Mitochondrial Dysfunction is Linked to Pathogenesis in the P497S UBQLN2 Mouse Model of ALS/FTD

Abstract

Ubiquilin-2 (UBQLN2) mutations cause amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD), but the mechanisms that drive disease pathogenesis remain unclear. Neurons have especially high energy requirements and consume copious amounts of ATP to support synaptic transmission and other complex processes. As such, mitochondrial dysfunction has been thought to play a pathogenic role in ALS. Recently, UBQLNs have been implicated in mitochondrial protein quality control whereby their inactivation in cells leads to the accumulation of cytostatic mitochondrial precursors. However, it is unclear what specific role UBQLN2 plays in maintaining mitochondrial proteostasis and how UBQLN2 mutations impact mitochondria physiology. In this thesis, I tested my hypothesis that the ALS-linked UBQLN2 P497S mutation causes mitochondrial dysfunction through loss of UBQLN2 chaperone function and impaired mitochondrial import. Our lab previously generated proteomic profiles of early-stage (8 weeks) hippocampal and spinal cord (SC) tissues isolated from non-transgenic (Non-Tg), wild-type (WT356), and P497S UBQLN2 mutant mice, whereby the mutant animal closely models human ALS/FTD. Gene ontology analysis revealed “mitochondrial proteins” as a major category altered in P497S animals. I immunoblotted SC lysates of Non-Tg, WT356 and P497S UBQLN2 animals for various mitochondrial proteins, and found decreased levels of many mitochondrial proteins, including those involved in oxidative phosphorylation (OXPHOS), network dynamics and import. I discovered through Seahorse respiration assays that mitochondria purified from the SC of P497S mice have age-dependent respiration deficits unlike those of age-matched Non-Tg and WT356 animals. Electron microscopy of spinal motor neurons in the P497S animals revealed distortions to mitochondria cristae. I demonstrated that mitochondrial alterations found in P497S mutant animals are recapitulated in UBQLN2 knock-out cells, suggesting loss of UBQLN2 function may underlie the mutation’s effects. Additionally, inactivation of UBQLN2 compromised proper targeting and processing of the mitochondrial import factor, TIMM44, which subsequently could be rescued by reexpression of WT UBQLN2, but not by mutant UBQLN2 proteins. ALS/FTD UBQLN2 mutants bind weaker to TIMM44 compared to WT UBQLN2, providing a possible mechanism for the mitochondrial import defects. Overall, these studies highlight a potential key role of UBQLN2 in maintaining mitochondrial health, and how its function is impaired by mutations in UBQLN2.