From their previous work, the Hayden group knew that getting rid of all five caspase cleavage sites in the N-terminal end of huntingtin mitigated the toxicity of polyQ-expanded huntingtin (Wellington et al., 2000), but they wanted to know if a particular site or caspase were key to the disease. To find out, lead author Rona Graham and coworkers generated transgenic mice expressing polyQ-expanded htt with mutations in the four potential caspase-3 sites and/or the single caspase-6 site.

Did the lack of caspase cleavage affect neurotoxicity? In the case of the caspase-6-resistant protein, the answer was a resounding yes. Though mice expressing polyQ-expanded htt had decreases in brain weight and striatal volume, and progressive motor dysfunction in rotarod or open field activity tests, the mice expressing the caspase-6-resistant protein performed just as well as wild-type mice. Pathology in caspase-3-resistant mice, however, was similar to that seen in animals with protease-susceptible polyQ-expanded huntingtin.

As might be expected, the caspase-3-resistant mice, but not the caspase-6-resistant animals, had the 586-amino-acid caspase-6 cleavage fragment in their brains. These htt fragments accumulated early in the nucleus of striatal cells, consistent with this peptide playing a key role in neurotoxicity. While the caspase-6-resistant mice did show some nuclear accumulation of htt fragments, their appearance was delayed. These tardy fragments, presumably derived from other proteases, were not associated with any toxicity in the cells.

Normal huntingtin is neuroprotective, and the researchers showed that the caspase-6-resistant form, even with polyQ expansion, also plays this role. The protein increased resistance of cultured neurons to NMDA excitotoxicity and protected mice against brain damage caused by injection of quinolinic acid in vivo. These results suggest a link between excitotoxicity, caspase-6 cleavage of mutant htt and Huntington disease.

The findings also echo studies on caspase cleavage of the amyloid precursor protein and tau in Alzheimer disease. Recent work from Dale Bredesen’s lab showed that a caspase cleavage site in the cytosolic tail of APP is required for synaptic toxicity and cognitive impairment in transgenic mice (see ARF related news story). Although that study did not identify the caspase responsible, a study on the neuroprotective capabilities of estrogen implicated caspase-6 in increasing APP processing and toxicity (see ARF related news story), while several years ago, another group found that caspase-3 cleavage of APP is associated with elevated Aβ production and the appearance of senile plaques (see Gervais et al., 1999). These findings, together with the potential role of caspase-6 in tau and now huntingtin pathology, highlight the possibility of blocking the production of pathogenic protein fragments as a favored therapeutic strategy.—Pat McCaffrey.

Reference:
Graham RK, Deng Y, Slow E, Haigh B, Bissada N, Lu G, Pearson J, Shehadeh J, Bertram L, Murphy Z, Warby SC, Doty CN, Roy S, Wellington CL, Leavitt BR, Raymond LA, Nicholson DW, Hayden MR. Cleavage at the caspase-6 site is required for neuronal dysfunction and degeneration due to mutant huntingtin. Cell. 2006 June 16; 125:1179-1191. Abstract