Embryonic Cortical Cells Research Articles

Age Dependent Integration of Cortical Progenitors Transplanted at the Adult CSF-Brain Interface.

There has been renewed interest in neural transplantation of cells and tissues for brain repair. Recent studies have demonstrated the ability of transplanted neural precursor cells and in vitro grown organoids to mature and locally integrate into host brain neural circuitry. Much effort has focused on how the transplant behaves and functions after the procedure, but the extent to which the host brain can properly innervate the transplant, particularly in the context of aging, is largely unexplored. Here we report that transplantation of rat embryonic cortical precursor cells into the cerebrospinal fluid-subventricular zone (CSF-SVZ) of adult rat brains generates a brain-like tissue (BLT) at an ectopic site. This model allows for the assessment of long-range connectivity and cellular interactions between the transplant and the host brain as a function of host age. The transplanted precursor cells initially proliferate, then differentiate, and develop into mature BLTs, which receive supportive cellular components from the host including blood vessels, microglia, astrocytes, and oligodendrocytes. There was integration of the BLT into the host brain which occurred at all ages studied, suggesting that host age does not affect the maturation and integration of the transplant-derived BLT. Long-range axonal projections from the BLT into the host brain were robust throughout the different aged recipients. However, long-distance innervation originating from the host brain into the BLT significantly declined with age. This work demonstrates the feasibility and utility of integrating new neural tissue structures at ectopic sites into adult brain circuits to study host-transplant interactions.

SnapHiC-G: identifying long-range enhancer-promoter interactions from single-cell Hi-C data via a global background model.

Harnessing the power of single-cell genomics technologies, single-cell Hi-C (scHi-C) and its derived technologies provide powerful tools to measure spatial proximity between regulatory elements and their target genes in individual cells. Using a global background model, we propose SnapHiC-G, a computational method, to identify long-range enhancer-promoter interactions from scHi-C data. We applied SnapHiC-G to scHi-C datasets generated from mouse embryonic stem cells and human brain cortical cells. SnapHiC-G achieved high sensitivity in identifying long-range enhancer-promoter interactions. Moreover, SnapHiC-G can identify putative target genes for noncoding genome-wide association study (GWAS) variants, and the genetic heritability of neuropsychiatric diseases is enriched for single-nucleotide polymorphisms (SNPs) within SnapHiC-G-identified interactions in a cell-type-specific manner. In sum, SnapHiC-G is a powerful tool for characterizing cell-type-specific enhancer-promoter interactions from complex tissues and can facilitate the discovery of chromatin interactions important for gene regulation in biologically relevant cell types.

Comprehensive characterization of the neurogenic and neuroprotective action of a novel TrkB agonist using mouse and human stem cell models of Alzheimer’s disease

BackgroundNeural stem cell (NSC) proliferation and differentiation in the mammalian brain decreases to minimal levels postnatally. Nevertheless, neurogenic niches persist in the adult cortex and hippocampus in rodents, primates and humans, with adult NSC differentiation sharing key regulatory mechanisms with development. Adult neurogenesis impairments have been linked to Alzheimer’s disease (AD) pathology. Addressing these impairments by using neurotrophic factors is a promising new avenue for therapeutic intervention based on neurogenesis. However, this possibility has been hindered by technical difficulties of using in-vivo models to conduct screens, including working with scarce NSCs in the adult brain and differences between human and mouse models or ethical limitations.MethodsHere, we use a combination of mouse and human stem cell models for comprehensive in-vitro characterization of a novel neurogenic compound, focusing on the brain-derived neurotrophic factor (BDNF) pathway. The ability of ENT-A011, a steroidal dehydroepiandrosterone derivative, to activate the tyrosine receptor kinase B (TrkB) receptor was tested through western blotting in NIH-3T3 cells and its neurogenic and neuroprotective action were assessed through proliferation, cell death and Amyloid-β (Aβ) toxicity assays in mouse primary adult hippocampal NSCs, mouse embryonic cortical NSCs and neural progenitor cells (NPCs) differentiated from three human induced pluripotent stem cell lines from healthy and AD donors. RNA-seq profiling was used to assess if the compound acts through the same gene network as BDNF in human NPCs.ResultsENT-A011 was able to increase proliferation of mouse primary adult hippocampal NSCs and embryonic cortical NSCs, in the absence of EGF/FGF, while reducing Aβ-induced cell death, acting selectively through TrkB activation. The compound was able to increase astrocytic gene markers involved in NSC maintenance, protect hippocampal neurons from Αβ toxicity and prevent synapse loss after Aβ treatment. ENT-A011 successfully induces proliferation and prevents cell death after Aβ toxicity in human NPCs, acting through a core gene network shared with BDNF as shown through RNA-seq.ConclusionsOur work characterizes a novel BDNF mimetic with preferable pharmacological properties and neurogenic and neuroprotective actions in Alzheimer’s disease via stem cell-based screening, demonstrating the promise of stem cell systems for short-listing competitive candidates for further testing.

