Human Neuronal Differentiation Research Articles

Neuronal splicing of the unmethylated histone H3K4 reader, PHF21A, prevents excessive synaptogenesis

PHF21A is a histone-binding protein that recognizes unmethylated histone H3K4, the reaction product of LSD1 histone demethylase. PHF21A and LSD1 form a complex, and both undergo neuron-specific microexon splicing. The PHF21A neuronal microexon interferes with nucleosome binding, whereas the LSD1 neuronal microexon weakens H3K4 demethylation activity and can alter the substrate specificity to H3K9 or H4K20. However, the temporal expression patterns of PHF21A and LSD1 splicing isoforms during brain development and their biological roles remain unknown. In this work, we report that neuronal PHF21A isoform expression precedes neuronal LSD1 expression during human neuron differentiation and mouse brain development. The asynchronous splicing events resulted in stepwise deactivation of the LSD1-PHF21A complex in reversing H3K4 methylation. An unbiased proteomics survey revealed that the enzymatically inactive LSD1-PHF21A complex interacts with neuron-specific binding partners, including MYT1-family transcription factors and post-transcriptional mRNA processing proteins such as VIRMA. The interaction with the neuron-specific components, however, did not require the PHF21A microexon, indicating that the neuronal proteomic milieu, rather than the microexon-encoded PHF21A segment, is responsible for neuron-specific complex formation. Finally, by using two Phf21a mutant mouse models, we found that Phf21a neuronal splicing prevents excess synapse formation that otherwise would occur when canonical PHF21A is expressed in neurons. These results suggest that the role of the PHF21A microexon is to dampen LSD1-mediated H3K4 demethylation, thereby containing aberrant synaptogenesis.

Satellite glial contact enhances differentiation and maturation of human iPSC-derived sensory neurons.

Sensory neurons generated from induced pluripotent stem cells (iSNs) are used to model human peripheral neuropathies, however current differentiation protocols produce sensory neurons with an embryonic phenotype. Peripheral glial cells contact sensory neurons early in development and contribute to formation of the canonical pseudounipolar morphology, but these signals are not encompassed in current iSN differentiation protocols. Here, we show that terminal differentiation of iSNs in co-culture with rodent Dorsal Root Ganglion satellite glia (rSG) advances their differentiation and maturation. Co-cultured iSNs develop a pseudounipolar morphology through contact with rSGs. This transition depends on semaphorin-plexin guidance cues and on glial gap junction signaling. In addition to morphological changes, iSNs terminally differentiated in co-culture exhibit enhanced spontaneous action potential firing, more mature gene expression, and increased susceptibility to paclitaxel induced axonal degeneration. Thus, iSNs differentiated in coculture with rSGs provide a better model for investigating human peripheral neuropathies.

Loss-of-function of the Zinc Finger Homeobox 4 (ZFHX4) gene underlies a neurodevelopmental disorder.

8q21.11 microdeletions encompassing the gene encoding transcription factor ZFHX4, have previously been associated by us with a syndromic form of intellectual disability, hypotonia, decreased balance and hearing loss. Here, we report on 57 individuals, 52 probands and 5 affected family members, with protein truncating variants (n=36), (micro)deletions (n=20) or an inversion (n=1) affecting ZFHX4 with variable developmental delay and intellectual disability, distinctive facial characteristics, morphological abnormalities of the central nervous system, behavioral alterations, short stature, hypotonia, and occasionally cleft palate and anterior segment dysgenesis. The phenotypes associated with 8q21.11 microdeletions and ZFHX4 intragenic loss-of-function variants largely overlap, identifying ZFHX4 as the main driver for the microdeletion syndrome, although leukocyte-derived DNA shows a mild common methylation profile for (micro)deletions only. We identify ZFHX4 as a transcription factor that is increasingly expressed during human brain development and neuronal differentiation. Furthermore, ZFHX4 interacting factors identified via IP-MS in neural progenitor cells, suggest an important role for ZFHX4 in cellular and developmental pathways, especially during histone modifications, cytosolic transport and development. Additionally, using CUT&RUN, we observed that ZFHX4 binds with the promoter regions of genes with crucial roles in embryonic, neuron and axon development. Since loss-of-function variants in ZFHX4 are found with consistent dysmorphic facial features, we investigated whether the disruption of zfhx4 causes craniofacial abnormalities in zebrafish. First-generation (F0) zfhx4 crispant zebrafish, (mosaic) mutant for zfhx4 loss-of-function variants, have significantly shorter Meckel's cartilages and smaller ethmoid plates compared to control zebrafish. Furthermore, behavioral assays show a decreased movement frequency in the zfhx4 crispant zebrafish in comparison with control zebrafish larvae. Although further research is needed, our in vivo work suggests a role for zfhx4 in facial skeleton patterning, palatal development and behavior.

