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Developmental and Epileptic Encephalopathies (DEE) are a genetically diverse group of severe, early onset seizure disorders. DEE are normally identified clinically in the first six months of life by the presence of frequent, difficult to control seizures and accompanying stalling or regression of development. DEE75 results from de novo mutations of the NEUROD2 gene that result in loss of activity of the encoded transcription factor, and the seizure phenotype was shown to be recapitulated in Xenopus tropicalis tadpoles. We used CRISPR/Cas9 to make a DEE75 model in Xenopus laevis , to further investigate the developmental etiology. NeuroD2.S CRISPR/Cas9 edited tadpoles were more active, swam faster on average, and had more seizures (C-shaped contractions resembling unprovoked C-start escape responses) than their sibling controls. Live imaging of Ca2+ signaling revealed prolongued, strong signals sweeping through the brain, indicative of neuronal hyperactivity. While the resulting tadpole brain appeared grossly normal, the blood-brain barrier (BBB) was found to be leakier than that of controls. Additionally, the TGFβ antagonist Losartan was shown to have a short-term protective effect, reducing neuronal hyperactivity and reducing permeability of the BBB. Treatment of NeuroD2 CRISPant tadpoles with 5 mM Losartan decreased seizure events by more than 4-fold compared to the baseline. Our results support a model of DEE75 resulting from reduced NeuroD2 activity during vertebrate brain development, and indicate that a leaky BBB contributes to epileptogenesis. [ABSTRACT FROM AUTHOR]

We recently identified PKN1 as a developmentally active gatekeeper of the transcription factor neuronal differentiation-2 (NeuroD2) in several brain areas. Since NeuroD2 plays an important role in amacrine cell (AC) and retinal ganglion cell (RGC) type formation, we aimed to study the expression of NeuroD2 in the postnatal retina of WT and Pkn1−/− animals, with a particular focus on these two cell types. We show that PKN1 is broadly expressed in the retina and that the gross retinal structure is not different between both genotypes. Postnatal retinal NeuroD2 levels were elevated upon Pkn1 knockout, with Pkn1−/− retinae showing more NeuroD2+ cells in the lower portion of the inner nuclear layer. Accordingly, immunohistochemical analysis revealed an increased amount of AC in postnatal and adult Pkn1−/− retinae. There were no differences in horizontal cell, bipolar cell, glial cell and RGC numbers, nor defective axon guidance to the optic chiasm or tract upon Pkn1 knockout. Interestingly, we did, however, see a specific reduction in SMI-32+ α-RGC in Pkn1−/− retinae. These results suggest that PKN1 is important for retinal cell type formation and validate PKN1 for future studies focusing on AC and α-RGC specification and development. [ABSTRACT FROM AUTHOR]

De novo heterozygous variants in the brain-specific transcription factor Neuronal Differentiation Factor 2 (NEUROD2) have been recently associated with early-onset epileptic encephalopathy and developmental delay. Here, we report an adolescent with developmental delay without seizures who was found to have a novel de novo heterozygous NEUROD2 missense variant, p.(Leu163Pro). Functional testing using an in vivo assay of neuronal differentiation in Xenopus laevis tadpoles demonstrated that the patient variant of NEUROD2 displays minimal protein activity, strongly suggesting a loss of function effect. In contrast, a second rare NEUROD2 variant, p.(Ala235Thr), identified in an adolescent with developmental delay but lacking parental studies for inheritance, showed normal in vivo NEUROD2 activity. We thus provide clinical, genetic, and functional evidence that NEUROD2 variants can lead to developmental delay without accompanying early-onset seizures, and demonstrate how functional testing can complement genetic data when determining variant pathogenicity.

Introduction: The molecular programs regulating human pancreatic endocrine cell induction and fate allocation are not well deciphered. Here, we investigated the spatiotemporal expression pattern and the function of the neurogenic differentiation factor 2 (NEUROD2) during human endocrinogenesis. Methods: Using Crispr-Cas9 gene editing, we generated a reporter knock-in transcription factor (TF) knock-out human inducible pluripotent stem cell (iPSC) line in which the open reading frame of both NEUROD2 alleles are replaced by a nuclear histone 2B-Venus reporter (NEUROD2nVenus/nVenus). Results: We identified a transient expression of NEUROD2 mRNA and its nuclear Venus reporter activity at the stage of human endocrine progenitor formation in an iPSC differentiation model. This expression profile is similar to what was previously reported in mice, uncovering an evolutionarily conserved gene expression pattern of NEUROD2 during endocrinogenesis. In vitro differentiation of the generated homozygous NEUROD2nVenus/nVenus iPSC line towards human endocrine lineages uncovered no significant impact upon the loss of NEUROD2 on endocrine cell induction. Moreover, analysis of endocrine cell specification revealed no striking changes in the generation of insulin-producing b cells and glucagon-secreting a cells upon lack of NEUROD2. Discussion: Overall, our results suggest that NEUROD2 is expendable for human b cell formation in vitro. [ABSTRACT FROM AUTHOR]

