Sunday, May 5, 2024
5/5/2024
1. Summary Huntington’s disease (HD) is an autosomal dominant, progressive neurodegenerative disease caused by abnormal polyglutamine (polyQ) repeat expansion in the huntingtin (HTT) protein. Despite the monogenic nature of the disease, HD pathogenesis is highly complex. Recent genome-wide association studies implicated strong connection between DNA repair deficiency and age dependent onset of HD, which is consistent with our earlier report showing that the loss of DNA repair activity of polynucleotide kinase 3’-phosphatase (PNKP), but not its protein level, is a key feature in HD patients. PNKP, an essential DNA end processing enzyme with 3’- phosphatase and 5’-kinase activities, is involved in multiple DNA strand-break repair pathways, including DNA double strand break repair (DSBR) via Classical-Non-Homologous End-Joining (C-NHEJ), the major DSBR pathway in postmitotic neurons. While investigating the biological basis of PNKP inactivation in HD, we isolated a multiprotein complex from chromatin extract of neuronal cells that involves inflammatory kinase IKK2/ß, a glycolytic enzyme, phosphofructokinase fructose-2,6-bisphosphatase 3 (PFKFB3) and HTT, along with PNKP and other C-NHEJ proteins. IKK2 was reported to inhibit PFKFB3 via phosphorylation, and we found that IKK2 also phosphorylates PNKP in vitro at serine 284, inactivating the protein. Surprisingly, an inhibitor of IKK2 restores PNKP activity and integrity of transcribed genome in HD-derived striatal neuronal cells. Moreover, non- specific (Lambda) phosphatase treatment of HD nuclear extract restored PNKP activity suggesting phosphorylation-mediated inactivation in HD. Notably, the level of both PFKFB3 and its enzymatic product fructose-2,6-bisphosphate (F2,6BP), a potent allosteric modulator of glycolysis, are remarkably low in the nuclear extract of HD patients’ brain tissue. Intriguingly, exogenous F2,6BP restored PNKP activity in such extract. F2,6BP also restores the activity of otherwise inactive phospho-mimic PNKP mutant (S284E). We thus postulate that loss of PNKP activity in HD patients is due to IKK2-mediated phosphorylation of PNKP at S284. The central focus of this renewal application is to unravel how specific posttranslational modifications of PNKP and PFKFB3 regulate protective vs. destructive roles under homeostatic vs. pathogenic conditions, respectively. We hypothesize that functional inactivation of PNKP and degradation of PFKFB3 via IKK2-mediated phosphorylation at specific residues under neuroinflammatory conditions in HD causes accumulation of DSBs in the transcribed genome of vulnerable neuronal and glial cells, triggering their death. To elucidate the causal pathway leading to HD, we will determine how IKK2 modulates PNKP activity in HD (Aim 1); and elucidate the role of PFKFB3 in modulation of PNKP activity in HD patients and animal models (Aim 2). We will further test, as a proof of principle, whether delivery of F2,6BP+ IKK2 inhibitor can rescue mutant HTT- induced toxicity. The knowledge gained from our studies will help understand the underlying cause of the HD and would help develop novel therapeutic interventions in HD and possibly other polyQ diseases.
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