Developmentally Regulated Ceramide Synthase 6 Increases Mitochondrial Ca2+ Loading Capacity and Promotes Apoptosis

Abstract

Ceramides, which are membrane sphingolipids and key mediators of cell-stress responses, are generated by a family of (dihydro) ceramide synthases (Lass1-6/CerS1-6). Here, we report that brain development features significant increases in sphingomyelin, sphingosine, and most ceramide species. In contrast, C 16:0 -ceramide was gradually reduced and CerS6 was down-regulated in mitochondria, thereby implicating CerS6 as a primary ceramide synthase generating C 16:0 -ceramide. Investigations into the role of CerS6 in mitochondria revealed that ceramide synthase down-regulation is associated with dramatically decreased mitochondrial Ca 2؉ -loading capacity, which could be rescued by addition of ceramide. Selective CerS6 complexing with the inner membrane component of the mitochondrial permeability transition pore was detected by immunoprecipitation. This suggests that CerS6-generated ceramide could prevent mitochondrial permeability transition pore opening, leading to increased Ca 2؉ accumulation in the mitochondrial matrix. We examined the effect of high CerS6 expression on cell survival in primary oligodendrocyte (OL) precursor cells, which undergo apoptotic cell death during early postnatal brain development. Exposure of OLs to glutamate resulted in apoptosis that was prevented by inhibitors of de novo ceramide biosynthesis, myriocin and fumonisin B1. Knockdown of CerS6 with siRNA reduced glutamate-triggered OL apoptosis, whereas knockdown of CerS5 had no effect: the pro-apoptotic role of CerS6 was not stimulus-specific. Knockdown of CerS6 with siRNA improved cell survival in response to nerve growth factor-induced OL apoptosis. Also, blocking mitochondrial Ca 2؉ uptake or decreasing Ca 2؉ -dependent protease calpain activity with specific inhibitors prevented OL apoptosis. Finally, knocking down CerS6 decreased calpain activation. Thus, our data suggest a novel role for CerS6 in the regulation of both mitochondrial Ca 2؉ homeostasis and calpain, which appears to be important in OL apoptosis during brain development.

Encephalopathy Caused by Ablation of Very Long Acyl Chain Ceramide Synthesis May Be Largely Due to Reduced Galactosylceramide Levels

“…1). Substantial levels of C16-ceramide and C16-SM are found immediately after birth and decrease during development in both CerS2-null and WT mice, consistent with the decreased expression of CerS6 during brain development (38). In contrast, the acyl chain composition of HexCer (which consists of both glucosylceramide (GlcCer) and galactosylceramide (GalCer)) changes dramatically during development.…”

Mitochondrial Ceramide in Stroke

“…Further sub-mitochondrial investigation of ceramide synthase activity revealed that both outer and inner mitochondrial membranes can synthesize ceramide (Bionda et al, 2004). Recent reports describing several ceramide synthase isoforms, including CerS1, CerS2, CerS4 and CerS6, in purified mouse brain mitochondria (Novgorodov et al, 2011a;Yu et al, 2007) support the notion that several ceramide synthesizing enzymes could be localized in mitochondria (Futerman, 2006). No such association was found in HeLa cells (Mesicek et al, 2010), suggesting that this might be a cell type/tissue specific event.…”

“…No such association was found in HeLa cells (Mesicek et al, 2010), suggesting that this might be a cell type/tissue specific event. The intramitochondrial localization of CerS was examined in purified brain mitochondria by immunoprecipitation (Novgorodov et al, 2011a). These studies reveal a selective CerS6 association with adenine nucleotide translocase, the inner membrane component of the mitochondrial permeability transition pore (MPTP).…”

“…Activity measurements indicated activation of CerS6 in ischemic mitochondria apparently via post-translational mechanisms; IR did not affect the CerS6 protein expression (Yu et al, 2007). It appears that CerS6 is developmentally regulated and primarily generates C 16:0 -ceramide in brain mitochondria (Novgorodov et al, 2011a). An investigation into the role of CerS6 in mitochondria revealed that ceramide synthase down-regulation during brain development is associated with dramatically decreased mitochondrial Ca 2+ -loading capacity (CLC) which could be rescued by addition of ceramide.…”

Silencing of CerS6 increases the invasion and glycolysis of melanoma WM35, WM451 and SK28 cell lines via increased GLUT1-induced downregulation of WNT5A

“…Senkal et al showed the anti-apoptotic role of CerS6 in HNSCC (23). However, developmentally regulated CerS6 was found to increase mitochondrial Ca 2+ loading capacity and promote apoptosis (32). Similarly, CerS6 knockdown inhibited TRAIL-induced apoptosis (22).…”