CROCCP2 acts as a human-specific modifier of cilia dynamics and mTOR signaling to promote expansion of cortical progenitors

The primary cilium is a central signaling component during embryonic development. Here we focus on CROCCP2, a hominid-specific gene duplicate from ciliary rootlet coiled coil (CROCC), also known as rootletin, that encodes the major component of the ciliary rootlet. We find that CROCCP2 is highly expressed in the human fetal brain and not in other primate species. CROCCP2 gain of function in the mouse embryonic cortex and human cortical cells and organoids results in decreased ciliogenesis and increased cortical progenitor amplification, particularly basal progenitors. CROCCP2 decreases ciliary dynamics by inhibition of the IFT20 ciliary trafficking protein, which then impacts neurogenesis through increased mTOR signaling. Loss of function of CROCCP2 in human cortical cells and organoids leads to increased ciliogenesis, decreased mTOR signaling, and impaired basal progenitor amplification. These data identify CROCCP2 as a human-specific modifier of cortical neurogenesis that acts through modulation of ciliary dynamics and mTOR signaling.

Valproic acid enhances neurosphere formation in cultured rat embryonic cortical cells through TGFβ1 signaling.

This study aimed to investigate the effect and mechanism of valproic acid (VPA) on the neurosphere formation in rat embryonic cortical cells. We used free-floating neurosphere formation as a model system to evaluate the VPA on the proliferation of neural stem cells (NSCs). We found a time- and dose-dependent increase in neurosphere formation and NSC proliferation after VPA treatment. Further RNA-seq analysis demonstrated that the upregulated TGFβ1 signaling might attribute to the effect of VPA on the neurosphere formation and NSC proliferation. Consistently, the neurosphere formation and NSC proliferation were blocked by the treatment with SB431542, an inhibitor of TGFβ1 receptor. Moreover, in a coculture system, NSCs treated with VPA significantly reduced the oxygen-glucose deprivation-induced neuronal apoptosis. Taken together, our results showed that VPA could enhance neurosphere formation and NSC proliferation by activating TGFβ1, which might be a novel therapeutic strategy for neurological disorders.

Maternal Ethanol Exposure Acutely Elevates Src Family Kinase Activity in the Fetal Cortex