M6a methylation orchestrates IMP1 regulation of microtubules during human neuronal differentiation

Neuronal differentiation requires building a complex intracellular architecture, and therefore the coordinated regulation of defined sets of genes. RNA-binding proteins (RBPs) play a key role in this regulation. However, while their action on individual mRNAs has been explored in depth, the mechanisms used to coordinate gene expression programs shaping neuronal morphology are poorly understood. To address this, we studied how the paradigmatic RBP IMP1 (IGF2BP1), an essential developmental factor, selects and regulates its RNA targets during the human neuronal differentiation. We perform a combination of system-wide and molecular analyses, revealing that IMP1 developmentally transitions to and directly regulates the expression of mRNAs encoding essential regulators of the microtubule network, a key component of neuronal morphology. Furthermore, we show that m6A methylation drives the selection of specific IMP1 mRNA targets and their protein expression during the developmental transition from neural precursors to neurons, providing a molecular principle for the onset of target selectivity.

REST and RCOR genes display distinct expression profiles in neurons and astrocytes using 2D and 3D human pluripotent stem cell models

Repressor element-1 silencing transcription factor (REST) is a transcriptional repressor involved in neurodevelopment and neuroprotection. REST forms a complex with the REST corepressors, CoREST1, CoREST2, or CoREST3 (encoded by RCOR1, RCOR2, and RCOR3, respectively). Emerging evidence suggests that the CoREST family can target unique genes independently of REST, in various neural and glial cell types during different developmental stages. However, there is limited knowledge regarding the expression and function of the CoREST family in human neurodevelopment. To address this gap, we employed 2D and 3D human pluripotent stem cell (hPSC) models to investigate REST and RCOR gene expression levels. Our study revealed a significant increase in RCOR3 expression in glutamatergic cortical and GABAergic ventral forebrain neurons, as well as mature functional NGN2-induced neurons. Additionally, a simplified astrocyte transdifferentiation protocol resulted in a significant decrease in RCOR2 expression following differentiation. REST expression was notably reduced in mature neurons and cerebral organoids. In summary, our findings provide the first insights into the cell-type-specific expression patterns of RCOR genes in human neuronal and glial differentiation. Specifically, RCOR3 expression increases in neurons, while RCOR2 levels decrease in astrocytes. The dynamic expression patterns of REST and RCOR genes during hPSC neuronal and glial differentiation underscore the potential distinct roles played by REST and CoREST proteins in regulating the development of these cell types in humans.

Transcriptomic profiling across human serotonin neuron differentiation via the FEV reporter system

BackgroundThe detailed transcriptomic profiles during human serotonin neuron (SN) differentiation remain elusive. The establishment of a reporter system based on SN terminal selector holds promise to produce highly-purified cells with an early serotonergic fate and help elucidate the molecular events during human SN development process.MethodsA fifth Ewing variant (FEV)-EGFP reporter system was established by CRISPR/Cas9 technology to indicate SN since postmitotic stage. FACS was performed to purify SN from the heterogeneous cell populations. RNA-sequencing analysis was performed for cells at four key stages of differentiation (pluripotent stem cells, serotonergic neural progenitors, purified postmitotic SN and purifed mature SN) to explore the transcriptomic dynamics during SN differentiation.ResultsWe found that human serotonergic fate specification may commence as early as day 21 of differentiation from human pluripotent stem cells. Furthermore, the transcriptional factors ZIC1, HOXA2 and MSX2 were identified as the hub genes responsible for orchestrating serotonergic fate determination.ConclusionsFor the first time, we exposed the developmental transcriptomic profiles of human SN via FEV reporter system, which will further our understanding for the development process of human SN.Graphical

