Characterization of intracellular aggregates using fluorescently-tagged polyglutamine-expanded androgen receptor.

Spinal bulbar muscular atrophy (SBMA) is a classic CAG-repeat neurodegenerative disease. It is caused by expansion of a polyglutamine (polyGln) tract in the androgen receptor (AR). Recent evidence has indicated a potential role for nuclear and cytoplasmic inclusions in the pathogenesis of these diseases. We have used blue and green fluorescently-tagged AR to show that both wild-type (WT) and poly-Gln-expanded full-length AR can form aggregates and that aggregation is not related to cytotoxicity. Twenty to thirty-five percent of all cell types transfected into COS cells showed aggregation containing both amino- and carboxy-terminal fluorescent tags. The aggregates reacted with (F39.4.1), an anti-AR antibody and with IC2, an expanded polyGln tract antibody. Western analysis of protein extracts revealed little evidence of proteolysis although some cleavage of the fusion proteins was seen. The general caspase inhibitor, Z-DEVD-FMK, did not affect aggregation in either wild type or polyGln-expanded GFP-AR transfected cells. Surprisingly, addition of Mibolerone a synthetic androgen significantly decreased inclusion formation in both WT and polyGln-expanded AR-transfected cells. Overall, we show that both WT and polyGln expanded full-length AR are found in aggregates and that proteolysis is not a requirement for aggregation. Our results also suggest that toxicity is not related to intracellular aggregation of polyGln expanded AR.