Stages Of Neural Differentiation Research Articles

Prenatal lipopolysaccharide exposure induces anxiety-like behaviour in male mouse offspring and aberrant glial differentiation of embryonic neural stem cells

BackgroundPrenatal infection has been implicated in the development of neuropsychiatric disorders in children. We hypothesised that exposure to lipopolysaccharide during prenatal development could induce anxiety-like behaviour and sensorineural hearing loss in offspring, as well as disrupt neural differentiation during embryonic neural development.MethodsWe simulated prenatal infection in FVB mice and mouse embryonic stem cell (ESC) lines, specifically 46C and E14Tg2a, through lipopolysaccharide treatment. Gene expression profiling analyses and behavioural tests were utilized to study the effects of lipopolysaccharide on the offspring and alterations in toll-like receptor (TLR) 2-positive and TLR4-positive cells during neural differentiation in the ESCs.ResultsExposure to lipopolysaccharide (25 µg/kg) on gestation day 9 resulted in anxiety-like behaviour specifically in male offspring, while no effects were detected in female offspring. We also found significant increases in the expression of GFAP and CNPase, as well as higher numbers of GFAP + astrocytes and O4+ oligodendrocytes in the prefrontal cortex of male offspring. Furthermore, increased scores for genes related to oligodendrocyte and lipid metabolism, particularly ApoE, were observed in the prefrontal cortex regions. Upon exposure to lipopolysaccharide during the ESC-to-neural stem cell (NSC) transition, Tuj1, Map2, Gfap, O4, and Oligo2 mRNA levels increased in the differentiated neural cells on day 14. In vitro experiments demonstrated that lipopolysaccharide exposure induced inflammatory responses, as evidenced by increased expression of IL1b and ApoB mRNA.ConclusionsOur findings suggest that prenatal infection at different stages of neural differentiation may result in distinct disturbances in neural differentiation during ESC—NSC transitions. Furthermore, early prenatal challenges with lipopolysaccharide selectively induce anxiety-like behaviour in male offspring. This behaviour may be attributed to the abnormal differentiation of astrocytes and oligodendrocytes in the brain, potentially mediated by ApoB/E signalling pathways in response to inflammatory stimuli.

The Oxygen and Glucose Deprivation of Immature Cells of the Nervous System Exerts Distinct Effects on Mitochondria, Mitophagy, and Autophagy, Depending on the Cells’ Differentiation Stage

Perinatal brain damage, one of the most common causes of lifelong impairment, is predominantly caused by a lack of oxygen and glucose during early development. These conditions, in turn, affect cells of the nervous tissue through various stages of their maturation. To quantify the influence of these factors on cell differentiation and mitochondrial parameters, we exposed neural cell precursors to oxygen and glucose deprivation (OGD) during three stages of their differentiation: day 1, day 7, and day 14 (D1, D7, and D14, respectively). The obtained results show that OGD slows down cellular differentiation and causes cell death. Regardless of the level of cell maturity, the overall area of the mitochondria, their length, and the branching of their filaments decreased uniformly when exposed to OGD-related stress. Moreover, the cells in all stages of differentiation exhibited an increase in ROS production, hyperpolarization of the mitochondrial membrane, and autophagy. Interestingly, day 7 was the only stage in which a significant increase in mitochondrial fission, along with measurable instances of mitophagy, were detected. Taken together, the results of this study suggest that, apart from common reactions to a sudden lack of oxygen and glucose, cells in specific stages of neural differentiation can also exhibit increased preferences for mitochondrial fission and mitophagy. Such findings could play a role in guiding the future development of novel therapeutic approaches targeting perinatal brain damage during specific stages of nervous system development.

Dynamics of Metabolic Pathways and Stress Response Patterns during Human Neural Stem Cell Proliferation and Differentiation.