IntroductionThe molecular programs regulating human pancreatic endocrine cell induction and fate allocation are not well deciphered. Here, we investigated the spatiotemporal expression pattern and the function of the neurogenic differentiation factor 2 (NEUROD2) during human endocrinogenesis.MethodsUsing Crispr-Cas9 gene editing, we generated a reporter knock-in transcription factor (TF) knock-out human inducible pluripotent stem cell (iPSC) line in which the open reading frame of both NEUROD2 alleles are replaced by a nuclear histone 2B-Venus reporter (NEUROD2nVenus/nVenus).ResultsWe identified a transient expression of NEUROD2 mRNA and its nuclear Venus reporter activity at the stage of human endocrine progenitor formation in an iPSC differentiation model. This expression profile is similar to what was previously reported in mice, uncovering an evolutionarily conserved gene expression pattern of NEUROD2 during endocrinogenesis. In vitro differentiation of the generated homozygous NEUROD2nVenus/nVenus iPSC line towards human endocrine lineages uncovered no significant impact upon the loss of NEUROD2 on endocrine cell induction. Moreover, analysis of endocrine cell specification revealed no striking changes in the generation of insulin-producing b cells and glucagon-secreting a cells upon lack of NEUROD2.DiscussionOverall, our results suggest that NEUROD2 is expendable for human b cell formation in vitro.

Abstract Background Epileptic spasms are a devastating form of early infantile epileptic encephalopathy (EIEE) with various etiologies. Early diagnosis and a shorter lead time to treatment are crucial to stop the seizures and optimize the neurodevelopmental outcome. Genetic testing has become an integral part of epilepsy care that directly guides management and family planning and discovers new targeted treatments. Neuronal differentiation Factor 2 (NEUROD2) variants have recently been a cause of neurodevelopmental disorders (NDDs) and EIEEs with distinctive features. However, there is limited information about the clinical and electroencephalographic response of epileptic spasm treatment in NEUROD2-related NDD syndrome. Case presentation We report a female patient of Southeast Asian ethnicity with global developmental delay and epileptic spasms commencing in the first few months of life. A novel de novo heterozygous pathogenic NEUROD2 variant, p. E130Q, was subsequently identified by whole-exome sequencing. Electroencephalogram before treatment showed multifocal independent spikes predominantly in both posterior head regions and demonstrated marked improvement following combined vigabatrin and high-dose prednisolone treatment. However, multiple courses of relapse occurred after weaning off the antiseizure medication. Conclusions We propose that epileptic spasms related to de novo NEUROD2 pathogenic variant respond well to combined vigabatrin and high-dose prednisolone therapy. These findings may imply the benefit of using combination therapy to treat epileptic spasms in NEUROD2-related NDD syndrome.

NEUROD2 , encoding the neurogenic differentiation factor 2, is essential for neurodevelopment. To date, heterozygous missense variants in this gene have been identified in eight patients (from six unrelated families) with epileptic encephalopathy and developmental delay. We describe a child with initial clinical suspicion of Rett/Rett-like syndrome, in whom exome sequencing detected a novel de novo variant (c.388G > A, p.Glu130Lys) in NEUROD2. Interestingly, a missense change affecting the same codon, c.388G > C (p.Glu130Gln), was previously identified in other two patients. Our results suggest that Glu130 might represent a potential mutational hotspot of NEUROD2. Furthermore, the clinical findings (especially the absence of clinically overt seizures) strengthen the NEUROD2- phenotypic spectrum, implying that developmental delay may also manifest isolatedly. We suggest inclusion of NEUROD2 -associated developmental and epileptic encephalopathies (DEEs) in the differential diagnosis of atypical Rett syndrome as well as gene panels related to autism spectrum disorder. [ABSTRACT FROM AUTHOR]

Aim: NeuroD2 transcription factor is a key regulator of neurogenin-NeuroD signaling network and induces neuronal development, differentiation, neurogenesis and calcium dependent signaling. NeuroD2 regulates expression of survival and plasticity related proteins in neurons. Surprisingly, inhibition of NeuroD2 causes an increase in apoptotic cell death. Even though previous studies found out important data about NeuroD2 function, molecular interactions of NeuroD2 behind all of these impacts remains elusive. For this reason, it was aimed to shed light on the proteome profile of NeuroD2 based changes in the N2A cell line. Materials and Methods: NeuroD2 over-expression and NeuroD2 inhibition groups were constructed via lentiviral vectors. Mouse N2A cell line was transfected with the given vectors and incubated for 6 hours. After incubation samples were prepared for proteomic analyses with Filter Aided Sample Preparation (FASP) protocol and LC-MS/MS analysis was carried out. Results: Under conditions of overexpression and inhibition, detected proteins were filtered according to significant cut off values. The filtered proteins were further investigated to exhibit a coherent expression in each situation. Eventually, increased NeuroD2 activity was accompanied by an increase in N-alpha-acetyltransferase 25 (NAA25), and Synaptobrevin homolog (YKT6). On the other hand, when NeuroD2 was suppressed, expression of Cytoplasmic Dynein 1 Light Intermediate Chain 1, Kinesin-Like Protein (KIF-11), Leucine-tRNA Ligase (LARS1), and Ubiquitin-Associated Protein 2 (UBA2) were found to be upregulated with a reverse action. Conclusion: Up-regulations of the proteins Cytoplasmic Dynein 1, KIF11, LARS1, and UBA2 suggested that these proteins might be controlled by inhibition of NeuroD2. In this contex it can be said that, axonal transport, neuronal signaling, and activity of PI3K/AKT pathway can be indirectly regulated by NeuroD2. [ABSTRACT FROM AUTHOR]

