RESUMO
The C2 domain containing protein extended synaptotagmin (E-Syt) plays important roles in both lipid homeostasis and the intracellular signaling; however, its role in physiology remains largely unknown. Here, we show that hypothalamic E-Syt3 plays a critical role in diet-induced obesity (DIO). E-Syt3 is characteristically expressed in the hypothalamic nuclei. Whole-body or proopiomelanocortin (POMC) neuron-specific ablation of E-Syt3 ameliorated DIO and related comorbidities, including glucose intolerance and dyslipidemia. Conversely, overexpression of E-Syt3 in the arcuate nucleus moderately promoted food intake and impaired energy expenditure, leading to increased weight gain. Mechanistically, E-Syt3 ablation led to increased processing of POMC to α-melanocyte-stimulating hormone (α-MSH), increased activities of protein kinase C and activator protein-1, and enhanced expression of prohormone convertases. These findings reveal a previously unappreciated role for hypothalamic E-Syt3 in DIO and related metabolic disorders.
Assuntos
Regulação da Expressão Gênica/fisiologia , Obesidade/induzido quimicamente , Obesidade/genética , Sinaptotagminas/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Predisposição Genética para Doença , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Pró-Proteína Convertase 1/genética , Pró-Proteína Convertase 1/metabolismo , Pró-Proteína Convertase 2/genética , Pró-Proteína Convertase 2/metabolismo , Sinaptotagminas/genéticaRESUMO
Adolescence is a remarkable period of brain development. Prenatal stress can increase the risk of various neuropsychiatric disorders. This research investigated neurochemical and behavioural changes in the offspring rats (especially adolescences) who were treated with repeated variable prenatal stress (PNS) during the third week of gestation. The study tested the concentration of brain-derived neurotrophic factor (BDNF), cluster of differentiation 68 (CD68), synaptotagmin-1(Syt-1), 5-hydroxytryptamine (5-HT), dopamine (DA), glucocorticoid receptors (GRs) and oestrogen receptors (ERs) in the PFC (prefrontal cortex). We also tested prepulse inhibition (PPI) of the acoustic startle reflex (ASR) (a measure of sensorimotor gating). The main results were as follows: PNS increased the BDNF and CD68 concentrations in adolescent females, and increased the Syt-1 concentration in adolescent males. The increases in BDNF/CD68 concentration (in females) and Syt-1/DA concentration (in males) with age were disturbed by PNS, and PNS changed the sex differences in CD68 concentration in adolescence and disturbed the sex differences in the Syt-1 concentration (in adolescence) and DA concentration (in adults). In conclusion, we found that PNS lead to Sex-dependent aberrant PFC development, and might accelerate the development of the adolescent PFC, and so that lessened the age difference (between adolescence and adulthood) and the sex difference.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Dopamina/metabolismo , Córtex Pré-Frontal/crescimento & desenvolvimento , Efeitos Tardios da Exposição Pré-Natal/patologia , Serotonina/metabolismo , Caracteres Sexuais , Estresse Psicológico/patologia , Estimulação Acústica , Animais , Feminino , Masculino , Córtex Pré-Frontal/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Inibição Pré-Pulso/fisiologia , Ratos , Receptores de Estrogênio/metabolismo , Receptores de Glucocorticoides/metabolismo , Reflexo de Sobressalto/fisiologia , Fatores Sexuais , Estresse Psicológico/metabolismo , Sinaptotagminas/metabolismoRESUMO
Neurotransmission is a tightly regulated Ca2+-dependent process. Upon Ca2+ influx, Synaptotagmin1 (Syt1) promotes fusion of synaptic vesicles (SVs) with the plasma membrane. This requires regulation at multiple levels, but the role of metabolites in SV release is unclear. Here, we uncover a role for isocitrate dehydrogenase 3a (idh3a), a Krebs cycle enzyme, in neurotransmission. Loss of idh3a leads to a reduction of the metabolite, alpha-ketoglutarate (αKG), causing defects in synaptic transmission similar to the loss of syt1. Supplementing idh3a flies with αKG suppresses these defects through an ATP or neurotransmitter-independent mechanism. Indeed, αKG, but not glutamate, enhances Syt1-dependent fusion in a reconstitution assay. αKG promotes interaction between the C2-domains of Syt1 and phospholipids. The data reveal conserved metabolic regulation of synaptic transmission via αKG. Our studies provide a synaptic role for αKG, a metabolite that has been proposed as a treatment for aging and neurodegenerative disorders.
