Tatyana I. Gudz

Direct Inhibition of Mitochondrial Respiratory Chain Complex III by Cell-permeable Ceramide

Ceramide is a lipid second messenger that mediates the effects of tumor necrosis factor α and other agents on cell growth and differentiation. Ceramide is believed to actvia activation of protein phosphatase, proline-directed protein kinase, or protein kinase C. Tumor necrosis factor α-induced common pathway of apoptosis is associated with an early impairment of mitochondria. Herein, we demonstrate that ceramide can directly inhibit mitochondrial respiratory chain function. In isolated mitochondria, a rapid decline of mitochondrial oxidative phosphorylation occurs in the presence of N-acetylsphingosine (C2-ceramide), a synthetic cell-permeable ceramide analog. An investigation of the site of ceramide action revealed that the activity of respiratory chain complex III is reduced by C2-ceramide with half-maximum effect at 5–7 μm. In contrast,N-acetylsphinganine (C2-dihydroceramide), which lacks a functionally critical double bond and is ineffective in cells, did not alter mitochondrial respiration or complex III activity. We suggest that these in vitro observations may set the stage for identifying a novel mechanism of regulation of mitochondrial function in vivo.

Glutamate Stimulates Oligodendrocyte Progenitor Migration Mediated via an αv Integrin/Myelin Proteolipid Protein Complex

In the mammalian CNS, oligodendrocyte precursor cells (OPCs) express most neurotransmitter receptors, but their function remains unclear. The current studies suggest a physiological role for glutamate (AMPA and/or kainate) receptors in OPC migration. AMPA stimulated αv integrin-mediated OPC migration by increasing both the rate of cell movement and the frequency of Ca2+ transients. A protein complex containing the myelin proteolipid protein (PLP) and αv integrin modulated the AMPA-stimulated migration, and stimulation of OPC AMPA receptors resulted in increased association of the AMPA receptor subunits themselves with the αv integrin/PLP complex. Thus, after AMPA receptor stimulation, an αv integrin/PLP/neurotransmitter receptor protein complex forms that reduces binding to the extracellular matrix and enhances OPC migration. To assess the extent to which PLP was involved in the AMPA-stimulated migration, OPCs from the myelin-deficient (MD) rat, which has a PLP gene mutation, were analyzed. OPCs from the MD rat had a normal basal migration rate, but AMPA did not stimulate the migration of these cells, suggesting that the PLP/αv integrin complex was important for the AMPA-mediated induction. AMPA-induced modulation of OPC migration was abolished by pertussis toxin, although baseline migration was normal. Thus, G-protein-dependent signaling is crucial for AMPA-stimulated migration of OPCs but not for basal OPC migration. Other signaling pathways involved in this AMPA-stimulated OPC migration were also determined. These studies highlight novel signaling determinants of OPC migration and suggest that glutamate could play a pivotal role in regulating integrin-mediated OPC migration.

Activation of sphingosine-1-phosphate receptor S1P5 inhibits oligodendrocyte progenitor migration

Sphingosine‐1‐phosphate (S1P) acts as an extracellular ligand for a family of G‐protein coupled receptors that are crucial in cell migration. S1P5 is exclusively expressed in oligodendrocytes and oligodendrocyte precursor cells (OPCs), which migrate considerable distances during brain development. The current studies suggest a physiological role for S1P and S1P5 in regulation of OPC migration. mRNA expression levels of S1P2 and S1P5 are comparable in OPCs, but S1P binding specifically to the S1P5 receptor blocked OPC migration (IC50 = 29 nM). Thus, knocking down S1P5 using siRNA prevented the S1P‐induced decrease in OPC migration, whereas knocking down S1P2 did not have any effect. S1P‐induced modulation of OPC migration was insensitive to pertussis toxin, suggesting that S1P5‐initiated signaling is not mediated by the Gαi‐protein coupled pathway. Furthermore, S1P5 appears to engage the Gα12/13 protein coupled Rho/ROCK signaling pathway to impede OPC migration. To modulate OPC motility, extracellular S1P could be derived from the export of intracellular S1P generated in response to glutamate treatment of OPCs. These studies suggest that S1P could be a part of the neuron‐oligodendroglial communication network regulating OPC migration and may provide directional guidance cues for migrating OPCs in the developing brain.—Novgorodov A. S., El‐Alwani, M., Bielawski, J., Obeid, L. M., Gudz T. I. Activation of sphingosine‐1‐phosphate receptor S1P5 inhibits oligodendrocyte progenitor migration. FASEB J. 21, 1503–1514 (2007)

JNK3 Signaling Pathway Activates Ceramide Synthase Leading to Mitochondrial Dysfunction

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.

CERAMIDE AND MITOCHONDRIA IN ISCHEMIA/REPERFUSION

A hallmark of tissue injury in various models of ischemia/reperfusion (IR) is mitochondrial dysfunction and the release of mitochondrial proapoptotic proteins leading to cell death. Although IR-induced mitochondrial injury has been extensively studied and key mitochondrial functions affected by IR are chiefly characterized, the nature of the molecule that causes loss of mitochondrial integrity and function remains obscure. It has become increasingly clear that ceramide, a membrane sphingolipid and a key mediator of cell stress responses, could play a critical role in IR-induced mitochondrial damage. Emerging data point to excessive ceramide accumulation in tissue and, specifically, in mitochondria after IR. Exogenously added to isolated mitochondria, ceramide could mimic some of the mitochondrial dysfunctions occurring in IR. The recent identification and characterization of major enzymes in ceramide synthesis is expected to contribute to the understanding of molecular mechanisms of ceramide involvement in mitochondrial damage in IR. This review will examine the experimental evidence supporting the important role of ceramide in mitochondrial dysfunction in IR to highlight potential targets for pharmacological manipulation of ceramide levels.

