Differentiation Of Neuronal Precursor Cells Research Articles

NTRK1/TrkA Activation Overrides the G2/M-Checkpoint upon Irradiation.

Simple SummaryNeuroblastoma (NB) is a solid childhood tumor and needs to be treated with multimodal therapy including radiation in advances stages. TrkA/NTRK1 expression is a hallmark of NB with excellent prognosis, but the impact of TrkA/NTRK1 on radiation response is largely unknown. Here, we report that human neuroblastoma cell lines engineered to express TrkA/NTRK1 in tightly controlled systems fail to activate the G2/M cell cycle checkpoint upon irradiation, which recapitulates the effects of ATM or ATR inhibition. Our findings point to a hitherto unrecognized TrkA/NTRK1-mediated wiring of the radiation response in NB cells.High expression of the receptor tyrosine kinase TrkA/NTRK1 is associated with a favorable outcome in several solid tumors of childhood including neuroblastoma. During development, TrkA/NTRK1 governs migration and differentiation of neuronal precursor cells, while it is associated with mitotic dysfunction and altered DNA damage response, among others, in neuroblastoma. Here, we used human neuroblastoma cell lines with inducible TrkA/NTRK1 expression to mechanistically explore the role of TrkA/NTRK1 signaling in checkpoint activation after DNA damage induced by ionizing radiation (IR). TrkA/NTRK1 activated cells showed increased short-term cell viability upon IR compared to vector control cells. This was accompanied by a deficient G2/M-checkpoint at both low (1 Gy) and high doses (4 Gy) of IR. In a tightly controlled setting, we confirmed that this effect was strictly dependent on activation of TrkA/NTRK1 by its ligand, nerve growth factor (NGF). TrkA/NTRK1-expressing cells displayed impaired ATM and CHK1 phosphorylation, resulting in stabilization of CDC25B. In line with these findings, ATM or ATR inhibition recapitulated the effects of TrkA/NTRK1 activation on the IR-induced G2/M-checkpoint. In conclusion, we here provide first evidence for a previously unrecognized function of NTRK signaling in checkpoint regulation and the response to IR.

Fingolimod for Irradiation-Induced Neurodegeneration.

BackgroundCranial irradiation is a common therapy for the treatment of brain tumors, but unfortunately patients suffer from side effects, particularly cognitive impairment, caused by neurodegenerative and neuroinflammatory mechanisms. Finding a therapeutic agent protecting hippocampal neurons would be beneficial. Fingolimod (FTY720), a sphingosine-1-phosphate receptor modulator approved for multiple sclerosis, is an immunosuppressant and known to enhance proliferation and differentiation of neuronal precursor cells (NPCs).ObjectivesTo investigate whether pre-treatment with FTY720 protects NPCs in vitro and in vivo from irradiation-induced damage.MethodsNeuronal precursor cells were isolated from E13 C57BL/6 wildtype mice, treated at day 0 of differentiation with FTY720 and irradiated on day 6 with 1 Gy. NPCs were analyzed for markers of cell death (PI, caspase-3), proliferation (Ki67), and differentiation (DCX, βIII-tubulin). Adult C57BL/6 wildtype mice were treated with FTY720 (1 mg/kg) and received a single dose of 6 Gy cranial irradiation at day 7. Using immunohistochemistry, we analyzed DCX and BrdU as markers of neurogenesis and Iba1, GFAP, and CD3 to visualize inflammation in the dentate gyrus (DG) and the subventricular zone (SVZ). B6(Cg)-Tyrc-2J/J DCX-luc reporter mice were used for bioluminescence imaging to evaluate the effect of FTY720 on neurogenesis in the DG and the spinal cord of naïve mice.ResultsFTY720 protected NPCs against irradiation induced cell death in vitro. Treatment with FTY720 dose-dependently reduced the number of PI+ cells 24 and 96 h after irradiation without effecting proliferation or neuronal differentiation. In vivo treatment resulted in a significant survival of DCX+ neurons in the DG and the SVZ 4 weeks after irradiation as well as a slight increase of proliferating cells. FTY720 inhibited microglia activation 24 h after X-ray exposure in the DG, while astrocyte activation was unaffected and no lymphocyte infiltrations were found. In naïve mice, FTY720 treatment for 4 weeks had no effect on neurogenesis.ConclusionFTY720 treatment of NPCs prior to X-ray exposure and of mice prior to cranial irradiation is neuroprotective. No effects on neurogenesis were found.