Assuntos
Ciclo do Ácido Cítrico , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/fisiologia , Isocitrato Desidrogenase/metabolismo , Mitocôndrias/metabolismo , Transmissão Sináptica , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Drosophila melanogaster/ultraestrutura , Ácidos Cetoglutáricos/metabolismo , Larva/metabolismo , Mitocôndrias/ultraestrutura , Junção Neuromuscular/metabolismo , Junção Neuromuscular/ultraestrutura , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Ligação Proteica , Domínios Proteicos , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , Sinaptotagminas/química , Sinaptotagminas/metabolismoRESUMO
The activities of mitochondrial enzymes, which are essential for neural function, decline with age and in age-related disease. In particular, the activity of cytochrome c oxidase (COX/complex IV) decreases in patients with Alzheimer's disease (AD). COX, a mitochondrial inner membrane protein complex that contains heme, plays an essential role in the electron transport chain that generates ATP. Heme synthesis begins with 5-aminolevulinic acid (5-ALA) in mitochondria. 5-ALA synthetase is the rate-limiting enzyme in heme synthesis, suggesting that supplementation with 5-ALA might help preserve mitochondrial activity in the aged brain. We administered a diet containing 5-ALA to triple-transgenic AD (3xTg-AD) model mice for 6 months, starting at 3 months of age. COX activity and protein expression, as well as mitochondrial membrane potential, were significantly higher in brains of 5-ALA-fed mice than in controls. Synaptotagmin protein levels were also significantly higher in 5-ALA-fed mice, suggesting improved preservation of synapses. Although brain Aß levels tended to decrease in 5-ALA-fed mice, we observed no other significant changes in other biochemical and pathological hallmarks of AD. Nevertheless, our study suggests that daily oral administration of 5-ALA could preserve mitochondrial enzyme activities in the brains of aged individuals, thereby contributing to the preservation of neural activity.
Assuntos
Doença de Alzheimer/prevenção & controle , Ácido Aminolevulínico/uso terapêutico , Suplementos Nutricionais , Modelos Animais de Doenças , Mitocôndrias/metabolismo , Neurônios/metabolismo , Nootrópicos/uso terapêutico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/enzimologia , Encéfalo/metabolismo , Encéfalo/patologia , Córtex Cerebral/enzimologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Imuno-Histoquímica , Masculino , Potencial da Membrana Mitocondrial , Camundongos Transgênicos , Mitocôndrias/enzimologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Neurônios/enzimologia , Neurônios/patologia , Caracteres Sexuais , Sinaptotagminas/metabolismoRESUMO
Flavonoids, a family of phenolic compounds, are widely present in our daily diet and exist in traditional Chinese medicines, in which they act as the major active functional ingredients. Different lines of evidence indicate that flavonoids have positive impacts on human health. Here, different subclasses of flavonoids were analyzed for their inductive roles in promoting the expression of synaptic proteins, synaptotagmin, and post-synaptic density protein-95 in cultured rat cortical neurons. Among the screened 65 flavonoids, (-)-catechin, luteolin, and isorhamnetin, in micromolar concentration, were found to induce the expression of synaptic proteins in a dose-dependent manner: the induction values were from 2- to 8-fold that of the control. Similar results were revealed in the flavonoid-treated hippocampal neurons. The identification of these synapse-promoting flavonoids could be very useful in finding potential drugs, or food supplements, for treating various neurodegenerative diseases, including Alzheimer's disease and depression.
