RESUMO
SMPD3 deficiency in the neutral sphingomyelinase (Smpd3-/-) mouse results in a novel form of juvenile dwarfism, suggesting smpd3 is a polygenetic determinant of body height. SMPD3 controls homeostasis of the sphingomyelin cycle in the Golgi compartment, essential for membrane remodeling, initiating multiform vesicle formation and transport in the Golgi secretory pathway. Using the unbiased Smpd3-/- genetic model, this study shows that the perturbed Golgi secretory pathway of chondrocytes of the epiphyseal growth zone leads to dysproteostasis, skeletal growth inhibition, malformation, and chondrodysplasia, but showed unimpaired mineralization in primary and secondary enchondral ossification centers. This has been elaborated by biochemical analyses and immunohistochemistry of long bones of Smpd3-/- mice. A more precise definition of the microarchitecture and three-dimensional structure of the bone was shown by peripheral quantitative computed tomography, high-resolution microcomputed tomography, and less precisely by dual-energy X-ray absorptiometry for osteodensitometry. Ablation of the Smpd3 locus as part of a 980-kb deletion on chromosome 8 in the fro/fro mutant, generated by chemical mutagenesis, is held responsible for skeletal hypomineralization, osteoporosis, and multiple fractures of long bones, which are hallmarks of human osteogenesis imperfecta. The phenotype of the genetically unbiased Smpd3-/- mouse, described here, precludes the proposed role of Smpd3 as a candidate gene of human osteogenesis imperfecta, but suggests SMPD3 deficiency as the pathogenetic basis of a novel form of chondrodysplasia.
Assuntos
Desenvolvimento Ósseo , Calcificação Fisiológica , Condrócitos/patologia , Osteocondrodisplasias/etiologia , Esfingomielina Fosfodiesterase/fisiologia , Animais , Condrócitos/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteocondrodisplasias/patologiaRESUMO
Mammalian ω3- and ω6-PUFAs are synthesized from essential fatty acids (EFAs) or supplied by the diet. PUFAs are constitutive elements of membrane architecture and precursors of lipid signaling molecules. EFAs and long-chain (LC)-PUFAs are precursors in the synthesis of endocannabinoid ligands of Gi/o protein-coupled cannabinoid receptor (CB)1 and CB2 in the endocannabinoid system, which critically regulate energy homeostasis as the metabolic signaling system in hypothalamic neuronal circuits and behavioral parameters. We utilized the auxotrophic fatty acid desaturase 2-deficient (fads2-/-) mouse, deficient in LC-PUFA synthesis, to follow the age-dependent dynamics of the PUFA pattern in the CNS-phospholipidome in unbiased dietary studies of three cohorts on sustained LC-PUFA-free ω6-arachidonic acid- and DHA-supplemented diets and their impact on the precursor pool of CB1 ligands. We discovered the transformation of eicosa-all cis-5,11,14-trienoic acid, uncommon in mammalian lipidomes, into two novel endocannabinoids, 20:35,11,14-ethanolamide and 2-20:35,11,14-glycerol. Their function as ligands of CB1 has been characterized in HEK293 cells. Labeling experiments excluded Δ8-desaturase activity and proved the position specificity of FADS2. The fads2-/- mutant might serve as an unbiased model in vivo in the development of novel CB1 agonists and antagonists.
Assuntos
Endocanabinoides/metabolismo , Ácidos Graxos Ômega-3/deficiência , Ácidos Graxos Ômega-6/deficiência , Receptor CB1 de Canabinoide/agonistas , Animais , Endocanabinoides/genética , Ácidos Graxos Dessaturases/deficiência , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-6/farmacologia , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismoRESUMO
Δ-6-fatty acid desaturase (FADS2) is the key enzyme in the biosynthesis of polyunsaturated fatty acids (PUFAs), the essential structural determinants of mammalian membrane lipid-bilayers. We developed the auxotrophic fads2(-/-) mouse mutant to assess the enigmatic role of ω3- and ω6-PUFAs in lipid homeostasis, membrane structure and function. Obesity resistance is another major phenotype of the fads2(-/-) mutant, the molecular basis of which is unknown. Phospholipidomic profiling of membrane systems of fads2(-/-)mice revealed diacylglycerol-structures, deprived of PUFAs but substituted with surrogate eicosa-5,11,14-trienoic acid. ω6-Arachidonic (AA) and ω3-docosahexaenoic acid (DHA) supplemented diets transformed fads2(-/-) into AA-fads2(-/-) and DHA-fads2(-/-) mutants. Severely altered phospholipid-bilayer structures of subcellular membranes of fads2(-/-) liver specifically interfered with maturation of transcription factor sterol-regulatory-element-binding protein, the key regulator of lipogenesis and lipid homeostasis. This study strengthens the concept that specific PUFA-substituted membrane phospholipid species are critical constituents of the structural platform operative in lipid homeostasis in normal and disease conditions.