Positive Regulation of Transcription by Human ZMYND8 through Its Association with P-TEFb Complex

Although human ZMYND8 has been implicated as a transcriptional co-repressor of multiple targets, global association of ZMYND8 with active genes and enhancer regions predicts otherwise. Here, we report an additional function of ZMYND8 in transcriptional activation through its association with the P-TEFb complex. Biochemical reconstitution analyses show that human ZMYND8, through direct association with CylcinT1, forms a minimal ZMYND8-P-TEFb complex. The importance of ZMYND8 in target gene activation, through P-TEFb complex recruitment, is demonstrated on chromosomally integrated reporter gene as well as native target genes invivo. Physiologically, we further show that the ZMYND8-P-TEFb complex-mediated transcriptional activation is required for all-trans retinoic acid (ATRA)-mediated differentiation of neuronal precursor cells. Finally, to detail the dual activator and repressor nature, mechanistically we show that, through its putative coiled-coil domain, ZMYND8 forms a homodimer that preferentially associates with the activator P-TEFb complex, whereas the monomer associates with the CHD4 subunit of repressor NuRD complex.

Mutation of neuron-specific chromatin remodeling subunit BAF53b: rescue of plasticity and memory by manipulating actin remodeling.

Recent human exome-sequencing studies have implicated polymorphic Brg1-associated factor (BAF) complexes (mammalian SWI/SNF chromatin remodeling complexes) in several intellectual disabilities and cognitive disorders, including autism. However, it remains unclear how mutations in BAF complexes result in impaired cognitive function. Post-mitotic neurons express a neuron-specific assembly, nBAF, characterized by the neuron-specific subunit BAF53b. Subdomain 2 of BAF53b is essential for the differentiation of neuronal precursor cells into neurons. We generated transgenic mice lacking subdomain 2 of Baf53b (BAF53bΔSB2). Long-term synaptic potentiation (LTP) and long-term memory, both of which are associated with phosphorylation of the actin severing protein cofilin, were assessed in these animals. A phosphorylation mimic of cofilin was stereotaxically delivered into the hippocampus of BAF53bΔSB2 mice in an effort to rescue LTP and memory. BAF53bΔSB2 mutant mice show impairments in phosphorylation of synaptic cofilin, LTP, and memory. Both the synaptic plasticity and memory deficits are rescued by overexpression of a phosphorylation mimetic of cofilin. Baseline physiology and behavior were not affected by the mutation or the experimental treatment. This study suggests a potential link between nBAF function, actin cytoskeletal remodeling at the dendritic spine, and memory formation. This work shows that a targeted manipulation of synaptic function can rescue adult plasticity and memory deficits caused by manipulations of nBAF, and thereby provides potential novel avenues for therapeutic development for multiple intellectual disability disorders.

Regulation of the Na+,Cl- Coupled Creatine Transporter CreaT (SLC6A8) by the Janus Kinase JAK3

Background: The creatine transporter CreaT (SLC6A8), a Na⁺,Cl^- coupled transporter is expressed in diverse tissues including the brain. Genetic defects of SLC6A8 result in mental retardation with seizures. The present study explored the regulation of CreaT by Janus kinase JAK3, which is expressed in a variety of tissues including the brain and participates in the regulation of cell survival and differentiation of neuronal precursor cells. Methods: CreaT was expressed in Xenopus laevis oocytes with or without wild-type JAK3, constitutively active ^A568VJAK3 and inactive ^K851AJAK3. Creatine transport in those oocytes was quantified utilizing dual electrode voltage clamp. Results: Electrogenic creatine transport was observed in CreaT expressing oocytes but not in water-injected oocytes. In CreaT expressing oocytes co-expression of JAK3 or ^A568VJAK3, but not co-expression of ^K851AJAK3 was followed by a significant decrease of creatine induced current. According to kinetic analysis JAK3 significantly decreased the maximal creatine transport rate. In CreaT and JAK3 expressing oocytes the creatine induced current was significantly increased by JAK3 inhibitor WHI-P154 (22 µM). Conclusion: JAK3 is a powerful negative regulator of the creatine transporter CreaT.

