Differentiation Of Cortical Neurons Research Articles

Herpes Simplex Virus 1 and 2 Infections during Differentiation of Human Cortical Neurons.

Herpes simplex virus 1 (HSV-1) and 2 (HSV-2) can infect the central nervous system (CNS) with dire consequences; in children and adults, HSV-1 may cause focal encephalitis, while HSV-2 causes meningitis. In neonates, both viruses can cause severe, disseminated CNS infections with high mortality rates. Here, we differentiated human induced pluripotent stem cells (iPSCs) towards cortical neurons for infection with clinical CNS strains of HSV-1 or HSV-2. Progenies from both viruses were produced at equal quantities in iPSCs, neuroprogenitors and cortical neurons. HSV-1 and HSV-2 decreased viability of neuroprogenitors by 36.0% and 57.6% (p < 0.0001), respectively, 48 h post-infection, while cortical neurons were resilient to infection by both viruses. However, in these functional neurons, both HSV-1 and HSV-2 decreased gene expression of two markers of synaptic activity, CAMK2B and ARC, and affected synaptic activity negatively in multielectrode array experiments. However, unaltered secretion levels of the neurodegeneration markers tau and NfL suggested intact axonal integrity. Viral replication of both viruses was found after six days, coinciding with 6-fold and 22-fold increase in gene expression of cellular RNA polymerase II by HSV-1 and HSV-2, respectively. Our results suggest a resilience of human cortical neurons relative to the replication of HSV-1 and HSV-2.

SVCT2 Overexpression and Ascorbic Acid Uptake Increase Cortical Neuron Differentiation, Which Is Dependent on Vitamin C Recycling between Neurons and Astrocytes.

During brain development, sodium–vitamin C transporter (SVCT2) has been detected primarily in radial glial cells in situ, with low-to-absent expression in cerebral cortex neuroblasts. However, strong SVCT2 expression is observed during the first postnatal days, resulting in increased intracellular concentration of vitamin C. Hippocampal neurons isolated from SVCT2 knockout mice showed shorter neurites and low clustering of glutamate receptors. Other studies have shown that vitamin C-deprived guinea pigs have reduced spatial memory, suggesting that ascorbic acid (AA) and SVCT2 have important roles in postnatal neuronal differentiation and neurite formation. In this study, SVCT2 lentiviral overexpression induced branching and increased synaptic proteins expression in primary cultures of cortical neurons. Analysis in neuroblastoma 2a (Neuro2a) and human subventricular tumor C3 (HSVT-C3) cells showed similar branching results. SVCT2 was mainly observed in the cell membrane and endoplasmic reticulum; however, it was not detected in the mitochondria. Cellular branching in neuronal cells and in a previously standardized neurosphere assay is dependent on the recycling of vitamin C or reduction in dehydroascorbic acid (DHA, produced by neurons) by glial cells. The effect of WZB117, a selective glucose/DHA transporter 1 (GLUT1) inhibitor expressed in glial cells, was also studied. By inhibiting GLUT1 glial cells, a loss of branching is observed in vitro, which is reproduced in the cerebral cortex in situ. We concluded that vitamin C recycling between neurons and astrocyte-like cells is fundamental to maintain neuronal differentiation in vitro and in vivo. The recycling activity begins at the cerebral postnatal cortex when neurons increase SVCT2 expression and concomitantly, GLUT1 is expressed in glial cells.

Major brain malformations: corpus callosum dysgenesis, agenesis of septum pellucidum and polymicrogyria in patients with BCORL1-related disorders.

