CNS sirtuins in radiation-induced vascular permeability and neurodegeneration

Smart, Deedee

Abstract

We have determined that Sirtuin 2 (Sirt2), the most common isoform of sirtuin in the central nervous system (CNS), interacts with beta-catenin. Sirt2 levels are increased during mouse glial stem cell gOlig2 differentiation, and beta-catenin and its targets are believed to be essential in the nervous system for differentiation and de-differentiation of both adult and stem cell populations, as a component of adherens junctions in cellular adhesion and signaling, as well as oncogenesis. The interaction of beta-catenin and Sirt2 increases in a directly proportional manner with radiation exposure, suggesting a possible role for ionizing radiation in differentiation, function, and repair of CNS cellular components. Transport of beta-catenin into the nucleus is dependent on the presence of Sirt2 as demonstrated by subcellular fractionation of Sirt2 wild-type and knockout mouse embryonic fibroblasts (MEFs). Likewise, the active form of beta-catenin that is phosphorylated on Tyrosine-142 is increased in Sirt2 knockout MEFs, indicating that Sirt2 is necessary for the activation and downregulation of beta-catenin targets. Although Sirt2 is a deacetylase, levels of acetylated beta-catenin do not differ between wild-type and knockout cells, suggesting that Sirt2 is not a direct acetylation target. GSK3beta, a kinase which consitiuitvely phosphorylates beta-catenin, and AKT are phosphorylated in Sirt2 knockout cells, and experiments are currently underway to determine the effect of Sirt2 on AKT and GSK3beta following radiation exposure. Sirt2 regulates expression of the Wnt target genes c-Myc, survivin, c-Jun, and Cyclin D1 as demonstrated by immunoblot and real-time PCR from wild-type and knockout cells. A PI3Kinase inhibitor decreases the expression of c-Jun, c-Myc, survivin, and phosphorylated AKT in the absence of Sirt2. Additionally, antisense RNA knockdown of Sirt2 in wild-type cells increases expression of Cyclin D1, c-Myc, and survivin by immunoblot and real-time PCR analysis. Taken together, along with the observation that Sirt2 regulated the transcriptional activity of beta-catenin, the data suggest that Sirt2 directly affects Wnt target genes and PI3kinase pathways. Beta-catenin directly binds to c-Myc, survivin, Cyclin D1 promoter. Sirt2 regulates the direct interaction of beta-catenin to these target promoters as demonstrated by chromatin immunoprecipitation analysis in wild-type and knockout cells. This suggests that Sirt2 may directly deactylates and alters DNA binding. In support of this, Sirt2 regulates the binding of acetylated histone H3 to the Wnt target promoters Cyclin D1, c-Myc and survivin as demonstrated by quantitative PCR. The data also suggest the possibility that Sirt2 is part of a complex of proteins and that Sirt2 may deacetylate an intermediary that then alters beta-catenin mediated transcription and signaling. On PCR array comparing wild-type and knockout Sirt2 cells, we have determined that Nkd1, Pitx1, Sfrp4 and Tle1 are negative regulators of the Wnt pathway that are Sirt2 dependent. Using Boyden chamber analysis of Sirt2 wild-type and knockout cells, we have determined that the presence of Sirt2 reduces cellular migration. Based on PCR an immunoblot analysis, we believe this occurs via Sirt2 regulation of matrix metalloproteinase 9 and a possible novel modification of E-cadherin. This data has been submitted for publication. We have also determined that Sirt2 functions as a constituitive radiosensitizer. Clonogenic survival assays performed on Sirt2 wild-type, Sirt2 knockdown, and Sirt2 knockout MEFs have demonstrated that increased levels of Sirt2 expression are associated with increased radiosensitivity. Tumor cells that have high constituitive levels of Sirt2, such as human prostate cancer PC3 cells, also demonstrate that radiosensitivity can be altered by altering the level of Sirt2 expression. Knockdown experiments in PC3 cells using siRNA for Sirt2 demonstrate that decreases in Sirt2 expression decrease the radiosensitivity of the cells. Accordingly, as gOlig2 precursor cells differentiate, and the levels of Sirt2 expression increase, there are associated increases in radiosensitivity as demonstrated by increased double-stranded DNA breaks measured by gamma H2AX ELISA. We hope to validate these using in vivo animal experiments with and without whole brain radiation on Sirt2 knockout mice in order to demonstrate that Sirt2 is necessary for regulating CNS response and repair from radiation therapy.