Stimulation of mGluR1/5 Improves Defective Internalization of AMPA Receptors in NPC1 Mutant Mouse.

Niemann-Pick type C1 (NPC1) disease is characterized by neurodegeneration caused by cholesterol accumulation in the late endosome/lysosome. In this study, a defective basal and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-stimulated internalization of GluR2-containing AMPA receptors in NPC1-/- cortical neurons was detected. Our results show that the amount of cholesterol and group I metabotropic glutamate receptors (mGluR1/5) in lipid rafts of NPC1-/- cortical tissue and neurons are decreased and their downstream signals of p-ERK are defective, which are restored by a rebalance of cholesterol homeostasis through ß-cyclodextrin (ß-CD) treatment. Application of 3,5-dihydroxyphenylglycine (DHPG)-a mGluR1/5 agonist-and ß-CD markedly increases the internalization of AMPA receptors and decreases over-influx of calcium in NPC1-/- neurons, respectively. Furthermore, the defective phosphorylated GluR2 and protein kinase C signals are ameliorated by the treatment with DHPG and ß-CD, respectively, suggesting an involvement of them in internalization dysfunction. Taken together, our data imply that abnormal internalization of AMPA receptors is a critical mechanism for neuronal dysfunction and the correction of dysfunctional mGluR1/5 is a potential therapeutic strategy for NPC1 disease.

Patient-Specific iPSC-Derived Neural Differentiated and Hepatocyte-like Cells, Carrying the Compound Heterozygous Mutation p.V1023Sfs*15/p.G992R, Present the "Variant" Biochemical Phenotype of Niemann-Pick Type C1 Disease.

Niemann-Pick disease type C1 (NP-C1) is a rare lysosomal storage disorder caused by autosomal recessive mutations in the NPC1 gene. Patients display a wide spectrum on the clinical as well as on the molecular level, wherein a so-called "variant" biochemical phenotype can be observed. Here, we report an in vitro analysis of fibroblasts obtained from an NP-C1 patient carrying the undescribed compound heterozygous mutation p.V1023Sfs*15/p.G992R. Since NP-C1 is a neurovisceral disease and the patient suffers from severe neurological as well as hepatic symptoms, we extended our study to neural differentiated and hepatocyte-like cells derived from patient-specific induced pluripotent stem cells. We detected slightly increased intracellular cholesterol levels compared to the control cell line in fibroblasts, neural differentiated and hepatocyte-like cells, suggesting a "variant" biochemical phenotype. Furthermore, the total NPC1 protein, as well as post-ER glycoforms of the NPC1 protein, tended to be reduced. In addition, colocalization analysis revealed a mild reduction of the NPC1 protein in the lysosomes. The patient was diagnosed with NP-C1 at the age of 34 years, after an initial misdiagnosis of schizophrenia. After years of mild and unspecific symptoms, such as difficulties in coordination and concentration, symptoms progressed and the patient finally presented with ataxia, dysarthria, dysphagia, vertical supranuclear gaze palsy, and hepatosplenomegaly. Genetic testing finally pointed towards an NP-C1 diagnosis, revealing the so-far undescribed compound heterozygous mutation p.V1023Sfs*15/p.G992R in the NPC1 gene. In light of these findings, this case provides support for the p.G992R mutation being causative for a "variant" biochemical phenotype leading to an adult-onset type of NP-C1 disease.

Pluripotent Stem Cells for Disease Modeling and Drug Discovery in Niemann-Pick Type C1.