BCORL1, a transcriptional co-repressor, has a role in cortical migration, neuronal differentiation, maturation, and cerebellar development. We describe BCORL1 as a new genetic cause for major brain malformations. We report three patients from two unrelated families with neonatal onset intractable epilepsy and profound global developmental delay. Brain MRI of two siblings from the first family depicted hypoplastic corpus callosum and septal agenesis (ASP) in the older brother and unilateral perisylvian polymicrogyria (PMG) in the younger one. MRI of the patient from the second family demonstrated complete agenesis of corpus callosum (CC). Whole Exome Sequencing revealed a novel hemizygous variant in NM_021946.5 (BCORL1):c.796C>T (p.Pro266Ser) in the two siblings from the first family and the NM_021946.5 (BCORL1): c.3376G>A; p.Asp1126Asn variant in the patient from the second family, both variants inherited from healthy mothers. We reviewed the patients' charts and MRIs and compared the phenotype to the other published BCORL1-related cases. Brain malformations have not been previously described in association with the BCORL1 phenotype. We discuss the potential influence of BCORL1 on brain development. We suggest that BCORL1 variants present with a spectrum of neurodevelopmental disorders and can lead to major brain malformations originating at different stages of fetal development. We suggest adding BCORL1 to the genetic causes of PMG, ASP, and CC dysgenesis.

Bisepoxide-Jeffamine microgel synthesis and application toward heterogeneous surface morphology for differential neuronal/non-neuronal cell responses in vitro

Herein, a new non-vinylic type of cationic microgels (MG) was readily prepared from ethylene glycol diglycidyl ether and Jeffamine T-403 in water. The MG was responsive to both temperature and pH, and oxidatively stable as demonstrated by the hydrogen peroxide study. Using glass as a model substrate, its surface was easily imparted with a heterogeneous morphology by simply adsorbing MG dispersed in basic solution. Specifically, the morphology features patches made of a monolayer of connected yet individually recognizable MG. Through in vitro cell studies, we show that a mere change of the extent of surface coverage by such a patchy morphology can strike a balance in promoting adhesion and differentiation of neuron-like PC-12 cells and primary cortical neurons of chick embryo, without soliciting proliferative response from non-neuronal cells of NIH3T3 fibroblast and CTX astrocyte. This simple yet unconventional surface morphology created by MG could be leveraged in the future as an alternative strategy for neural interface engineering.

Activation of HERV-K(HML-2) disrupts cortical patterning and neuronal differentiation by increasing NTRK3.

The biological function and disease association of human endogenous retroviruses (HERVs) are largely elusive. HERV-K(HML-2) has been associated with neurotoxicity, but there is no clear understanding of its role or mechanistic basis. We addressed the physiological functions of HERV-K(HML-2) in neuronal differentiation using CRISPR engineering to activate or repress its expression levels in a human-pluripotent-stem-cell-based system. We found that elevated HERV-K(HML-2) transcription is detrimental for the development and function of cortical neurons. These effects are cell-type-specific, as dopaminergic neurons are unaffected. Moreover, high HERV-K(HML-2) transcription alters cortical layer formation in forebrain organoids. HERV-K(HML-2) transcriptional activation leads to hyperactivation of NTRK3 expression and other neurodegeneration-related genes. Direct activation of NTRK3 phenotypically resembles HERV-K(HML-2) induction, and reducing NTRK3 levels in context of HERV-K(HML-2) induction restores cortical neuron differentiation. Hence, these findings unravel a cell-type-specific role for HERV-K(HML-2) in cortical neuron development.

Whole-exome sequencing analysis to identify novel potential pathogenetic mutations in fetuses with abnormal brain structure.

BackgroundGenetic mutations in genes related to the production, migration, or differentiation of cortical neurons can result in malformations of cortical development (MCDs). However, a large number of MCD-related pathogenetic mutations remain unknown. This study aimed to investigate the genetic cause of MCDs and to identify the new MCD-associated mutations through whole‐exome sequencing (WES) in fetuses with abnormal brain structure.MethodsCord venous blood samples were collected from 11 fetuses with MCDs. Whole-genome DNA was extracted from the blood, and WES was performed. Single nucleotide substitutions, insertions, and deletions were detected by bioinformatics analysis. Genetic mutations in genes associated with MCD were identified.ResultsA total of 1035 genes with high-impact genetic variants in at least 1 fetus were identified. The results of gene ontology enrichment analysis were consistent with those of previous studies and also indicated new potential MCD-related pathogenetic genetic mutations. Genes with high-impact mutations in multiple fetuses, such as CTDSP2 and C-terminal binding protein 2 (CTBP2), were more likely to be the genes affecting normal brain development.ConclusionsThis study has characterized variations in fetuses with MCDs and identified potential genetic mutations causing MCDs. Our findings extend the mutation spectrum of MCDs and provide a promising source for the identification of MCD-related pathogenetic mutations.

