Mapping the ALS Citrullinome: A Proteomic Perspective on Neurodegeneration, Aggregation, and Protein Dysfunction

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that is characterized by progressive motor neuron loss, muscle wasting, paralysis, and death. Ninety percent of the cases are sporadic (sALS), while the remaining ten percent are familial (fALS). A hallmark of ALS neuropathology is aberrant protein aggregation and inclusion body formation in neurons leading to eventual degeneration of motor neurons in the brain and spinal cord. Previously, we showed that protein citrullination (PC), a post-translational modification (PTM), and peptidyl arginine deiminase 2 (PAD2) expression are altered dynamically in the spinal cord during ALS disease progression, increasing in astrocytes while decreasing in neurons (PMID: 36076282, 38253209). Interestingly, the citrullinated proteins accumulate in myelin protein aggregates, suggesting a role of PC in protein aggregation. Here, we applied proteomic methods to identify citrullinated proteins in an ALS mouse model expressing mutant SOD1G93A. The ALS citrullinome profiles disease progression distinctly from normal aging in mice and highlighted an increase in citrullinated glial proteins and a decrease in citrullinated neuronal proteins, validating PC as a marker of reactive astrogliosis and neurodegeneration. Additional analyses found that soluble citrullinated proteins were enriched in inflammation, membrane traffic and metabolic pathways, whereas for insoluble citrullinated proteins, myelin proteins were enriched. The findings in the soluble fraction were validated in analysis of two human ALS proteomic datasets. Among the highly citrullinated proteins in ALS are heat shock proteins and MBP. PC severely compromises these proteins’ structure and function. These results demonstrate the impact of PC in protein function, and furthermore, suggest that PC could provide candidate biomarkers of early-stage ALS and be targeted with novel ALS therapeutics.