The lysosomal storage disorders Niemann-Pick disease Type C1 (NPC1) and Type C2 (NPC2) are rare diseases caused by mutations in the NPC1 or NPC2 gene. Both NPC1 and NPC2 are proteins responsible for the exit of cholesterol from late endosomes and lysosomes (LE/LY). Consequently, mutations in one of the two proteins lead to the accumulation of unesterified cholesterol and glycosphingolipids in LE/LY, displaying a disease hallmark. A total of 95% of cases are due to a deficiency of NPC1 and only 5% are caused by NPC2 deficiency. Clinical manifestations include neurological symptoms and systemic symptoms, such as hepatosplenomegaly and pulmonary manifestations, the latter being particularly pronounced in NPC2 patients. NPC1 and NPC2 are rare diseases with the described neurovisceral clinical picture, but studies with human primary patient-derived neurons and hepatocytes are hardly feasible. Obviously, induced pluripotent stem cells (iPSCs) and their derivatives are an excellent alternative for indispensable studies with these affected cell types to study the multisystemic disease NPC1. Here, we present a review focusing on studies that have used iPSCs for disease modeling and drug discovery in NPC1 and draw a comparison to commonly used NPC1 models.

Pathophysiological In Vitro Profile of Neuronal Differentiated Cells Derived from Niemann-Pick Disease Type C2 Patient-Specific iPSCs Carrying the NPC2 Mutations c.58G>T/c.140G>T.

Niemann-Pick type C2 (NP-C2) disease is a rare hereditary disease caused by mutations in the NPC2 gene. NPC2 is a small, soluble protein consisting of 151 amino acids, primarily expressed in late endosomes and lysosomes (LE/LY). Together with NPC1, a transmembrane protein found in these organelles, NPC2 accomplishes the exclusion of cholesterol; thus, both proteins are essential to maintain cellular cholesterol homeostasis. Consequently, mutations in the NPC2 or NPC1 gene result in pathophysiological accumulation of cholesterol and sphingolipids in LE/LY. The vast majority of Niemann-Pick type C disease patients, 95%, suffer from a mutation of NPC1, and only 5% display a mutation of NPC2. The biochemical phenotype of NP-C1 and NP-C2 appears to be indistinguishable, and both diseases share several commonalities in the clinical manifestation. Studies of the pathological mechanisms underlying NP-C2 are mostly based on NP-C2 animal models and NP-C2 patient-derived fibroblasts. Recently, we established induced pluripotent stem cells (iPSCs), derived from a donor carrying the NPC2 mutations c.58G>T/c.140G>T. Here, we present a profile of pathophysiological in vitro features, shared by NP-C1 and NP-C2, of neural differentiated cells obtained from the patient specific iPSCs. Profiling comprised a determination of the NPC2 protein level, detection of cholesterol accumulation by filipin staining, analysis of oxidative stress, and determination of autophagy. As expected, the NPC2-deficient cells displayed a significantly reduced amount of NPC2 protein, and, accordingly, we observed a significantly increased amount of cholesterol. Most notably, NPC2-deficient cells displayed only a slight increase of reactive oxygen species (ROS), suggesting that they do not suffer from oxidative stress and express catalase at a high level. As a site note, comparable NPC1-deficient cells suffer from a lack of catalase and display an increased level of ROS. In summary, this cell line provides a valuable tool to gain deeper understanding, not only of the pathogenic mechanism of NP-C2, but also of NP-C1.

Oxidative Stress and Alterations in the Antioxidative Defense System in Neuronal Cells Derived from NPC1 Patient-Specific Induced Pluripotent Stem Cells.

