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.
Theproductionofmisfolded,aggregation-prone,proteinsinneurons,eitherthroughdisease-associatedmutationor as a consequence of reduced protein clearance capacity, is causally linked to the development of a host of devastating neurodegenerative diseases, including ALS/FTD. In this project we are using the fruit fly, Drosophila melanogaster,tounderstandhowmutationsinaproteinthatcriticallyparticipatesinproteinclearance?ubiquilin2 (UQ2)?trigger neurodegeneration. We have found that ALS/FTD-associated mutants of UQ2 disrupt neuronal function in Drosophila and have made progress toward understanding the molecular events through which this occurs. By carrying out genetic modifier screens in Drosophila, we hope to identify pathways whose perturbation either rescue or worsen UQ2-mediated neurodegeneration. In the future, this information may be leveraged to developchemicalsthatattenuateALS/FTD.
| Anderson, Eric N; Gochenaur, Lauren; Singh, Aditi et al. (2018) Traumatic injury induces stress granule formation and enhances motor dysfunctions in ALS/FTD models. Hum Mol Genet 27:1366-1381 |
| Kim, Sang Hwa; Stiles, Shannon G; Feichtmeier, Joseph M et al. (2018) Mutation-dependent aggregation and toxicity in a Drosophila model for UBQLN2-associated ALS. Hum Mol Genet 27:322-337 |