Fetal alcohol syndrome (FAS) is characterized by disrupted fetal brain development and postnatal cognitive impairment. The targets of alcohol are diverse, and it is not clear whether there are common underlying molecular mechanisms producing these disruptions. Prior work established that acute ethanol exposure causes a transient increase in tyrosine phosphorylation of multiple proteins in cultured embryonic cortical cells. In this study, we show that a similar tyrosine phosphorylation transient occurs in the fetal brain after maternal dosing with ethanol. Using phospho-specific antibodies and immunohistochemistry, we mapped regions of highest tyrosine phosphorylation in the fetal cerebral cortex and found that areas of dendritic and axonal growth showed elevated tyrosine phosphorylation 10 min after maternal ethanol exposure. These were also areas of Src expression and Src family kinase (SFK) activation loop phosphorylation (pY416) expression. Importantly, maternal pretreatment with the SFK inhibitor dasatinib completely prevents both the pY416 increase and the tyrosine phosphorylation response. The phosphorylation response was observed in the perisomatic region and neurites of immature migrating and differentiating primary neurons. Importantly, the initial phosphotyrosine transient (~ 30 min) targets both Src and Dab1, two critical elements in Reelin signaling, a pathway required for normal cortical development. This initial phosphorylation response is followed by sustained reduction in Ser3 phosphorylation of n-cofilin, a critical actin severing protein and an identified downstream effector of Reelin signaling. This biochemical disruption is associated with sustained reduction of F-actin content and disrupted Golgi apparatus morphology in developing cortical neurons. The finding outlines a model in which the initial activation of SFKs by ethanol has the potential to disrupt multiple developmentally important signaling systems for several hours after maternal exposure.

Interfacing Neurons with Nanostructured Electrodes Modulates Synaptic Circuit Features.

Understanding neural physiopathology requires advances in nanotechnology-based interfaces, engineered to monitor the functional state of mammalian nervous cells. Such interfaces typically contain nanometer-size features for stimulation and recording as in cell-non-invasive extracellular microelectrode arrays. In such devices, it turns crucial to understand specific interactions of neural cells with physicochemical features of electrodes, which could be designed to optimize performance. Herein, versatile flexible nanostructured electrodes covered by arrays of metallic nanowires are fabricated and used to investigate the role of chemical composition and nanotopography on rat brain cells in vitro. By using Au and Ni as exemplary materials, nanostructure and chemical composition are demonstrated to play major roles in the interaction of neural cells with electrodes. Nanostructured devices are interfaced to rat embryonic cortical cells and postnatal hippocampal neurons forming synaptic circuits. It is shown that Au-based electrodes behave similarly to controls. Contrarily, Ni-based nanostructured electrodes increase cell survival, boost neuronal differentiation, and reduce glial cells with respect to flat counterparts. Nonetheless, Au-based electrodes perform superiorly compared to Ni-based ones. Under electrical stimulation, Au-based nanostructured substrates evoke intracellular calcium dynamics compatible with neural networks activation. These studies highlight the opportunity for these electrodes to excite a silent neural network by direct neuronal membranes depolarization.

Abstract TP274: Post-stroke Treatment With Docosanoids Promote Functional Recovery, Neurogenesis and Angiogenesis, Attenuate Blood-brain Barrier After Experimental Ischemic Stroke

Objective: Ischemic injury induces neurogenesis in the subventricular zone (SVZ) of the lateral ventricles and subgranular zone (SGZ) of dentate gyrus (DG) promotes the migration of neuroblasts, guided by blood vessels, into the ischemic damaged area. Neurogenesis leads the functional and histological recovery in the ischemic-damaged brain through the proliferation, migration, differentiation and integration of newly generated neural cells into the existing neural circuit. Recently, we have shown that Docosahexaenoic acid (DHA) and Neuroprotectin D1 (NPD1) therapy improves functional and histological outcomes following experimental stroke. It has also been shown that DHA promotes neurite outgrowth of immature neurons, such as embryonic hippocampal cells and embryonic cortical cells. The objective of this study was to explore underlying mechanisms of docosanoid neuroprotection after cerebral ischemia. Methods: Male Sprague-Dawley rats were anesthetized with isoflurane/nitrous oxide, underwent 2h of middle cerebral artery occlusion (MCAo) and treated with DHA (5mg/kg, IV) or NPD1 (5μg/per rat, ICV) and vehicles 1h after. Neuro-behavioral assessments were conducted on days 1, 2, 3 and on weeks 1, 2, 3 or 4. BrdU was injected on days 4, 5 and 6, immunohistochemistry was performed on week 2 or 4, MRI on day 7 and lipidomic analysis at 4 and 5h after onset of stroke. Results: DHA improved short- and long-term behavioral functions and reduced cortical, subcortical and total infarct volumes (by 42%, 47% and 31%, respectively) after 2 weeks and reduced tissue loss by 50% after 4 weeks. DHA increased the number of BrdU + /Ki-67 + , BrdU + /DCX + and BrdU + /NeuN + cells in the cortex, subventricular zone and dentate gyrus and potentiated NPD1 synthesis in the penumbra at 5h after MCAo. NPD1 improved behavior, reduced lesion volumes, protected ischemic penumbra, increased NeuN, GFAP, SMI-71 positive cells and vessels, axonal regeneration in the penumbra and attenuated blood-brain barrier (BBB) after MCAo. Conclusion: We conclude that docosanoid administration increases neurogenesis, angiogenesis, activates NPD1 synthesis in the penumbra and diminishes BBB, which correlates to long-term neurobehavioral recovery after experimental ischemic stroke.

