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Project description:The ability of XIST to dosage compensate a trisomic autosome presents unique experimental opportunities and potentially transformative therapeutic prospects. However, it is currently thought that XIST requires the natural context surrounding pluripotency to initiate chromosome silencing. Here, we demonstrate that XIST RNA induced in differentiated neural cells can trigger chromosome-wide silencing of chromosome 21 in Down syndrome patient-derived cells. Use of this tightly controlled system revealed a deficiency in differentiation of trisomic neural stem cells to neurons, correctible by inducing XIST at different stages of neurogenesis. Single-cell transcriptomics and other analyses strongly implicate elevated Notch signaling due to trisomy 21, thereby promoting neural stem cell cycling that delays terminal differentiation. These findings have significance for illuminating the epigenetic plasticity of cells during development, the understanding of how human trisomy 21 effects Down syndrome neurobiology, and the translational potential of XIST, a unique non-coding RNA.
| S-EPMC7055611 | biostudies-literature
Project description:Although Down Syndrome (DS) is the leading genetic cause of intellectual disability in children, the developmental pathogenesis remains largely unknown, and better strategies are needed to investigate this. We previously showed that one copy of chromosome 21 can be epigenetically silenced in DS iPSCs by insertion of an XIST transgene, which produces a non-coding RNA that normally silences one X chromosome in female cells. XIST was shown to induce heterochromatin and silence transcription across chromosome 21 in pluripotent stem cells, the natural developmental context of XIST function. Prior literature indicated that initiation of chromosome silencing is only possible within 48 hours of mES cell differentiation, however it would be highly advantageous experimentally if trisomy silencing could be initiated in differentiated cells, and this is critical for any therapeutic potential of XIST. Here we use RNAseq and molecular cytology to investigate the effectiveness of XIST for trisomy silencing in cells undergoing in vitro neural differentiation and examine the potential cell phenotypic effects of chromosomal silencing. Induction of XIST from the onset of differentiation resulted in comprehensive silencing of chromosome 21 genes, providing a powerful approach to examine effects of trisomy on neurogenesis. To determine whether human neural stem cells can initiate XIST-mediated silencing, we induced XIST at several times during neural differentiation. We demonstrate for the first time that differentiated normal human cells can initiate chromosome silencing in response to XIST expression. While this process only takes a few days in pluripotent cells, we show that it takes 2-3 weeks in differentiated cells. Importantly, single-cell RNAseq revealed that cells which express XIST preferentially differentiate into neurons, providing evidence of phenotypic improvement with trisomy silencing, which is seen even when XIST is initiated weeks into differentiation. We are currently investigating whether silenced neurons show other non-chromosome 21 transcriptional changes suggestive of potentially improved function. These studies have important implications for the understanding of XIST biology and DS neurobiology, and also further open the possibility of a chromosomal therapy for DS using a single gene: XIST.
2020-01-23 | GSE125837 | GEO
Project description:Although Down Syndrome (DS) is the leading genetic cause of intellectual disability in children, the developmental pathogenesis remains largely unknown, and better strategies are needed to investigate this. We previously showed that one copy of chromosome 21 can be epigenetically silenced in DS iPSCs by insertion of an XIST transgene, which produces a non-coding RNA that normally silences one X chromosome in female cells. XIST was shown to induce heterochromatin and silence transcription across chromosome 21 in pluripotent stem cells, the natural developmental context of XIST function. Prior literature indicated that initiation of chromosome silencing is only possible within 48 hours of mES cell differentiation, however it would be highly advantageous experimentally if trisomy silencing could be initiated in differentiated cells, and this is critical for any therapeutic potential of XIST. Here we use RNAseq and molecular cytology to investigate the effectiveness of XIST for trisomy silencing in cells undergoing in vitro neural differentiation and examine the potential cell phenotypic effects of chromosomal silencing. Induction of XIST from the onset of differentiation resulted in comprehensive silencing of chromosome 21 genes, providing a powerful approach to examine effects of trisomy on neurogenesis. To determine whether human neural stem cells can initiate XIST-mediated silencing, we induced XIST at several times during neural differentiation. We demonstrate for the first time that differentiated normal human cells can initiate chromosome silencing in response to XIST expression. While this process only takes a few days in pluripotent cells, we show that it takes 2-3 weeks in differentiated cells. Importantly, single-cell RNAseq revealed that cells which express XIST preferentially differentiate into neurons, providing evidence of phenotypic improvement with trisomy silencing, which is seen even when XIST is initiated weeks into differentiation. We are currently investigating whether silenced neurons show other non-chromosome 21 transcriptional changes suggestive of potentially improved function. These studies have important implications for the understanding of XIST biology and DS neurobiology, and also further open the possibility of a chromosomal therapy for DS using a single gene: XIST.