Background: Epileptic spasms are a devastating form of early infantile epileptic encephalopathy (EIEE) with various etiologies. Early diagnosis and a shorter lead time to treatment are crucial to stop the seizures and optimize the neurodevelopmental outcome. Genetic testing has become an integral part of epilepsy care that directly guides management and family planning and discovers new targeted treatments. Neuronal differentiation Factor 2 (NEUROD2) variants have recently been a cause of neurodevelopmental disorders (NDDs) and EIEEs with distinctive features. However, there is limited information about the clinical and electroencephalographic response of epileptic spasm treatment in NEUROD2-related NDD syndrome. Case presentation: We report a female patient of Southeast Asian ethnicity with global developmental delay and epileptic spasms commencing in the first few months of life. A novel de novo heterozygous pathogenic NEUROD2 variant, p. E130Q, was subsequently identified by whole-exome sequencing. Electroencephalogram before treatment showed multifocal independent spikes predominantly in both posterior head regions and demonstrated marked improvement following combined vigabatrin and high-dose prednisolone treatment. However, multiple courses of relapse occurred after weaning off the antiseizure medication. Conclusions: We propose that epileptic spasms related to de novo NEUROD2 pathogenic variant respond well to combined vigabatrin and high-dose prednisolone therapy. These findings may imply the benefit of using combination therapy to treat epileptic spasms in NEUROD2-related NDD syndrome. [ABSTRACT FROM AUTHOR]

Yongfeng Pang, Mingfei Shi Department of Rehabilitation, Tiantai People’s Hospital of Zhejiang Province, Zhengjiang, 317200, People’s Republic of ChinaCorrespondence: Mingfei ShiDepartment of Rehabilitation, Tiantai People’s Hospital of Zhejiang Province, No. 1 Kangning Middle Road, Tiantai County, Zhengjiang, 317200, People’s Republic of ChinaTel +86-13516877456Email ming_fly141@yeah.netBackground: Mild cognitive impairment (MCI) is a typical symptom of early Alzheimer’s disease (AD) and is driven by the dysfunction of microRNAs (miRs). Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive technique for handling neuropsychiatric disorders and has universally effects on the functions of miRs. In the current study, the improvement effects of rTMS on MCI associated with AD were explored by focusing on miR-567/NEUROD2/PSD95 axis.Methods: MCI was induced in mice using scopolamine and was treated with rTMS of two frequencies (1 Hz and 10 Hz). The changes in cognitive function, brain structure, neurotrophic factor levels, and activity of miR-567/NEUROD2/PSD95 axis were assessed. The interaction between rTMS and miR-567 was further verified by inducing the level of miR-567 in AD mice.Results: The administrations of rTMS improved the cognitive function of AD mice and attenuated brain tissue destruction, which were associated with the restored production of BDNF and NGF. Additionally, rTMS administrations suppressed the expression of miR-567 and up-regulated the expressions of NEUROD2 and PSD95, which contributed to the improved condition in central nerve system. With the induced level of miR-567, the effects of rTMS were counteracted: the learning and memorizing abilities of mice were impaired, the brain neuron viability was suppressed, and the production of neurotrophic factors was suppressed even under the administration of rTMS. The changes in brain function and tissues were associated with the inhibited expressions of NEUROD2 and PSD95.Conclusion: The findings outlined in the current study demonstrated that rTMS treatment could protect brain against AD-induced MCI without significant side effects, and the function depended on the inhibition of miR-567.Keywords: Alzheimer’s disease, mild cognitive impairment, miR-567, NEUROD2, repetitive transcranial magnetic stimulation