Novel Pathway of Ceramide Production in Mitochondria THIOESTERASE AND NEUTRAL CERAMIDASE PRODUCE CERAMIDE FROM SPHINGOSINE AND ACYL-CoA

Reports suggest that excessive ceramide accumulation in mitochondria is required to initiate the intrinsic apoptotic pathway and subsequent cell death, but how ceramide accumulates is unclear. Here we report that liver mitochondria exhibit ceramide formation from sphingosine and palmitoyl-CoA and from sphingosine and palmitate. Importantly, this activity was markedly decreased in liver from neutral ceramidase (NCDase)-deficient mice. Moreover, the levels of ceramide were dissimilar in liver mitochondria of WT and NCDase KO mice. These results suggest that NCDase is a key participant of ceramide formation in liver mitochondria. We also report that highly purified liver mitochondria have ceramidase, reverse ceramidase, and thioesterase activities. Increased accessibility of palmitoyl-CoA to the mitochondrial matrix with the pore-forming peptide zervamicin IIB resulted in 2-fold increases in palmitoyl-CoA hydrolysis by thioesterase. This increased hydrolysis was accompanied by an increase in ceramide formation, demonstrating that both outer membrane and matrix localized thioesterases can regulate ceramide formation. Also, ceramide formation might occur both in the outer mitochondrial membrane and in the mitochondrial matrix, suggesting the existence of distinct ceramide pools. Taken together, these results suggest that the reverse activity of NCDase contributes to sphingolipid homeostasis in this organelle in vivo.

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

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, C16:0-ceramide was gradually reduced and CerS6 was down-regulated in mitochondria, thereby implicating CerS6 as a primary ceramide synthase generating C16:0-ceramide. Investigations into the role of CerS6 in mitochondria revealed that ceramide synthase down-regulation is associated with dramatically decreased mitochondrial Ca2+-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 Ca2+ 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 Ca2+ uptake or decreasing Ca2+-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 Ca2+ homeostasis and calpain, which appears to be important in OL apoptosis during brain development.

Long-chain Ceramide Is a Potent Inhibitor of the Mitochondrial Permeability Transition Pore

The sphingolipid ceramide has been implicated in mediating cell death that is accompanied by mitochondrial functional alterations. Moreover, ceramide has been shown to accumulate in mitochondria upon induction of apoptotic processes. In this study, we sought to evaluate the effects of natural, highly hydrophobic long-chain ceramides on mitochondrial function in vitro. Ceramide in a dodecane/ethanol delivery system inhibited the opening of the mitochondrial permeability transition pore (PTP) induced by either oxidative stress, SH group cross-linking, or high Ca2+ load, suggesting that the inhibitory point is at a level at which major PTP regulatory pathways converge. Moreover, ceramide had no effect on well known mitochondrial components that modulate PTP activity, such as cyclophilin D, voltage-dependent anion channel, adenine nucleotide transporter, and ATP synthase. The inhibitory effect of ceramide on PTP was not stereospecific, nor was there a preference for ceramide over dihydroceramide. However, the effect of ceramide on PTP was significantly influenced by the fatty acid moiety chain length. These studies are the first to show that long-chain ceramide can influence PTP at physiologically relevant concentrations, suggesting that it is the only known potent natural inhibitor of PTP. These results suggest a novel mechanism of ceramide regulation of mitochondrial function.

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

Integrins govern cellular adhesion and transmit signals leading to activation of intracellular signaling pathways aimed to prevent apoptosis. Herein we report that attachment of oligodendrocytes (OLs) to fibronectin via αvβ3 integrin receptors rendered the cells more resistant to apoptosis than the cells attached to laminin via α6β1 integrins. Investigation of molecular mechanisms involved in αvβ3 integrin-mediated cell survival revealed that ligation of the integrin with fibronectin results in higher expression of activated Lyn kinase. Both in OLs and in the mouse brain, Lyn selectively associates with αvβ3 integrin, not with αvβ5 integrin, leading to suppression of acid sphingomyelinase activity and preventing ceramide-mediated apoptosis. In OLs, knockdown of Lyn with small interfering RNA resulted in OL apoptosis with concomitant accumulation of C16-ceramide due to activation of acid sphingomyelinase (ASMase) and sphingomyelin hydrolysis. Knocking down ASMase partially protected OLs from apoptosis. In the brain, ischemia/reperfusion (IR) triggered rearrangements in the αvβ3 integrin-Lyn kinase complex leading to disruption of Lyn kinase-mediated suppression of ASMase activity. Thus, co-immunoprecipitation studies revealed an increased association of αvβ3 integrin-Lyn kinase complex with ionotropic glutamate receptor subunits, GluR2 and GluR4, after cerebral IR. Sphingolipid analysis of the brain demonstrated significant accumulation of ceramide and sphingomyelin hydrolysis. The data suggest a novel mechanism for regulation of ASMase activity during cell adhesion in which Lyn acts as a key upstream kinase that may play a critical role in cerebral IR injury.

SIRT3 Deacetylates Ceramide Synthases: Implications for Mitochondrial Dysfunction and Brain Injury

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.