Assuntos
Linoleoil-CoA Desaturase/deficiência , Lipogênese , Obesidade/enzimologia , Adipócitos Brancos/patologia , Tecido Adiposo Branco/patologia , Animais , Ácido Araquidônico/metabolismo , Tamanho Celular , Resistência à Doença , Ácidos Docosa-Hexaenoicos/metabolismo , Fígado Gorduroso/enzimologia , Feminino , Ácido Linoleico/metabolismo , Linoleoil-CoA Desaturase/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Fosfolipídeos/metabolismo , Transcriptoma , Aumento de PesoRESUMO
Mitochondrial enoyl-CoA isomerase (ECI1) is an auxiliary enzyme involved in unsaturated fatty acid oxidation. In contrast to most of the other enzymes involved in fatty acid oxidation, a deficiency of ECI1 has yet to be identified in humans. We used wild-type (WT) and Eci1-deficient knockout (KO) mice to explore a potential presentation of human ECI1 deficiency. Upon food withdrawal, Eci1-deficient mice displayed normal blood ß-hydroxybutyrate levels (WT 1.09 mM vs. KO 1.10 mM), a trend to lower blood glucose levels (WT 4.58 mM vs. KO 3.87 mM, P=0.09) and elevated blood levels of unsaturated acylcarnitines, in particular C12:1 acylcarnitine (WT 0.03 µM vs. KO 0.09 µM, P<0.01). Feeding an olive oil-rich diet induced an even greater increase in C12:1 acylcarnitine levels (WT 0.01 µM vs. KO 0.04 µM, P<0.01). Overall, the phenotypic presentation of Eci1-deficient mice is mild, possibly caused by the presence of a second enoyl-CoA isomerase (Eci2) in mitochondria. Knockdown of Eci2 in Eci1-deficient fibroblasts caused a more pronounced accumulation of C12:1 acylcarnitine on incubation with unsaturated fatty acids (12-fold, P<0.05). We conclude that Eci2 compensates for Eci1 deficiency explaining the mild phenotype of Eci1-deficient mice. Hypoglycemia and accumulation of C12:1 acylcarnitine might be diagnostic markers to identify ECI1 deficiency in humans.
Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Ácidos Graxos Insaturados/metabolismo , Mitocôndrias/enzimologia , Animais , Glicemia/metabolismo , Isomerases de Ligação Dupla Carbono-Carbono/genética , Carnitina/análogos & derivados , Carnitina/sangue , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Dodecenoil-CoA Isomerase , Immunoblotting , Espectrometria de Massas , Camundongos , Camundongos Knockout , Oxirredução , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Astrocytes show large morphological and functional heterogeneity and are involved in many aspects of neural function. Progress in defining astrocyte subpopulations has been hampered by the lack of a suitable antibody for their direct detection and isolation. Here, we describe a new monoclonal antibody, ACSA-1, which was generated by immunization of GLAST1 knockout mice. The antibody specifically detects an extracellular epitope of the astrocyte-specific L-glutamate/L-aspartate transporter GLAST (EAAT1, Slc1a3). As shown by immunohistochemistry, immunocytochemistry, and flow cytometry, ACSA-1 was cross-reactive for mouse, human, and rat. It labeled virtually all astrocytes positive for GFAP, GS, BLBP, RC2, and Nestin, including protoplastic, fibrous, and reactive astrocytes as well as Bergmann glia, Müller glia, and radial glia. Oligodendrocytes, microglia, neurons, and neuronal progenitors were negative for ACSA-1. Using an immunomagnetic approach, we established a method for the isolation of GLAST-positive cells with high purity. Binding of the antibody to GLAST and subsequent sorting of GLAST-positive cells neither interfered with cellular glutamate transport nor compromised astrocyte viability in vitro. The ACSA-1 antibody is not only a valuable tool to identify and track astrocytes by immunostaining, but also provides the possibility of separation and further analysis of pure astrocytes.