Alterations in the processes that control α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) expression, assembly and trafficking are closely linked to psychiatric and neurodegenerative disorders. We have recently shown that the serine/threonine kinase Protein kinase N1 (PKN1) is a developmentally active regulator of cerebellar synaptic maturation by inhibiting AKT and the neurogenic transcription factor neurogenic differentiation factor-2 (NeuroD2). NeuroD2 is involved in glutamatergic synaptic maturation by regulating expression levels of various synaptic proteins. Here we aimed to study the effect of Pkn1 knockout on AKT phosphorylation and NeuroD2 levels in the hippocampus and the subsequent expression levels of the NeuroD2 targets and AMPAR subunits: glutamate receptor 1 (GluA1) and GluA2/3. We show that PKN1 is expressed throughout the hippocampus. Interestingly, not only postnatal but also adult hippocampal phospho-AKT and NeuroD2 levels were significantly elevated upon Pkn1 knockout. Postnatal and adult Pkn1–/– hippocampi showed enhanced expression of the AMPAR subunit GluA1, particularly in area CA1. Surprisingly, GluA2/3 levels were not different between both genotypes. In addition to higher protein levels, we also found an enhanced GluA1 content in the membrane fraction of postnatal and adult Pkn1–/– animals, while GluA2/3 levels remained unchanged. This points toward a very specific regulation of GluA1 expression and/or trafficking by the novel PKN1-AKT-NeuroD2 axis. Considering the important role of GluA1 in hippocampal development as well as the pathophysiology of several disorders, ranging from Alzheimer’s, to depression and schizophrenia, our results validate PKN1 for future studies into neurological disorders related to altered AMPAR subunit expression in the hippocampus.

Orofacial pain and headache disorders are among the most debilitating pain conditions. While the pathophysiological basis of these disorders may be diverse, it is generally accepted that a common mechanism behind the arising pain is the sensitization of extra- and intracranial trigeminal primary afferents. In the present study we investigated gene expression changes in the trigeminal ganglia (TRG), trigeminal nucleus caudalis (TNC) and peripheral blood mononuclear cells (PBMC) evoked by Complete Freund's Adjuvant (CFA)-induced orofacial inflammation in rats, as a model of trigeminal sensitization. Microarray analysis revealed 512 differentially expressed genes between the ipsi- and contralateral TRG samples 7 days after CFA injection. Time-dependent expression changes of G-protein coupled receptor 39 (Gpr39), kisspeptin-1 receptor (Kiss1r), kisspeptin (Kiss1), as well as synaptic plasticity-associated Lkaaear1 (Lkr) and Neurod2 mRNA were described on the basis of qPCR results. The greatest alterations were observed on day 3 ipsilaterally, when orofacial mechanical allodynia reached its maximum. This corresponded well with patterns of neuronal (Fosb), microglia (Iba1), and astrocyte (Gfap) activation markers in both TRG and TNC, and interestingly also in PBMCs. This is the first description of up- and downregulated genes both in primary and secondary sensory neurones of the trigeminovascular system that might play important roles in neuroinflammatory activation mechanisms. We are the first to show transcriptomic alterations in the PBMCs that are similar to the neuronal changes. These results open new perspectives and initiate further investigations in the research of trigeminal pain disorders.

OBJECTIVE: The aim of this study was to explore the specific role of miR-219-5p in spinal cord injury (SCI), and to investigate its underlying mechanism. MATERIALS AND METHODS: The SCI model was first constructed in mice, and the motor function of each mouse was evaluated by the Basso Beattie Bresnahan (BBB) method. The protein and mRNA expression levels of miR-219- 5p and NEUROD2 in SCI mice were detected by Western blot and quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), respectively. Subsequently, all mice were assigned into 3 groups, including the sham operation group, the SCI group and the SCI+miR-219-5p group. The levels of inflammatory factors (TNF-α, IL-1β and IL-6) were detected by Western blot and qRTPCR. Meanwhile, reactive oxygen species (ROS) were detected by flow cytometry. Target genes of miR-219-5p were predicted by TargetScan and verified by Luciferase reporter gene assay. For in vitro experiments, the possible molecule mechanism of miR-219-5p in regulating NEUROD2 was detected by Western blot. RESULTS: MiR-219-5p was significantly downregulated after SCI. The expression level of miR- 219-5p was decreased in the SCI group than that of the sham operation group in a time-dependent manner, which reached the lowest level on the 7th day. Besides, the mRNA and protein levels of NEUROD2 in the SCI group were both remarkably increased in a time-dependent manner, which reached a peak on the 7th day. The levels of inflammatory factors (TNF-α, IL-1β and IL-6) and ROS were significantly higher in the SCI group, which could be reversed by miR-219- 5p mimics transfection in SCI mice. Meanwhile, the BBB score in the SCI group was remarkably lower than that of the SCI + miR-219-5p group from the 4th day after SCI. TargetScan predicted that NEUROD2 was the target gene of miRNA- 219-5p. In addition, Western blot results indicated that miR-219-5p could regulate NEUROD2, eventually promoting the recovery of SCI. CONCLUSIONS: Overexpressed miR-219-5p promotes SCI recovery and motor function elevation via alleviating NEUROD2-regulated inflammation and oxidative stress. [ABSTRACT FROM AUTHOR]

