Drosophila models for ubiquilin-associated ALS

University Of Wisconsin-Madison, Madison WI

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Abstract

Project Summary The overarching goal of this study is to use the fruit fly, Drosophila melanogaster, to understand how mutations in the Ubiquilin 2 (UBQLN2) gene cause amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). UBQLN2 (UQ2) and closely related UBQLN1 (UQ1) belong to a family of eukaryotic ubiquitin (Ub)-binding proteins that function, in part, as chaperone factors for proteins that are destined for degradation by the proteasome. UQ1 and UQ2 share 74% amino acid identity, with the most striking difference being a proline-rich-repeat (PRR) domain that is unique to UQ2. Rare missense mutations within the UQ2 PRR cause familial, X-linked, forms of ALS/FTD, whereas ubiquilin histopathology, comprised of dense aggregates of UQ2 and UQ1, are observed in most instances of ALS/FTD regardless of UBQLN2 mutation status. To address pathomechanisms of UQ2-associated ALS/FTD we exploited the upstream activating sequence (UAS)/GAL4 system to generate isogenic Drosophila strains expressing wild-type (WT) and ALS mutant forms of UQ2 in different tissues and cell types. We found that UQ2ALS mutants elicited dose-dependent phenotypes?including eye degeneration, motor defects, and lifespan shortening?that were more severe than phenotypes caused by equivalent expression of UQ2WT. UQ2ALS mutants, but not UQ2WT, formed intraneuronal aggregates characteristic of ubiquilin inclusions found in ALS/FTD patients. The formation of these inclusions required the Ub-binding activity of UQ2, raising the possibility that UQ2 aggregation?and potentially its toxicity?are coupled to its functions as an Ub chaperone. Finally, we provide evidence that expression of UQ2ALS mutants worsens degenerative phenotypes associated with hexanucleotide repeat expansions in the C9ORF72 gene that comprise the single most common genetic cause of ALS/FTD. In this exploratory R21 grant proposal we will leverage the Drosophila UQ2-ALS model to answer the following questions concerning the mechanisms of UQ2 toxicity: (i) Do UQ2ALS mutants perturb neuronal regulation by disrupting proteostasis and Ub homeostasis? (ii) What are the relationships between Ub binding, aggregation, and neurotoxicity of UQ2ALS mutants; can we identify additional domains that required for neurotoxicity? (iii) What pathways contribute to UQ2-mediated neurodegeneration? The proposed experiments will illuminate intra- and intermolecular determinants of UQ2-mediated neurodegeneration and may identify protein interfaces that can be targeted with small molecules to disrupt pathologic ubiquilin aggregation and toxicity in ALS/FTD.