cell (hESC)-derived neuronal cells is critical for the application of hESCs in treating neurodegenerative disorders. To study the potential functionality of hESC-derived neurons, we cultured and monitored the development of hESC-derived neuronal networks on microelectrode arrays. Immunocytochemical studies revealed that these networks were positive for the neuronal marker proteins beta-tubulin(III) and microtubule-associated protein 2 (MAP-2). The hESC-derived neuronal networks were spontaneously active and exhibited a multitude of electrical impulse firing patterns. Synchronous bursts of electrical activity similar to those reported for hippocampal neurons and rodent embryonic stem cell-derived neuronal networks were recorded from the differentiated cultures until up to 4 months. The dependence of the observed neuronal network activity on sodium ion channels was examined using tetrodotoxin (TTX). Antagonists for the glutamate receptors NMDA [D(-)-2-amino-5-phosphonopentanoic acid] and AMPA/kainate [6-cyano-7-nitroquinoxaline-2,3-dione], and for GABAA receptors [(-)-bicuculline methiodide] modulated the spontaneous electrical activity, indicating that pharmacologically susceptible neuronal networks with functional synapses had been generated. The findings indicate that hESC-derived neuronal cells can generate spontaneously active networks with synchronous communication in vitro, and are therefore suitable for use in developmental and drug screening studies, as well as for regenerative medicine.

  15. Fibroblast Growth Factor Receptor-2 Contributes to the Basic Fibroblast Growth Factor-Induced Neuronal Differentiation in Canine Bone Marrow Stromal Cells via Phosphoinositide 3-Kinase/Akt Signaling Pathway

    PubMed Central

    Nakano, Rei; Edamura, Kazuya; Nakayama, Tomohiro; Narita, Takanori; Okabayashi, Ken; Sugiya, Hiroshi

    2015-01-01

    Bone marrow stromal cells (BMSCs) are considered as candidates for regenerative therapy and a useful model for studying neuronal differentiation. The role of basic fibroblast growth factor (bFGF) in neuronal differentiation has been previously studied; however, the signaling pathway involved in this process remains poorly understood. In this study, we investigated the signaling pathway in the bFGF-induced neuronal differentiation of canine BMSCs. bFGF induced the mRNA expression of the neuron marker, microtubule associated protein-2 (MAP2) and the neuron-like morphological change in canine BMSCs. In the presence of inhibitors of fibroblast growth factor receptors (FGFR), phosphatidylinositol 3-kinase (PI3K) and Akt, i.e., SU5402, LY294002, and MK2206, respectively, bFGF failed to induce the MAP2 mRNA expression and the neuron-like morphological change. bFGF induced Akt phosphorylation, but it was attenuated by the FGFR inhibitor SU5402 and the PI3K inhibitor LY294002. In canine BMSCs, expression of FGFR-1 and FGFR-2 was confirmed, but only FGFR-2 activation was detected by cross-linking and immunoprecipitation analysis. Small interfering RNA-mediated knockdown of FGFR-2 in canine BMSCs resulted in the attenuation of bFGF-induced Akt phosphorylation. These results suggest that the FGFR-2/PI3K/Akt signaling pathway is involved in the bFGF-induced neuronal differentiation of canine BMSCs. PMID:26523832

  16. N-acetylaspartate (NAA) induces neuronal differentiation of SH-SY5Y neuroblastoma cell line and sensitizes it to chemotherapeutic agents

    PubMed Central

    Mazzoccoli, Carmela; Ruggieri, Vitalba; Tataranni, Tiziana; Agriesti, Francesca; Laurenzana, Ilaria; Fratello, Angelo; Capitanio, Nazzareno; Piccoli, Claudia

    2016-01-01

    Neuroblastoma is the most commonly extra-cranial solid tumor of childhood frequently diagnosed. The nervous system-specific metabolite N-acetylaspartate (NAA) is synthesized from aspartate and acetyl-CoA in neurons, it is among the most abundant metabolites present in the central nervous system (CNS) and appears to be involved in many CNS disorders. The functional significance of the high NAA concentration in the brain remains uncertain, but it confers to NAA a unique clinical significance exploited in magnetic resonance spectroscopy. In the current study, we show that treatment of SH-SY5Y neuroblastoma-derived cell line with sub-cytotoxic physiological concentrations of NAA inhibits cell growth. This effect is partly due to enhanced apoptosis, shown by decrease of the anti-apoptotic factors survivin and Bcl-xL, and partly to arrest of the cell-cycle progression, linked to enhanced expression of the cyclin-inhibitors p53, p21Cip1/Waf1 and p27Kip1. Moreover, NAA-treated SH-SY5Y cells exhibited morphological changes accompanied with increase of the neurogenic markers TH and MAP2 and down-regulation of the pluripotency markers OCT4 and CXCR4/CD184. Finally, NAA-pre-treated SH-SY5Y cells resulted more sensitive to the cytotoxic effect of the chemotherapeutic drugs Cisplatin and 5-fluorouracil. To our knowledge, this is the first study demonstrating the neuronal differentiating effects of NAA in neuroblastoma cells. NAA may be a potential preconditioning or adjuvant compound in chemotherapeutic treatment. PMID:27036033

  17. N-acetylaspartate (NAA) induces neuronal differentiation of SH-SY5Y neuroblastoma cell line and sensitizes it to chemotherapeutic agents.

    PubMed

    Mazzoccoli, Carmela; Ruggieri, Vitalba; Tataranni, Tiziana; Agriesti, Francesca; Laurenzana, Ilaria; Fratello, Angelo; Capitanio, Nazzareno; Piccoli, Claudia

    2016-05-01

    Neuroblastoma is the most commonly extra-cranial solid tumor of childhood frequently diagnosed. The nervous system-specific metabolite N-acetylaspartate (NAA) is synthesized from aspartate and acetyl-CoA in neurons, it is among the most abundant metabolites present in the central nervous system (CNS) and appears to be involved in many CNS disorders. The functional significance of the high NAA concentration in the brain remains uncertain, but it confers to NAA a unique clinical significance exploited in magnetic resonance spectroscopy. In the current study, we show that treatment of SH-SY5Y neuroblastoma-derived cell line with sub-cytotoxic physiological concentrations of NAA inhibits cell growth. This effect is partly due to enhanced apoptosis, shown by decrease of the anti-apoptotic factors survivin and Bcl-xL, and partly to arrest of the cell-cycle progression, linked to enhanced expression of the cyclin-inhibitors p53, p21Cip1/Waf1 and p27Kip1. Moreover, NAA-treated SH-SY5Y cells exhibited morphological changes accompanied with increase of the neurogenic markers TH and MAP2 and down-regulation of the pluripotency markers OCT4 and CXCR4/CD184. Finally, NAA-pre-treated SH-SY5Y cells resulted more sensitive to the cytotoxic effect of the chemotherapeutic drugs Cisplatin and 5-fluorouracil.To our knowledge, this is the first study demonstrating the neuronal differentiating effects of NAA in neuroblastoma cells. NAA may be a potential preconditioning or adjuvant compound in chemotherapeutic treatment.

  18. N-acetylaspartate (NAA) induces neuronal differentiation of SH-SY5Y neuroblastoma cell line and sensitizes it to chemotherapeutic agents.

    PubMed

    Mazzoccoli, Carmela; Ruggieri, Vitalba; Tataranni, Tiziana; Agriesti, Francesca; Laurenzana, Ilaria; Fratello, Angelo; Capitanio, Nazzareno; Piccoli, Claudia

    2016-05-01

    Neuroblastoma is the most commonly extra-cranial solid tumor of childhood frequently diagnosed. The nervous system-specific metabolite N-acetylaspartate (NAA) is synthesized from aspartate and acetyl-CoA in neurons, it is among the most abundant metabolites present in the central nervous system (CNS) and appears to be involved in many CNS disorders. The functional significance of the high NAA concentration in the brain remains uncertain, but it confers to NAA a unique clinical significance exploited in magnetic resonance spectroscopy. In the current study, we show that treatment of SH-SY5Y neuroblastoma-derived cell line with sub-cytotoxic physiological concentrations of NAA inhibits cell growth. This effect is partly due to enhanced apoptosis, shown by decrease of the anti-apoptotic factors survivin and Bcl-xL, and partly to arrest of the cell-cycle progression, linked to enhanced expression of the cyclin-inhibitors p53, p21Cip1/Waf1 and p27Kip1. Moreover, NAA-treated SH-SY5Y cells exhibited morphological changes accompanied with increase of the neurogenic markers TH and MAP2 and down-regulation of the pluripotency markers OCT4 and CXCR4/CD184. Finally, NAA-pre-treated SH-SY5Y cells resulted more sensitive to the cytotoxic effect of the chemotherapeutic drugs Cisplatin and 5-fluorouracil.To our knowledge, this is the first study demonstrating the neuronal differentiating effects of NAA in neuroblastoma cells. NAA may be a potential preconditioning or adjuvant compound in chemotherapeutic treatment. PMID:27036033

  19. Reprogramming RPE to differentiate towards retinal neurons with Sox2

    PubMed Central

    Ma, Wenxin; Yan, Run-Tao; Li, Xiumei; Wang, Shu-Zhen

    2009-01-01

    Guiding non-neural, retinal pigment epithelium (RPE) to produce retinal neurons may offer a source of developing neurons for cell-replacement. Sox2 plays important roles in maintaining neural progenitor/stem cell properties and in converting fibroblasts into pluripotent stem cells. This study tests the possibility of using Sox2 to reprogram RPE to differentiate towards retinal neurons in vivo and in vitro. Expression of Sox2 in the chick retina was detected in progenitor cells, in cells at a discrete location in the layers of amacrine and ganglion cells, and in Műller glia. Overexpression of Sox2 in the developing eye resulted in hypo-pigmentation of the RPE. In the affected regions, expression of retinal ganglion cell markers became apparent in the RPE layer. In RPE cell culture, Sox2 promoted the expression of retinal ganglion and amacrine markers and suppressed the expression of genes associate with RPE properties. Mechanistic investigation using the developing retina revealed a co-expression of Sox2 and bFGF, a growth factor commonly used in stem cell culture and capable of inducing RPE-to-retina transdifferentiation (or reprogramming) during early development. Similar patterns of changes in Sox2 expression and in bFGF expression were observed in atrophic retina and in injured retina. In RPE cell culture, Sox2 and bFGF mutually enhanced one another's expression. Up-regulation of bFGF expression by Sox2 also occurred in the retina. These results suggest that Sox2 can initiate a reprogramming of RPE cells to differentiate towards retinal neurons and may engage bFGF during the process. PMID:19489100

  20. A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem Cells (hNSC).

    PubMed

    Alessandri, Kevin; Feyeux, Maxime; Gurchenkov, Basile; Delgado, Christophe; Trushko, Anastasiya; Krause, Karl-Heinz; Vignjević, Daniela; Nassoy, Pierre; Roux, Aurélien

    2016-04-26

    We present here a microfluidic device that generates sub-millimetric hollow hydrogel spheres, encapsulating cells and coated internally with a layer of reconstituted extracellular matrix (ECM) of a few microns thick. The spherical capsules, composed of alginate hydrogel, originate from the spontaneous instability of a multi-layered jet formed by co-extrusion using a coaxial flow device. We provide a simple design to manufacture this device using a DLP (digital light processing) 3D printer. Then, we demonstrate how the inner wall of the capsules can be decorated with a continuous ECM layer that is anchored to the alginate gel and mimics the basal membrane of a cellular niche. Finally, we used this approach to encapsulate human Neural Stem Cells (hNSC) derived from human Induced Pluripotent Stem Cells (hIPSC), which were further differentiated into neurons within the capsules with negligible loss of viability. Altogether, we show that these capsules may serve as cell micro-containers compatible with complex cell culture conditions and applications. These developments widen the field of research and biomedical applications of the cell encapsulation technology. PMID:27025278

  1. Pituitary adenoma-neuronal choristoma is a pituitary adenoma with ganglionic differentiation.

    PubMed

    Nguyen, Michaela T; Lavi, Ehud

    2015-12-01

    The presence of ganglion cells within an endocrine pituitary tumor has been named hamartoma, choristoma, gangliocytoma, or most recently pituitary adenoma-neuronal choristoma (PANCH). The presence of neuronal differentiation in regular pituitary adenomas has been previously suggested, however, its origin, the extent of its presence, and the relationship between the neuronal elements and the pituitary adenoma remain uncertain. Thus, to further explore the neuronal potential of pituitary tumors, we used immunohistochemistry on pituitary tumors of different grades, with a neuronal antigen protein (NeuN) antibody as a specific marker for mature neuronal differentiation. We found NeuN expression in 26.47% (9/34) cases of pituitary tumors without ganglionic differentiation (7 adenomas, 1 atypical adenoma and 1 pituitary carcinoma), in addition to NeuN expression in pituitary adenomas with ganglionic cells (2/2). Thus, neuronal expression is an innate property of pituitary adenomas. We propose that the rare presence of ganglionic cells in pituitary adenomas is not the result of a separate lesion or "collision sellar tumors", as previously suggested, but a ganglionic neuronal differentiation in an endocrine neoplasm. The ganglionic cells may be arising from uncommitted stem/progenitor cells that contain both neuronal and endocrine properties. A label of "pituitary adenoma with ganglionic differentiation" would better reflect the dual differentiation in a neuroendocrine tumor than the current label "PANCH".

  2. Neuronal differentiation distinguishes supratentorial and infratentorial childhood ependymomas.

    PubMed

    Andreiuolo, Felipe; Puget, Stéphanie; Peyre, Matthieu; Dantas-Barbosa, Carmela; Boddaert, Nathalie; Philippe, Cathy; Mauguen, Audrey; Grill, Jacques; Varlet, Pascale

    2010-11-01

    Ependymomas are glial neoplasms occurring in any location throughout the central nervous system and supposedly are derived from radial glia cells. Recent data suggest that these tumors may have different biological and clinical behaviors according to their location. Pediatric supratentorial and infratentorial ependymoma (SE and IE) were compared with respect to clinical and radiological parameters and immunohistochemistry (IHC). Neuronal markers were specifically assessed by IHC and quantitative PCR (qPCR). No single morphological or radiological characteristic was associated with location or any neuronal marker. However, there was a significant overexpression of neuronal markers in SE compared with IE: neurofilament light polypeptide 70 (NEFL)-positive tumor cells were found in 23 of 34 SE and in only 4 of 32 IE (P < .001). Among SE, 10 of 34 exhibited high expression of NEFL, defined as more than 5% positive cells. qPCR confirmed the upregulation of neuronal markers (NEFL, LHX2, FOXG1, TLX1, and NPTXR) in SE compared with IE. In addition, strong NEFL expression in SE was correlated with better progression-free survival (P = .007). Our results support the distinction of SE and IE. SEs are characterized by neuronal differentiation, which seems to be associated with better prognosis.

  3. [Differential diagnosis using artificial neuronal networks].

    PubMed

    Shchetinin, V G; Komarov, V T

    1998-11-01

    In order to rule out the effects of subjective factors and decrease the number of diagnostic errors, artificial neuron nets are proposed. By means of these nets the subjective and half-empirical heuristics are replaced with rational diagnostic information based on quantitative and logic analysis. The proposed method was used for deriving the decisive regularities ensuring the differential diagnosis between infective endocarditis and active rheumatic fever, infective endocarditis and systemic lupus erythematosus, and systemic lupus erythematosus and active rheumatic fever. Reliability of diagnostic tables representing the decisive regularities in the usual form is confirmed by clinical data.

  4. Hydrogel-encapsulated 3D microwell array for neuronal differentiation.

    PubMed

    Bae, Jun Hyuk; Lee, Jong Min; Chung, Bong Geun

    2016-02-01

    We developed a photo-crosslinkable hydrogel-encapsulated three-dimensional (3D) microwell array for studying embryonic stem (ES) cell-derived neuronal differentiation. ES cells were cultured for 5 d in microwells and were subsequently encapsulated by photo-crosslinkable gelatin methacrylate (GelMA) and polyethylene glycol (PEG) hydrogels for an additional 7 d. We observed that ES cells cultured in PEG microwells became uniform-sized embryoid bodies (EBs) compared to those in GelMA microwells. Although ES cells were encapsulated by photo-crosslinkable GelMA and PEG hydrogels, they were highly viable. We demonstrated that uniform-sized EBs encapsulated by GelMA hydrogels in PEG microwells are largely differentiated into neuronal cells. It was revealed that neurites at the periphery of EBs in PEG microwells largely extended into the interface between GelMA hydrogels and PEG microwells for generating neuronal networks. Therefore, this photo-crosslinkable GelMA hydrogel-encapsulated PEG microwell array could be a potentially powerful tool for neurodegenerative disease applications.

  5. Differential effects of impaired mitochondrial energy production on the function of mu and delta opioid receptors in neuronal SK-N-SH cells.

    PubMed

    Raut, Atul; Iglewski, Myriam; Ratka, Anna

    2006-08-14

    Oxidative stress contributes to changes in neurosensory processing, including pain, that occur during aging and neurodegeneration. The effects of neuronal oxidation on the opioid system are poorly understood. In this in vitro study, oxidative stress was induced by 3-nitroproprionic acid (3-NPA) in opioid-responsive differentiated SK-N-SH cells. Changes in the inhibitory effects of opioid receptor agonists on intracellular cAMP were used as a marker of the function of mu and delta opioid receptors (MOR and DOR, respectively). Cells were treated with morphine and selective MOR and DOR agonists and antagonists to characterize the function of each receptor subtype. Cyclic AMP (cAMP) was measured by enzyme immunoassay. Levels of reactive oxygen species (ROS) were assessed using the 2',7'-dichlorofluorescin diacetate assay. Exposure of cells to 3-NPA resulted in an increase in ROS. After 3-NPA exposure, there was a significant attenuation of the inhibitory effect of morphine and DAMGO but not of DPDPE on cAMP. In cells pretreated with CTOP, 3-NPA did not change the inhibitory effect on cAMP. These findings demonstrate for the first time that under conditions of mitochondrial damage, the function of MOR is significantly decreased, while the function of DOR does not change, suggesting that the effect of 3-NPA on opioid receptors is subtype-specific.

  6. Differential effects of "Advanced glycation endproducts" and beta-amyloid peptide on glucose utilization and ATP levels in the neuronal cell line SH-SY5Y.

    PubMed

    Kuhla, B; Loske, C; Garcia De Arriba, S; Schinzel, R; Huber, J; Münch, G

    2004-03-01

    Beta-amyloid peptide (Abeta) and "Advanced glycation endproducts" (AGEs) are components of the senile plaques in Alzheimer's disease patients. It has been proposed that both AGEs and Abeta exert many of their effects, which include the upregulation of pro-inflammatory cytokines, through RAGE ("receptor for advanced glycation endproducts"). To investigate whether Abeta and AGEs cause similar or identical effects on cell survival and energy metabolism, we have compared the effects of a model-AGE and Abeta on cell viability, ATP level, glucose consumption and lactate production in the neuroblastoma cell line SH-SY5Y. The results show that AGEs and Abeta increase glucose consumption and decrease ATP levels in a dose dependent manner. Furthermore, both compounds decrease mitochondrial activity measured by the MTT assay. However, only AGEs decrease the number of cells and significantly increase lactate production. These data indicate that both AGEs and Abeta can cause differential disturbances in neuronal metabolism, which may contribute to the pathophysiological findings in Alzheimer's disease. However, their signalling pathways are apparently quite distinct, a fact which should stimulate a more detailed investigation in this field, e.g. for the purpose of a rational design of potential "neuroprotective" RAGE antagonists. PMID:14991463

  7. Interaction of leech neurons with topographical gratings: comparison with rodent and human neuronal lines and primary cells

    PubMed Central

    Tonazzini, Ilaria; Pellegrini, Monica; Pellegrino, Mario; Cecchini, Marco

    2014-01-01

    Controlling and improving neuronal cell migration and neurite outgrowth are critical elements of tissue engineering applications and development of artificial neuronal interfaces. To this end, a promising approach exploits nano/microstructured surfaces, which have been demonstrated to be capable of tuning neuronal differentiation, polarity, migration and neurite orientation. Here, we investigate the neurite contact guidance of leech neurons on plastic gratings (GRs; anisotropic topographies composed of alternating lines of grooves and ridges). By high-resolution microscopy, we quantitatively evaluate the changes in tubulin cytoskeleton organization and cell morphology and in the neurite and growth cone development. The topography-reading process of leech neurons on GRs is mediated by filopodia and is more responsive to 4-µm-period GRs than to smaller period GRs. Leech neuron behaviour on GRs is finally compared and validated with several other neuronal cells, from murine differentiated embryonic stem cells and primary hippocampal neurons to differentiated human neuroblastoma cells. PMID:24501675

  8. FOXP2 drives neuronal differentiation by interacting with retinoic acid signaling pathways

    PubMed Central

    Devanna, Paolo; Middelbeek, Jeroen; Vernes, Sonja C.