Modeling Single-Molecule Conformations of the HoxD Region in Mouse Embryonic Stem and Cortical Neuronal Cells

Complex architectural rearrangements are associated to the control of the HoxD genes in different cell types; yet, how they are implemented in single cells remains unknown. By use of polymer models, we dissect the locus 3D structure at the single DNA molecule level in mouse embryonic stem and cortical neuronal cells, as the HoxD cluster changes from a poised to a silent state. Our model describes published Hi-C, 3-way 4C, and FISH data with high accuracy and is validated against independent 4C data on the Nsi-SB 0.5-Mb duplication and on triple contacts. It reveals the mode of action of compartmentalization on the regulation of the HoxD genes that have gene- and cell-type-specific multi-way interactions with their regulatory elements and high cell-to-cell variability. It shows that TADs and higher-order 3D structures, such as metaTADs, associate with distinct combinations of epigenetic factors, including but not limited to CCCTC-binding factor (CTCF) and histone marks.

SYM 2081 Exerts Neuroprotective Effect Modulating Anti-Apoptosis in the Hypoxic-Ischemic Brain Injury of Neonatal Rats

Objective: SYM 2081 ((2S,4R)-4-methylglutamic acid), an agonist and functional antagonist of the kainite receptor, has shown a neuroprotective effect in neurodegenerative diseases in adults but little is known concerning perinatal hypoxic-ischemic encephalopathy. This study is designed to evaluate whether SYM 2081 has preventive mechanisms via anti-apoptosis to gain further insight into neuroprotective roles of SYM 2081. Methods: In an in vivo animal model, the left carotid artery was ligated in 7-day-old Sprague-Dawley rat pups. The pups were divided into six groups: normoxia (N), hypoxia (8% O2, 92% N2) (H), H with shamoperation, H with operation (HO), HO treated with vehicle and HO treated with SYM 2081. In an in vitro model, the cultured embryonic cortical neuronal cells were divided into three groups: normoxia (95% air, 5% CO2), hypoxia (94% N2, 5% CO2) (Hc), and Hc treated with SYM 2081 before a hypoxic insult. Apoptosis was assessed using western blots and real-time polymerase chain reaction with Bcl-2, Bax, and caspase-3 antibodies and mRNAs. Results: In both in vitro and in vivo studies, Bcl-2 expression increased, whereas expressions of Bax, caspase-3, and the ratio of Bax/Bcl-2 were reduced with SYM 2081 treatment resulting in improved cell survival. Conclusion: This study showed that SYM 2081 exerts a neuroprotective effect against hypoxic-ischemic injury through modulating apoptotic signaling pathways.

