RESUMO
Traumatic brain injury (TBI) is one of the leading causes of disability worldwide and a prominent risk factor for neurodegenerative diseases. The expansion of nervous tissue damage after the initial trauma involves a multifactorial cascade of events, including excitotoxicity, oxidative stress, inflammation, and deregulation of sphingolipid metabolism that further mitochondrial dysfunction and secondary brain damage. Here, we show that a posttranscriptional activation of an acid sphingomyelinase (ASM), a key enzyme of the sphingolipid recycling pathway, resulted in a selective increase of sphingosine in mitochondria during the first week post-TBI that was accompanied by reduced activity of mitochondrial cytochrome oxidase and activation of the Nod-like receptor protein 3 inflammasome. TBI-induced mitochondrial abnormalities were rescued in the brains of ASM KO mice, which demonstrated improved behavioral deficit recovery compared with WT mice. Furthermore, an elevated autophagy in an ASM-deficient brain at the baseline and during the development of secondary brain injury seems to foster the preservation of mitochondria and brain function after TBI. Of note, ASM deficiency attenuated the early stages of reactive astrogliosis progression in an injured brain. These findings highlight the crucial role of ASM in governing mitochondrial dysfunction and brain-function impairment, emphasizing the importance of sphingolipids in the neuroinflammatory response to TBI.
Assuntos
Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Técnicas de Inativação de Genes , Mitocôndrias/patologia , Recuperação de Função Fisiológica , Esfingomielina Fosfodiesterase/deficiência , Animais , Lesões Encefálicas/enzimologia , Lesões Encefálicas/genética , Cognição , Ativação Enzimática , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Esfingomielina Fosfodiesterase/genéticaRESUMO
Inhibiting the glutamate/cystine antiporter system xc-, a key antioxidant defense machinery in the CNS, could trigger a novel form of regulated necrotic cell death, ferroptosis. The underlying mechanisms of system xc--dependent cell demise were elucidated using primary oligodendrocytes (OLs) treated with glutamate to block system xc- function. Pharmacological analysis revealed ferroptosis as a major contributing factor to glutamate-initiated OL death. A sphingolipid profile showed elevations of ceramide species and sphingosine that were preventable by inhibiting of an acid sphingomyelinase (ASM) activity. OL survival was enhanced by both downregulating ASM expression and blocking ASM activity. Glutamate-induced ASM activation seems to involve posttranscriptional mechanisms and was associated with a decreased GSH level. Further investigation of the mechanisms of OL response to glutamate revealed enhanced reactive oxygen species production, augmented lipid peroxidation, and opening of the mitochondrial permeability transition pore that were attenuated by hindering ASM. Of note, knocking down sirtuin 3, a deacetylase governing the mitochondrial antioxidant system, reduced OL survival. The data highlight the importance of the mitochondrial compartment in regulated necrotic cell death and accentuate the novel role of ASM in disturbing mitochondrial functions during OL response to glutamate toxicity, which is essential for pathobiology in stroke and traumatic brain injury.
Assuntos
Ácido Glutâmico/farmacologia , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Esfingomielina Fosfodiesterase/metabolismo , Animais , Células Cultivadas , Feminino , Camundongos , Mitocôndrias/efeitos dos fármacos , Necrose/metabolismo , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Lesões Encefálicas/enzimologia , Proteínas de Membrana/metabolismo , Mitocôndrias/enzimologia , Sirtuína 3/metabolismo , Esfingosina N-Aciltransferase/metabolismo , Animais , Apoptose , Lesões Encefálicas/genética , Lesões Encefálicas/metabolismo , Lesões Encefálicas/fisiopatologia , Ceramidas/metabolismo , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Estresse Oxidativo , Sirtuína 3/genética , Esfingosina N-Aciltransferase/genéticaRESUMO
