RESUMO
We previously demonstrated that oligomeric amyloid ß42 (oAß42) inhibits the mevalonate pathway impairing cholesterol synthesis and protein prenylation. Enzymes of the mevalonate pathway are regulated by the transcription factor SREBP-2. Here, we show that in several neuronal types challenged with oAß42, SREBP-2 activation is reduced. Moreover, SREBP-2 activation is also decreased in the brain cortex of the Alzheimer's disease (AD) mouse model, TgCRND8, suggesting that SREBP-2 may be affected in vivo early in the disease. We demonstrate that oAß42 does not affect enzymatic cleavage of SREBP-2 per se, but may impair SREBP-2 transport from the endoplasmic reticulum (ER) to the Golgi. Trafficking of SREBP-2 from the ER to the Golgi requires protein kinase B (Akt) activation. oAß42 significantly reduces Akt phosphorylation and this decrease is responsible for the decline in SREBP-2 activation. Overexpression of constitutively active Akt prevents the effect of oAß42 on SREBP-2 and the downstream inhibition of cholesterol synthesis and protein prenylation. Our work provides a novel mechanistic link between Aß and the mevalonate pathway, which will impact the views on issues related to cholesterol, isoprenoids, and statins in AD. We also identify SREBP-2 as an indirect target of Akt in neurons, which may play a role in the cross-talk between AD and diabetes.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Feminino , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Accumulation of ß-amyloid (Aß) inside brain neurons is an early and crucial event in Alzheimer's disease (AD). Studies in brains of AD patients and mice models of AD suggested that cholesterol homeostasis is altered in neurons that accumulate Aß. Here we directly investigated the role of intracellular oligomeric Aß(42) (oAß(42)) in neuronal cholesterol homeostasis. We report that oAß(42) induces cholesterol sequestration without increasing cellular cholesterol mass. Several features of AD, such as endosomal abnormalities, brain accumulation of Aß and neurofibrillary tangles, and influence of apolipoprotein E genotype, are also present in Niemann-Pick type C, a disease characterized by impairment of intracellular cholesterol trafficking. These common features and data presented here suggest that a pathological mechanism involving abnormal cholesterol trafficking could take place in AD. Cholesterol sequestration in Aß-treated neurons results from impairment of intracellular cholesterol trafficking secondary to inhibition of protein prenylation. oAß(42) reduces sterol regulatory element-binding protein-2 (SREBP-2) cleavage, causing decrease of protein prenylation. Inhibition of protein prenylation represents a mechanism of oAß(42)-induced neuronal death. Supply of the isoprenoid geranylgeranyl pyrophosphate to oAß(42)-treated neurons recovers normal protein prenylation, reduces cholesterol sequestration, and prevents Aß-induced neurotoxicity. Significant to AD, reduced levels of protein prenylation are present in the cerebral cortex of the TgCRND8 mouse model. In conclusion, we demonstrate a significant inhibitory effect of Aß on protein prenylation and identify SREBP-2 as a target of oAß(42), directly linking Aß to cholesterol homeostasis impairment.
