Sunday, May 18, 2025
5/18/2025
The interplay of the heat shock cascade and Rett syndrome - PROJECT SUMMARY / ABSTRACT Rett syndrome (RTT) is a neurodevelopmental disorder that is associated with loss-of-function mutations in the Methyl CPG Binding Protein 2 (MeCP2) gene. MeCP2 is a methyl-reader protein that forms a bridge between methylated DNA and larger chromatin structure. As a result, mutations in MeCP2 result in the disruption of thousands of genes, making it difficult to identify those that are pathogenic and those that are transcriptional noise. Traditionally, RTT is first diagnosed clinically and then confirmed through the identification of mutations in the MECP2 gene. However, ~5% of RTT patients are found to be MeCP2-mutation-negative (atypical RTT), despite sharing overlapping symptomology with typical RTT. We hypothesized that genes with conserved disruption in typical and atypical patient populations would identify those most critical to their shared clinical presentation. Using a cohort of 44 autopsy samples from RTT patients, we identified 5 that were MeCP2- mutation-negative. We then conducted differential RNA sequencing on these samples relative to 6 typical RTT samples and 9 neurotypical controls. These experiments revealed that pathways associated with heat shock (HS) signaling are dramatically elevated in both populations. When we assessed all 44 autopsy samples, elevated HS signaling was enriched in typical RTT patients with severe, truncating mutations and not mild, missense mutations. As both patient groups exhibited the same protracted death process, the differential expression of HS proteins suggests that it is not an artifact, but rather a bona fide component of RTT. We hypothesize that increased HS signaling contributes to severe phenotypes in RTT, and we will test this hypothesis in Aim 1 by 1) genetically diminishing the HS response (HSR) from conception in a mouse model of RTT to assess whether phenotypes become milder, and 2) pharmacologically normalizing the HSR to determine whether it rescues existing phenotypes. Mechanistically, MeCP2 is known to regulate baseline gene expression by controlling the rate of transcriptional initiation. We hypothesize that MeCP2 regulates the expression of activity-dependent genes, like HSPs, in an identical manner. In Aim 2 we will first determine the scope of gene disruption following thermal stimulation using RNA-seq (Aim 2a), and then mirror those experiments with Pro- seq on mice treated with the elongation inhibitor flavopiridol (Aim 2b). Together, these experiments will identify which stress response genes are affected by the loss of MeCP2, and establish whether their change in expression is associated with impairments in transcriptional initiation, thereby providing a mechanistic complement to the therapeutically relevant experiments outlined in Aim 1. This proposal has been designed to provide extensive training in neuropharmacology, behavioral neuroscience, and bioinformatics which I have identified as critical for my career development. My mentors and I have developed a training plan composed of didactic and technical training to prepare me for an independent career in academic research.
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