Oxidative stress (OS) represents a state of an imbalanced amount of reactive oxygen species (ROS) and/or a hampered efficacy of the antioxidative defense system. Cells of the central nervous system are particularly sensitive to OS, as they have a massive need of oxygen to maintain proper function. Consequently, OS represents a common pathophysiological hallmark of neurodegenerative diseases and is discussed to contribute to the neurodegeneration observed amongst others in Alzheimer's disease and Parkinson's disease. In this context, accumulating evidence suggests that OS is involved in the pathophysiology of Niemann-Pick type C1 disease (NPC1). NPC1, a rare hereditary neurodegenerative disease, belongs to the family of lysosomal storage disorders. A major hallmark of the disease is the accumulation of cholesterol and other glycosphingolipids in lysosomes. Several studies describe OS both in murine in vivo and in vitro NPC1 models. However, studies based on human cells are limited to NPC1 patient-derived fibroblasts. Thus, we analyzed OS in a human neuronal model based on NPC1 patient-specific induced pluripotent stem cells (iPSCs). Higher ROS levels, as determined by DCF (dichlorodihydrofluorescein) fluorescence, indicated oxidative stress in all NPC1-deficient cell lines. This finding was further supported by reduced superoxide dismutase (SOD) activity. The analysis of mRNA and protein levels of SOD1 and SOD2 did not reveal any difference between control cells and NPC1-deficient cells. Interestingly, we observed a striking decrease in catalase mRNA and protein levels in all NPC1-deficient cell lines. As catalase is a key enzyme of the cellular antioxidative defense system, we concluded that the lack of catalase contributes to the elevated ROS levels observed in NPC1-deficient cells. Thus, a restitution of a physiological catalase level may pose an intervention strategy to rescue NPC1-deficient cells from the repercussions of oxidative stress contributing to the neurodegeneration observed in NPC1.

Identification of Brain-Specific Treatment Effects in NPC1 Disease by Focusing on Cellular and Molecular Changes of Sphingosine-1-Phosphate Metabolism.

Niemann-Pick type C1 (NPC1) is a lysosomal storage disorder, inherited as an autosomal-recessive trait. Mutations in the Npc1 gene result in malfunction of the NPC1 protein, leading to an accumulation of unesterified cholesterol and glycosphingolipids. Beside visceral symptoms like hepatosplenomegaly, severe neurological symptoms such as ataxia occur. Here, we analyzed the sphingosine-1-phosphate (S1P)/S1P receptor (S1PR) axis in different brain regions of Npc1-/- mice and evaluated specific effects of treatment with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) together with the iminosugar miglustat. Using high-performance thin-layer chromatography (HPTLC), mass spectrometry, quantitative real-time PCR (qRT-PCR) and western blot analyses, we studied lipid metabolism in an NPC1 mouse model and human skin fibroblasts. Lipid analyses showed disrupted S1P metabolism in Npc1-/- mice in all brain regions, together with distinct changes in S1pr3/S1PR3 and S1pr5/S1PR5 expression. Brains of Npc1-/- mice showed only weak treatment effects. However, side effects of the treatment were observed in Npc1+/+ mice. The S1P/S1PR axis seems to be involved in NPC1 pathology, showing only weak treatment effects in mouse brain. S1pr expression appears to be affected in human fibroblasts, induced pluripotent stem cells (iPSCs)-derived neural progenitor and neuronal differentiated cells. Nevertheless, treatment-induced side effects make examination of further treatment strategies indispensable.

Alteration of GABAergic Input Precedes Neurodegeneration of Cerebellar Purkinje Cells of NPC1-Deficient Mice.

Niemann-Pick Disease Type C1 (NPC1) is a rare hereditary neurodegenerative disease belonging to the family of lysosomal storage disorders. NPC1-patients suffer from, amongst other symptoms, ataxia, based on the dysfunction and loss of cerebellar Purkinje cells. Alterations in synaptic transmission are believed to contribute to a pathological mechanism leading to the progressive loss of Purkinje cells observed in NPC1-deficient mice. With regard to inhibitory synaptic transmission, alterations of GABAergic synapses are described but functional data are missing. For this reason, we have examined here the inhibitory GABAergic synaptic transmission of Purkinje cells of NPC1-deficient mice (NPC1-/-). Patch clamp recordings of inhibitory post-synaptic currents (IPSCs) of Purkinje cells revealed an increased frequency of GABAergic IPSCs in NPC1-/- mice. In addition, Purkinje cells of NPC1-/- mice were less amenable for modulation of synaptic transmission via the activation of excitatory NMDA-receptors (NMDA-Rs). Western blot testing disclosed a reduced protein level of phosphorylated alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs) subunit GluA2 in the cerebella of NPC1-/- mice, indicating a disturbance in the internalization of GluA2-containing AMPA-Rs. Since this is triggered by the activation of NMDA-Rs, we conclude that a disturbance in the synaptic turnover of AMPA-Rs underlies the defective inhibitory GABAergic synaptic transmission. While these alterations precede obvious signs of neurodegeneration of Purkinje cells, we propose a contribution of synaptic malfunction to the initiation of the loss of Purkinje cells in NPC1. Thus, a prevention of the disturbance of synaptic transmission in early stages of the disease might display a target with which to avert progressive neurodegeneration in NPC1.

Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test.

Niemann-Pick Type C (NP-C) is a rare disorder of lipid metabolism caused by mutations within the NPC1 and NPC2 genes. NP-C is a neurovisceral disease leading to a heterogeneous, multisystemic spectrum of symptoms in those affected. Until now, there is no investigative tool to demonstrate the significance of single variants within the NPC genes. Hence, the aim of the study was to establish a test that allows for an objective assessment of the pathological potential of NPC1 gene variants. Chinese hamster ovary cells defective in the NPC1 gene accumulate cholesterol in lysosomal storage organelles. The cells were transfected with NPC1-GFP plasmid vectors carrying distinct sequence variants. Filipin staining was used to test for complementation of the phenotype. The known variant p.Ile1061Thr showed a significantly impaired cholesterol clearance after 12 and 24 h compared to the wild type. Among the investigated variants, p.Ser954Leu and p.Glu1273Lys showed decelerated cholesterol clearance as well. The remaining variants p.Gln60His, p.Val494Met, and p.Ile787Val showed a cholesterol clearance indistinguishable from wild type. Further, p.Ile1061Thr acquired an enhanced clearance ability upon 25-hydroxycholesterol treatment. We conclude that the variants that caused an abnormal clearance phenotype are highly likely to be of clinical relevance. Moreover, we present a system that can be utilized to screen for new drugs.

Decreased calcium flux in Niemann-Pick type C1 patient-specific iPSC-derived neurons due to higher amount of calcium-impermeable AMPA receptors.

Niemann-Pick disease type C1 (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene, resulting mainly in the accumulation of cholesterol and the ganglioside GM2. Recently, we described accumulations of these lipids in neuronal differentiated cells derived from NPC1 patient-specific induced pluripotent stem cells (iPSCs). As these lipids are essential for proper cell membrane composition, we were interested in the expression and function of voltage-gated ion channels and excitatory AMPA receptors (AMPARs) in neurons derived from three patient-specific iPSC lines. By means of patch clamp recordings and microfluorimetric measurements of calcium (Ca2+), we examined the expression of voltage-gated ion channels and AMPARs. Cells of the three used cell lines carrying the c.1836A>C/c.1628delC, the c.1180T>C or the c.3182T>C mutation demonstrated a significantly reduced AMPA-induced Ca2+-influx, suggesting an altered expression profile of these receptors. RT-qPCR revealed a significant upregulation of mRNA for the AMPA receptor subunits GluA1 and GluA2 and western blot analysis showed increased protein level of GluA2. Thus, we conclude that the observed reduced Ca2+-influx is based on an increase of GluA2 containing Ca2+-impermeable AMPARs. An attenuated function of GluRs in neurons potentially contributes to the progressive neurodegeneration observed in NPC1 and might represent an objective in regard of the development of new therapeutic approaches in NPC1.

Non-canonical pathway induced by Wnt3a regulates ß-catenin via Pyk2 in differentiating human neural progenitor cells.