Molecular effect of ethanol during neural differentiation of human embryonic stem cells in vitro.

Potential teratogenic effects of alcohol on fetal development have been documented. Especially studies have demonstrated deleterious effect of ethanol exposure on neuronal development in animal models and on the maintenance and differentiation of neuronal precursor cells derived from stem cells. To better understand the molecular effect of alcohol on the process of neural differentiation, we have performed gene expression microarray analysis on human embryonic stem cells being directed to neural rosettes and neural precursor cells in the presence of ethanol treatment. Here we provide detailed experimental methods, analysis and information associated with our data deposited into Gene Expression Omnibus (GEO) under GSE56906. Our data provide scientific insight on potential molecular effects of fetal alcohol exposure on neural differentiation of early embryo development.

Effects of histone deacetylase inhibition on the survival, proliferation and migration of Schwann cells, as well as on the expression of neurotrophic factors and genes associated with myelination.

Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, has been shown to have neuroprotective, neurotrophic and anti-inflammatory properties in both animal and cellular models of neurodegenerative disorders. In a previous study of ours, we demonstrated that TSA inhibited the proliferation and increased the differentiation of neuronal precursor cells (NPCs). However, the effects of TSA on Schwann cells (SCs) have not yet been fully elucidated. Thus, in the present study, using SCs derived from adult rat sciatic nerves, we investigated the effects of TSA on the survival, proliferation, migration and myelination of SCs. We found that TSA significantly induced SC death when used at high concentrations. We also observed that TSA promoted the proliferation of SCs in a time-dependent manner. In addition, TSA inhibited the migration of SCs. Moreover, RT-PCR revealed that TSA increased the mRNA expression of several neurotrophic factors and inhibited the expression of genes associated with myelination, including myelin basic protein (MBP) and myelin protein zero (MPZ). Taken together, our results suggest that TSA plays an important role in regulating the growth and biological function of SCs. These data may contribute to our understanding of TSA-based treatment of neurodegenerative diseases.

BDNF: a regulator of learning and memory processes with clinical potential

Memories are believed to be represented by facilitated synaptic transmission of electrical signals in neuronal networks. The ability to acquire new memories or to change old memory content results from the plastic properties of the brain. Molecular changes in synaptic plasticity of neuronal networks are considered to be the cellular correlates of learning and memory, and the neurotrophin brain-derived neurotropic factor (BDNF) plays an important role in these processes. This neurotrophic factor coordinates a multitude of biological functions. In addition to its role in neuronal plasticity processes, such as long-term potentiation of synaptic transmission, the protein regulates the differentiation of neuronal precursor cells, synaptogenesis, and neuronal survival. Cellular processes like BDNF protein processing, anterograde and retrograde transport, as well as exocytosis and endocytosis of BDNF vesicles are necessary to enable the protein to fulfill its neuroprotective and plasticity-related functions in its target areas. Therefore, deficits in one of these functions, resulting in a reduction or a lack of BDNF supply, can result in dysfunctional or reduced synaptic plasticity in virtually every brain area. Since cognitive processes and mental health require the intact formation and modification of memory traces, a change in BDNF turnover is considered as a contributing factor to a number of neurodegenerative and psychological disorders. This review summarizes the current knowledge regarding the connection between BDNF, its role in synaptic plasticity and its role in brain.

