RESUMO
Cellular mechanisms that mediate steatohepatitis, an increasingly prevalent condition in the Western world for which no therapies are available, are poorly understood. Despite the fact that its synthetic agonists induce fatty liver, the liver X receptor (LXR) transcription factor remains a target of interest because of its anti-atherogenic, cholesterol removal, and anti-inflammatory activities. Here we show that tetratricopeptide repeat domain protein 39B (Ttc39b, C9orf52) (T39), a high-density lipoprotein gene discovered in human genome-wide association studies, promotes the ubiquitination and degradation of LXR. Chow-fed mice lacking T39 (T39(-/-)) display increased high-density lipoprotein cholesterol levels associated with increased enterocyte ATP-binding cassette transporter A1 (Abca1) expression and increased LXR protein without change in LXR messenger RNA. When challenged with a high fat/high cholesterol/bile salt diet, T39(-/-) mice or mice with hepatocyte-specific T39 deficiency show increased hepatic LXR protein and target gene expression, and unexpectedly protection from steatohepatitis and death. Mice fed a Western-type diet and lacking low-density lipoprotein receptor (Ldlr(-/-)T39(-/-)) show decreased fatty liver, increased high-density lipoprotein, decreased low-density lipoprotein, and reduced atherosclerosis. In addition to increasing hepatic Abcg5/8 expression and limiting dietary cholesterol absorption, T39 deficiency inhibits hepatic sterol regulatory element-binding protein 1 (SREBP-1, ADD1) processing. This is explained by an increase in microsomal phospholipids containing polyunsaturated fatty acids, linked to an LXRα-dependent increase in expression of enzymes mediating phosphatidylcholine biosynthesis and incorporation of polyunsaturated fatty acids into phospholipids. The preservation of endogenous LXR protein activates a beneficial profile of gene expression that promotes cholesterol removal and inhibits lipogenesis. T39 inhibition could be an effective strategy for reducing both steatohepatitis and atherosclerosis.
Assuntos
Aterosclerose/genética , Fígado Gorduroso/genética , Lipoproteínas HDL/deficiência , Lipoproteínas HDL/genética , Receptores Nucleares Órfãos/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Aterosclerose/prevenção & controle , Aterosclerose/terapia , Ácidos e Sais Biliares/metabolismo , Colesterol na Dieta/metabolismo , HDL-Colesterol/metabolismo , Dieta Hiperlipídica , Ácidos Graxos Insaturados/metabolismo , Fígado Gorduroso/prevenção & controle , Fígado Gorduroso/terapia , Feminino , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Ligantes , Lipogênese/genética , Lipoproteínas/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Receptores X do Fígado , Masculino , Camundongos , Receptores Nucleares Órfãos/genética , Fosfatidilcolinas/biossíntese , Fosfatidilcolinas/metabolismo , Estabilidade Proteica , Proteólise , Receptores de LDL/deficiência , Receptores de LDL/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , UbiquitinaçãoRESUMO
In the amyloidogenic pathway associated with Alzheimer disease (AD), the amyloid precursor protein (APP) is cleaved by ß-secretase to generate a 99-aa C-terminal fragment (C99) that is then cleaved by γ-secretase to generate the ß-amyloid (Aß) found in senile plaques. In previous reports, we and others have shown that γ-secretase activity is enriched in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) and that ER-mitochondrial connectivity and MAM function are upregulated in AD We now show that C99, in addition to its localization in endosomes, can also be found in MAM, where it is normally processed rapidly by γ-secretase. In cell models of AD, however, the concentration of unprocessed C99 increases in MAM regions, resulting in elevated sphingolipid turnover and an altered lipid composition of both MAM and mitochondrial membranes. In turn, this change in mitochondrial membrane composition interferes with the proper assembly and activity of mitochondrial respiratory supercomplexes, thereby likely contributing to the bioenergetic defects characteristic of AD.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Linhagem Celular , Respiração Celular , Retículo Endoplasmático/ultraestrutura , Humanos , Membranas Intracelulares/ultraestrutura , Camundongos , Mitocôndrias/ultraestrutura , Mutação/genética , Consumo de Oxigênio , Presenilinas/genética , Transporte Proteico , Esfingolipídeos/metabolismo , Regulação para CimaRESUMO
Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated Δ-super-tether (Δ-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in Δ-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in Δ-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of Δ-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity.
Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Fosfolipídeos/metabolismo , Esteróis/metabolismo , Lipídeos/biossíntese , Proteínas de Membrana/metabolismo , Receptores de Esteroides/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , LevedurasRESUMO
Membrane phase behavior has been well characterized in model membranes in vitro under thermodynamic equilibrium state. However, the widely observed differences between biological membranes and their in vitro counterparts are placing more emphasis on nonequilibrium factors, including influx and efflux of lipid molecules. The endoplasmic reticulum (ER) is the largest cellular membrane system and also the most metabolically active organelle responsible for lipid synthesis. However, how the nonequilibrium metabolic activity modulates ER membrane phase has not been investigated. Here, we studied the phase behavior of functional ER in the context of lipid metabolism. Utilizing advanced vibrational imaging technique, that is, stimulated Raman scattering microscopy, we discovered that metabolism of palmitate, a prevalent saturated fatty acid (SFA), could drive solid-like domain separation from the presumably uniformly fluidic ER membrane, a previously unknown phenomenon. The potential of various fatty acids to induce solid phase can be predicted by the transition temperatures of their major metabolites. Interplay between saturated and unsaturated fatty acids is also observed. Hence, our study sheds light on cellular membrane biophysics by underscoring the nonequilibrium metabolic status of living cell.
Assuntos
Retículo Endoplasmático/metabolismo , Microdomínios da Membrana/metabolismo , Animais , Células COS , Chlorocebus aethiops , Retículo Endoplasmático/ultraestrutura , Ácidos Graxos/metabolismo , Células HeLa , HumanosRESUMO
Bile acids (BAs) are cholesterol derivatives that regulate lipid metabolism, through their dual abilities to promote lipid absorption and activate BA receptors. However, different BA species have varying abilities to perform these functions. Eliminating 12α-hydroxy BAs in mice via Cyp8b1 knockout causes low body weight and improved glucose tolerance. The goal of this study was to determine mechanisms of low body weight in Cyp8b1-/- mice. We challenged Cyp8b1-/- mice with a Western-type diet and assessed body weight and composition. We measured energy expenditure, fecal calories, and lipid absorption and performed lipidomic studies on feces and intestine. We investigated the requirement for dietary fat in the phenotype using a fat-free diet. Cyp8b1-/- mice were resistant to Western diet-induced body weight gain, hepatic steatosis, and insulin resistance. These changes were associated with increased fecal calories, due to malabsorption of hydrolyzed dietary triglycerides. This was reversed by treating the mice with taurocholic acid, the major 12α-hydroxylated BA species. The improvements in body weight and steatosis were normalized by feeding mice a fat-free diet. The effects of BA composition on intestinal lipid handling are important for whole body energy homeostasis. Thus modulating BA composition is a potential tool for obesity or diabetes therapy.
Assuntos
Dieta Ocidental/efeitos adversos , Gorduras na Dieta/metabolismo , Fígado Gorduroso/genética , Absorção Intestinal/genética , Metabolismo dos Lipídeos/genética , Esteroide 12-alfa-Hidroxilase/genética , Aumento de Peso/genética , Animais , Ácidos e Sais Biliares/metabolismo , Dieta Hiperlipídica , Fígado Gorduroso/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Accumulating evidence from genetic and biochemical studies implicates dysfunction of the autophagic-lysosomal pathway as a key feature in the pathogenesis of Parkinson's disease (PD). Most studies have focused on accumulation of neurotoxic α-synuclein secondary to defects in autophagy as the cause of neurodegeneration, but abnormalities of the autophagic-lysosomal system likely mediate toxicity through multiple mechanisms. To further explore how endolysosomal dysfunction causes PD-related neurodegeneration, we generated a murine model of Kufor-Rakeb syndrome (KRS), characterized by early-onset Parkinsonism with additional neurological features. KRS is caused by recessive loss-of-function mutations in the ATP13A2 gene encoding the endolysosomal ATPase ATP13A2. We show that loss of ATP13A2 causes a specific protein trafficking defect, and that Atp13a2 null mice develop age-related motor dysfunction that is preceded by neuropathological changes, including gliosis, accumulation of ubiquitinated protein aggregates, lipofuscinosis, and endolysosomal abnormalities. Contrary to predictions from in vitro data, in vivo mouse genetic studies demonstrate that these phenotypes are α-synuclein independent. Our findings indicate that endolysosomal dysfunction and abnormalities of α-synuclein homeostasis are not synonymous, even in the context of an endolysosomal genetic defect linked to Parkinsonism, and highlight the presence of α-synuclein-independent neurotoxicity consequent to endolysosomal dysfunction.