Understanding early nervous system stress response mechanisms is crucial for studying developmental neurotoxicity and devising neuroprotective treatments. We used hiPSC-derived long-term self-renewing neuroepithelial stem (lt-NES) cells differentiated for up to 12 weeks as an in vitro model of human neural development. Following a transcriptome analysis to identify pathway alterations, we induced acute oxidative stress (OS) using tert-butyl hydroperoxide (TBHP) and assessed cell viability at different stages of neural differentiation. We studied NRF2 activation, autophagy, and proteasomal function to explore the contribution and interplay of these pathways in the acute stress response. With increasing differentiation, lt-NES cells showed changes in the expression of metabolic pathway-associated genes with engagement of the pentose phosphate pathway after 6 weeks, this was accompanied by a decreased susceptibility to TBHP-induced stress. Microarray analysis revealed upregulation of target genes of the antioxidant response KEAP1–NRF2–ARE pathway after 6 weeks of differentiation. Pharmacological inhibition of NRF2 confirmed its vital role in the increased resistance to stress. While autophagy was upregulated alongside differentiation, it was not further increased upon oxidative stress and had no effect on stress-induced cell loss and the activation of NRF2 downstream genes. In contrast, proteasome inhibition led to the aggravation of the stress response resulting in decreased cell viability, derangement of NRF2 and KEAP1 protein levels, and lacking NRF2-pathway activation. Our data provide detailed insight into the dynamic regulation and interaction of pathways involved in modulating stress responses across defined time points of neural differentiation.

Evaluation of atrazine neurodevelopment toxicity in vitro-application of hESC-based neural differentiation model

Atrazine is one of the widely used herbicides in the world and most of the current researches on atrazine neurodevelopment toxicity have focused on rodents or zebrafish models in vivo, resulting in relatively high cost, time consumption, and lower translational value to identify its hazard for the developing brain. Major international initiatives have pushed forward to convert the traditional animal-based developmental toxicity tests to in vitro assays using human cells to detect and predict chemical health hazards. In this study, we presented a human neural differentiation model based on human embryonic stem cells (hESC) that can be used to test toxicity at different stages of neural differentiation in vitro. hESC were differentiated into neural stem cells (NSC) and then terminally differentiated towards mixed neurons and glial cells for 21 days. Cell survival, proliferation, cell cycle, apoptosis, and gene expression levels were examined. Our results demonstrated that atrazine inhibited the proliferation of hESC and NSC, and showed different toxic sensitivity on these two kinds of cells. Also, atrazine blocked the NSC cell cycle G1 phase via down-regulating CCND1, CDK2, and CDK4, with no obvious effect on apoptosis. In addition, atrazine curbed EB spontaneous differentiation and NSC-induced neurons and glia cells differentiation. Atrazine altered genes expression levels of PAX6, TUBB3, NCAM1, GFAP, TH, NR4A1, and GRIA1. From the data we obtained, we recognized that the dopaminergic system was not the only target of atrazine neurotoxicity, glutamatergic neurons and astrocytes were also adversely affected.

Nestin Expression as a Diagnostic and Prognostic Marker in Colorectal Cancer and Other Tumors.

Lung cancer, colon cancer, breast cancer, and prostate cancer are the leading causes of death in developed countries. Many cancers display non-specific signs in the early stage of the disease, thus making early diagnosis often difficult. We focused on nestin as a new biomarker of possible clinical importance in the early diagnosis and monitoring of cancer. The expression of nestin takes place at an early stage of neural differentiation, but no expression of the nestin gene can be revealed in normal, mature adult tissues. Nestin plays an important role in the development of the central nervous system and contributes to the organization and maintenance of cell shape. Nestin was found to be a marker of microvessel density, which in turn has proven to be a reliable prognostic factor for neoplastic malignancies in patients. Nestin expression correlates with an increased aggressiveness of tumor cells. The role of nestin in cancers of the colon and rectum, liver, central nervous system, lung cancer, breast cancer, melanoma, and other cancers has been reviewed in the literature. Associations between nestin expression and prognosis or drug-resistance may help in disease management. More research is needed to understand the molecular mechanisms of nestin expression and its role in possible targeted therapy.

Origin of the Induced Pluripotent Stem Cells Affects Their Differentiation into Dopaminergic Neurons.