Assuntos
Anticorpos Monoclonais/metabolismo , Astrócitos/metabolismo , Encéfalo/citologia , Transportador 1 de Aminoácido Excitatório/imunologia , Transportador 1 de Aminoácido Excitatório/metabolismo , Animais , Animais Recém-Nascidos , Ácido Ascórbico , Ácido Aspártico/metabolismo , Encéfalo/metabolismo , Antígeno CD11b/metabolismo , Células Cultivadas , Eletroporação/métodos , Transportador 1 de Aminoácido Excitatório/deficiência , Transportador 1 de Aminoácido Excitatório/farmacologia , Feminino , Citometria de Fluxo , Gangliosídeos/metabolismo , Glutamato-Amônia Ligase/metabolismo , Humanos , Magnésio , Camundongos , Camundongos Knockout , Proteínas da Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito , Proteínas do Tecido Nervoso/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neurônios/metabolismo , Ratos , Ácidos Siálicos/metabolismo , Trítio/metabolismo , Vitamina B 6RESUMO
OBJECTIVE: An increased ω6/ω3-polyunsaturated fatty acid ratio in the current Western diet is regarded as a critical epigenetic nutritional factor in the pathogenesis of several human lifestyle diseases, metabolic syndrome, cardiovascular disease, the central nervous system and the female and male reproductive systems. The impact of nutrient ω3-and ω6-PUFAs in the pathogenesis of dyslipoproteinemia and atherosclerosis has been a topic of intense efforts for several decades. Cellular homeostasis of the ω3-and ω6- PUFA pool is maintained by the synthesis of ω3-and ω6-PUFAs from essential fatty acids (EFA) (linoleic and α-linolenic acid) and their dietary supply. In this study, we used the auxotrophic Δ6-fatty acid desaturase- (FADS2) deficient mouse (fads2-/-), an unbiased model congenial for stringent feeding experiments, to investigate the molecular basis of the proposed protective role of dietary ω3-and ω6-PUFAs (Western diet) in the pathogenesis of multifactorial dyslipoproteinemia and atherosclerosis. We focused on the metabolic axis-liver endoplasmic reticulum (ER), serum lipoprotein system (Lp) and aorta vessel wall. Furthermore, we addressed the impact of the inactivated fads2-locus with inactivated PUFA synthesis on the development and progression of extended atherosclerosis in two different mouse mutants with disrupted cholesterol homeostasis, using the apoe-/- and ldlr-/- mutants and the fads2-/- x apoe-/- and fads2-/- x ldlr-/- double mutants. METHODS: Cohorts of +/+ and fads2-/- mice underwent two long-term dietary regimens: a) a PUFA-free standard chow diet containing only EFAs, essential for viability, and b) a high fat/high cholesterol (HFHC) diet, a mimicry of the human atherogenic "Western" diet. c) To study the molecular impact of PUFA synthesis deficiency on the development and progression of atherosclerosis in the hypercholesterolemic apoe-/- and ldlr-/- mouse models fed PUFA-free regular and sustained HFHC diets, we generated the fads2-/- x apoe-/- and the fads2-/- x ldlr-/- double knockout mutants. We assessed essential molecular, biochemical and cell biological links between the diet-induced modified lipidomes of the membrane systems of the endoplasmic reticulum/Golgi complex, the site of lipid synthesis, the PL monolayer and neutral lipid core of LD and serum-Lp profiles and cellular reactions in the aortic wall. RESULTS: ω3-and ω6-PUFA synthesis deficiency in the fads2-/- mouse causes a) hypocholesterolemia and hypotriglyceridemia, b) dyslipoproteinemia with a shift of high-density lipoprotein (HDL) to very low-density lipoprotein (VLDL)-enriched Lp-pattern and c) altered liver lipid droplet structures. d) Long-term HFHC diet does not trigger atherosclerotic plaque formation in the aortic arc, the thoracic and abdominal aorta of PUFA-deficient fads2-/- mice. Inactivation of the fads2-/- locus, abolishing systemic PUFA synthesis in the fads2-/- x apoe-/- and fads2-/- x ldlr-/- double knockout mouse lines. CONCLUSIONS: Deficiency of ω3-and ω6-PUFA in the fads2-/- mutant perturbs liver lipid metabolism, causes hypocholesterolemia and hypotriglyceridemia and renders the fads2-/- mutant resistant to sustained atherogenic HFHC diet. Neither PUFA-free regular nor long-term HFHC-diet impacts the apoe- and LDL-receptor deficiency-provoked hypercholesterolemia and atherosclerotic plaque formation, size and distribution in the aorta. Our study strongly suggests that the absence of PUFAs as highly vulnerable chemical targets of autoxidation attenuates inflammatory responses and the formation of atherosclerotic lesions. The cumulative data and insight into the molecular basis of the pleiotropic functions of PUFAs challenge a differentiated view of PUFAs as culprits or benefactors during a lifespan, pivotal for legitimate dietary recommendations.
Assuntos
Aterosclerose/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Ômega-3/biossíntese , Ácidos Graxos Ômega-6/biossíntese , Receptores de LDL/metabolismo , Animais , Colesterol na Dieta/efeitos adversos , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos Dessaturases/deficiência , Ácidos Graxos Dessaturases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/deficiênciaRESUMO
The CNS-restricted versican splice-variant V2 is a large chondroitin sulfate proteoglycan incorporated in the extracellular matrix surrounding myelinated fibers and particularly accumulating at nodes of Ranvier. In vitro, it is a potent inhibitor of axonal growth and therefore considered to participate in the reduction of structural plasticity connected to myelination. To study the role of versican V2 during postnatal development, we designed a novel isoform-specific gene inactivation approach circumventing early embryonic lethality of the complete knock-out and preventing compensation by the remaining versican splice variants. These mice are viable and fertile; however, they display major molecular alterations at the nodes of Ranvier. While the clustering of nodal sodium channels and paranodal structures appear in versican V2-deficient mice unaffected, the formation of the extracellular matrix surrounding the nodes is largely impaired. The conjoint loss of tenascin-R and phosphacan from the perinodal matrix provide strong evidence that versican V2, possibly controlled by a nodal receptor, organizes the extracellular matrix assembly in vivo.