Assuntos
Adenosina Trifosfatases/deficiência , Encéfalo/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/deficiência , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/patologia , alfa-Sinucleína/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/ultraestrutura , Animais , Encéfalo/patologia , Encéfalo/ultraestrutura , Citosol/metabolismo , Citosol/ultraestrutura , Modelos Animais de Doenças , Neurônios Dopaminérgicos/patologia , Endossomos/metabolismo , Endossomos/ultraestrutura , Comportamento Exploratório/fisiologia , Elevação dos Membros Posteriores/psicologia , Concentração de Íons de Hidrogênio , Lipídeos/análise , Lisossomos/ultraestrutura , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/genética , Proteínas do Tecido Nervoso/metabolismo , Transtornos Parkinsonianos/fisiopatologia , Equilíbrio Postural/genética , ATPases Translocadoras de PrótonsRESUMO
Plasma membrane-derived vesicles are being used in biophysical and biochemical research as a simple, yet native-like model of the cellular membrane. Here we report on the characterization of vesicles produced via two different vesiculation methods from CHO and A431 cell lines. The first method is a recently developed method which utilizes chloride salts to induce osmotic vesiculation. The second is a well established chemical vesiculation method which uses DTT and formaldehyde. We show that both vesiculation methods produce vesicles which contain the lipid species previously reported in the plasma membrane of these cell lines. The two methods lead to small but statistically significant differences in two lipid species only; phosphatidylcholine (PC) and plasmalogen phosphatidylethanolamine (PEp). However, highly significant differences were observed in the degree of incorporation of a membrane receptor and in the degree of retention of soluble cytosolic proteins within the vesicles.
Assuntos
Membrana Celular/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Western Blotting , Células CHO , Linhagem Celular Tumoral , Membrana Celular/química , Colesterol/metabolismo , Cromatografia Líquida , Cricetinae , Cricetulus , Ditiotreitol/farmacologia , Receptores ErbB/metabolismo , Formaldeído/farmacologia , Humanos , Espectrometria de Massas , Microscopia Confocal , Pressão Osmótica , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Plasmalogênios/metabolismo , Vesículas Transportadoras/química , Vesículas Transportadoras/efeitos dos fármacosRESUMO
Peroxisome proliferator-activated receptors (PPARs) are ligand-mediated transcription factors, which control both lipid and energy metabolism and inflammation pathways. PPARγ agonists are effective in the treatment of metabolic diseases and, more recently, neurodegenerative diseases, in which they show promising neuroprotective effects. We studied the effects of the pan-PPAR agonist bezafibrate on tau pathology, inflammation, lipid metabolism and behavior in transgenic mice with the P301S human tau mutation, which causes familial frontotemporal lobar degeneration. Bezafibrate treatment significantly decreased tau hyperphosphorylation using AT8 staining and the number of MC1-positive neurons. Bezafibrate treatment also diminished microglial activation and expression of both inducible nitric oxide synthase and cyclooxygenase 2. Additionally, the drug differentially affected the brain and brown fat lipidome of control and P301S mice, preventing lipid vacuoles in brown fat. These effects were associated with behavioral improvement, as evidenced by reduced hyperactivity and disinhibition in the P301S mice. Bezafibrate therefore exerts neuroprotective effects in a mouse model of tauopathy, as shown by decreased tau pathology and behavioral improvement. Since bezafibrate was given to the mice before tau pathology had developed, our data suggest that bezafibrate exerts a preventive effect on both tau pathology and its behavioral consequences. Bezafibrate is therefore a promising agent for the treatment of neurodegenerative diseases associated with tau pathology.