An Autism-Related, Nonsense Foxp1 Mutant Induces Autophagy and Delays Radial Migration of the Cortical Neurons.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder that has a strong genetic component. Disruptions of FOXP1, a transcription factor expressed in the developing cerebral cortex, were associated with ASD. FOXP1(R525X) is a de novo heterozygous mutation found in patients with autism and severe mental retardation. To explore the neuronal basis of FOXP1(R525X) in ASD, we created Foxp1(R521X), a mouse homolog of the human variant. Ectopic expression of Foxp1(R521X) led to cytoplasmic aggregates and activated macroautophagy in neuroblastoma N2a cells and the developing neuronal cells. Cortical neurons expressing Foxp1(R521X) exhibited delayed migration and altered dendritic morphology. As a control, mutant Y435X that was expressed diffusively in the cytoplasm did not induce autophagy and migration delay in the cortex. The embryonic cortical cells had a minimal activity of nonsense-mediated mRNA decay (NMD) as assayed by a splicing-dependent NMD reporter. We hypothesize that the developing neuronal cells use autophagy but not NMD as a safeguard mechanism against nonsense mutant aggregates, resulting in impairment of the cortical development. This study suggests a novel mechanism other than heterozygous loss of FOXP1 for the development of ASD and may advance our understanding of the complex relationships between gene mutation and the related psychiatric disorders.

Abstract TP260: A Novel Neurogenic-Stem Cell Stimulating Curcumin Analog to Treat Acute Ischemic Stroke

While nerve cells rapidly die in stroke, and patients do initiate a neurogenic response to replace these cells, there has been no attempt to develop pleiotropic cytoprotective compounds to further promote neurogenesis in stroke patients, and there are no neurogenic or cytoprotective drugs approved by the FDA. In this study, we focused on the parent compound J-147, a curcumin derivative that is a 5-LOX inhibitor, and has potent neuroprotective and neurogenic activities. J-147 rescues HT22 cells from iodoacetic acid challenge (EC 50 50 nM), and oxytosis (EC 50 115 nM). J-147 is neuroprotective against glutamate excitotoxcity in E18 cortical neuron culture (EC 50 27 nM), and prevents cell death in an embryonic cortical cell trophic factor withdrawal assay (EC 50 25 nM). J-147 can rescue a clone of the hippocampal nerve cell line HT22 expressing TrkB, from serum starvation under conditions where the cells can be protected by BDNF (EC 50 50 nM). In mice, J-147 is neurogenic and can stimulate cell division in germinal regions of the brain (increase BrdU + DCX + labeled cells), enhances endogenous neurotrophic factors and energy metabolism. With this basis, we tested J-147 in 2 translational stroke models. First, using a rodent intraluminal suture model, in combination with a quantal analysis method to determine the effect of increasing ischemia duration (5-90 minutes) on behavioral deficits, IV J-147 treatment (10 mg/kg) administered 5 minutes following the end of occlusion (5-90 minutes), significantly (P=0.045) reduced stroke-induced behavioral deficits and increased the P 50 value to 36.37 ± 1.72 (n=19) minutes from P 50 = 20.1 ± 4.07 minutes (n=19) for the control group. J-147 also reduced infarct volume following IV administration (p<0.05). Second, using the standardized rabbit small clot embolic stroke model, IV J-147 treatment (10mg/kg) administered 1 hour post-embolization increased the P 50 value (behavior) to 1.88 ± 0.10 mg (n=20) compared to P 50 = 1.07 ± 0.07 (n=19) for control(p<0.05). Additional studies showed that J-147 was not mitogenic, or cytotoxic using the CeeTox assay (Raking 90μM) and had a good IV PK profile in rabbits/rats (t 1/2 5.89-8.56 hours). These data support the development of J-147 and neurogenic analogs as a novel class of drug to treat stroke.

Regulation of neural stem cell fate decisions by mitochondrial dynamics.

Regulation of neural stem cell fate decisions by mitochondrial dynamics.

A Central Nervous System Axonal Myelination Assay for High-Throughput Screening.

