Title: Screening of a custom RNAi library to identify genes underlying mitochondrial dysfunction in Rett syndrome astrocytes
Legend: (A) Left (clockwise): Representative image of GFAP (green) and DAPI (blue) staining of astrocytes differentiated from RTT IPSCs. Representative image of astrocytes transfected with positive control siRNA. Representative JC-10 asssay images of in R294X mutant (MT) and wild type (WT) astrocytes. Right: Representative JC-10 assay images of in R294X MT astrocytes transfected with siRNA against candidate genes. Scale bar = 100μm. (B) Scatter plot distribution showing the JC-10 assay results from a high-throughput screening of custom siRNA library, containing 336 genes previously found to be upregulated in RTT astrocytes. Blue dots mark 12 hits with rescuing effects that are 2 standard deviations from the mean. Gene e shows rescuing effect that is 3 standard deviations from the mean. (C) Left: Representative JC-10 assay images of untreated R294X WT astrocytes, untreated R294X MT astrocytes, and gene e siRNA treated R294X MT astrocytes. Scale bar = 10μm. F590/520 ratio shown Gene e RNAi significantly rescued MMP in cell body analysis. Right: Quantification of the JC-10 assay results. Statistical significances between groups were determined by two-tailed T-test (NS, no significance, *p<0.05, **p<0.01, ***p<0.001)
Abstract: Rett syndrome (RTT) is a severe X chromosome-linked debilitating neurodevelopmental disorder caused by mutations in the MECP2 gene affects 1 in 10,000-15,000 girls with no effective treatment. Our lab has been using RTT induced pluripotent stem cells (iPSC) and neurons and astrocytes differentiated from iPSC as a platform to understand RTT disease mechanisms and develop treatment. Recently, we have identified a series of mitochondrial phenotypes in RTT astrocytes. Among these, the reduction of mitochondrial membrane potential (MMP) can be readily and consistently measured by high throughput fluorescence assay (JC-10). To better understand disease mechanism and develop treatment, we screened a custom RNAi library consisting of 336 candidate genes known to be up-regulated in RTT astrocytes, using the JC-10 assay to measure MMP. We identified 12 genes that, when knocked down in RTT astrocytes, resulted in significant improvement in MMP. Among those 12 genes, TRPC4 is a gene identified in our recent work (Dong et al, eLife, 2018) to be up-regulated in RTT astrocytes, which contributes to many cellular phenotypes in those cells, including reduced MMP. The identification of TRPC4 as hit suggests the screen worked well. We are currently performing more experiments to validate our screening results and reveal how those identified genes contribute to mitochondrial defects in RTT astrocytes.
About the Lab: The long-term goal of the Chang lab is to understand the molecular mechanism underlying DNA methylation-dependent epigenetic regulation of brain functions. His current focus is on the central role of MeCP2 (methyl-CpG binding protein 2), a molecular linker between DNA methylation and chromatin remodeling and transcriptional control, in the development and function of the nervous system. The functional significance of such a molecular linker is highlighted by the fact that mutations in the MECP2 gene cause Rett syndrome (RTT), a debilitating neurodevelopmental disorder that shares many features with autism. His studies include basic research aimed at understanding the function of MeCP2 and translational research aimed at understanding disease pathology and developing effective treatment. These two types of research are tightly interwoven—the need to solve a practical problem in translational research will always influence the direction of basic research; and fundamental mechanisms revealed by basic research will ultimately guide the effort in treating/curing the disease.