Assuntos
Comportamento Animal/efeitos dos fármacos , Bezafibrato/farmacologia , Tauopatias/metabolismo , Proteínas tau/metabolismo , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Animais , Bezafibrato/administração & dosagem , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Ácidos Graxos/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Transgênicos , Oxirredução , Estresse Oxidativo , Fosforilação/efeitos dos fármacos , Tauopatias/tratamento farmacológicoRESUMO
Lipids are key regulators of brain function and have been increasingly implicated in neurodegenerative disorders including Alzheimer disease (AD). Here, a systems-based approach was employed to determine the lipidome of brain tissues affected by AD. Specifically, we used liquid chromatography-mass spectrometry to profile extracts from the prefrontal cortex, entorhinal cortex, and cerebellum of late-onset AD (LOAD) patients, as well as the forebrain of three transgenic familial AD (FAD) mouse models. Although the cerebellum lacked major alterations in lipid composition, we found an elevation of a signaling pool of diacylglycerol as well as sphingolipids in the prefrontal cortex of AD patients. Furthermore, the diseased entorhinal cortex showed specific enrichment of lysobisphosphatidic acid, sphingomyelin, the ganglioside GM3, and cholesterol esters, all of which suggest common pathogenic mechanisms associated with endolysosomal storage disorders. Importantly, a significant increase in cholesterol esters and GM3 was recapitulated in the transgenic FAD models, suggesting that these mice are relevant tools to study aberrant lipid metabolism of endolysosomal dysfunction associated with AD. Finally, genetic ablation of phospholipase D(2), which rescues the synaptic and behavioral deficits of an FAD mouse model, fully normalizes GM3 levels. These data thus unmask a cross-talk between the metabolism of phosphatidic acid, the product of phospholipase D(2), and gangliosides, and point to a central role of ganglioside anomalies in AD pathogenesis. Overall, our study highlights the hypothesis generating potential of lipidomics and identifies novel region-specific lipid anomalies potentially linked to AD pathogenesis.
Assuntos
Doença de Alzheimer/metabolismo , Química Encefálica , Cerebelo/metabolismo , Metabolismo dos Lipídeos , Lipídeos , Doença de Alzheimer/genética , Animais , Cerebelo/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos TransgênicosRESUMO
Type I phosphatidylinositol-4-phosphate 5-kinase (PIPKI) is the main enzyme generating the lipid second messenger phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which has critical functions in many cellular processes, such as cytoskeletal reorganization, membrane trafficking, and signal transduction. All three members of the PIPKI family are activated by phosphatidic acid (PA). However, how PA regulates the activity and functions of PIPKI have not been fully elucidated. In this study, we identify a PA-binding site on PIPKIγ. Mutation of this site inhibited the PA-stimulated activity and membrane localization of PIPKIγ as well as the formation of actin comets and foci induced by PIPKIγ. We also demonstrate that phospholipase D (PLD) generates a pool of PA involved in PIPKIγ regulation by showing that PLD inhibitors blocked the membrane localization of PIPKIγ and its ability to induce actin cytoskeletal reorganization. Targeting the PIPKIγ PA-binding-deficient mutant to membranes by a membrane localization sequence failed to restore the actin reorganization activity of PIPKIγ, suggesting that PA binding is not only involved in recruiting PIPKIγ to membranes but also may induce a conformational change. Taken together, these results reveal a new molecular mechanism through which PA regulates PIPKI and provides direct evidence that PA is important for the localization and functions of PIPKI in intact cells.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Ácidos Fosfatídicos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Actinas/efeitos dos fármacos , Animais , Sítios de Ligação/efeitos dos fármacos , Células COS , Células Cultivadas , Chlorocebus aethiops , Citoesqueleto/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Mutação , Fosfolipase D/antagonistas & inibidores , Fosfolipase D/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Relação Estrutura-AtividadeRESUMO
