Cytoplasmic retention of polyglutamine-expanded androgen receptor ameliorates disease via autophagy in a mouse model of spinal and bulbar muscular atrophy

Abstract

The nucleus is the primary site of protein aggregation in many polyglutamine diseases, suggesting a central role in pathogenesis. In SBMA, the nucleus is further implicated by the critical role for disease of androgens, which promote the nuclear translocation of the mutant androgen receptor (AR). To clarify the importance of the nucleus in SBMA, we genetically manipulated the nuclear localization signal of the polyglutamine-expanded AR. Transgenic mice expressing this mutant AR displayed inefficient nuclear translocation and substantially improved motor function compared with SBMA mice. While we found that nuclear localization of polyglutamine-expanded AR is required for SBMA, we also discovered, using cell models of SBMA, that it is insufficient for both aggregation and toxicity and requires androgens for these disease features. Through our studies of cultured motor neurons, we further found that the autophagic pathway was able to degrade cytoplasmically retained expanded AR and represents an endogenous neuroprotective mechanism. Moreover, pharmacologic induction of autophagy rescued motor neurons from the toxic effects of even nuclear-residing mutant AR, suggesting a therapeutic role for autophagy in this nucleus-centric disease. Thus, our studies firmly establish that polyglutamine-expanded AR must reside within nuclei in the presence of its ligand to cause SBMA. They also highlight a mechanistic basis for the requirement for nuclear localization in SBMA neurotoxicity, namely the lack of mutant AR removal by the autophagic protein degradation pathway.

Nuclear location of an endogenously expressed antigen, EBNA1, restricts access to macroautophagy and the range of CD4 epitope display

“…Furthermore, although the processing route was not investigated, the tumor antigen CDC27 is better presented to CD4 + T cells when located in the cytoplasm rather than the nucleus (24). In addition, two polyglutamine-expanded proteins involved in the pathogenesis of neurologic disease, ataxin-5 (25) and androgen receptor (26), resist degradation by autophagy in the nucleus but not the cytoplasm. Such findings are consistent with autophagy being predominantly a cytoplasmic process, with autophagosomes mainly forming in the cell periphery far away from the nucleus (21).…”

Proteasome-mediated Proteolysis of the Polyglutamine-expanded Androgen Receptor Is a Late Event in Spinal and Bulbar Muscular Atrophy (SBMA) Pathogenesis

“…In addition, observations that expression of a truncated AR results in substantially greater toxicity (18,31,41) than does expression of the fulllength expanded polyglutamine protein (7,11,29) suggested that proteolysis of the polyglutamine-expanded AR produces a toxic and aggregation-prone fragment. We therefore sought to explore the mechanism and timing of AR cleavage.…”

“…Immunofluorescence-PC12 cells, primary neuronal cultures, and mouse tissue sections were fixed and stained as described previously (11). Antibodies used included AR(H280) (1:100), AR(441) (1:50) (Santa Cruz Biotechnology), 3B5H10 (1:4000) (a kind gift from Steve Finkbeiner, Gladstone Institute of Neurological Disease), anti-FLAG antibody (1:100) (Pierce), and SMI32 (1:1000) (Sternberger Monoclonals Inc., Baltimore, MD).…”

“…Primary Dissociated Spinal Cord Cultures and AAV Infection-Dissociated spinal cord cultures were established according to Roy et al (28) with modifications as described (11,29,30). Briefly, spinal cords from non-transgenic 13.5-day-old mouse embryos were dissected on ice in dissection medium (0.1% dextrose, 2% sucrose, 6.8 mM NaCl, 0.27 mM KCl, 8.4 M Na 2 HPO 4 , 11 M KH 2 PO 4 , and 9.9 mM HEPES), pooled, dissociated with trypsin, plated on poly-D-lysine (Sigma)-coated German glass coverslips (Chemglass Life Sciences), and cultured for 3 weeks in glial conditioned medium (minimum essential medium, 3% charcoal-stripped horse serum, 35 mM NaHCO 3 , 0.5% dextrose, 1% N3, 10 nM 2.5S nerve growth factor).…”

“…While signs of androgen insensitivity are sometimes seen in SBMA, the disease is not due to a loss of AR function but rather to a gain of a toxic property of the mutant polyglutamine-expanded androgen receptor. Although the precise mechanistic pathway leading to AR misfolding, aggregation, and its subsequent toxicity has not been fully elucidated, it is known that binding of one of the natural AR ligands, testosterone or 5␣-dihydrotestosterone, and nuclear localization are required for both motor dysfunction and neuropathology (9,11,12).…”