Neuronal differentiation of human induced pluripotent stem (iPS) cells, both in 2D models and 3D systems in vitro, allows for the study of disease pathomechanisms and the development of novel therapies. To verify if the origin of donor cells used for reprogramming to iPS cells can influence the differentiation abilities of iPS cells, peripheral blood mononuclear cells (PBMC) and keratinocytes were reprogrammed to iPS cells using the Sendai viral vector and were subsequently checked for pluripotency markers and the ability to form teratomas in vivo. Then, iPS cells were differentiated into dopaminergic neurons in 2D and 3D cultures. Both PBMC and keratinocyte-derived iPS cells were similarly reprogrammed to iPS cells, but they displayed differences in gene expression profiles and in teratoma compositions in vivo. During 3D organoid formation, the origin of iPS cells affected the levels of FOXA2 and LMX1A only in the first stages of neural differentiation, whereas in the 2D model, differences were detected at the levels of both early and late neural markers FOXA2, LMX1A, NURR1, TUBB and TH. To conclude, the origin of iPS cells may significantly affect iPS differentiation abilities in teratomas, as well as exerting effects on 2D differentiation into dopaminergic neurons and the early stages of 3D midbrain organoid formation.

Transcriptome Analysis of iPSC-Derived Neurons from Rubinstein-Taybi Patients Reveals Deficits in Neuronal Differentiation

Rubinstein-Taybi syndrome (RSTS) is a rare multisystem developmental disorder with moderate to severe intellectual disability caused by heterozygous mutations of either CREBBP or EP300 genes encoding CBP/p300 chromatin regulators. We explored the gene programs and processes underlying the morphological and functional alterations shown by iPSC-derived neurons modeling RSTS to bridge the molecular changes resulting from defective CBP/p300 to cognitive impairment. By global transcriptome analysis, we compared the differentially expressed genes (DEGs) marking the transition from iPSC-derived neural progenitors to cortical neurons (iNeurons) of five RSTS patients carrying private CREBBP/EP300 mutations and manifesting differently graded neurocognitive signs with those of four healthy controls. Our data shows a defective and altered neuroprogenitor to neuron transcriptional program in the cells from RSTS patients. First, transcriptional regulation is weaker in RSTS as less genes than in controls are modulated, including genes of key processes of mature functional neurons, such as those for voltage-gated channels and neurotransmitters and their receptors. Second, regulation is subverted as genes acting at pre-terminal stages of neural differentiation in cell polarity and adhesive functions (members of the cadherin family) and axon extension/guidance (members of the semaphorins and SLIT receptors families) are improperly upregulated. Impairment or delay of RSTS neuronal differentiation program is also evidenced by decreased modulation of the overall number of neural differentiation markers, significantly impacting the initial and final stages of the differentiation cascade. Last, extensive downregulation of genes for RNA/DNA metabolic processes confirms that RSTS is a global transcription disorder, consistent with a syndrome driven by chromatin dysregulation. Interestingly, the morphological and functional alterations we have previously appointed as biomarkers of RSTS iNeurons provide functional support to the herein designed transcriptome profile pointing to key dysregulated neuronal genes as main contributors to patients’ cognitive deficit. The impact of RSTS transcriptome may go beyond RSTS as comparison of dysregulated genes across modeled neurodevelopmental disorders could unveil convergent genes of cognitive impairment.

Reference Gene Validation via RT\u2013qPCR for Human iPSC-Derived Neural Stem Cells and Neural Progenitors

Correct selection of the reference gene(s) is the most important step in gene expression analysis. The aims of this study were to identify and evaluate the panel of possible reference genes in neural stem cells (NSC), early neural progenitors (eNP) and neural progenitors (NP) obtained from human-induced pluripotent stem cells (hiPSC). The stability of expression of genes commonly used as the reference in cells during neural differentiation is variable and does not meet the criteria for reference genes. In the present work, we evaluated the stability of expression of 16 candidate reference genes using the four most popular algorithms: the ΔCt method, BestKeeper, geNorm and NormFinder. All data were analysed using the online tool RefFinder to obtain a comprehensive ranking. Our results indicate that NormFinder is the best tool for reference gene selection in early stages of hiPSC neural differentiation. None of the 16 tested genes is suitable as reference gene for all three stages of development. We recommend using different genes (panel of genes) to normalise RT–qPCR data for each of the neural differentiation stages.