Engrailed 1 deficiency induces changes in ciliogenesis during human neuronal differentiation

A key pathological feature of Parkinson’s Disease (PD) is the progressive degeneration of dopaminergic neurons (DAns) in the substantia nigra pars compacta. Considering the major role of EN1 in the development and maintenance of these DAns and the implications from En1 mouse models, it is highly interesting to study the molecular and protective effect of EN1 also in a human cellular model. Therefore, we generated EN1 knock-out (ko) human induced pluripotent stem cell (hiPSCs) lines and analyzed these during neuronal differentiation. Although the EN1 ko didn’t interfere with neuronal differentiation and generation of tyrosine hydroxylase positive (TH+) neurons per se, the neurons exhibited shorter neurites. Furthermore, mitochondrial respiration, as well as mitochondrial complex I abundance was significantly reduced in fully differentiated neurons. To understand the implications of an EN1 ko during differentiation, we performed a transcriptome analysis of human neuronal precursor cells (hNPCs) which unveiled alterations in cilia-associated pathways. Further analysis of ciliary morphology revealed an elongation of primary cilia in EN1-deficient hNPCs. Besides, also Wnt signaling pathways were severely affected. Upon stimulating hNPCs with Wnt which drastically increased EN1 expression in WT lines, the phenotypes concerning mitochondrial function and cilia were exacerbated in EN1 ko hNPCs. They failed to enhance the expression of the complex I subunits NDUFS1 and 3, and now displayed a reduced mitochondrial respiration. Furthermore, Wnt stimulation decreased ciliogenesis in EN1 ko hNPCs but increased ciliary length even further. This further highlights the relevance of primary cilia next to mitochondria for the functionality and correct maintenance of human DAns and provides new possibilities to establish neuroprotective therapies for PD.

Primary cilia promote the differentiation of human neurons through the WNT signaling pathway.

Primary cilia emanate from most human cell types, including neurons. Cilia are important for communicating with the cell's immediate environment: signal reception and transduction to/from the ciliated cell. Deregulation of ciliary signaling can lead to ciliopathies and certain neurodevelopmental disorders. In the developing brain cilia play well-documented roles for the expansion of the neural progenitor cell pool, while information about the roles of cilia during post-mitotic neuron differentiation and maturation is scarce. We employed ciliated Lund Human Mesencephalic (LUHMES) cells in time course experiments to assess the impact of ciliary signaling on neuron differentiation. By comparing ciliated and non-ciliated neuronal precursor cells and neurons in wild type and in RFX2 -/- mutant neurons with altered cilia, we discovered an early-differentiation "ciliary time window" during which transient cilia promote axon outgrowth, branching and arborization. Experiments in neurons with IFT88 and IFT172 ciliary gene knockdowns, leading to shorter cilia, confirm these results. Cilia promote neuron differentiation by tipping WNT signaling toward the non-canonical pathway, in turn activating WNT pathway output genes implicated in cyto-architectural changes. We provide a mechanistic entry point into when and how ciliary signaling coordinates, promotes and translates into anatomical changes. We hypothesize that ciliary alterations causing neuron differentiation defects may result in "mild" impairments of brain development, possibly underpinning certain aspects of neurodevelopmental disorders.