Assuntos
Flavonoides/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/efeitos dos fármacos , Proteínas de Membrana/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Sinaptotagminas/efeitos dos fármacos , Animais , Catequina/química , Catequina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteína 4 Homóloga a Disks-Large , Relação Dose-Resposta a Droga , Flavonoides/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Luteolina/química , Luteolina/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Estrutura Molecular , Neurônios/metabolismo , Quercetina/análogos & derivados , Quercetina/química , Quercetina/farmacologia , Ratos , Sinapses/metabolismo , Sinaptotagminas/genética , Sinaptotagminas/metabolismoRESUMO
The protozoan parasite Trypanosoma cruzi, the aetiological agent of Chagas' disease, has two infective life cycle stages, trypomastigotes and amastigotes. While trypomastigotes actively enter mammalian cells, highly infective extracellular amastigotes (type Iâ T. cruzi) rely on actin-mediated uptake, which is generally inefficient in non-professional phagocytes. We found that extracellular amastigotes (EAs) of T. cruziâ G strain (type I), but not Y strain (type II), were taken up 100-fold more efficiently than inert particles. Mammalian cell lines showed levels of parasite uptake comparable to macrophages, and extensive actin recruitment and polymerization was observed at the site of entry. EA uptake was not dependent on parasite-secreted molecules and required the same molecular machinery utilized by professional phagocytes during large particle phagocytosis. Transcriptional silencing of synaptotagmin VII and CD63 significantly inhibited EA internalization, demonstrating that delivery of supplemental lysosomal membrane to form the phagosome is involved in parasite uptake. Importantly, time-lapse live imaging using fluorescent reporters revealed phagosome-associated modulation of phosphoinositide metabolism during EA uptake that closely resembles what occurs during phagocytosis by macrophages. Collectively, our results demonstrate that T. cruziâ EAs are potent inducers of phagocytosis in non-professional phagocytes, a process that may facilitate parasite persistence in infected hosts.
Assuntos
Doença de Chagas/fisiopatologia , Células HeLa/parasitologia , Estágios do Ciclo de Vida/fisiologia , Fagocitose/fisiologia , Trypanosoma cruzi/crescimento & desenvolvimento , Actinas/metabolismo , Animais , Doença de Chagas/metabolismo , Humanos , Metabolismo dos Lipídeos/fisiologia , Fosfatidilinositóis/metabolismo , Sinaptotagminas/metabolismo , Tetraspanina 30/metabolismo , Trypanosoma cruzi/patogenicidadeRESUMO
Ca(2+)-dependent activator protein for secretion (CAPS) is an essential factor for regulated vesicle exocytosis that functions in priming reactions before Ca(2+)-triggered fusion of vesicles with the plasma membrane. However, the precise events that CAPS regulates to promote vesicle fusion are unclear. In the current work, we reconstituted CAPS function in a SNARE-dependent liposome fusion assay using VAMP2-containing donor and syntaxin-1/SNAP-25-containing acceptor liposomes. The CAPS stimulation of fusion required PI(4,5)P(2) in acceptor liposomes and was independent of Ca(2+), but Ca(2+) dependence was restored by inclusion of synaptotagmin. CAPS stimulated trans-SNARE complex formation concomitant with the stimulation of full membrane fusion at physiological SNARE densities. CAPS bound syntaxin-1, and CAPS truncations that competitively inhibited syntaxin-1 binding also inhibited CAPS-dependent fusion. The results revealed an unexpected activity of a priming protein to accelerate fusion by efficiently promoting trans-SNARE complex formation. CAPS may function in priming by organizing SNARE complexes on the plasma membrane.