Assuntos
Sistema Nervoso Central/citologia , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Nós Neurofibrosos/metabolismo , Versicanas/metabolismo , Potenciais de Ação/genética , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Contactinas , Matriz Extracelular/genética , Matriz Extracelular/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Canal de Potássio Kv1.2/genética , Canal de Potássio Kv1.2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.6 , Proteínas do Tecido Nervoso/metabolismo , Condução Nervosa/genética , Isoformas de Proteínas/genética , Nós Neurofibrosos/ultraestrutura , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Canais de Sódio/metabolismo , Tenascina/genética , Tenascina/metabolismo , Versicanas/classificação , Versicanas/deficiênciaRESUMO
OBJECTIVE: Polyunsaturated fatty acids (PUFAs), including essential fatty acids linoleic and α-linolenic acid and derived long chain and very long chain ω3-and ω6-polyunsaturated fatty acids, are vital structures in mammalian membrane systems and signaling molecules, pivotal in brain development, lipid, and energy metabolism and in female and male fertility during human evolution. Numerous nutritional studies suggest imbalance of PUFA metabolism as a critical factor in the pathogenesis of several human lifestyle diseases: dyslipoproteinemia, obesity, cardiovascular and neurodegenerative diseases, and infertility. The lack of unbiased animal models impedes molecular interpretation of the role of synthesized and dietary supplied PUFAs in these conditions. In this study, we used a Δ6 fatty acid desaturase (FADS2) deficient mouse mutant lacking key enzyme activity in the biosynthesis of ω3-and ω6-PUFAs from EFAs to address the molecular role of PUFAs in female and male fertility. Infertility is a hallmark of the pleiotropic but auxotrophic fads2-/- phenotype and is therefore helpful for stringent dietary studies on the role of individual PUFAs. METHODS: Feeding regimens: Age- and gender-matched infertile fads2-/- mice were maintained on defined diets, normal diet containing essential fatty acids, and supplemented with ω6-arachidonic acid, ω3-docosahexaenoic acid, and arachidonic/docosahexaenoic acid, starting (a) after weaning and (b) initiated in 4-month-old female and male fads2-/- mice. Phospho- and sphingolipidomes of ovarian and testicular membrane lipid bilayers in each cohort were established and the impact on the expression and topology of membrane marker proteins, membrane morphology, germ cell development, and female and male fertility in the respective cohorts was elaborated. RESULTS: PUFA synthesis deficiency caused a halt to folliculogenesis, atresia of oocytes, and infertility of fads2-/- female mice. A PUFA-deficient membrane lipid bilayer core structure led to the disassembly of the gap junction network of the follicular granulosa cells. In fads2-/- testis, the blood-testis barrier was disrupted and spermatogenesis arrested, leading to infertility. Sustained supply of combined AA and DHA remodeled the PUFA-deficient ovarian and testicular membrane lipidomes, facilitating the reassembly of the functional gap junction network for regular ovarian cycles and the reconstitution of the blood-testis barrier in Sertoli cells, reconstituting fertility not only in developing newborns, but surprisingly also in adult infertile fads2-/- mice. CONCLUSIONS: These findings demonstrate the previously unrecognized membrane structure-based molecular link between nutrient ω3-and ω6-PUFAs, gonadal membrane structures, and female and male fertility and might foster studies of the pivotal role of dietary PUFAs in human fertility.
Assuntos
Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Insaturados/metabolismo , Fertilidade/fisiologia , Animais , Dieta , Suplementos Nutricionais , Ácidos Graxos Dessaturases/deficiência , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Feminino , Gônadas/efeitos dos fármacos , Gônadas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , ObesidadeRESUMO
Neutral sphingomyelinase smpd3 is most abundantly expressed in neurons of brain. The function of SMPD3 has remained elusive. Here, we report a pathogenetic nexus between absence of SMPD3 in the Golgi compartment (GC) of neurons of the smpd3-/- mouse brain, inhibition of Golgi vesicular protein transport and progressive cognitive impairment. Absence of SMPD3 activity in the Golgi sphingomyelin cycle impedes remodeling of the lipid bilayer, essential for budding and multivesicular body formation. Importantly, we show that inhibition of the Golgi vesicular protein transport causes accumulation of neurotoxic proteins APP, Aß and phosphorylated Tau, dysproteostasis, unfolded protein response, and apoptosis, which ultimately manifests in progressive cognitive decline, similar to the pathognomonic signatures of familial and sporadic forms of Alzheimer´s disease. This discovery might contribute to the search for other primary pathogenic mechanisms, which link perturbed lipid bilayer structures and protein processing and transport in the neuronal Golgi compartment and neurodegeneration and cognitive deficits.