Haploinsufficiency of ZFHX3, encoding a key player in neuronal development, causes syndromic intellectual disability

Neurodevelopmental disorders (NDDs) result from impaired development and functioning of the brain. Here, we identify loss-of-function (LoF) variation in ZFHX3 as a cause for syndromic intellectual disability (ID). ZFHX3 is a zinc-finger homeodomain transcription factor involved in various biological processes, including cell differentiation and tumorigenesis. We describe 42 individuals with protein-truncating variants (PTVs) or (partial) deletions of ZFHX3, exhibiting variable intellectual disability and autism spectrum disorder, recurrent facial features, relative short stature, brachydactyly, and, rarely, cleft palate. ZFHX3 LoF associates with a specific methylation profile in whole blood extracted DNA. Nuclear abundance of ZFHX3 increases during human brain development and neuronal differentiation. ZFHX3 was found to interact with the chromatin remodeling BRG1/Brm-associated factor complex and the cleavage and polyadenylation complex, suggesting a function in chromatin remodeling and mRNA processing. Furthermore, ChIP-seq for ZFHX3 revealed that it predominantly binds promoters of genes involved in nervous system development. We conclude that loss-of-function variants in ZFHX3 are a cause of syndromic ID associating with a specific DNA methylation profile.

Rapid and high-purity differentiation of human medium spiny neurons reveals LMNB1 hypofunction and subtype necessity in modeling Huntington’s disease

BackgroundDifferent neural subtypes are selectively lost in diverse neurodegenerative diseases. Huntington’s disease (HD) is an inherited neurodegenerative disease characterized by motor abnormalities that primarily affect the striatum. The Huntingtin (HTT) mutation involves an expanded CAG repeat, leading to insoluble polyQ, which renders GABA+ medium spiny neurons (MSN) more venerable to cell death. Human pluripotent stem cells (hPSCs) technology allows for the construction of disease-specific models, providing valuable cellular models for studying pathogenesis, drug screening, and high-throughput analysis.MethodsIn this study, we established a method that allows for rapid and efficient generation of MSNs (> 90%) within 21 days from hPSC-derived neural progenitor cells, by introducing a specific combination of transcription factors.ResultsWe efficiently induced several neural subtypes, in parallel, based on the same cell source, and revealed that, compared to other neural subtypes, MSNs exhibited higher polyQ aggregation propensity and overexpression toxicity, more severe dysfunction in BDNF/TrkB signaling, greater susceptibility to BDNF withdrawal, and more severe disturbances in nucleocytoplasmic transport (NCT). We further found that the nuclear lamina protein LMNB1 was greatly reduced in HD neurons and mislocalized to the cytoplasm and axons. Knockdown of HTT or treatment with KPT335, an orally selective inhibitor of nuclear export (SINE), effectively attenuated the pathological phenotypes and alleviated neuronal death caused by BDNF withdrawal.ConclusionsThis study thus establishes an effective method for obtaining MSNs and underscores the necessity of using high-purity MSNs to study HD pathogenesis, especially the MSN-selective vulnerability.

Directed Differentiation of Neurons from Human iPSCs for Modeling Neurological Disorders.

Human-induced pluripotent stem cell (hiPSC) technology has enabledcomprehensive human cell-based disease modeling in vitro. Due to limited accessibility of primary human neurons as well as species-specific divergence between human and rodent brain tissues, hiPSC-derived neurons have become a popular tool for studying neuronal biology in a dish. Here, we provide methods for transcription factor-driven directed differentiation of neurons from hiPSCs via a neural progenitor cell (NPC) intermediate. Doxycycline-inducible expression of neuron fate-determining transcription factors neurogenin 2 (NGN2) and achaete-scute homolog 1 (ASCL1) enables rapid and controllable differentiation of human neurons for disease modeling applications. The provided method is also designed to improve the reproducibility of human neuron differentiation by reducing the batch-to-batch variation of NPC differentiation and lentiviral transduction.

