Regulation of sphingomyelinases in cells of the oligodendrocyte lineage

Abstract

Controversy exists regarding the nature of the "executioner" sphingomyelinase (SMase) in cells and its subcellular localization. A new fluorescence-based assay with the substrate 6-hexadecanoylamino-4-methylumbelliferyl-phosphorylcholine allowed rapid and reliable microassays of neutral (N) and acid (A) SMase activity in cell extracts from primary cultures of neonatal rat oligodendrocytes (OPC) and a human oligodendroglioma cell line (HOG). Total SMase activity was much higher in OPC than in HOG cells. Both staurosporine and tumor necrosis factor-alpha (TNF-alpha) induced apoptosis and activated NSMase in a multiphasic manner in both OPC and HOG cells. The increase in caspase 8 activity preceded the 1 hr peak of NSMase activation, which was followed by caspase 3 activation. In contrast, ASMase activity, which constituted >90% of the total SMase activity, was unresponsive to proapoptotic drugs. Neither reducing ASMase levels by 50% by pretreatment with desipramine nor inhibiting sphingolipid synthesis by 50% with fumonisin B1 had any effect on cell death. Isolation of sphingolipid-rich plasma membrane microdomains (rafts) from the cells by sucrose density gradient ultracentrifugation revealed an enrichment of sphingomyelin, ceramide, and caspase 8. Proapoptotic drugs such as staurosporine promoted the translocation of NSMase to the raft fraction. In contrast, ASMase, other lysosomal hydrolases, and caspase 3 remained absent from rafts even after staurosporine treatment. The staurosporine-induced concomitant increase of ceramide in the raft fraction and caspase 3 in the cytosol could be mimicked by the addition of exogenous bacterial SMase. We conclude that caspase 8 activates NSMase in rafts in oligodendrocytes and that the downstream apoptotic signal is via caspase 3.

Cytokine-induced release of ceramide-enriched exosomes as a mediator of cell death signaling in an oligodendroglioma cell line

“…With respect to the intracellular source of Cer, previous studies with cytokine-treated HOG cells have indicated the major role of raft-associated neutral SMase in SM hydrolysis without the participation of the more abundant acid SMase (10). This is consistent with the ascribed role of neutral SMase in Cer-dependent exosome biogenesis (25).…”

The deficit in long-term potentiation induced by chronic administration of amyloid-β is attenuated by treatment of rats with a novel phospholipid-based drug formulation, VP025

“…The coupled increases in activation of these enzymes is consistent with the fact that the executioner caspase 3 is a major substrate for the initiator caspase 8 (Stennicke et al, 1998), with the evidence which indicated that caspase 8 can activate sphingomyelinase (Heinrich et al, 2004) and with the demonstration that TNF -induced apoptosis in oligodendrocytes is dependent on sequential activation of caspase 8, neutral sphingomyelinase and caspase 3 (Testai et al, 2004). Numerous studies have reported that A increases caspase 3 activity (Awasthi et al, 2005;Boland and Campbell, 2004) and caspase 8 activity (Awasthi et al, 2005) in cultured neurons while increases in both enzymes have also been reported in the AD brain (Rohn et al, 2002).…”

Integrin-associated Lyn Kinase Promotes Cell Survival by Suppressing Acid Sphingomyelinase Activity

“…Knocking down either ␣ v subunit of integrin or Lyn kinase triggered activation of ASMase about 2-or 1.7-fold, respectively. There was no change in neutral sphingomyelinase activity, which was 5.64 Ϯ 0.39 pmol/g/h, which constitutes less than 10% of total sphingomyelinase activity in these cells (47). The results of these studies suggest that ␣ v ␤ 3 integrin receptor-Lyn-mediated signaling blocks ASMase activity in OLs grown on fibronectin.…”

“…To determine what sphingomyelinase is regulated by ␣ v ␤ 3 integrin-Lyn kinase signaling, we focused first on ASMase, which is a major sphingomyelinase enzyme in OLs (47). The activity of ASMase was measured after OL transfection with siRNA targeting the ␣ v subunit of the integrin receptor or Lyn kinase (Fig.…”