Orofacial pain and headache disorders are among the most debilitating pain conditions. While the pathophysiological basis of these disorders may be diverse, it is generally accepted that a common mechanism behind the arising pain is the sensitization of extra- and intracranial trigeminal primary afferents. In the present study we investigated gene expression changes in the trigeminal ganglia (TRG), trigeminal nucleus caudalis (TNC) and peripheral blood mononuclear cells (PBMC) evoked by Complete Freund's Adjuvant (CFA)-induced orofacial inflammation in rats, as a model of trigeminal sensitization. Microarray analysis revealed 512 differentially expressed genes between the ipsi- and contralateral TRG samples 7 days after CFA injection. Time-dependent expression changes of G-protein coupled receptor 39 (Gpr39), kisspeptin-1 receptor (Kiss1r), kisspeptin (Kiss1), as well as synaptic plasticity-associated Lkaaear1 (Lkr) and Neurod2 mRNA were described on the basis of qPCR results. The greatest alterations were observed on day 3 ipsilaterally, when orofacial mechanical allodynia reached its maximum. This corresponded well with patterns of neuronal (Fosb), microglia (Iba1), and astrocyte (Gfap) activation markers in both TRG and TNC, and interestingly also in PBMCs. This is the first description of up- and downregulated genes both in primary and secondary sensory neurones of the trigeminovascular system that might play important roles in neuroinflammatory activation mechanisms. We are the first to show transcriptomic alterations in the PBMCs that are similar to the neuronal changes. These results open new perspectives and initiate further investigations in the research of trigeminal pain disorders. [ABSTRACT FROM AUTHOR]

The factors involved in cell differentiation have recently garnered interest for their role in inhibition of pathogenesis in various tumors. However, their role in glioblastoma (GBM) remains poorly understood. We analyzed The Cancer Genome Atlas (TCGA) GBM data and found significant downregulation of neurogenic differentiation factor NeuroD2 in GBM patients. Low levels of NeuroD2 were found to be correlated with poor overall survival of GBM patients in TCGA dataset as well as in our cohort. Interestingly, NeuroD2 was shown to be transcriptionally induced by p53 and post‐transcriptionally targeted by hypoxia‐ inducible miRNA, miR‐210. NeuroD2 overexpression diminished GBM aggressiveness by inhibiting cell proliferation, migration and promoting apoptosis under hypoxia. NeuroD2 overexpressing GBM cells failed to form three‐dimensional (3D)‐tumor spheroids and displayed reduced migration in a 3D gelatin matrix. NeuroD2 gene signature was enriched in pathways belonging to cytokine–cytokine receptor interaction, TNF‐signaling, PI3K‐AKT signaling, focal adhesion and ECM–receptor interaction. Overall, our study identifies a novel role of NeuroD2 as a tumor suppressor and prognostic biomarker in GBM the levels of which are tightly regulated by p53 and miR‐210. Overexpressing NeuroD2 may potentially be a simple and efficient therapeutic strategy to inhibit the malignant phenotype of GBM cells. [ABSTRACT FROM AUTHOR]

Cell proliferation and differentiation are interdependent processes. Here, we have asked to what extent the two processes of neural progenitor cell amplification and differentiation are functionally separated. Thus, we analyzed whether it is possible to rescue a defect of terminal differentiation in progenitor cells of the dentate gyrus, where new neurons are generated throughout life, by inducing their proliferation and/or their differentiation with different stimuli appropriately timed. As a model we used the Tis21 knockout mouse, whose dentate gyrus neurons, as demonstrated by us and others, have an intrinsic defect of terminal differentiation. We first tested the effect of two proliferative as well as differentiative neurogenic stimuli, one pharmacological (fluoxetine), the other cognitive (the Morris water maze (MWM) training). Both effectively enhanced the number of new dentate gyrus neurons produced, and fluoxetine also reduced the S-phase length of Tis21 knockout dentate gyrus progenitor cells and increased the rate of differentiation of control cells, but neither factor enhanced the defective rate of differentiation. In contrast, the defect of terminal differentiation was fully rescued by in vivo infection of proliferating dentate gyrus progenitor cells with retroviruses either silencing Id3, an inhibitor of neural differentiation, or expressing NeuroD2, a proneural gene expressed in terminally differentiated dentate gyrus neurons. This is the first demonstration that NeuroD2 or the silencing of Id3 can activate the differentiation of dentate gyrus neurons, complementing a defect of differentiation. It also highlights how the rate of differentiation of dentate gyrus neurons is regulated genetically at several levels and that a neurogenic stimulus for amplification of neural stem/progenitor cells may not be sufficient in itself to modify this rate.