Precisely controlling endogenous protein dosage in hPSCs and derivatives to model FOXG1 syndrome

Dosage of key regulators impinge on developmental disorders such as FOXG1 syndrome. Since neither knock-out nor knock-down strategy assures flexible and precise protein abundance control, to study hypomorphic or haploinsufficiency expression remains challenging. We develop a system in human pluripotent stem cells (hPSCs) using CRISPR/Cas9 and SMASh technology, with which we can target endogenous proteins for precise dosage control in hPSCs and at multiple stages of neural differentiation. We also reveal FOXG1 dose-dependently affect the cellular constitution of human brain, with 60% mildly affect GABAergic interneuron development while 30% thresholds the production of MGE derived neurons. Abnormal interneuron differentiation accounts for various neurological defects such as epilepsy or seizures, which stimulates future innovative cures of FOXG1 syndrome. By means of its robustness and easiness, dosage-control of proteins in hPSCs and their derivatives will update the understanding and treatment of additional diseases caused by abnormal protein dosage.

The collagen scaffold supports hiPSC-derived NSC growth and restricts hiPSC.

The human induced pluripotent stem cells (hiPSC) are one of the promising candidates as patient specific cell source for autologous transplantation or modeling of diseases. The collagen (Col) scaffolds have been shown suitable to create in vitro biomimetic microenvironment for human neural stem cells, but their ability to accommodate stem cells at different stages of neural differentiation has not been verified yet. In this paper we compare lineage related hiPSC during neural differentiation for their ability to colonize Col scaffold. We have also focused on modification of collagen physicochemical properties with improved mechanical and thermal stability, without loss of its biological activity. The hiPSC expressing markers of pluripotency (OCT4, SOX2, NANOG) after neural commitment are NESTIN, GFAP, PDGFR alpha, beta- TUBULIN III, MAP-2, DCX, GalC positive. We have shown, that Col scaffold was not preferable for hiPSC culture, while the neurally committed population after seeding on Col scaffolds revealed good adhesion, viability, proliferation, along with sustaining markers of neuronal and glial differentiation. The Col scaffold-based 3D culture of hiPSC-NSCs may serve as a research tool for further translational studies.

Single-cell RNA-seq reveals dynamic transcriptome profiling in human early neural differentiation.

BackgroundInvestigating cell fate decision and subpopulation specification in the context of the neural lineage is fundamental to understanding neurogenesis and neurodegenerative diseases. The differentiation process of neural-tube-like rosettes in vitro is representative of neural tube structures, which are composed of radially organized, columnar epithelial cells and give rise to functional neural cells. However, the underlying regulatory network of cell fate commitment during early neural differentiation remains elusive.ResultsIn this study, we investigated the genome-wide transcriptome profile of single cells from six consecutive reprogramming and neural differentiation time points and identified cellular subpopulations present at each differentiation stage. Based on the inferred reconstructed trajectory and the characteristics of subpopulations contributing the most toward commitment to the central nervous system lineage at each stage during differentiation, we identified putative novel transcription factors in regulating neural differentiation. In addition, we dissected the dynamics of chromatin accessibility at the neural differentiation stages and revealed active cis-regulatory elements for transcription factors known to have a key role in neural differentiation as well as for those that we suggest are also involved. Further, communication network analysis demonstrated that cellular interactions most frequently occurred in the embryoid body stage and that each cell subpopulation possessed a distinctive spectrum of ligands and receptors associated with neural differentiation that could reflect the identity of each subpopulation.ConclusionsOur study provides a comprehensive and integrative study of the transcriptomics and epigenetics of human early neural differentiation, which paves the way for a deeper understanding of the regulatory mechanisms driving the differentiation of the neural lineage.

