Biofluid Proteomic Biomarker Discovery in a Large Animal Model of CLN2 Neuronal Ceroid Lipofuscinosis.

CLN2 disease is a fatal lysosomal storage disorder caused by deficiency of the lysosomal enzyme tripeptidyl peptidase 1 (TPP1), resulting in progressive neurodegeneration and non-neuronal pathology. While enzyme replacement therapy has improved disease management, there remains a critical need for objective molecular biomarkers capable of monitoring disease progression and therapeutic response. The purpose of this study was to utilize a canine model of CLN2 disease to investigate proteomic changes in cerebrospinal fluid (CSF) and in minimally invasive biofluids tears and plasma to identify candidate disease biomarkers. Untargeted proteomic analysis was conducted using these biofluids from unaffected controls and CLN2-affected dogs at presymptomatic, symptomatic, and advanced disease stages. CSF demonstrated the greatest degree of proteomic alterations over the disease progression. Proteins associated with cytoskeletal organization, synaptic signaling, lysosomal biology, mitochondrial metabolism, lipid homeostasis, and inflammatory responses were significantly altered. Tear fluid demonstrated overlapping biological themes with CSF, particularly involving mitochondrial metabolism, lysosomal function, and lipid-associated pathways. In contrast, plasma proteomic alterations were comparatively modest but supported systemic changes associated with extracellular signaling, inflammation, and lipid metabolism. Collectively, these findings demonstrate progressive and coordinated proteomic alterations across multiple biofluids in CLN2 disease that can serve as candidate biomarkers of disease progression.