Mutant huntingtin inhibits clathrin-independent endocytosis and causes accumulation of cholesterol in vitro and in vivo
1 Department of Molecular Pharmacology and Experimental Therapeutics, 2 Department of Biochemistry and Molecular Biology, 3 Department of Internal Medicine and 4 Department of Physiology, Gastroenterology Research Unit and Tumor Biology Program, 5 Molecular Neuroscience Program and 6 Thoracic Diseases Research Unit Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA and 7 Institute for Molecular Bioscience and Centre for Microscopy and Microanalysis, University of Queensland, Queensland 4072, Australia
* To whom correspondence should be addressed. Tel: +1 5072841597; Fax: +1 5072849111; Email: mcmurray.cynthia{at}mayo.edu
Received October 16, 2006; Accepted November 9, 2006
We show that the mutant Huntington's disease (HD) protein (mhtt) specifically inhibits endocytosis in primary striatal neurons. Unexpectedly, mhtt does not inhibit clathrin-dependent endocytosis as was anticipated based on known interacting partners. Instead, inhibition occurs through a non-clathrin, caveolar-related pathway. Expression of mhtt inhibited internalization of BODIPY-lactosylceramide (LacCer), which is internalized by a caveolar-related mechanism. In contrast, endocytosis of Alexa Fluor 594-transferrin (Tfn) and epidermal growth factor, internalized through clathrin pathway, was unaffected by mhtt expression. Caveolin-1 (cav1), the major structural protein of caveolae binds cholesterol and is responsible for its trafficking inside cells. Mhtt interacts with cav-1 and caused a striking accumulation of intracellular cholesterol. Cholesterol accumulated in cultured neurons expressing mhtt in vitro and in brains of mhtt-expressing animals in vivo, and was observed after induction of mhtt expression in PC-12 cell lines. The accumulation occurred only when mhtt and cav1 were simultaneously expressed in cells. Knockdown of cav1 in mhtt-expressing neurons blocked cholesterol accumulation and restored LacCer endocytosis. Thus, mhtt and cav1 functionally interact to cause both cellular defects. These data provide the first direct link between mhtt and caveolar-related endocytosis and also suggest a possible mechanism for HD neurotoxicity where cholesterol homeostasis is perturbed.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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