Neurogenic differentiation factor 2 (NeuroD2) is a highly expressed transcription factor in the developing central nervous system. In newborn neurons, NeuroD2-mediated gene expression promotes differentiation, maturation, and survival. In addition to these early, cell-intrinsic developmental processes, NeuroD2 in postmitotic neurons also regulates synapse growth and ion channel expression to control excitability. While NeuroD2 transactivation can be induced in an activity-dependent manner, little is known about how expression of NeuroD2 itself is regulated. Using genome-wide, mRNA-based microarray analysis, we found that NeuroD2 is actually one of hundreds of genes whose mRNA levels are suppressed by synaptic activity, in a manner dependent upon N-methyl d-aspartate receptor (NMDAR) activation. We confirmed this observation both in vitro and in vivo and provide evidence that this happens at the level of transcription and not mRNA stability. Our experiments further indicate that suppression of NeuroD2 message by NMDARs likely involves both CaMKII and MAPK but not voltage-gated calcium channels, in contrast to its mechanism of transactivation. We predict from these data that NMDARs may transduce information about the level of synaptic activity a developing neuron receives, to down-regulate NeuroD2 and allow proper maturation of cortical circuits by suppressing expression of neurite and synaptic growth promoting gene products. [ABSTRACT FROM AUTHOR]

Cover illustration: The cover image, by Fading Chen and Benjamin J. Hall, is based on the Research Article Synaptic activity suppresses expression of neurogenic differentiation factor 2 in an NMDA receptor‐dependent manner, DOI: 10.1002/syn.21986. [ABSTRACT FROM AUTHOR]

Cell proliferation and differentiation are interdependent processes. Here, we have asked to what extent the two processes of neural progenitor cell amplification and differentiation are functionally separated. Thus, we analyzed whether it is possible to rescue a defect of terminal differentiation in progenitor cells of the dentate gyrus, where new neurons are generated throughout life, by inducing their proliferation and/or their differentiation with different stimuli appropriately timed. As a model we used the Tis21 knockout mouse, whose dentate gyrus neurons, as demonstrated by us and others, have an intrinsic defect of terminal differentiation. We first tested the effect of two proliferative as well as differentiative neurogenic stimuli, one pharmacological (fluoxetine), the other cognitive (the Morris water maze (MWM) training). Both effectively enhanced the number of new dentate gyrus neurons produced, and fluoxetine also reduced the S-phase length of Tis21 knockout dentate gyrus progenitor cells and increased the rate of differentiation of control cells, but neither factor enhanced the defective rate of differentiation. In contrast, the defect of terminal differentiation was fully rescued by in vivo infection of proliferating dentate gyrus progenitor cells with retroviruses either silencing Id3, an inhibitor of neural differentiation, or expressing NeuroD2, a proneural gene expressed in terminally differentiated dentate gyrus neurons. This is the first demonstration that NeuroD2 or the silencing of Id3 can activate the differentiation of dentate gyrus neurons, complementing a defect of differentiation. It also highlights how the rate of differentiation of dentate gyrus neurons is regulated genetically at several levels and that a neurogenic stimulus for amplification of neural stem/progenitor cells may not be sufficient in itself to modify this rate. [ABSTRACT FROM AUTHOR]

Introduction:NEUROD2 is a neurospecific helix–loop–helix transcription factor which has an impact on the regulation of glutamatergic and GABAergic genes. We investigated an association of NEUROD2 with neurocognitive dysfunctions in schizophrenia and schizoaffective disorder patients before and during treatment with different second-generation antipsychotics. Methods:Patients were genotyped for four different polymorphisms of the NEUROD2 gene ((rs9889354(A/G), rs1877032(C/T), rs12453682(C/T) and rs11078918(C/G)). Cognitive function was assessed at baseline and week 8. Results of individual neuropsychological tests were assigned to six cognitive domains (reaction time and quality; executive function; working, verbal and visual memory) and a general cognitive index. Results:167 patients were included in the study. The NEUROD2 exonic polymorphism rs11078918 showed significant associations with verbal memory and executive functions, whereas the NEUROD2 polymorphism rs12453682 was significantly associated with working and verbal memory, executive functions and with a cognitive index. Significant associations were found at baseline and after eight weeks. Moreover, significant associations between the change in neuropsychological test results during antipsychotic treatment and the NEUROD2 polymorphisms rs11078918 and rs12453682 were observed. Conclusions:Our findings suggest that the NEUROD2 gene could play a role in the pathophysiology of neurocognitive dysfunctions as well as in the change of cognitive symptoms under antipsychotic treatment in schizophrenia and schizoaffective disorder. [ABSTRACT FROM PUBLISHER]

The expression of neurogenesis marker - NeuroD2 transcription factor - in the hippocampal dentate gyrus was studied in rats exposed to severe destructive hypoxia, a single or three episodes of moderate hypobaric hypoxia, preconditioned severe hypoxia, and severe hypoxia followed by 3 sessions of postconditioning by moderate hypobaric hypoxia. All the studied hypoxic exposure modes led to an increase of NeuroD2 level. Three-fold moderate hypoxia per se and in the preconditioning mode (followed by exposure to severe hypoxia) produced most pronounced up-regulatory effect on NeuroD2 expression. The results indicated that stimulation of neurogenesis processes seemed to be one of the aspects of the neuroprotective effect of three-fold preconditioning moderate hypoxia, but not of hypoxic postconditioning. [ABSTRACT FROM AUTHOR]