Interspecies chimerism with human embryonic stem cells generates functional human dopamine neurons at low efficiency

Interspecies chimeras offer great potential for regenerative medicine and the creation of human disease models. Whether human pluripotent stem cell-derived neurons in an interspecies chimera can differentiate into functional neurons and integrate into host neural circuity is not known. Here, we show, using Engrailed 1 (En1) as a development niche, that human naive-like embryonic stem cells (ESCs) can incorporate into embryonic and adult mouse brains. Human-derived neurons including tyrosine hydroxylase (TH)+ neurons integrate into the mouse brain at low efficiency. These TH+ neurons have electrophysiologic properties consistent with their human origin. In addition, these human-derived neurons in the mouse brain accumulate pathologic phosphorylated α-synuclein in response to α-synuclein preformed fibrils. Optimization of human/mouse chimeras could be used to study human neuronal differentiation and human brain disorders.

Multiomics analysis identifies novel facilitators of human dopaminergic neuron differentiation

Midbrain dopaminergic neurons (mDANs) control voluntary movement, cognition, and reward behavior under physiological conditions and are implicated in human diseases such as Parkinson’s disease (PD). Many transcription factors (TFs) controlling human mDAN differentiation during development have been described, but much of the regulatory landscape remains undefined. Using a tyrosine hydroxylase (TH) human iPSC reporter line, we here generate time series transcriptomic and epigenomic profiles of purified mDANs during differentiation. Integrative analysis predicts novel regulators of mDAN differentiation and super-enhancers are used to identify key TFs. We find LBX1, NHLH1 and NR2F1/2 to promote mDAN differentiation and show that overexpression of either LBX1 or NHLH1 can also improve mDAN specification. A more detailed investigation of TF targets reveals that NHLH1 promotes the induction of neuronal miR-124, LBX1 regulates cholesterol biosynthesis, and NR2F1/2 controls neuronal activity.

METTL3-Dependent N6-Methyladenosine Modification Programs Human Neural Progenitor Cell Proliferation.

METTL3, a methyltransferase responsible for N6-methyladenosine (m6A) modification, plays key regulatory roles in mammal central neural system (CNS) development. However, the specific epigenetic mechanisms governing human CNS development remain poorly elucidated. Here, we generated small-molecule-assisted shut-off (SMASh)-tagged hESC lines to reduce METTL3 protein levels, and found that METTL3 is not required for human neural progenitor cell (hNPC) formation and neuron differentiation. However, METTL3 deficiency inhibited hNPC proliferation by reducing SLIT2 expression. Mechanistic studies revealed that METTL3 degradation in hNPCs significantly decreased the enrichment of m6A in SLIT2 mRNA, consequently reducing its expression. Our findings reveal a novel functional target (SLIT2) for METTL3 in hNPCs and contribute to a better understanding of m6A-dependent mechanisms in hNPC proliferation.

High content screening miniaturization and single cell imaging of mature human feeder layer-free iPSC-derived neurons.

Human induced pluripotent stem cell (iPSC)-derived neurons are being increasingly used for high content imaging and screening. However, iPSC-derived neuronal differentiation and maturation is time-intensive, often requiring >8 weeks. Unfortunately, the differentiating and maturing iPSC-derived neuronal cultures also tend to migrate and coalesce into ganglion-like clusters making single-cell analysis challenging, especially in miniaturized formats. Using our defined extracellular matrix and low oxygen culturing conditions for the differentiation and maturation of human cortical neurons, we further modified neuronal progenitor cell seeding densities and feeder layer-free culturing conditions in miniaturized formats (i.e., 96 well) to decrease neuronal clustering, enhance single-cell identification and reduce edge effects usually observed after extended neuronal cell culture. Subsequent algorithm development refined capabilities to distinguish and identify single mature neurons, as identified by NeuN expression, from large cellular aggregates, which were excluded from image analysis. Incorporation of astrocyte conditioned medium during differentiation and maturation periods significantly increased the percentage (i.e., ∼10% to ∼30%) of mature neurons (i.e., NeuN+) detected at 4-weeks post-differentiation. Pilot, proof of concept studies using this optimized assay system yielded negligible edge effects and robust Z-factors in population-based as well as image-based neurotoxicity assay formats. Moreover, moxidectin, an FDA-approved drug with documented neurotoxic adverse effects, was identified as a hit using both screening formats. This miniaturized, feeder layer-free format and image analysis algorithm provides a foundational imaging and screening platform, which enables quantitative single-cell analysis of differentiated human neurons.