Insufficient Apaf-1 expression in early stages of neural differentiation of human embryonic stem cells might protect them from apoptosis

Recent evidence suggests that mitochondrial apoptosis regulators and executioners may regulate differentiation, without being involved in cell death. However, the involved factors and their roles in differentiation and apoptosis are still not fully determined. In the present study, we compared mitochondrial pathway of cell death during early neural differentiation from human embryonic stem cells (hESCs). Our results demonstrated that ROS generation, cytosolic cytochrome c release, caspases activation and rise in p53 protein level occurred upon either neural or apoptosis induction in hESCs. However, unlike apoptosis, no remarkable increase in apoptotic protease activating factor-1 (Apaf-1) level at early stages of differentiation was observed. Also the caspase-like activity of caspase-9 and caspase-3/7 were seen less than apoptosis. The results suggest that low levels of Apaf-1 as an adaptor protein might be considered as a possible regulatory barrier by which differentiating cells control cell death upon rise in ROS production and cytochrome c release from mitochondria. Better understanding of mechanisms via which mitochondria-mediated apoptotic pathway promote neural differentiation can result in development of novel therapeutic approaches.

VPA Inhibits P19 Neural Differentiation through Redox Dysregulation and Oxidative Stress

Valproic acid (VPA), a common anti-epileptic drug and migraine reducer, causes deficiencies in developing nervous systems and can be manifested as neural tube defects (NTDs). The appearance of these defects is believed to be due to the disruption of the redox state of differentiating cells. P19 cells are embryo carcinoma cells and are an established model for neurogenesis. Preliminary work in P19 cells shows that during neural differentiation, the glutathione (GSH) redox potential (Eh) shifts from a reducing state to a more oxidizing one. The effects of VPA exposure during distinct stages of neural differentiation are unknown. The purpose of this study was to determine if specific states of neural differentiation are more susceptible to redox disruption. In undifferentiated cells, GSH Eh were much more easily disrupted with VPA treatment than differentiated cells, suggesting earlier periods of development are more susceptible to redox dysregulation. Cellular proteins in undifferentiated P19 cells also demonstrate a greater degree of redox dysregulation, where VPA altered protein redox states to a greater extent in undifferentiated cells. Furthermore, P19 cells treated early in pro-differentiation conditions with VPA decreased the expression of neurogenic terminal differentiation markers (B III tubulin) compared to control, untreated cultures. Interestingly, pretreatment with D3T, a Nrf2 inducer, prior to VPA exposure, diminished GSH Eh oxidative shifts, increased differentiation and reduced alterations to protein redox states. Together, these data suggest that VPA-induced oxidative stress is a critical factor regulating P19 neurogenesis and may serve as an important mediator in VPA-induced NTDs. Clearly, VPA-induced oxidative stress in the embryo elicits further study.

Mir-29b Mediates the Neural Tube versus Neural Crest Fate Decision during Embryonic Stem Cell Neural Differentiation.

SummaryDuring gastrulation, the neuroectoderm cells form the neural tube and neural crest. The nervous system contains significantly more microRNAs than other tissues, but the role of microRNAs in controlling the differentiation of neuroectodermal cells into neural tube epithelial (NTE) cells and neural crest cells (NCCs) remains unknown. Using embryonic stem cell (ESC) neural differentiation systems, we found that miR-29b was upregulated in NTE cells and downregulated in NCCs. MiR-29b promoted the differentiation of ESCs into NTE cells and inhibited their differentiation into NCCs. Accordingly, the inhibition of miR-29b significantly inhibited the differentiation of NTE cells. A mechanistic study revealed that miR-29b targets DNA methyltransferase 3a (Dnmt3a) to regulate neural differentiation. Moreover, miR-29b mediated the function of Pou3f1, a critical neural transcription factor. Therefore, our study showed that the Pou3f1-miR-29b-Dnmt3a regulatory axis was active at the initial stage of neural differentiation and regulated the determination of cell fate.