Assuntos
Encéfalo/metabolismo , Disfunção Cognitiva/genética , Neurônios/metabolismo , Proteostase/genética , Esfingomielina Fosfodiesterase/genética , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Apoptose/genética , Encéfalo/patologia , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/patologia , Modelos Animais de Doenças , Progressão da Doença , Regulação da Expressão Gênica , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/fisiologia , Neurônios/patologia , Cultura Primária de Células , Teste de Desempenho do Rota-Rod , Transdução de Sinais , Esfingomielina Fosfodiesterase/deficiência , Resposta a Proteínas não Dobradas , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Sphingomyelin is a major lipid in the bilayer of subcellular membranes of eukaryotic cells. Different sphingomyelinases catalyze the initial step in the catabolism of sphingomyelin, the hydrolysis to phosphocholine and ceramide. Sphingomyelinases have been postulated to generate ceramide as a lipophilic second messenger in intracellular signaling pathways involved in cell proliferation, differentiation, or apoptosis. To elucidate the function of the first cloned Mg(2+)-dependent, neutral sphingomyelinase (nSMase 1) in sphingomyelin catabolism and its potential role in signaling processes in a genetic and molecular approach, we have generated an nSMase 1-null mutant mouse line by gene targeting. The nSMase 1-deficient mice show an inconspicuous phenotype and no accumulation or changed metabolism of sphingomyelin or other lipids, despite grossly reduced nSMase activity in all organs except brain. We also addressed the recent proposal that nSMase 1 possesses lysophospholipase C activity. The unaltered metabolism of lysophosphatidylcholine or lyso-platelet-activating factor excludes the proposed role of nSMase 1 as a lysophospholipase C.
Assuntos
Esfingomielina Fosfodiesterase/deficiência , Animais , Clonagem Molecular , Feminino , Marcação de Genes , Metabolismo dos Lipídeos , Erros Inatos do Metabolismo Lipídico/enzimologia , Erros Inatos do Metabolismo Lipídico/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças de Niemann-Pick/enzimologia , Doenças de Niemann-Pick/genética , Fenótipo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/fisiologiaRESUMO
Stearoyl-CoA desaturase 1 (SCD1) is the dominant member of the SCD-isozyme family, regarded as a major regulator of lipid and energy metabolism in liver and adipose tissue. SCD1 deficiency impairs the desaturation of de novo-synthesized palmitoyl- and stearoyl-CoA to palmitoleoyl- and oleoyl-CoA. Scd1-/- mice develop metabolic waste syndrome and skin lesions: epidermal barrier disruption, alopecia, and degeneration of sebaceous glands. The unifying molecular link between the two divergent traits remains incompletely understood. Here we show the absence of palmitoleic acid (9Z-16:1) in the lipidome of the scd1-null mouse, which prohibits posttranslational O-palmitoleoylation of Wnt3a protein, essential for Wnt3a/ß-catenin signaling in stem cell lineage decision in development of the epidermal barrier, hair growth cycle, and sebaceous glands. Substitution of the disrupted epidermal lipid barrier by an inert hydrocarbon coat prevents excessive transepidermal water loss, normalizes thermogenesis and metabolic parameters, and surprisingly leads to the activation of hair bulge progenitor cells and reprograming of a regular hair growth cycle and development of a regular fur in scd1-/- mice. Progenitor sebocytes are not activated. Independent of age, application or removal of the artificial lipid barrier allows the reversible telogen-anagen reentry and exit of the hair growth cycle.
Assuntos
Metabolismo Energético , Folículo Piloso/crescimento & desenvolvimento , Metabolismo dos Lipídeos , Glândulas Sebáceas/metabolismo , Estearoil-CoA Dessaturase/deficiência , Proteína Wnt3A/metabolismo , Tecido Adiposo/metabolismo , Animais , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Folículo Piloso/metabolismo , Imuno-Histoquímica , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Estearoil-CoA Dessaturase/biossíntese , Estearoil-CoA Dessaturase/genéticaRESUMO
Systemic loss of neutral sphingomyelinase (SMPD3) in mice leads to a novel form of systemic, juvenile hypoplasia (dwarfism). SMPD3 deficiency in mainly two growth regulating cell types contributes to the phenotype, in chondrocytes of skeletal growth zones to skeletal malformation and chondrodysplasia, and in hypothalamic neurosecretory neurons to systemic hypothalamus-pituitary-somatotropic hypoplasia. The unbiased smpd3-/- mouse mutant and derived smpd3-/- primary chondrocytes were instrumental in defining the enigmatic role underlying the systemic and cell autonomous role of SMPD3 in the Golgi compartment. Here we describe the unprecedented role of SMPD3. SMPD3 deficiency disrupts homeostasis of sphingomyelin (SM), ceramide (Cer) and diacylglycerol (DAG) in the Golgi SMPD3-SMS1 (SM-synthase1) cycle. Cer and DAG, two fusogenic intermediates, modify the membrane lipid bilayer for the initiation of vesicle formation and transport. Dysproteostasis, unfolded protein response, endoplasmic reticulum stress and apoptosis perturb the Golgi secretory pathway in the smpd3-/- mouse. Secretion of extracellular matrix proteins is arrested in chondrocytes and causes skeletal malformation and chondrodysplasia. Similarly, retarded secretion of proteo-hormones in hypothalamic neurosecretory neurons leads to hypothalamus induced combined pituitary hormone deficiency. SMPD3 in the regulation of the protein vesicular secretory pathway may become a diagnostic target in the etiology of unknown forms of juvenile growth and developmental inhibition.