Wnt/ß-catenin and Wnt/Ca2+ pathways are involved in cellular processes during embryonic development and the interaction between them in the same cell decides the outcome of cellular functions. In this study, we showed that Wnt3a triggers the Wnt/Ca2+ signaling pathway, indicated by an increase of cytosolic free calcium ([Ca2+]i) and activation of calmodulin dependent kinase II (CaMKII) during the differentiation of human neuronal progenitor cells (hNPCs). Wnt3a via the increase of [Ca2+]i activates proline-rich tyrosine kinase 2 (Pyk2), which subsequently regulates phosphorylation of glycogen synthase kinase 3ß (GSK3ß) and ß-catenin stabilization. Our findings suggest that Pyk2 plays an important role in the coordination of stabilization of ß-catenin in the crosstalk between Wnt/ß-catenin and Wnt/Ca2+ signaling pathways upon Wnt3a stimulation in differentiating hNPCs.

The novel arylindolylmaleimide PDA-66 displays pronounced antiproliferative effects in acute lymphoblastic leukemia cells.

BACKGROUND: Prognosis of adult patients suffering from acute lymphoblastic leukemia (ALL) is still unsatisfactory. Targeted therapy via inhibition of deregulated signaling pathways appears to be a promising therapeutic option for the treatment of ALL. Herein, we evaluated the influence of a novel arylindolylmaleimide (PDA-66), a potential GSK3ß inhibitor, on several ALL cell lines. METHODS: ALL cell lines (SEM, RS4;11, Jurkat and MOLT4) were exposed to different concentrations of PDA-66. Subsequently, proliferation, metabolic activity, apoptosis and necrosis, cell cycle distribution and protein expression of Wnt and PI3K/Akt signaling pathways were analyzed at different time points. RESULTS: PDA-66 inhibited the proliferation of ALL cells significantly by reduction of metabolic activity. The 72 h IC50 values ranged between 0.41 to 1.28 µM PDA-66. Additionally, caspase activated induction of apoptosis could be detected in the analyzed cell lines. PDA-66 influenced the cell cycle distribution of ALL cell lines differently. While RS4;11 and MOLT4 cells were found to be arrested in G2 phase, SEM cells showed an increased cell cycle in G0/1 phase. CONCLUSION: PDA-66 displays significant antileukemic activity in ALL cells and classifies as candidate for further evaluation as a potential drug in targeted therapy of ALL.

FUS ALS neurons activate major stress pathways and reduce translation as an early protective mechanism against neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder causing progressive loss of motor neurons. Mutations in Fused in sarcoma (FUS) leading to its cytoplasmic mislocalization cause a subset of ALS. Under stress, mutant FUS localizes to stress granules (SGs)-cytoplasmic condensates composed of RNA and various proteins. Aberrant dynamics of SGs is linked to the pathology of ALS. Here, using motor neurons (MNs) derived from human induced pluripotent stem cells, we show that, in mutant FUS, MN dynamics of SGs is disturbed. Additionally, heat-shock response (HSR) and integrated stress response (ISR) involved in the regulation of SGs are upregulated in mutant MNs. HSR activation correlates with the amount of cytoplasmic FUS mislocalization. While inhibition of SG formation, translation, or ISR does not influence survival of FUS ALS neurons, proteotoxicity that cannot be compensated with the activation of stress pathways is the main driver of neurodegeneration in early FUS ALS.

Generation of the human iPSC lines AKOSi011-A carrying the mutation p.Pro65Ser/p.Asp35T and AKOSi012-A, carrying the mutation p.Tyr231His, derived from FAHN patient fibroblasts.

Fatty acid hydroxylase-associated neurodegeneration (FAHN) is a hereditary neurodegenerative disease caused by mutations in the FA2H gene. Patients show a wide range of neurological symptoms and an abnormal myelination. Here we describe the generation of the human induced pluripotent stem cell (hiPSC) lines AKOSi011-A and AKOSi012-A, derived from FAHN-patient fibroblasts, carrying the compound heterozygous mutation p.Pro65Ser/p.Asp35Tyr and the homozygous mutation p.Tyr231His, respectively. The hiPSC lines were generated using a non-integrating Sendai virus. The obtained hiPSCs show an unobtrusive karyotype, carry the mutations of the original fibroblasts, express pluripotency markers and can differentiate into cells of the three germ layers.

