Abstract. Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by mutant huntingtin (Htt) with an expanded polyglutamine tract. It has been reported that Htt regulates autophagy. However, it remains unclear whether mutant Htt affects chaperone-mediated autophagy (CMA). Our study aimed to investigate the effect of mutant Htt on CMA activity in cultured HEK293T cells. A HEK293T cell model of HD was produced by transient transfection of wild-type (20Q) or mutant (120Q) Htt plasmids. The effect of mutant Htt on two CMA components, lysosomal-associated membrane protein 2a (Lamp2a) and heat-shock cognate protein 70 (Hsc70), was determined by western blotting and immunofluorescent staining. We observed that mutant Htt did not significantly alter the expression of Lamp2a and Hsc70 when compared to normal Htt. These findings suggest that mutant Htt does not reduce CMA activity and that enhancing CMA activity to clear mutant Htt may be a novel strategy for the management of HD.
IntroductionHuntington's disease (HD) is a progressive, autosomal dominant, neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat (>35 repeats) in the huntingtin (Htt) gene, which is translated into an expanded polyglutamine tract in the N-terminus of the Htt protein (1). The most obvious characteristic of HD is uncontrolled movement, dementia, emotional disturbance and early death, which often occur in middle age (2,3). These symptoms are associated with neuronal loss that preferentially occurs in medium-sized spiny neurons of the striatum and also extends to other brain regions during the late stages of the disease.Wild-type Htt is considered to possess a number of cellular functions, such as protein transport, vesicle trafficking and cytoskeletal anchoring. When Htt is mutated with an expanded polyglutamine tract (>36 glutamines), Htt becomes misfolded to induce gain of toxic function and may also lose its normal function. Mutant Htt appears to affect a variety of cellular functions, including intracellular signaling pathways (4), the protein degradation pathway (5) and the nuclear transcription and axon transport (6,7), resulting in degeneration of neuronal cells in the striatum and other brain regions.Clearing mutant Htt may be of value in preventing the pathogenesis and development of HD. In eukaryotic cells, there exist two systems of protein degradation, the ubiquitin-proteasome system and autophagy. Autophagy is a non-specific bulk degradation pathway for long-lived cytoplasmic proteins, protein complexes, or damaged organelles. There are three types of autophagy, namely macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). Macroautophagy is considered to promote the degradation of Htt and clearance of aggregates. This is supported by several lines of evidence that rapamycin (an activator of autophagy) (8) and its several small molecular enhancers (9-11) may enhance the degradation of mutant Htt and reduce its toxicity. It is also important to investigate whether mu...