Poly-Glutamine Induces Bax-Mediated Cell Death by Dissociating Ku70 from Bax through Ku70-Acetylation.

Poly-glutamine (PolyQ) diseases (e.g. Huntington’s disease and Machado-Joseph disease) are caused by the amplification of CAG repeats in the genes of proteins normally expressed in the cell. PolyQ induces caspase-independent cell death associated with vacuolization in the cytoplasm that is distinct from the previously known cell death types such as apoptosis and necrosis. PolyQ-induced cell death is considered a new type of cell death and the elucidation of its mechanism is being awaited. At present, there is no effective treatment to rescue cells from polyQ’s toxicity. Previously, we developed cell permeable cytoprotective peptides designed form Bax-inhibiting domain of Ku70, and these peptides were named Bax Inhibiting Peptides (BIPs). We found that BIP (50 – 200 uM range) blocked polyQ-induced cell death in human epithelial kidney (HEK) 293T cells and human neuroblastoma cell line (Neuro2). PolyQ was expressed by plasmid transfection and cell death was detected by trypan blue exclusion and the release of Lactose Dehydrogenease (LDH) from dead cells into the medium. BIP almost completely suppressed polyQ-induced cell death and vacuolization while caspase-inhibitor (z-VAD-fmk) did not. In Bax-deficient human prostate cancer cell (Du145), polyQ did not induce cell death, but the expression of sub-lethal level of Bax restored the toxicity of polyQ in Du145. Bax changes its conformation when it is activated to mediate cell death and the “active” Bax can be detected by a specific monoclonal antibody (6A7 antibody). By immunohistochemistry Bax activation was confirmed in the neurons of human patients suffering from PolyQ disease as well as in the neurons of a transgenic mouse model of the disease. Previously we found that Ku70 binds and inhibits Bax activation, and that the acetylation of Ku70 releases Bax from Ku70. We found that polyQ binds Ku70 and a Histone Acetyl-Transferase (Creb Binding Protein-CBP), and thus polyQ mediates Ku70 acetylation in the cells. We confirmed that Ku70 overexpression protects cells from polyQ expression whereas Ku70 mutants mimicking acetylated Ku70 did not. Based on these in vivo and in vitro studies, we propose that Bax plays a key role in polyQ-induced cell death and that BIP and Ku70 may be utilized to rescue cells from this kind of death in patients suffering from polyQ diseases.