Assuntos
Peptídeos beta-Amiloides/fisiologia , Colesterol/metabolismo , Fragmentos de Peptídeos/fisiologia , Prenilação de Proteína/fisiologia , Proteína de Ligação a Elemento Regulador de Esterol 2/antagonistas & inibidores , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Peptídeos beta-Amiloides/antagonistas & inibidores , Animais , Anticolesterolemiantes/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Morte Celular , Células Cultivadas , Feminino , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/genética , Espaço Intracelular/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/patologia , Fragmentos de Peptídeos/antagonistas & inibidores , Prenilação de Proteína/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Since the discovery that apolipoprotein E, a cholesterol transport protein, is a major risk factor for Alzheimer's disease (AD) development, there has been a remarkable interest in understanding the many facets of the relationship between cholesterol and AD. Several lines of evidence have demonstrated the importance of cholesterol in amyloid beta peptide (Aß) production and metabolism, as well as the involvement of Aß in cholesterol homeostasis. The emerging picture is complex and still incomplete. This review discusses findings that indicate that a reciprocal regulation exists between Aß and cholesterol at the subcellular level. The pathological impact of such regulation is highlighted.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Colesterol/metabolismo , Animais , Apolipoproteínas E/metabolismo , HumanosRESUMO
Extracellular vesicles (EVs) are involved in a multitude of physiological functions and play important roles in health and disease. The largest proportion of studies on EVs is based on the analysis and characterization of EVs secreted in the cell culture medium. These studies remain challenging due to the small size of the EV particles, a lack of universal EV markers, and sample loss or technical artifacts that are often associated with EV labeling for single particle tracking and/or separation techniques. To address these problems, we characterized and validated a method for in-cell EV labeling with fluorescent lipids coupled with direct analysis of lipid-labeled EVs in the conditioned medium by imaging flow cytometry (IFC). This approach significantly reduces sample processing and loss compared to established methods for EV separation and labeling in vitro, resulting in improved detection of quantitative changes in EV secretion and subpopulations compared to protocols that rely on EV separation by size-exclusion chromatography and ultracentrifugation. Our optimized protocol for in-cell EV labeling and analysis of the conditioned medium reduces EV sample processing and loss, and is well-suited for cell biology studies that focus on modulation of EV secretion by cells in culture.
Assuntos
Vesículas Extracelulares , Meios de Cultivo Condicionados/análise , Meios de Cultivo Condicionados/farmacologia , Vesículas Extracelulares/química , Citometria de Fluxo/métodos , Manejo de Espécimes , Ultracentrifugação/métodosRESUMO
Glia modulate neuronal activity by releasing transmitters in a process called gliotransmission. The role of this process in controlling the activity of neuronal networks underlying motor behavior is unknown. ATP features prominently in gliotransmission; it also contributes to the homeostatic ventilatory response evoked by low oxygen through mechanisms that likely include excitation of preBötzinger complex (preBötC) neural networks, brainstem centers critical for breathing. We therefore inhibited glial function in rhythmically active inspiratory networks in vitro to determine whether glia contribute to preBötC ATP sensitivity. Glial toxins markedly reduced preBötC responses to ATP, but not other modulators. Furthermore, since preBötC glia responded to ATP with increased intracellular Ca(2+) and glutamate release, we conclude that glia contribute to the ATP sensitivity of preBötC networks, and possibly the hypoxic ventilatory response. Data reveal a role for glia in signal processing within brainstem motor networks that may be relevant to similar networks throughout the neuraxis.
Assuntos
Trifosfato de Adenosina/fisiologia , Inalação/fisiologia , Rede Nervosa/fisiologia , Neuroglia/fisiologia , Periodicidade , Receptores Purinérgicos/fisiologia , Centro Respiratório/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Animais Recém-Nascidos , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/fisiologia , Células Cultivadas , Inalação/efeitos dos fármacos , Rede Nervosa/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Agonistas Purinérgicos , Ratos , Ratos Sprague-Dawley , Centro Respiratório/efeitos dos fármacosRESUMO
BACKGROUND: Myocardial energy metabolism is a strong predictor of postoperative cardiac function. This study profiled the metabolites and metabolic changes in the myocardium exposed to sevoflurane, propofol, and Intralipid and investigated the underlying molecular mechanisms. METHODS: Sevoflurane (2 vol%) and propofol (10 and 100 microM) in the formulation of 1% Diprivan (AstraZeneca Inc., Mississauga, ON, Canada) were compared for their effects on oxidative energy metabolism and contractility in the isolated working rat heart model. Intralipid served as a control. Substrate flux through the major pathways for adenosine triphosphate generation in the heart, that is, fatty acid and glucose oxidation, was measured using [H]palmitate and [C]glucose. Biochemical analyses of nucleotides, acyl-CoAs, ceramides, and 32 acylcarnitine species were used to profile individual metabolites. Lipid rafts were isolated and used for Western blotting of the plasma membrane transporters CD36 and glucose transporter 4. RESULTS: Metabolic profiling of the hearts exposed to sevoflurane and propofol revealed distinct regulation of fatty acid and glucose oxidation. Sevoflurane selectively decreased fatty acid oxidation, which was closely related to a marked reduction in left ventricular work. In contrast, propofol at 100 microM but not 10 microM increased glucose oxidation without affecting cardiac work. Sevoflurane decreased fatty acid transporter CD36 in lipid rafts/caveolae, whereas high propofol increased pyruvate dehydrogenase activity without affecting glucose transporter 4, providing mechanisms for the fuel shifts in energy metabolism. Propofol increased ceramide formation, and Intralipid increased hydroxy acylcarnitine species. CONCLUSIONS: Anesthetics and their solvents elicit distinct metabolic profiles in the myocardium, which may have clinical implications for the already jeopardized diseased heart.
