Monoallelic and bi-allelic variants in NCDN cause neurodevelopmental delay, intellectual disability, and epilepsy.

Neurochondrin (NCDN) is a cytoplasmatic neural protein of importance for neural growth, glutamate receptor (mGluR) signaling, and synaptic plasticity. Conditional loss of Ncdn in mice neural tissue causes depressive-like behaviors, impaired spatial learning, and epileptic seizures. We report on NCDN missense variants in six affected individuals with variable degrees of developmental delay, intellectual disability (ID), and seizures. Three siblings were found homozygous for a NCDN missense variant, whereas another three unrelated individuals carried different de novo missense variants in NCDN. We assayed the missense variants for their capability to rescue impaired neurite formation in human neuroblastoma (SH-SY5Y) cells depleted of NCDN. Overexpression of wild-type NCDN rescued the neurite-phenotype in contrast to expression of NCDN containing the variants of affected individuals. Two missense variants, associated with severe neurodevelopmental features and epilepsy, were unable to restore mGluR5-induced ERK phosphorylation. Electrophysiological analysis of SH-SY5Y cells depleted of NCDN exhibited altered membrane potential and impaired action potentials at repolarization, suggesting NCDN to be required for normal biophysical properties. Using available transcriptome data from human fetal cortex, we show that NCDN is highly expressed in maturing excitatory neurons. In combination, our data provide evidence that bi-allelic and de novo variants in NCDN cause a clinically variable form of neurodevelopmental delay and epilepsy, highlighting a critical role for NCDN in human brain development.

Single cell analysis of autism patient with bi-allelic NRXN1-alpha deletion reveals skewed fate choice in neural progenitors and impaired neuronal functionality.

We generated human iPS derived neural stem cells and differentiated cells from healthy control individuals and an individual with autism spectrum disorder carrying bi-allelic NRXN1-alpha deletion. We investigated the expression of NRXN1-alpha during neural induction and neural differentiation and observed a pivotal role for NRXN1-alpha during early neural induction and neuronal differentiation. Single cell RNA-seq pinpointed neural stem cells carrying NRXN1-alpha deletion shifting towards radial glia-like cell identity and revealed higher proportion of differentiated astroglia. Furthermore, neuronal cells carrying NRXN1-alpha deletion were identified as immature by single cell RNA-seq analysis, displayed significant depression in calcium signaling activity and presented impaired maturation action potential profile in neurons investigated with electrophysiology. Our observations propose NRXN1-alpha plays an important role for the efficient establishment of neural stem cells, in neuronal differentiation and in maturation of functional excitatory neuronal cells.

Transcriptomes of Dravet syndrome iPSC derived GABAergic cells reveal dysregulated pathways for chromatin remodeling and neurodevelopment.

Dravet syndrome (DS) is an early onset refractory epilepsy typically caused by de novo heterozygous variants in SCN1A encoding the α-subunit of the neuronal sodium channel Nav1.1. The syndrome is characterized by age-related progression of seizures, cognitive decline and movement disorders. We hypothesized that the distinct neurodevelopmental features in DS are caused by the disruption of molecular pathways in Nav1.1 haploinsufficient cells resulting in perturbed neural differentiation and maturation. Here, we established DS-patient and control induced pluripotent stem cell derived neural progenitor cells (iPSC NPC) and GABAergic inter-neuronal (iPSC GABA) cells. The DS-patient iPSC GABA cells showed a shift in sodium current activation and a perturbed response to induced oxidative stress. Transcriptome analysis revealed specific dysregulations of genes for chromatin structure, mitotic progression, neural plasticity and excitability in DS-patient iPSC NPCs and DS-patient iPSC GABA cells versus controls. The transcription factors FOXM1 and E2F1, positive regulators of the disrupted pathways for histone modification and cell cycle regulation, were markedly up-regulated in DS-iPSC GABA lines. Our study highlights transcriptional changes and disrupted pathways of chromatin remodeling in Nav1.1 haploinsufficient GABAergic cells, providing a molecular framework that overlaps with that of neurodevelopmental disorders and other epilepsies.

ZEB2 haploinsufficient Mowat-Wilson syndrome induced pluripotent stem cells show disrupted GABAergic transcriptional regulation and function.