Organelle mapping in dendrites of human iPSC-derived neurons reveals dynamic functional dendritic Golgi structures

Secretory pathways within dendrites of neurons have been proposed for local transport of newly synthesized proteins. However, little is known about the dynamics of the local secretory system and whether the organelles are transient or stable structures. Here, we quantify the spatial and dynamic behavior of dendritic Golgi and endosomes during differentiation of human neurons generated from induced pluripotent stem cells (iPSCs). In early neuronal development, before and during migration, the entire Golgi apparatus transiently translocates from the soma into dendrites. In mature neurons, dynamic Golgi elements, containing cis and trans cisternae, are transported from the soma along dendrites, in an actin-dependent process. Dendritic Golgi outposts are dynamic and display bidirectional movement. Similar structures were observed in cerebral organoids. Using the retention using selective hooks (RUSH) system, Golgi resident proteins are transported efficiently into Golgi outposts from the endoplasmic reticulum. This study reveals dynamic, functional Golgi structures in dendrites and a spatial map for investigating dendrite trafficking in human neurons.

Differentiation, Transcriptomic Profiling, and Calcium Imaging of Human Hypothalamic Neurons.

Neurons in the hypothalamus orchestrate homeostatic physiological processes and behaviors essential for life. Human pluripotent stem cells (hPSCs) can be differentiated into many types of hypothalamic neurons, progenitors, and glia. This updated unit includes published studies and protocols with new advances in the differentiation, maturation, and interrogation by transcriptomic profiling and calcium imaging of human hypothalamic cell populations. Specifically, new methods to freeze and thaw hypothalamic progenitors after they have been patterned and before substantial neurogenesis has occurred are provided that will facilitate experimental flexibility and planning. Also included are updated recipes and protocols for neuronal maturation, with details on the equipment and methods for examining their transcriptomic response and cell-autonomous properties in culture in the presence of synaptic blockers. Together, these protocols facilitate the adoption and use of this model system for fundamental biological discovery and therapeutic translation to human diseases such as obesity, diabetes, sleep disorders, infertility, and chronic stress. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: hPSC maintenance Basic Protocol 2: Hypothalamic neuron differentiation Support Protocol 1: Cortical neuron (control) differentiation Basic Protocol 3: Neuronal maturation Support Protocol 2: Cryopreservation and thawing of neuronal progenitors Support Protocol 3: Quality control: Confirmation of hypothalamic patterning and neurogenesis Support Protocol 4: Bulk RNA sequencing of hypothalamic cultures Basic Protocol 4: Calcium imaging of hypothalamic neurons using Fura-2 AM Alternate Protocol: Calcium imaging of green fluorescent hypothalamic neurons using Rhod-3 AM.

Circular RNAs arising from synaptic host genes during human neuronal differentiation are modulated by SFPQ RNA-binding protein

BackgroundCircular RNA (circRNA) molecules, generated through non-canonical back-splicing of exon-exon junctions, have recently been implicated in diverse biological functions including transcriptional regulation and modulation of protein interactions. CircRNAs are emerging as a key component of the complex neural transcriptome implicated in brain development. However, the specific expression patterns and functions of circRNAs in human neuronal differentiation have not been explored.ResultsUsing total RNA sequencing analysis, we identified expressed circRNAs during the differentiation of human neuroepithelial stem (NES) cells into developing neurons and discovered that many circRNAs originated from host genes associated with synaptic function. Interestingly, when assessing population data, exons giving rise to circRNAs in our dataset had a higher frequency of genetic variants. Additionally, screening for RNA-binding protein sites identified enrichment of Splicing Factor Proline and Glutamine Rich (SFPQ) motifs in increased circRNAs, several of which were reduced by SFPQ knockdown and enriched in SFPQ ribonucleoprotein complexes.ConclusionsOur study provides an in-depth characterisation of circRNAs in a human neuronal differentiation model and highlights SFPQ as both a regulator and binding partner of circRNAs elevated during neuronal maturation.