Growing evidence implicates aberrant lipid signaling in Alzheimer's disease (AD). While phospholipases A2 and C have been recently shown to mediate key actions of amyloid ß-peptide (Aß) through a dysregulation of arachidonic acid and phosphatidylinositol-4,5-bisphosphate metabolism, respectively, the role of phospholipase D (PLD) has so far remained elusive. PLD produces phosphatidic acid (PA), a bioactive lipid involved in multiple aspects of cell physiology, including signaling and membrane trafficking processes. Here we show that oligomeric Aß enhances PLD activity in cultured neurons and that this stimulatory effect does not occur upon ablation of PLD2 via gene targeting. Aß fails to suppress long-term potentiation in PLD2-deficient hippocampal slices, suggesting that PLD2 is required for the synaptotoxic action of this peptide. In vivo PLD activity, as assessed by detection of phosphatidylethanol levels using mass spectrometry (MS) following ethanol injection, is also increased in the brain of a transgenic mouse model of AD (SwAPP). Furthermore, Pld2 ablation rescues memory deficits and confers synaptic protection in SwAPP mice despite a significant Aß load. MS-based lipid analysis of Pld2 mutant brains in the presence or absence of the SwAPP transgene unmasks striking crosstalks between different PA species. This lipid analysis shows an exquisite acyl chain specificity and plasticity in the perturbation of PA metabolism. Collectively, our results point to specific molecular species of PA as key modulators of AD pathogenesis and identify PLD2 as a novel potential target for therapeutics.
Assuntos
Doença de Alzheimer/complicações , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/patologia , Fosfolipase D/deficiência , Sinapses/genética , Fatores Etários , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/farmacologia , Precursor de Proteína beta-Amiloide/genética , Animais , Comportamento Animal , Linhagem Celular Transformada , Transtornos Cognitivos/genética , Condicionamento Psicológico/fisiologia , Modelos Animais de Doenças , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Estrenos/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Medo/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Humanos , Técnicas In Vitro , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/genética , Espectrometria de Massas/métodos , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Células PC12/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Fosfolipase D/genética , Pirrolidinonas/farmacologia , Ratos , Fatores de TempoRESUMO
STARD4, a member of the evolutionarily conserved START gene family, is a soluble sterol transport protein implicated in cholesterol sensing and maintenance of cellular homeostasis. STARD4 is widely expressed and has been shown to transfer sterol between liposomes as well as organelles in cells. However, STARD4 knockout mice lack an obvious phenotype, so the overall role of STARD4 is unclear. To model long term depletion of STARD4 in cells, we use short hairpin RNA technology to stably decrease STARD4 expression in human U2OS osteosarcoma cells (STARD4-KD). We show that STARD4-KD cells display increased total cholesterol, slower cholesterol trafficking between the plasma membrane and the endocytic recycling compartment, and increased plasma membrane fluidity. These effects can all be rescued by transient expression of a short hairpin RNA-resistant STARD4 construct. Some of the cholesterol increase was due to excess storage in late endosomes or lysosomes. To understand the effects of reduced STARD4, we carried out transcriptional and lipidomic profiling of control and STARD4-KD cells. Reduction of STARD4 activates compensatory mechanisms that alter membrane composition and lipid homeostasis. Based on these observations, we propose that STARD4 functions as a critical sterol transport protein involved in sterol sensing and maintaining lipid homeostasis.
