Abstract
Background: N-terminal cleavage products of mutant huntingtin (htt) generate pathologic neuronal inclusion bodies. The precise length of the htt fragment, termed Cp-A/1, that produces HD pathologic inclusions is unknown. Objective: We sought to elucidate the protein sequence elements within the N-terminus of htt that mediate its proteolysis based on a model in which engineered htt fragments terminating at residue 171 are cleaved to produce Cp-A/1 fragments. Methods: We expressed htt N171 cDNAs harboring a series of experimental mutations in the presumptive cleavage site that generates Cp-A/1 in cells to identify cleavage resistant mutants of htt N171. One of these constructs was expressed in mice, followed by analysis using immunoblots of brain extracts and immunohistochemistry of transgenic mouse brain tissues. Results: Using the HEK293 cell model, mutagenesis studies mapped the cleavage site in htt N171 to sequences between residues 105–114. Mutation of 8 positively charged residues (H, K, R) located between residues 88 and 114 to alanine to neutralize the charge also blocked the generation of Cp-A/1 like fragments. Transgenic mice expressing this latter construct, termed N171-82Q-N8, developed phenotypes similar to previously characterized N171-82Q transgenic mice, including rotarod deficiency, intranuclear inclusions, and premature death. Surprisingly, the N171-82Q-N8 protein was efficiently cleaved in vivo to produce Cp-A/1 fragments that accumulated as insoluble inclusions. Conclusion: Mutagenesis of htt to identify critical amino acids that direct its cleavage predicted a role for charged residues in the sequence flanking the presumptive cleavage site. However, the role for these residues could not be confirmed in vivo. The basis for the discrepancy between predicted outcomes in HEK293 cells and the mouse models remain unresolved, but the data provide another validation of the hypothesis that Cp-A/1 fragments of mutant htt can induce HD-like phenotypes.