Grafted Neuronal Precursor Cells Differentiate and Integrate in Injured Hippocampus in Experimental Pneumococcal Meningitis

Bacterial meningitis (BM) frequently causes persisting neurofunctional sequelae. Autopsy studies in patients dying from BM show characteristic apoptotic brain injury to the stem cell niche in the subgranular zone of the hippocampal dentate gyrus (DG), and this form of brain damage is associated with learning and memory deficits in experimental BM. With an eye to potential regenerative therapies, the survival, migration, and differentiation of neuronal precursor cells (NPCs) were evaluated after engraftment into the injured hippocampus in vitro and in vivo in an infant rat model of pneumococcal meningitis. Green fluorescent protein (GFP)-expressing NPCs were grafted into the DG of organotypic hippocampal slice cultures injured by challenge with live Streptococcus pneumoniae. Seven days after engraftment, NPCs had migrated from the site of injection into the injured granular layer of the DG and electro-functionally integrated into the hippocampal network. In vivo, GFP-expressing NPCs migrated within 1 week from the injection site in the hilus region to the injured granular layer of the hippocampal DG and showed neuronal differentiation at 2 and 4 weeks after transplantation. Hippocampal injury induced by BM guides grafted NPCs to the area of brain damage and provides a microenvironment for neuronal differentiation and functional integration.

Innovative Treatment Approaches in Schizophrenia Enhancing Neuroplasticity: Aerobic Exercise, Erythropoetin and Repetitive Transcranial Magnetic Stimulation

Schizophrenia is a brain disorder associated with subtle, but replicable cerebral volume loss mostly prevalent in frontal and temporal brain regions. Post-mortem studies of the hippocampus point to a reduction of the neuropil constituting mainly of synapses associated with changes of molecules mediating plastic responses of neurons during development and learning. Derived from animal studies interventions to enhance neuroplasticity by inducing adult neurogenesis, synaptogenesis, angiogenesis and long-term potentiation (LTP) were developed and the results translated into clinical studies in schizophrenia. Out of these interventions aerobic exercise has been shown to increase hippocampal volume, elevate N-acetyl-aspartate in the hippocampus as neuronal marker, and improve short-term memory in schizophrenia. The hematopoietic growth factor erythropoetin (EPO) is involved in brain development and associated with the production and differentiation of neuronal precursor cells. A first study demonstrated a positive effect of EPO application on cognition in schizophrenia patients. In randomised controlled studies with small sample size, the efficacy of repetitive transcranial magnetic stimulation (rTMS), a biological intervention focussing on the enhancement of LTP, has been shown for the improvement of positive and negative symptoms in schizophrenia,. The putative underlying neurobiological mechanisms of these interventions including the role of neurotrophic factors are outlined and implications for future research regarding neuroprotection strategies to improve schizophrenia are discussed.

Neurocognitive, adaptive, and behavioral functioning of individuals with Costello syndrome: A review

Costello syndrome is a rare rasopathy resulting from germline mutations of the proto-oncogene HRAS. Its phenotype includes severe failure-to-thrive, cardiac abnormalities, a predisposition to benign and malignant tumors, hypotonia, and developmental delay. Costello syndrome is associated with cognitive impairment, including intellectual functioning generally in the mild to moderate range of disability, commensurate adaptive functioning, and increased anxiety. Relative strengths have been found for nonverbal fluid reasoning (FR). Gender effects have been reported, with females showing better adaptive functioning across domains. Developmentally, nonverbal skills plateau in late childhood/early adolescence, whereas the rate of vocabulary acquisition may increase in adolescence into early adulthood. Here we review the literature assessing cognitive, adaptive, and behavioral functioning in Costello syndrome, and we provide data from an ongoing longitudinal study. Severity of cognitive impairment may depend upon the specific HRAS mutation, as three individuals with the p.G13C change showed average nonverbal FR skills and borderline-to-low average overall nonverbal IQ. Further, separation anxiety is more common in Costello syndrome than in the general population, affecting 39% of this cohort, and males are more often overly anxious than females. Interrelations between anxiety and cognitive and adaptive functioning were found, pointing to functional difficulties as a likely source of stress and anxiety. Taking into account data from animal models, cognitive and behavioral changes likely originate from abnormal differentiation of neuronal precursor cells, which result in structural and functional brain differences.

Flavones from Root of Scutellaria Baicalensis Georgi: Drugs of the Future in Neurodegeneration?

