Summary UBQLN2 mutations cause X-linked dominant inheritance of amyotrophic lateral sclerosis with frontotemporal dementia (ALS/FTD). The encoded ubiquilin-2 protein (UBQLN2) belongs to a small family of conserved proteins that function to maintain proteostasis by binding and disposing ubiquitinated proteins through the proteasome and autophagy-lysosomal degradation systems. There is increasing evidence that disruption in proteostasis by interference in either of these clearing systems cause neurodegeneration. Therefore, understanding how UBQLN2 proteins function and dysfunction has broad implications for neurodegenerative diseases. The mechanisms by which UBQLN2 mutations cause pathogenesis are emerging. Towards this goal, we generated transgenic (Tg) mouse models for the P497S and P506T UBQLN2 mutations, showing that both mutant lines recapitulate central features of the human disease, including deposition of UBQLN2 inclusions, cognitive deficits, motor neuron disease and TDP-43 pathology. Through proteomic and immunoblot analysis we found that P497S mutant mice have major alterations in proteins involved in autophagy and in proteins required for mitochondrial health. Using a novel reporter system in combination with UBQLN2 knockout (KO) cells, we found ALS/FTD-linked mutations in UBQLN2 impede autophagy by blocking autophagosome acidification. We tied the defect to a novel function of UBQLN2 in regulation of the vacuolar(H+)-ATPase pump. Respiration assays of mitochondria purified from the spinal cord revealed mutant UBQLN2 animals have an age-dependent decline in oxidative phosphorylation. Similar defects were found in UBQLN2 KO cells, suggesting the functional decline in mitochondrial activity may stem from loss of UBQLN2 function. In support of this idea, we found that wild type (WT) UBQLN2 rescued the mitochondrial function deficits whereas UBQLN2 bearing an ALS/FTD mutation did not. Furthermore, we found WT UBQLN2 is required and regulates mitochondrial protein import whereas ALS/FTD mutant UBQLN2 proteins are deficient of the activity. For this renewal, we propose 4 aims that will capitalize on these exciting discoveries, as well as other important findings made during the last funding period. In Aim 1 we will determine the molecular mechanisms by which UBQLN2 functions in vacuolar-ATPase regulation and clarify why ALS/FTD UBQLN2 mutant proteins are disrupted in the activity. In Aim 2 we will determine the molecular mechanisms underlying UBQLN2 function and dysfunction in mitochondrial protein import and activity. Aim 3 is to test whether overexpression of UBQLN1 alleviates disease in SOD1 mouse models of ALS. Aim 4 is to recover the two mutant UBQLN2 mouse lines from sperm cryo-stocks for better phenotypes. The results of this research are likely to considerably advance our knowledge of the mechanisms by which UBQLN2 proteins function in health and disease, the lessons of which could be exploited for therapeutic intervention in not only...