2020-01-23 | GSE125838 | GEO
Project description:Silencing Trisomy 21 with XIST in Neural Stem Cells Promotes Neuronal Differentiation
| PRJNA517686 | ENA
Project description:Silencing Trisomy 21 with XIST in Neural Stem Cells Promotes Neuronal Differentiation (bulk)
| PRJNA517690 | ENA
Project description:Silencing Trisomy 21 with XIST in Neural Stem Cells Promotes Neuronal Differentiation (single cells)
| PRJNA517689 | ENA
Project description:Hippocalcin (HPCA) is a calcium-binding protein that is restricted to nervous tissue and contributes to neuronal activity. Here we report that, in addition to inducing neurogenesis, HPCA inhibits astrocytic differentiation of neural stem cells. It promotes neurogenesis by regulating protein kinase Cα (PKCα) activation by translocating to the membrane and binding to phosphoinositide-dependent protein kinase 1 (PDK1), which induces PKCα phosphorylation. We also found that phospholipase D1 (PLD1) is implicated in the HPCA-mediated neurogenesis pathway; this enzyme promotes dephosphorylation of signal transducer and activator of transcription 3 (STAT3[Y705]), which is necessary for astrocytic differentiation. Moreover, we found that the SH2-domain-containing tyrosine phosphatase 1 (SHP-1) acts upstream of STAT3. Importantly, this SHP-1-dependent STAT3-inhibitory mechanism is closely involved in neurogenesis and suppression of gliogenesis by HPCA. Taken together, these observations suggest that HPCA promotes neuronal differentiation through activation of the PKCα/PLD1 cascade followed by activation of SHP-1, which dephosphorylates STAT3(Y705), leading to inhibition of astrocytic differentiation.
| S-EPMC5233403 | biostudies-literature
Project description:IntroductionDown syndrome, caused by trisomy 21, is a complex developmental disorder associated with intellectual disability and reduced growth of multiple organs. Structural pathologies are present at birth, reflecting embryonic origins. A fundamental unanswered question is how an extra copy of human chromosome 21 contributes to organ-specific pathologies that characterize individuals with Down syndrome, and, relevant to the hallmark intellectual disability in Down syndrome, how trisomy 21 affects neural development. We tested the hypothesis that trisomy 21 exerts effects on human neural development as early as neural induction.MethodsBulk RNA sequencing was performed on isogenic trisomy 21 and euploid human induced pluripotent stem cells (iPSCs) at successive stages of neural induction: embryoid bodies at Day 6, early neuroectoderm at Day 10, and differentiated neuroectoderm at Day 17.ResultsGene expression analysis revealed over 1,300 differentially expressed genes in trisomy 21 cells along the differentiation pathway compared to euploid controls. Less than 5% of the gene expression changes included upregulated chromosome 21 encoded genes at every timepoint. Genes involved in specific growth factor signaling pathways (WNT and Notch), metabolism (including oxidative stress), and extracellular matrix were altered in trisomy 21 cells. Further analysis uncovered heterochronic expression of genes.ConclusionTrisomy 21 impacts discrete developmental pathways at the earliest stages of neural development. The results suggest that metabolic dysfunction arises early in embryogenesis in trisomy 21 and may affect development and function more broadly.
| S-EPMC10865501 | biostudies-literature
Project description:Neural stem cells (NSCs) are self-renewing cells that can differentiate into multiple neural lineages and repopulate regions of the brain after injury. We have investigated the role of endocannabinoids (eCBs), endogenous cues that modulate neuronal functions including neurogenesis, and their receptors CB(1) and CB(2) in mouse NSCs. Real-time PCR and Western blot analyses indicated that CB(1) is present at higher levels than CB(2) in NSCs. The eCB anandamide (AEA) or the CB(1)-specific agonist ACEA enhanced NSC differentiation into neurons, but not astrocytes and oligodendrocytes, whereas the CB(2)-specific agonist JWH133 was ineffective. Conversely, the effect of AEA was inhibited by CB(1), but not CB(2), antagonist, corroborating the specificity of the response. CB(1) activation also enhanced maturation of neurons, as indicated by morphometric analysis of neurites. CB(1) stimulation caused long-term inhibition of the ERK1/2 pathway. Consistently, pharmacological inhibition of the ERK1/2 pathway recapitulated the effects exerted by CB(1) activation on neuronal differentiation and maturation. Lastly, gene array profiling showed that CB(1) activation augmented the expression of genes involved in neuronal differentiation while decreasing that of stemness genes. These results highlight the role of CB(1) in the regulation of NSC fate and suggest that its activation may represent a pro-neuronal differentiation signal.
| S-EPMC3553153 | biostudies-literature
Project description:The ubiquitin E3 ligase RNF220 and its co-factor ZC4H2 are required for multiple neural developmental processes through different targets, including spinal cord patterning and the development of the cerebellum and the locus coeruleus. Here, we explored the effects of loss of ZC4H2 and RNF220 on the proliferation and differentiation of neural stem cells (NSCs) derived from mouse embryonic cortex. We showed that loss of either ZC4H2 or RNF220 inhibits the proliferation and promotes the differentiation abilities of NSCs in vitro. RNA-Seq profiling revealed 132 and 433 differentially expressed genes in the ZCH2-/- and RNF220-/- NSCs, compared to wild type (WT) NSCs, respectively. Specifically, Cend1, a key regulator of cell cycle exit and differentiation of neuronal precursors, was found to be upregulated in both ZCH2-/- and RNF220-/- NSCs at the mRNA and protein levels. The targets of Cend1, such as CyclinD1, Notch1 and Hes1, were downregulated both in ZCH2-/- and RNF220-/- NSCs, whereas p53 and p21 were elevated. ZCH2-/- and RNF220-/- NSCs showed G0/G1 phase arrest compared to WT NSCs in cell cycle analysis. These results suggested that ZC4H2 and RNF220 are likely involved in the regulation of neural stem cell proliferation and differentiation through Cend1.
| S-EPMC7408363 | biostudies-literature