SIRT3 Deacetylates Ceramide Synthases: IMPLICATIONS FOR MITOCHONDRIAL DYSFUNCTION AND BRAIN INJURY

Sergei A Novgorodov,Christopher L Riley,Jarryd A Keffler,Jin Yu,Mark S Kindy,Wendy B Macklin,David B Lombard,Tatyana I Gudz

doi:10.1074/jbc.m115.668228

Abstract

Experimental evidence supports the role of mitochondrial ceramide accumulation as a cause of mitochondrial dysfunction and brain injury after stroke. Herein, we report that SIRT3 regulates mitochondrial ceramide biosynthesis via deacetylation of ceramide synthase (CerS) 1, 2, and 6. Reciprocal immunoprecipitation experiments revealed that CerS1, CerS2, and CerS6, but not CerS4, are associated with SIRT3 in cerebral mitochondria. Furthermore, CerS1, -2, and -6 are hyperacetylated in the mitochondria of SIRT3-null mice, and SIRT3 directly deacetylates the ceramide synthases in a NAD(+)-dependent manner that increases enzyme activity. Investigation of the SIRT3 role in mitochondrial response to brain ischemia/reperfusion (IR) showed that SIRT3-mediated deacetylation of ceramide synthases increased enzyme activity and ceramide accumulation after IR. Functional studies demonstrated that absence of SIRT3 rescued the IR-induced blockade of the electron transport chain at the level of complex III, attenuated mitochondrial outer membrane permeabilization, and decreased reactive oxygen species generation and protein carbonyls in mitochondria. Importantly, Sirt3 gene ablation reduced the brain injury after IR. These data support the hypothesis that IR triggers SIRT3-dependent deacetylation of ceramide synthases and the elevation of ceramide, which could inhibit complex III, leading to increased reactive oxygen species generation and brain injury. The results of these studies highlight a novel mechanism of SIRT3 involvement in modulating mitochondrial ceramide biosynthesis and suggest an important role of SIRT3 in mitochondrial dysfunction and brain injury after experimental stroke.

Highlights

There were no significant changes in ceramide, sphingosine, and sphingosine 1-phosphate (S1P) in tissue homogenate from SIRT3-deficient mouse brain compared with wild type (WT) (Fig. 1A)
Our data suggest that cerebral IR triggers SIRT3-mediated deacetylation of CerS1, -2, and -6, which results in the accumulation of ceramide, mitochondrial dysfunction, and generation of reactive oxygen species (ROS) promoting brain injury (Fig. 12)
Given the significance of mitochondria in regulation of neural cell function and survival, the characterization of SIRT3mediated mitochondrial impairment triggered by cerebral IR is a valuable contribution to our understanding of the pathophysiological mechanisms of brain damage after stroke

Summary

IMPLICATIONS FOR MITOCHONDRIAL DYSFUNCTION AND BRAIN INJURY*

Sirt gene ablation reduced the brain injury after IR These data support the hypothesis that IR triggers SIRT3-dependent deacetylation of ceramide synthases and the elevation of ceramide, which could inhibit complex III, leading to increased reactive oxygen species generation and brain injury. We show that Sirt gene ablation decreases ceramide, preserves mitochondrial respiratory chain function, reduces ROS generation and oxidative protein damage, and attenuates brain tissue injury in the experimental stroke mouse model. These studies emphasize a crucial role of SIRT3, a novel regulator of mitochondrial ceramide biosynthesis, in brain response to IR

Experimental Procedures

Results

Ceramide species content of WT mouse brain mitochondria

Discussion