The role and timing of aberrant polyglutamine-expanded androgen receptor proteolysis in cell models of spinal and bulbar muscular atrophy
Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease resulting from a CAG repeat expansion, encoding a polyglutamine tract in the amino-terminus of the androgen receptor (AR). A histopathological hallmark is the presence of intranuclear inclusions containing amino-terminal portions of the polyglutamine-expanded AR. The aberrant protein cleavage frequently seen in polyglutamine repeat diseases has long been thought a critical step to pathogenesis, but the precise role of proteolysis and whether the resulting fragment is itself toxic or an indicator of disease in SBMA remains unknown. A canonical model describes proteolysis of the mutant protein producing a soluble "toxic fragment" inducing neuronal dysfunction and death; toxicity of the fragment is buffered by incorporation into inclusions. In contrast, we show, using cell models and biochemical techniques, that in SBMA, proteolysis of the mutant AR to a toxic fragment does not occur. Instead, the mutant AR aggregates as a full-length protein, becoming proteolyzed to an amino-terminal fragment through a proteasome dependent process after its incorporation into intranuclear inclusions. Moreover, the toxicity-predicting antibody 3B5H10 binds to soluble full-length AR species but not to fragment-containing nuclear inclusions. We also identify a specific region of aberrant cleavage in a PC12 cell model of SBMA using mass spectrometry to identify carboxyl-terminal residues, and address the necessity of this cleavage for toxicity and inclusion formation. We show that AR fragments from purified inclusions terminate at residue L163 and/or D146. Mutation of these sites does not prevent inclusion formation or AR cleavage, but does prevent DHT-dependent toxicity in a cell model of SBMA, suggesting a critical role for these residues in the toxicity of polyglutamine-expanded AR. In addition, we show that inclusion formation is dependent on the protease activity of the proteasome, suggesting its involvement in aberrant cleavage and inclusion formation. Taken together, these data fundamentally alter the order of events involving the AR in SBMA, placing cleavage downstream of both toxicity and aggregation. This redefines the role of AR proteolysis in SBMA pathogenesis and describes a critical role for the proteasome in these processes, redirecting attention to full-length AR as the toxic species in SBMA.
Heine, Erin M, "The role and timing of aberrant polyglutamine-expanded androgen receptor proteolysis in cell models of spinal and bulbar muscular atrophy" (2013). ETD Collection for Thomas Jefferson University. AAI3603210.