Flavonoids are natural, plant-derived compounds which exert diverse biological activities, also valuable neuroprotective actions within the brain and currently are intensively studied as agents able to modulate neuronal function and to prevent age-related neurodegeneration. Among them, flavones isolated from Scutellaria baicalensis root exhibit strong neuroprotective effects on the brain and are not toxic in the broad range of tested doses. Their neuroprotective potential has been shown in both oxidative stress-induced and amyloid-beta and alpha-synuclein-induced neuronal death models. Baicalein, the main flavone present in Scutellaria baicalensis root, strongly inhibited aggregation of neuronal amyloidogenic proteins in vitro and induces dissolution of amyloid deposits. It exerts strong antioxidative and anti-inflammatory activities and also exhibits anti-convulsive, anxiolytic, and mild sedative actions. Importantly, baicalein, and also another flavone: oroxylin A, markedly enhanced cognitive and mnestic functions in animal models of aging brains and neurodegeneration. In the preliminary study, wogonin, another flavone from Scutellaria baicalensis root, has been shown to stimulate brain tissue regeneration, inducing differentiation of neuronal precursor cells. This concise review provides the main examples of neuroprotective activities of the flavones and reveals their potential in prevention and therapyof neurodegenerative diseases.

CXCR4 promotes differentiation of oligodendrocyte progenitors and remyelination

Multiple sclerosis is a neurodegenerative disease characterized by episodes of autoimmune attack of oligodendrocytes leading to demyelination and progressive functional deficits. Because many patients exhibit functional recovery in between demyelinating episodes, understanding mechanisms responsible for repair of damaged myelin is critical for developing therapies that promote remyelination and prevent disease progression. The chemokine CXCL12 is a developmental molecule known to orchestrate the migration, proliferation, and differentiation of neuronal precursor cells within the developing CNS. Although studies suggest a role for CXCL12 in oligodendroglia ontogeny in vitro, no studies have investigated the role of CXCL12 in remyelination in vivo in the adult CNS. Using an experimental murine model of demyelination mediated by the copper chelator cuprizone, we evaluated the expression of CXCL12 and its receptor, CXCR4, within the demyelinating and remyelinating corpus callosum (CC). CXCL12 was significantly up-regulated within activated astrocytes and microglia in the CC during demyelination, as were numbers of CXCR4+NG2+ oligodendrocyte precursor cells (OPCs). Loss of CXCR4 signaling via either pharmacological blockade or in vivo RNA silencing led to decreased OPCs maturation and failure to remyelinate. These data indicate that CXCR4 activation, by promoting the differentiation of OPCs into oligodendrocytes, is critical for remyelination of the injured adult CNS.

LEF1/β-Catenin Complex Regulates Transcription of the Cav3.1 Calcium Channel Gene (Cacna1g) in Thalamic Neurons of the Adult Brain

beta-Catenin, together with LEF1/TCF transcription factors, activates genes involved in the proliferation and differentiation of neuronal precursor cells. In mature neurons, beta-catenin participates in dendritogenesis and synaptic function as a component of the cadherin cell adhesion complex. However, the transcriptional activity of beta-catenin in these cells remains elusive. In the present study, we found that in the adult mouse brain, beta-catenin and LEF1 accumulate in the nuclei of neurons specifically in the thalamus. The particular electrophysiological properties of thalamic neurons depend on T-type calcium channels. Cav3.1 is the predominant T-type channel subunit in the thalamus, and we hypothesized that the Cacna1g gene encoding Cav3.1 is a target of the LEF1/beta-catenin complex. We demonstrated that the expression of Cacna1g is high in the thalamus and is further increased in thalamic neurons treated in vitro with LiCl or WNT3A, activators of beta-catenin. Luciferase reporter assays confirmed that the Cacna1G promoter is activated by LEF1 and beta-catenin, and footprinting analysis revealed four LEF1 binding sites in the proximal region of this promoter. Chromatin immunoprecipitation demonstrated that the Cacna1g proximal promoter is occupied by beta-catenin in vivo in the thalamus, but not in the hippocampus. Moreover, WNT3A stimulation enhanced T-type current in cultured thalamic neurons. Together, our data indicate that the LEF1/beta-catenin complex regulates transcription of Cacna1g and uncover a novel function for beta-catenin in mature neurons. We propose that beta-catenin contributes to neuronal excitability not only by a local action at the synapse but also by activating gene expression in thalamic neurons.