Assuntos
Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Exocitose/fisiologia , Homeostase , Lecitinas/metabolismo , Lipossomos/metabolismo , Fusão de Membrana/fisiologia , Células PC12/fisiologia , Fosfatidilserinas/metabolismo , Ratos , Sinaptotagminas/metabolismo , Sintaxina 1/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
Our aging society is confronted with a dramatic increase of patients suffering from tauopathies, which include Alzheimer disease and certain frontotemporal dementias. These disorders are characterized by typical neuropathological lesions including hyperphosphorylation and subsequent aggregation of TAU protein and neuronal cell death. Currently, no mechanism-based cures are available. We generated fluorescently labeled TAU transgenic zebrafish, which rapidly recapitulated key pathological features of tauopathies, including phosphorylation and conformational changes of human TAU protein, tangle formation, neuronal and behavioral disturbances, and cell death. Due to their optical transparency and small size, zebrafish larvae are well suited for both in vivo imaging and drug development. TAU-induced neuronal cell death was imaged by time-lapse microscopy in vivo. Furthermore, we used this zebrafish model to identify compounds targeting the TAU kinase glycogen synthase kinase 3beta (GSK3beta). We identified a newly developed highly active GSK3beta inhibitor, AR-534, by rational drug design. AR-534 reduced TAU phosphorylation in TAU transgenic zebrafish. This transgenic zebrafish model may become a valuable tool for further studies of the neuropathology of dementia.
Assuntos
Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Neurônios/patologia , Tauopatias/tratamento farmacológico , Tauopatias/patologia , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Morte Celular , Desenho de Fármacos , Embrião não Mamífero/anormalidades , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Reação de Fuga , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/química , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Larva/anatomia & histologia , Larva/efeitos dos fármacos , Larva/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Moleculares , Estrutura Molecular , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Conformação Proteica , Alinhamento de Sequência , Medula Espinal/metabolismo , Medula Espinal/patologia , Sinaptotagminas/metabolismo , Tauopatias/metabolismo , Peixe-Zebra/genética , Proteínas tau/genética , Proteínas tau/metabolismo , Proteína Vermelha FluorescenteRESUMO
The two universally required components of the intracellular membrane fusion machinery, SNARE and SM (Sec1/Munc18-like) proteins, play complementary roles in fusion. Vesicular and target membrane-localized SNARE proteins zipper up into an alpha-helical bundle that pulls the two membranes tightly together to exert the force required for fusion. SM proteins, shaped like clasps, bind to trans-SNARE complexes to direct their fusogenic action. Individual fusion reactions are executed by distinct combinations of SNARE and SM proteins to ensure specificity, and are controlled by regulators that embed the SM-SNARE fusion machinery into a physiological context. This regulation is spectacularly apparent in the exquisite speed and precision of synaptic exocytosis, where synaptotagmin (the calcium-ion sensor for fusion) cooperates with complexin (the clamp activator) to control the precisely timed release of neurotransmitters that initiates synaptic transmission and underlies brain function.
Assuntos
Fusão de Membrana , Proteínas Munc18/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Motivos de Aminoácidos , Animais , Proteínas Munc18/química , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/química , Sinapses/fisiologia , Transmissão Sináptica , Vesículas Sinápticas/fisiologia , Sinaptotagminas/metabolismo , Proteínas de Transporte Vesicular/químicaRESUMO
Calpains are Ca2+-dependent proteinases that mediate protein turnover in crustacean skeletal muscles. We used an antibody directed against lobster muscle-specific calpain (Ha-CalpM) to examine its distribution in differentiating juvenile lobster claw muscles. These muscles are comprised of both fast and slow fibers early in development, but become specialized into predominantly fast or exclusively slow muscles in adults. The transition into adult muscle types requires that myofibrillar proteins specific for fast or slow muscles to be selectively removed and replaced by the appropriate proteins. Using immunohistochemistry, we observed a distinct staining pattern where staining was preferentially localized in the fiber periphery along one side of the fiber. Immunolabeling with an antibody directed against synaptotagmin revealed that the calpain staining was greatest in the cytoplasm adjacent to synaptic terminals. In complementary analyses, we used sequence-specific primers with real-time PCR to quantify the levels of Ha-CalpM in whole juvenile claw muscles. These expression levels were not significantly different between cutter and crusher claws, but were positively correlated with the expression of fast myosin heavy chain. The anatomical localization of Ha-CalpM near motor endplates, coupled with the correlation with fast myofibrillar gene expression, suggests a role for this intracellular proteinase in fiber type switching.
