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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. [ABSTRACT FROM AUTHOR]

In addition to immediate brain damage, traumatic brain injury (TBI) initiates a cascade of pathophysiological events producing secondary injury. The biochemical and cellular mechanisms that comprise secondary injury are not entirely understood. Herein, we report a substantial deregulation of cerebral sphingolipid metabolism in a mouse model of TBI. Sphingolipid profile analysis demonstrated increases in sphingomyelin species and sphingosine concurrently with up-regulation of intermediates of de novo sphingolipid biosynthesis in the brain. Investigation of intracellular sites of sphingosine accumulation revealed an elevation of sphingosine in mitochondria due to the activation of neutral ceramidase (NCDase) and the reduced activity of sphingosine kinase 2 (SphK2). The lack of change in gene expression suggested that post-translational mechanisms are responsible for the shift in the activities of both enzymes. Immunoprecipitation studies revealed that SphK2 is complexed with NCDase and cytochrome oxidase (COX) subunit 1 in mitochondria and that brain injury hindered SphK2 association with the complex. Functional studies showed that sphingosine accumulation resulted in a decreased activity of COX, a rate-limiting enzyme of the mitochondrial electron transport chain. Knocking down NCDase reduced sphingosine accumulation in mitochondria and preserved COX activity after the brain injury. Also, NCDase knockdown improved brain function recovery and lessened brain contusion volume after trauma. These studies highlight a novel mechanism of secondary TBI involving a disturbance of sphingolipid-metabolizing enzymes in mitochondria and suggest a critical role for mitochondrial sphingosine in promoting brain injury after trauma. [ABSTRACT FROM AUTHOR]