Impact of Organelle Transport Deficits on Mitophagy and Autophagy in Niemann-Pick Disease Type C.

Defective mitochondria are pathophysiological features of a number of neurodegenerative diseases. Here, we investigated mitochondrial dysfunction in the context of the rare lysosomal storage diseases Niemann-Pick disease type C1 and type C2 (NP-C1 and NP-C2). Mutations in either the NPC1 or NPC2 gene lead to cholesterol accumulation in late endosomes and lysosomes, resulting in impaired cholesterol homeostasis. The extent to which this may lead to mitochondrial dysfunction has been poorly studied so far. Therefore, we investigated the morphology, function, and transport of mitochondria, as well as their degradation via mitophagy, in a disease-associated human neural cell model of NP-C. By performing live cell imaging, we observed markedly reduced mitochondrial transport, although morphology and function were not appreciably altered. However, we observed a defective mitophagy induction shown by a reduced capability to elevate parkin expression and engulf mitochondria in autophagosomes after treatment with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). This was accompanied by defects in autophagy induction, exhibited by a hampered p62 expression and progression, shown by increased LC3BII levels and a defective fusion of autophagosomes and lysosomes. The latter might have been additionally influenced by the observed reduced lysosomal transport. Hence, we hypothesized that a reduced recycling of mitochondria contributes to the pathophysiology of NP-C.

Assessment of FDA-Approved Drugs as a Therapeutic Approach for Niemann-Pick Disease Type C1 Using Patient-Specific iPSC-Based Model Systems.

Niemann-Pick type C1 (NP-C1) is a fatal, progressive neurodegenerative disease caused by mutations in the NPC1 gene. Mutations of NPC1 can result in a misfolded protein that is subsequently marked for proteasomal degradation. Such loss-of-function mutations lead to cholesterol accumulation in late endosomes and lysosomes. Pharmacological chaperones (PCs) are described to protect misfolded proteins from proteasomal degradation and are being discussed as a treatment strategy for NP-C1. Here, we used a combinatorial approach of high-throughput in silico screening of FDA-approved drugs and in vitro biochemical assays to identify potential PCs. The effects of the hit compounds identified by molecular docking were compared in vitro with 25-hydroxycholesterol (25-HC), which is known to act as a PC for NP-C1. We analyzed cholesterol accumulation, NPC1 protein content, and lysosomal localization in patient-specific fibroblasts, as well as in neural differentiated and hepatocyte-like cells derived from patient-specific induced pluripotent stem cells (iPSCs). One compound, namely abiraterone acetate, showed comparable results to 25-HC and restored NPC1 protein level, corrected the intracellular localization of NPC1, and consequently decreased cholesterol accumulation in NPC1-mutated fibroblasts and iPSC-derived neural differentiated and hepatocyte-like cells. The discovered PC altered not only the pathophysiological phenotype of cells carrying the I1061T mutation- known to be responsive to treatment with PCs-but an effect was also observed in cells carrying other NPC1 missense mutations. Therefore, we hypothesize that the PCs studied here may serve as an effective treatment strategy for a large group of NP-C1 patients.

Generation of the human iPSC line AKOSi010-A from fibroblasts of a female FAHN patient, carrying the compound heterozygous mutation p.Gly45Arg/p.His319Arg.

Fatty acid hydroxylase-associated neurodegeneration (FAHN) is a rare childhood onset neurodegenerative disease caused by mutations in the FA2H gene. Patients display abnormal myelination, cerebellar atrophy and some have iron deposition in the central nervous system. Here we describe the generation of AKOSi010-A, a human induced pluripotent stem cell (hiPSC) line derived from fibroblasts of a female patient carrying the compound heterozygous p.Gly45Arg/p.His319Arg, using non-integrating Sendai virus. The generated iPSCs express pluripotency markers, can differentiate into cell types of the three germ layers and show a normal karyotype. This cell line displays a unique source to study the pathophysiology of FAHN.

