Abstract
A cardinal feature of brain tissue injury in stroke is mitochondrial dysfunction leading to cell death, yet remarkably little is known about the mechanisms underlying mitochondrial injury in cerebral ischemia/reperfusion (IR). Ceramide, a naturally occurring membrane sphingolipid, functions as an important second messenger in apoptosis signaling and is generated by de novo synthesis, sphingomyelin hydrolysis, or recycling of sphingolipids. In this study, cerebral IR-induced ceramide elevation resulted from ceramide biosynthesis rather than from hydrolysis of sphingomyelin. Investigation of intracellular sites of ceramide accumulation revealed the elevation of ceramide in mitochondria because of activation of mitochondrial ceramide synthase via post-translational mechanisms. Furthermore, ceramide accumulation appears to cause mitochondrial respiratory chain damage that could be mimicked by exogenously added natural ceramide to mitochondria. The effect of ceramide on mitochondria was somewhat specific; dihydroceramide, a structure closely related to ceramide, did not inflict damage. Stimulation of ceramide biosynthesis seems to be under control of JNK3 signaling: IR-induced ceramide generation and respiratory chain damage was abolished in mitochondria of JNK3-deficient mice, which exhibited reduced infarct volume after IR. These studies suggest that the hallmark of mitochondrial injury in cerebral IR, respiratory chain dysfunction, is caused by the accumulation of ceramide via stimulation of ceramide synthase activity in mitochondria, and that JNK3 has a pivotal role in regulation of ceramide biosynthesis in cerebral IR.
Highlights
Mitochondria are known to be involved in both necrotic and apoptotic cell death, both of which have been identified in the ischemia/reperfusion (IR)2-injured brain [1,2,3]
We present evidence that ceramide biosynthesis rather than sphingomyelin hydrolysis mediates the accumulation of ceramide in mitochondria
The results of this study indicate that IR-induced stimulation of mitochondrial ceramide biosynthesis is under control of JNK3-mediated signaling
Summary
Mitochondria are known to be involved in both necrotic and apoptotic cell death, both of which have been identified in the ischemia/reperfusion (IR)2-injured brain [1,2,3]. Generation of ceramide in the plasma membrane, endoplasmic reticulum (ER), or Golgi apparatus by bacterial sphingomyelinase targeted to these compartments did not effect cell viability [25]. De novo synthesis of ceramide is thought to occur in the endoplasmic reticulum (ER), recent studies describe activity in semi-purified liver mitochondria [26, 27]. (Dihydro)ceramide synthase (EC 2.3.1.24) is a key enzyme in de novo synthesis of ceramide [28, 29], and in yeast, longevity assurance gene 1 (Lag1) was identified as a component of ceramide synthase. The protein exhibiting the ceramide synthase activity has been partially purified from liver mitochondria [27], the expression of LASS proteins in mitochondria remains to be elucidated
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