CLN5 disease-causing mutations impact lysosomal biology by affecting intracellular degradation and protein trafficking.

Ceroid lipofuscinosis neuronal 5 (CLN5) disease is a subtype of neuronal ceroid lipofuscinosis (NCL, commonly known as Batten disease) that is caused by mutations in the CLN5 gene. While 70 distinct CLN5 disease-causing mutations have been documented, the pathological effects of these mutations are largely unknown. In this study, we used the model eukaryote Dictyostelium discoideum to examine the molecular and cellular effects of five CLN5 disease-causing mutations (p.Cys77Tyr, p.Trp158Ser, p.Tyr209Asp, p.Glu303*, and p.Tyr343*). We used informatics tools to show that the five mutations alter the predicted structure of the protein. We then introduced these mutations into Dictyostelium Cln5 and examined their effects on the localization and secretion of the protein, as well as proteostasis and lysosomal activity. We observed that the mutations alter the cellular distribution of Cln5 and intracellular catabolic mechanisms, including 20S proteasome-mediated protein degradation and lysosomal enzyme-mediated breakdown. The mutations also affect vesicles within the endo-lysosome pathway and the release of Cln5 and other lysosomal enzymes from cells, which impacts extracellular enzyme activity. Finally, while cell proliferation and aggregation are not affected by mutated Cln5, loss of the signal peptide in Cln5 delays aggregation, supporting an extracellular role for the protein. This study, which is the first to comprehensively examine the effects of the p.Cys77Tyr, p.Trp158Ser, p.Tyr209Asp, p.Glu303*, and p.Tyr343* mutations on cellular function, enhances our understanding of the effects of mutations in CLN5 on endo-lysosomal trafficking and lysosomal biology, as well as the pathological mechanisms underlying CLN5 disease.