Mowat-Wilson syndrome (MWS) is a severe neurodevelopmental disorder caused by heterozygous variants in the gene encoding transcription factor ZEB2. Affected individuals present with structural brain abnormalities, speech delay and epilepsy. In mice, conditional loss of Zeb2 causes hippocampal degeneration, altered migration and differentiation of GABAergic interneurons, a heterogeneous population of mainly inhibitory neurons of importance for maintaining normal excitability. To get insights into GABAergic development and function in MWS we investigated ZEB2 haploinsufficient induced pluripotent stem cells (iPSC) of MWS subjects together with iPSC of healthy donors. Analysis of RNA-sequencing data at two time points of GABAergic development revealed an attenuated interneuronal identity in MWS subject derived iPSC with enrichment of differentially expressed genes required for transcriptional regulation, cell fate transition and forebrain patterning. The ZEB2 haploinsufficient neural stem cells (NSCs) showed downregulation of genes required for ventral telencephalon specification, such as FOXG1, accompanied by an impaired migratory capacity. Further differentiation into GABAergic interneuronal cells uncovered upregulation of transcription factors promoting pallial and excitatory neurons whereas cortical markers were downregulated. The differentially expressed genes formed a neural protein-protein network with extensive connections to well-established epilepsy genes. Analysis of electrophysiological properties in ZEB2 haploinsufficient GABAergic cells revealed overt perturbations manifested as impaired firing of repeated action potentials. Our iPSC model of ZEB2 haploinsufficient GABAergic development thus uncovers a dysregulated gene network leading to immature interneurons with mixed identity and altered electrophysiological properties, suggesting mechanisms contributing to the neuropathogenesis and seizures in MWS.

DNA methylation changes in Down syndrome derived neural iPSCs uncover co-dysregulation of ZNF and HOX3 families of transcription factors.

BACKGROUND: Down syndrome (DS) is characterized by neurodevelopmental abnormalities caused by partial or complete trisomy of human chromosome 21 (T21). Analysis of Down syndrome brain specimens has shown global epigenetic and transcriptional changes but their interplay during early neurogenesis remains largely unknown. We differentiated induced pluripotent stem cells (iPSCs) established from two DS patients with complete T21 and matched euploid donors into two distinct neural stages corresponding to early- and mid-gestational ages. RESULTS: Using the Illumina Infinium 450K array, we assessed the DNA methylation pattern of known CpG regions and promoters across the genome in trisomic neural iPSC derivatives, and we identified a total of 500 stably and differentially methylated CpGs that were annotated to CpG islands of 151 genes. The genes were enriched within the DNA binding category, uncovering 37 factors of importance for transcriptional regulation and chromatin structure. In particular, we observed regional epigenetic changes of the transcription factor genes ZNF69, ZNF700 and ZNF763 as well as the HOXA3, HOXB3 and HOXD3 genes. A similar clustering of differential methylation was found in the CpG islands of the HIST1 genes suggesting effects on chromatin remodeling. CONCLUSIONS: The study shows that early established differential methylation in neural iPSC derivatives with T21 are associated with a set of genes relevant for DS brain development, providing a novel framework for further studies on epigenetic changes and transcriptional dysregulation during T21 neurogenesis.

Generation of three human induced pluripotent stem cell (iPSC) lines from three patients with Dravet syndrome carrying distinct SCN1A gene mutations.

Dravet syndrome (DS) is a childhood epilepsy syndrome caused by heterozygous mutations in the SCN1A gene encoding voltage-gated sodium channel Nav1.1. We generated iPSCs from fibroblasts of three DS patients carrying distinct SCN1A mutations (c.5502-5509dupGCTTGAAC, c.2965G>C and c.651C>G). The iPSC lines were genetically stable and each line retained the SCN1A gene mutation of the donor fibroblasts. Characterization of the iPSC lines confirmed expression of pluripotency markers, absence of exogenous vector expression and trilineage differentiation potential. These iPSC lines offer a useful resource to investigate the molecular mechanisms underlying Nav1.1 haploinsufficiency and for drug development to improve treatment of DS patients.

Generation of human induced pluripotent stem cell (iPSC) lines from three patients with von Hippel-Lindau syndrome carrying distinct VHL gene mutations.

Von Hippel-Lindau (VHL) syndrome is a familial cancer syndrome caused by mutations in the tumor suppressor gene VHL. We generated human iPSC lines from primary dermal fibroblasts of three VHL syndrome patients carrying distinct VHL germ line mutations (c.194C>G, c.194C>T and nt440delTCT, respectively). Characterization of the iPSC lines confirmed expression of pluripotency markers, trilineage differentiation potential and absence of exogenous vector expression. The three hiPSC lines were genetically stable and retained the VHL mutation of each donor. These iPSC lines, the first derived from VHL syndrome patients, offer a useful resource to study disease pathophysiology and for anti-cancer drug development.

Mowat-Wilson syndrome: Generation of two human iPS cell lines (UUIGPi004A and UUIGPi005A) from siblings with a truncating ZEB2 gene variant.

