Although htt cleavage products have been found in postmortem HD patients (Kim et al., 2001;Mende-Mueller et al., 2001) and development of a rapid neurological phenotype is well established in mice expressing Glumetinib (SCC-244) truncated htt (Mangiarini et al., 1996; Bates et al., 1998), our study is the first to demonstrate that htt cleavage precedes the onset of the HD phenotypetreatment with caspase-3. projection neurons, suggesting that accumulation of expanded htt fragments in these neurons may lead to corticostriatal dysfunction as an early event in the pathogenesis of HD. Keywords: Huntington’s disease, huntingtin, caspase cleavage, and models (Rigamonti et al., 2000; Leavitt et al., 2001; Zuccato et al., 2001). These observations suggest that inhibition of htt proteolysis would both reduce the load of toxic N-terminal fragments and maintain the neuroprotective function of intact htt. Despite numerous and studies that support a correlation between htt Glumetinib (SCC-244) length and toxicity, little is known about the proteolytic processing of htt has not been demonstrated conclusively, in part because the site of htt cleavage has not been determined precisely in any study to date. Indeed, there is controversy in the literature about whether Glumetinib (SCC-244) expanded htt is even susceptible to cleavage compared with htt with a normal polyglutamine tract (Dyer and McMurray, 2001). Furthermore, whether cleavage of htt is an early Rabbit polyclonal to GLUT1 or late event in HD pathogenesis has not been specifically addressed. Finally, the cellular populations that may be particularly enriched in cleaved htt are not known. To unequivocally detect caspase cleavage products of htt, we have developed novel antibodies specifically designed to detect N-terminal htt fragments generated by caspase cleavage at amino acid 513 or 552. Here, we provide direct evidence for caspase-mediated cleavage of htt. In early grade HD postmortem tissue as well as in young transgenic mice expressing expanded human htt, both normal and expanded htt are cleaved at amino acid 552 before the onset of gross striatal atrophy. Cleaved htt was also observed in control brain, suggesting that caspase cleavage of htt may be part of its normal regulated catabolism. The observation of htt cleavage most abundantly in cortical projection neurons suggests that disruption of the corticostriatal pathway may be an early event in the pathogenesis of HD. MATERIALS AND METHODS Human HD and control brain postmortem tissues were obtained from the Canadian Brain Tissue Bank (Toronto, Canada) and the Harvard Brain Tissue Resource Center (Cambridge, MA). Tissue from a presymptomatic patient was obtained from Emory University as described previously (Gutekunst et al., 1999). All human tissues were obtained after ethical review. Yeast artificial chromosome (YAC) transgenic animals expressing human htt with 72 (CAG) repeats (Hodgson et al., 1999) were used as a source of murine tissues according to the University of British Columbia animal protocol A00-0254. Line 2511 contains one to two copies of the YAC and has been described previously (Hodgson et al., 1999). Line 44 was generated by independent microinjection and contains two to four copies of the YAC transgene. The number of CAG repeats in htt from patient brain was determined by nonradioactive PCR using standards of known CAG size as described previously (Andrew et al., 1993; Bruland et Glumetinib (SCC-244) al., 1999). Antibodies specific for the C-terminal ends of htt caspase cleavage products ending at amino acid 513 or 552 were prepared using the immunizing peptides KLH-CHTLQADSVD and KLH-CSDPAMDLND, respectively. Antibodies were affinity purified as described previously (Gervais et al., 1999). The polyclonal N-terminal htt antibody BKP1 has been described previously (Kalchman et al., 1997). In addition to polyclonal BKP1, we have also generated a monoclonal version of BKP1 that recognizes the N-terminal 17 aa of htt. Polyclonal and monoclonal BKP1 were used interchangeably. Monoclonal antibodies specific for glyceraldehyde phosphate dehydrogenase (GAPDH) and expanded polyglutamine tracts (1C2) were purchased from Chemicon(Temecula, CA). EM48, an antibody that recognizes htt aggregates, has been described previously (Gutekunst et al., 1999; Li et al, 1999). Human embryonic kidney (HEK) 293T cells were cultured and transfected by calcium phosphate as described previously, using the htt constructs pRcCMV-3949-15 and pRcCMV-3949-128 (Wellington et al., 2000). The constructs pRcCMV-3949-15-514X and pRcCMV-3949-128-514X were generated by inserting a translation terminal codon at amino acid 514. Apoptosis in cultured cells was induced by the addition of 35 m tamoxifen as described previously (Hackam et al., 1998; Ellerby et al., 1999). Murine and human tissues were homogenized in a buffer containing 0.25 m sucrose, 20 mm Tris HCl, pH Glumetinib (SCC-244) 7.2,.
