Project Summary Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that causes motor neuron degeneration, muscle weakness, paralysis, and death. The mechanism of motor neuron degeneration is incompletely understood, and currently, no therapy can arrest or reverse the disease progression. To understand the disease and open new avenues for therapy, exploring new mechanisms of the disease is needed. To this end, the Xu and Thompson labs have joined forces to investigate the role of protein citrullination, which is catalyzed by Protein Arginine Deiminases (PADs), in ALS. Citrullination removes positive charges from proteins. Therefore, this modification can alter the protein function and its interaction with other proteins, membranes, and nucleic acids. Mammals have five PADs: PADs 1–4 and PAD6. PAD2 is the dominant form in the central nervous system (CNS). Previous studies have shown that PAD2 and protein citrullination are increased in neurodegenerative diseases such as Alzheimer's disease and Prion disease. However, no studies have linked PADs and protein citrullination with ALS. Furthermore, no studies have investigated the functional role of PAD2 and protein citrullination in neurodegeneration. To fill these knowledge gaps, the Xu and Thompson labs have applied their combined expertise in ALS and protein citrullination and initiated this investigation. Our preliminary studies show unequivocal evidence that PAD2 expression (but not PAD3, 4) and protein citrullination are spatially and temporally altered in two ALS mouse models, one expressing mutant SOD1G93A and the other expressing mutant PFN1C71G. While PAD2 expression is increased in astrocytes, its expression is decreased in neurons during the disease progression. By proteomics, we identified several hundred citrullinated proteins in the spinal cord. In ALS mice, the early disease stage is dominated by decreased protein citrullination. In contrast, the late disease stage shows increased citrullination in one-half of the proteins and decreased citrullination in the other half. Intriguingly, the highly citrullinated proteins are enriched in the insoluble fractions in the late stage. We propose to further our investigation by answering the following seven questions: (1) How is protein citrullination altered in ALS? (2) What are the citrullinated proteins in the spinal cord, and how is citrullination altered in these proteins in ALS? (3) What are the sites of citrullination on ALS-associated proteins? (4) Is PAD2 responsible for the altered protein citrullination in ALS? (5) How does protein citrullination impact protein aggregation? (6) How does the citrullination of ALS-associated targets impact their protein function? (7) How is protein citrullination altered in human ALS? By answering these questions, we will enter and investigate a hitherto unexplored research area in ALS, thereby opening a new dimension in understanding the mechanism of neurodegeneration in ALS. ...