Exploring Heterochromatin Alterations in C9 FTD - PROJECT SUMMARY Frontotemporal Dementia (FTD), an Alzheimer’s Disease Related Dementia, is the leading cause of dementia in people under 65. FTD is a cortical neuron degenerative disease that exists on a disease continuum with Amyotrophic Lateral Sclerosis (ALS), a motor neuron degenerative disease. Hexanucleotide repeat expansions in Chromosome 9 Open Reading Frame 72 (C9orf72) leading to dipeptide repeat proteins are the most frequent genetic alteration in FTD and ALS. Despite growing evidence of their importance in this context, epigenetic mechanisms remain an understudied aspect in the etiology of neurodegenerative disorders. Histone proteins, which DNA complexes with to make chromatin, undergo post-translational modifications (PTMs) that comprise a code that is installed by protein ‘writers’, interacted on by protein ‘readers’, and removed by ‘eraser’ proteins. Chromatin is categorized into two states: euchromatin, which is transcriptionally active, and heterochromatin, which is transcriptionally inactive. Changes in chromatin structure are gravely understudied in the context of FTD. Exploiting C9 ALS patient-derived fibroblasts, we have revealed increases in euchromatin-linked histone PTM H3S10ph. Furthermore, we have revealed increased micrococcal nuclease (MNase) accessibility linked to H3S10ph increases in C9 fibroblasts compared to healthy controls, which suggests that changes to chromatin state may underlie C9 proteinopathy. We hypothesize that alterations in histone PTMs connect to an observable loss of heterochromatin condensed regions and alterations of epigenomic and transcriptomic profiles that are associated with C9 expansions. To test this hypothesis, we propose to: (1) characterize C9 expansions and DPRs, connect them to H3S10ph and H3K9me3 changes, and explore the role of epigenetics in neurodegenerative pathology such as ER stress via genotyping, dot blotting, immunoblotting, and chemical inhibition studies; (2) examine changes to the distribution of heterochromatin structural components such as condensed chromocenters, H3K9me3+ heterochromatin and Heterochromatin Protein 1 levels and localization using microscopy; (3) delineate alterations in chromatin accessibility patterns, transcriptomic profiles and genetic targets of key heterochromatin factors, HP1𝛼 and H3K9me3 by enzymatic accessibility assay, RNA-seq, and CUT&RUN. This project will leverage a dual model approach, utilizing both C9 FTD patient-derived fibroblasts and induced pluripotent stem cells (iPSCs) differentiated into cortical neurons for cost and time efficiency. Building on our previous knowledge, this project provides a plethora of technical and professional development opportunities that will expand our understanding of FTD and other ADRDs as well as bring me closer to my ultimate career goal of becoming an independent teacher-scholar.