Sphingolipids have been implicated as key mediators of cell-stress responses and effectors of mitochondrial function. To investigate potential mechanisms underlying mitochondrial dysfunction, an important contributor to diabetic cardiomyopathy, we examined alterations of cardiac sphingolipid metabolism in a mouse with streptozotocin-induced type 1 diabetes. Diabetes increased expression of desaturase 1, (dihydro)ceramide synthase (CerS)2, serine palmitoyl transferase 1, and the rate of ceramide formation by mitochondria-resident CerSs, indicating an activation of ceramide biosynthesis. However, the lack of an increase in mitochondrial ceramide suggests concomitant upregulation of ceramide-metabolizing pathways. Elevated levels of lactosylceramide, one of the initial products in the formation of glycosphingolipids were accompanied with decreased respiration and calcium retention capacity (CRC) in mitochondria from diabetic heart tissue. In baseline mitochondria, lactosylceramide potently suppressed state 3 respiration and decreased CRC, suggesting lactosylceramide as the primary sphingolipid responsible for mitochondrial defects in diabetic hearts. Moreover, knocking down the neutral ceramidase (NCDase) resulted in an increase in lactosylceramide level, suggesting a crosstalk between glucosylceramide synthase- and NCDase-mediated ceramide utilization pathways. These data suggest the glycosphingolipid pathway of ceramide metabolism as a promising target to correct mitochondrial abnormalities associated with type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Lactosilceramidas/metabolismo , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Animais , Respiração Celular , Diabetes Mellitus Tipo 1/enzimologia , Diabetes Mellitus Tipo 1/fisiopatologia , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Coração/fisiopatologia , Hidrólise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ceramidase Neutra/deficiência , Ceramidase Neutra/genética , Ceramidase Neutra/metabolismoRESUMO
In addition to immediate brain damage, traumatic brain injury (TBI) initiates a cascade of pathophysiological events producing secondary injury. The biochemical and cellular mechanisms that comprise secondary injury are not entirely understood. Herein, we report a substantial deregulation of cerebral sphingolipid metabolism in a mouse model of TBI. Sphingolipid profile analysis demonstrated increases in sphingomyelin species and sphingosine concurrently with up-regulation of intermediates of de novo sphingolipid biosynthesis in the brain. Investigation of intracellular sites of sphingosine accumulation revealed an elevation of sphingosine in mitochondria due to the activation of neutral ceramidase (NCDase) and the reduced activity of sphingosine kinase 2 (SphK2). The lack of change in gene expression suggested that post-translational mechanisms are responsible for the shift in the activities of both enzymes. Immunoprecipitation studies revealed that SphK2 is complexed with NCDase and cytochrome oxidase (COX) subunit 1 in mitochondria and that brain injury hindered SphK2 association with the complex. Functional studies showed that sphingosine accumulation resulted in a decreased activity of COX, a rate-limiting enzyme of the mitochondrial electron transport chain. Knocking down NCDase reduced sphingosine accumulation in mitochondria and preserved COX activity after the brain injury. Also, NCDase knockdown improved brain function recovery and lessened brain contusion volume after trauma. These studies highlight a novel mechanism of secondary TBI involving a disturbance of sphingolipid-metabolizing enzymes in mitochondria and suggest a critical role for mitochondrial sphingosine in promoting brain injury after trauma.
Assuntos
Ceramidase Alcalina/metabolismo , Lesões Encefálicas/metabolismo , Encéfalo/metabolismo , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Esfingosina/metabolismo , Ceramidase Alcalina/genética , Animais , Encéfalo/patologia , Lesões Encefálicas/genética , Lesões Encefálicas/patologia , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Proteínas do Tecido Nervoso/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/genéticaRESUMO
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.
Assuntos
Apoptose/fisiologia , Encéfalo/enzimologia , Cálcio/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Oligodendroglia/enzimologia , Esfingosina N-Aciltransferase/metabolismo , Células-Tronco/enzimologia , Animais , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Calpaína/genética , Calpaína/metabolismo , Células Cultivadas , Feminino , Técnicas de Silenciamento de Genes , Homeostase/fisiologia , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas do Tecido Nervoso/genética , Oligodendroglia/citologia , Ratos , Ratos Sprague-Dawley , Esfingosina N-Aciltransferase/genética , Células-Tronco/citologiaRESUMO
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.
Assuntos
Ceramidas/metabolismo , Metabolismo dos Lipídeos/fisiologia , Mitocôndrias Hepáticas/enzimologia , Proteínas Mitocondriais/metabolismo , Ceramidase Neutra/metabolismo , Palmitoil Coenzima A/metabolismo , Esfingosina/metabolismo , Animais , Ceramidas/genética , Hidrólise/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/genética , Proteínas Mitocondriais/genética , Ceramidase Neutra/genética , Palmitoil Coenzima A/genética , Palmitoil-CoA Hidrolase , Peptaibols/farmacologia , Ratos , Ratos Sprague-Dawley , Esfingosina/genéticaRESUMO
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.