    2014-01-01

    FOXP2 was the first gene shown to cause a Mendelian form of speech and language disorder. Although developmentally expressed in many organs, loss of a single copy of FOXP2 leads to a phenotype that is largely restricted to orofacial impairment during articulation and linguistic processing deficits. Why perturbed FOXP2 function affects specific aspects of the developing brain remains elusive. We investigated the role of FOXP2 in neuronal differentiation and found that FOXP2 drives molecular changes consistent with neuronal differentiation in a human model system. We identified a network of FOXP2 regulated genes related to retinoic acid signaling and neuronal differentiation. FOXP2 also produced phenotypic changes associated with neuronal differentiation including increased neurite outgrowth and reduced migration. Crucially, cells expressing FOXP2 displayed increased sensitivity to retinoic acid exposure. This suggests a mechanism by which FOXP2 may be able to increase the cellular differentiation response to environmental retinoic acid cues for specific subsets of neurons in the brain. These data demonstrate that FOXP2 promotes neuronal differentiation by interacting with the retinoic acid signaling pathway and regulates key processes required for normal circuit formation such as neuronal migration and neurite outgrowth. In this way, FOXP2, which is found only in specific subpopulations of neurons in the brain, may drive precise neuronal differentiation patterns and/or control localization and connectivity of these FOXP2 positive cells. PMID:25309332

  9. FOXP2 drives neuronal differentiation by interacting with retinoic acid signaling pathways.

    PubMed

    Devanna, Paolo; Middelbeek, Jeroen; Vernes, Sonja C

    2014-01-01

    FOXP2 was the first gene shown to cause a Mendelian form of speech and language disorder. Although developmentally expressed in many organs, loss of a single copy of FOXP2 leads to a phenotype that is largely restricted to orofacial impairment during articulation and linguistic processing deficits. Why perturbed FOXP2 function affects specific aspects of the developing brain remains elusive. We investigated the role of FOXP2 in neuronal differentiation and found that FOXP2 drives molecular changes consistent with neuronal differentiation in a human model system. We identified a network of FOXP2 regulated genes related to retinoic acid signaling and neuronal differentiation. FOXP2 also produced phenotypic changes associated with neuronal differentiation including increased neurite outgrowth and reduced migration. Crucially, cells expressing FOXP2 displayed increased sensitivity to retinoic acid exposure. This suggests a mechanism by which FOXP2 may be able to increase the cellular differentiation response to environmental retinoic acid cues for specific subsets of neurons in the brain. These data demonstrate that FOXP2 promotes neuronal differentiation by interacting with the retinoic acid signaling pathway and regulates key processes required for normal circuit formation such as neuronal migration and neurite outgrowth. In this way, FOXP2, which is found only in specific subpopulations of neurons in the brain, may drive precise neuronal differentiation patterns and/or control localization and connectivity of these FOXP2 positive cells.

  10. Pain related channels are differentially expressed in neuronal and non-neuronal cells of glabrous skin of fabry knockout male mice.

    PubMed

    Lakomá, Jarmila; Rimondini, Roberto; Donadio, Vincenzo; Liguori, Rocco; Caprini, Marco

    2014-01-01

    Fabry disease (FD) is one of the X-linked lysosomal storage disorders caused by deficient functioning of the alpha-galactosidase A (α-GalA) enzyme. The α-GalA deficiency leads to multi-systemic clinical manifestations caused by the preferential accumulation of globotriaosylceramide in the endothelium and vascular smooth muscles. A hallmark symptom of FD patients is peripheral pain that appears in the early stage of the disease. Pain in FD patients is a peripheral small-fiber idiopathic neuropathy, with intra-epidermal fiber density and integrity being used for diagnosing FD in humans. However, the molecular correlates underlying pain sensation in FD remain elusive. Here, we have employed the α-GalA gene KO mouse as a model of FD in rodents to investigate molecular changes in their peripheral nervous system that may account for their algesic symptoms. The α-GalA null mice display neuropathic pain as evidenced by thermal hyperalgesia and mechanical allodynia, with histological analyses showing alterations in cutaneous innervation. Additionally, KO mice showed a decreased and scattered pattern of neuronal terminations consistent with the reduction in neuronal terminations in skin biopsies of patients with small fiber neuropathies. At the molecular level KO animals showed an increase in the expression of TRPV1 and Nav1.8, and a decrease in the expression of TRPM8. Notably, these alterations are observed in young animals. Taken together, our findings imply that the α-GalA KO mouse is a good model in which to study the peripheral small fiber neuropathy exhibited by FD patients, and provides molecular evidence for a hyperexcitability of small nociceptors in FD. PMID:25337704

  11. Pain Related Channels Are Differentially Expressed in Neuronal and Non-Neuronal Cells of Glabrous Skin of Fabry Knockout Male Mice

    PubMed Central

    Lakomá, Jarmila; Rimondini, Roberto; Donadio, Vincenzo; Liguori, Rocco; Caprini, Marco

    2014-01-01

    Fabry disease (FD) is one of the X-linked lysosomal storage disorders caused by deficient functioning of the alpha-galactosidase A (α-GalA) enzyme. The α-GalA deficiency leads to multi-systemic clinical manifestations caused by the preferential accumulation of globotriaosylceramide in the endothelium and vascular smooth muscles. A hallmark symptom of FD patients is peripheral pain that appears in the early stage of the disease. Pain in FD patients is a peripheral small-fiber idiopathic neuropathy, with intra-epidermal fiber density and integrity being used for diagnosing FD in humans. However, the molecular correlates underlying pain sensation in FD remain elusive. Here, we have employed the α-GalA gene KO mouse as a model of FD in rodents to investigate molecular changes in their peripheral nervous system that may account for their algesic symptoms. The α-GalA null mice display neuropathic pain as evidenced by thermal hyperalgesia and mechanical allodynia, with histological analyses showing alterations in cutaneous innervation. Additionally, KO mice showed a decreased and scattered pattern of neuronal terminations consistent with the reduction in neuronal terminations in skin biopsies of patients with small fiber neuropathies. At the molecular level KO animals showed an increase in the expression of TRPV1 and Nav1.8, and a decrease in the expression of TRPM8. Notably, these alterations are observed in young animals. Taken together, our findings imply that the α-GalA KO mouse is a good model in which to study the peripheral small fiber neuropathy exhibited by FD patients, and provides molecular evidence for a hyperexcitability of small nociceptors in FD. PMID:25337704

  12. Neuronal differentiation modulates the dystrophin Dp71d binding to the nuclear matrix

    SciTech Connect

    Rodriguez-Munoz, Rafael; Villarreal-Silva, Marcela; Gonzalez-Ramirez, Ricardo; Garcia-Sierra, Francisco; Mondragon, Monica; Mondragon, Ricardo; Cerna, Joel; Cisneros, Bulmaro

    2008-10-24

    The function of dystrophin Dp71 in neuronal cells remains unknown. To approach this issue, we have selected the PC12 neuronal cell line. These cells express both a Dp71f cytoplasmic variant and a Dp71d nuclear isoform. In this study, we demonstrated by electron and confocal microscopy analyses of in situ nuclear matrices and Western blotting evaluation of cell extracts that Dp71d associates with the nuclear matrix. Interestingly, this binding is modulated during NGF-induced neuronal differentiation of PC12 cells with a twofold increment in the differentiated cells, compared to control cells. Also, distribution of Dp71d along the periphery of the nuclear matrix observed in the undifferentiated cells is replaced by intense fluorescent foci localized in Center of the nucleoskeletal structure. In summary, we revealed that Dp71d is a dynamic component of nuclear matrix that might participate in the nuclear modeling occurring during neuronal differentiation.

  13. Asarone from Acori Tatarinowii Rhizoma Potentiates the Nerve Growth Factor-Induced Neuronal Differentiation in Cultured PC12 Cells: A Signaling Mediated by Protein Kinase A

    PubMed Central

    Lam, Kelly Y. C.; Chen, Jianping; Lam, Candy T. W.; Wu, Qiyun; Yao, Ping; Dong, Tina T. X.; Lin, Huangquan; Tsim, Karl W. K.

    2016-01-01

    Acori Tatarinowii Rhizoma (ATR), the rhizome of Acorus tatarinowii Schott, is being used clinically to treat neurological disorders. The volatile oil of ATR is being considered as an active ingredient. Here, α-asarone and β-asarone, accounting about 95% of ATR oil, were evaluated for its function in stimulating neurogenesis. In cultured PC12 cells, application of ATR volatile oil, α-asarone or β-asarone, stimulated the expression of neurofilaments, a bio-marker for neurite outgrowth, in a concentration-dependent manner. The co-treatment of ATR volatile oil, α-asarone or β-asarone, with low concentration of nerve growth factor (NGF) potentiated the NGF-induced neuronal differentiation in cultured PC12 cells. In addition, application of protein kinase A inhibitors, H89 and KT5720, in cultures blocked the ATR-induced neurofilament expression, as well as the phosphorylation of cAMP-responsive element binding protein (CREB). In the potentiation of NGF-induced signaling in cultured PC12 cells, α-asarone and β-asarone showed synergistic effects. These results proposed the neurite-promoting asarone, or ATR volatile oil, could be useful in finding potential drugs for treating various neurodegenerative diseases, in which neurotrophin deficiency is normally involved. PMID:27685847

  14. Electrophysiological differentiation of new neurons in the olfactory bulb.

    PubMed

    Belluzzi, Ottorino; Benedusi, Mascia; Ackman, James; LoTurco, Joseph J

    2003-11-12

    The subventricular zone produces neuroblasts that migrate to the olfactory bulb (OB) and differentiate into interneurons throughout postnatal life (Altman and Das, 1966; Hinds, 1968; Altman, 1969; Kishi et al., 1990; Luskin, 1993; Lois and Alvarez-Buylla, 1994). Although such postnatally generated interneurons have been characterized morphologically, their physiological differentiation has not been thoroughly described. Combining retroviral-mediated labeling of newly generated neurons with patch-clamp electrophysiology, we demonstrated that soon after new cells enter the layers of the olfactory bulb, they display voltage-dependent currents typical of more mature neurons. We also show that these "newcomers" express functional GABA and glutamate receptor channels, respond synaptically to stimulation of the olfactory nerve, and may establish both axodendritic and dendrodendritic synaptic contacts within the olfactory bulb. These data provide a basic description of the physiology of newly generated cells in the OB and show that such new cells are functional neurons that synaptically integrate into olfactory bulb circuitry soon after their arrival.

  15. The Effects of Controlled Release of Neurotrophin-3 from PCLA Scaffolds on the Survival and Neuronal Differentiation of Transplanted Neural Stem Cells in a Rat Spinal Cord Injury Model

    PubMed Central

    Shi, Bo; Qu, Yanzhen; Huang, Zeyu; Lin, Qiang; Guo, Xiaodong; Pei, Fuxing

    2014-01-01

    Neural stem cells (NSCs) have emerged as a potential source for cell replacement therapy following spinal cord injury (SCI). However, poor survival and low neuronal differentiation remain major obstacles to the use of NSCs. Biomaterials with neurotrophic factors are promising strategies for promoting the proliferation and differentiation of NSCs. Silk fibroin (SF) matrices were demonstrated to successfully deliver growth factors and preserve their potency. In this study, by incorporating NT-3 into a SF coating, we successfully developed NT-3-immobilized scaffolds (membranes and conduits). Sustained release of bioactive NT-3 from the conduits for up to 8 weeks was achieved. Cell viability was confirmed using live/dead staining after 14 days in culture. The efficacy of the immobilized NT-3 was confirmed by assessing NSC neuronal differentiation in vitro. NSC neuronal differentiation was 55.2±4.1% on the NT-3-immobilized membranes, which was significantly higher than that on the NT-3 free membrane. Furthermore, 8 weeks after the NSCs were seeded into conduits and implanted in rats with a transected SCI, the conduit+NT-3+NSCs group achieved higher NSC survival (75.8±15.1%) and neuronal differentiation (21.5±5.2%) compared with the conduit+NSCs group. The animals that received the conduit+NT-3+NSCs treatment also showed improved functional outcomes, as well as increased axonal regeneration. These results indicate the feasibility of fabricating NT-3-immobilized scaffolds using the adsorption of NT-3/SF coating method, as well as the potential of these scaffolds to induce SCI repair by promoting survival and neuronal differentiation of transplanted NSCs. PMID:25215612

  16. Cell cycle markers have different expression and localization patterns in neuron-like PC12 cells and primary hippocampal neurons.

    PubMed

    Negis, Yesim; Unal, Aysegul Yildiz; Korulu, Sirin; Karabay, Arzu

    2011-06-01

    Neuron-like PC12 cells are extensively used in place of neurons in published studies. Aim of this paper has been to compare mRNA and protein expressions of cell cycle markers; cyclinA, B, D, E; Cdk1, 2 and 4; and p27 in post-mitotic primary hippocampal neurons, mitotically active PC12 cells and NGF-differentiated post-mitotic PC12 cells. Contrary to PC12 cells, in neurons, the presence of all these markers was detected only at mRNA level; except for cyclinA, cyclinE and Cdk4, which were detectable also at protein levels. In both NGF-treated PC12 cells and neurons, cyclinE was localized only in the nucleus. In NGF-treated PC12 cells cyclinD and Cdk4 were localized in the nucleus while, in neurons cyclinD expression was not detectable; Cdk4 was localized in the cytoplasm. In neurons, cyclinA was nuclear, whereas in NGF-treated PC12 cells, it was localized in the cell body and along the processes. These results suggest that PC12 cells and primary neurons are different in terms of cell cycle protein expressions and localizations. Thus, it may not be very appropriate to use these cells as neuronal model system in order to understand neuronal physiological activities, upstream of where may lie cell cycle activation triggered events.

  17. Localization of heat shock protein HSPA6 (HSP70B') to sites of transcription in cultured differentiated human neuronal cells following thermal stress.

    PubMed

    Khalouei, Sam; Chow, Ari M; Brown, Ian R

    2014-12-01

    Heat shock proteins (Hsps) are a set of highly conserved proteins that are involved in cellular repair and protective mechanisms. In order to identify potential stress-sensitive sites in differentiated SH-SY5Y human neuronal cells, localization of two inducible members of the HSPA (HSP70) family was investigated, namely HSPA6 (HSP70B') and HSPA1A (HSP70-1). Following heat shock, yellow fluorescent protein (YFP)-tagged HSPA6 and HSPA1A proteins localized to nuclear speckles that are enriched in RNA splicing factors (identified by SC35 and SON marker proteins) and then to the granular component of the nucleolus (identified by nucleophosmin). Subsequently, YFP-HSPA6 protein, but not YFP-HSPA1A, localized to the periphery of nuclear speckles that are sites of RNA transcription. The HSPA6 gene is present in the human genome but not in genomes of rat and mouse. Hence, current animal models of neurodegenerative diseases are lacking a potentially protective member of the HSPA family. Potential stress-sensitive sites were identified in differentiated human SH-SY5Y cells by the localization of HSPA6 (HSP70B') and HSPA1A (HSP70-1) to nuclear components following heat shock. HSPA6 and HSPA1A rapidly moved to nuclear speckles, enriched in RNA splicing factors, then to the granular layer of the nucleolus. Subsequently, HSPA6 exhibited a novel localization not observed for the more widely studied HSPA1A, namely association with the periphery of nuclear speckles that are sites of transcription. HS = heat shock; HSPA6 = HSP70B' protein; HSPA1A = HSP70-1 protein.

  18.  Neuronal Differentiation of Rat Hair Follicle Stem Cells: the Involvement of the Neuroprotective Factor Seladin-1 (DHCR24)

    PubMed Central

    Gilanchi, Samira; Esmaeilzade, Banafshe; Eidi, Akram; Barati, Mahmood; Mehrabi, Soraya; Moghani Ghoroghi, Fatima; Nobakht, Maliheh

    2014-01-01

    Background: The seladin-1 (selective Alzheimer disease indicator-1), also known as DHCR24, is a gene found to be down-regulated in brain region affected by Alzheimer disease (AD). Whereas, hair follicle stem cells (HFSC), which are affected in with neurogenic potential, it might to hypothesize that this multipotent cell compartment is the predominant source of seladin-1. Our aim was to evaluate seladin-1 gene expression in hair follicle stem cells. Methods: In this study, bulge area of male Wistar rat HFSC were cultured and then characterized with Seladin-1 immunocytochemistry and flow cytometry on days 8 to 14. Next, 9-11-day cells were evaluated for seladin-1 gene expression by real-time PCR. Results: Our results indicated that expression of the seladin-1 gene (DHCR24) on days 9, 10, and 11 may contribute to the development of HFSC. However, the expression of this gene on day 11 was more than day 10 and on 10th day was more than day 9. Also, we assessed HFSC on day 14 and demonstrated these cells were positive for β-ш tubulin, and seladin-1 was not expressed in this day. Conclusion: HFSC express seladin-1 and this result demonstrates that these cells might be used to cell therapy for AD in future. PMID:24842139

  19. Impact of 60-GHz millimeter waves on stress and pain-related protein expression in differentiating neuron-like cells.

    PubMed

    Haas, Alexis J; Le Page, Yann; Zhadobov, Maxim; Boriskin, Artem; Sauleau, Ronan; Le Dréan, Yves

    2016-10-01

    Millimeter waves (MMW) will be increasingly used for future wireless telecommunications. Previous studies on skin keratinocytes showed that MMW could impact the mRNA expression of Transient Receptor Potential cation channel subfamily Vanilloid, member 2 (TRPV2). Here, we investigated the effect of MMW exposure on this marker, as well as on other membrane receptors such as Transient Receptor Potential cation channel subfamily Vanilloid, member 1 (TRPV1) and purinergic receptor P2X, ligand-gated ion channel, 3 (P2 × 3). We exposed the Neuroscreen-1 cell line (a PC12 subclone), in order to evaluate if acute MMW exposures could impact expression of these membrane receptors at the protein level. Proteotoxic stress-related chaperone protein Heat Shock Protein 70 (HSP70) expression level was also assessed. We used an original high-content screening approach, based on fluorescence microscopy, to allow cell-by-cell analysis and to detect any cell sub-population responding to exposure. Immunocytochemistry was done after 24 h MMW exposure of cells at 60.4 GHz, with an incident power density of 10 mW/cm(2) . Our results showed no impact of MMW exposure on protein expressions of HSP70, TRPV1, TRPV2, and P2 × 3. Moreover, no specific cell sub-populations were found to express one of the studied markers at a different level, compared to the rest of the cell populations. However, a slight insignificant increase in HSP70 expression and an increase in protein expression variability within cell population were observed in exposed cells, but controls showed that this was related to thermal effect. Bioelectromagnetics. 37:444-454, 2016. © 2016 Wiley Periodicals, Inc. PMID:27483046

  20. Impact of 60-GHz millimeter waves on stress and pain-related protein expression in differentiating neuron-like cells.

    PubMed

    Haas, Alexis J; Le Page, Yann; Zhadobov, Maxim; Boriskin, Artem; Sauleau, Ronan; Le Dréan, Yves

    2016-10-01

    Millimeter waves (MMW) will be increasingly used for future wireless telecommunications. Previous studies on skin keratinocytes showed that MMW could impact the mRNA expression of Transient Receptor Potential cation channel subfamily Vanilloid, member 2 (TRPV2). Here, we investigated the effect of MMW exposure on this marker, as well as on other membrane receptors such as Transient Receptor Potential cation channel subfamily Vanilloid, member 1 (TRPV1) and purinergic receptor P2X, ligand-gated ion channel, 3 (P2 × 3). We exposed the Neuroscreen-1 cell line (a PC12 subclone), in order to evaluate if acute MMW exposures could impact expression of these membrane receptors at the protein level. Proteotoxic stress-related chaperone protein Heat Shock Protein 70 (HSP70) expression level was also assessed. We used an original high-content screening approach, based on fluorescence microscopy, to allow cell-by-cell analysis and to detect any cell sub-population responding to exposure. Immunocytochemistry was done after 24 h MMW exposure of cells at 60.4 GHz, with an incident power density of 10 mW/cm(2) . Our results showed no impact of MMW exposure on protein expressions of HSP70, TRPV1, TRPV2, and P2 × 3. Moreover, no specific cell sub-populations were found to express one of the studied markers at a different level, compared to the rest of the cell populations. However, a slight insignificant increase in HSP70 expression and an increase in protein expression variability within cell population were observed in exposed cells, but controls showed that this was related to thermal effect. Bioelectromagnetics. 37:444-454, 2016. © 2016 Wiley Periodicals, Inc.

  1. Evidence for heterogeneous groups of neuronal differentiation of Ewing's sarcoma.

    PubMed

    Hara, S; Adachi, Y; Kaneko, Y; Fujimoto, J; Hata, J

    1991-12-01

    We have investigated the capability of differentiation of Ewing's sarcoma (ES) towards a neuronal direction through the establishment of four extraosseous ES cell lines and by in vitro stimulation with dibutyryladenosine cyclic monophosphate (db-cAMP) of eight ES lines. All except one of the lines expressed the molecule defined by 5C11, the antibody specifically reactive with ES. Two ES lines expressed a 200 kilodalton (kD) neurofilament protein (NFP) although their original tumours were negative for NFP. Elongation of cytoplasmic processes and increased NFP expression were observed after db-cAMP treatment of these lines and microtubules in the cytoplasmic processes were ultrastructurally demonstrated. Six lines were NFP negative, but three lines changed their morphology after induction of 200 kD NFP expression by db-cAMP treatment. The other three showed no definitive differentiation after db-cAMP treatment. Chromosomal analysis of the new ES lines showed the typical t(11;22) in one line and a +der(22) in two lines. No correlation was observed between the chromosomal abnormality and the differentiation capability. We conclude that ES is a heterogeneous group of tumours with respect to capability of differentiation into the neuronal lineage, but it is clearly distinguished from peripheral primitive neuroectodermal tumours by its 5C11 reactivity.

  2. Evidence for heterogeneous groups of neuronal differentiation of Ewing's sarcoma.

    PubMed Central

    Hara, S.; Adachi, Y.; Kaneko, Y.; Fujimoto, J.; Hata, J.

    1991-01-01

    We have investigated the capability of differentiation of Ewing's sarcoma (ES) towards a neuronal direction through the establishment of four extraosseous ES cell lines and by in vitro stimulation with dibutyryladenosine cyclic monophosphate (db-cAMP) of eight ES lines. All except one of the lines expressed the molecule defined by 5C11, the antibody specifically reactive with ES. Two ES lines expressed a 200 kilodalton (kD) neurofilament protein (NFP) although their original tumours were negative for NFP. Elongation of cytoplasmic processes and increased NFP expression were observed after db-cAMP treatment of these lines and microtubules in the cytoplasmic processes were ultrastructurally demonstrated. Six lines were NFP negative, but three lines changed their morphology after induction of 200 kD NFP expression by db-cAMP treatment. The other three showed no definitive differentiation after db-cAMP treatment. Chromosomal analysis of the new ES lines showed the typical t(11;22) in one line and a +der(22) in two lines. No correlation was observed between the chromosomal abnormality and the differentiation capability. We conclude that ES is a heterogeneous group of tumours with respect to capability of differentiation into the neuronal lineage, but it is clearly distinguished from peripheral primitive neuroectodermal tumours by its 5C11 reactivity. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:1764362

  3. Co-infusion of autologous adipose tissue derived neuronal differentiated mesenchymal stem cells and bone marrow derived hematopoietic stem cells, a viable therapy for post-traumatic brachial plexus injury: a case report.

