Cacace, A. | Search bioRxiv

bioRxiv2019

Small molecule inhibitors of G9a reactivate the maternal PWS genes in Prader-Willi-Syndrome patient derived neural stem cells and differentiated neurons

Abstract/SummaryPatients with Prader-Willi-Syndrome (PWS) display intellectual impairment, hyperphagia, and various behavioral problems during childhood that converge on a neurologic deficit. The majority of PWS patients have genetic deletions of the paternal 15q11-q13 chromosomal region, with their maternal PWS locus intact but epigenetically silenced by hypermethylation and repressive histone modulation of the PWS imprinting center (PWS-IC). Inhibition of the euchromatin histone methyltransferase G9a by small molecules has been recently reported to reactivate PWS genes in patient fibroblasts and a mouse model. However, it is unknown if inhibition of G9a could have similar effect in human PWS neural cells, the cell types that have direct pathophysiological relevance to PWS. Here, we use neural progenitor cells (NPCs) and cortical excitatory neurons derived from a patient iPSC to model PWS, and quantitatively profile the expression of PWS genes using a NanoString panel. We demonstrated that the methylation of the PWS-IC is stable during neuronal lineage conversion, and that the maternal PWS genes remain silenced in PWS NPCs and neurons. Multiple small molecule inhibitors of G9a activate maternal PWS genes in a dose dependent manner in both NPCs and neurons. In addition, G9a inhibitors induce GNRH1 and HTR2C, two neuronal specific genes that contribute to PWS pathology in neurons. Interestingly, distinct from 5-Azacytidine, G9a inhibition does not induce methylation changes of the maternal PWS-IC, indicating that disruption of the histone repressive complex alone is sufficient to drive an open chromatin state at the PWS-IC that leads to partial reactivation of PWS genes. HighlightsO_LIModeling PWS disease in a dish using patient derived NPCs and neurons C_LIO_LIG9a inhibition activates maternal PWS genes in patient-derived neural cells C_LIO_LIG9a inhibition activates maternal SNORD116 and other PWS genes in patient-derived neurons C_LIO_LIInhibition of G9a induces PWS downstream genes GNRH1 and HTR2C in PWS neurons C_LI

cell biology↗

bioRxiv2019

Partial FMRP expression is sufficient to normalize neuronal hyperactivity in Fragile X neurons

Fragile X Syndrome (FXS) is the most common genetic form of intellectual disability caused by a CGG repeat expansion in the 5-UTR of the Fragile X mental retardation gene FMR1, triggering epigenetic silencing and the subsequent absence of the protein, FMRP. Reactivation of FMR1 represents an attractive therapeutic strategy targeting the genetic root cause of FXS. However, largely missing in the FXS field is an understanding of how much FMR1 reactivation is required to rescue FMRP-dependent mutant phenotypes. Here, we utilize FXS patient derived excitatory neurons to model FXS in vitro and confirm that the absence of FMRP leads to neuronal hyperactivity. We further determined the levels of FMRP and the percentage of FMRP positive cells necessary to correct this phenotype utilizing a mixed and mosaic neuronal culture system and a combination of CRISPR, antisense and expression technologies to titrate FMRP in FXS and WT neurons. Our data demonstrate that restoration of greater than 5% of overall FMRP expression levels or greater than 20% FMRP expressing neurons in a mosaic pattern is sufficient to normalize a FMRP-dependent, hyperactive phenotype in FXS iPSC-derived neurons.\n\nHighlightsO_LICRISPR gene editing to generate FMRP KO and CGG-deleted isogenic iPSCs\nC_LIO_LIMEA as an approach to identify FMR1 dependent phenotype in NGN2 neurons derived from FXS and FMRP KO iPSCs\nC_LIO_LICell mixing paradigm as mosaicism in a dish to rescue phenotype\nC_LIO_LIMinimal level of FMRP determined by FMR1 mRNA and targeted demethylation of CGG repeats to correct the hyperactive phenotype in FXS neurons\nC_LIO_LIASO titration-validated partial expression of FMRP is sufficient to normalize increased neuronal activity\nC_LI

neuroscience↗