Mowat-Wilson syndrome (MWS) is a complex developmental syndrome caused by heterozygous mutations in the Zinc finger E-box-binding homeobox 2 gene (ZEB2). We generated the first human iPSC lines from primary fibroblasts of two siblings with MWS carrying a heterozygous ZEB2 stop mutation (c.1027C > T; p.Arg343*) using the Sendai virus reprogramming system. Both iPSC lines were free from reprogramming vector genes, expressed pluripotency markers and showed potential to differentiate into the three germ layers. Genetic analysis confirmed normal karyotypes and a preserved stop mutation. These iPSC lines will provide a useful resource to study altered neural lineage fate and neuropathophysiology in MWS.

Transcriptome and Proteome Profiling of Neural Induced Pluripotent Stem Cells from Individuals with Down Syndrome Disclose Dynamic Dysregulations of Key Pathways and Cellular Functions.

Down syndrome (DS) or trisomy 21 (T21) is a leading genetic cause of intellectual disability. To gain insights into dynamics of molecular perturbations during neurogenesis in DS, we established a model using induced pluripotent stem cells (iPSC) with transcriptome profiles comparable to that of normal fetal brain development. When applied on iPSCs with T21, transcriptome and proteome signatures at two stages of differentiation revealed strong temporal dynamics of dysregulated genes, proteins and pathways belonging to 11 major functional clusters. DNA replication, synaptic maturation and neuroactive clusters were disturbed at the early differentiation time point accompanied by a skewed transition from the neural progenitor cell stage and reduced cellular growth. With differentiation, growth factor and extracellular matrix, oxidative phosphorylation and glycolysis emerged as major perturbed clusters. Furthermore, we identified a marked dysregulation of a set of genes encoded by chromosome 21 including an early upregulation of the hub gene APP, supporting its role for disturbed neurogenesis, and the transcription factors OLIG1, OLIG2 and RUNX1, consistent with deficient myelination and neuronal differentiation. Taken together, our findings highlight novel sequential and differentiation-dependent dynamics of disturbed functions, pathways and elements in T21 neurogenesis, providing further insights into developmental abnormalities of the DS brain.

Ataxia in Patients With Bi-Allelic NFASC Mutations and Absence of Full-Length NF186.

The etiology of hereditary ataxia syndromes is heterogeneous, and the mechanisms underlying these disorders are often unknown. Here, we utilized exome sequencing in two siblings with progressive ataxia and muscular weakness and identified a novel homozygous splice mutation (c.3020-1G > A) in neurofascin (NFASC). In RNA extracted from fibroblasts, we showed that the mutation resulted in inframe skipping of exon 26, with a deprived expression of the full-length transcript that corresponds to NFASC isoform NF186. To further investigate the disease mechanisms, we reprogrammed fibroblasts from one affected sibling to induced pluripotent stem cells, directed them to neuroepithelial stem cells and finally differentiated to neurons. In early neurogenesis, differentiating cells with selective depletion of the NF186 isoform showed significantly reduced neurite outgrowth as well as fewer emerging neurites. Furthermore, whole-cell patch-clamp recordings of patient-derived neuronal cells revealed a lower threshold for openings, indicating altered Na+ channel kinetics, suggesting a lower threshold for openings as compared to neuronal cells without the NFASC mutation. Taken together, our results suggest that loss of the full-length NFASC isoform NF186 causes perturbed neurogenesis and impaired neuronal biophysical properties resulting in a novel early-onset autosomal recessive ataxia syndrome.

Recurrent Sleep Fragmentation Induces Insulin and Neuroprotective Mechanisms in Middle-Aged Flies.

Lack of quality sleep increases central nervous system oxidative stress and impairs removal of neurotoxic soluble metabolites from brain parenchyma. During aging poor sleep quality, caused by sleep fragmentation, increases central nervous system cellular stress. Currently, it is not known how organisms offset age-related cytotoxic metabolite increases in order to safeguard neuronal survival. Furthermore, it is not understood how age and sleep fragmentation interact to affect oxidative stress protection pathways. We demonstrate sleep fragmentation increases systems that protect against oxidative damage and neuroprotective endoplasmic reticulum molecular chaperones, as well as neuronal insulin and dopaminergic expression in middle-aged Drosophila males. Interestingly, even after sleep recovery the expression of these genes was still upregulated in middle-aged flies. Finally, sleep fragmentation generates higher levels of reactive oxygen species (ROS) in middle-aged flies and after sleep recovery these levels remain significantly higher than in young flies. The fact that neuroprotective pathways remain upregulated in middle-aged flies beyond sleep fragmentation suggests it might represent a strong stressor for the brain during later life.