Neuronal differentiation by analogs of staurosporine

RGC-5 cells are transformed cells that express several surface markers characteristic of neuronal precursor cells, but resemble glial cells morphologically and divide in culture. When treated with the apoptosis-inducing agent staurosporine, RGC-5 cells assume a neuronal morphology, extend neurites, stop dividing, and express ion channels without acute signs of apoptosis. This differentiation with staurosporine is similar to what has been described for certain other neuronal cell lines, and occurs by a mechanism not yet understood. Inhibition of several kinases known to be inhibited by staurosporine fails to differentiate RGC-5 cells, and examination of the kinome associated with staurosporine-dependent differentiation has been unhelpful so far. To better understand the mechanism of staurosporine-mediated differentiation of neuronal precursor cells, we studied the effects of the following structurally similar molecules on differentiation of neuronal and non-neuronal cell lines, comparing them to staurosporine: 9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid, 2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-, methyl ester, (9S,10R,12R)-(K252a), (5R,6S,8S)-6-hydroxy-5-methyl-13-oxo-6,7,8,13,14,15-hexahydro-5H-16-oxa-4b,8a,14-triaza-5,8-methanodibenzo[b,h]cycloocta[jkl]cyclopenta[e]-as-indacene-6-carboxylic acid (K252b), staurosporine aglycone (K252c), 7-hydroxystaurosporine (UCN-01), and 4'-N-benzoylstaurosporine (PKC-412). Morphological differentiation, indicated by neurite extension and somal rounding, was quantitatively assessed with NeuronJ. We found that the critical structural component for differentiation in RGC-5 cells is a basic amine adjacent to an accessible methoxy group at the 3' carbon. Given that UCN-01 and similar compounds are potent anti-cancer drugs, examination of molecules that share similar structural features may yield insights into the design of other drugs for differentiation.

Endothelium-Induced Proliferation and Electrophysiological Differentiation of Human Embryonic Stem Cell-Derived Neuronal Precursors

Neurogenesis occurs in a stem cell niche in which vascular elements, including endothelial cells (ECs), are thought to play an important role. Using co-culture experiments, we investigated the effect of ECs on proliferation and functional neuronal differentiation of human embryonic stem (ES) cellderived neuronal precursor cells (NPCs). NPCs were cultured for 5 days in medium containing fibroblast growth factor-2 (FGF-2), with or without ECs. FGF-2 and ECs were then removed, and NPCs were maintained in culture for additional periods. Compared to control NPC cultures, EC-treated NPC cultures showed increased cell proliferation at short intervals (5 days) after withdrawal of FGF-2 and larger tetrodotoxin-sensitive inward membrane currents at longer intervals (10-14 days), but a similar pattern of development of neuronal differentiation markers. The effects of ECs appeared to result from the release of soluble factors rather than from cell contact, because they were observed despite the physical separation of NPCs from ECs by a cell-impermeable membrane. These findings indicate that ECs can regulate the proliferation and electrophysiological neuronal differentiation of human NPCs.

The expression level of the orphan nuclear receptor GCNF (germ cell nuclear factor) is critical for neuronal differentiation.

The germ cell nuclear factor (GCNF) is essential for normal embryonic development and gametogenesis. To test the prediction that GCNF is additionally required for neuronal differentiation, we used the mouse embryonal carcinoma cell line PCC7-Mz1, which represents an advantageous model to study neuronal cells from the stage of fate choice until the acquirement of functional competence. We generated stable transfectants that express gcnf sense or antisense RNA under the control of a tetracycline-regulated promoter. After retinoic acid-induced withdrawal from the cell cycle, sense clones developed a neuron network with changed properties, and the time course of neuron maturation was shortened. Consistent with these data, differentiation of neuronal precursor cells was impaired in antisense cultures. This involved a delay in 1) the down-regulation of nestin, a marker for undifferentiated neuroepithelial cells and stem cells of the central nervous system, and 2) up-regulation of the somatodendritic protein microtubule-associated protein 2 and the synaptic vesicle protein synaptophysin. Neuronal cells in the antisense cultures acquired functional competence, although with a significant delay. Our data propose that the level of GCNF is critical for differentiation and maturation of neuronal precursor cells.