Interpretation of epileptogenic lesions: Its limitations in medico-legal experts’ reports

Focal cortical dysplasia (FCD) is the third leading cause of pharmacoresistant epilepsy diagnosed by epilepsy surgery and is a common malformation of cerebral cortical development. It is marked by abnormalities of cortical lamination, neuronal differentiation, and neuronal maturation. These epileptogenic lesions have proven anatomical and clinical correlations. Only one case of incidental FCD had been described. Incidental findings highlight infrequent lesions that go unnoticed when a person is alive. It can be an important finding for pathologists as well as for the forensic expert. The difficulty of the expertise will be in cases where these incidental lesions are known to cause symptoms that may be the cause of death. We wish to argue if they can be used in a medico-legal procedure. We report two cases demonstrating discrepancies between anatomical and clinical features, which should encourage the medical expert to use caution when interpreting neuropathological reports. La dysplasie corticale focale (DCF) est la troisième cause d’épilepsie pharmacorésistante diagnostiquée par chirurgie de l’épilepsie. Il s’agit d’une malformation courante du développement cortical cérébral. Elle est marquée par des anomalies de la stratification corticale, de la différenciation neuronale et de la maturation neuronale. Ces lésions épileptogènes ont des corrélations anatomiques et cliniques prouvées. Un seul cas de DCF de découverte fortuite à l’imagerie a été décrit dans la littérature. Ces découvertes fortuites mettent en évidence des lésions peu fréquentes qui passent inaperçues lorsqu’une personne est en vie. Or, ces lésions sont une découverte importante pour les pathologistes ainsi que pour l’expert médico-légal. La difficulté de l’expertise se situera dans les cas où ces lésions incidentelles sont connues pour provoquer des symptômes pouvant être la cause du décès. Nous souhaitons discuter s’ils peuvent être utilisés dans une procédure médico-légale. Nous rapportons deux cas démontrant des divergences entre les caractéristiques anatomiques et cliniques, ce qui devrait inciter l’expert médical à faire preuve de prudence dans l’interprétation des rapports neuropathologiques.

Roles of developmentally regulated KIF2A alternative isoforms in cortical neuron migration and differentiation.

KIF2A is a kinesin motor protein with essential roles in neural progenitor division and axonal pruning during brain development. However, how different KIF2A alternative isoforms function during development of the cerebral cortex is not known. Here, we focus on three Kif2a isoforms expressed in the developing cortex. We show that Kif2a is essential for dendritic arborization in mice and that the functions of all three isoforms are sufficient for this process. Interestingly, only two of the isoforms can sustain radial migration of cortical neurons; a third isoform, lacking a key N-terminal region, is ineffective. By proximity-based interactome mapping for individual isoforms, we identify previously known KIF2A interactors, proteins localized to the mitotic spindle poles and, unexpectedly, also translation factors, ribonucleoproteins and proteins that are targeted to organelles, prominently to the mitochondria. In addition, we show that a KIF2A mutation, which causes brain malformations in humans, has extensive changes to its proximity-based interactome, with depletion of mitochondrial proteins identified in the wild-type KIF2A interactome. Our data raises new insights about the importance of alternative splice variants during brain development.

Reg-1α Promotes Differentiation of Cortical Progenitors via Its N-Terminal Active Domain.

