Proteasome-mediated proteolysis of the polyglutamine-expanded androgen receptor is a late event in spinal and bulbar muscular atrophy (SBMA) pathogenesis.

Proteolysis of polyglutamine-expanded proteins is thought to be a required step in the pathogenesis of several neurodegenerative diseases. The accepted view for many polyglutamine proteins is that proteolysis of the mutant protein produces a toxic fragment that induces neuronal dysfunction and death in a soluble form; toxicity of the fragment is buffered by its incorporation into amyloid-like inclusions. In contrast to this view, we show that, in the polyglutamine disease spinal and bulbar muscular atrophy, proteolysis of the mutant androgen receptor (AR) is a late event. Immunocytochemical and biochemical analyses revealed that the mutant AR aggregates as a full-length protein, becoming proteolyzed to a smaller fragment through a process requiring the proteasome after it is incorporated into intranuclear inclusions. Moreover, the toxicity-predicting conformational antibody 3B5H10 bound to soluble full-length AR species but not to fragment-containing nuclear inclusions. These data suggest that the AR is toxic as a full-length protein, challenging the notion of polyglutamine protein fragment-associated toxicity by redefining the role of AR proteolysis in spinal and bulbar muscular atrophy pathogenesis

Using cell and animal models to understand pathogenic mechanisms in spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is a late onset neurodegenerative disease caused by a polyglutamine expansion in the androgen receptor. In cell and animal models of SBMA, disease is dependent on hormone and nuclear localization of the AR. However, drugs that lower androgen levels were moderately effective in clinical trials. Moreover, the decreased androgen levels caused decreased mental wellbeing in patients. Therefore, an ideal therapy would prevent the toxicity of expanded-polyglutamine AR while maintaining AR function. A better understanding of disease pathogenesis will enable the development of such targeted therapies. ^ Here, we investigated the role of an interdomain interaction of the AR, between the amino-terminal FxxLF motif and carboxyl-terminal AF-2 domain (N/C interaction), in disease in a mouse model of SBMA. We created transgenic mice that express expanded-polyglutamine AR with a mutation in the FxxLF motif (F23A) to prevent the N/C interaction. We found that preventing the AR N/C interaction delayed disease onset, restored phosphorylation alterations in spinal motor neurons, and reduced aggregation of AR in the ventral horn of the spinal cord compared to male N/C-intact AR-expressing mice. Using cell models of SBMA, we determined that the decreased aggregation and toxicity afforded by the AR F23A mutation were dependent on serine 16 of the AR; mutating this residue abolished the protective effects of the AR F23A. ^ We next sought to examine whether cellular dysfunction was involved in disease pathogenesis. Prior studies in a mouse model of SBMA found substantial behavioral deficits without neuronal loss, suggesting that cellular dysfunction and not cell loss caused behavioral deficits in this mouse model of SBMA. Therefore we examined the neuromuscular junction at a late stage of disease progression for signs of synaptic dysfunction. We found substantial changes in endplate morphology and a decrease in the number of fully innervated endplates in the AR 112Q soleus compared to the non-transgenic soleus, suggesting a possible decrease in synaptic activity. Therefore, pharmacologically increasing synaptic activity could potentially improve patient outcome. ^ Together this thesis identified two specific therapeutic targets for treating SBMA and improving patient outcomes. Further studies should confirm the efficacy of these targets in treating this debilitating neurodegenerative disease.

Preventing the Androgen Receptor N/C Interaction Delays Disease Onset in a Mouse Model of SBMA

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by a polyglutamine expansion in the androgen receptor (AR) and is associated with misfolding and aggregation of the mutant AR. We investigated the role of an interdomain interaction between the amino (N)-terminal FxxLF motif and carboxyl (C)-terminal AF-2 domain in a mouse model of SBMA. Male transgenic mice expressing polyQ-expanded AR with a mutation in the FxxLF motif (F23A) to prevent the N/C interaction displayed substantially improved motor function compared with N/C-intact AR-expressing mice and showed reduced pathological features of SBMA. Serine 16 phosphorylation was substantially enhanced by the F23A mutation; moreover, the protective effect of AR F23A was dependent on this phosphorylation. These results reveal an important role for the N/C interaction on disease onset in mice and suggest that targeting AR conformation could be a therapeutic strategy for patients with SBMA