Abstract Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two fatal neurodegenerative conditions with no current treatment to prevent, decelerate or stop neuronal death in patients. ALS and FTLD are clinically distinct but show an overlap in postmortem brain pathology and genetic factors: nuclear clearance and cytoplasmic accumulation of TDP-43 in affected central nervous system (CNS) regions is observed in 98% of ALS and 50% of FTLD patients. While initial symptoms lead to the diagnosis of either ALS or FTLD, up to 50% of ALS patients eventually develop symptoms of FTLD, with ~15% of patients ultimately receiving both diagnoses (FTLD with motor neuron disease, FTLD/MND). Mutations in the gene encoding TDP-43 (TARDBP) lead to rare cases of ALS, while TDP-43 pathology is observed in patients carrying more prevalent mutations, such as a pathological C9orf72 hexanucleotide repeat expansion (C9orf72+)—the most common genetic cause of ALS and FTLD identified thus far. TDP-43 therefore appears to be a pivotal and convergent factor in the pathogenesis of both ALS and FTLD. Despite this, however, the reasons for selective vulnerability of motor neurons, the mechanisms responsible for TDP-43 mislocalization, and the impact on neuronal health of nuclear TDP-43 exclusion and aberrant liquid-liquid phase separation underlying cytoplasmic demixing remain unknown. To address this challenge, in Aim 1, we systematically profile the transcriptional and epigenomic alterations of ALS and FTLD/MND patients at single-cell resolution using post-mortem CNS samples. In Aim 2, we integrate the resulting datasets to study the link between genetic, epigenomic, transcriptional, and cellular signatures of ALS and FTLD/MND. We associate these links with available clinical information, elucidate the genes and biological pathways altered in each, and predict new therapeutic targets. In Aim 3, we validate the molecular and cellular effects of these targets by assessing their impact on neuronal viability and TDP-43 functions/aggregation using high-throughput directed perturbation experiments. We study both cell-autonomous and non-cell-autonomous effects of these perturbations in human dura fibroblast-derived iPSC neurons and astroglia. In Aim 4, we perform neuropathological analyses of TDP-43 modifiers in ALS and FTLD/MND postmortem tissues, and endeavor to rescue in vivo pathology and phenotypes in a mouse model. The resulting datasets, analyses, and dura-derived iPSCs will provide an invaluable resource to understand the mechanisms of TDP-43 pathology in ALS and FTLD/MND, and may reveal putative therapeutic targets able to mitigate TDP-43 pathology through genetic manipulation.

Narrative Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two devastating and fatal neurodegenerative disorders that are clinically distinct but show an overlap in genetic factors and central nervous system (CNS) neuropathology in the form of cytoplasmic TDP-43 depositions, but the mechanistic basis of their shared and distinct circuitry remains unknown at the molecular level, preventing the identification of needed therapeutic targets. Here, we use single-cell high-resolution profiling of epigenomic and transcriptional alterations in post-mortem CNS samples from ALS and FTLD with motor neuron disease (FTLD/MND) patients to elucidate their underlying mechanism of action, shed light on their common and distinct circuitry, and identify genetic and cellular factors that drive TDP-43 neuropathology and corresponding neurotoxicity, using patient derived cells and in vivo model systems. This ambitious proposal has the potential to (a) provide mechanistic insights into the development of ALS and FTLD/MND, (b) identify genetic modifiers of TDP-43 pathobiology, and (c) reveal functional risk variants and target genes for therapeutic intervention using gene modifying therapies.