Assuntos
Ceramidas/metabolismo , Mitocôndrias/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Encéfalo/enzimologia , Encéfalo/metabolismo , Encéfalo/patologia , Morte Celular , Ceramidas/biossíntese , Humanos , Rim/enzimologia , Rim/metabolismo , Rim/patologia , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Miocárdio/enzimologia , Miocárdio/metabolismo , Miocárdio/patologia , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/patologia , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismoRESUMO
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 G alpha(i)-protein coupled pathway. Furthermore, S1P5 appears to engage the G alpha(12/13) 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.
Assuntos
Movimento Celular , Oligodendroglia/citologia , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Sequência de Bases , Movimento Celular/efeitos dos fármacos , Técnicas de Cocultura , Primers do DNA , Inativação Gênica , Ácido Glutâmico/farmacologia , Lisofosfolipídeos/farmacologia , RNA Mensageiro/genética , Ratos , Receptores de Lisoesfingolipídeo/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização por Electrospray , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Espectrometria de Massas em TandemRESUMO
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 alphav 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 alphav integrin modulated the AMPA-stimulated migration, and stimulation of OPC AMPA receptors resulted in increased association of the AMPA receptor subunits themselves with the alphav integrin/PLP complex. Thus, after AMPA receptor stimulation, an alphav 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/alphav 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.
Assuntos
Movimento Celular/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Integrina alfaV/fisiologia , Proteína Proteolipídica de Mielina/fisiologia , Oligodendroglia/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Análise de Variância , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Benzodiazepinas/farmacologia , Western Blotting/métodos , Cálcio/metabolismo , Carbacol/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Células Cultivadas , Quelantes/farmacologia , Agonistas Colinérgicos/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Fibronectinas/metabolismo , Fibronectinas/farmacologia , Imuno-Histoquímica/métodos , Imunoprecipitação/métodos , Ionóforos/farmacologia , Ácido Caínico/farmacologia , Modelos Biológicos , Bainha de Mielina/genética , Toxina Pertussis/farmacologia , Quinoxalinas/farmacologia , Ratos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de AMPA/metabolismo , Rutênio Vermelho/farmacologia , Tapsigargina/farmacologia , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
Myelination of axons in the CNS by oligodendrocytes is a process critical to rapid and efficient impulse conduction. A new role for the myelin proteolipid protein (PLP), the most abundant protein of CNS myelin, has been identified, in studies showing PLP interaction with signaling proteins in oligodendrocytes. In particular, these studies suggest that the PLP protein may be involved in signaling through integrins in oligodendrocytes. Stimulation of muscarinic acetylcholine receptors on oligodendrocytes induced formation of a tripartite complex containing PLP, calreticulin, and alpha(v)-integrin. PLP interacted directly with the cytoplasmic domain of the alpha(v)-integrin. Complex formation was mediated by phospholipase C and Ca2+ binding to the high affinity binding site on calreticulin. This complex appears important for binding of fibronectin to oligodendrocytes. These data establish a novel function for PLP as a part of the integrin signaling complex in oligodendrocytes and suggest that neurotransmitter-mediated integrin receptor signaling may be involved in myelinogenesis.
Assuntos
Integrinas/metabolismo , Proteína Proteolipídica de Mielina/metabolismo , Oligodendroglia/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Animais , Antígenos CD/metabolismo , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Calreticulina , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Proteínas da Matriz Extracelular/metabolismo , Integrina alfaV , Substâncias Macromoleculares , Agonistas Muscarínicos/farmacologia , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Testes de Precipitina , Ligação Proteica , Ratos , Ribonucleoproteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
Pelizaeus Merzbacher disease is an X-linked dysmyelinating disorder of the CNS, resulting from mutations in the proteolipid protein (PLP) gene. An animal model for this disorder, the myelin-deficient (MD) rat, carries a point mutation in the PLP gene and exhibits a phenotype similar to the fatal, connatal disease, including extensive dysmyelination, tremors, ataxia, and death at approximately postnatal day 21 (P21). We postulated that early death might result from disruption of myelinated neural pathways in the caudal brainstem and altered ventilatory response to oxygen deprivation or hypercapnic stimulus. Using barometric plethysmography to measure respiratory function, we found that the MD rat develops lethal hypoxic depression of breathing at P21, but hypercapnic ventilatory response is normal. Histologic examination of the caudal brainstem in the MD rat at this age showed extensive dysmyelination and downregulation of NMDA and to a lesser extent GABA(A) receptors on neurons in the nucleus tractus solitarius, hypoglossal nucleus, and dorsal motor nucleus of the vagus. Unexpectedly, immunoreactive PLP/DM20 was detected in neurons in the caudal brainstem. Not all biosynthetic functions and structural elements were altered in these neurons, because phosphorylated and nonphosphorylated neurofilament and choline acetyltransferase expression were comparable between MD and wild-type rats. These findings suggest that PLP is expressed in neurons in the developing brainstem and that PLP gene mutation can selectively disrupt central processing of afferent neural input from peripheral chemoreceptors, leaving the central chemosensory system for hypercapnia intact.