Assuntos
Complexo de Golgi/metabolismo , Via Secretória , Esfingomielina Fosfodiesterase/deficiência , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ceramidas/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Detergentes/farmacologia , Diglicerídeos/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Perfilação da Expressão Gênica , Complexo de Golgi/efeitos dos fármacos , Células HEK293 , Homeostase/efeitos dos fármacos , Humanos , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Camundongos Endogâmicos C57BL , Via Secretória/efeitos dos fármacos , Solubilidade , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Fosfolipases Tipo C/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
The high affinity, Na(+)-dependent, electrogenic glial L-glutamate transporters GLAST1 and GLT1, and two neuronal EAAC1 and EAAT4, regulate the neurotransmitter concentration in excitatory synapses of the central nervous system. We dissected the function of the individual transporters in the monogenic null allelic mouse lines, glast1(-/-) and eaac1(-/-), and the derived double mutant glast(-/-)eaac1(-/-). Unexpectedly, the biochemical analysis and the behavioral phenotypes of these null allelic mouse lines were inconspicuous. Inhibition studies of the Na(+)-dependent glutamate transport by plasma membrane vesicles and by isolated astrocytes of wt and glast1(-/-) mouse brains indicated the pivotal compensatory role of GLT1 in the absence particularly of GLAST1 and GLAST1 and EAAC1 mutant mice. In electrophysiological studies, the decay rate of excitatory postsynaptic currents (EPSCs) of Purkinje cells (PC) after selective activation of parallel and climbing fibers proved to be similar in wt and eaac1(-/-), but was significantly prolonged in glast1(-/-) PCs. Bath application of the glutamate uptake blocker SYM2081 prolonged EPSC decay profiles in both wt and double mutant glast1(-/-)eaac1(-/-) PCs by 286% and 229%, respectively, indicating a prominent role of compensatory glutamate transport in shaping glast1(-/-)eaac1(-/-) EPSCs.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/fisiologia , Transportador 1 de Aminoácido Excitatório/deficiência , Células de Purkinje/fisiologia , Transmissão Sináptica/fisiologia , Sistema X-AG de Transporte de Aminoácidos/genética , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/efeitos da radiação , Western Blotting/métodos , Encéfalo/citologia , Encéfalo/fisiologia , Células Cultivadas , Estimulação Elétrica/métodos , Embrião de Mamíferos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Transportador 1 de Aminoácido Excitatório/genética , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos da radiação , Glutamatos/farmacologia , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Imuno-Histoquímica/métodos , Marcação In Situ das Extremidades Cortadas/métodos , Técnicas In Vitro , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp/métodos , Piperazinas/farmacologia , Quinoxalinas/farmacologia , RNA/isolamento & purificação , Células-Tronco/fisiologia , Transmissão Sináptica/genética , Fatores de TempoRESUMO
Biochemical and ultrastructural studies of ceramide galactosyltransferase (CGT) in a CGT-deficient mouse line (cgt-/-) were complemented by nerve conduction velocity (NCV) measurements in motor nerves (sciatic nerve in the hind limbs) of wild type (wt) and cgt-/- mice. Stimulation and recording electrodes were adapted to the small size of developing mice during their myelination period. Motor NCVs in wt mice ranged between 16 and 26 m/s but in cgt-/- mice between 6 and 13 m/s, which corresponds to the conductance of unmyelinated peripheral nerves. These electrophysiologic data provide additional functional parameters to the neuropathology of a new form of a dysmyelinosis.
Assuntos
Galactosiltransferases/deficiência , Condução Nervosa/fisiologia , Nervo Isquiático/fisiopatologia , Potenciais de Ação/genética , Potenciais de Ação/fisiologia , Animais , Sistema Nervoso Central/patologia , Sistema Nervoso Central/ultraestrutura , Estimulação Elétrica/métodos , Eletrofisiologia/métodos , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , N-Acilesfingosina Galactosiltransferase , Condução Nervosa/genética , Nervos Periféricos/patologia , Nervos Periféricos/ultraestrutura , Tempo de Reação/genética , Tempo de Reação/fisiologia , Nervo Isquiático/efeitos da radiação , Nervo Isquiático/ultraestruturaRESUMO
PURPOSE: The tolerability, safety, and visual comfort of two new tear film substitutes were studied in a phase I clinical study. METHODS: Two amphipathic lipids (phosphatidylcholine, cholesterol, sphingomyelin, and gangliosides) containing solutions (I and II) of well-defined stoichiometry, free of preservatives, were studied in 20 eyes of 20 healthy volunteers (age range, 26-32 years) in a randomized, double-blind, crossover study. Randomization was achieved by treating one eye of each volunteer four times daily for 7 days with composite solution I. The contralateral eye served as a control. After a washout interval of 7 days, the same eye was treated similarly four times daily with the solution II under randomizing conditions. Slit-lamp biomicroscopy, visual acuity, visual analog scales, and side effects were monitored at the beginning of the study weekly and for 3 weeks. RESULTS: The tear substitutes proved to have no influence on the visual acuity and were safe and well tolerated. No allergic reactions or any other side effects such as hyperemia, corneal disturbance, and foreign body deposits were observed in any volunteer. CONCLUSION: The biophysical properties of the amphipathic lipids comprising the two preservative-free tear film substitutes were studied in monolayer experiments. They form reversibly compressible and expandable monomolecular films at the air-water interface, a prerequisite for the mimicry of the tear film produced normally by meibomian glands. The efficacy and safety of both medications will be investigated in patients with keratoconjunctivitis sicca and dry eye syndrome in future experiments.