Mitochondrial biogenesis and neural differentiation of human iPSC is modulated by idebenone in a developmental stage-dependent manner

Idebenone, the synthetic analog of coenzyme Q10 can improve electron transport in mitochondria. Therefore, it is used in the treatment of Alzheimer’s disease and other cognitive impairments. However, the mechanism of its action on neurodevelopment is still to be elucidated. Here we demonstrate that the cellular response of human induced pluripotent stem cells (hiPSC) to idebenone depends on the stage of neural differentiation. When: neural stem cells (NSC), early neural progenitors (eNP) and advanced neural progenitors (NP) have been studied a significant stimulation of mitochondrial biogenesis was observed only at the eNP stage of development. This coexists with the enhancement of cell viability and increase in total cell number. In addition, we report novel idebenone properties in a possible regulation of neural stem cells fate decision: only eNP stage responded with up-regulation of both neuronal (MAP2), astrocytic (GFAP) markers, while at NSC and NP stages significant down-regulation of MAP2 expression was observed, promoting astrocyte differentiation. Thus, idebenone targets specific stages of hiPSC differentiation and may influence the neural stem cell fate decision.

Sensitivity of hiPSC-derived neural stem cells (NSC) to Pyrroloquinoline quinone depends on their developmental stage

Pyrroloquinoline quinone (PQQ) is a factor influencing on the mitochondrial biogenesis. In this study the PQQ effect on viability, total cell number, antioxidant capacity, mitochondrial biogenesis and differentiation potential was investigated in human induced Pluripotent Stem Cells (iPSC) - derived: neural stem cells (NSC), early neural progenitors (eNP) and neural progenitors (NP). Here we demonstrated that sensitivity to PQQ is dependent upon its dose and neural stage of development. Induction of the mitochondrial biogenesis by PQQ at three stages of neural differentiation was evaluated at mtDNA, mRNA and protein level. Changes in NRF1, TFAM and PPARGC1A gene expression were observed at all developmental stages, but only at eNP were correlated with the statistically significant increase in the mtDNA copy numbers and enhancement of SDHA, COX-1 protein level. Thus, the “developmental window” of eNP for PQQ-evoked mitochondrial biogenesis is proposed. This effect was independent of high antioxidant capacity of PQQ, which was confirmed in all tested cell populations, regardless of the stage of hiPSC neural differentiation. Furthermore, a strong induction of GFAP, with down regulation of MAP2 gene expression upon PQQ treatment was observed. This indicates a possibility of shifting the balance of cell differentiation in the favor of astroglia, but more research is needed at this point.

Correlation between receptor-interacting protein 140 expression and directed differentiation of human embryonic stem cells into neural stem cells.

Overexpression of receptor-interacting protein 140 (RIP140) promotes neuronal differentiation of N2a cells via extracellular regulated kinase 1/2 (ERK1/2) signaling. However, involvement of RIP140 in human neural differentiation remains unclear. We found both RIP140 and ERK1/2 expression increased during neural differentiation of H1 human embryonic stem cells. Moreover, RIP140 negatively correlated with stem cell markers Oct4 and Sox2 during early stages of neural differentiation, and positively correlated with the neural stem cell marker Nestin during later stages. Thus, ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural differentiation to eventually affect the number of neurons produced.

Identification of β-Dystrobrevin as a Direct Target of miR-143: Involvement in Early Stages of Neural Differentiation.