A high ratio of linoleic acid (n-6 PUFA) to alpha-linolenic acid (n-3 PUFA) adversely affects early stage of human neuronal differentiation and electrophysiological activity of glutamatergic neurons in vitro.

Introduction: There is a growing interest in the possibility of dietary supplementation with polyunsaturated fatty acids (PUFAs) for treatment and prevention of neurodevelopmental and neuropsychiatric disorders. Studies have suggested that of the two important classes of polyunsaturated fatty acids, omega-6 (n-6) and omega-3 (n-3), n-3 polyunsaturated fatty acids support brain development and function, and when used as a dietary supplement may have beneficial effects for maintenance of a healthy brain. However, to date epidemiological studies and clinical trials on children and adults have been inconclusive regarding treatment length, dosage and use of specific n-3 polyunsaturated fatty acids. The aim of this study is to generate a simplified in vitro cell-based model system to test how different n-6 to n-3 polyunsaturated fatty acids ratios affect human-derived neurons activity as a cellular correlate for brain function and to probe the mechanism of their action. Methods: All experiments were performed by use of human induced pluripotent stem cells (iPSCs). In this study, we examined the effect of different ratios of linoleic acid (n-6) to alpha-linolenic acid in cell growth medium on induced pluripotent stem cell proliferation, generation of neuronal precursors and electrophysiology of cortical glutamatergic neurons by multielectrode array (MEA) analysis. Results: This study shows that at a n-6:n-3 ratio of 5:1 polyunsaturated fatty acids induce stem cell proliferation, generating a large increase in number of cells after 72h treatment; suppress generation of neuronal progenitor cells, as measured by decreased expression of FOXG1 and Nestin in neuronal precursor cells (NPC) after 20days of development; and disrupt neuronal activity in vitro, increasing spontaneous neuronal firing, reducing synchronized bursting receptor subunits. We observed no significant differences for neuronal precursor cells treated with ratios 1:3 and 3:1, in comparison to 1:1 control ratio, but higher ratios of n-6 to n-3 polyunsaturated fatty acids adversely affect early stages of neuronal differentiation. Moreover, a 5:1 ratio in cortical glutamatergic neurons induce expression of GABA receptors which may explain the observed abnormal electrophysiological activity.

Glycolysis mediates neuron specific histone acetylation in valproic acid-induced human excitatory neuron differentiation.

Pregnancy exposure of valproic acid (VPA) is widely adopted as a model of environmental factor induced autism spectrum disorder (ASD). Increase of excitatory/inhibitory synaptic transmission ratio has been proposed as the mechanism of VPA induced ASD. How this happened, particularly at the level of excitatory neuron differentiation in human neural progenitor cells (NPCs) remains largely unclear. Here, we report that VPA exposure remarkably inhibited human NPC proliferation and induced excitatory neuronal differentiation without affecting inhibitory neurons. Following VPA treatment, mitochondrial dysfunction was observed before neuronal differentiation, as showed by ultrastructural changes, respiratory complex activity, mitochondrial membrane potential and oxidation levels. Meanwhile, extracellular acidification assay revealed an elevation of glycolysis by VPA stimulation. Interestingly, inhibiting glycolysis by 2-deoxy-d-glucose-6-phosphate (2-DG) efficiently blocked the excitatory neuronal differentiation of human NPCs induced by VPA. Furthermore, 2-DG treatment significantly compromised the VPA-induced expression of H3ac and H3K9ac, and the VPA-induced binding of H3K9ac on the promoter of Ngn2 and Mash1, two key transcription factors of excitatory neuron fate determination. These data, for the first time, demonstrated that VPA biased excitatory neuron differentiation by glycolysis-mediated histone acetylation of neuron specific transcription factors.