Assuntos
Colesterol/administração & dosagem , Gangliosídeos/administração & dosagem , Soluções Oftálmicas/administração & dosagem , Fosfatidilcolinas/administração & dosagem , Esfingomielinas/administração & dosagem , Adulto , Colesterol/efeitos adversos , Colesterol/química , Córnea/efeitos dos fármacos , Estudos Cross-Over , Método Duplo-Cego , Hipersensibilidade a Drogas , Quimioterapia Combinada , Síndromes do Olho Seco/tratamento farmacológico , Feminino , Gangliosídeos/efeitos adversos , Gangliosídeos/química , Humanos , Masculino , Soluções Oftálmicas/efeitos adversos , Soluções Oftálmicas/química , Fosfatidilcolinas/efeitos adversos , Fosfatidilcolinas/química , Conservantes Farmacêuticos , Segurança , Esfingomielinas/efeitos adversos , Esfingomielinas/química , Lágrimas/química , Acuidade VisualRESUMO
Various studies point to a crucial role of the high-affinity sodium-coupled glutamate aspartate transporter GLAST-1 for modulation of excitatory transmission as shown in the retina and the CNS. While 2-4-month-old GLAST-1 null mice did not show any functional vestibular abnormality, we observed profound circling behavior in older (7 months) animals lacking GLAST-1. An unchanged total number of otoferlin-positive vestibular hair cells (VHCs), similar ribbon numbers in VHCs, and an unchanged VGLUT3 expression in type II VHCs were detected in GLAST-1 null compared to wild-type mice. A partial loss of supporting cells and an apparent decline of a voltage-gated channel potassium subunit (KCNQ4) was observed in postsynaptic calyceal afferents contacting type I VHCs, together with a reduction of neurofilament- (NF200-) and vesicular glutamate transporter 1- (VGLUT1-) positive calyces in GLAST-1 null mice. Taken together, GLAST-1 deletion appeared to preferentially affect the maintenance of a normal postsynaptic/neuronal phenotype, evident only with increasing age.
Assuntos
Transportador 1 de Aminoácido Excitatório/fisiologia , Vestíbulo do Labirinto/fisiologia , Animais , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Células Receptoras Sensoriais/fisiologia , Vestíbulo do Labirinto/anatomia & histologiaRESUMO
Mammalian cell viability is dependent on the supply of the essential fatty acids (EFAs) linoleic and alpha-linolenic acid. EFAs are converted into omega3- and omega6-polyunsaturated fatty acids (PUFAs), which are essential constituents of membrane phospholipids and precursors of eicosanoids, anandamide and docosanoids. Whether EFAs, PUFAs and eicosanoids are essential for cell viability has remained elusive. Here, we show that deletion of delta6-fatty acid desaturase (FADS2) gene expression in the mouse abolishes the initial step in the enzymatic cascade of PUFA synthesis. The lack of PUFAs and eicosanoids does not impair the normal viability and lifespan of male and female fads2 -/- mice, but causes sterility. We further provide the molecular evidence for a pivotal role of PUFA-substituted membrane phospholipids in Sertoli cell polarity and blood-testis barrier, and the gap junction network between granulosa cells of ovarian follicles. The fads2 -/- mouse is an auxotrophic mutant. It is anticipated that FADS2 will become a major focus in membrane, haemostasis, inflammation and atherosclerosis research.
