RESUMO
Traumatic brain injury (TBI) is an established risk factor for neurodegenerative diseases. In this study, we used the Closed Head Injury Model of Engineered Rotational Acceleration (CHIMERA) to investigate the effects of a single high-energy TBI in rTg4510 mice, a mouse model of tauopathy. Fifteen male rTg4510 mice (4 mo) were impacted at 4.0 J using interfaced CHIMERA and were compared to sham controls. Immediately after injury, the TBI mice showed significant mortality (7/15; 47%) and a prolonged duration of loss of the righting reflex. At 2 mo post-injury, surviving mice displayed significant microgliosis (Iba1) and axonal injury (Neurosilver). Western blotting indicated a reduced p-GSK-3ß (S9):GSK-3ß ratio in TBI mice, suggesting chronic activation of tau kinase. Although longitudinal analysis of plasma total tau suggested that TBI accelerates the appearance of tau in the circulation, there were no significant differences in brain total or p-tau levels, nor did we observe evidence of enhanced neurodegeneration in TBI mice compared to sham mice. In summary, we showed that a single high-energy head impact induces chronic white matter injury and altered GSK-3ß activity without an apparent change in post-injury tauopathy in rTg4510 mice.
Assuntos
Lesões Encefálicas Traumáticas , Traumatismos Cranianos Fechados , Tauopatias , Camundongos , Masculino , Animais , Glicogênio Sintase Quinase 3 beta/genética , Lesões Encefálicas Traumáticas/genética , Encéfalo/metabolismo , Tauopatias/genética , Modelos Animais de Doenças , Aceleração , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
INTRODUCTION: The neurovascular unit (NVU) - the interaction between the neurons and the cerebrovasculature - is increasingly important to interrogate through human-based experimental models. Although advanced models of cerebral capillaries have been developed in the last decade, there is currently no in vitro 3-dimensional (3D) perfusible model of the human cortical arterial NVU. METHOD: We used a tissue-engineering technique to develop a scaffold-directed, perfusible, 3D human NVU that is cultured in native-like flow conditions that mimics the anatomy and physiology of cortical penetrating arteries. RESULTS: This system, composed of primary human vascular cells (endothelial cells, smooth muscle cells and astrocytes) and induced pluripotent stem cell (iPSC) derived neurons, demonstrates a physiological multilayer organization of the involved cell types. It reproduces key characteristics of cortical neurons and astrocytes and enables formation of a selective and functional endothelial barrier. We provide proof-of-principle data showing that this in vitro human arterial NVU may be suitable to study neurovascular components of neurodegenerative diseases such as Alzheimer's disease (AD), as endogenously produced phosphorylated tau and beta-amyloid accumulate in the model over time. Finally, neuronal and glial fluid biomarkers relevant to neurodegenerative diseases are measurable in our arterial NVU model. CONCLUSION: This model is a suitable research tool to investigate arterial NVU functions in healthy and disease states. Further, the design of the platform allows culture under native-like flow conditions for extended periods of time and yields sufficient tissue and media for downstream immunohistochemistry and biochemistry analyses.
Assuntos
Artérias/metabolismo , Astrócitos/metabolismo , Células Endoteliais/metabolismo , Doenças Neurodegenerativas/metabolismo , Doença de Alzheimer/metabolismo , Artérias/fisiopatologia , Barreira Hematoencefálica/metabolismo , Técnicas de Cocultura , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Doenças Neurodegenerativas/patologia , Neurônios/metabolismoRESUMO
Alzheimer's disease (AD), the leading cause of dementia, is a chronic neurodegenerative disease. Apolipoprotein E (apoE), which carries lipids in the brain in the form of lipoproteins, plays an undisputed role in AD pathophysiology. A high-throughput phenotypic screen was conducted using a CCF-STTG1 human astrocytoma cell line to identify small molecules that could upregulate apoE secretion. AZ7235, a previously discovered Axl kinase inhibitor, was identified to have robust apoE activity in brain microglia, astrocytes and pericytes. AZ7235 also increased expression of ATP-binding cassette protein A1 (ABCA1), which is involved in the lipidation and secretion of apoE. Moreover, AZ7235 did not exhibit Liver-X-Receptor (LXR) activity and stimulated apoE and ABCA1 expression in the absence of LXR. Target validation studies using AXL-/- CCF-STTG1 cells showed that Axl is required to mediate AZ7235 upregulation of apoE and ABCA1. Intriguingly, apoE expression and secretion was significantly attenuated in AXL-deficient CCF-STTG1 cells and reconstitution of Axl or kinase-dead Axl significantly restored apoE baseline levels, demonstrating that Axl also plays a role in maintaining apoE homeostasis in astrocytes independent of its kinase activity. Lastly, these effects may require human apoE regulatory sequences, as AZ7235 exhibited little stimulatory activity toward apoE and ABCA1 in primary murine glia derived from neonatal human APOE3 targeted-replacement mice. Collectively, we identified a small molecule that exhibits robust apoE and ABCA1 activity independent of the LXR pathway in human cells and elucidated a novel relationship between Axl and apoE homeostasis in human astrocytes.
