Amyotrophic lateral sclerosis (ALS) is a devastating human neurodegenerative disorder that is manifested in the degeneration of upper and lower motor neurons. ALS has higher incidence in U.S. military Veterans than in the general population and is considered a service-connected condition. Understanding the biological basis of ALS remains a major challenge, which is largely due to the complexity of the human central nervous system, which contain vast numbers of specialized cell types. Whereas the original focus of ALS research was concentrated on motor neurons, the non-neuronal cell types have also been suggested to play a crucial role in motor neuron death. Previous studies used genome-wide analysis of gene expression in bulk brain tissues to assess transcriptional changes associated with ALS. However, information on key changes that could affect different cell types in ALS brain remains limited. One reason is that changes affecting a particular cell type cannot be reliably inferred from data on bulk brain specimens that conflate signals from all cell types. The majority of ALS cases (~90%) occur sporadically (sALS) with unknown etiology, while ~10% of cases are classified as familial (fALS). To date, mutations in more than 50 genes have been linked to fALS. Expansion of the hexanucleotide repeat in C9orf72 (C9) gene is the most common cause of ALS and another neurodegenerative disorder, frontotemporal dementia (FTD), accounting for ~ 11% of all ALS and ~13% of all FTD cases. We recently performed single nucleus (sn)RNA-seq analysis using autopsied motor and prefrontal cortices from ALS and FTD cases with a C9 mutation and from controls. We identified disease-related changes in many cell types, including shared effects in ALS and FTD, and numerous disease-specific alterations. Among other findings, we detected changes in gene expression in endothelial cells, astrocytes, and excitatory neurons from C9-ALS cases that suggest a specific intercellular pathway that might, at least in part, underlie an ALS- associated glutamate (Glu) excitotoxicity. Our application aims to address the following issues: (1) Our snRNA- seq studies were limited to C9 cases; therefore, it is not known if the observed cell-type-dependent deficits are specific for C9-ALS or are also present in patients with sALS. (2) Although ALS typically leads to death within 3 to 5 years after initial symptom onset, approximately 10% of patients with ALS live significantly longer (>10 years after symptom onset; hereafter named “long duration ALS”) [10]. The molecular underpinnings of these differences have not been investigated. To address these issues, we propose the following Aims: Aim 1: To study cell-type-specific transcriptional dysregulation in the brains of U.S. military Veterans with sALS in single cell resolution. Hypothesis: Transcriptional deficits that we identified in C9-ALS patients and which, at least in part, explain the ALS-associated Glu excitotoxicity, are also present...