Mechanism of ALS/FTD determined by multimodal single cell spatial transcriptomics - PROJECT SUMMARY Abnormal cytoplasmic accumulation and nuclear depletion of the RNA-binding protein TDP-43, collectively referred to as TDP-43 proteinopathy, has been reported in 40% of frontal temporal dementia (FTD), 97% of instances of amyotrophic lateral sclerosis (ALS), 50% of Alzheimer's disease (AD), and 100% of limbic- predominant age-related TDP-43 encephalopathy (LATE). Across this disease spectrum, neurons develop distinct stages of TDP-43 mislocalization and aggregation. A major genetic cause of FTD and ALS is a GGGGCC (G4C2) hexanucleotide expansion in the C9ORF72 gene (C9orf72 in mice). While a normal human C9ORF72 allele has less than 20 G4C2 repeats, pathogenic repeats in disease are in the hundreds, with somatic expansion up to 4000 repeats. There is consensus that TDP-43 nuclear loss and cytoplasmic aggregation are hallmarks of C9ORF72 repeat expansion-mediated neurodegenerative disease. However, the precise mechanism by which an expanded G4C2 repeat initiates pathogenesis is not established. Three primary mechanisms have been proposed: 1) haploinsufficiency of C9ORF72 protein, 2) toxic RNA foci generated from sense and/or antisense repeat-containing transcripts, and/or 3) accumulation of dipeptide repeat (DPR) protein products. To identify mechanisms of pathogenesis in TDP-43 proteinopathies, we have developed a Multimodal MERFISH (Multiplexed Error-Robust Fluorescence in situ Hybridization) approach, enabling the measurement of key candidate proteins (including TDP-43 and DPRs) to determine cellular pathologic state while simultaneously measuring any effect on the corresponding transcriptome. By integrating this approach with single-nuclear RNA sequencing, our extensions allow for the determination of nearly complete transcriptomes in up to 80,000 individual cells within a single intact tissue slice. This approach preserves the critical spatial context within intact tissue and enables correlating pathologic protein mislocalization with measurements of somatic repeat expansion and single-cell transcriptomic RNA profiles. With this technology, we will determine which cell types and genes are affected by TDP-43 proteinopathy, first in sporadic ALS. Next, we will extend this analysis to C9ORF72-ALS/FTD to determine which cell types and genes are affected by repeat expansion-mediated C9ORF72 pathological hallmarks, TDP-43 proteinopathy, as well as the presence and extent of somatic repeat expansion. Finally, we will determine whether any genes and/or disease mechanisms are exacerbated by reduced C9orf72 activity. By generating and examining cohorts of mice with ALS-like motor neuron degeneration from expression of a G4C2 hexanucleotide repeat with normal, reduced, or complete absence of C9orf72 protein, we will leverage Multimodal MERFISH to determine RNA transcriptomes in all cells as well as the presence of TDP-43 proteinopathy, sense/antisense RNA foci, and DPR accumulation from early to late disease stages. This analysis will determine whether, and if so how, C9orf72 loss of function affects repeat expansion-linked toxicity.