Assuntos
Colesterol/metabolismo , Metabolismo dos Lipídeos , Proteínas de Membrana Transportadoras/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Endocitose , Humanos , Lipidômica , Proteínas de Membrana Transportadoras/genética , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Lipids are major constituents of the brain largely implicated in physiological and pathological processes. The hippocampus is a complex brain structure involved in learning, memory and emotional responses, and its functioning is also affected in various disorders. Despite conserved intrinsic circuitry, behavioral and anatomical studies suggest the existence of a structural and functional gradient along the hippocampal longitudinal axis. Here, we used an unbiased mass spectrometry approach to characterize the lipid composition of distinct hippocampal subregions. In addition, we evaluated the susceptibility of each area to lipid modulation by corticosterone (CORT), an important mediator of the effects of stress. We confirmed a great similarity between hippocampal subregions relatively to other brain areas. Moreover, we observed a continuous molecular gradient along the longitudinal axis of the hippocampus, with the dorsal and ventral extremities differing significantly from each other, particularly in the relative abundance of sphingolipids and phospholipids. Also, whereas chronic CORT exposure led to remodeling of triacylglycerol and phosphatidylinositol species in both hippocampal poles, our study suggests that the ventral hippocampus is more sensitive to CORT-induced changes, with regional modulation of ceramide, dihydrosphingomyelin and phosphatidic acid. Thus, our results confirm a multipartite molecular view of dorsal-ventral hippocampal axis and emphasize lipid metabolites as candidate effectors of glucocorticoid signaling, mediating regional susceptibility to neurological disorders associated with stress.
Assuntos
Corticosterona/fisiologia , Hipocampo/química , Lipídeos/química , Estresse Psicológico/fisiopatologia , Animais , Masculino , Ratos , Ratos WistarRESUMO
Defects in endolysosomal and autophagic functions are increasingly viewed as key pathological features of neurodegenerative disorders. A master regulator of these functions is phosphatidylinositol-3-phosphate (PI3P), a phospholipid synthesized primarily by class III PI 3-kinase Vps34. Here we report that disruption of neuronal Vps34 function in vitro and in vivo impairs autophagy, lysosomal degradation as well as lipid metabolism, causing endolysosomal membrane damage. PI3P deficiency also promotes secretion of unique exosomes enriched for undigested lysosomal substrates, including amyloid precursor protein C-terminal fragments (APP-CTFs), specific sphingolipids, and the phospholipid bis(monoacylglycero)phosphate (BMP), which normally resides in the internal vesicles of endolysosomes. Secretion of these exosomes requires neutral sphingomyelinase 2 and sphingolipid synthesis. Our results reveal a homeostatic response counteracting lysosomal dysfunction via secretion of atypical exosomes eliminating lysosomal waste and define exosomal APP-CTFs and BMP as candidate biomarkers for endolysosomal dysfunction associated with neurodegenerative disorders.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Exossomos/metabolismo , Lipídeos/análise , Lisossomos/metabolismo , Neurônios/metabolismo , Precursor de Proteína beta-Amiloide/química , Animais , Autofagia/genética , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Classe III de Fosfatidilinositol 3-Quinases/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Células HEK293 , Humanos , Lisofosfolipídeos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monoglicerídeos/metabolismo , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismoRESUMO
Parkinson's disease (PD) is a common neurodegenerative disease whose pathological hallmark is the accumulation of intracellular α-synuclein aggregates in Lewy bodies. Lipid metabolism dysregulation may play a significant role in PD pathogenesis; however, large plasma lipidomic studies in PD are lacking. In the current study, we analyzed the lipidomic profile of plasma obtained from 150 idiopathic PD patients and 100 controls, taken from the 'Spot' study at Columbia University Medical Center in New York. Our mass spectrometry based analytical panel consisted of 520 lipid species from 39 lipid subclasses including all major classes of glycerophospholipids, sphingolipids, glycerolipids and sterols. Each lipid species was analyzed using a logistic regression model. The plasma concentrations of two lipid subclasses, triglycerides and monosialodihexosylganglioside (GM3), were different between PD and control participants. GM3 ganglioside concentration had the most significant difference between PD and controls (1.531±0.037 pmol/µl versus 1.337±0.040 pmol/µl respectively; p-value = 5.96E-04; q-value = 0.048; when normalized to total lipid: p-value = 2.890E-05; q-value = 2.933E-03). Next, we used a collection of 20 GM3 and glucosylceramide (GlcCer) species concentrations normalized to total lipid to perform a ROC curve analysis, and found that these lipids compare favorably with biomarkers reported in previous studies (AUC = 0.742 for males, AUC = 0.644 for females). Our results suggest that higher plasma GM3 levels are associated with PD. GM3 lies in the same glycosphingolipid metabolic pathway as GlcCer, a substrate of the enzyme glucocerebrosidase, which has been associated with PD. These findings are consistent with previous reports implicating lower glucocerebrosidase activity with PD risk.
