Pharmacologic and genetic inhibition of hsp90-dependent trafficking reduces aggregation and promotes degradation of the expanded glutamine androgen receptor without stress protein induction

Abstract

The molecular chaperone hsp90 has emerged as an important therapeutic target in cancer and neurodegenerative diseases, including the polyglutamine expansion disorders, because of its ability to regulate the activity, turnover and trafficking of many proteins. For neurodegenerative disorders associated with protein aggregation, the rationale has been that inhibition of hsp90 by geldanamycin and related compounds activates heat shock factor 1 (HSF1) to induce the production of the chaperones hsp70 and hsp40 that promote disaggregation and protein degradation. However, we show here that geldanamycin blocks the development of aggregates of the expanded glutamine androgen receptor (AR112Q) of Kennedy disease in Hsf1(-/-) mouse embryonic fibroblasts where these chaperones are not induced. Geldanamycin is additionally known to inhibit hsp90-dependent protein trafficking and to promote proteasomal degradation of client proteins. Overexpression of the hsp90 cochaperone p23 also promotes AR112Q degradation, and inhibits both AR trafficking and AR112Q aggregation without altering levels of hsp70 or hsp40. The hsp90-dependent trafficking mechanism has been defined, and it is shown that key immunophilin (IMM) components of the trafficking machinery are present in polyglutamine aggregates in cell and mouse models of Kennedy disease. Our results indicate that inhibition of the hsp90-dependent trafficking mechanism prevents aggregation of the expanded glutamine androgen receptor, thereby opening a variety of novel therapeutic approaches to these neurodegenerative disorders.

Small Ubiquitin-like Modifier (SUMO) Modification of the Androgen Receptor Attenuates Polyglutamine-mediated Aggregation

“…As can be seen in Fig. 1B and consistent with previous reports (3,4), agonist treatment of HeLa cells expressing a polyglutamine-expanded form of AR leads to the appearance of punctate cytoplasmic inclusions in a substantial fraction (ϳ40%) of AR-expressing cells. In addition, significant diffuse staining of AR, likely reflecting smaller scale aggregates, is detectable in the cytoplasm.…”

“…Although this likely reflects the reduced formation of smaller scale aggregates, this may also indicate that SUMOylation-dependent alterations in nucleocytoplasmic transport could contribute to the protective effects. Enhanced nuclear translocation, however, is not directly consistent with the reduced aggregation observed upon inhibition of HSP90-dependent transport (4).…”

“…The HA-tagged version of the preprocessed protease-resistant form of SUMO3 (HA-SUMO3-Q89P-GGstop) and the surface mutant (HA-SUMO3-Q89P-K33E/K42E-GGstop) were generated by sitedirected mutagenesis using as a template the pCMV-driven (pcDNA3) expression vector for HA-tagged SUMO3 described previously (36). The reporter plasmid p⌬(TAT) 4 -Luc harbors four copies of a minimal response element from the tyrosine aminotransferase (TAT) gene upstream of a minimal Drosophila distal alcohol dehydrogenase promoter (Ϫ33 to ϩ55) driving the luciferase gene (40,59). The FKBP5 luciferase reporter, which harbors a 500-bp genomic region centered on the AR binding region in the first intron of the human FKBP5 gene, was a kind gift of Dr Keith Yamamoto (60).…”

“…fasciculations followed by progressive weakness and atrophy of the proximal limb and bulbar muscles (3)(4)(5). The causative genetic alteration is an expansion in the length of a CAG trinucleotide repeat within the coding sequence of the androgen receptor (AR) gene, leading to an expanded polyglutamine tract in the N-terminal transcriptional regulatory domain of the receptor.…”

“…In addition, alterations in cellular transport mechanisms, which are critical to the function of motoneurons, have been also implicated (3,4). Although the dominant inheritance of SBMA indicates the gain of a toxic function, an expanded polyglutamine tract in AR is also associated with a partial loss of function in its transcriptional properties (17,18), and other polyglutamine diseases are also associated with alterations in transcriptional programs, presumably by altering the activity of transcriptional coregulators (10,19,20).…”