Assuntos
Antígenos CD36/metabolismo , Ácidos Graxos/metabolismo , Glucose/metabolismo , Coração/efeitos dos fármacos , Éteres Metílicos/farmacologia , Miocárdio/metabolismo , Propofol/farmacologia , Anestésicos Intravenosos/farmacologia , Animais , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Coração/fisiologia , Técnicas In Vitro , Masculino , Metaboloma/efeitos dos fármacos , Metaboloma/fisiologia , Miocárdio/enzimologia , Oxirredução/efeitos dos fármacos , Complexo Piruvato Desidrogenase/metabolismo , Ratos , Ratos Sprague-Dawley , SevofluranoRESUMO
Simple sphingolipids such as ceramide, sphingosine and sphingosine 1-phosphate are key regulators of diverse cellular functions. Their roles in the nervous system are supported by extensive evidence derived primarily from studies in cultured cells. More recently animal studies and studies with human samples have revealed the importance of ceramide and its metabolites in the development and progression of neurodegenerative disorders. The roles of sphingolipids in neurons and glial cells are complex, cell dependent, and many times contradictory. In this review I will summarize the effects elicited by ceramide and ceramide metabolites in cells of the nervous system, in particular those effects related to cell survival and death, emphasizing the molecular mechanisms involved. I also discuss recent evidence for the implication of sphingolipids in the development and progression of certain dementias.
Assuntos
Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Regeneração Nervosa/fisiologia , Sistema Nervoso/citologia , Esfingolipídeos/fisiologia , Animais , Ceramidas/metabolismo , Células PC12 , RatosRESUMO
In sympathetic neurons, C6-ceramide, as well as endogenous ceramides, blocks apoptosis elicited by NGF (nerve growth factor) deprivation. The mechanism(s) involved in ceramide-induced neuronal survival are poorly understood. Few direct targets for the diverse cellular effects of ceramide have been identified. Amongst those proposed is PP-1c, the catalytic subunit of serine/threonine PP-1 (protein phosphatase-1). Here, we present the first evidence of PP-1c activation by ceramide in live cells, namely NGF-deprived sympathetic neurons. We first determined PP activity in cellular lysates from sympathetic neurons treated with exogenous ceramide and demonstrated a 2-3-fold increase in PP activity. PP activation was completely blocked by the addition of the specific type-1 PP inhibitor protein I-2 as well as by tautomycin, but unaffected by 2 nM okadaic acid, strongly indicating that the ceramide-activated phosphatase activity was PP-1c. Inhibition of PP activity by phosphatidic acid (which has been reported to be a selective inhibitor of PP-1c) and tautomycin (a PP-1 and PP-2A inhibitor), but not by 10 nM okadaic acid, abolished the anti-apoptotic effect of ceramide in NGF-deprived neurons, suggesting that activation of PP-1c is required for ceramide-induced neuronal survival. Ceramide was able to prevent pRb (retinoblastoma gene product) hyperphosphorylation by a mechanism dependent on PP-1c activation, suggesting that two consequences of NGF deprivation in sympathetic neurons are inhibition of PP-1c and subsequent hyperphosphorylation of pRb protein. These findings suggest a novel mechanism for ceramide-induced survival, and implicate the involvement of PPs in apoptosis induced by NGF deprivation.
