ALS/FTD-Linked UBQLN2 Mutations Result in Aberrant Accumulation and Aggregation of Serine Protease Inhibitor Proteins

Abstract

Mutations in the protein UBQLN2, which functions in protein quality control, cause amyotrophic lateral sclerosis with frontotemporal dementia (ALS/FTD). However, the mechanism(s) by which the mutations cause disease remains unclear. In this study, we used proteomic profiles generated from 8-week-old transgenic mice expressing either P497S mutant (P497S line) or wildtype UBQLN2 (WT356 line), and non-transgenic (Non-Tg) mice to gain insight into the pathophysiologic mechanisms driving disease. The P497S line recapitulates key features of human ALS/FTD, including motor neuron disease, cognitive impairments, and pathologic accumulation of UBQLN2 inclusions; symptoms and pathology which are largely absent in the WT356 line. Comparison of proteomic changes in the hippocampus and spinal cord of these animals revealed several members of the serine protease inhibitor (serpin) protein family are more highly expressed in P497S animals compared to either WT356 or Non-Tg controls. Serpins function in the regulation of proteolytic cascades by entrapping and destroying proteases through a conformational switch of the protein. However, this metastability makes serpins highly prone to misfolding and polymerization, placing a high demand on protein degradation systems to remove the misfolded proteins. Thus, we tested the hypothesis that ALS/FTD mutations in UBQLN2 perturb proteostasis, causing aberrant accumulation of misfolded serpin proteins. Double immunofluorescent staining revealed colocalization of Serpin A1, C1 and I1 with UBQLN2 inclusions that form in the brain and spinal cord of P497S mice. To further investigate the aberrant accumulation and mislocalization of serpins, we performed aggregation assays on cortical tissue from 8- and 32-week-old P497S, WT356, and Non-Tg animals, which revealed that serpin proteins have an increased tendency to aggregate in P497S animals compared to control mice. We also studied the effects of altered UBQLN2 expression on serpin protein levels and aggregation and found that UBQLN2 knockout induces serpin aggregation while overexpression of WT or mutant UBQLN2 oppositely affected serpin protein levels. These results are consistent with ALS/FTD mutations causing aberrant serpin accumulation through a loss-of-function mechanism. Taken together, this study identifies a novel role for UBQLN2 in the proper regulation of serpin expression and suggests that misaggregation of serpin proteins may be a pathologic consequence of UBQLN2 mutations.