Assuntos
Hipóxia/fisiopatologia , Proteína Proteolipídica de Mielina/genética , Bainha de Mielina/genética , Proteínas do Tecido Nervoso , Doença de Pelizaeus-Merzbacher/fisiopatologia , Respiração/genética , Animais , Tronco Encefálico/patologia , Tronco Encefálico/fisiopatologia , Contagem de Células , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Hipercapnia/fisiopatologia , Masculino , Camundongos , Camundongos Transgênicos , Proteína Proteolipídica de Mielina/biossíntese , Bainha de Mielina/patologia , Vias Neurais/patologia , Vias Neurais/fisiopatologia , Proteínas de Neurofilamentos/biossíntese , Neurônios/patologia , Oligodendroglia/patologia , Doença de Pelizaeus-Merzbacher/genética , Doença de Pelizaeus-Merzbacher/patologia , Fosforilação , Mutação Puntual , Ratos , Receptores de GABA-A/biossíntese , Receptores de N-Metil-D-Aspartato/biossínteseRESUMO
BACKGROUND: Dihydroceramide desaturase 1 (DES) is the enzyme responsible for converting dihydroceramide into ceramide in the de novo sphingolipid biosynthesis pathway. Dihydroceramide can inhibit ceramide channel formation to interfere with apoptosis. We have shown that following ceramide synthase knockdown, photodynamic therapy (PDT), a cancer treatment modality, is associated with decreased levels of ceramides and dihydroceramides in cells that are resistant to apoptosis. AIM: Here we investigated the effect of DES knockdown on the sphingolipid profile and apoptosis in human head and neck squamous carcinoma cells after PDT with the silicon phthalocyanine Pc 4. MATERIALS AND METHODS: Following siRNA transfection and PDT treatment, quantitative real-time polymerase chain reaction for quantification of DES mRNA, immunoblotting for protein expression, mass spectrometry for sphingolipid analysis, spectrofluorometry for caspase 3-like (DEVDase) activity, flow cytometry for apoptosis detection, and trypan blue assay for cell viability evaluation, were performed. RESULTS: Down-regulation of DES led to a substantial increase in levels of dihydroceramides without affecting ceramide levels. PDT-induced accumulation of individual dihydroceramides and global ceramides was increased by DES knockdown. Concomitantly, mitochondrial depolarization, DEVDase activation, late-apoptosis and cell death were attenuated by DES knockdown. Early apoptosis, however, was enhanced. CONCLUSION: Our findings support the following: (i) dihydroceramide reduces pro-apoptotic effects of ceramide; (ii) cells adapt to DES knockdown to become more sensitive to ceramide and early-apoptosis; (iii) DES is a potential molecular target for regulating apoptotic resistance to PDT.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Oxirredutases , Fotoquimioterapia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/terapia , Linhagem Celular Tumoral , Ceramidas/metabolismo , Ceramidas/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/terapia , Humanos , Indóis/administração & dosagem , Terapia de Alvo Molecular , Oxirredutases/antagonistas & inibidores , Oxirredutases/genética , Oxirredutases/metabolismo , RNA Interferente Pequeno , Esfingolipídeos/metabolismoRESUMO
Photodynamic therapy (PDT) is not always effective as an anticancer treatment, therefore, PDT is combined with other anticancer agents for improved efficacy. The combination of dasatinib and PDT with the silicone phthalocyanine photosensitizer Pc 4 was assessed for increased killing of SCCVII mouse squamous cell carcinoma cells, a preclinical model of head and neck squamous cell carcinoma, using apoptotic markers and colony formation as experimental end-points. Because each of these treatments regulates the metabolism of the sphingolipid ceramide, their effects on mRNA