Background: Cellular differentiation programs are controlled, to a large extent, by the combinatorial functioning of specific transcription factors. Cortical projection neurons constitute the major excitatory neuron population within the cortex and mediate long distance communication between the cortex and other brain regions. Our understanding of effector transcription factors and their downstream transcriptional programs that direct the differentiation process of cortical projection neurons is far from complete. Results: In this study, we carried out a ChIP-Seq (chromatin-immunoprecipitation and sequencing) analysis of NEUROD2, an effector transcription factor expressed in lineages of cortical projection neurons during the peak of cortical excitatory neurogenesis. Our results suggest that during cortical development NEUROD2 targets key genes that are required for Reelin signaling, a major pathway that regulates the migration of neurons from germinal zones to their final layers of residence within the cortex. We also find that NEUROD2 binds to a large set of genes with functions in layer-specific differentiation and in axonal pathfinding of cortical projection neurons. Conclusions: Our analysis of in vivo NEUROD2 target genes offers mechanistic insight into signaling pathways that regulate neuronal migration and axon guidance and identifies genes that are likely to be required for proper cortical development. [ABSTRACT FROM AUTHOR]

Abstract: Pro-neural basic helix loop helix (bHLH) transcription factors are involved in many aspects of normal neuronal development, and over-expression of genes for several of these factors has been shown to induce aspects of neuronal differentiation in cell lines and stem cells. Here we show that over-expression of NeuroD2 (ND2), Neurogenin1 and 2 leads to morphological differentiation of N18-RE-105 neuroblastoma cells and increased expression of synaptic proteins. Particularly ND2 induced neurite formation and increases in the expression of synaptic proteins such as synaptotagmin, that is not expressed normally in this cell type, as well as the redistribution of another synaptic protein, SNAP25, to a cell membrane location. Infection of human neural progenitor cells using adeno associated viral (AAV) vectors also promoted neuronal differentiation. Over-expressing cells demonstrated a significant increase in the neuron specific form of tubulin as well as increased expression of synaptotagmin. Genetic modification of neural progenitor cell with bHLH factors such as ND2 may be a viable strategy to enhance differentiation of these cells into replacement neurons for human disease. [Copyright &y& Elsevier]

Abstract: Results of a human post mortem study performed by our own group have suggested that the transcription factor NEUROD2, which plays a role in neuronal development, as well as in the development of anxiety and risk behavior in mice, might be a susceptibility factor for addictive disorders. Therefore the aim of the present study was to analyze a possible relation between genetic variants in the NEUROD2 gene and alcohol dependence in a sample of the Munich Gene Bank of Alcoholism (MGBA). We performed single SNP (single nucleotide polymorphism) and haplotype studies in 430 alcohol-dependent patients and 365 healthy controls with four SNPs covering the gene region of NEUROD2. Neither single SNP nor haplotype analysis could detect significant associations with alcohol dependence. Additionally we could not detect any relation of the analyzed genetic variants to Cloninger''s Type 1/2 or Babor''s Type A/B classification, to the age of onset or to the amount of alcohol intake. Our results do not provide evidence for an involvement of NEUROD2 polymorphisms in the pathophysiology of alcohol dependence. Further association studies are needed to confirm our findings. [Copyright &y& Elsevier]

Abstract: For a progenitor cell to become a neuron, three activities must occur: neuronal differentiation program must be activated, elements repressing neuronal differentiation must be deactivated and competing differentiation programs must be silenced. It is known that NeuroD2 and related bHLH transcription factors induce neuronal differentiation, REST represses neuronal differentiation, and Zfhx1a prevents myogenic gene expression. We demonstrate that NeuroD2 suppresses REST during differentiation in culture. In the hippocampus of NeuroD2 knockout mice, higher level of REST is detected. Functional significance of NeuroD2-REST interplay is uncovered by showing that forced expression of REST interferes with neuronal differentiation in culture. NeuroD2 inhibits REST indirectly by involving the inhibitor of myogenic genes, Zfhx1a, which binds response elements in REST 5′-UTR. Our study supports a model wherein NeuroD2 induces transcription of neuronal genes and Zfhx1a, which in turn de-represses neuronal differentiation by down-regulating REST, and suppresses competing myogenic fate. [Copyright &y& Elsevier]

Abstract: A basic helix-loop-helix transcriptional factor, NeuroD2, plays important roles in neuronal differentiation and survival. We introduced the tetracycline-dependent NeuroD2 expression system to embryonic stem (ES) cells and studied the role of NeuroD2 in the neuronal differentiation. The addition of doxycycline induced the expression of NeuroD2 after 24h and the differentiation to neurons after 3 days in ES cells, which are transfected with vectors composed of reverse tetracycline-controlled transactivator with cytomegarovirus promoter and NeuroD2 with tetracycline response element. Treatment with doxycycline for 3 days induced neuronal differentiation, but not within 1 day; furthermore NeuroD2 was detected in the nucleus 3 days after treatment, but also not within 1 day. The results suggest that the expression of NeuroD2 requires an appropriate period of about 3 days to elicit neuronal differentiation in ES cells. [Copyright &y& Elsevier]