The formation of new myelin in persistent multiple sclerosis (MS) lesions is compromised, leading to a reduction in neuron function and subject to degeneration and death. Current MS therapies can control autoimmune-mediated demyelination, but none directly promote the regeneration of myelin in the central nervous system (CNS). To identify new drugs that stimulate remyelination, we established a high-throughput cell-based assay to identify compounds that promote myelination. Methods were developed for initiating myelination in vitro using a preparation of primary embryonic rat cortical cells. We developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. The assay scalability and consistency was validated by screening the NIH clinical collection library of 727 compounds and identified ten compounds that promote myelination (Lariosa-Willingham etal., BMC Neurosci 17:16, 2016). Here, we present the detailed methods for a high capacity in vitro assay that assesses myelination of live axons.

Receptor-stimulated transamidation induces activation of Rac1 and Cdc42 and the regulation of dendritic spines

Regulation of dendritic spines is an important component of synaptic function and plasticity whereas dendritic spine dysregulation is related to several psychiatric and neurological diseases. In the present study, we tested the hypothesis that serotonin (5-HT)2A/2C receptor-induced Rho family transamidation and activation regulates dendritic spine morphology and that activation of multiple types of receptors can induce transglutaminase (TGase)-catalyzed transamidation of small G proteins. We previously reported a novel 5-HT2A receptor downstream effector, TGase-catalyzed serotonylation of the small G protein Rac1 in A1A1v cells, a rat embryonic cortical cell line. We now extend these findings to rat primary cortical cultures which develop dendritic spines; stimulation of 5-HT2A/2C receptors increased transamidation of Rac1 and Cdc42, but not RhoA. Inhibition of TGases significantly decreased transamidation and activation of Rac1 and Cdc42, suggesting that transamidation led to their activation. In primary cortical cultures, stimulation of 5-HT2A/2C receptors by 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) caused a transient dendritic spine enlargement, which was blocked by TGase inhibition. Stimulation of both 5-HT2A and 5-HT2C receptors contributed to DOI-induced Rac1 transamidation in primary cortical cultures as demonstrated by selective antagonists. Furthermore, stimulation of muscarinic acetylcholine receptors and NMDA receptors also increased TGase-catalyzed Rac1 activation in SH-SY5Y cells and N2a cells, respectively. Receptor-stimulated TGase-catalyzed transamidation of Rac1 occurs at Q61, a site previously reported to be important in the inactivation of Rac1. These studies demonstrate that TGase-catalyzed transamidation and activation of small G proteins results from stimulation of multiple types of receptors and this novel signaling pathway can regulate dendritic spine morphology and plasticity.

Recombinant Human Erythropoietin Exerts Neuroprotective Effects via Modulation of Nitric Oxide Synthase on Hypoxic-Ischemic Brain Injury in Neonatal Rats

Objective: Erythropoietin (EPO) has neuroprotective effects in many animal models of brain injury including hypoxic-ischemic (HI) encephalopathy, trauma- and excitotoxicity. Current studies have demonstrated the neuroprotective effects of EPO, but there are limited the same consequences oc-curring during neonatal periods. Here, we investigated whether recombinant human EPO (rHuEPO) can protect the developing rat brain from HI injury via the modulation of nitric oxide synthase. Methods: The in vitro model involved culturing embryonic cortical neuronal cells of Sprague- Dawley (SD) rats at 19 days gestation. The cultured cells were divided into five groups: normoxia (N), hypoxia (H), 1, 10 and 100 IU/mL rHuEPO-treated (H+E1, H+E10, and H+E100). In the in vivo model, left carotid artery ligation was performed in 7-day-old SD rat pups. The animals were divided into six groups: normoxia control, normoxia Sham-operated, hypoxia only (H), hypoxia+vehicle, hypoxia+ rHuEPO before a hypoxic insult (HE-B), and hypoxia+rHuEPO after a hypoxic insult (HE-A). Western blotting and real-time polymerase chain reaction using antibodies and primers of inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS) were performed. Results: The rHuEPO-treated group in the in vitro model showed increased expressions of iNOS and eNOS and decreased expression of nNOS compared to those of the H group. In the HE-A and HE-B groups of the in vivo model of the, the results were similar as aforementioned. Conclusion: rHuEPO exerts neuroprotective properties over perinatal HI brain injuries through the modulation of nitric oxide synthase.