Reg-1α belongs to the Reg family of small, secreted proteins expressed in both pancreas and nervous system. Reg-1α is composed of two domains, an insoluble C-type lectin domain and a short soluble N-terminal peptide, which is released from the molecule upon proteolytic N-terminal processing, although the biological significance of this proteolysis remains unclear. We have previously shown that binding of Reg-1α to its receptor Extl3 stimulates axonal outgrowth. Reg-1α and Extl3 genes are expressed in the developing cortex but their expression decreases in adulthood, pointing to a possible function of this signaling system at the early developmental stages. Here, we demonstrate that recombinant Reg-1α increases migration and differentiation of cultured embryonic rat telencephalic progenitors via the activation of GSK-3β activity. In vivo overexpression of Reg-1α by in utero electroporation, has a similar effect, favoring premature differentiation of cortical progenitors. Notably, the N-terminal soluble domain, but not the C-type lectin domain, is largely responsible for Reg-1α effects on cortical neuronal differentiation. We thus conclude that Reg-1α via its proteolytically generated N-terminal domain is required for basic development processes.

Deficiency of TRPM2 leads to embryonic neurogenesis defects in hyperthermia.

Temperature homeostasis is critical for fetal development. The heat sensor protein TRPM2 (transient receptor potential channel M2) plays crucial roles in the heat response, but its function and specific mechanism in brain development remain largely unclear. Here, we observe that TRPM2 is expressed in neural stem cells. In hyperthermia, TRPM2 knockdown and knockout reduce the proliferation of neural progenitor cells (NPCs) and, accordingly, increase premature cortical neuron differentiation. In terms of the mechanism, TRPM2 regulates neural progenitor self-renewal by targeting SP5 (specificity protein 5) via inhibiting the phosphorylation of β-catenin and increasing β-catenin expression. Furthermore, the constitutive expression of TRPM2 or SP5 partly rescues defective NPC proliferation in the TRPM2-deficient embryonic brain. Together, the data suggest that TRPM2 has a critical function in maintaining the NPC pool during heat stress, and the findings provide a framework for understanding how the disruption of the TRPM2 gene may contribute to neurological disorders.

Changes in the morphofunctional development of the neuronal network in a dissociated cell culture of rat cerebral cortical neurons

Introduction. Study of the morphofunctional neuronal development in a dissociated cerebrocortical cell culture, using modern cell technologies, is a priority in experimental neurology, which is required for successful in vitro modelling of acute and chronic forms of cerebral pathology. Aim. A morphofunctional study of the in vitro changes in neuronal differentiation of rat cerebral cortical neurons, using a range of analysis methods, including immunohistochemistry, fluorescence, and electrophysiology. Materials and methods. We investigated the degree of culture differentiation on day 3–4 and day 10–11 of in vitro cultivation, measured by the intensity of the PSA-NCAM protein expression and the level of neuronal glutamate-induced calcium overload. That was then compared with the functional activity of the neuronal network cultivated on a microelectrode array, and with changes of the neuronal network’s activity in response to glutamate receptor overstimulation. Results. A significant glutamate-induced increase of the intracellular calcium concentration was typical for mature neurons (day 10–11 of cultivation), along with a lack of PSA-NCAM paranuclear accumulation, which was only found in immature cells (day 3–4 of cultivation). There was a glutamate suppression of the neuronal network burst activity, formed in vitro by day 10–11, with had no effect on the generation of single action potentials. At the same time, kainate, the exogenous selective agonist of the one of the glutamate subtypes, completely blocked spontaneous activity of the mature neurons. Conclusion. Neocortical rat neurons reach the differentiation level necessary for the modelling of the cerebral pathologies by day 10–11 of in vitro cultivation. At this point, the process of disruption of the microelectrode array cultivated neuronal network by the glutamate receptor overactivation, has become multilayered: excitotoxic glutamate-induced damage produces selective disruption of neuronal burst activity, and with the greater cytotoxicity caused by kainate, spontaneous bioelectrical activity is completely blocked.

