The overarching goal of this study is to use complementary Drosophila and inducible pluripotent stem cells (iPSC)-derived neuronal models to understand how mutations in the Ubiquilin 2 (UBQLN2) gene cause amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). UBQLN2 and closely related UBQLN1 belong to a family of eukaryotic ubiquitin (Ub)-binding proteins that function, in part, as chaperones for proteins that are destined for proteasomal degradation. Missense mutations within a unique, functionally orphan proline-repeat region (PRR) of UBQLN2 cause X-linked, forms of ALS/FTD, with some patients exhibiting a pure dementia onset. In addition, ubiquilin histopathology, comprised of dense aggregates of UBQLN2 and UBQLN1 are observed in most instances of ALS/FTD regardless of UBQLN2 mutation status. To address pathomechanisms of UBQLN2-associated FTD we exploited the upstream activating sequence (UAS)/GAL4 system to generate isogenic Drosophila strains expressing wild-type (WT) and ALS mutant forms of UBQLN2 in different tissues and cell types. We found that UBQLN2ALS mutants elicited dose-dependent phenotypes? including eye degeneration, motor defects, and lifespan shortening?that were more severe than phenotypes caused by equivalent expression of UBQLN2WT. UBQLN2ALSmutants, but not UBQLN2WT, formed intraneuronal aggregates characteristic of ubiquilin inclusions found in ALS/FTD patients. Unbiased genetic screens identified more than 30 genetic intervals that either reduced or enhanced UBQLN2ALS mutant toxicity. Gene mapping studies suggest that endolysosomal pathways are centrally involved in UBQLN2ALS-mediated neurodegeneration and point toward axon guidance genes and neuronal dependence receptors as potentially novel disease modifiers. In this R01 grant proposal we will continue genetic studies of UBQLN2-mediated neurodegeneration in Drosophila, focusing on endolysosomal trafficking and axon guidance as lead pathways for discovery efforts. Modifier pathways identified in Drosophila will, in turn, inform studies of iPSC-derived motor neurons (iMNs) expressing endogenous UBQLN2ALS alleles. The specific objectives of Aim 1 are to: (a) investigate the Rab5 gene and endolysosomal defects in UBQLN2ALS flies; (b) investigate the contributions of the Unc-5 axon guidance pathway to UBQLN2-mediated neurodegeneration; and (c) map and validate of UBQLN2ALS modifier genes in Drosophila. The specific objectives of Aim 2 are to: (a) establish functional defects in UBQLN2ALS iMNs; (b) carry out proteomic analysis of UBQLN2ALS iMNs; and (c) evaluate genes emerging from Drosophila screens in Aim 1 as genetic modifiers of UBQLN2ALS toxicity in iMNs. The combined genetic, cellular, and biochemical studies using Drosophila and mammalian iMN models will provide important new insights into UBQLN2-associated neurodegeneration in ALS/FTD. In addition, pathways identified in our study are likely to overlap with and inform toxicity pathways instigated by other aggregation-prone, ALS/FTD- associated genes.
The production of misfolded, aggregation-prone, proteins in neurons, either through disease-associated mutation or as a consequence of reduced protein clearance capacity, is causally linked to the development of devastating neurodegenerative diseases, including frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In this project we are using the fruit fly, Drosophila melanogaster, to discover pathways that diminish toxicity of an aggregation-prone protein termed UBQLN2 whose mutation is linked to familial forms of FTD and ALS. Discoveries made in flies will be leveraged to understand how UBQLN2 mutations instigate toxicity in stem cell-derived human neurons. In the future, this information may facilitate the development of drugs that attenuate ALS/FTD.