SUMMARY The combined degeneration of both “lower” motor neurons in the brainstem and spinal cord and “upper” motor neurons (UMNs) in the cerebral cortex is an important hallmark of ALS. Almost all cases of ALS are eventually fatal, and the rapid progression of the disease makes it particularly terrible, with over 80% of patients dying within five years of diagnosis. No cure exists for ALS and the only available treatments slow disease progression by merely a few months. Therefore, a great need exists for more effective and specific therapies that can stop or even reverse neurodegeneration. Innovation for such therapies will only arise from a better understanding of the molecular mechanisms underlying the pathological process. Most genes linked to ALS are ubiquitously expressed yet only specific populations of cells degenerate. Understanding why certain cells are uniquely vulnerable and mapping cell type specific pathways that are dysregulated during disease are crucial milestones for developing innovative therapies. This has posed a considerable challenge for the spinal cord-projecting upper motor neurons (UMNs) since they are difficult to distinguish from other pyramidal cell types and are therefore often overlooked in preclinical studies. Because of this, the basis for their selective vulnerability to disease-causing mutations has remained a mystery. The proposed study aims to overcome this by building on recent work that identified two highly similar yet molecularly distinct subpopulations of projection neurons in layer 5b of motor cortex, where UMNs reside. These populations have overlapping projections to pons, but non-overlapping projections to the spinal cord or thalamus. Examining these cells in preclinical models of ALS revealed that the corticospinal projecting neurons (CSTNs) were vulnerable to degeneration, while the corticopontine-only population (CPN) did not degenerate. The selective vulnerability of the CSTNs was likely due to dysregulation of mitochondrial function since a dramatic upregulation of genes related to oxidative phosphorylation and mitophagy was observed at symptomatic stages of disease. Aim 1 of this grant will employ an integrative multi-omics approach to address whether differences in the properties of mitochondria between CSTNs and CPNs drive differential responses to disease using a novel, viral-based strategy to isolate cell type specific mitochondria during disease progression in two preclinical ALS models, SOD1G93A and FUSP525L. Aim 2 focuses on characterizing the cellular role of identified candidate genes that are enriched in CSTNs, shown to localize to mitochondria, and have been directly linked to clinical cases of ALS and other neurodegenerative disorders. To increase the translational significance of this work, Aim 3 will leverage novel markers for CSTNs and CPNs for a detailed anatomical analysis of postmortem tissue from ALS patients perform transcriptional profiling on cell type specific nuclei isol...