Summary Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder associated with loss of upper and lower motor neurons. Some ALS patients also develop frontotemporal dementia (FTD). Genetic findings have linked mutations in different genes to the range of symptoms seen in ALS. This proposal focuses on UBQLN2, missense mutations in which cause dominant inheritance of ALS-FTD. UBQLN2 is one of four ubiquilin (UBQLN) proteins found in humans. UBQLN proteins function to clear misfolded proteins from cells through the proteasome and autophagy pathways. This function could be important in neurodegenerative diseases, where a build up of misfolded proteins is frequently seen. Therefore, understanding how UBQLN proteins function and dysfunction has broad implications for neurodegenerative diseases. Animal models of the disease are useful for understanding the mechanisms of pathogenesis and for therapeutic studies. Toward such a goal, we generated and characterized transgenic mice carrying Thy1.2 promoter-driven expression of human UBQLN2 proteins encoding either the wild type (WT) or the P497S or P506S mutants that cause ALS- FTD. Mouse lines carrying each of the mutations were found to develop motor neuron disease and cognitive deficits, mimicking the human disease, whereas the WT mice were devoid of motor neuron disease. Immunoblots of spinal cord proteins revealed a dramatic reduction in TBK1 levels in animals with end-stage disease in both the mutant UBQLN2 mouse lines compared to non-transgenic animals. The reduction could be significant because haploinsufficiency of TBK1 expression caused by TBK1 mutations were recently linked to ALS-FTD. Prompted by these relationships, we examined whether TBK1 binds with UBQLN2. Double immunofluorescence staining indicated TBK1 and UBQLN colocalize in cells in autophagosomes. Furthermore, by both immunoprecipitation and GST-pulldown assays we found WT UBQLN2 binds TBK1, but the ALS UBQLN2 mutant proteins bind more TBK1. We hypothesize that the increased binding with UBQLN2 mutants alters one or both of the protein's functions. Accordingly, we propose experiments in Aim 1 to study the functional significance of the interaction, and how mutations in UBQLN2 affect this interaction especially with regard to the function of the proteins in autophagy, which we found is disturbed in our mutant UBQLN2 mouse lines. In Aim 2, we will determine whether transgenic overexpression of TBK1, in an effort to restore its levels in our UBQLN2 mice, will extend survival and delay ALS-FTD symptoms. In Aim 3, will investigate the exciting possibility, supported by our preliminary studies in double transgenic mice, that overexpression of UBQLN1 in our mtUBQLN2 lines will delay ALS-FTD symptoms. The outcome of this work is likely to be important both in terms of its implications for our understanding of the underlying mechanisms involved in ALS-FTD pathogenesis and because it could reveal whether modulati...