Structural and Functional Features of the Polyglutamine-Expanded Androgen Receptor in Spinal and Bulbar Muscular Atrophy
Polyglutamine-repeat disorders are part of a larger family of neurodegenerative diseases characterized by protein misfolding and aggregation. A leading hypothesis for many polyglutamine proteins is that proteolysis of the mutant protein produces a polyglutamine-containing fragment that is most toxic in a soluble form, prior to fragment accumulation into insoluble inclusions. Despite this view, recent data demonstrate that the native function of the protein affected by polyglutamine-expansion is important in disease. The focus of this work was to further our understanding of the relationship between native AR function, aggregation, and toxicity using cell and mouse models of SBMA that express the full-length polyglutamine-expanded AR protein. To investigate the role of native AR function in protein aggregation, we developed sensitive biochemical assays in order to compare the temporal formation of soluble and insoluble aggregation species with that of nuclear inclusions and motor neuron degeneration. We demonstrated that soluble conformations of polyglutamine-expanded AR aggregation species form early in the course of hormone treatment and contain the full-length polyglutamine-expanded AR protein; proteasome-mediated cleavage to an amino-terminal fragment was only observed after the formation of insoluble protein species and nuclear inclusions, suggesting that early aggregation events precede detectable AR proteolysis. Additionally, soluble, full-length polyglutamine-expanded AR aggregation species were demonstrated to bind the conformation-specific and toxicity-predicting antibody, 3B5H10. We found that soluble 3B5H10-reactive aggregation species exist in low-density conformations and are larger by atomic force microscopy, suggesting that they may be less compact than later-stage insoluble aggregates. We further showed that these species exist in vivo and demonstrated correlations with toxicity in vitro, using a cell line that expresses a protective mutation of the polyglutamine-expanded AR in an otherwise toxic native functional domain. To further explore the relationship between AR native function and toxicity, we investigated the role of AR transactivation, a major native function of the AR, in SBMA. Using 2 different cell models of SBMA, we demonstrated that cells expressing DNA-binding deficient polyglutamine-expanded AR still display hormone-dependent toxicity, suggesting that AR transcriptional activity is not absolutely necessary for the toxicity observed in SBMA. Taken together, the data presented in this thesis contribute to a novel model of aggregation in SBMA, based on the incorporation of full-length polyglutamine-expanded AR protein into early aggregation species, with unique structural features.
Berger, Tamar R, "Structural and Functional Features of the Polyglutamine-Expanded Androgen Receptor in Spinal and Bulbar Muscular Atrophy" (2016). ETD Collection for Thomas Jefferson University. AAI10044510.