Assuntos
Ceramidas/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Fosfoproteínas Fosfatases/metabolismo , Sistema Nervoso Simpático/citologia , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fator de Crescimento Neural/deficiência , Fator de Crescimento Neural/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Ácidos Fosfatídicos/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 1 , Ratos , Ratos Sprague-Dawley , Proteína do Retinoblastoma/metabolismo , Serina/metabolismo , Treonina/metabolismoRESUMO
C6-ceramide protects sympathetic neurons from apoptosis caused by nerve growth factor (NGF) deprivation. Here, we report for the first time that ceramide generated "de novo" is also anti-apoptotic. Moreover, C6-ceramide is converted to long-chain ceramides in a process inhibited by fumonisin B1. The anti-apoptotic effect of C6-ceramide is due to the short analogue as to the long-chain ceramides. C6-ceramide shares mechanisms of action with NGF. C6-ceramide induces TrkA phosphorylation and selective activation of the phosphatidyl inositol 3-kinase (PI3-kinase)/Akt pathway but not the MAPK/ERK pathway. Importantly, the PI3-kinase inhibitor LY294002 abolishes the pro-survival effect of C6-ceramide. We identified a novel way to activate retrograde-mediated neuronal survival in the absence of NGF. Using compartmented cultures we show that addition of C6-ceramide exclusively to distal axons is sufficient to abort nuclear apoptosis. Our system offers a very unique alternative to understand the molecular bases of retrograde signaling in the absence of retrograde transport of neurotrophins. In search for a natural ligand that leads to ceramide generation we examined the activation of the sphingomyelin (SM) cycle downstream the p75 neurotrophin receptor (p75NTR). We found that in sympathetic neurons, selective activation of p75NTR by brain-derived neurotrophin factor or NGF plus K252a induces elevation of ceramide that correlates with SM hydrolysis. However, p75NTR activation does not generate sufficient ceramide to block apoptosis probably due to the rapid decrease in p75NTR expression that occurs upon NGF withdrawal.
Assuntos
Fibras Adrenérgicas/fisiologia , Apoptose/fisiologia , Ceramidas/fisiologia , Inibição Neural/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Receptores de Fator de Crescimento Neural/metabolismo , Fibras Adrenérgicas/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Ceramidas/metabolismo , Ceramidas/farmacologia , Fator de Crescimento Neural/antagonistas & inibidores , Fator de Crescimento Neural/farmacologia , Inibição Neural/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptor de Fator de Crescimento Neural , Receptores de Fator de Crescimento Neural/genéticaRESUMO
The IGF-II/mannose 6-phosphate (M6P) receptor is a single-transmembrane domain glycoprotein that plays an important role in the intracellular trafficking of lysosomal enzymes and endocytosis-mediated degradation of IGF-II. The receptor may also mediate certain biological effects in response to IGF-II binding by interacting with G proteins. However, the nature of the IGF-II/M6P receptor's interaction with the G protein or with G protein-coupled receptor (GPCR) interacting proteins such as ß-arrestin remains unclear. Here we report that [(125)I]IGF-II receptor binding in the rat hippocampal formation is sensitive to guanosine-5'-[γ-thio]triphosphate, mastoparan, and Mas-7, which are known to interfere with the coupling of the classical GPCR with G protein. Monovalent and divalent cations also influenced [(125)I]IGF-II receptor binding. The IGF-II/M6P receptor, as observed for several GPCRs, was found to be associated with ß-arrestin 2, which exhibits sustained ubiquitination after stimulation with Leu(27)IGF-II, an IGF-II analog that binds rather selectively to the IGF-II/M6P receptor. Activation of the receptor by Leu(27)IGF-II induced stimulation of extracellular signal-related kinase 1/2 via a pertussis toxin-dependent pathway. Additionally, we have shown that IGF-II/M6P receptors under normal conditions are associated mostly with detergent-resistant membrane domains, but after stimulation with Leu(27)IGF-II, are translocated to the detergent-soluble fraction along with a portion of ß-arrestin 2. Collectively these results suggest that the IGF-II/M6P receptor may interact either directly or indirectly with G protein as well as ß-arrestin 2, and activation of the receptor by an agonist can lead to alteration in its subcellular distribution along with stimulation of an intracellular signaling cascade.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Hipocampo/metabolismo , Receptor IGF Tipo 2/metabolismo , Animais , Arrestinas/genética , Arrestinas/metabolismo , Colesterol/metabolismo , Proteínas de Ligação ao GTP/genética , Hipocampo/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Peptídeos/farmacologia , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptor IGF Tipo 2/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Venenos de Vespas/farmacologia , beta-Arrestina 2 , beta-ArrestinasRESUMO
In the brain, the amyloid ß peptide (Aß) exists extracellularly and inside neurons. The intracellular accumulation of Aß in Alzheimer's disease brain has been questioned for a long time. However, there is now sufficient strong evidence indicating that accumulation of Aß inside neurons plays an important role in the pathogenesis of Alzheimer's disease. Intraneuronal Aß originates from intracellular cleavage of APP and from Aß internalization from the extracellular milieu. We discuss here the different molecular mechanisms that are responsible for Aß internalization in neurons and the links between Aß internalization and neuronal dysfunction and death. A brief description of Aß uptake by glia is also presented.