    PubMed

    Thakkar, Umang G; Vanikar, Aruna V; Trivedi, Hargovind L

    2014-01-01

    Stem cell therapy is emerging as a viable approach in regenerative medicine. A 31-year-old male with brachial plexus injury had complete sensory-motor loss since 16 years with right pseudo-meningocele at C5-D1 levels and extra-spinal extension up to C7-D1, with avulsion on magnetic resonance imaging and irreversible damage. We generated adipose tissue derived neuronal differentiated mesenchymal stem cells (N-AD-MSC) and bone marrow derived hematopoietic stem cells (HSC-BM). Neuronal stem cells expressed β-3 tubulin and glial fibrillary acid protein which was confirmed on immunofluorescence. On day 14, 2.8 ml stem cell inoculum was infused under local anesthesia in right brachial plexus sheath by brachial block technique under ultrasonography guidance with a 1.5-inch-long 23 gauge needle. Nucleated cell count was 2 × 10 4 /μl, CD34+ was 0.06%, and CD45-/90+ and CD45-/73+ were 41.63% and 20.36%, respectively. No untoward effects were noted. He has sustained recovery with re-innervation over a follow-up of 4 years documented on electromyography-nerve conduction velocity study. PMID:25116721

  4. Excitatory Projection Neuron Subtypes Differentially Control the Distribution of Local Inhibitory Interneurons in the Cerebral Cortex

    PubMed Central

    Lodato, Simona; Rouaux, Caroline; Quast, Kathleen B.; Jantrachotechatchawan, Chanati; Studer, Michèle; Hensch, Takao K.; Arlotta, Paola

    2011-01-01

    In the mammalian cerebral cortex, the developmental events governing the integration of excitatory projection neurons and inhibitory interneurons into balanced local circuitry are poorly understood. We report that different subtypes of projection neurons uniquely and differentially determine the laminar distribution of cortical interneurons. We find that in Fezf2−/− cortex, the exclusive absence of subcerebral projection neurons and their replacement by callosal projection neurons cause distinctly abnormal lamination of interneurons and altered GABAergic inhibition. In addition, experimental generation of either corticofugal neurons or callosal neurons below the cortex is sufficient to recruit cortical interneurons to these ectopic locations. Strikingly, the identity of the projection neurons generated, rather than strictly their birthdate, determines the specific types of interneurons recruited. These data demonstrate that in the neocortex individual populations of projection neurons cell-extrinsically control the laminar fate of interneurons and the assembly of local inhibitory circuitry. PMID:21338885

  5. Differential transcriptional profiling of damaged and intact adjacent dorsal root ganglia neurons in neuropathic pain.

    PubMed

    Reinhold, A K; Batti, L; Bilbao, D; Buness, A; Rittner, H L; Heppenstall, P A

    2015-01-01

    Neuropathic pain, caused by a lesion in the somatosensory system, is a severely impairing mostly chronic disease. While its underlying molecular mechanisms are not thoroughly understood, neuroimmune interactions as well as changes in the pain pathway such as sensitization of nociceptors have been implicated. It has been shown that not only are different cell types involved in generation and maintenance of neuropathic pain, like neurons, immune and glial cells, but, also, intact adjacent neurons are relevant to the process. Here, we describe an experimental approach to discriminate damaged from intact adjacent neurons in the same dorsal root ganglion (DRG) using differential fluorescent neuronal labelling and fluorescence-activated cell sorting (FACS). Two fluorescent tracers, Fluoroemerald (FE) and 1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI), were used, whose properties allow us to distinguish between damaged and intact neurons. Subsequent sorting permitted transcriptional analysis of both groups. Results and qPCR validation show a strong regulation in damaged neurons versus contralateral controls as well as a moderate regulation in adjacent neurons. Data for damaged neurons reveal an mRNA expression pattern consistent with established upregulated genes like galanin, which supports our approach. Moreover, novel genes were found strongly regulated such as corticotropin-releasing hormone (CRH), providing novel targets for further research. Differential fluorescent neuronal labelling and sorting allows for a clear distinction between primarily damaged neuropathic neurons and "bystanders," thereby facilitating a more detailed understanding of their respective roles in neuropathic processes in the DRG. PMID:25880204

  6. Differential transcriptional profiling of damaged and intact adjacent dorsal root ganglia neurons in neuropathic pain.

    PubMed

    Reinhold, A K; Batti, L; Bilbao, D; Buness, A; Rittner, H L; Heppenstall, P A

    2015-01-01

    Neuropathic pain, caused by a lesion in the somatosensory system, is a severely impairing mostly chronic disease. While its underlying molecular mechanisms are not thoroughly understood, neuroimmune interactions as well as changes in the pain pathway such as sensitization of nociceptors have been implicated. It has been shown that not only are different cell types involved in generation and maintenance of neuropathic pain, like neurons, immune and glial cells, but, also, intact adjacent neurons are relevant to the process. Here, we describe an experimental approach to discriminate damaged from intact adjacent neurons in the same dorsal root ganglion (DRG) using differential fluorescent neuronal labelling and fluorescence-activated cell sorting (FACS). Two fluorescent tracers, Fluoroemerald (FE) and 1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI), were used, whose properties allow us to distinguish between damaged and intact neurons. Subsequent sorting permitted transcriptional analysis of both groups. Results and qPCR validation show a strong regulation in damaged neurons versus contralateral controls as well as a moderate regulation in adjacent neurons. Data for damaged neurons reveal an mRNA expression pattern consistent with established upregulated genes like galanin, which supports our approach. Moreover, novel genes were found strongly regulated such as corticotropin-releasing hormone (CRH), providing novel targets for further research. Differential fluorescent neuronal labelling and sorting allows for a clear distinction between primarily damaged neuropathic neurons and "bystanders," thereby facilitating a more detailed understanding of their respective roles in neuropathic processes in the DRG.

  7. Differential Transcriptional Profiling of Damaged and Intact Adjacent Dorsal Root Ganglia Neurons in Neuropathic Pain

    PubMed Central

    Reinhold, A. K.; Batti, L.; Bilbao, D.; Buness, A.; Rittner, H. L.; Heppenstall, P. A.

    2015-01-01

    Neuropathic pain, caused by a lesion in the somatosensory system, is a severely impairing mostly chronic disease. While its underlying molecular mechanisms are not thoroughly understood, neuroimmune interactions as well as changes in the pain pathway such as sensitization of nociceptors have been implicated. It has been shown that not only are different cell types involved in generation and maintenance of neuropathic pain, like neurons, immune and glial cells, but, also, intact adjacent neurons are relevant to the process. Here, we describe an experimental approach to discriminate damaged from intact adjacent neurons in the same dorsal root ganglion (DRG) using differential fluorescent neuronal labelling and fluorescence-activated cell sorting (FACS). Two fluorescent tracers, Fluoroemerald (FE) and 1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI), were used, whose properties allow us to distinguish between damaged and intact neurons. Subsequent sorting permitted transcriptional analysis of both groups. Results and qPCR validation show a strong regulation in damaged neurons versus contralateral controls as well as a moderate regulation in adjacent neurons. Data for damaged neurons reveal an mRNA expression pattern consistent with established upregulated genes like galanin, which supports our approach. Moreover, novel genes were found strongly regulated such as corticotropin-releasing hormone (CRH), providing novel targets for further research. Differential fluorescent neuronal labelling and sorting allows for a clear distinction between primarily damaged neuropathic neurons and “bystanders,” thereby facilitating a more detailed understanding of their respective roles in neuropathic processes in the DRG. PMID:25880204

  8. p62 modulates Akt activity via association with PKC{zeta} in neuronal survival and differentiation

    SciTech Connect

    Joung, Insil . E-mail: ijoung@hanseo.ac.kr; Kim, Hak Jae; Kwon, Yunhee Kim . E-mail: kimyh@khu.ac.kr

    2005-08-26

    p62 is a ubiquitously expressed phosphoprotein that interacts with a number of signaling molecules and a major component of neurofibrillary tangles in the brain of Alzheimer's disease patients. It has been implicated in important cellular functions such as cell proliferation and anti-apoptotic pathways. In this study, we have addressed the potential role of p62 during neuronal differentiation and survival using HiB5, a rat neuronal progenitor cell. We generated a recombinant adenovirus encoding T7-epitope tagged p62 to reliably transfer p62 cDNA into the neuronal cells. The results show that an overexpression of p62 led not only to neuronal differentiation, but also to decreased cell death induced by serum withdrawal in HiB5 cells. In this process p62-dependent Akt phosphorylation occurred via the release of Akt from PKC{zeta} by association of p62 and PKC{zeta}, which is known as a negative regulator of Akt activation. These findings indicate that p62 facilitates cell survival through novel signaling cascades that result in Akt activation. Furthermore, we found that p62 expression was induced during neuronal differentiation. Taken together, the data suggest p62 is a regulator of neuronal cell survival and differentiation.

  9. Onset of electrical excitability during a period of circus plasma membrane movements in differentiating Xenopus neurons.

    PubMed

    Olson, E C

    1996-08-15

    Living neurons are usually first identifiable in primary cultures at the time of neurite initiation, and studies of excitability have been restricted largely to the subsequent period. A morphological early marker is described that identifies neurons for whole-cell voltage-clamp recordings before neurite initiation. Video time-lapse recordings of cultured cells dissociated from neurectoderm of Xenopus neural plate stage embryos reveal cells demonstrating circus movements, in which blebs of plasma membrane propagate around the cell circumference within a period of several minutes. All neurons demonstrate circus movements before morphological differentiation; the fraction of cells exhibiting circus movements that differentiate morphologically depends on the substrate on which they are cultured. Blockade of circus activity with cytochalasin B does not prevent neuronal differentiation. Circus movements are not neurectoderm-specific because they similarly predict differentiation of myocytes developing in mesodermal cultures. Initially inexcitable, neurons develop voltage-dependent K+, Na+, and Ca2+ currents during the period of several hours in which they exhibit circus movements. The early development of depolarization-induced elevations of [Ca2+]i several hours before morphological differentiation corresponds to the previously described onset of functionally significant spontaneous elevations of [Ca2+]i in these neurons and demonstrates a role for early expression of voltage-dependent ion channels.

  10. Differential Tiam1/Rac1 activation in hippocampal and cortical neurons mediates differential spine shrinkage in response to oxygen/glucose deprivation

    PubMed Central

    Blanco-Suárez, Elena; Fiuza, Maria; Liu, Xun; Chakkarapani, Elavazhagan; Hanley, Jonathan G

    2014-01-01

    Distinct neuronal populations show differential sensitivity to global ischemia, with hippocampal CA1 neurons showing greater vulnerability compared to cortical neurons. The mechanisms that underlie differential vulnerability are unclear, and we hypothesize that intrinsic differences in neuronal cell biology are involved. Dendritic spine morphology changes in response to ischemic insults in vivo, but cell type-specific differences and the molecular mechanisms leading to such morphologic changes are unexplored. To directly compare changes in spine size in response to oxygen/glucose deprivation (OGD) in cortical and hippocampal neurons, we used separate and equivalent cultures of each cell type. We show that cortical neurons exhibit significantly greater spine shrinkage compared to hippocampal neurons. Rac1 is a Rho-family GTPase that regulates the actin cytoskeleton and is involved in spine dynamics. We show that Rac1 and the Rac guanine nucleotide exchange factor (GEF) Tiam1 are differentially activated by OGD in hippocampal and cortical neurons. Hippocampal neurons express more Tiam1 than cortical neurons, and reducing Tiam1 expression in hippocampal neurons by shRNA enhances OGD-induced spine shrinkage. Tiam1 knockdown also reduces hippocampal neuronal vulnerability to OGD. This work defines fundamental differences in signalling pathways that regulate spine morphology in distinct neuronal populations that may have a role in the differential vulnerability to ischemia. PMID:25248834

  11. Effect of matrix composition on differentiation of nestin-positive neural progenitors from circulation into neurons

    NASA Astrophysics Data System (ADS)

    Jose, Anumol; Krishnan, Lissy K.

    2010-06-01

    The human peripheral blood mononuclear cell has a mixture of progenitor cells with potential to differentiate into a wide range of lineages. The ability of hematopoietic tissue-derived adult stem cells to differentiate into neural progenitor cells offers an alternative to embryonic stem cells as a viable source for cell transplantation therapies to cure neurodegenerative diseases. This approach could lead to the use of autologous progenitors from blood circulation; however, due to the limited numbers available, in vitro cell expansion may be indispensable. In addition, for successful transplantation there is the requirement of a delivery matrix on which cells can survive and differentiate. In this context we carried out this study to identify a suitable biodegradable matrix on which progenitor cells can home, multiply and differentiate. We designed different compositions of the biomimetic matrix containing fibrin, fibronectin, gelatin, growth factors, laminin and hyaluronic acid. The attached cells expressed proliferation markers in initial periods of culture and between days 6 and 9 in culture they differentiated into neurons and/or astrocytes. The differentiation of progenitors into neurons and asterocyte on the composed matrix was established by morphological and immunochemical analysis. Flow cytometric analysis of cells in culture was employed to track development of neurons which expressed an early marker β-tubulin3 and a terminal marker microtubule-associated protein-2 at a later culture period. In vitro experiments indicate that a highly specific niche consisting of various components of the extracellular matrix, including hyaluronic acid, promote cell homing, survival and differentiation.

  12. Characterization of Differentiated SH-SY5Y as Neuronal Screening Model Reveals Increased Oxidative Vulnerability.

    PubMed

    Forster, J I; Köglsberger, S; Trefois, C; Boyd, O; Baumuratov, A S; Buck, L; Balling, R; Antony, P M A

    2016-06-01

    The immortalized and proliferative cell line SH-SY5Y is one of the most commonly used cell lines in neuroscience and neuroblastoma research. However, undifferentiated SH-SY5Y cells share few properties with mature neurons. In this study, we present an optimized neuronal differentiation protocol for SH-SY5Y that requires only two work steps and 6 days. After differentiation, the cells present increased levels of ATP and plasma membrane activity but reduced expression of energetic stress response genes. Differentiation results in reduced mitochondrial membrane potential and decreased robustness toward perturbations with 6-hydroxydopamine. We are convinced that the presented differentiation method will leverage genetic and chemical high-throughput screening projects targeting pathways that are involved in the selective vulnerability of neurons with high energetic stress levels.

  13. Characterization of Differentiated SH-SY5Y as Neuronal Screening Model Reveals Increased Oxidative Vulnerability

    PubMed Central

    Forster, J. I.; Köglsberger, S.; Trefois, C.; Boyd, O.; Baumuratov, A. S.; Buck, L.; Balling, R.; Antony, P. M. A.

    2016-01-01

    The immortalized and proliferative cell line SH-SY5Y is one of the most commonly used cell lines in neuroscience and neuroblastoma research. However, undifferentiated SH-SY5Y cells share few properties with mature neurons. In this study, we present an optimized neuronal differentiation protocol for SH-SY5Y that requires only two work steps and 6 days. After differentiation, the cells present increased levels of ATP and plasma membrane activity but reduced expression of energetic stress response genes. Differentiation results in reduced mitochondrial membrane potential and decreased robustness toward perturbations with 6-hydroxydopamine. We are convinced that the presented differentiation method will leverage genetic and chemical high-throughput screening projects targeting pathways that are involved in the selective vulnerability of neurons with high energetic stress levels. PMID:26738520

  14. Neuronal cell lines as model dorsal root ganglion neurons

    PubMed Central

    Yin, Kathleen; Baillie, Gregory J

    2016-01-01

    Background Dorsal root ganglion neuron-derived immortal cell lines including ND7/23 and F-11 cells have been used extensively as in vitro model systems of native peripheral sensory neurons. However, while it is clear that some sensory neuron-specific receptors and ion channels are present in these cell lines, a systematic comparison of the molecular targets expressed by these cell lines with those expressed in intact peripheral neurons is lacking. Results In this study, we examined the expression of RNA transcripts in the human neuroblastoma-derived cell line, SH-SY5Y, and two dorsal root ganglion hybridoma cell lines, F-11 and ND7/23, using Illumina next-generation sequencing, and compared the results with native whole murine dorsal root ganglions. The gene expression profiles of these three cell lines did not resemble any specific defined dorsal root ganglion subclass. The cell lines lacked many markers for nociceptive sensory neurons, such as the Transient receptor potential V1 gene, but expressed markers for both myelinated and unmyelinated neurons. Global gene ontology analysis on whole dorsal root ganglions and cell lines showed similar enrichment of biological process terms across all samples. Conclusions This paper provides insights into the receptor repertoire expressed in common dorsal root ganglion neuron-derived cell lines compared with whole murine dorsal root ganglions, and illustrates the limits and potentials of these cell lines as tools for neuropharmacological exploration. PMID:27130590

  15. GATA2 IS REQUIRED FOR MIGRATION AND DIFFERENTIATION OF RETINORECIPIENT NEURONS IN THE SUPERIOR COLLICULUS

    PubMed Central

    Willett, Ryan T.; Greene, Lloyd A.

    2011-01-01

    The superior colliculus (SC)/optic tectum of the dorsal mesencephalon plays a major role in responses to visual input, yet regulation of neuronal differentiation within this layered structure is only partially understood. Here, we show that the zinc finger transcription factor Gata2 is required for normal SC development. Starting at e15 (corresponding to the times at which neurons of the outer and intermediate layers of the SC are generated), Gata2 is transiently expressed in the rat embryonic dorsal mesencephalon within a restricted region between proliferating cells of the ventricular zone and the deepest neuronal layers of the developing SC. The Gata2 positive cells are post-mitotic and lack markers of differentiated neurons, but express markers for immature neuronal precursors including Ascl1 and Pax3/7. In utero electroporation with Gata2 shRNAs at e16 into cells along the dorsal mesencephalic ventricle interferes with their normal migration into the SC and maintains them in a state characterized by retention of Pax3 expression and the absence of mature neuronal markers. Collectively, these findings indicate that Gata2 plays a required role in the transition of post-mitotic neuronal precursor cells of the retinorecipient layers of the SC into mature neurons and that loss of Gata2 arrests them at an intermediate stage of differentiation. PMID:21430145

  16. Sequential transcriptional waves direct the differentiation of newborn neurons in the mouse neocortex.

    PubMed

    Telley, Ludovic; Govindan, Subashika; Prados, Julien; Stevant, Isabelle; Nef, Serge; Dermitzakis, Emmanouil; Dayer, Alexandre; Jabaudon, Denis

    2016-03-25

    During corticogenesis, excitatory neurons are born from progenitors located in the ventricular zone (VZ), from where they migrate to assemble into circuits. How neuronal identity is dynamically specified upon progenitor division is unknown. Here, we study this process using a high-temporal-resolution technology allowing fluorescent tagging of isochronic cohorts of newborn VZ cells. By combining this in vivo approach with single-cell transcriptomics in mice, we identify and functionally characterize neuron-specific primordial transcriptional programs as they dynamically unfold. Our results reveal early transcriptional waves that instruct the sequence and pace of neuronal differentiation events, guiding newborn neurons toward their final fate, and contribute to a road map for the reverse engineering of specific classes of cortical neurons from undifferentiated cells. PMID:26940868

  17. The Drosophila Transcription Factor Dimmed Affects Neuronal Growth and Differentiation in Multiple Ways Depending on Neuron Type and Developmental Stage

    PubMed Central

    Liu, Yiting; Luo, Jiangnan; Nässel, Dick R.

    2016-01-01

    Growth of postmitotic neurons occurs during different stages of development, including metamorphosis, and may also be part of neuronal plasticity and regeneration. Recently we showed that growth of post-mitotic neuroendocrine cells expressing the basic helix loop helix (bHLH) transcription factor Dimmed (Dimm) in Drosophila could be regulated by insulin/IGF signaling and the insulin receptor (dInR). Dimm is also known to confer a secretory phenotype to neuroendocrine cells and can be part of a combinatorial code specifying terminal differentiation in peptidergic neurons. To further understand the mechanisms of Dimm function we ectopically expressed Dimm or Dimm together with dInR in a wide range of Dimm positive and Dimm negative peptidergic neurons, sensory neurons, interneurons, motor neurons, and gut endocrine cells. We provide further evidence that dInR mediated cell growth occurs in a Dimm dependent manner and that one source of insulin-like peptide (DILP) for dInR mediated cell growth in the CNS is DILP6 from glial cells. Expressing both Dimm and dInR in Dimm negative neurons induced growth of cell bodies, whereas dInR alone did not. We also found that Dimm alone can regulate cell growth depending on specific cell type. This may be explained by the finding that the dInR is a direct target of Dimm. Conditional gene targeting experiments showed that Dimm alone could affect cell growth in certain neuron types during metamorphosis or in the adult stage. Another important finding was that ectopic Dimm inhibits apoptosis of several types of neurons normally destined for programmed cell death (PCD). Taken together our results suggest that Dimm plays multiple transcriptional roles at different developmental stages in a cell type-specific manner. In some cell types ectopic Dimm may act together with resident combinatorial code transcription factors and affect terminal differentiation, as well as act in transcriptional networks that participate in long term maintenance

  18. Leukemia inhibitory factor (LIF) enhances MAP2 + and HUC/D + neurons and influences neurite extension during differentiation of neural progenitors derived from human embryonic stem cells.

    EPA Science Inventory

    Leukemia Inhibitory Factor (L1F), a member of the Interleukin 6 cytokine family, has a role in differentiation of Human Neural Progenitor (hNP) cells in vitro. hNP cells, derived from Human Embryonic Stem (hES) cells, have an unlimited capacity for self-renewal in monolayer cultu...

  19. Prostate Cancer and Neuroendocrine Differentiation: More Neuronal, Less Endocrine?

    PubMed Central

    Grigore, Alexandru Dan; Ben-Jacob, Eshel; Farach-Carson, Mary C.