Abstract: NeuroD2, one of the neurospecific basic helix–loop–helix transcription factors, has the ability to induce neural differentiation in undifferentiated cells. In this paper, we show that transduction of NeuroD2 protein induced mouse neuroblastoma cell line N1E-115 into neural differentiation. NeuroD2 has two basic-rich domains, one is nuclear localization signal (NLS) and the other is basic region of basic helix–loop–helix (basic). We constructed some mutants of NeuroD2, ND2Δ100–115 (lack of NLS), ND2Δ123–134 (lack of basic) and ND2Δ100–134 (lack of both NLS and basic) for transduction experiments. Using these proteins, we have shown that NLS region of NeuroD2 plays a role of protein transduction. Continuous addition of NeuroD2 protein resulted in N1E-115 cells adopting neural morphology after 4 days and Tau mRNA expression was increased. These results suggest that neural differentiation can be induced by direct addition of NeuroD2 protein. [Copyright &y& Elsevier]

The basic helix–loop–helix (bHLH) transcription factors NEUROD1, NEUROD2 and ATH2 are expressed during first trimester human placental development. We determined the transactivation potential of each of these factors in trophoblasts by measuring changes in the endogenous gene activity using absolute quantification by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) after transient transfection. In these assays, NEUROD1 was found to transiently transactivate NEUROD2 in trophoblast cells. Promotor truncation assays, using luciferase constructs, showed the presence of two domains in the NEUROD2 promotor, which showed increased activity after NeuroD1 transfection. Each of these NeuroD1-responsive domains contains an E-box sequence. The NEUROD2 transactivation data fit with the spatial expression pattern of NEUROD1 and NEUROD2, since they are expressed in endovascular trophoblasts. This expression pattern, as well as the present transactivation results, might suggest the presence of a NEUROD differentiation cascade during first trimester human placental development. [Copyright &y& Elsevier]

The basic helix–loop–helix (bHLH) transcription factor, neuroD2, induces neuronal differentiation and promotes neuronal survival. Reduced levels of neuroD2 were previously shown to cause motor deficits, ataxia, and seizure propensity. Because neuroD2 levels may be critical for brain function, we studied the regulation of neuroD2 gene in cell culture and transgenic mouse models. In transgenic mice, a 10-kb fragment of the neuroD2 promoter fully recapitulated the endogenous neuroD2 staining pattern. A 1-kb fragment of the neuroD2 promoter drove reporter gene expression in most, but not all neuroD2-positive neuronal populations. Mutation of two critical E-boxes, E4 and E5 (E4 and E5 situated 149 and 305 bp upstream of the transcriptional start site) eliminated gene expression. NeuroD2 expression was diminished in mice lacking neurogenin1 demonstrating that neurogenin1 regulates neuroD2 during murine brain development. These studies demonstrate that neuroD2 expression is highly dependent on bHLH-responsive E-boxes in the proximal promoter region, that additional distal regulatory elements are important for neuroD2 expression in a subset of cortical neurons, and that neurogenin1 regulates neuroD2 expression during mouse brain development. [Copyright &y& Elsevier]

Regulation of cell differentiation is an important assignment for cellular engineering. One of the techniques for regulation is gene transfection into undifferentiated cells. Transient expression of NeuroD2, one of neural bHLH transcription factors, converted mouse N1E-115 neuroblastoma cells into differentiated neurons. The regulation of neural bHLH expression should be a novel strategy for cell differentiation. In this study, we tried to regulate neural differentiation by NeuroD2 gene inserted under the control of heat shock protein-70 (HSP) promoter, which can be activated by electrical stimulation. Mouse neuroblastoma cell line, N1E-115, was stably transfected with expression vector containing mouse NeuroD2 cDNA under HSP promoter. Transfected cells were cultured on the electrode surface and applied electrical stimulation. After stimulation, NeuroD2 expression was induced, and transfected cells adopt a neuronal morphology at 3 days after stimulation. These results suggest that neural differentiation can be induced by electrically stimulated gene expression of NeuroD2. [Copyright &y& Elsevier]

During early human pregnancy, extravillous trophoblast cells invade the maternal tissue of the uterus in a way similar to invasion by cancer cells. However, the process of trophoblast invasion is regulated in a time and place restricted way, in contrast to cancer invasion. We screened first trimester placental tissue enriched by extravillous invasive trophoblasts for the expression of proneural basic helix–loop–helix (bHLH) transcription factors, which are important controllers of cell fate. Surprisingly, the presence of NEUROD1, NEUROD2 and ATH2 transcripts was found by reverse transcriptase polymerase chain reaction (RT-PCR) analysis in first trimester placentabed. Of these genes, the proneural genes NEUROD1 and NEUROD2 are expressed in different subsets of invasive trophoblasts. NEUROD1 expression is found in interstitial and endovascular invasive cells, while NEUROD2 expression is observed mainly in endovascular invasive cells, respectively. These data suggest that in addition to the involvement of proneural genes in neuron, neurendocrine and pancreas differentiation, these genes are involved in trophoblast differentation during progression of invasion. [Copyright &y& Elsevier]