A Glo1-Methylglyoxal Pathway that Is Perturbed in Maternal Diabetes Regulates Embryonic and Adult Neural Stem Cell Pools in Murine Offspring

Maternal diabetes is known to adversely influence brain development in offspring. Here, we provide evidence that this involves the circulating metabolite methylglyoxal, which is increased in diabetes, and its detoxifying enzyme, glyoxalase 1 (Glo1), which when mutated is associated with neurodevelopmental disorders. Specifically, when Glo1 levels were decreased in embryonic mouse cortical neural precursor cells (NPCs), this led to premature neurogenesis and NPC depletion embryonically and long-term alterations in cortical neurons postnatally. Increased circulating maternal methylglyoxal caused similar changes in embryonic cortical precursors andneurons and long-lasting changes in cortical neurons and NPCs in adult offspring. Depletion of embryonic and adult NPCs was also observed in murine offspring exposed to a maternal diabetic environment. Thus, the Glo1-methylglyoxal pathway integrates maternal and NPC metabolism to regulateneural development, and perturbations in this pathway lead to long-lasting alterations in adult neurons and NPC pools.

Development of a central nervous system axonal myelination assay for high throughput screening.

BackgroundRegeneration of new myelin is impaired in persistent multiple sclerosis (MS) lesions, leaving neurons unable to function properly and subject to further degeneration. Current MS therapies attempt to ameliorate autoimmune-mediated demyelination, but none directly promote the regeneration of lost and damaged myelin of the central nervous system (CNS). Development of new drugs that stimulate remyelination has been hampered by the inability to evaluate axonal myelination in a rapid CNS culture system.ResultsWe established a high throughput cell-based assay to identify compounds that promote myelination. Culture methods were developed for initiating myelination in vitro using primary embryonic rat cortical cells. We developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. Using γ-secretase inhibitors as promoters of myelination, the optimal growth, time course and compound treatment conditions were established in a 96 well plate format. We have characterized the cortical myelination assay by evaluating the cellular composition of the cultures and expression of markers of differentiation over the time course of the assay. We have validated the assay scalability and consistency by screening the NIH clinical collection library of 727 compounds and identified ten compounds that promote myelination. Half maximal effective concentration (EC50) values for these compounds were determined to rank them according to potency.ConclusionsWe have designed the first high capacity in vitro assay that assesses myelination of live axons. This assay will be ideal for screening large compound libraries to identify new drugs that stimulate myelination. Identification of agents capable of promoting the myelination of axons will likely lead to the development of new therapeutics for MS patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12868-016-0250-2) contains supplementary material, which is available to authorized users.

Study of the impact of Tau phosphorylation on its internalization and processing in cultured neuronal cells

Study of the impact of Tau phosphorylation on its internalization and processing in cultured neuronal cells

Gabapentin Enhances Neurogenesis in E14 Rat Embryonic Neocortex Stem Cells.

Many anticonvulsant drugs have been studied for their non conventional therapeutic effects on neurodegerative diseases but merely a few demonstrated potential neurogenic characteristic. Gabapentin as a well-known mood stabilizer was studied for its potential capability to promote neurogenesis in embryonic rat cortical stem cells. Rat E14 cortical stem cells were exposed to gabapentin during differentiation for 7 days and subjected to immunocytochemistry. The phenotypic changes were evaluated in the ultimately survived and differentiated cells. Gabapentin (16 µg/ml) exposure significantly increased the number and percentage of MAP2 immunopositive neurons with no significant alterations in nestin or GFAP immunopositivity in neural or glial progenitors. The enhanced number of neurons by therapeutic doses of gabapentin via augmentation of the neuronal differentiation in neural stem cells may participate to the therapeutic properties of gabapentin in the treatment of mood disorder.