    2015-01-01

    Neuroendocrine differentiation (NED) marks a structural and functional feature of certain cancers, including prostate cancer (PCa), whereby the malignant tissue contains a significant proportion of cells displaying neuronal, endocrine, or mixed features. NED cells produce, and can secrete, a cocktail of mediators commonly encountered in the nervous system, which may stimulate and coordinate cancer growth. In PCa, NED appears during advanced stages, subsequent to treatment, and accompanies treatment resistance and poor prognosis. However, the term “neuroendocrine” in this context is intrinsically vague. This article seeks to provide a framework on which a unified view of NED might emerge. First, we review the mutually beneficial interplay between PCa and neural structures, mainly supported by cell biology experiments and neurological conditions. Next, we address the correlations between PCa and neural functions, as described in the literature. Based upon the integration of clinical and basic observations, we suggest that it is legitimate to seek for true neural differentiation, or neuromimicry, in cancer progression, most notably in PCa cells exhibiting what is commonly described as NED. PMID:25785244

  20. A natural diarylheptanoid promotes neuronal differentiation via activating ERK and PI3K-Akt dependent pathways.

    PubMed

    Tang, G; Dong, X; Huang, X; Huang, X-J; Liu, H; Wang, Y; Ye, W-C; Shi, L

    2015-09-10

    Neuronal differentiation is a critical developmental process that determines accurate synaptic connection and circuit wiring. A wide variety of naturally occurring compounds have been shown as promising drug leads for the generation and differentiation of neurons. Here we report that a diarylheptanoid from the plant Alpinia officinarum, 7-(4-hydroxyphenyl)-1-phenyl-4E-hepten-3-one (Cpd 1), exhibited potent activities in neuronal differentiation and neurite outgrowth. Cpd 1 induced differentiation of neuroblastoma Neuro-2a cells into a neuron-like morphology, and accelerated the establishment of axon-dendrite polarization of cultured hippocampal neurons. Moreover, Cpd 1 promoted neurite extension in both Neuro-2a cells and neurons. We showed that the effects of Cpd 1 on neuronal differentiation and neurite growth were specifically dependent on the activation of extracellular signal-regulated kinases (ERKs) and phosphoinositide 3-kinase (PI3K)-Akt signaling pathways. Importantly, intraperitoneal administration of Cpd 1 promoted the differentiation of new-born progenitor cells into mature neurons in the adult hippocampal dentate gyrus. Collectively, this study identifies a naturally occurring diarylheptanoid with beneficial effects on neuronal differentiation and neurite outgrowth in vitro and in vivo.

  1. Neurons within the same network independently achieve conserved output by differentially balancing variable conductance magnitudes.

    PubMed

    Ransdell, Joseph L; Nair, Satish S; Schulz, David J

    2013-06-12

    Biological and theoretical evidence suggest that individual neurons may achieve similar outputs by differentially balancing variable underlying ionic conductances. Despite the substantial amount of data consistent with this idea, a direct biological demonstration that cells with conserved output, particularly within the same network, achieve these outputs via different solutions has been difficult to achieve. Here we demonstrate definitively that neurons from native neural networks with highly similar output achieve this conserved output by differentially tuning underlying conductance magnitudes. Multiple motor neurons of the crab (Cancer borealis) cardiac ganglion have highly conserved output within a preparation, despite showing a 2-4-fold range of conductance magnitudes. By blocking subsets of these currents, we demonstrate that the remaining conductances become unbalanced, causing disparate output as a result. Therefore, as strategies to understand neuronal excitability become increasingly sophisticated, it is important that such variability in excitability of neurons, even among those within the same individual, is taken into account. PMID:23761890

  2. Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons.

    PubMed

    Machado, Carolina Barcellos; Kanning, Kevin C; Kreis, Patricia; Stevenson, Danielle; Crossley, Martin; Nowak, Magdalena; Iacovino, Michelina; Kyba, Michael; Chambers, David; Blanc, Eric; Lieberam, Ivo

    2014-02-01

    Air breathing is an essential motor function for vertebrates living on land. The rhythm that drives breathing is generated within the central nervous system and relayed via specialised subsets of spinal motor neurons to muscles that regulate lung volume. In mammals, a key respiratory muscle is the diaphragm, which is innervated by motor neurons in the phrenic nucleus. Remarkably, relatively little is known about how this crucial subtype of motor neuron is generated during embryogenesis. Here, we used direct differentiation of motor neurons from mouse embryonic stem cells as a tool to identify genes that direct phrenic neuron identity. We find that three determinants, Pou3f1, Hoxa5 and Notch, act in combination to promote a phrenic neuron molecular identity. We show that Notch signalling induces Pou3f1 in developing motor neurons in vitro and in vivo. This suggests that the phrenic neuron lineage is established through a local source of Notch ligand at mid-cervical levels. Furthermore, we find that the cadherins Pcdh10, which is regulated by Pou3f1 and Hoxa5, and Cdh10, which is controlled by Pou3f1, are both mediators of like-like clustering of motor neuron cell bodies. This specific Pcdh10/Cdh10 activity might provide the means by which phrenic neurons are assembled into a distinct nucleus. Our study provides a framework for understanding how phrenic neuron identity is conferred and will help to generate this rare and inaccessible yet vital neuronal subtype directly from pluripotent stem cells, thus facilitating subsequent functional investigations.

  3. Bradykinin-induced inhibition of proliferation rate during neurosphere differentiation: consequence or cause of neuronal enrichment?

    PubMed

    Pillat, Micheli M; Cheffer, Arquimedes; de Andrade, Cinthia M; Morsch, Vera M; Schetinger, Maria R C; Ulrich, Henning

    2015-10-01

    Neural stem cells proliferate and differentiate into neurons and glial cells, being responsible for embryonic and postnatal development of the central nervous system (CNS) as well as for regeneration in the adult brain. These cells also play a key role in maintaining the physiological integrity of the CNS in face of injury or disease. The previous study has demonstrated that bradykinin (BK) treatment simultaneously induces neuronal enrichment (indicating that BK contributes to neurogenesis) and reduced proliferation rates during in vitro differentiation of rat embryonic telencephalon neural precursor cells (NPCs). Here, we provide a mechanism for the unresolved question whether (i) the low rate of proliferation is owed to enhanced neurogenesis or, conversely, (ii) the alteration of the population ratio could result from low proliferation of NPCs and glial cells. In agreement with the previous study, BK promoted neuron-specific β3-tubulin and MAP2 expression in differentiating embryonic mouse neurospheres, whereas glial protein expression and global proliferation rates decreased. Furthermore, BK augmented the global frequency of cells in G0 -phase of cell cycle after differentiation. Heterogeneous cell populations were observed at this stage, including neurons that always remaining a quiescent state (G0 -phase). It is noteworthy that BK did not interfere with proliferation of any particular cell type, evidenced by coimmunostaining for nestin, β3-tubulin, glial fibrillary acidic protein (GFAP), and 5-ethynyl-2'-deoxyuridine (EdU). Thus, we conclude that neuronal enrichment is owing only to the fostering of neurogenesis, and that the low proliferation rate on the seventh day of differentiation is a consequence and not the cause of BK-induced neuronal enrichment.

  4. Small Molecules Greatly Improve Conversion of Human-Induced Pluripotent Stem Cells to the Neuronal Lineage

    PubMed Central

    Mak, Sally K.; Huang, Y. Anne; Iranmanesh, Shifteh; Vangipuram, Malini; Sundararajan, Ramya; Nguyen, Loan; Langston, J. William; Schüle, Birgitt

    2012-01-01

    Efficient in vitro differentiation into specific cell types is more important than ever after the breakthrough in nuclear reprogramming of somatic cells and its potential for disease modeling and drug screening. Key success factors for neuronal differentiation are the yield of desired neuronal marker expression, reproducibility, length, and cost. Three main neuronal differentiation approaches are stromal-induced neuronal differentiation, embryoid body (EB) differentiation, and direct neuronal differentiation. Here, we describe our neurodifferentiation protocol using small molecules that very efficiently promote neural induction in a 5-stage EB protocol from six induced pluripotent stem cells (iPSC) lines from patients with Parkinson's disease and controls. This protocol generates neural precursors using Dorsomorphin and SB431542 and further maturation into dopaminergic neurons by replacing sonic hedgehog with purmorphamine or smoothened agonist. The advantage of this approach is that all patient-specific iPSC lines tested in this study were successfully and consistently coaxed into the neural lineage. PMID:22567022

  5. Afadin Regulates Puncta Adherentia Junction Formation and Presynaptic Differentiation in Hippocampal Neurons

    PubMed Central

    Toyoshima, Daisaku; Mandai, Kenji; Maruo, Tomohiko; Supriyanto, Irwan; Togashi, Hideru; Inoue, Takahito; Mori, Masahiro; Takai, Yoshimi

    2014-01-01

    The formation and remodeling of mossy fiber-CA3 pyramidal cell synapses in the stratum lucidum of the hippocampus are implicated in the cellular basis of learning and memory. Afadin and its binding cell adhesion molecules, nectin-1 and nectin-3, together with N-cadherin, are concentrated at puncta adherentia junctions (PAJs) in these synapses. Here, we investigated the roles of afadin in PAJ formation and presynaptic differentiation in mossy fiber-CA3 pyramidal cell synapses. At these synapses in the mice in which the afadin gene was conditionally inactivated before synaptogenesis by using nestin-Cre mice, the immunofluorescence signals for the PAJ components, nectin-1, nectin-3 and N-cadherin, disappeared almost completely, while those for the presynaptic components, VGLUT1 and bassoon, were markedly decreased. In addition, these signals were significantly decreased in cultured afadin-deficient hippocampal neurons. Furthermore, the interevent interval of miniature excitatory postsynaptic currents was prolonged in the cultured afadin-deficient hippocampal neurons compared with control neurons, indicating that presynaptic functions were suppressed or a number of synapse was reduced in the afadin-deficient neurons. Analyses of presynaptic vesicle recycling and paired recordings revealed that the cultured afadin-deficient neurons showed impaired presynaptic functions. These results indicate that afadin regulates both PAJ formation and presynaptic differentiation in most mossy fiber-CA3 pyramidal cell synapses, while in a considerable population of these neurons, afadin regulates only PAJ formation but not presynaptic differentiation. PMID:24587018

  6. Early-stage development of human induced pluripotent stem cell-derived neurons.

    PubMed

    Ohara, Yuki; Koganezawa, Noriko; Yamazaki, Hiroyuki; Roppongi, Reiko T; Sato, Kaoru; Sekino, Yuko; Shirao, Tomoaki

    2015-12-01

    Recent advances in human induced pluripotent stem cells (hiPSCs) offer new possibilities for biomedical research and clinical applications. Differentiated neurons from hiPSCs are expected to be useful for developing novel methods of treatment for various neurological diseases. However, the detailed process of functional maturation of hiPSC-derived neurons (hiPS neurons) remains poorly understood. This study analyzes development of hiPS neurons, focusing specifically on early developmental stages through 48 hr after cell seeding; development was compared with that of primary cultured neurons derived from the rat hippocampus. At 5 hr after cell seeding, neurite formation occurs in a similar manner in both neuronal populations. However, very few neurons with axonal polarization were observed in the hiPS neurons even after 48 hr, indicating that hiPS neurons differentiate more slowly than rat neurons. We further investigated the elongation speed of axons and found that hiPS neuronal axons were slower. In addition, we characterized the growth cones. The localization patterns of skeletal proteins F-actin, microtubule, and drebrin were similar to those of rat neurons, and actin depolymerization by cytochalasin D induced similar changes in cytoskeletal distribution in the growth cones between hiPS neurons and rat neurons. These results indicate that, during the very early developmental stage, hiPS neurons develop comparably to rat hippocampal neurons with regard to axonal differentiation, but the growth of axons is slower.

  7. Probing Mechanoregulation of Neuronal Differentiation by Plasma Lithography Patterned Elastomeric Substrates

    PubMed Central

    Nam, Ki-Hwan; Jamilpour, Nima; Mfoumou, Etienne; Wang, Fei-Yue; Zhang, Donna D.; Wong, Pak Kin

    2014-01-01

    Cells sense and interpret mechanical cues, including cell-cell and cell-substrate interactions, in the microenvironment to collectively regulate various physiological functions. Understanding the influences of these mechanical factors on cell behavior is critical for fundamental cell biology and for the development of novel strategies in regenerative medicine. Here, we demonstrate plasma lithography patterning on elastomeric substrates for elucidating the influences of mechanical cues on neuronal differentiation and neuritogenesis. The neuroblastoma cells form neuronal spheres on plasma-treated regions, which geometrically confine the cells over two weeks. The elastic modulus of the elastomer is controlled simultaneously by the crosslinker concentration. The cell-substrate mechanical interactions are also investigated by controlling the size of neuronal spheres with different cell seeding densities. These physical cues are shown to modulate with the formation of focal adhesions, neurite outgrowth, and the morphology of neuroblastoma. By systematic adjustment of these cues, along with computational biomechanical analysis, we demonstrate the interrelated mechanoregulatory effects of substrate elasticity and cell size. Taken together, our results reveal that the neuronal differentiation and neuritogenesis of neuroblastoma cells are collectively regulated via the cell-substrate mechanical interactions. PMID:25376886

  8. Probing Mechanoregulation of Neuronal Differentiation by Plasma Lithography Patterned Elastomeric Substrates

    NASA Astrophysics Data System (ADS)

    Nam, Ki-Hwan; Jamilpour, Nima; Mfoumou, Etienne; Wang, Fei-Yue; Zhang, Donna D.; Wong, Pak Kin

    2014-11-01

    Cells sense and interpret mechanical cues, including cell-cell and cell-substrate interactions, in the microenvironment to collectively regulate various physiological functions. Understanding the influences of these mechanical factors on cell behavior is critical for fundamental cell biology and for the development of novel strategies in regenerative medicine. Here, we demonstrate plasma lithography patterning on elastomeric substrates for elucidating the influences of mechanical cues on neuronal differentiation and neuritogenesis. The neuroblastoma cells form neuronal spheres on plasma-treated regions, which geometrically confine the cells over two weeks. The elastic modulus of the elastomer is controlled simultaneously by the crosslinker concentration. The cell-substrate mechanical interactions are also investigated by controlling the size of neuronal spheres with different cell seeding densities. These physical cues are shown to modulate with the formation of focal adhesions, neurite outgrowth, and the morphology of neuroblastoma. By systematic adjustment of these cues, along with computational biomechanical analysis, we demonstrate the interrelated mechanoregulatory effects of substrate elasticity and cell size. Taken together, our results reveal that the neuronal differentiation and neuritogenesis of neuroblastoma cells are collectively regulated via the cell-substrate mechanical interactions.

  9. Tanshinone II A, a multiple target neuroprotectant, promotes caveolae-dependent neuronal differentiation.

    PubMed

    Zhao, Yuming; Xu, Pingxiang; Hu, Shengquan; Du, Libo; Xu, Zhiqing; Zhang, Huan; Cui, Wei; Mak, Shinghung; Xu, Daping; Shen, Jianggang; Han, Yifan; Liu, Yang; Xue, Ming

    2015-10-15

    Neuron loss is one fundamental features of neurodegenerative diseases. Stimulating endogenous neurogenesis, especially neuronal differentiation, might potentially provide therapeutic effects to these diseases. In this study, tanshinone II A (TIIA), a multiple target neuroprotectant, was demonstrated to promote dose-dependent neuronal differentiation in three cell models of immortalized C17.2 neuronal stem cells, rat embryonic cortical neural stem cells (NSCs) and rat PC12 pheochromocytoma cells. In particular, TIIA exerted promising effects on NSCs even at the dose of 3 nM. In PC12 cells, TIIA activated mitogen-activated protein kinase 42/44 (MAPK42/44) and its downstream transcription factor, cAMP response element-binding protein (CREB). In addition, TIIA up-regulated the expressions of brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF). The MEK inhibitor and the antagonist to the receptors of NGF and BDNF could partially attenuate the differentiation effects, indicating that MAPK42/44 mediated BDNF and NGF signals were involved in TIIA's differentiation effects. Caveolin-1 (CAV-1), the major functional protein of membrane caveolae, plays critical roles in the endocytosis of exogenous materials. CAV1, which was activated by TIIA, might help TIIA transport across cell membrane to initiate its differentiation effects. It was proven by the evidences that suppressing the function of caveolin inhibited the differentiation effects of TIIA. Therefore, we concluded that TIIA promoted neuronal differentiation partially through MAPK42/44 mediated BDNF and NGF signals in a caveolae-dependent manner. PMID:26363255

  10. Quantification of dopaminergic neuron differentiation and neurotoxicity via a genetic reporter

    PubMed Central

    Cui, Jun; Rothstein, Megan; Bennett, Theo; Zhang, Pengbo; Xia, Ninuo; Reijo Pera, Renee A.

    2016-01-01

    Human pluripotent stem cells provide a powerful human-genome based system for modeling human diseases in vitro and for potentially identifying novel treatments. Directed differentiation of pluripotent stem cells produces many specific cell types including dopaminergic neurons. Here, we generated a genetic reporter assay in pluripotent stem cells using newly-developed genome editing technologies in order to monitor differentiation efficiency and compare dopaminergic neuron survival under different conditions. We show that insertion of a luciferase reporter gene into the endogenous tyrosine hydroxylase (TH) locus enables rapid and easy quantification of dopaminergic neurons in cell culture throughout the entire differentiation process. Moreover, we demonstrate that the cellular assay is effective in assessing neuron response to different cytotoxic chemicals and is able to be scaled for high throughput applications. These results suggest that stem cell-derived terminal cell types can provide an alternative to traditional immortal cell lines or primary cells as a quantitative cellular model for toxin evaluation and drug discovery. PMID:27121904

  11. Modulation of the Isoprenoid/Cholesterol Biosynthetic Pathway During Neuronal Differentiation In Vitro.

    PubMed

    Cartocci, Veronica; Segatto, Marco; Di Tunno, Ilenia; Leone, Stefano; Pfrieger, Frank W; Pallottini, Valentina

    2016-09-01

    During differentiation, neurons acquire their typical shape and functional properties. At present, it is unclear, whether this important developmental step involves metabolic changes. Here, we studied the contribution of the mevalonate (MVA) pathway to neuronal differentiation using the mouse neuroblastoma cell line N1E-115 as experimental model. Our results show that during differentiation, the activity of 3-hydroxy 3-methylglutaryl Coenzyme A reductase (HMGR), a key enzyme of MVA pathway, and the level of Low Density Lipoprotein receptor (LDLr) decrease, whereas the level of LDLr-related protein-1 (LRP1) and the dimerization of Scavanger Receptor B1 (SRB-1) rise. Pharmacologic inhibition of HMGR by simvastatin accelerated neuronal differentiation by modulating geranylated proteins. Collectively, our data suggest that during neuronal differentiation, the activity of the MVA pathway decreases and we postulate that any interference with this process impacts neuronal morphology and function. Therefore, the MVA pathway appears as an attractive pharmacological target to modulate neurological and metabolic symptoms of developmental neuropathologies. J. Cell. Biochem. 117: 2036-2044, 2016. © 2016 Wiley Periodicals, Inc. PMID:27392312

  12. Modulation of the Isoprenoid/Cholesterol Biosynthetic Pathway During Neuronal Differentiation In Vitro.

    PubMed

    Cartocci, Veronica; Segatto, Marco; Di Tunno, Ilenia; Leone, Stefano; Pfrieger, Frank W; Pallottini, Valentina

    2016-09-01

    During differentiation, neurons acquire their typical shape and functional properties. At present, it is unclear, whether this important developmental step involves metabolic changes. Here, we studied the contribution of the mevalonate (MVA) pathway to neuronal differentiation using the mouse neuroblastoma cell line N1E-115 as experimental model. Our results show that during differentiation, the activity of 3-hydroxy 3-methylglutaryl Coenzyme A reductase (HMGR), a key enzyme of MVA pathway, and the level of Low Density Lipoprotein receptor (LDLr) decrease, whereas the level of LDLr-related protein-1 (LRP1) and the dimerization of Scavanger Receptor B1 (SRB-1) rise. Pharmacologic inhibition of HMGR by simvastatin accelerated neuronal differentiation by modulating geranylated proteins. Collectively, our data suggest that during neuronal differentiation, the activity of the MVA pathway decreases and we postulate that any interference with this process impacts neuronal morphology and function. Therefore, the MVA pathway appears as an attractive pharmacological target to modulate neurological and metabolic symptoms of developmental neuropathologies. J. Cell. Biochem. 117: 2036-2044, 2016. © 2016 Wiley Periodicals, Inc.

  13. Regenerative therapy for neuronal diseases with transplantation of somatic stem cells.

    PubMed

    Kanno, Hiroshi

    2013-10-26

    Pluripotent stem cells, which are capable of differentiating in various species of cells, are hoped to be donor cells in transplantation in regenerative medicine. Embryonic stem (ES) cells and induced pluripotent stem cells have the potential to differentiate in approximately all species of cells. However, the proliferating ability of these cells is high and the cancer formation ability is also recognized. In addition, ethical problems exist in using ES cells. Somatic stem cells with the ability to differentiate in various species of cells have been used as donor cells for neuronal diseases, such as amyotrophic lateral sclerosis, spinal cord injury, Alzheimer disease, cerebral infarction and congenital neuronal diseases. Human mesenchymal stem cells derived from bone marrow, adipose tissue, dermal tissue, umbilical cord blood and placenta are usually used for intractable neuronal diseases as somatic stem cells, while neural progenitor/stem cells and retinal progenitor/stem cells are used for a few congenital neuronal diseases and retinal degenerative disease, respectively. However, non-treated somatic stem cells seldom differentiate to neural cells in recipient neural tissue. Therefore, the contribution to neuronal regeneration using non-treated somatic stem cells has been poor and various differential trials, such as the addition of neurotrophic factors, gene transfer, peptide transfer for neuronal differentiation of somatic stem cells, have been performed. Here, the recent progress of regenerative therapies using various somatic stem cells is described.

