Sirtuin family members (Sirtuin 1 to 7) are highly conserved proteins, both structurally and functionally, and have either NAD+ dependent enzymatic activity (Sirtuin 1,2, 3, 5 and 7) for deacetylation of proteins or ADP ribosyl transferase activity (Sirtuin 4 and 6). Their functions in metabolic processes and chromatin remodeling have been conserved throughout evolution. Sirtuin 1 and Sirtuin 2 have been shown to be linked with several neurological disorders. The role of other Sirtuins in neurodegenerative disorders is not known. Radiation therapy (RT), one of the preferred and widely accepted treatments for intracranial primary and secondary metastatic tumors, may lead to long-term irreversible neurotoxicity in susceptible individuals with cognitive dysfunction similar to that observed in other neurodegenerative disorders. There are ample data which demonstrate that aberrant autophagic process and dysfunctional mitochondria are important factors leading to several neurological diseases, such as Huntington's disease and Parkinson's dementia. Among the Sirtuins, Sirtuin 3, 4 and 5 are localized within mitochondria, however, their role in radiation-induced neurotoxicity has not been evaluated.
Using Sirtuin 3 knockout (KO) mice generated through genomic deletion allowed us the opportunity to study its role in autophagy and mitochondrial function during exposure to radiation. Preliminary reports show Sirtuin 3 in the adult mouse brain to be localized primarily within hippocampus and subcortical plate. Through flow cytometry and immunofluorescence stainin of mouse embryonic fibroblasts (MEFs) derived from Sirtuin 3 KO mice, we demonstrate that loss of Sirtuin 3 alters rates of basal autophagy. Furthermore, immunoblot analysis of autophagic marker LC3B I &II in the presence and absence of hydrolase inhibitors (E64d & Pepstatin A) exhibits that loss of Sirtuin 3 stimulates autophagy. Radiation treatment of Sirtuin 3 wild-type and knockout MEFs exhibited dose dependent induction of autophagy. Staining with mitotracker red and JC1 illustrates that MEFs lacking Sirtuin 3 retain mitochondria with abnormal morphology and disturbed membrane potential which can further enhanced by radiation treatment and exposure to the ionophore Carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Enrichment of lower form of LC3B in Sirtuin 3 KO cells upon treatment with CCCP and radiation suggests that loss of Sirtuin 3 sensitizes cells towards enhanced autophagy via mitophagy. Taken together, this study indicates that Sirtuin 3 is an important player in deciding cell fate via mitophagy during radiation treatment and has important implications in further studies of radiation-induced neurotoxicity.
Citation Format: Sadhanshu Shukla, DeeDee Smart. Radiation-induced autophagy is dependent on Sirtuin 3. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1682. doi:10.1158/1538-7445.AM2013-1682