High glucose concentrations mask cellular phenotypes in a stem cell model of tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder caused by deletions in the TSC1 or TSC2 genes that is associated with epilepsy in up to 90% of patients. Seizures are suggested to start in benign brain tumors, cortical tubers, or in the perituberal tissue making these tubers an interesting target for further research into mechanisms underlying epileptogenesis in TSC. Animal models of TSC insufficiently capture the neurodevelopmental biology of cortical tubers, and hence, human stem cell-based in vitro models of TSC are being increasingly explored in attempts to recapitulate tuber development and epileptogenesis in TSC. However, in vitro culture conditions for stem cell-derived neurons do not necessarily mimic physiological conditions. For example, very high glucose concentrations of up to 25 mM are common in culture media formulations. As TSC is potentially caused by a disruption of the mechanistic target of rapamycin (mTOR) pathway, a main integrator of metabolic information and intracellular signaling, we aimed to examine the impact of different glucose concentrations in the culture media on cellular phenotypes implicated in tuber characteristics. Here, we present preliminary data from a pilot study exploring cortical neuronal differentiation on human embryonic stem cells (hES) harboring a TSC2 knockout mutation (TSC2−/−) and an isogenic control line (TSC2+/+). We show that the commonly used high glucose media profoundly mask cellular phenotypes in TSC2−/− cultures during neuronal differentiation. These phenotypes only become apparent when differentiating TSC2+/+ and TSC2−/− cultures in more physiologically relevant conditions of 5 mM glucose suggesting that the careful consideration of culture conditions is vital to ensuring biological relevance and translatability of stem cell models for neurological disorders such as TSC.This article is part of the Special Issue “Proceedings of the 7th London-Innsbruck Colloquium on Status Epilepticus and Acute Seizures".

Reprogramming of DNA methylation at NEUROD2-bound sequences during cortical neuron differentiation.

The characteristics of DNA methylation changes that occur during neurogenesis in vivo remain unknown. We used whole-genome bisulfite sequencing to quantitate DNA cytosine modifications in differentiating neurons and their progenitors isolated from mouse brain at the peak of embryonic neurogenesis. Localized DNA hypomethylation was much more common than hypermethylation and often occurred at putative enhancers within genes that were upregulated in neurons and encoded proteins crucial for neuronal differentiation. The hypomethylated regions strongly overlapped with mapped binding sites of the key neuronal transcription factor NEUROD2. The 5-methylcytosine oxidase ten-eleven translocation 2 (TET2) interacted with NEUROD2, and its reaction product 5-hydroxymethylcytosine accumulated at the demethylated regions. NEUROD2-targeted differentially methylated regions retained higher methylation levels in Neurod2 knockout mice, and inducible expression of NEUROD2 caused TET2-associated demethylation at its in vivo binding sites. The data suggest that the reorganization of DNA methylation in developing neurons involves NEUROD2 and TET2-mediated DNA demethylation.

Long non-coding RNA LncKdm2b regulates cortical neuronal differentiation by cis-activating Kdm2b

ABSTRACTThe mechanisms underlying spatial and temporal control of cortical neurogenesis of the brain are largely elusive. Long non-coding RNAs (lncRNAs) have emerged as essential cell fate regulators. Here we found LncKdm2b (also known as Kancr), a lncRNA divergently transcribed from a bidirectional promoter of Kdm2b, is transiently expressed during early differentiation of cortical projection neurons. Interestingly, Kdm2b’s transcription is positively regulated in cis by LncKdm2b, which has intrinsic-activating function and facilitates a permissive chromatin environment at the Kdm2b’s promoter by associating with hnRNPAB. Lineage tracing experiments and phenotypic analyses indicated LncKdm2b and Kdm2b are crucial in proper differentiation and migration of cortical projection neurons. These observations unveiled a lncRNA-dependent machinery in regulating cortical neuronal differentiation.