Multiple Effector Domains within SNT1 Coordinate ERK Activation and Neuronal Differentiation of PC12 Cells

Differentiation of neuronal precursor cells in response to neurotrophic differentiation factors is accompanied by the activation of membrane-anchored SNT signaling adaptor proteins. Two classes of differentiation factors, the neurotrophins and fibroblast growth factors, induce rapid tyrosine phosphorylation of SNT1(FRS2alpha), which in turn enables SNT1 to recruit Shp2 tyrosine phosphatase and Grb2 adaptor protein in complex with the Ras GDP/GTP exchange factor Sos. To determine effector functions of SNT that promote neuronal differentiation of PC12 pheochromocytoma cells, we engineered a chimeric protein, SNT1(IRS)CX, bearing the effector region of SNT1 and the insulin receptor recognition domains of IRS2. Insulin promoted tyrosine phosphorylation of SNT1(IRS)CX in transfected PC12 cells accompanied by sustained activation of ERK1/2 mitogen-activated protein kinases and neuronal differentiation. The SNT1(IRS)CX-mediated response was dependent on endogenous Ras, MEK, and Shp2 activities. Mutagenesis of SNT1(IRS)CX identified three classes of effector motifs within SNT critical for both sustained ERK activation and neuronal differentiation: 1) four phosphotyrosine motifs that mediate recruitment of Grb2, 2) two phosphotyrosine motifs that mediate recruitment of Shp2, and 3) a C-terminal motif that functions by helping to recruit Sos. We discuss possible mechanisms by which three functionally distinct SNT effector motifs collaborate to promote a downstream biochemical and biological response.

Effect of fatty acid supplementation on growth and differentiation of human IMR-32 neuroblastoma cells in vitro.

Polyunsaturated fatty acids play a critical role in the structure and function of the developing nervous system. It has been proposed that fatty acids may effect a variety of biologic processes through the activation of the peroxisome proliferator activated receptors (PPARs)-ligand activated transcription factors. In this report, we demonstrate that fatty acids can inhibit the proliferation of the human neuronal cell line IMR-32. The fatty acids linoleate, alpha-linoleate, arachidonate, docosahexaenoate, and oleate all inhibited [(3)H]thymidine incorporation of IMR-32 cells after 72 h. Fatty acid supplementation also led to the morphologic differentiation of the IMR-32 cells. Linoleate and arachidonate, fatty acids of the n-6 series, induced the most extensive differentiation. Furthermore, the addition of fatty acids to IMR-32 cells led to PPAR activation, suggesting that PPAR activation may be an important event in fatty acid modulation of IMR-32 cell growth. In support of this hypothesis, clofibric acid, a specific ligand of PPARalpha, also inhibited IMR-32 cell proliferation and strongly induced PPAR activation. Together these results suggest that fatty acids may play an important role in the development of neuronal precursor cells and that activation of the PPARs may be one pathway by which fatty acids modulate the growth and differentiation of neuronal precursor cells.

Reduced Pteridine Derivatives Induce Apoptosis in Human Neuronal NT2/HNT Cells

Elevated concentrations of the pteridine compound neopterin, usually accompanied by 7,8- dihydroneopterin were found in cerebrospinal fluids of patients with neurodegenerative diseases and central nervous system infections. Here, the potential of pteridines to induce apoptosis of the human neuronal cell line (NT2) was investigated. Reduced neopterin, biopterin- and folate derivatives led to a time-dependent increase of apoptosis of cells. In contrast, non-reduced pteridines did not significantly alter cell survival. After differentiation of neuronal precursor cells to neurons and astrocyte-like cells, similar effects were detected. Antioxidants partly protected NT2 from pteridines-induced apoptosis, suggesting the involvement of reactive oxygen intermediates. In vitro experiments using dichlorofluorescin-diacetate further indicated a direct formation of reactive oxygen species in cells. Results implicate that high concentrations of reduced pteridines, might contribute to the loss of neuronal cells in neurodegenerative diseases.