Background Caspase-6 involvement in Huntington's disease (HD) is now well established, as caspase-6 has a key role in the apoptotic events seen in HD. It has been shown that caspase-6 is activated early in the disease course, inducing different cellular dysfunctions. Importantly, caspase-6 proteolysis of mutant huntingtin is known to be an essential process in generating toxic N-terminal fragments, which leads to neurodegeneration and symptoms onset. Therefore, interfering with caspase-6 activity was suggested as a promising therapeutic target for reducing mutant huntingtin toxicity, and rescuing neuronal cells from degeneration.

Aims The aim of this study is to develop a novel peptide (ED11), based on huntingtin Caspase-6 cleavage site, and fused to a cell penetrating peptide, which will serve as an efficient competitive inhibitor of caspase-6 cleavage, thus reducing HD related toxicity.

Methods Using purified caspase-6 activity luminescence assay, the peptide (ED11) caspase-inhibition ability was measured. To test ED11 ability to protect cells from HD related toxicity, neuronal (SH-SY5Y) cells were placed under glutamate toxicity and three parameters were tested: Caspase-6 activity was measured using FLICA in-cell caspase activity assay, cell viability was measured by Alamar blue assay, and cell apoptosis was assessed using annexin V-PI flow-cytometry analysis.

Results We have demonstrated that ED11 significantly reduces caspase-6 specific activity. Moreover, in neuronal cells it inhibited caspase-6 activity under stress, and protected the cells from glutamate toxicity, an important factor in the pathogenesis of HD. Importantly, ED11 was found to be non-toxic to neuronal cells even in high doses.

Conclusions ED11 was shown to inhibit caspase-6 activity and provided protection against glutamate toxicity in vitro. These results show the potential of ED11 as a novel therapeutic compound in HD.