levels of ceramide synthase, a ceramide-producing enzyme, and the sphingolipid profile were determined. PDT + dasatinib induced an additive loss of clonogenicity. Unlike PDT alone or PDT + dasatinib, dasatinib induced zVAD-fmk-dependent cell killing. PDT or dasatinib-induced caspase-3 activation was potentiated after the combination. PDT alone induced mitochondrial depolarization, and the effect was inhibited after the combination. Annexin V+ and propidium iodide+ cells remained at control levels after treatments. In contrast to PDT alone, dasatinib induced upregulation of ceramide synthase 1 mRNA, and the effect was enhanced after the combination. Dasatinib induced a modest increase in C20:1- and C22-ceramide but had no effect on total ceramide levels. PDT increased the levels of 12 individual ceramides and total ceramides, and the addition of dasatinib did not affect these increases. PDT alone decreased substantially sphingosine levels and inhibited the activity of acid ceramidase, an enzyme that converts ceramide to sphingosine. The data suggest that PDT-induced increases in ceramide levels do not correlate with ceramide synthase mRNA levels but rather with inhibition of ceramidase. Cell killing was zVAD-fmk-sensitive after dasatinib but not after either PDT or the combination and enhanced cell killing after the combination correlated with potentiated caspase-3 activation and upregulation of ceramide synthase 1 mRNA but not the production of ceramide. The data imply potential significance of the combination for cancer treatment.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Indóis/uso terapêutico , Oxirredutases/genética , Fotoquimioterapia/métodos , Pirimidinas/uso terapêutico , Tiazóis/uso terapêutico , Neoplasias Abdominais/tratamento farmacológico , Ceramidase Ácida/antagonistas & inibidores , Ceramidase Ácida/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Anexina A5/metabolismo , Apoptose , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Ceramidas/biossíntese , Ceramidas/metabolismo , Dasatinibe , Ativação Enzimática , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C3H , Mitocôndrias/metabolismo , Propídio , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , RNA Mensageiro/biossíntese , Esfingosina/metabolismoRESUMO
BACKGROUND: The effectiveness of photodynamic therapy (PDT) for cancer treatment correlates with apoptosis. We previously observed that the knockdown of ceramide synthase 6, an enzyme from the de novo sphingolipid biosynthesis pathway, is associated with marked reduction in C18-dihydroceramide and makes cells resistant to apoptosis post-PDT. Down-regulation of ceramide synthase 1 (CERS1) can also render cells resistant to anticancer drugs. AIM: To explore the impact of CERS1 knockdown on apoptosis and the sphingolipid profile, post-PDT, with the silicone phthalocyanine Pc 4, in a human head and neck squamous carcinoma cell line. MATERIALS AND METHODS: Besides siRNA transfection and PDT treatment, the following methods were used: immunoblotting for protein expression, mass spectrometry for sphingolipid analysis, spectroflurometry and flow cytometry for apoptosis detection, and trypan blue assay for cell viability evaluation. RESULTS: CERS1 knockdown led to inhibition of PDT-induced caspase 3-like (DEVDase) activation, of apoptosis and cell death. CERS1 knockdown was associated with global and selective decreases in ceramides and dihydroceramides, in particular C18-, C18:1- and C20-ceramide post-PDT. CONCLUSION: Our novel findings are consistent with the notion that CERS1 regulates apoptotic resistance to PDT, partly via C18- and C20-ceramide, and that CERS1 is a molecular target for controlling resistance to PDT.