  14. B35 neuroblastoma cells: an easily transfected, cultured cell model of central nervous system neurons.

    PubMed

    Otey, Carol A; Boukhelifa, Malika; Maness, Patricia

    2003-01-01

    A panel of neuronal cell lines was derived from tumors of the neonatal rat central nervous system (CNS) in 1974, and two of these lines are in wide use today. Both the B35 and B50 lines offer a number of advantages to researchers who study CNS neurons in culture: they are simple to grow, to differentiate, and to transfect. B50 cells have been used extensively in the study of neuronal cell death, toxicology, and differentiation, whereas B35 cells have proven useful in the molecular analysis of endocytosis and of signaling pathways, in particular those that guide axonal outgrowth and cell motility. This chapter provides protocols for growing and transfecting B35 cells, selecting stable transfectants, exploring protein function using an antisense approach, and assaying cell motility in a Transwell chamber. All of these protocols have been written for researchers who have some skill in basic cell culture techniques, but previous experience with cultured neurons is not required.

  15. Tracing Synaptic Connectivity onto Embryonic Stem Cell-Derived Neurons

    PubMed Central

    Garcia, Isabella; Huang, Longwen; Ung, Kevin; Arenkiel, Benjamin R.

    2012-01-01

    Transsynaptic circuit tracing using genetically modified Rabies virus (RV) is an emerging technology for identifying synaptic connections between neurons. Complementing this methodology, it has become possible to assay the basic molecular and cellular properties of neuronal lineages derived from embryonic stem (ES) cells in vitro, and these properties are under intense investigation towards devising cell replacement therapies. Here, we report the generation of a novel mouse ES cell (mESC) line that harbors the genetic elements to allow RV-mediated transsynaptic circuit tracing in ES cell-derived neurons and their synaptic networks. To facilitate transsynaptic tracing, we have engineered a new reporter allele by introducing cDNA encoding tdTomato, the Rabies-G glycoprotein, and the avian TVA receptor into the ROSA26 locus by gene targeting. We demonstrate high-efficiency differentiation of these novel mESCs into functional neurons, show their capacity to functionally connect with primary neuronal cultures as evidenced by immunohistochemistry and electrophysiological recordings, and show their ability to act as source cells for presynaptic tracing of neuronal networks in vitro and in vivo. Together, our data highlight the potential for using genetically engineered stem cells to investigate fundamental mechanisms of synapse and circuit formation with unambiguous identification of presynaptic inputs onto neuronal populations of interest. PMID:22996827

  16. Differential expression of id genes and their potential regulator znf238 in zebrafish adult neural progenitor cells and neurons suggests distinct functions in adult neurogenesis.

    PubMed

    Diotel, Nicolas; Beil, Tanja; Strähle, Uwe; Rastegar, Sepand

    2015-01-01

    Teleost fish display a remarkable ability to generate new neurons and to repair brain lesions during adulthood. They are, therefore, a very popular model to investigate the molecular mechanisms of constitutive and induced neurogenesis in adult vertebrates. In this study, we investigated the expression patterns of inhibitor of DNA binding (id) genes and of their potential transcriptional repressor, znf238, in the whole brain of adult zebrafish. We show that while id1 is exclusively expressed in ventricular cells in the whole brain, id2a, id3 and id4 genes are expressed in broader areas. Interestingly, znf238 was also detected in these regions, its expression overlapping with id2a, id3 and id4 expression. Further detailed characterization of the id-expressing cells demonstrated that (a) id1 is expressed in type 1 and type 2 neural progenitors as previously published, (b) id2a in type 1, 2 and 3 neural progenitors, (c) id3 in type 3 neural progenitors and (d) id4 in postmitotic neurons. Our data provide a detailed map of id and znf238 expression in the brain of adult zebrafish, supplying a framework for studies of id genes function during adult neurogenesis and brain regeneration in the zebrafish.

  17. Abeta40 promotes neuronal cell fate in neural progenitor cells.

    PubMed

    Chen, Y; Dong, C

    2009-03-01

    Sequential cleavage of the amyloid precursor protein (APP) by beta- and then gamma- secretase gives rise to Abeta(1-40) (Abeta40), a major species of Abeta (beta-amyloid) produced by neurons under physiological conditions. Abeta(1-42) (Abeta42), a minor species of Abeta, is also produced by a similar but less understood mechanism of the gamma-secretase. The physiological functions of these Abeta species remain to be defined. In this report, we demonstrate that freshly prepared soluble Abeta40 significantly promotes neurogenesis in primary neural progenitor cells (NPCs). First, Abeta40 increases neuronal markers as determined by NeuN expression and Tuj1 promoter activity, differing from Abeta42, which induces astrocyte markers in NPCs. Second, Abeta40 induces neuronal differentiation at the end of S-phase in the cell cycle. Third, Abeta40 promotes NPC entry into S-phase, playing a role in NPC self-renewal. Interestingly, Abeta40 does not significantly increase apoptotic indexes such as DNA condensation and DNA fragmentation. In addition, Abeta40 does not augment caspase-3 activation in NeuN(+) or nestin(+) cells. Collectively, this report provides strong evidence that Abeta40 is a neurogenic factor and suggests that the debilitated function of Abeta40 in neurogenesis may account for the shortage of neurons in Alzheimer's disease.

  18. Glycine and Glycine Receptor Signalling in Non-Neuronal Cells

    PubMed Central

    den Eynden, Jimmy Van; Ali, Sheen Saheb; Horwood, Nikki; Carmans, Sofie; Brône, Bert; Hellings, Niels; Steels, Paul; Harvey, Robert J.; Rigo, Jean-Michel

    2009-01-01

    Glycine is an inhibitory neurotransmitter acting mainly in the caudal part of the central nervous system. Besides this neurotransmitter function, glycine has cytoprotective and modulatory effects in different non-neuronal cell types. Modulatory effects were mainly described in immune cells, endothelial cells and macroglial cells, where glycine modulates proliferation, differentiation, migration and cytokine production. Activation of glycine receptors (GlyRs) causes membrane potential changes that in turn modulate calcium flux and downstream effects in these cells. Cytoprotective effects were mainly described in renal cells, hepatocytes and endothelial cells, where glycine protects cells from ischemic cell death. In these cell types, glycine has been suggested to stabilize porous defects that develop in the plasma membranes of ischemic cells, leading to leakage of macromolecules and subsequent cell death. Although there is some evidence linking these effects to the activation of GlyRs, they seem to operate in an entirely different mode from classical neuronal subtypes. PMID:19738917

  19. Differential roles of NF-Y transcription factor in ER chaperone expression and neuronal maintenance in the CNS

    PubMed Central

    Yamanaka, Tomoyuki; Tosaki, Asako; Miyazaki, Haruko; Kurosawa, Masaru; Koike, Masato; Uchiyama, Yasuo; Maity, Sankar N.; Misawa, Hidemi; Takahashi, Ryosuke; Shimogori, Tomomi; Hattori, Nobutaka; Nukina, Nobuyuki

    2016-01-01

    The mammalian central nervous system (CNS) contains various types of neurons with different neuronal functions. In contrast to established roles of cell type-specific transcription factors on neuronal specification and maintenance, whether ubiquitous transcription factors have conserved or differential neuronal function remains uncertain. Here, we revealed that inactivation of a ubiquitous factor NF-Y in different sets of neurons resulted in cell type-specific neuropathologies and gene downregulation in mouse CNS. In striatal and cerebellar neurons, NF-Y inactivation led to ubiquitin/p62 pathologies with downregulation of an endoplasmic reticulum (ER) chaperone Grp94, as we previously observed by NF-Y deletion in cortical neurons. In contrast, NF-Y inactivation in motor neurons induced neuronal loss without obvious protein deposition. Detailed analysis clarified downregulation of another ER chaperone Grp78 in addition to Grp94 in motor neurons, and knockdown of both ER chaperones in motor neurons recapitulated the pathology observed after NF-Y inactivation. Finally, additional downregulation of Grp78 in striatal neurons suppressed ubiquitin accumulation induced by NF-Y inactivation, implying that selective ER chaperone downregulation mediates different neuropathologies. Our data suggest distinct roles of NF-Y in protein homeostasis and neuronal maintenance in the CNS by differential regulation of ER chaperone expression. PMID:27687130

  20. Differentiation state determines neural effects on microvascular endothelial cells

    SciTech Connect

    Muffley, Lara A.; Pan, Shin-Chen; Smith, Andria N.; Ga, Maricar; Hocking, Anne M.; Gibran, Nicole S.

    2012-10-01

    Growing evidence indicates that nerves and capillaries interact paracrinely in uninjured skin and cutaneous wounds. Although mature neurons are the predominant neural cell in the skin, neural progenitor cells have also been detected in uninjured adult skin. The aim of this study was to characterize differential paracrine effects of neural progenitor cells and mature sensory neurons on dermal microvascular endothelial cells. Our results suggest that neural progenitor cells and mature sensory neurons have unique secretory profiles and distinct effects on dermal microvascular endothelial cell proliferation, migration, and nitric oxide production. Neural progenitor cells and dorsal root ganglion neurons secrete different proteins related to angiogenesis. Specific to neural progenitor cells were dipeptidyl peptidase-4, IGFBP-2, pentraxin-3, serpin f1, TIMP-1, TIMP-4 and VEGF. In contrast, endostatin, FGF-1, MCP-1 and thrombospondin-2 were specific to dorsal root ganglion neurons. Microvascular endothelial cell proliferation was inhibited by dorsal root ganglion neurons but unaffected by neural progenitor cells. In contrast, microvascular endothelial cell migration in a scratch wound assay was inhibited by neural progenitor cells and unaffected by dorsal root ganglion neurons. In addition, nitric oxide production by microvascular endothelial cells was increased by dorsal root ganglion neurons but unaffected by neural progenitor cells. -- Highlights: Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell proliferation. Black-Right-Pointing-Pointer Neural progenitor cells, not dorsal root ganglion neurons, regulate microvascular endothelial cell migration. Black-Right-Pointing-Pointer Neural progenitor cells and dorsal root ganglion neurons do not effect microvascular endothelial tube formation. Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate

  1. FOXG1-Dependent Dysregulation of GABA/Glutamate Neuron Differentiation in Autism Spectrum Disorders.

    PubMed

    Mariani, Jessica; Coppola, Gianfilippo; Zhang, Ping; Abyzov, Alexej; Provini, Lauren; Tomasini, Livia; Amenduni, Mariangela; Szekely, Anna; Palejev, Dean; Wilson, Michael; Gerstein, Mark; Grigorenko, Elena L; Chawarska, Katarzyna; Pelphrey, Kevin A; Howe, James R; Vaccarino, Flora M

    2015-07-16

    Autism spectrum disorder (ASD) is a disorder of brain development. Most cases lack a clear etiology or genetic basis, and the difficulty of re-enacting human brain development has precluded understanding of ASD pathophysiology. Here we use three-dimensional neural cultures (organoids) derived from induced pluripotent stem cells (iPSCs) to investigate neurodevelopmental alterations in individuals with severe idiopathic ASD. While no known underlying genomic mutation could be identified, transcriptome and gene network analyses revealed upregulation of genes involved in cell proliferation, neuronal differentiation, and synaptic assembly. ASD-derived organoids exhibit an accelerated cell cycle and overproduction of GABAergic inhibitory neurons. Using RNA interference, we show that overexpression of the transcription factor FOXG1 is responsible for the overproduction of GABAergic neurons. Altered expression of gene network modules and FOXG1 are positively correlated with symptom severity. Our data suggest that a shift toward GABAergic neuron fate caused by FOXG1 is a developmental precursor of ASD. PMID:26186191

  2. A short upstream promoter region mediates transcriptional regulation of the mouse doublecortin gene in differentiating neurons

    PubMed Central

    2010-01-01

    Background Doublecortin (Dcx), a MAP (Microtubule-Associated Protein), is transiently expressed in migrating and differentiating neurons and thereby characterizes neuronal precursors and neurogenesis in developing and adult neurogenesis. In addition, reduced Dcx expression during development has been related to appearance of brain pathologies. Here, we attempt to unveil the molecular mechanisms controlling Dcx gene expression by studying its transcriptional regulation during neuronal differentiation. Results To determine and analyze important regulatory sequences of the Dcx promoter, we studied a putative regulatory region upstream from the mouse Dcx coding region (pdcx2kb) and several deletions thereof. These different fragments were used in vitro and in vivo to drive reporter gene expression. We demonstrated, using transient expression experiments, that pdcx2kb is sufficient to control specific reporter gene expression in cerebellar cells and in the developing brain (E14.5). We determined the temporal profile of Dcx promoter activity during neuronal differentiation of mouse embryonic stem cells (mESC) and found that transcriptional activation of the Dcx gene varies along with neuronal differentiation of mESC. Deletion experiments and sequence comparison of Dcx promoters across rodents, human and chicken revealed the importance of a highly conserved sequence in the proximal region of the promoter required for specific and strong expression in neuronal precursors and young neuronal cells. Further analyses revealed the presence in this short sequence of several conserved, putative transcription factor binding sites: LEF/TCF (Lymphoid Enhancer Factor/T-Cell Factor) which are effectors of the canonical Wnt pathway; HNF6/OC2 (Hepatocyte Nuclear Factor-6/Oncecut-2) members of the ONECUT family and NF-Y/CAAT (Nuclear Factor-Y). Conclusions Studies of Dcx gene regulatory sequences using native, deleted and mutated constructs suggest that fragments located upstream of the

  3. Neuronize: a tool for building realistic neuronal cell morphologies

    PubMed Central

    Brito, Juan P.; Mata, Susana; Bayona, Sofia; Pastor, Luis; DeFelipe, Javier; Benavides-Piccione, Ruth

    2013-01-01

    This study presents a tool, Neuronize, for building realistic three-dimensional models of neuronal cells from the morphological information extracted through computer-aided tracing applications. Neuronize consists of a set of methods designed to build 3D neural meshes that approximate the cell membrane at different resolution levels, allowing a balance to be reached between the complexity and the quality of the final model. The main contribution of the present study is the proposal of a novel approach to build a realistic and accurate 3D shape of the soma from the incomplete information stored in the digitally traced neuron, which usually consists of a 2D cell body contour. This technique is based on the deformation of an initial shape driven by the position and thickness of the first order dendrites. The addition of a set of spines along the dendrites completes the model, building a final 3D neuronal cell suitable for its visualization in a wide range of 3D environments. PMID:23761740

  4. Transcription factor activating protein 2 beta (TFAP2B) mediates noradrenergic neuronal differentiation in neuroblastoma.

    PubMed

    Ikram, Fakhera; Ackermann, Sandra; Kahlert, Yvonne; Volland, Ruth; Roels, Frederik; Engesser, Anne; Hertwig, Falk; Kocak, Hayriye; Hero, Barbara; Dreidax, Daniel; Henrich, Kai-Oliver; Berthold, Frank; Nürnberg, Peter; Westermann, Frank; Fischer, Matthias

    2016-02-01

    Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2'-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma. PMID:26598443

  5. Generation of serotonin neurons from human pluripotent stem cells.

    PubMed

    Lu, Jianfeng; Zhong, Xuefei; Liu, Huisheng; Hao, Ling; Huang, Cindy Tzu-Ling; Sherafat, Mohammad Amin; Jones, Jeffrey; Ayala, Melvin; Li, Lingjun; Zhang, Su-Chun

    2016-01-01

    Serotonin neurons located in the raphe nucleus of the hindbrain have crucial roles in regulating brain functions and have been implicated in various psychiatric disorders. Yet functional human serotonin neurons are not available for in vitro studies. Through manipulation of the WNT pathway, we demonstrate efficient differentiation of human pluripotent stem cells (hPSCs) to cells resembling central serotonin neurons, primarily those located in the rhombomeric segments 2-3 of the rostral raphe, which participate in high-order brain functions. The serotonin neurons express a series of molecules essential for serotonergic development, including tryptophan hydroxylase 2, exhibit typical electrophysiological properties and release serotonin in an activity-dependent manner. When treated with the FDA-approved drugs tramadol and escitalopram oxalate, they release or uptake serotonin in a dose- and time-dependent manner, suggesting the utility of these cells for the evaluation of drug candidates.

  6. Generation of serotonin neurons from human pluripotent stem cells

    PubMed Central

    Lu, Jianfeng; Zhong, Xuefei; Liu, Huisheng; Hao, Ling; Huang, Cindy Tzu-Ling; Sherafat, Mohammad Amin; Jones, Jeffrey; Ayala, Melvin; Li, Lingjun; Zhang, Su-Chun

    2016-01-01

    Serotonin neurons located in the raphe nucleus of the hindbrain have crucial roles in regulating brain functions and have been implicated in various psychiatric disorders. Yet functional human serotonin neurons are not available for in vitro studies. Through manipulation of the WNT pathway, we demonstrate efficient differentiation of human pluripotent stem cells (hPSCs) to cells resembling central serotonin neurons, primarily those located in the rhombomeric segments 2–3 of the rostral raphe, which participate in high-order brain functions. The serotonin neurons express a series of molecules essential for serotonergic development, including tryptophan hydroxylase 2, exhibit typical electrophysiological properties and release serotonin in an activity-dependent manner. When treated with the FDA-approved drugs tramadol and escitalopram oxalate, they release or uptake serotonin in a dose- and time-dependent manner, suggesting the utility of these cells for the evaluation of drug candidates. PMID:26655496

  7. Manganese inhibits the ability of astrocytes to promote neuronal differentiation

    SciTech Connect

    Giordano, Gennaro; Pizzurro, Daniella; VanDeMark, Kathryn; Guizzetti, Marina; Costa, Lucio G.

    2009-10-15

    Manganese (Mn) is a known neurotoxicant and developmental neurotoxicant. As Mn has been shown to accumulate in astrocytes, we sought to investigate whether Mn would alter astrocyte-neuronal interactions, specifically the ability of astrocytes to promote differentiation of neurons. We found that exposure of rat cortical astrocytes to Mn (50-500 {mu}M) impaired their ability to promote axonal and neurite outgrowth in hippocampal neurons. This effect of Mn appeared to be mediated by oxidative stress, as it was reversed by antioxidants (melatonin and PBN) and by increasing glutathione levels, while it was potentiated by glutathione depletion in astrocytes. As the extracellular matrix protein fibronectin plays an important role in astrocyte-mediated neuronal neurite outgrowth, we also investigated the effect of Mn on fibronectin. Mn caused a concentration-dependent decrease of fibronectin protein and mRNA in astrocytes lysate and of fibronectin protein in astrocyte medium; these effects were also antagonized by antioxidants. Exposure of astrocytes to two oxidants, H{sub 2}O{sub 2} and DMNQ, similarly impaired their neuritogenic action, and led to a decreased expression of fibronectin. Mn had no inhibitory effect on neurite outgrowth when applied directly onto hippocampal neurons, where it actually caused a small increase in neuritogenesis. These results indicate that Mn, by targeting astrocytes, affects their ability to promote neuronal differentiation by a mechanism which is likely to involve oxidative stress.

  8. Phenotypic characteristics of hybrid cells generated by transferring neuronal nuclei into bone marrow stromal cell cytoplasts.

    PubMed

    Zhou, Zhujuan; Xu, Yan; Zhong, Qi; Zheng, Jian

    2012-02-10

    Bone marrow stromal cells (BMSCs) are promising donor cells for transplantation therapies for a variety of diseases. However, there still lack efficient ways to induce directional differentiation of BMSCs to promote their practical use in transplantation therapy. In this study, we constructed hybrid cells by transferring neuronal nuclei into BMSC cytoplasts and investigated the proliferative capacity and phenotypic characteristics of the hybrid cells. The neuronal nuclei were labeled with Hoechst 33342 before the transfer process, and the cell membrane antigen CD71 was used as a marker of BMSC cytoplasts. The BMSC cytoplasts and neuronal karyoplasts were separated by Ficoll density gradient ultracentrifugation. The hybrid cells were generated by the polyethylene glycol-mediated fusion of BMSC cytoplasts with neuronal karyoplasts. The hybrid cells exhibited Hoechst 33342 staining in their nuclei and CD71 staining on their cytomembranes, which confirmed the success of cell fusion. The hybrid cells were positive for BrdU immunostaining. Viability analysis of the cultured hybrid cells by the MTT assay demonstrated their proliferative ability. Immunocytochemical staining revealed the expression of the neuron-specific markers NeuN and MAP2 in the third passage hybrid cells, which indicated their neuronal phenotypic characteristics. The results demonstrated that the hybrid cells produced by fusing neuronal karyoplasts with BMSC cytoplasts had proliferative capability and expressed the neuron-specific markers. Further study is required to investigate the phenotype of the hybrid cells both structurally and functionally.

  9. Purine Metabolism During Neuronal Differentiation: The Relevance of Purine Synthesis and Recycling

    PubMed Central

    Göttle, Martin; Burhenne, Heike; Sutcliffe, Diane; Jinnah, H. A.

