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Abstract
Spinal and bulbar muscular atrophy (SBMA) is one of a group of human inherited neurodegenerative diseases caused by polyglutamine expansion. We have previously demonstrated that the SBMA gene product, the androgen receptor protein, is toxic and aggregates when truncated. Heat shock proteins function as molecular chaperones, which recognize and renaturate misfolded protein (aggregate). We thus assessed the effect of a variety of chaperones in a cultured neuronal cell model of SBMA. Overexpression of chaperones reduces aggregate formation and suppresses apoptosis in a cultured neuronal cell model of SBMA to differing degrees depending on the chaperones and their combinations. Combination of Hsp70 and Hsp40 was the most effective among the chaperones in reducing aggregate formation and providing cellular protection, reflecting that Hsp70 and Hsp40 act together in chaperoning mutant and disabled proteins. Although Hdj2/Hsdj chaperone has been previously reported to suppress expanded polyglutamine tract-formed aggregate, Hsdj/Hdj2 showed little effect in our system. These findings indicate that chaperones may be one of the key factors in the developing of CAG repeat disease and suggested that increasing expression level or enhancing the function of chaperones will provide an avenue for the treatment of CAG repeat disease.
Highlights
Spinal and bulbar muscular atrophy (SBMA)1 is an X-linked neurodegenerative disease caused by the expansion of a CAG repeat in the first exon of the androgen receptor (AR) gene [1]
The neuronal cultured cell model has shown nuclear aggregates, which are pathological hallmark of CAG repeat disease, and an apoptosis, which might be mediated by cellular toxicity of expanded polyglutamine tract
These results indicate that this neuronal cultured cell model could be utilized as a cell model of SBMA
Summary
Truncated AR—Human AR cDNAs containing 24 or 97 CAG repeats [18] were subcloned into pcDNA3.1 (Invitrogen, Carlsbad, CA). After the constructs were digested with AflII and blunt-ended, the polymerase chain reaction-amplified coding sequence of GFP (0.8 kilobase) was inserted into the digested constructs to create truncated AR constructs (24 CAG repeats, 215 N-terminal amino acids; 97 CAG repeats, 442 N-terminal amino acids of AR). For the construction of pRC-Hsp, a 1.5-kilobase EcoRI fragment containing the entire coding region of human Hsp40 [43] was excised from the cDNA clone and subcloned in pRC/CMV (Invitrogen). For the construction of pRC-Hsdj, the polymerase chain reaction-amplified coding sequence of Hsdj (1.1 kilobase) [44] was subcloned in pRC/CMV. Anti-Hsp Ab (rabbit polyclonal IgG) [45] and anti-Hsp Ab (rabbit polyclonal IgG) [46] were previously described.
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