Assuntos
Gangliosídeo G(M3)/sangue , Doença de Parkinson/sangue , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/fisiopatologia , Caracteres SexuaisRESUMO
Neuronal synapses are the fundamental units of neural signal transduction and must maintain exquisite signal fidelity while also accommodating the plasticity that underlies learning and development. To achieve these goals, the molecular composition and spatial organization of synaptic terminals must be tightly regulated; however, little is known about the regulation of lipid composition and organization in synaptic membranes. Here we quantify the comprehensive lipidome of rat synaptic membranes during postnatal development and observe dramatic developmental lipidomic remodeling during the first 60 postnatal days, including progressive accumulation of cholesterol, plasmalogens, and sphingolipids. Further analysis of membranes associated with isolated postsynaptic densities (PSDs) suggests the PSD-associated postsynaptic plasma membrane (PSD-PM) as one specific location of synaptic remodeling. We analyze the biophysical consequences of developmental remodeling in reconstituted synaptic membranes and observe remarkably stable microdomains, with the stability of domains increasing with developmental age. We rationalize the developmental accumulation of microdomain-forming lipids in synapses by proposing a mechanism by which palmitoylation of the immobilized scaffold protein PSD-95 nucleates domains at the postsynaptic plasma membrane. These results reveal developmental changes in lipid composition and palmitoylation that facilitate the formation of postsynaptic membrane microdomains, which may serve key roles in the function of the neuronal synapse.
Assuntos
Densidade Pós-Sináptica/fisiologia , Sinapses/fisiologia , Animais , Membrana Celular/fisiologia , Feminino , Hipocampo/metabolismo , Lipídeos , Lipoilação , Masculino , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurogênese , Plasticidade Neuronal , Terminações Pré-Sinápticas/metabolismo , Ratos , Ratos Sprague-Dawley , Sinapses/metabolismo , Membranas Sinápticas/metabolismoRESUMO
Inhibition of VLDL secretion reduces plasma levels of atherogenic apolipoprotein B (apoB) lipoproteins but can also cause hepatic steatosis. Approaches targeting apoB synthesis, which lies upstream of VLDL secretion, have potential to effectively reduce dyslipidemia but can also lead to hepatic accumulation of unsecreted triglycerides (TG). Here, we found that treating mice with apoB antisense oligonucleotides (ASOs) for 6 weeks decreased VLDL secretion and plasma cholesterol without causing steatosis. The absence of steatosis was linked to an increase in ER stress in the first 3 weeks of ASO treatment, followed by development of ER autophagy at the end of 6 weeks of treatment. The latter resulted in increased fatty acid (FA) oxidation that was inhibited by both chloroquine and 3-methyl adenine, consistent with trafficking of ER TG through the autophagic pathway before oxidation. These findings support the concept that inhibition of apoB synthesis traps lipids that have been transferred to the ER by microsomal TG transfer protein (MTP), inducing ER stress. ER stress then triggers ER autophagy and subsequent lysosomal lipolysis of TG, followed by mitochondrial oxidation of released FA, leading to prevention of steatosis. The identification of this pathway indicates that inhibition of VLDL secretion remains a viable target for therapies aiming to reduce circulating levels of atherogenic apoB lipoproteins.