Assuntos
Calpaína/metabolismo , Diferenciação Celular , Casco e Garras/metabolismo , Placa Motora/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Nephropidae/metabolismo , Envelhecimento/metabolismo , Animais , Western Blotting , Calpaína/genética , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Casco e Garras/citologia , Casco e Garras/crescimento & desenvolvimento , Imuno-Histoquímica , Placa Motora/citologia , Placa Motora/crescimento & desenvolvimento , Desenvolvimento Muscular/genética , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Cadeias Pesadas de Miosina/metabolismo , Nephropidae/citologia , Nephropidae/genética , Nephropidae/crescimento & desenvolvimento , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sinaptotagminas/metabolismoRESUMO
Trafficking of the vesicular acetylcholine transporter (VAChT) to synaptic vesicles has the potential to regulate storage and release of acetylcholine. We used the C-terminal tail of the vesicular acetylcholine transporter as bait for the screening of a brain cDNA library by yeast-two hybrids. Here we report an interaction uncovered in this screening with SEC14L1, a mammalian SEC14-like protein that may function as a phospholipid transfer protein. The interaction of VAChT and SEC14L1 occurred through the GOLD domain found in the latter and was confirmed in mammalian cells. In addition, we also found that SEC14L1 co-immunoprecipitates with the high affinity choline transporter (CHT1), but not with synaptophysin or synaptotagmin. In cultured cells SEC14L1 was predominantly found in the cytosol with little or no localization in defined organelles. In contrast, overexpression of VAChT or CHT1 with SEC14L1 recruited the latter to large intracellular organelles similar to vesicles or vesicle aggregates. Finally, we find that overexpression of SEC14L1 modestly decreases high affinity choline transport activity. We suggest that interaction of cholinergic transporters with proteins containing the GOLD domain may be relevant for transporter function.
Assuntos
Proteínas de Transporte/metabolismo , Lipoproteínas/metabolismo , Transativadores/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Sequência de Aminoácidos , Animais , Química Encefálica/genética , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular , Células Cultivadas , Colina/metabolismo , Clonagem Molecular , Citosol/metabolismo , DNA Complementar/genética , Imunofluorescência , Biblioteca Gênica , Humanos , Imunoprecipitação , Microscopia Confocal , Dados de Sequência Molecular , Células PC12 , Proteínas de Transferência de Fosfolipídeos/metabolismo , Plasmídeos/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transmissão Sináptica/fisiologia , Sinaptofisina/metabolismo , Sinaptotagminas/metabolismo , TransfecçãoRESUMO
Synaptotagmin (Syt) VII is a ubiquitously expressed member of the Syt family of Ca2+ sensors. It is present on lysosomes in several cell types, where it regulates Ca2+-dependent exocytosis. Because [Ca2+]i and exocytosis have been associated with phagocytosis, we investigated the phagocytic ability of macrophages from Syt VII-/- mice. Syt VII-/- macrophages phagocytose normally at low particle/cell ratios but show a progressive inhibition in particle uptake under high load conditions. Complementation with Syt VII rescues this phenotype, but only when functional Ca2+-binding sites are retained. Reinforcing a role for Syt VII in Ca2+-dependent phagocytosis, particle uptake in Syt VII-/- macrophages is significantly less dependent on [Ca2+]i. Syt VII is concentrated on peripheral domains of lysosomal compartments, from where it is recruited to nascent phagosomes. Syt VII recruitment is rapidly followed by the delivery of Lamp1 to phagosomes, a process that is inhibited in Syt VII-/- macrophages. Thus, Syt VII regulates the Ca2+-dependent mobilization of lysosomes as a supplemental source of membrane during phagocytosis.