Quantitative and kinetic profile of Wnt/ß-catenin signaling components during human neural progenitor cell differentiation.

ReNcell VM is an immortalized human neural progenitor cell line with the ability to differentiate in vitro into astrocytes and neurons, in which the Wnt/ß-catenin pathway is known to be involved. However, little is known about kinetic changes of this pathway in human neural progenitor cell differentiation. In the present study, we provide a quantitative profile of Wnt/ß-catenin pathway dynamics showing its spatio-temporal regulation during ReNcell VM cell differentiation. We show first that T-cell factor dependent transcription can be activated by stabilized ß-catenin. Furthermore, endogenous Wnt ligands, pathway receptors and signaling molecules are temporally controlled, demonstrating changes related to differentiation stages. During the first three hours of differentiation the signaling molecules LRP6, Dvl2 and ß-catenin are spatio-temporally regulated between distinct cellular compartments. From 24 h onward, components of the Wnt/ß-catenin pathway are strongly activated and regulated as shown by mRNA up-regulation of Wnt ligands (Wnt5a and Wnt7a), receptors including Frizzled-2, -3, -6, -7, and -9, and co-receptors, and target genes including Axin2. This detailed temporal profile of the Wnt/ß-catenin pathway is a first step to understand, control and to orientate, in vitro, human neural progenitor cell differentiation.

Erythropoietin and the effect of oxygen during proliferation and differentiation of human neural progenitor cells.

BACKGROUND: Hypoxia plays a critical role in various cellular mechanisms, including proliferation and differentiation of neural stem and progenitor cells. In the present study, we explored the impact of lowered oxygen on the differentiation potential of human neural progenitor cells, and the role of erythropoietin in the differentiation process. RESULTS: In this study we demonstrate that differentiation of human fetal neural progenitor cells under hypoxic conditions results in an increased neurogenesis. In addition, expansion and proliferation under lowered oxygen conditions also increased neuronal differentiation, although proliferation rates were not altered compared to normoxic conditions. Erythropoietin partially mimicked these hypoxic effects, as shown by an increase of the metabolic activity during differentiation and protection of differentiated cells from apoptosis. CONCLUSION: These results provide evidence that hypoxia promotes the differentiation of human fetal neural progenitor cells, and identifies the involvement of erythropoietin during differentiation as well as different cellular mechanisms underlying the induction of differentiation mediated by lowered oxygen levels.

Differentiation of human neural progenitor cells regulated by Wnt-3a.

Wnt ligands play pivotal roles in the control of cell growth and differentiation during central nervous system development via the Wnt signaling pathway. In this study, we investigated the effects of Wnt-3a and ß-catenin on the differentiation of ReNcell VM human neural progenitor cells. After overexpression of Wnt-3a or mutant-stabilized ß-catenin in ReNcell VM cells, their effects on TCF-mediated transcription, Wnt target gene expression and differentiation into neuronal and glial cells were investigated. Our results show that activation of Wnt/ß-catenin signaling increases TCF-mediated transcription and the expression of the Wnt target genes Axin2, LEF1 and CyclinD1 in ReNcell VM cells. In contrast to mutant-stabilized ß-catenin, Wnt-3a increases neurogenesis during the differentiation of ReNcell VM cells. Thus, our data suggest that neurogenesis induced by Wnt-3a is independent of the transcriptional activity of Wnt/ß-catenin pathway in ReNcell VM cells.

A new facile synthesis of 3-amidoindole derivatives and their evaluation as potential GSK-3beta inhibitors.

3-Amidoindoles were synthesized from commercially available arylhydrazines and propargylamines over Zn-salt mediated one pot procedure in excellent regioselectivity and up to 94% yield.