INVESTIGATING AB-TAU INTERACTION IN TAU-DEFICIENT HUMAN NEURONS

Alzheimer's disease (AD) is an age-related neurodegenerative disease that leads to insidious loss of neurons, and subsequently to dementia. Amyloid-b (Ab) and tau are the two pathological hallmarks, and both are known to be dysregulated in AD leading to widespread proteinaceous aggregation in the brain. We now know from tau-deficient animal models that tau does not merely facilitate Ab-induced toxicity but is also essential in that particular signalling cascade. However, a tau-deficient human in vitro model has not been available to corroborate the Ab-tau interaction. Here we report generation of the first MAPT knockout (tau-KO) human induced pluripotent stem cell (iPSC) lines in order to study Ab-tau interaction in human biological context. We also characterised a versatile and scalable cortical neuron differentiation protocol that successfully produced a heterogeneous population of functional neurons manifesting cortical identity. Furthermore, we extracted brain homogenate from AD patient to serve as a source of Ab. Application of brain homogenate, which contains soluble Ab in pM range, in iPSC-derived neuronal culture resulted in neurite degeneration and cytotoxicity within 72 hours. We then asked if the human tau-KO genetic background could exhibit neuroprotective effects upon Ab exposure to the neuronal culture. We have initiated an investigation into the interaction between Ab and tau in human iPSC tau-KO cortical neurons utilising patient brain homogenate as the source of Ab to mimic human AD pathology in vitro.

Modelling the neuropathology of lysosomal storage disorders through disease-specific human induced pluripotent stem cells

Mucopolysaccharidosis II (MPS II) is a lysosomal storage disorder (LSD), caused by iduronate 2-sulphatase (IDS) enzyme dysfunction. The neuropathology of the disease is not well understood, although the neural symptoms are currently incurable. MPS II-patient derived iPSC lines were established and differentiated to neuronal lineage. The disease phenotype was confirmed by IDS enzyme and glycosaminoglycan assay. MPS II neuronal precursor cells (NPCs) showed significantly decreased self-renewal capacity, while their cortical neuronal differentiation potential was not affected. Major structural alterations in the ER and Golgi complex, accumulation of storage vacuoles, and increased apoptosis were observed both at protein expression and ultrastructural level in the MPS II neuronal cells, which was more pronounced in GFAP + astrocytes, with increased LAMP2 expression but unchanged in their RAB7 compartment. Based on these finding we hypothesize that lysosomal membrane protein (LMP) carrier vesicles have an initiating role in the formation of storage vacuoles leading to impaired lysosomal function. In conclusion, a novel human MPS II disease model was established for the first time which recapitulates the in vitro neuropathology of the disorder, providing novel information on the disease mechanism which allows better understanding of further lysosomal storage disorders and facilitates drug testing and gene therapy approaches.

Choice of Alternative Polyadenylation Sites, Mediated by the RNA-Binding Protein Elavl3, Plays a Role in Differentiation of Inhibitory Neuronal Progenitors.

Alternative polyadenylation (APA) is a widespread mechanism involving about half of the expressed genes, resulting in varying lengths of the 3′ untranslated region (3′UTR). Variations in length and sequence of the 3′UTR may underlie changes of post-transcriptional processing, localization, miRNA targeting and stability of mRNAs. During embryonic development a large array of mRNAs exhibit APA, with a prevalence of the longer 3′UTR versions in differentiating cells. Little is known about polyA+ site usage during differentiation of mammalian neural progenitors. Here we exploit a model of adherent neural stem (ANS) cells, which homogeneously and efficiently differentiate into GABAergic neurons. RNAseq data shows a global trend towards lengthening of the 3′UTRs during differentiation. Enriched expression of the longer 3′UTR variants of Pes1 and Gng2 was detected in the mouse brain in areas of cortical and subcortical neuronal differentiation, respectively, by two-probes fluorescent in situ hybridization (FISH). Among the coding genes upregulated during differentiation of ANS cells we found Elavl3, a neural-specific RNA-binding protein homologous to Drosophila Elav. In the insect, Elav regulates polyA+ site choice while interacting with paused Pol-II promoters. We tested the role of Elavl3 in ANS cells, by silencing Elavl3 and observed consistent changes in 3′UTR length and delayed neuronal differentiation. These results indicate that choice of the polyA+ site and lengthening of 3′UTRs is a possible additional mechanism of posttranscriptional RNA modification involved in neuronal differentiation.

Sprouty1 regulates neuritogenesis and survival of cortical neurons.