    2013-01-01

    Purines are a class of small organic molecules that are essential for all cells. They play critical roles in neuronal differentiation and function. Their importance is highlighted by several inherited disorders of purine metabolism, such as the Lesch-Nyhan disease, which is caused by a deficiency of the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt). Despite the known importance of purines in the nervous system, knowledge regarding their metabolism in neurons is limited. In the current studies, purine pools and their metabolism were examined in rat PC6-3 cells, a PC12 pheochromocytoma subclone that undergoes robust differentiation with nerve growth factor. The results were compared with five new independent PC6-3 subclones with defective purine recycling due to different mutations affecting HGprt enzyme activity. The results demonstrate an increase in most purines and in energy state following neuronal differentiation, as well as specific abnormalities when purine recycling is lost. The loss of HGprt-mediated purine recycling also is associated with significant loss of dopamine and related metabolites in the mutant PC6-3 lines, suggesting an important connection between purine and dopamine pathways. These results provide insights into how purine pools and metabolism change with neuronal differentiation, and how specific enzyme defects may cause neuronal dysfunction. PMID:23859490

  10. Illumination controls differentiation of dopamine neurons regulating behaviour.

    PubMed

    Dulcis, Davide; Spitzer, Nicholas C

    2008-11-13

    Specification of the appropriate neurotransmitter is a crucial step in neuronal differentiation because it enables signalling among populations of neurons. Experimental manipulations demonstrate that both autonomous and activity-dependent genetic programs contribute to this process during development, but whether natural environmental stimuli specify transmitter expression in a neuronal population is unknown. We investigated neurons of the ventral suprachiasmatic nucleus that regulate neuroendocrine pituitary function in response to light in teleosts, amphibia and primates. Here we show that altering light exposure, which changes the sensory input to the circuit controlling adaptation of skin pigmentation to background, changes the number of neurons expressing dopamine in larvae of the amphibian Xenopus laevis in a circuit-specific and activity-dependent manner. Neurons newly expressing dopamine then regulate changes in camouflage colouration in response to illumination. Thus, physiological activity alters the numbers of behaviourally relevant amine-transmitter-expressing neurons in the brain at postembryonic stages of development. The results may be pertinent to changes in cognitive states that are regulated by biogenic amines. PMID:19005547

  11. Loss of aPKCλ in Differentiated Neurons Disrupts the Polarity Complex but Does Not Induce Obvious Neuronal Loss or Disorientation in Mouse Brains

    PubMed Central

    Yamanaka, Tomoyuki; Tosaki, Asako; Kurosawa, Masaru; Akimoto, Kazunori; Hirose, Tomonori; Ohno, Shigeo; Hattori, Nobutaka; Nukina, Nobuyuki

    2013-01-01

    Cell polarity plays a critical role in neuronal differentiation during development of the central nervous system (CNS). Recent studies have established the significance of atypical protein kinase C (aPKC) and its interacting partners, which include PAR-3, PAR-6 and Lgl, in regulating cell polarization during neuronal differentiation. However, their roles in neuronal maintenance after CNS development remain unclear. Here we performed conditional deletion of aPKCλ, a major aPKC isoform in the brain, in differentiated neurons of mice by camk2a-cre or synapsinI-cre mediated gene targeting. We found significant reduction of aPKCλ and total aPKCs in the adult mouse brains. The aPKCλ deletion also reduced PAR-6β, possibly by its destabilization, whereas expression of other related proteins such as PAR-3 and Lgl-1 was unaffected. Biochemical analyses suggested that a significant fraction of aPKCλ formed a protein complex with PAR-6β and Lgl-1 in the brain lysates, which was disrupted by the aPKCλ deletion. Notably, the aPKCλ deletion mice did not show apparent cell loss/degeneration in the brain. In addition, neuronal orientation/distribution seemed to be unaffected. Thus, despite the polarity complex disruption, neuronal deletion of aPKCλ does not induce obvious cell loss or disorientation in mouse brains after cell differentiation. PMID:24391875

  12. Differentiation of postmitotic neuroblasts into substance P-immunoreactive sensory neurons in dissociated cultures of chick dorsal root ganglion.

    PubMed

    Barakat, I; Droz, B

    1987-07-01

    Counts performed on dissociated cell cultures of E10 chick embryo dorsal root ganglia (DRG) showed after 4-6 days of culture a pronounced decline of the neuronal population in neuron-enriched cultures and a net gain in the number of ganglion cells in mixed DRG cell cultures (containing both neurons and nonneuronal cells). In the latter case, the increase in the number of neurons was found to depend on NGF and to average 119% in defined medium or 129% in horse serum-supplemented medium after 6 days of culture. The lack of [3H]thymidine incorporation into the neuronal population indicated that the newly formed ganglion cells were not generated by proliferation. On the contrary, the differentiation of postmitotic neuroblasts present in the nonneuronal cell compartment was supported by sequential microphotographs of selected fields taken every hour for 48-55 hr after 3 days of culture. Apparently nonneuronal flat dark cells exhibited morphological changes and gradually evolved into neuronal ovoid and refringent cell bodies with expanding neurites. The ultrastructural organization of these evolving cells corresponded to that of primitive or intermediate neuroblasts. The neuronal nature of these rounding up cell bodies was indeed confirmed by the progressive expression of various neuronal cell markers (150 and 200-kDa neurofilament triplets, neuron specific enolase, and D2/N-CAM). Besides a constant lack of immunoreactivity for tyrosine hydroxylase, somatostatin, parvalbumin, and calbindin-D 28K and a lack of cytoenzymatic activity for carbonic anhydrase, all the newly produced neurons expressed three main phenotypic characteristics: a small cell body, a strong immunoreactivity to MAG, and substance P. Hence, ganglion cells newly differentiated in culture would meet characteristics ascribed to small B sensory neurons and more specifically to a subpopulation of ganglion cells containing substance P-immunoreactive material.

  13. Ablation of BRaf impairs neuronal differentiation in the postnatal hippocampus and cerebellum.

    PubMed

    Pfeiffer, Verena; Götz, Rudolf; Xiang, Chaomei; Camarero, Guadelupe; Braun, Attila; Zhang, Yina; Blum, Robert; Heinsen, Helmut; Nieswandt, Bernhard; Rapp, Ulf R

    2013-01-01

    This study focuses on the role of the kinase BRaf in postnatal brain development. Mice expressing truncated, non-functional BRaf in neural stem cell-derived brain tissue demonstrate alterations in the cerebellum, with decreased sizes and fuzzy borders of the glomeruli in the granule cell layer. In addition we observed reduced numbers and misplaced ectopic Purkinje cells that showed an altered structure of their dendritic arborizations in the hippocampus, while the overall cornus ammonis architecture appeared to be unchanged. In male mice lacking BRaf in the hippocampus the size of the granule cell layer was normal at postnatal day 12 (P12) but diminished at P21, as compared to control littermates. This defect was caused by a reduced ability of dentate gyrus progenitor cells to differentiate into NeuN positive granule cell neurons. In vitro cell culture of P0/P1 hippocampal cells revealed that BRaf deficient cells were impaired in their ability to form microtubule-associated protein 2 positive neurons. Together with the alterations in behaviour, such as autoaggression and loss of balance fitness, these observations indicate that in the absence of BRaf all neuronal cellular structures develop, but neuronal circuits in the cerebellum and hippocampus are partially disturbed besides impaired neuronal generation in both structures. PMID:23505473

  14. C-terminal trans-activation sub-region of VP16 is uniquely required for forskolin-induced herpes simplex virus type 1 reactivation from quiescently infected-PC12 cells but not for replication in neuronally differentiated-PC12 cells.

    PubMed

    Danaher, Robert J; Cook, Ross K; Wang, Chunmei; Triezenberg, Steven J; Jacob, Robert J; Miller, Craig S

    2013-02-01

    The HSV-1 tegument protein VP16 contains a trans-activation domain (TAD) that is required for induction of immediate early (IE) genes during lytic infection and induced reactivation from latency. Here we report the differential contributions of the two sub-regions of the TAD in neuronal and non-neuronal cells during activation of IE gene expression, virus replication, and reactivation from quiescently infected (QIF)-PC12 cells. Our studies show that VP16- and chemical (hexamethylenebisacetamide)-induced IE gene activation is attenuated in neuronal cells. Irrespective of neuronal or non-neuronal cell backgrounds, IE gene activation demonstrated a greater requirement for the N-terminal sub-region of VP16 TAD (VP16N) than the C-terminal sub-region (VP16C). In surprising contrast to these findings, a recombinant virus (RP4) containing the VP16N deletion was capable of modest forskolin-induced reactivation whereas a recombinant (RP3) containing a deletion of VP16C was incapable of stress-induced reactivation from QIF-PC12 cells. These unique process-dependent functions of the VP16 TAD sub-regions may be important during particular stages of the virus life cycle (lytic, entrance, and maintenance of a quiescent state and reactivation) when viral DNA would be expected to be differentially modified. PMID:23192733

  15. Midline thalamic neurons are differentially engaged during hippocampus network oscillations.

    PubMed

    Lara-Vásquez, Ariel; Espinosa, Nelson; Durán, Ernesto; Stockle, Marcelo; Fuentealba, Pablo

    2016-01-01

    The midline thalamus is reciprocally connected with the medial temporal lobe, where neural circuitry essential for spatial navigation and memory formation resides. Yet, little information is available on the dynamic relationship between activity patterns in the midline thalamus and medial temporal lobe. Here, we report on the functional heterogeneity of anatomically-identified thalamic neurons and the differential modulation of their activity with respect to dorsal hippocampal rhythms in the anesthetized mouse. Midline thalamic neurons expressing the calcium-binding protein calretinin, irrespective of their selective co-expression of calbindin, discharged at overall low levels, did not increase their activity during hippocampal theta oscillations, and their firing rates were inhibited during hippocampal sharp wave-ripples. Conversely, thalamic neurons lacking calretinin discharged at higher rates, increased their activity during hippocampal theta waves, but remained unaffected during sharp wave-ripples. Our results indicate that the midline thalamic system comprises at least two different classes of thalamic projection neuron, which can be partly defined by their differential engagement by hippocampal pathways during specific network oscillations that accompany distinct behavioral contexts. Thus, different midline thalamic neuronal populations might be selectively recruited to support distinct stages of memory processing, consistent with the thalamus being pivotal in the dialogue of cortical circuits. PMID:27411890

  16. Midline thalamic neurons are differentially engaged during hippocampus network oscillations

    PubMed Central

    Lara-Vásquez, Ariel; Espinosa, Nelson; Durán, Ernesto; Stockle, Marcelo; Fuentealba, Pablo

    2016-01-01

    The midline thalamus is reciprocally connected with the medial temporal lobe, where neural circuitry essential for spatial navigation and memory formation resides. Yet, little information is available on the dynamic relationship between activity patterns in the midline thalamus and medial temporal lobe. Here, we report on the functional heterogeneity of anatomically-identified thalamic neurons and the differential modulation of their activity with respect to dorsal hippocampal rhythms in the anesthetized mouse. Midline thalamic neurons expressing the calcium-binding protein calretinin, irrespective of their selective co-expression of calbindin, discharged at overall low levels, did not increase their activity during hippocampal theta oscillations, and their firing rates were inhibited during hippocampal sharp wave-ripples. Conversely, thalamic neurons lacking calretinin discharged at higher rates, increased their activity during hippocampal theta waves, but remained unaffected during sharp wave-ripples. Our results indicate that the midline thalamic system comprises at least two different classes of thalamic projection neuron, which can be partly defined by their differential engagement by hippocampal pathways during specific network oscillations that accompany distinct behavioral contexts. Thus, different midline thalamic neuronal populations might be selectively recruited to support distinct stages of memory processing, consistent with the thalamus being pivotal in the dialogue of cortical circuits. PMID:27411890

  17. Signals from the AT2 (angiotensin type 2) receptor of angiotensin II inhibit p21ras and activate MAPK (mitogen-activated protein kinase) to induce morphological neuronal differentiation in NG108-15 cells.

    PubMed

    Gendron, L; Laflamme, L; Rivard, N; Asselin, C; Payet, M D; Gallo-Payet, N

    1999-09-01

    In a previous study, we had shown that activation of the AT2 (angiotensin type 2) receptor of angiotensin II (Ang II) induced morphological differentiation of the neuronal cell line NG108-15. In the present study, we investigated the nature of the possible intracellular mediators involved in the AT2 effect. We found that stimulation of AT2 receptors in NG108-15 cells resulted in time-dependent modulation of tyrosine phosphorylation of a number of cytoplasmic proteins. Stimulation of NG108-15 cells with Ang II induced a decrease in GTP-bound p21ras but a sustained increase in the activity of p42mapk and p44mapk as well as neurite outgrowth. Similarly, neurite elongation, increased polymerized tubulin levels, and increased mitogen-activated protein kinase (MAPK) activity were also observed in a stably transfected NG108-15 cell line expressing the dominant-negative mutant of p21ras, RasN17. These results support the observation that inhibition of p21ras did not impair the effect of Ang II on its ability to stimulate MAPK activity. While 10 microM of the MEK inhibitor, PD98059, only moderately affected elongation, 50 microM PD98059 completely blocked the Ang II- and the RasN17-mediated induction of neurite outgrowth. These results demonstrate that some of the events associated with the AT2 receptor-induced neuronal morphological differentiation of NG108-15 cells not only include inhibition of p21ras but an increase in MAPK activity as well, which is essential for neurite outgrowth.

  18. Epigenetic regulation contributes to urocortin-enhanced midbrain dopaminergic neuron differentiation.

    PubMed

    Huang, Hsin-Yi; Chiu, Tsung-Lang; Chang, Hui-Fen; Hsu, Hui-Ru; Pang, Cheng-Yoong; Liew, Hock-Kean; Wang, Mei-Jen

    2015-05-01

    The production of midbrain dopaminergic (mDA) neurons requires precise extrinsic inductive signals and intrinsic transcriptional cascade at a specific time point in development. Urocortin (UCN) is a peptide of the corticotropin-releasing hormone family that mediates various responses to stress. UCN was first cloned from adult rat midbrain. However, the contribution of UCN to the development of mDA neurons is poorly understood. Here, we show that UCN is endogenously expressed in the developing ventral midbrain (VM) and its receptors are exhibited in Nurr1(+) postmitotic mDA precursors and TH(+) neurons, suggesting possible roles in regulating their terminal differentiation. UCN treatment increased DA cell numbers in rat VM precursor cultures by promoting the conversion of Nurr1(+) precursors into DA neurons. Furthermore, neutralization of secreted UCN with anti-UCN antibody resulted in a reduction in the number of DA neurons. UCN induced an abundance of acetylated histone H3 and enhanced late DA regulator Nurr1, Foxa2, and Pitx3 expressions. Using pharmacological and RNA interference approaches, we further demonstrated that histone deacetylase (HDAC) inhibition and late transcriptional factors upregulation contribute to UCN-mediated DA neuron differentiation. Chromatin immunoprecipitation analyses revealed that UCN promoted histone acetylation of chromatin surrounding the TH promoter by directly inhibiting HDAC and releasing of methyl CpG binding protein 2-CoREST-HDAC1 repressor complex from the promoter, ultimately leading to an increase in Nurr1/coactivators-mediated transcription of TH gene. Moreover, UCN treatment in vivo also resulted in increased DA neuron differentiation. These findings suggest that UCN might contribute to regulate late mDA neuron differentiation during VM development.

  19. Morphological homogeneity of neurons: searching for outlier neuronal cells.

    PubMed

    Zawadzki, Krissia; Feenders, Christoph; Viana, Matheus P; Kaiser, Marcus; Costa, Luciano da F

    2012-10-01

    We report a morphology-based approach for the automatic identification of outlier neurons, as well as its application to the NeuroMorpho.org database, with more than 5,000 neurons. Each neuron in a given analysis is represented by a feature vector composed of 20 measurements, which are then projected into a two-dimensional space by applying principal component analysis. Bivariate kernel density estimation is then used to obtain the probability distribution for the group of cells, so that the cells with highest probabilities are understood as archetypes while those with the smallest probabilities are classified as outliers. The potential of the methodology is illustrated in several cases involving uniform cell types as well as cell types for specific animal species. The results provide insights regarding the distribution of cells, yielding single and multi-variate clusters, and they suggest that outlier cells tend to be more planar and tortuous. The proposed methodology can be used in several situations involving one or more categories of cells, as well as for detection of new categories and possible artifacts. PMID:22615032

  20. Innervation of Cochlear Hair Cells by Human Induced Pluripotent Stem Cell-Derived Neurons In Vitro.

    PubMed

    Gunewardene, Niliksha; Crombie, Duncan; Dottori, Mirella; Nayagam, Bryony A

    2016-01-01

    Induced pluripotent stem cells (iPSCs) may serve as an autologous source of replacement neurons in the injured cochlea, if they can be successfully differentiated and reconnected with residual elements in the damaged auditory system. Here, we explored the potential of hiPSC-derived neurons to innervate early postnatal hair cells, using established in vitro assays. We compared two hiPSC lines against a well-characterized hESC line. After ten days' coculture in vitro, hiPSC-derived neural processes contacted inner and outer hair cells in whole cochlear explant cultures. Neural processes from hiPSC-derived neurons also made contact with hair cells in denervated sensory epithelia explants and expressed synapsin at these points of contact. Interestingly, hiPSC-derived neurons cocultured with hair cells at an early stage of differentiation formed synapses with a higher number of hair cells, compared to hiPSC-derived neurons cocultured at a later stage of differentiation. Notable differences in the innervation potentials of the hiPSC-derived neurons were also observed and variations existed between the hiPSC lines in their innervation efficiencies. Collectively, these data illustrate the promise of hiPSCs for auditory neuron replacement and highlight the need to develop methods to mitigate variabilities observed amongst hiPSC lines, in order to achieve reliable clinical improvements for patients.

  1. Innervation of Cochlear Hair Cells by Human Induced Pluripotent Stem Cell-Derived Neurons In Vitro

    PubMed Central

    Gunewardene, Niliksha; Crombie, Duncan; Dottori, Mirella; Nayagam, Bryony A.

    2016-01-01

    Induced pluripotent stem cells (iPSCs) may serve as an autologous source of replacement neurons in the injured cochlea, if they can be successfully differentiated and reconnected with residual elements in the damaged auditory system. Here, we explored the potential of hiPSC-derived neurons to innervate early postnatal hair cells, using established in vitro assays. We compared two hiPSC lines against a well-characterized hESC line. After ten days' coculture in vitro, hiPSC-derived neural processes contacted inner and outer hair cells in whole cochlear explant cultures. Neural processes from hiPSC-derived neurons also made contact with hair cells in denervated sensory epithelia explants and expressed synapsin at these points of contact. Interestingly, hiPSC-derived neurons cocultured with hair cells at an early stage of differentiation formed synapses with a higher number of hair cells, compared to hiPSC-derived neurons cocultured at a later stage of differentiation. Notable differences in the innervation potentials of the hiPSC-derived neurons were also observed and variations existed between the hiPSC lines in their innervation efficiencies. Collectively, these data illustrate the promise of hiPSCs for auditory neuron replacement and highlight the need to develop methods to mitigate variabilities observed amongst hiPSC lines, in order to achieve reliable clinical improvements for patients. PMID:26966437

  2. Notch/Rbpjκ signaling regulates progenitor maintenance and differentiation of hypothalamic arcuate neurons

    PubMed Central

    Aujla, Paven K.; Naratadam, George T.; Xu, Liwen; Raetzman, Lori T.

    2013-01-01

    The hypothalamic arcuate nucleus (Arc), containing pro-opoiomelanocortin (POMC), neuropeptide Y (NPY) and growth hormone releasing hormone (GHRH) neurons, regulates feeding, energy balance and body size. Dysregulation of this homeostatic mediator underlies diseases ranging from growth failure to obesity. Despite considerable investigation regarding the function of Arc neurons, mechanisms governing their development remain unclear. Notch signaling factors such as Hes1 and Mash1 are present in hypothalamic progenitors that give rise to Arc neurons. However, how Notch signaling controls these progenitor populations is unknown. To elucidate the role of Notch signaling in Arc development, we analyzed conditional loss-of-function mice lacking a necessary Notch co-factor, Rbpjκ, in Nkx2.1-cre-expressing cells (Rbpjκ cKO), as well as mice with expression of the constitutively active Notch1 intracellular domain (NICD) in Nkx2.1-cre-expressing cells (NICD Tg). We found that loss of Rbpjκ results in absence of Hes1 but not of Hes5 within the primordial Arc at E13.5. Additionally, Mash1 expression is increased, coincident with increased proliferation and accumulation of Arc neurons at E13.5. At E18.5, Rbpjκ cKO mice have few progenitors and show increased numbers of differentiated Pomc, NPY and Ghrh neurons. By contrast, NICD Tg mice have increased hypothalamic progenitors, show an absence of differentiated Arc neurons and aberrant glial differentiation at E18.5. Subsequently, both Rbpjκ cKO and NICD Tg mice have changes in growth and body size during postnatal development. Taken together, our results demonstrate that Notch/Rbpjκ signaling regulates the generation and differentiation of Arc neurons, which contribute to homeostatic regulation of body size. PMID:23884446

  3. The Timing of Differentiation of Adult Hippocampal Neurons Is Crucial for Spatial Memory

    PubMed Central

    Cinà, Irene; Aceti, Massimiliano; Micheli, Laura; Bacci, Alberto; Cestari, Vincenzo; Tirone, Felice

    2008-01-01

    Adult neurogenesis in the dentate gyrus plays a critical role in hippocampus-dependent spatial learning. It remains unknown, however, how new neurons become functionally integrated into spatial circuits and contribute to hippocampus-mediated forms of learning and memory. To investigate these issues, we used a mouse model in which the differentiation of adult-generated dentate gyrus neurons can be anticipated by conditionally expressing the pro-differentiative gene PC3 (Tis21/BTG2) in nestin-positive progenitor cells. In contrast to previous studies that affected the number of newly generated neurons, this strategy selectively changes their timing of differentiation. New, adult-generated dentate gyrus progenitors, in which the PC3 transgene was expressed, showed accelerated differentiation and significantly reduced dendritic arborization and spine density. Functionally, this genetic manipulation specifically affected different hippocampus-dependent learning and memory tasks, including contextual fear conditioning, and selectively reduced synaptic plasticity in the dentate gyrus. Morphological and functional analyses of hippocampal neurons at different stages of differentiation, following transgene activation within defined time-windows, revealed that the new, adult-generated neurons up to 3–4 weeks of age are required not only to acquire new spatial information but also to use previously consolidated memories. Thus, the correct unwinding of these key memory functions, which can be an expression of the ability of adult-generated neurons to link subsequent events in memory circuits, is critically dependent on the correct timing of the initial stages of neuron maturation and connection to existing circuits. PMID:18842068

  4. Alternative Splicing of Neuronal Differentiation Factor TRF2 Regulated by HNRNPH1/H2.

    PubMed

    Grammatikakis, Ioannis; Zhang, Peisu; Panda, Amaresh C; Kim, Jiyoung; Maudsley, Stuart; Abdelmohsen, Kotb; Yang, Xiaoling; Martindale, Jennifer L; Motiño, Omar; Hutchison, Emmette R; Mattson, Mark P; Gorospe, Myriam