Assuntos
Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Linoleoil-CoA Desaturase/deficiência , Animais , Barreira Hematotesticular/fisiologia , Metabolismo dos Carboidratos , Polaridade Celular/fisiologia , Sobrevivência Celular/fisiologia , Eicosanoides/biossíntese , Feminino , Infertilidade/enzimologia , Infertilidade/genética , Infertilidade/patologia , Linoleoil-CoA Desaturase/genética , Linoleoil-CoA Desaturase/metabolismo , Metabolismo dos Lipídeos , Macrófagos/enzimologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Folículo Ovariano/fisiologia , Células de Sertoli/metabolismo , Células de Sertoli/patologia , Tromboembolia/enzimologia , Tromboembolia/genética , Tromboembolia/prevenção & controleRESUMO
Targeted deletion of the stearoyl-CoA desaturase 1 gene (scd1) in mouse causes obesity resistance and a severe skin phenotype. Here, we demonstrate that SCD1 deficiency disrupts the epidermal lipid barrier and leads to uncontrolled transepidermal water loss, breakdown of adaptive thermoregulation and cold resistance, as well as a metabolic wasting syndrome. The loss of omega-hydroxylated very long-chain fatty acids (VLCFA) and ceramides substituted with omega-hydroxylated VLCFA covalently linked to corneocyte surface proteins leads to the disruption of the epidermal lipid barrier in scd1-/- mutants. Artificial occlusion of the skin by topical lipid application largely reconstituted the epidermal barrier and also reversed dysregulation of thermogenesis and cold resistance, as well as the metabolic disturbances. Interestingly, SCD1 deficiency abolished expression of the key transcription factor Lef1, which is essential for interfollicular epidermis, sebaceous glands, and hair follicle development. Finally, the occurrence of SCD1 and a newly described hSCD5 (ACOD4) gene in humans suggests that the scd1-/- mouse mutant might be a valuable animal model for the study of human skin diseases associated with epidermal barrier defects.
Assuntos
Regulação da Temperatura Corporal/fisiologia , Epiderme/fisiologia , Lipídeos/fisiologia , Obesidade/prevenção & controle , Estearoil-CoA Dessaturase/deficiência , Administração Tópica , Animais , Ceramidas/metabolismo , Colesterol/sangue , Temperatura Baixa/efeitos adversos , Regulação para Baixo , Metabolismo Energético , Epiderme/química , Fígado/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/biossíntese , Camundongos , Camundongos Knockout , Vaselina/administração & dosagem , Vaselina/uso terapêutico , Transdução de Sinais , Absorção Cutânea/fisiologia , Dermatopatias Genéticas/fisiopatologia , Fatores de Transcrição/biossíntese , Triglicerídeos/sangue , Síndrome de Emaciação/etiologia , Perda Insensível de ÁguaRESUMO
Neutral sphingomyelinase SMPD3 (nSMase2), a sphingomyelin phosphodiesterase, resides in the Golgi apparatus and is ubiquitously expressed. Gene ablation of smpd3 causes a generalized prolongation of the cell cycle that leads to late embryonic and juvenile hypoplasia because of the SMPD3 deficiency in hypothalamic neurosecretory neurons. We show here that this novel form of combined pituitary hormone deficiency is characterized by the perturbation of the hypothalamus-pituitary growth axis, associated with retarded chondrocyte development and enchondral ossification in the epiphyseal growth plate. To study the contribution by combined pituitary hormone deficiency and by the local SMPD3 deficiency in the epiphyseal growth plate to the skeletal phenotype, we introduced the full-length smpd3 cDNA transgene under the control of the chondrocyte-specific promoter Col2a1. A complete rescue of the smpd3(-/-) mouse from severe short-limbed skeletal dysplasia was achieved. The smpd3(-/-) mouse shares its dwarf and chondrodysplasia phenotype with the most common form of human achondrodysplasia, linked to the fibroblast-growth-factor receptor 3 locus, not linked to deficits in the hypothalamic-pituitary epiphyseal growth plate axis. The rescue of smpd3 in vivo has implications for future research into dwarfism and, particularly, growth and development of the skeletal system and for current screening and future treatment of combined dwarfism and chondrodysplasia.
Assuntos
Desenvolvimento Ósseo/genética , Colágeno Tipo II/fisiologia , Nanismo/genética , Crescimento/genética , Osteocondrodisplasias/genética , Esfingomielina Fosfodiesterase/fisiologia , Animais , Exostose Múltipla Hereditária/genética , Sistema Hipotálamo-Hipofisário/fisiologia , Camundongos , Camundongos Knockout , Esfingomielina Fosfodiesterase/genética , TransgenesRESUMO
Neutral sphingomyelinases sphingomyelin phosphodiesterase (SMPD)2 and -3 hydrolyze sphingomyelin to phosphocholine and ceramide. smpd2 is expressed ubiquitously, and smpd3 is expressed predominantly in neurons of the CNS. Their activation and the functions of the released ceramides have been associated with signaling pathways in cell growth, differentiation, and apoptosis. However, these cellular responses remain poorly understood. Here we describe the generation and characterization of the smpd3(-/-) and smpd2(-/-)smpd3(-/-) double mutant mouse, which proved to be devoid of neutral sphingomyelinase activity. SMPD3 plays a pivotal role in the control of late embryonic and postnatal development: the smpd3-null mouse develops a novel form of dwarfism and delayed puberty as part of a hypothalamus-induced combined pituitary hormone deficiency. Our studies suggest that SMPD3 is segregated into detergent-resistant subdomains of Golgi membranes of hypothalamic neurosecretory neurons, where its transient activation modifies the lipid bilayer, an essential step in the Golgi secretory pathway. The smpd3(-/-) mouse might mimic a form of human combined pituitary hormone deficiency.