Duchenne Muscular Dystrophy, a genetic disorder that results in a gradual breakdown of muscle, is associated to mild to severe cognitive impairment in about one-third of dystrophic patients. The brain dysfunction is independent of the muscular pathology, occurs early, and is most likely due to defects in the assembly of the Dystrophin-associated Protein Complex (DPC) during embryogenesis. We have recently described the interaction of the DPC component β-dystrobrevin with members of complexes that regulate chromatin dynamics, and suggested that β-dystrobrevin may play a role in the initiation of neuronal differentiation. Since oxygen concentrations and miRNAs appear as well to be involved in the cellular processes related to neuronal development, we have studied how these factors act on β-dystrobrevin and investigated the possibility of their functional interplay using the NTera-2 cell line, a well-established model for studying neurogenesis. We followed the pattern of expression and regulation of β-dystrobrevin during the early stages of neuronal differentiation induced by exposure to retinoic acid (RA) under hypoxia as compared with normoxia, and found that β-dystrobrevin expression is regulated during RA-induced differentiation of NTera-2 cells. We also found that β-dystrobrevin pattern is delayed under hypoxic conditions, together with a delay in the differentiation and an increase in the proliferation rate of cells. We identified miRNA-143 as a direct regulator of β-dystrobrevin expression, demonstrated that β-dystrobrevin is expressed in the nucleus and showed that, in line with our previous in vitro results, β-dystrobrevin is a repressor of synapsin I in live cells. Altogether the newly identified regulatory pathway miR-143/β-dystrobrevin/synapsin I provides novel insights into the functions of β-dystrobrevin and opens up new perspectives for elucidating the molecular mechanisms underlying the neuronal involvement in muscular dystrophy.

MiR-744 and miR-224 Downregulate Npas4 and Affect Lineage Differentiation Potential and Neurite Development During Neural Differentiation of Mouse Embryonic Stem Cells.

Neuronal PAS domain protein 4 (Npas4) is a brain-specific transcription factor whose expression is enriched in neurogenic regions of the brain. In addition, it was demonstrated that Npas4 expression is dynamic and highly regulated during neural differentiation of embryonic stem cells (ESCs). While these findings implicate a role for Npas4 in neurogenesis, the underlying mechanisms of regulation remain unknown. Given that growing evidence suggests that microRNAs (miRNAs) play important roles in both embryonic and adult neurogenesis, we reasoned that miRNAs are good candidates for regulating Npas4 expression during neural differentiation of ESCs. In this study, we utilized the small RNA sequencing method to profile miRNA expression during neural differentiation of mouse ESCs. Two differentially expressed miRNAs were identified to be able to significantly reduce reporter gene activity by targeting the Npas4 3'UTR, namely miR-744 and miR-224. More importantly, ectopic expression of these miRNAs during neural differentiation resulted in downregulation of endogenous Npas4 expression. Subsequent functional analysis revealed that overexpression of either miR-744 or miR-224 delayed early neural differentiation, reduced GABAergic neuron production and inhibited neurite outgrowth. Collectively, our findings indicate that Npas4 not only functions at the early stages of neural differentiation but may also, in part, contribute to neuronal subtype specification and neurite development.

Involvement of NFκB signaling in mediating the effects of GRK5 on neural stem cells

Nuclear factor κB (NFκB) signaling plays ubiquitous roles in inflammation, immune response and neurogenesis. G protein-coupled receptor kinase 5 (GRK5) can protect neurons from degeneration. GRK5 also mediates tumor necrosis factor-α (TNFα)-induced NFκB signaling through the phosphorylation of IκBα. Here, we show that NFκB signaling is involved in neural stem cell (NSC) differentiation. The IκBα/p65 pathway was activated by phorbol myristate acetate (PMA), a stimulator of protein kinase C (PKC). Once the NFκB was activated, the initial stage of neural differentiation was induced, with an increased level of GRK5 in NSCs. This finding was reversed in response to the NFκB inhibitor N-acetyl cysteine (NAC). To evaluate the effect of GRK5-NFκB signaling crosstalk on NSC neurogenesis and apoptosis, GRK5 was knocked down by siRNAs in cell culture. SiRNAs against GRK5 not only impaired neural differentiation and axogenesis, but also induced apoptosis of NSC. GRK5 knockdown affected the transcription of NFκB, phosphorylation of the liver kinase B1 (LKB1) and the activity of caspase 3, thereby modulated neurogenesis and apoptosis. Taken together, our findings reveal a novel function of GRK5 in neurogenesis and provide insight into the molecular mechanisms underlying neurodevelopmental disorders and neurodegenerative diseases.