Assuntos
Carcinoma de Células Escamosas/terapia , Neoplasias de Cabeça e Pescoço/terapia , Proteínas de Membrana/genética , Fotoquimioterapia/métodos , RNA Interferente Pequeno/administração & dosagem , Esfingosina N-Aciltransferase/genética , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Regulação para Baixo , Técnicas de Silenciamento de Genes , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/enzimologia , Neoplasias de Cabeça e Pescoço/genética , Humanos , Immunoblotting , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Peptídeo Hidrolases/metabolismo , RNA Interferente Pequeno/genética , Esfingolipídeos/análise , Esfingolipídeos/metabolismo , Esfingosina N-Aciltransferase/deficiência , Esfingosina N-Aciltransferase/metabolismo , Transfecção , Azul Tripano/análiseRESUMO
BACKGROUND: The effectiveness of photodynamic therapy (PDT) for cancer treatment correlates with apoptosis. We observed that suppression of de novo-generated sphingolipids, e.g. ceramide, renders cells resistant to apoptosis post-PDT. Ceramide synthase 6 (CerS6) has been implicated in apoptosis after various stimuli. AIM: To investigate the involvement of down-regulation of CerS6 in apoptosis and its impact on the sphingolipid profile post-PDT with the silicone phthalocyanine Pc 4 in a human head and neck squamous carcinoma cell line. MATERIALS AND METHODS: Besides siRNA transfections and PDT treatment, immunoblotting for protein expression, mass spectrometry for sphingolipid analysis, spectroflurometry and flow cytometry for apoptotic marker detection, and trypan blue assay for cytotoxicity assessment, were used. RESULTS: CerS6 knockdown led to reduction in PDT-induced DEVDase activation, mitochondrial depolarization, apoptosis and cell death. CerS6 knockdown was associated with selective decreases in ceramides and dihydroceramides, markedly of C18-dihydroceramide, post-PDT. CONCLUSION: CerS6 might be a novel therapeutic target for regulating apoptotic resistance to PDT.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Proteínas de Membrana/metabolismo , Fotoquimioterapia , Esfingosina N-Aciltransferase/metabolismo , Sequência de Bases , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/patologia , Primers do DNA , Técnicas de Silenciamento de Genes , Neoplasias de Cabeça e Pescoço/enzimologia , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Proteínas de Membrana/genética , Esfingosina N-Aciltransferase/genéticaRESUMO
Sphingolipids are essential structural components of cellular membranes, playing prominent roles in signal transduction that governs cell proliferation, differentiation and apoptosis. Ceramides, a family of distinct molecular species characterized by various acyl chains, are synthesized de novo at the cytosolic side of the endoplasmic reticulum serving as precursors for the biosynthesis of sphingolipids in the Golgi. Recently, mitochondria emerged as an important intracellular compartment of sphingolipid metabolism. Thus, several sphingolipid-metabolizing enzymes were found to be associated with mitochondria, including neutral ceramidase, novel neutral sphingomyelinase, and (dihydro) ceramide synthase, an important ceramide-generating enzyme in de novo ceramide synthesis and recycling pathway. Mitochondrial dysfunction appears to be essential in tissue damage after brain ischemia/reperfusion (IR). Mitochondria are known to be involved in both the necrosis and apoptosis detected in animal models of ischemic stroke, and treatments that ameliorate tissue infarction were associated with better recovery of mitochondrial function. Although mitochondrial injury in stroke has been extensively studied and key mitochondrial functions affected by IR are mainly characterized, the nature of the molecule that causes loss of mitochondrial integrity and function remains obscure. Emerging data indicate a deregulation of ceramide metabolism in mitochondria damaged by IR suggesting that ceramides could play critical roles in cerebral IR-induced mitochondrial damage. This review will examine the experimental evidence supporting the key role of ceramides in mitochondrial dysfunction in cerebral IR and highlight potential targets for development of novel therapeutic approaches for stroke treatment.
RESUMO
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.
Assuntos
Ceramidas/farmacologia , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Animais , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Ceramidas/metabolismo , Peptidil-Prolil Isomerase F , Ciclofilinas/metabolismo , Masculino , Poro de Transição de Permeabilidade Mitocondrial , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas de Transporte de Nucleosídeos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ratos , Ratos Sprague-Dawley , Esfingolipídeos/metabolismo , Esfingolipídeos/farmacologiaRESUMO
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 alpha(v)beta(3) integrin receptors rendered the cells more resistant to apoptosis than the cells attached to laminin via alpha(6)beta(1) integrins. Investigation of molecular mechanisms involved in alpha(v)beta(3) 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 alpha(v)beta(3) integrin, not with alpha(v)beta(5) 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 C(16)-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 alpha(v)beta(3) integrin-Lyn kinase complex leading to disruption of Lyn kinase-mediated suppression of ASMase activity. Thus, co-immunoprecipitation studies revealed an increased association of alpha(v)beta(3) 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.