In multicellular organisms, receptor tyrosine kinases (RTKs) control a variety of cellular processes, including cell proliferation, differentiation, migration, and survival. Sprouty (SPRY) proteins represent an important class of ligand-inducible inhibitors of RTK-dependent signaling pathways. Here, we investigated the role of SPRY1 in cells of the central nervous system (CNS). Expression of SPRY1 was substantially higher in neural stem cells than in cortical neurons and was increased during neuronal differentiation of cortical neurons. We found that SPRY1 was a direct target gene of the CNS-specific microRNA, miR-124 and miR-132. In primary cultures of cortical neurons, the neurotrophic factors brain-derived neurotrophic factor (BDNF) and Basic fibroblast growth factor (FGF2) downregulated SPRY1 expression to positively regulate their own functions. In immature cortical neurons and mouse N2 A cells, we found that overexpression of SPRY1 inhibited neurite development, whereas knockdown of SPRY1 expression promoted neurite development. In mature neurons, overexpression of SPRY1 inhibited the prosurvival effects of both BDNF and FGF2 on glutamate-mediated neuronal cell death. SPRY1 was also upregulated upon glutamate treatment in mature neurons and partially contributed to the cytotoxic effect of glutamate. Together, our results indicate that SPRY1 contributes to the regulation of CNS functions by influencing both neuronal differentiation under normal physiological processes and neuronal survival under pathological conditions.

Development of Cortical Pyramidal Cell and Interneuronal Dendrites: a Role for Kainate Receptor Subunits and NETO1.

During neuronal development, AMPA receptors (AMPARs) and NMDA receptors (NMDARs) are important for neuronal differentiation. Kainate receptors (KARs) are closely related to AMPARs and involved in the regulation of cortical network activity. However, their role for neurite growth and differentiation of cortical neurons is unclear. Here, we used KAR agonists and overexpression of selected KAR subunits and their auxiliary neuropilin and tolloid-like proteins, NETOs, to investigate their influence on dendritic growth and network activity in organotypic cultures of rat visual cortex. Kainate at 500nM enhanced network activity and promoted development of dendrites in layer II/III pyramidal cells, but not interneurons. GluK2 overexpression promoted dendritic growth in pyramidal cells and interneurons. GluK2 transfectants were highly active and acted as drivers for network activity. GluK1 and NETO1 specifically promoted dendritic growth of interneurons. Our study provides new insights for the roles of KARs and NETOs in the morphological and physiological development of the visual cortex.

Expression and secretion of synaptic proteins during stem cell differentiation to cortical neurons

Synaptic function and neurotransmitter release are regulated by specific proteins. Cortical neuronal differentiation of human induced pluripotent stem cells (hiPSC) provides an experimental model to obtain more information about synaptic development and physiology in vitro. In this study, expression and secretion of the synaptic proteins, neurogranin (NRGN), growth-associated protein-43 (GAP-43), synaptosomal-associated protein-25 (SNAP-25) and synaptotagmin-1 (SYT-1) were analyzed during cortical neuronal differentiation. Protein levels were measured in cells, modeling fetal cortical development and in cell-conditioned media which was used as a model of cerebrospinal fluid (CSF), respectively. Human iPSC-derived cortical neurons were maintained over a period of at least 150 days, which encompasses the different stages of neuronal development. The differentiation was divided into the following stages: hiPSC, neuro-progenitors, immature and mature cortical neurons. We show that NRGN was first expressed and secreted by neuro-progenitors while the maximum was reached in mature cortical neurons. GAP-43 was expressed and secreted first by neuro-progenitors and its expression increased markedly in immature cortical neurons. SYT-1 was expressed and secreted already by hiPSC but its expression and secretion peaked in mature neurons. SNAP-25 was first detected in neuro-progenitors and the expression and secretion increased gradually during neuronal stages reaching a maximum in mature neurons. The sensitive analytical techniques used to monitor the secretion of these synaptic proteins during cortical development make these data unique, since the secretion of these synaptic proteins has not been investigated before in such experimental models. The secretory profile of synaptic proteins, together with low release of intracellular content, implies that mature neurons actively secrete these synaptic proteins that previously have been associated with neurodegenerative disorders, including Alzheimer's disease. These data support further studies of human neuronal and synaptic development in vitro, and would potentially shed light on the mechanisms underlying altered concentrations of the proteins in bio-fluids in neurodegenerative diseases.