Epigenetically Preserved iNSCs to Study dsRNA-Mediated Inflammation in C9orf72-ALS/FTD - PROJECT SUMMARY Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal, age-associated neurodegenerative disorders that exist along a clinical and genetic continuum. Nearly all ALS and approximately half of FTD cases are marked by pathological aggregation and nuclear depletion of TDP-43, an essential RNA- binding protein whose dysfunction alters RNA metabolism and mitochondrial integrity. The most common genetic cause of ALS/FTD is a hexanucleotide (G₄C₂) repeat expansion in the C9orf72 gene, which contributes to both gain- and loss-of-function mechanisms, including impaired autophagy, toxic RNA foci, dipeptide repeat proteins (DPRs), and heightened inflammation. Mitochondrial dysfunction and innate immune activation are now recognized as early and potentially synergistic events in ALS/FTD, but the upstream signals linking them remain poorly understood. This proposal tests the central hypothesis that mitochondrial dysfunction in C9orf72-ALS/FTD leads to the accumulation and cytosolic release of immunogenic double-stranded RNA (dsRNA), which activates RIG-I-like innate immune receptors (RLRs) and drives a maladaptive inflammatory cascade. To study this, we utilize a novel disease model: induced neural stem cells (iNSCs) directly transdifferentiated from patient fibroblasts, which retain age-associated epigenetic features typically erased in iPSC-based models. Preliminary data in C9 iNSCs reveal hallmarks of disease, including phosphorylated TDP-43, elevated interferon signaling, and cytosolic dsRNA accumulation. Aim 1 will characterize canonical neurodegenerative hallmarks in age- preserved C9 iNSCs, including epigenetic age signatures (via whole-genome bisulfite sequencing), TDP-43 pathology (including cryptic exon splicing defects), type-I interferon responses, and mitochondrial dysfunction (quantified by oxygen consumption and reactive oxygen species production). Critically, this aim will also define the subcellular localization and abundance of dsRNA, using immunocytochemistry, confocal microscopy, and biochemical fractionation. Aim 2 will identify the intracellular source and innate immune potency of dsRNA in C9 iNSCs. We will use RIG-I-Apex2 proximity biotinylation and sequencing to profile RNAs physically bound to RIG- I and determine whether they originate from mitochondria, cryptic exons, transposable elements, or repeat- derived hairpins. Functional relevance will be assessed by selectively depleting candidate RNA sources using small molecule inhibitors or CRISPR interference and measuring the impact on dsRNA accumulation and downstream inflammatory signaling. By integrating age-appropriate modeling with mechanistic dissection of the dsRNA-RIG-I axis, this project will define a novel, mitochondria-driven mechanism of neuroinflammation in ALS/FTD. Findings may reveal new targets for therapeutic modulation of innate immune signaling in neurodegeneration.