    2016-05-01

    During neuronal differentiation, use of an alternative splice site on the rat telomere repeat-binding factor 2 (TRF2) mRNA generates a short TRF2 protein isoform (TRF2-S) capable of derepressing neuronal genes. However, the RNA-binding proteins (RBPs) controlling this splicing event are unknown. Here, using affinity pull-down analysis, we identified heterogeneous nuclear ribonucleoproteins H1 and H2(HNRNPH) as RBPs specifically capable of interacting with the spliced RNA segment (exon 7) of Trf2 pre-mRNA. HNRNPH proteins prevent the production of the short isoform of Trf2 mRNA, as HNRNPH silencing selectively elevates TRF2-S levels. Accordingly, HNRNPH levels decline while TRF2-S levels increase during neuronal differentiation. In addition, CRISPR/Cas9-mediated deletion of hnRNPH2 selectively accelerates the NGF-triggered differentiation of rat pheochromocytoma cells into neurons. In sum, HNRNPH is a splicing regulator of Trf2 pre-mRNA that prevents the expression of TRF2-S, a factor implicated in neuronal differentiation. PMID:27117401

  5. Gammaherpesvirus Infection of Human Neuronal Cells

    PubMed Central

    Jha, Hem Chandra; Mehta, Devan; Lu, Jie; El-Naccache, Darine; Shukla, Sanket K.; Kovacsics, Colleen; Kolson, Dennis

    2015-01-01

    ABSTRACT Gammaherpesviruses human herpesvirus 4 (HHV4) and HHV8 are two prominent members of the herpesvirus family associated with a number of human cancers. HHV4, also known as Epstein-Barr virus (EBV), a ubiquitous gammaherpesvirus prevalent in 90 to 95% of the human population, is clinically associated with various neurological diseases such as primary central nervous system lymphoma, multiple sclerosis, Alzheimer’s disease, cerebellar ataxia, and encephalitis. However, the possibility that EBV and Kaposi’s sarcoma-associated herpesvirus (KSHV) can directly infect neurons has been largely overlooked. This study has, for the first time, characterized EBV infection in neural cell backgrounds by using the Sh-Sy5y neuroblastoma cell line, teratocarcinoma Ntera2 neurons, and primary human fetal neurons. Furthermore, we also demonstrated KSHV infection of neural Sh-Sy5y cells. These neuronal cells were infected with green fluorescent protein-expressing recombinant EBV or KSHV. Microscopy, genetic analysis, immunofluorescence, and Western blot analyses for specific viral antigens supported and validated the infection of these cells by EBV and KSHV and showed that the infection was efficient and productive. Progeny virus produced from infected neuronal cells efficiently infected fresh neuronal cells, as well as peripheral blood mononuclear cells. Furthermore, acyclovir was effective at inhibiting the production of virus from neuronal cells similar to lymphoblastoid cell lines; this suggests active lytic replication in infected neurons in vitro. These studies represent a potentially new in vitro model of EBV- and KSHV-associated neuronal disease development and pathogenesis. PMID:26628726

  6. Efficient and cost-effective generation of mature neurons from human induced pluripotent stem cells.

    PubMed

    Badja, Cherif; Maleeva, Galyna; El-Yazidi, Claire; Barruet, Emilie; Lasserre, Manon; Tropel, Philippe; Binetruy, Bernard; Bregestovski, Piotr; Magdinier, Frédérique

    2014-12-01

    For years, our ability to study pathological changes in neurological diseases has been hampered by the lack of relevant models until the recent groundbreaking work from Yamanaka's group showing that it is feasible to generate induced pluripotent stem cells (iPSCs) from human somatic cells and to redirect the fate of these iPSCs into differentiated cells. In particular, much interest has focused on the ability to differentiate human iPSCs into neuronal progenitors and functional neurons for relevance to a large number of pathologies including mental retardation and behavioral or degenerative syndromes. Current differentiation protocols are time-consuming and generate limited amounts of cells, hindering use on a large scale. We describe a feeder-free method relying on the use of a chemically defined medium that overcomes the need for embryoid body formation and neuronal rosette isolation for neuronal precursors and terminally differentiated neuron production. Four days after induction, expression of markers of the neurectoderm lineage is detectable. Between 4 and 7 days, neuronal precursors can be expanded, frozen, and thawed without loss of proliferation and differentiation capacities or further differentiated. Terminal differentiation into the different subtypes of mature neurons found in the human brain were observed. At 6-35 days after induction, cells express typical voltage-gated and ionotrophic receptors for GABA, glycine, and acetylcholine. This specific and efficient single-step strategy in a chemically defined medium allows the production of mature neurons in 20-40 days with multiple applications, especially for modeling human pathologies.

  7. Merkel cells and neurons keep in touch

    PubMed Central

    Woo, Seung-Hyun; Lumpkin, Ellen A.; Patapoutian, Ardem

    2014-01-01

    The Merkel cell-neurite complex is a unique vertebrate touch receptor comprising two distinct cell types in the skin. Its presence in touch-sensitive skin areas was recognized more than a century ago, but the functions of each cell type in sensory transduction have been unclear. Three recent studies demonstrate that Merkel cells are mechanosensitive cells that function in touch transduction via Piezo2. One study concludes that Merkel cells rather than sensory neurons are principal sites of mechanotransduction, whereas the other two studies report that both Merkel cells and neurons encode mechanical inputs. Together, these studies settle a longstanding debate on whether Merkel cells are mechanosensory cells, and enable future investigations of how these skin cells communicate with neurons. PMID:25480024

  8. Regulation of differentiation flux by Notch signalling influences the number of dopaminergic neurons in the adult brain

    PubMed Central

    Trujillo-Paredes, Niurka; Valencia, Concepción; Guerrero-Flores, Gilda; Arzate, Dulce-María; Baizabal, José-Manuel; Guerra-Crespo, Magdalena; Fuentes-Hernández, Ayari; Zea-Armenta, Iván; Covarrubias, Luis

    2016-01-01

    ABSTRACT Notch signalling is a well-established pathway that regulates neurogenesis. However, little is known about the role of Notch signalling in specific neuronal differentiation. Using Dll1 null mice, we found that Notch signalling has no function in the specification of mesencephalic dopaminergic neural precursor cells (NPCs), but plays an important role in regulating their expansion and differentiation into neurons. Premature neuronal differentiation was observed in mesencephalons of Dll1-deficient mice or after treatment with a Notch signalling inhibitor. Coupling between neurogenesis and dopaminergic differentiation was indicated from the coincident emergence of neuronal and dopaminergic markers. Early in differentiation, decreasing Notch signalling caused a reduction in NPCs and an increase in dopaminergic neurons in association with dynamic changes in the proportion of sequentially-linked dopaminergic NPCs (Msx1/2+, Ngn2+, Nurr1+). These effects in differentiation caused a significant reduction in the number of dopaminergic neurons produced. Accordingly, Dll1 haploinsufficient adult mice, in comparison with their wild-type littermates, have a consistent reduction in neuronal density that was particularly evident in the substantia nigra pars compacta. Our results are in agreement with a mathematical model based on a Dll1-mediated regulatory feedback loop between early progenitors and their dividing precursors that controls the emergence and number of dopaminergic neurons. PMID:26912775

  9. A new method to effectively and rapidly generate neurons from SH-SY5Y cells.

    PubMed

    Yang, HongNa; Wang, Jing; Sun, JinHua; Liu, XiaoDun; Duan, Wei-Ming; Qu, TingYu

    2016-01-01

    It is well known that neurons differentiated from SH-SY5Y cells can serve as cell models for neuroscience research; i.e., neurotoxicity and tolerance to morphine in vitro. To differentiate SH-SY5Y cells into neurons, RA (retinoic acid) is commonly used to produce the inductive effect. However, the percentage of neuronal cells produced from SH-SY5Y cells is low, either from the use of RA treatment alone or from the combined application of RA and other chemicals. In the current study, we used CM-hNSCs (conditioned medium of human neural stem cells) as the combinational inducer with RA to prompt neuronal differentiation of SH-SY5Y cells. We found that neuronal differentiation was improved and that neurons were greatly increased in the differentiated SH-SY5Y cells using a combined treatment of CM-hNSCs and RA compared to RA treatment alone. The neuronal percentage was higher than 80% (about 88%) on the 3rd day and about 91% on the 7th day examined after a combined treatment with CM-hNSCs and RA. Cell maturation and neurite growth of these neuronal cells were also improved. In addition, the use of CM-hNSCs inhibited the apoptosis of RA-treated SH-SY5Y cells in culture. We are the first to report the use of CM-hNSCs in combination with RA to induce neuronal differentiation of RA-treated SH-SY5Y cells. Our method can rapidly and effectively promote the neuronal production of SH-SY5Y cells in culture conditions.

  10. A new method to effectively and rapidly generate neurons from SH-SY5Y cells.

    PubMed

    Yang, HongNa; Wang, Jing; Sun, JinHua; Liu, XiaoDun; Duan, Wei-Ming; Qu, TingYu

    2016-01-01

    It is well known that neurons differentiated from SH-SY5Y cells can serve as cell models for neuroscience research; i.e., neurotoxicity and tolerance to morphine in vitro. To differentiate SH-SY5Y cells into neurons, RA (retinoic acid) is commonly used to produce the inductive effect. However, the percentage of neuronal cells produced from SH-SY5Y cells is low, either from the use of RA treatment alone or from the combined application of RA and other chemicals. In the current study, we used CM-hNSCs (conditioned medium of human neural stem cells) as the combinational inducer with RA to prompt neuronal differentiation of SH-SY5Y cells. We found that neuronal differentiation was improved and that neurons were greatly increased in the differentiated SH-SY5Y cells using a combined treatment of CM-hNSCs and RA compared to RA treatment alone. The neuronal percentage was higher than 80% (about 88%) on the 3rd day and about 91% on the 7th day examined after a combined treatment with CM-hNSCs and RA. Cell maturation and neurite growth of these neuronal cells were also improved. In addition, the use of CM-hNSCs inhibited the apoptosis of RA-treated SH-SY5Y cells in culture. We are the first to report the use of CM-hNSCs in combination with RA to induce neuronal differentiation of RA-treated SH-SY5Y cells. Our method can rapidly and effectively promote the neuronal production of SH-SY5Y cells in culture conditions. PMID:26497914

  11. Embryonic stem cell-derived neural stem cells fuse with microglia and mature neurons.

    PubMed

    Cusulin, Carlo; Monni, Emanuela; Ahlenius, Henrik; Wood, James; Brune, Jan Claas; Lindvall, Olle; Kokaia, Zaal

    2012-12-01

    Transplantation of neural stem cells (NSCs) is a novel strategy to restore function in the diseased brain, acting through multiple mechanisms, for example, neuronal replacement, neuroprotection, and modulation of inflammation. Whether transplanted NSCs can operate by fusing with microglial cells or mature neurons is largely unknown. Here, we have studied the interaction of a mouse embryonic stem cell-derived neural stem (NS) cell line with rat and mouse microglia and neurons in vitro and in vivo. We show that NS cells spontaneously fuse with cocultured cortical neurons, and that this process requires the presence of microglia. Our in vitro data indicate that the NS cells can first fuse with microglia and then with neurons. The fused NS/microglial cells express markers and retain genetic and functional characteristics of both parental cell types, being able to respond to microglia-specific stimuli (LPS and IL-4/IL-13) and to differentiate to neurons and astrocytes. The NS cells fuse with microglia, at least partly, through interaction between phosphatidylserine exposed on the surface of NS cells and CD36 receptor on microglia. Transplantation of NS cells into rodent cortex results in fusion with mature pyramidal neurons, which often carry two nuclei, a process probably mediated by microglia. The fusogenic role of microglia could be even more important after NSC transplantation into brains affected by neurodegenerative diseases associated with microglia activation. It remains to be elucidated how the occurrence of the fused cells will influence the functional outcome after NSC transplantation in the diseased brain.

  12. Differential Sensitivity of Specific Neuronal Populations of the Rat Hypothalamus to Prolactin Action

    PubMed Central

    Sapsford, Tony J.; Kokay, Ilona C.; Östberg, Lovisa; Bridges, Robert S.; Grattan, David R.

    2014-01-01

    Prolactin stimulates dopamine release from neuroendocrine dopaminergic (NEDA) neurons in the hypothalamic arcuate nucleus (ARC) to maintain low levels of serum prolactin. Elevated prolactin levels during pregnancy and lactation may mediate actions in other hypothalamic regions such as the paraventricular nucleus (PVN) and rostral preoptic area (rPOA). We predicted that NEDA neurons would be more sensitive prolactin targets than neurons in other regions because they are required to regulate basal prolactin secretion. Moreover, differences in the accessibility of the ARC to prolactin in blood may influence the responsiveness of this population. Therefore, we compared prolactin-induced signaling in different hypothalamic neuronal populations following either systemic or intracerebroventricular (icv) prolactin administration. Phosphorylation of the signal transduction factor, STAT5 (pSTAT5), was used to identify prolactin-responsive neurons. In response to systemic prolactin, pSTAT5-labeled cells were widely observed in the ARC but absent from the rPOA and PVN. Many of these responsive cells in the ARC were identified as NEDA neurons. The lowest icv prolactin dose (10 ng) induced pSTAT5 in the ARC, but with higher doses (>500 ng) pSTAT5 was detected in numerous regions, including the rPOA and PVN. NEDA neurons were maximally labeled with nuclear pSTAT5 in response to 500 ng prolactin and appeared to be more sensitive than dopaminergic neurons in the rPOA. Subpopulations of oxytocin neurons in the hypothalamus were also found to be differentially sensitive to prolactin. These data suggest that differences in the accessibility of the arcuate nucleus to prolactin, together with intrinsic differences in the NEDA neurons, may facilitate homeostatic feedback regulation of prolactin release. PMID:21953590

  13. Cells from the adult corneal stroma can be reprogrammed to a neuron-like cell using exogenous growth factors

    SciTech Connect

    Greene, Carol Ann Chang, Chuan-Yuan; Fraser, Cameron J.; Nelidova, Dasha E.; Chen, Jing A.; Lim, Angela; Brebner, Alex; McGhee, Jennifer; Sherwin, Trevor; Green, Colin R.

    2014-03-10

    Cells thought to be stem cells isolated from the cornea of the eye have been shown to exhibit neurogenic potential. We set out to uncover the identity and location of these cells within the cornea and to elucidate their neuronal protein and gene expression profile during the process of switching to a neuron-like cell. Here we report that every cell of the adult human and rat corneal stroma is capable of differentiating into a neuron-like cell when treated with neurogenic differentiation specifying growth factors. Furthermore, the expression of genes regulating neurogenesis and mature neuronal structure and function was increased. The switch from a corneal stromal cell to a neuron-like cell was also shown to occur in vivo in intact corneas of living rats. Our results clearly indicate that lineage specifying growth factors can affect changes in the protein and gene expression profiles of adult cells, suggesting that possibly many adult cell populations can be made to switch into another type of mature cell by simply modifying the growth factor environment. - Highlights: • Adult corneal stromal cells can differentiated into neuron-like cells. • Neuronal specification of the adult stromal cell population is stochastic. • Neuronal specification in an adult cell population can be brought about by growth factors.

  14. Differential Modulation of Excitatory and Inhibitory Neurons during Periodic Stimulation

    PubMed Central

    Mahmud, Mufti; Vassanelli, Stefano

    2016-01-01

    Non-invasive transcranial neuronal stimulation, in addition to deep brain stimulation, is seen as a promising therapeutic and diagnostic approach for an increasing number of neurological diseases such as epilepsy, cluster headaches, depression, specific type of blindness, and other central nervous system disfunctions. Improving its effectiveness and widening its range of use may strongly rely on development of proper stimulation protocols that are tailored to specific brain circuits and that are based on a deep knowledge of different neuron types response to stimulation. To this aim, we have performed a simulation study on the behavior of excitatory and inhibitory neurons subject to sinusoidal stimulation. Due to the intrinsic difference in membrane conductance properties of excitatory and inhibitory neurons, we show that their firing is differentially modulated by the wave parameters. We analyzed the behavior of the two neuronal types for a broad range of stimulus frequency and amplitude and demonstrated that, within a small-world network prototype, parameters tuning allow for a selective enhancement or suppression of the excitation/inhibition ratio. PMID:26941602

  15. The function of the neuronal proteins Shc and huntingtin in stem cells and neurons: pharmacologic exploitation for human brain diseases.

    PubMed

    Zuccato, Chiara; Conti, Luciano; Reitano, Erika; Tartari, Marzia; Cattaneo, Elena

    2005-05-01

    The identification of intracellular molecules and soluble factors that are important for neuronal differentiation and survival are of critical importance for development of therapeutic strategies for brain diseases. First, the activity of these factors/molecules may be enhanced in vivo in the attempt to induce proper neuronal differentiation and integration of the resident stem cells. Second, these factors may be applied ex vivo to increase the recovery of neurons from stem cells. Third, for those intracellular molecules that play crucial roles in neuronal survival, identification of their downstream targets may give us the chance to develop drug screening assays that use these targets for therapeutic purposes. In recent years, it has become evident that intracellular signaling processes are critical mediators of the responses of neural stem cells and neurons to growth factors. Analysis of the mechanisms of signal transduction has led to the striking finding that a handful of conserved signaling pathways appear to be used in different combinations to specify a wide variety of tissues or cells. This review will focus on the mechanisms by which specific molecules control the transition from proliferation to differentiation of neural progenitor cells and the subsequent survival of postmitotic neurons; it also discusses how this knowledge may be exploited to increase the potential efficacy of stem cell replacement in the damaged brain. PMID:15965106

  16. Interactive properties of human glioblastoma cells with brain neurons in culture and neuronal modulation of glial laminin organization.

    PubMed

    Faria, Jane; Romão, Luciana; Martins, Sheila; Alves, Tércia; Mendes, Fabio A; de Faria, Giselle Pinto; Hollanda, Rosenilde; Takiya, Christina; Chimelli, Leila; Morandi, Veronica; de Souza, Jorge Marcondes; Abreu, Jose Garcia; Moura Neto, Vivaldo

    2006-12-01

    The harmonious development of the central nervous system depends on the interactions of the neuronal and glial cells. Extracellular matrix elements play important roles in these interactions, especially laminin produced by astrocytes, which has been shown to be a good substrate for neuron growth and axonal guidance. Glioblastomas are the most common subtypes of primary brain tumors and may be astrocytes in origin. As normal laminin-producing glial cells are the preferential substrate for neurons, and glial tumors have been shown to produce laminin, we questioned whether glioblastoma retained the same normal glial-neuron interactive properties with respect to neuronal growth and differentiation. Then, rat neurons were co-cultured onto rat normal astrocytes or onto three human glioblastoma cell lines obtained from neurosurgery. The co-culture confirmed that human glioblastoma cells as well as astrocytes maintained the ability to support neuritogenesis, but non-neural normal or tumoral cells failed to do so. However, glioblastoma cells did not distinguish embryonic from post-natal neurons in relation to neurite pattern in the co-cultures, as normal astrocytes did. Further, the laminin organization on both normal and tumoral glial cells was altered from a filamentous arrangement to a mixed punctuate/filamentous pattern when in co-culture with neurons. Together, these results suggest that glioblastoma cells could identify neuronal cells as partners, to support their growth and induce complex neurites, but they lost the normal glia property to distinguish neuronal age. In addition, our results show for the first time that neurons modulate the organization of astrocytes and glioblastoma laminin on the extracellular matrix.

  17. Human neural progenitors differentiate into astrocytes and protect motor neurons in aging rats.

    PubMed

    Das, Melanie M; Avalos, Pablo; Suezaki, Patrick; Godoy, Marlesa; Garcia, Leslie; Chang, Christine D; Vit, Jean-Philippe; Shelley, Brandon; Gowing, Genevieve; Svendsen, Clive N

    2016-06-01

    Age-associated health decline presents a significant challenge to healthcare, although there are few animal models that can be used to test potential treatments. Here, we show that there is a significant reduction in both spinal cord motor neurons and motor function over time in the aging rat. One explanation for this motor neuron loss could be reduced support from surrounding aging astrocytes. Indeed, we have previously shown using in vitro models that aging rat astrocytes are less supportive to rat motor neuron function and survival over time. Here, we test whether rejuvenating the astrocyte niche can improve the survival of motor neurons in an aging spinal cord. We transplanted fetal-derived human neural progenitor cells (hNPCs) into the aging rat spinal cord and found that the cells survive and differentiate into astrocytes with a much higher efficiency than when transplanted into younger animals, suggesting that the aging environment stimulates astrocyte maturation. Importantly, the engrafted astrocytes were able to protect against motor neuron loss associated with aging, although this did not result in an increase in motor function based on behavioral assays. We also transplanted hNPCs genetically modified to secrete glial cell line-derived neurotrophic factor (GDNF) into the aging rat spinal cord, as this combination of cell and protein delivery can protect motor neurons in animal models of ALS. During aging, GDNF-expressing hNPCs protected motor neurons, though to the same extent as hNPCs alone, and again had no effect on motor function. We conclude that hNPCs can survive well in the aging spinal cord, protect motor neurons and mature faster into astrocytes when compared to transplantation into the young spinal cord. While there was no functional improvement, there were no functional deficits either, further supporting a good safety profile of hNPC transplantation even into the older patient population. PMID:27032721

  18. Replication of lymphocytic choriomeningitis virus is restricted in terminally differentiated neurons.

    PubMed Central

    de la Torre, J C; Rall, G; Oldstone, C; Sanna, P P; Borrow, P; Oldstone, M B

    1993-01-01

    We have investigated the replication of lymphocytic choriomeningitis virus (LCMV) before and after the nerve growth factor (NGF)-induced transdifferentiation of PC12 cells from the chromaffin to the neuron-like phenotype. Untreated and NGF-treated cells were equally susceptible to LCMV infection; however, the viral yield was found to be 1,000-fold lower in NGF-differentiated PC12 cells. The reduced viral yield correlated with restricted LCMV replication and transcription within the infected cell, which was not caused by the lack of cell proliferation in the NGF-treated cells but rather was related to the induction or changes in expression levels of specific gene product(s) associated with the cell commitment to a neuronal phenotype. The return to the chromaffin phenotype after withdrawal of NGF restored normal LCMV yields as well as levels of viral replication and transcription. The finding of reduced viral replication in terminally differentiated neuronal cells has important implications for understanding the mechanism by which neurotropic viruses, such as LCMV, are able to establish a long-term persistent infection in the central nervous system in the absence of severe pathological changes. Images PMID:8230458

  19. Zeb1 controls neuron differentiation and germinal zone exit by a mesenchymal-epithelial-like transition.

    PubMed

    Singh, Shalini; Howell, Danielle; Trivedi, Niraj; Kessler, Ketty; Ong, Taren; Rosmaninho, Pedro; Raposo, Alexandre Asf; Robinson, Giles; Roussel, Martine F; Castro, Diogo S; Solecki, David J

    2016-05-14

    In the developing mammalian brain, differentiating neurons mature morphologically via neuronal polarity programs. Despite discovery of polarity pathways acting concurrently with differentiation, it's unclear how neurons traverse complex polarity transitions or how neuronal progenitors delay polarization during development. We report that zinc finger and homeobox transcription factor-1 (Zeb1), a master regulator of epithelial polarity, controls neuronal differentiation by transcriptionally repressing polarity genes in neuronal progenitors. Necessity-sufficiency testing and functional target screening in cerebellar granule neuron progenitors (GNPs) reveal that Zeb1 inhibits polarization and retains progenitors in their germinal zone (GZ). Zeb1 expression is elevated in the Sonic Hedgehog (SHH) medulloblastoma subgroup originating from GNPs with persistent SHH activation. Restored polarity signaling promotes differentiation and rescues GZ exit, suggesting a model for future differentiative therapies. These results reveal unexpected parallels between neuronal differentiation and mesenchymal-to-epithelial transition and suggest that active polarity inhibition contributes to altered GZ exit in pediatric brain cancers.

