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Reports

N-Terminal Proteolysis of Full-Length Mutant Huntingtin in an Inducible PC12 Cell Model of Huntington’s Disease

Tamara Ratovitski, Masayuki Nakamura, James D'Ambola, Ekaterine Chighladze, Yideng Liang, Wenfei Wang, Rona Graham, Michael R. Hayden, David R. Borchelt, Ricky R. Hirschhorn and Christopher A. Ross

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Proteolytic cleavage of mutant huntingtin may play a key role in the pathogenesis of Huntington’s disease; however the steps in huntingtin proteolysis are not fully understood. Huntingtin was shown to be cleaved by caspases and calpains within a region between 460-600 amino acids from the N-terminus. Two smaller N-terminal fragments produced by unknown protease have been previously described as cp-A and cp-B. To further investigate the huntingtin proteolytic pathway, we used an inducible PC12 cell model expressing full-length huntingtin with either normal or expanded polyglutamine. This cell model recapitulates several steps of huntingtin proteolysis: proteolysis mediated by caspases within the region previously mapped for caspase cleavage, and cleavage generating two novel N-terminal fragments (cp-1 approximately 90-105 residues long and cp-2 extending beyond 115-129 epitope of huntingtin). Interestingly, the deletion of amino acids 105-114 (mapped previously as a cleavage site for cp-A) failed to affect the production of cp-1 or cp-2. Therefore, we conclude that these new fragments are distinct from previously described cp-A and cp-B. We demonstrate that cp-1 and cp-2 fragments are produced and accumulate within nuclear and cytoplasmic inclusions prior to huntingtin-induced cell toxicity, and these fragments can be formed by caspase-independent proteolytic cleavage of huntingtin in PC12 cells. In addition, inhibition of calpains leads to decreased subsequent degradation of cp-1 and cp-2 fragments, and accelerated formation of inclusions. Further delineation of huntingtin cleavage events may lead to novel therapeutic targets for HD.

Authors

Tamara Ratovitski

Johns Hopkins University School of Medicine; Baltimore, MD

Masayuki Nakamura

Johns Hopkins University School of Medicine; Baltimore, MD

James D'Ambola

Johns Hopkins University School of Medicine; Baltimore, MD

Ekaterine Chighladze

Johns Hopkins University School of Medicine; Baltimore, MD

Yideng Liang

Johns Hopkins University School of Medicine; Baltimore, MD

Wenfei Wang

Johns Hopkins University School of Medicine; Baltimore, MD

Rona Graham

University of British Columbia; Vancouver, British Columbia, Canada

Michael R. Hayden

University of British Columbia; Vancouver, British Columbia, Canada

David R. Borchelt

University of Florida; Gainesville, FL

Ricky R. Hirschhorn

Johns Hopkins University School of Medicine; Baltimore, MD

Christopher A. Ross

Johns Hopkins University School of Medicine; Baltimore, MD

Purchase article for $19

Subscribe to this journal for $129/year