Investigating the Molecular and Cellular Basis of RNA Exosome-linked Pontocerebellar Hypoplasia Type1b in Human 3D Brain Organoids - PROJECT SUMMARY The diversity of neuronal cell types within the mammalian brain is derived from neural stem cells that sequentially express specific transcriptional programs. Control of gene expression programs is critically important for proper neural stem cell differentiation and subtype specification, which consists of many regulatory processes, including transcriptional and post-transcriptional regulatory events that balance both RNA synthesis and degradation. Intriguingly, mutations in essential and ubiquitous post-transcriptional RNA-regulatory proteins cause neurodevelopmental disorders (NDDs) characterized by distinct cell type and/or brain-region-specific pathology, thus underscoring the importance of post-transcriptional regulation of gene expression to support proper brain development. This proposal will focus on an essential and ubiquitous RNA processing and decay machine, the RNA exosome, and its role during cell fate determination in neuronal cells. The RNA exosome is critically important for the precise processing and turnover of various RNAs, including rRNAs. Recessive missense mutations in the EXOSC3 gene, which encodes a structural subunit of the RNA exosome, cause Pontocerebellar Hypoplasia Type 1b (PCH1b). PCH1b is a severe NDD clinically characterized by atrophy of the brainstem and cerebellar structures. The severity of PCH1b pathology is influenced by EXOSC3 allelic heterogeneity, suggesting a genotype-phenotype correlation. Most RNA exosome-associated diseases include neurological phenotypes, suggesting tissue-specific function(s) for the RNA exosome. These observations reveal a vital role for the RNA exosome within the nervous system and provide a rationale to characterize the function of the complex within the brain. The central hypothesis underlying the proposed work is that the RNA exosome processes specific target RNAs essential for proper progenitor proliferation and neuronal differentiation within the brain. We have generated human induced pluripotent stem cells (hiPSCs) with two distinct PCH1b disease- causing EXOSC3 mutations linked to moderate or severe disease via CRISPR engineering to reveal the molecular and cellular basis of PCH1b. My preliminary data in hiPSC-derived cerebellar organoids modeling PCH1b mutations show cellular defects and increased steady-state levels of multiple functionally important neuronal transcripts, including synaptic regulator Arc, compared to controls. Utilizing this model, I aim to 1) define cell type-specific defects, including progenitor proliferation and differentiation and genotype-phenotype correlations, and 2) analyze gene expression dynamics in hiPSC-derived cerebellar organoids modeling PCH1b- linked EXOSC3 missense mutations compared to controls. Alongside the work proposed here, we will perform functional assays to test the neuronal function of mutant cerebellar organoids compared to controls. This project is designed to elucidate the role of the RNA exosome in cell fate determination within the cerebellum and establish whether defective differentiation is a critical component of PCH1b pathogenesis.