RESUMO
Simple sphingolipids such as ceramide and sphingomyelin (SM) as well as more complex glycosphingolipids play very important roles in cell function under physiological conditions and during disease development and progression. Sphingolipids are particularly abundant in the nervous system. Due to their amphiphilic nature they localize to cellular membranes and many of their roles in health and disease result from membrane reorganization and from lipid interaction with proteins within cellular membranes. In this review we discuss some of the functions of sphingolipids in processes that entail cellular membranes and their role in neurodegenerative diseases, with an emphasis on SM, ceramide and gangliosides.
Assuntos
Membrana Celular/fisiologia , Gangliosídeos/fisiologia , Doenças do Sistema Nervoso/fisiopatologia , Sistema Nervoso/metabolismo , Esfingolipídeos/fisiologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Envelhecimento/fisiologia , Animais , Membrana Celular/patologia , Gangliosídeos/química , Gangliosídeos/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Modelos Biológicos , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Doenças do Sistema Nervoso/patologia , Fenômenos Fisiológicos do Sistema Nervoso , Organelas/metabolismo , Organelas/patologia , Organelas/fisiologia , Esfingolipídeos/química , Esfingolipídeos/metabolismoRESUMO
Adult neuronal phenotype is maintained, at least in part, by the sensitivity of individual neurons to a specific selection of neurotrophic factors and the availability of such factors in the neurons' environment. Nerve growth factor (NGF) increases the functional expression of Na(+) channel currents (I(Na)) and both N- and L-type Ca(2+) currents (I(Ca,N) and I(Ca,L)) in adult bullfrog sympathetic ganglion (BFSG) B-neurons. The effects of NGF on I(Ca) involve the mitogen-activated protein kinase (MAPK) pathway. Prolonged exposure to the ganglionic neurotransmitter luteinizing hormone releasing hormone (LHRH) also increases I(Ca,N) but the transduction mechanism remains to be elucidated as does the transduction mechanism for NGF regulation of Na(+) channels. We therefore exposed cultured BFSG B-neurons to chicken II LHRH (0.45 microM; 6-9 days) or to NGF (200 ng/ml; 9-10 days) and used whole cell recording, immunoblot analysis, and ras or rap-1 pulldown assays to study effects of various inhibitors and activators of transduction pathways. We found that 1) LHRH signals via ras-MAPK to increase I(Ca,N), 2) this effect is mediated via protein kinase C-beta (PKC-beta-IotaIota), 3) protein kinase A (PKA) is necessary but not sufficient to effect transduction, 4) NGF signals via phosphatidylinositol 3-kinase (PI3K) to increase I(Na), and 5) long-term exposure to LHRH fails to affect I(Na). Thus downstream signaling from LHRH has access to the ras-MAPK pathway but not to the PI3K pathway. This allows for differential retrograde and anterograde neurotrophic regulation of sodium and calcium channels in an adult sympathetic neuron.