  20. Induction of human umbilical Wharton's jelly-derived mesenchymal stem cells toward motor neuron-like cells.

    PubMed

    Bagher, Zohreh; Ebrahimi-Barough, Somayeh; Azami, Mahmoud; Mirzadeh, Hamid; Soleimani, Mansooreh; Ai, Jafar; Nourani, Mohammad Reza; Joghataei, Mohammad Taghi

    2015-10-01

    The most important property of stem cells from different sources is the capacity to differentiate into various cells and tissue types. However, problems including contamination, normal karyotype, and ethical issues cause many limitations in obtaining and using these cells from different sources. The cells in Wharton's jelly region of umbilical cord represent a pool source of primitive cells with properties of mesenchymal stem cells (MSCs). The aim of this study was to determine the potential of human Wharton's jelly-derived mesenchymal stem cells (WJMSCs) for differentiation to motor neuron cells. WJMSCs were induced to differentiate into motor neuron-like cells by using different signaling molecules and neurotrophic factors in vitro. Differentiated neurons were then characterized for expression of motor neuron markers including nestin, PAX6, NF-H, Islet 1, HB9, and choline acetyl transferase (ChAT) by quantitative reverse transcription PCR and immunocytochemistry. Our results showed that differentiated WJMSCs could significantly express motor neuron biomarkers in RNA and protein levels 15 d post induction. These results suggested that WJMSCs can differentiate to motor neuron-like cells and might provide a potential source in cell therapy for neurodegenerative disease.

  1. Osthole promotes neuronal differentiation and inhibits apoptosis via Wnt/β-catenin signaling in an Alzheimer's disease model.

    PubMed

    Yao, Yingjia; Gao, Zhong; Liang, Wenbo; Kong, Liang; Jiao, Yanan; Li, Shaoheng; Tao, Zhenyu; Yan, Yuhui; Yang, Jingxian

    2015-12-15

    Neurogenesis is the process by which neural stem cells (NSCs) proliferate and differentiate into neurons. This is diminished in several neurodegenerative disorders such as Alzheimer's disease (AD), which is characterized by the deposition of amyloid (A)β peptides and neuronal loss. Stimulating NSCs to replace lost neurons is therefore a promising approach for AD treatment. Our previous study demonstrated that osthole modulates NSC proliferation and differentiation, and may reduce Aβ protein expression in nerve cells. Here we investigated the mechanism underlying the effects of osthole on NSCs. We found that osthole enhances NSC proliferation and neuronal differentiation while suppressing apoptosis, effects that were exerted via activation of Wnt/β-catenin signaling. These results provide evidence that osthole can potentially be used as a therapeutic agent in the treatment of AD and other neurodegenerative disorders.

  2. The Shh coreceptor Cdo is required for differentiation of midbrain dopaminergic neurons.

    PubMed

    Kwon, Yu-Rim; Jeong, Myong-Ho; Leem, Young-Eun; Lee, Sang-Jin; Kim, Hyun-Jin; Bae, Gyu-Un; Kang, Jong-Sun

    2014-09-01

    Sonic hedgehog (Shh) signaling is required for numerous developmental processes including specification of ventral cell types in the central nervous system such as midbrain dopaminergic (DA) neurons. The multifunctional coreceptor Cdo increases the signaling activity of Shh which is crucial for development of forebrain and neural tube. In this study, we investigated the role of Cdo in midbrain DA neurogenesis. Cdo and Shh signaling components are induced during neurogenesis of embryonic stem (ES) cells. Cdo(-/-) ES cells show reduced neuronal differentiation accompanied by increased cell death upon neuronal induction. In addition, Cdo(-/-) ES cells form fewer tyrosine hydroxylase (TH) and microtubule associated protein 2 (MAP2)-positive DA neurons correlating with the decreased expression of key regulators of DA neurogenesis, such as Shh, Neurogenin2, Mash1, Foxa2, Lmx1a, Nurr1 and Pitx3, relative to the Cdo(+/+) ES cells. Consistently, the Cdo(-/-) embryonic midbrain displays a reduction in expression of TH and Nurr1. Furthermore, activation of Shh signaling by treatment with Purmorphamine (Pur) restores the DA neurogenesis of Cdo(-/-) ES cells, suggesting that Cdo is required for the full Shh signaling activation to induce efficient DA neurogenesis.

  3. Decay in survival motor neuron and plastin 3 levels during differentiation of iPSC-derived human motor neurons

    PubMed Central

    Boza-Morán, María G; Martínez-Hernández, Rebeca; Bernal, Sara; Wanisch, Klaus; Also-Rallo, Eva; Le Heron, Anita; Alías, Laura; Denis, Cécile; Girard, Mathilde; Yee, Jiing-Kuan; Tizzano, Eduardo F.; Yáñez-Muñoz, Rafael J

    2015-01-01

    Spinal muscular atrophy (SMA) is a neuromuscular disease caused by mutations in Survival Motor Neuron 1 (SMN1), leading to degeneration of alpha motor neurons (MNs) but also affecting other cell types. Induced pluripotent stem cell (iPSC)-derived human MN models from severe SMA patients have shown relevant phenotypes. We have produced and fully characterized iPSCs from members of a discordant consanguineous family with chronic SMA. We differentiated the iPSC clones into ISL-1+/ChAT+ MNs and performed a comparative study during the differentiation process, observing significant differences in neurite length and number between family members. Analyses of samples from wild-type, severe SMA type I and the type IIIa/IV family showed a progressive decay in SMN protein levels during iPSC-MN differentiation, recapitulating previous observations in developmental studies. PLS3 underwent parallel reductions at both the transcriptional and translational levels. The underlying, progressive developmental decay in SMN and PLS3 levels may lead to the increased vulnerability of MNs in SMA disease. Measurements of SMN and PLS3 transcript and protein levels in iPSC-derived MNs show limited value as SMA biomarkers. PMID:26114395

  4. Decay in survival motor neuron and plastin 3 levels during differentiation of iPSC-derived human motor neurons.

    PubMed

    Boza-Morán, María G; Martínez-Hernández, Rebeca; Bernal, Sara; Wanisch, Klaus; Also-Rallo, Eva; Le Heron, Anita; Alías, Laura; Denis, Cécile; Girard, Mathilde; Yee, Jiing-Kuan; Tizzano, Eduardo F; Yáñez-Muñoz, Rafael J

    2015-06-26

    Spinal muscular atrophy (SMA) is a neuromuscular disease caused by mutations in Survival Motor Neuron 1 (SMN1), leading to degeneration of alpha motor neurons (MNs) but also affecting other cell types. Induced pluripotent stem cell (iPSC)-derived human MN models from severe SMA patients have shown relevant phenotypes. We have produced and fully characterized iPSCs from members of a discordant consanguineous family with chronic SMA. We differentiated the iPSC clones into ISL-1+/ChAT+ MNs and performed a comparative study during the differentiation process, observing significant differences in neurite length and number between family members. Analyses of samples from wild-type, severe SMA type I and the type IIIa/IV family showed a progressive decay in SMN protein levels during iPSC-MN differentiation, recapitulating previous observations in developmental studies. PLS3 underwent parallel reductions at both the transcriptional and translational levels. The underlying, progressive developmental decay in SMN and PLS3 levels may lead to the increased vulnerability of MNs in SMA disease. Measurements of SMN and PLS3 transcript and protein levels in iPSC-derived MNs show limited value as SMA biomarkers.

  5. Retinoic acid-induced neural differentiation of embryonal carcinoma cells.

    PubMed Central

    Jones-Villeneuve, E M; Rudnicki, M A; Harris, J F; McBurney, M W

    1983-01-01

    We have previously shown that the P19 line of embryonal carcinoma cells develops into neurons, astroglia, and fibroblasts after aggregation and exposure to retinoic acid. The neurons were initially identified by their morphology and by the presence of neurofilaments within their cytoplasm. We have more fully documented the neuronal nature of these cells by showing that their cell surfaces display tetanus toxin receptors, a neuronal cell marker, and that choline acetyl-transferase and acetyl cholinesterase activities appear coordinately in neuron-containing cultures. Several days before the appearance of neurons, there is a marked decrease in the amount of an embryonal carcinoma surface antigen, and at the same time there is a substantial decrease in the volumes of individual cells. Various retinoids were able to induce the development of neurons in cultures of aggregated P19 cells, but it did not appear that polyamine metabolism was involved in the effect. We have isolated a mutant clone which does not differentiate in the presence of any of the drugs which are normally effective in inducing differentiation of P19 cells. This mutant and others may help to elucidate the chain of events triggered by retinoic acid and other differentiation-inducing drugs. Images PMID:6656766

  6. Gene Expression Profile of NF-κB, Nrf2, Glycolytic, and p53 Pathways During the SH-SY5Y Neuronal Differentiation Mediated by Retinoic Acid.

    PubMed

    de Bittencourt Pasquali, Matheus Augusto; de Ramos, Vitor Miranda; Albanus, Ricardo D Oliveira; Kunzler, Alice; de Souza, Luis Henrinque Trentin; Dalmolin, Rodrigo Juliani Siqueira; Gelain, Daniel Pens; Ribeiro, Leila; Carro, Luigi; Moreira, José Cláudio Fonseca

    2016-01-01

    SH-SY5Y cells, a neuroblastoma cell line that is a well-established model system to study the initial phases of neuronal differentiation, have been used in studies to elucidate the mechanisms of neuronal differentiation. In the present study, we investigated alterations of gene expression in SH-SY5Y cells during neuronal differentiation mediated by retinoic acid (RA) treatment. We evaluated important pathways involving nuclear factor kappa B (NF-κB), nuclear E2-related factor 2 (Nrf2), glycolytic, and p53 during neuronal differentiation. We also investigated the involvement of reactive oxygen species (ROS) in modulating the gene expression profile of those pathways by antioxidant co-treatment with Trolox®, a hydrophilic analogue of α-tocopherol. We found that RA treatment increases levels of gene expression of NF-κB, glycolytic, and antioxidant pathway genes during neuronal differentiation of SH-SY5Y cells. We also found that ROS production induced by RA treatment in SH-SY5Y cells is involved in gene expression profile alterations, chiefly in NF-κB, and glycolytic pathways. Antioxidant co-treatment with Trolox® reversed the effects mediated by RA NF-κB, and glycolytic pathways gene expression. Interestingly, co-treatment with Trolox® did not reverse the effects in antioxidant gene expression mediated by RA in SH-SY5Y. To confirm neuronal differentiation, we quantified endogenous levels of tyrosine hydroxylase, a recognized marker of neuronal differentiation. Our data suggest that during neuronal differentiation mediated by RA, changes in profile gene expression of important pathways occur. These alterations are in part mediated by ROS production. Therefore, our results reinforce the importance in understanding the mechanism by which RA induces neuronal differentiation in SH-SY5Y cells, principally due this model being commonly used as a neuronal cell model in studies of neuronal pathologies.

  7. Regulation and direction of umbilical cord blood mesenchymal stem cells to adopt neuronal fate.

    PubMed

    Wang, Lei; Lu, Ming

    2014-03-01

    Umbilical cord blood mesenchymal stem cells (UCB-MSCs) transplantation is becoming a promising and attractive cell-based treatment modality for repairing the damaged central nervous system due to its advantages of low immunogenicity, wide range of sources, and less ethical controversy. One of the limitations of this approach is that the proportion of neurons differentiated from UCB-MSCs still remains at low level. Thus, to induce UCB-MSCs to differentiate into neuron-like cells with a higher proportion is one of the key technologies of regenerative medicine and tissue engineering. Many induction protocols with remarkably higher differentiation rate to neurons have been reported. However, each protocol has its pros and cons and whether the neurons differentiated from UCB-MSCs under a certain protocol has normal nerve function remains controversial. Therefore, to guarantee the success of future clinical applications of UCB-MSCs, more investigations should be performed to improve the induction method and differentiation efficiency.

  8. Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

    SciTech Connect

    Kilcoyne, Michelle; Sharma, Shashank; McDevitt, Niamh; O'Leary, Claire; Joshi, Lokesh; McMahon, Siobhan S.

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer Carbohydrates are important in the CNS and ChABC has been used for spinal cord injury (SCI) treatment. Black-Right-Pointing-Pointer Neuronal glycosylation in injury and after ChABC treatment is unknown. Black-Right-Pointing-Pointer In silico mining verified that glyco-related genes were differentially regulated after SCI. Black-Right-Pointing-Pointer In vitro model system revealed abnormal sialylation in an injured environment. Black-Right-Pointing-Pointer The model indicated a return to normal neuronal glycosylation after ChABC treatment. -- Abstract: Glycosylation is found ubiquitously throughout the central nervous system (CNS). Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellular matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration. Several CSPGs are up-regulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA). Usually {alpha}-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8. Treatment

  9. [Human pluripotent stem cell and neural differentiation].

    PubMed

    Wataya, Takafumi; Muguruma, Keiko; Sasai, Yoshiki

    2008-10-01

    Recovery of lost brain function is an important issue in medical studies because neurons of the central nervous system (CNS) have poor potential for regeneration. Since few CNS diseases can be treated completely by medicines, regenerative therapy by using stem cells should be studied as a new type of therapeutic intervention. The efficacy of cell replacement therapy in Parkinson's disease has been well investigated. Several studies on fetal tissue transplantation have revealed that quantity and purity of transplanted cells are necessary for recovery of symptoms. SFEB (Serum-free floating culture of embryoid body-like aggregates) method is capable of inducing multi-potential CNS progenitors that can be steered to differentiate into region-specific tissues. On the basis of the existing knowledge of embryology, we have succeeded in the generating of various types of neurons such as telencephalic, cerebeller (Purkinje and granule cells), retinal (photoreceptor cells) and hypothalamic neurons. Application of this culture method to human ES (hES) cells is necessary for clinical purpose: however, poor survival of hES cells in SFEB culture might limit the possibility of using these cells for future medical applications. We found that a selective Rho-associated kinase (ROCK) inhibitor, Y-27632, markedly diminished the dissociation-induced apoptosis of hES cells and enabled the cells to form aggregates in SFEB culture. For both mouse and human ES cells, SFEB culture is a favorable method that can generate large amounts of region-specific neurons. However, stem cell-based therapy continues to face several obstacles. It is important that researchers in the basic sciences and clinical medicine should discuss these problems together to overcome both scientific and ethical issues related to stem cells.

  10. Major epigenetic development distinguishing neuronal and non-neuronal cells occurs postnatally in the murine hypothalamus.

    PubMed

    Li, Ge; Zhang, Wenjuan; Baker, Maria S; Laritsky, Eleonora; Mattan-Hung, Natalia; Yu, Dahai; Kunde-Ramamoorthy, Govindarajan; Simerly, Richard B; Chen, Rui; Shen, Lanlan; Waterland, Robert A

    2014-03-15

    Prenatal and early postnatal environment can persistently alter one's risk of obesity. Environmental effects on hypothalamic developmental epigenetics constitute a likely mechanism underlying such 'developmental programming' of energy balance regulation. To advance our understanding of these processes, it is essential to develop approaches to disentangle the cellular and regional heterogeneity of hypothalamic developmental epigenetics. We therefore performed genome-scale DNA methylation profiling in hypothalamic neurons and non-neuronal cells at postnatal day 0 (P0) and P21 and found, surprisingly, that most of the DNA methylation differences distinguishing these two cell types are established postnatally. In particular, neuron-specific increases in DNA methylation occurred extensively at genes involved in neuronal development. Quantitative bisulfite pyrosequencing verified our methylation profiling results in all 15 regions examined, and expression differences were associated with DNA methylation at several genes. We also identified extensive methylation differences between the arcuate (ARH) and paraventricular nucleus of the hypothalamus (PVH). Integrating these two data sets showed that genomic regions with PVH versus ARH differential methylation strongly overlap with those undergoing neuron-specific increases from P0 to P21, suggesting that these developmental changes occur preferentially in either the ARH or PVH. In particular, neuron-specific methylation increases at the 3' end of Shh localized to the ARH and were positively associated with gene expression. Our data indicate a key role for DNA methylation in establishing the gene expression potential of diverse hypothalamic cell types, and provide the novel insight that early postnatal life is a critical period for cell type-specific epigenetic development in the murine hypothalamus.

  11. Ensemble Recording of Electrical Activity in Neurons Derived from P19 Embryonal Carcinoma Cells

    NASA Astrophysics Data System (ADS)

    Takayama, Yuzo; Saito, Atushi; Moriguchi, Hiroyuki; Kotani, Kiyoshi; Jimbo, Yasuhiko

    Regeneration of the central nervous system (CNS) is one of the most important research themes in neuroscience and neuroengineering. It is essential to replenish the lost neurons and to establish appropriate functional neuronal networks using pluripotent stem cells. Little is known, however, about the properties of stem cell-derived neuronal networks, particularly under the differentiation and development processes. In this work, we cultured P19 embryonal carcinoma cells on micro-electrode arrays (MEAs). P19 cells were differentiated into neurons by retinoic acid application and formed densely connected networks. Spontaneous electrical activity was extracellulary recorded through substrate electrodes and analyzed. Synchronized periodic bursts, which were the characteristic features in primary cultured CNS neurons, were observed. Pharmacological studies demonstrated that the glutamatergic excitatory synapses and the GABAergic inhibitory synapses were active in these P19-derived neuronal networks. The results suggested that MEA-based recording was useful for monitoring differentiation processes of stem cells. P19-derived neuronal networks had quite similar network properties to those of primary cultured neurons, and thus provide a novel model system to investigate stem cell-based neuronal regeneration.

  12. Differential regulation of proliferation and neuronal differentiation in adult rat spinal cord neural stem/progenitors by ERK1/2, Akt, and PLCγ

    PubMed Central

    Chan, Wai Si; Sideris, Alexandra; Sutachan, Jhon J.; Montoya G, Jose V.; Blanck, Thomas J. J.; Recio-Pinto, Esperanza

    2013-01-01

    Proliferation of endogenous neural stem/progenitor cells (NSPCs) has been identified in both normal and injured adult mammalian spinal cord. Yet the signaling mechanisms underlying the regulation of adult spinal cord NSPCs proliferation and commitment toward a neuronal lineage remain undefined. In this study, the role of three growth factor-mediated signaling pathways in proliferation and neuronal differentiation was examined. Adult spinal cord NSPCs were enriched in the presence of fibroblast growth factor 2 (FGF2). We observed an increase in the number of cells expressing the microtubule-associated protein 2 (MAP2) over time, indicating neuronal differentiation in the culture. Inhibition of the mitogen-activated protein kinase or extracellular signal-regulated kinase (ERK) kinase 1 and 2/ERK 1 and 2 (MEK/ERK1/2) or the phosphoinositide 3-kinase (PI3K)/Akt pathways suppressed active proliferation in adult spinal cord NSPC cultures; whereas neuronal differentiation was negatively affected only when the ERK1/2 pathway was inhibited. Inhibition of the phospholipase Cγ (PLCγ) pathway did not affect proliferation or neuronal differentiation. Finally, we demonstrated that the blockade of either the ERK1/2 or PLCγ signaling pathways reduced neurite branching of MAP2+ cells derived from the NSPC cultures. Many of the MAP2+ cells expressed synaptophysin and had a glutamatergic phenotype, indicating that over time adult spinal cord NSPCs had differentiated into mostly glutamatergic neurons. Our work provides new information regarding the contribution of these pathways to the proliferation and neuronal differentiation of NSPCs derived from adult spinal cord cultures, and emphasizes that the contribution of these pathways is dependent on the origin of the NSPCs. PMID:23986655

  13. Transcription factor-induced lineage programming of noradrenaline and motor neurons from embryonic stem cells.

    PubMed

    Mong, Jamie; Panman, Lia; Alekseenko, Zhanna; Kee, Nigel; Stanton, Lawrence W; Ericson, Johan; Perlmann, Thomas

    2014-03-01

    An important goal in stem cell biology is to