Neutral sphingomyelinase activation precedes NADPH oxidase‐dependent damage in neurons exposed to the proinflammatory cytokine tumor necrosis factor‐α

Abstract

Inflammation accompanied by severe oxidative stress plays a vital role in the orchestration and progression of neurodegeneration prevalent in chronic and acute CNS pathologies as well as in aging. The proinflammatory cytokine tumor necrosis factor alpha (TNFα) elicits the formation of the bioactive ceramide by stimulating the hydrolysis of the membrane lipid sphingomyelin by sphingomyelinase activities. Ceramide stimulates the formation of reactive oxygen species (ROS) and apoptotic mechanisms in both neurons and non-neuronal cells establishing a link between sphingolipid metabolism and oxidative stress. We demonstrated in SH-SY5Y human neuroblastoma cells and primary cortical neurons, that TNFα is a potent stimulator of Mg2+-dependent neutral sphingomyelinase (Mg2+-nSMase) activity and sphingomyelin hydrolysis rather than de novo synthesis, was the predominant source of ceramide increases. Mg2+-nSMase activity preceded an accumulation of ROS by a neuronal NADPH oxidase (NOX). Notably, TNFα provoked a NOX-dependent oxidative damage to sphingosine kinase-1, which generates sphingosine-1-phosphate, a ceramide metabolite associated with neurite outgrowth. Indeed, ceramide and ROS inhibited neurite outgrowth of DRG neurons by disrupting growth cone motility. Blunting ceramide and ROS formation both rescued sphingosine kinase-1 activity and neurite outgrowth. Our studies suggest that TNFα-mediated activation of Mg2+-nSMase and NOX in neuronal cells not only produced the neurotoxic intermediates ceramide and ROS but also directly antagonized neuronal survival mechanisms thus accelerating neurodegeneration.

Structure of human nSMase2 reveals an interdomain allosteric activation mechanism for ceramide generation

“…Neutral sphingomyelinase 2 (nSMase2, product of the SMPD3 gene) is the major neutral sphingomyelinase (N-SMase) in mammalian cells for the stressinduced generation of ceramide (4). TNF-α (9-15), cell confluence (16,17), oxidative stress (18)(19)(20), and inflammation (20,21) are all potent activators of nSMase2.…”

Regulation of the Sphingosine Kinase/Sphingosine 1-Phosphate Pathway

“…2 and Table 1) (Du et al 2012). Lastly, TNF-α was shown to negatively regulate SK/S1P signaling via promotion of NADPH oxidase (Nox)-mediated oxidative damage to SK1 in neuronal cells (Barth et al 2012). Discussed in the next chapter, the SK/S1P pathway has been shown to mediate a large portion of TNF-α-mediated events, supporting TNF-α induced "inside-out" S1P signaling.…”

Ceramide 1-Phosphate: A Mediator of Inflammatory Responses

“…On the other hand, C1P has been demonstrated to mediate inflammatory responses (Chalfant and Spiegel 2005;HinkovskaGalcheva et al 2005) and to promote cell migration Granado et al 2009b;Karapetyan et al 2013;Kim et al 2012;Ratajczak et al 2010), an action that is also associated to inflammation. Utilizing a combination of pharmacological and molecular approaches, Barth and co-coworkers demonstrated that in human neuroblastoma cells, CerK activity was strictly required for activation of various oxidoreductases, including reactive oxygen species (ROS) producing NADPH oxidase (NOX), cyclooxygenase (COX), and 5-lipooxygenase (5-LOX), which are enzymes involved in pro-inflammatory processes (Barth et al 2012). The latter study also showed that activation of CerK caused cPLA2 activation and subsequent eicosanoid biosynthesis and that elimination of CerK rescued neuronal viability in the presence of pro-inflammatory TNF-a.…”