Assuntos
Apolipoproteínas B/biossíntese , Autofagia , Retículo Endoplasmático/metabolismo , Fígado Gorduroso/terapia , Animais , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Aterosclerose/etiologia , Aterosclerose/patologia , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Células Cultivadas , Dislipidemias/complicações , Dislipidemias/patologia , Estresse do Retículo Endoplasmático , Ácidos Graxos/metabolismo , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Técnicas de Silenciamento de Genes , Lipogênese , Fígado/metabolismo , Fígado/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligonucleotídeos Antissenso/genética , Oxirredução , Biossíntese de Proteínas , Triglicerídeos/metabolismoRESUMO
Parental behavioural traits can be transmitted by non-genetic mechanisms to the offspring. Although trait transmission via sperm has been extensively researched, epidemiological studies indicate the exclusive/prominent maternal transmission of many non-genetic traits. Since maternal conditions impact the offspring during gametogenesis and through fetal/early-postnatal life, the resultant phenotype is likely the aggregate of consecutive germline and somatic effects; a concept that has not been previously studied. Here, we dissected a complex maternally transmitted phenotype, reminiscent of comorbid generalized anxiety/depression, to elementary behaviours/domains and their transmission mechanisms in mice. We show that four anxiety/stress-reactive traits are transmitted via independent iterative-somatic and gametic epigenetic mechanisms across multiple generations. Somatic/gametic transmission alters DNA methylation at enhancers within synaptic genes whose functions can be linked to the behavioural traits. Traits have generation-dependent penetrance and sex specificity resulting in pleiotropy. A transmission-pathway-based concept can refine current inheritance models of psychiatric diseases and facilitate the development of better animal models and new therapeutic approaches.
Assuntos
Comportamento Animal/fisiologia , Epigênese Genética , Células Germinativas/fisiologia , Herança Materna/fisiologia , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Ansiedade/genética , Ansiedade/psicologia , Metilação de DNA/genética , Modelos Animais de Doenças , Feminino , Gametogênese/fisiologia , Impressão Genômica/fisiologia , Hipotermia/induzido quimicamente , Hipotermia/genética , Hipotermia/psicologia , Masculino , Metabolômica/métodos , Camundongos , Camundongos Knockout , Modelos Animais , Penetrância , Fenótipo , Receptor 5-HT1A de Serotonina/genética , Receptor 5-HT1A de Serotonina/metabolismo , Agonistas do Receptor de Serotonina/farmacologia , Estresse Psicológico/genética , Estresse Psicológico/psicologiaRESUMO
A fundamental question is how autophagosome formation is regulated. Here we show that the PX domain protein HS1BP3 is a negative regulator of autophagosome formation. HS1BP3 depletion increased the formation of LC3-positive autophagosomes and degradation of cargo both in human cell culture and in zebrafish. HS1BP3 is localized to ATG16L1- and ATG9-positive autophagosome precursors and we show that HS1BP3 binds phosphatidic acid (PA) through its PX domain. Furthermore, we find the total PA content of cells to be significantly upregulated in the absence of HS1BP3, as a result of increased activity of the PA-producing enzyme phospholipase D (PLD) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 regulates autophagy by modulating the PA content of the ATG16L1-positive autophagosome precursor membranes through PLD1 activity and localization. Our findings provide key insights into how autophagosome formation is regulated by a novel negative-feedback mechanism on membrane lipids.
Assuntos
Autofagia/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Ácidos Fosfatídicos/metabolismo , Animais , Animais Geneticamente Modificados , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Linhagem Celular , Cortactina/metabolismo , Células HEK293 , Células HeLa , Humanos , Lipídeos de Membrana/metabolismo , Modelos Biológicos , Proteínas do Tecido Nervoso/química , Fosfolipase D/metabolismo , Domínios Proteicos , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Vibrio cholerae, responsible for acute gastroenteritis secretes a large multifunctional-autoprocessing repeat-in-toxin (MARTX) toxin linked to evasion of host immune system, facilitating colonization of small intestine. Unlike other effector domains of the multifunctional toxin that target cytoskeleton, the function of alpha-beta hydrolase (ABH) remained elusive. This study demonstrates that ABH is an esterase/lipase with catalytic Ser-His-Asp triad. ABH binds with high affinity to phosphatidylinositol-3-phosphate (PtdIns3P) and cleaves the fatty acid in PtdIns3P at the sn1 position in vitro making it the first PtdIns3P-specific phospholipase A1 (PLA1). Expression of ABH in vivo reduces intracellular PtdIns3P levels and its PtdIns3P-specific PLA1 activity blocks endosomal and autophagic pathways. In accordance with recent studies acknowledging the potential of extracellular pathogens to evade or exploit autophagy to prevent their clearance and facilitate survival, this is the first report highlighting the role of ABH in inhibiting autophagy and endosomal trafficking induced by extracellular V. cholerae.