Assuntos
Doença de Alzheimer/metabolismo , Apolipoproteínas E/metabolismo , Astrócitos/efeitos dos fármacos , Astrocitoma/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Apolipoproteínas E/genética , Astrocitoma/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Técnicas de Silenciamento de Genes , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptor Tirosina Quinase AxlRESUMO
BACKGROUND: Alzheimer's disease (AD) is defined by amyloid beta (Aß) plaques and neurofibrillary tangles and characterized by neurodegeneration and memory loss. The majority of AD patients also have Aß deposition in cerebral vessels known as cerebral amyloid angiopathy (CAA), microhemorrhages, and vascular co-morbidities, suggesting that cerebrovascular dysfunction contributes to AD etiology. Promoting cerebrovascular resilience may therefore be a promising therapeutic or preventative strategy for AD. Plasma high-density lipoproteins (HDL) have several vasoprotective functions and are associated with reduced AD risk in some epidemiological studies and with reduced Aß deposition and Aß-induced inflammation in 3D engineered human cerebral vessels. In mice, deficiency of apoA-I, the primary protein component of HDL, increases CAA and cognitive dysfunction, whereas overexpression of apoA-I from its native promoter in liver and intestine has the opposite effect and lessens neuroinflammation. Similarly, acute peripheral administration of HDL reduces soluble Aß pools in the brain and some studies have observed reduced CAA as well. Here, we expand upon the known effects of plasma HDL in mouse models and in vitro 3D artery models to investigate the interaction of amyloid, astrocytes, and HDL on the cerebrovasculature in APP/PS1 mice. METHODS: APP/PS1 mice deficient or hemizygous for Apoa1 were aged to 12 months. Plasma lipids, amyloid plaque deposition, Aß protein levels, protein and mRNA markers of neuroinflammation, and astrogliosis were assessed using ELISA, qRT-PCR, and immunofluorescence. Contextual and cued fear conditioning were used to assess behavior. RESULTS: In APP/PS1 mice, complete apoA-I deficiency increased total and vascular Aß deposition in the cortex but not the hippocampus compared to APP/PS1 littermate controls hemizygous for apoA-I. Markers of both general and vascular neuroinflammation, including Il1b mRNA, ICAM-1 protein, PDGFRß protein, and GFAP protein, were elevated in apoA-I-deficient APP/PS1 mice. Additionally, apoA-I-deficient APP/PS1 mice had elevated levels of vascular-associated ICAM-1 in the cortex and hippocampus and vascular-associated GFAP in the cortex. A striking observation was that astrocytes associated with cerebral vessels laden with Aß or associated with Aß plaques showed increased reactivity in APP/PS1 mice lacking apoA-I. No behavioral changes were observed. CONCLUSIONS: ApoA-I-containing HDL can reduce amyloid pathology and astrocyte reactivity to parenchymal and vascular amyloid in APP/PS1 mice.
Assuntos
Doença de Alzheimer/sangue , Doença de Alzheimer/patologia , Apolipoproteína A-I/genética , Angiopatia Amiloide Cerebral/sangue , Angiopatia Amiloide Cerebral/patologia , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Angiopatia Amiloide Cerebral/genética , Modelos Animais de Doenças , Encefalite/metabolismo , Encefalite/patologia , Feminino , Gliose/metabolismo , Hipocampo/patologia , Lipoproteínas HDL/sangue , Masculino , Camundongos Transgênicos , Placa Amiloide/metabolismoRESUMO
PURPOSE OF REVIEW: We review current knowledge regarding HDL and Alzheimer's disease, focusing on HDL's vasoprotective functions and potential as a biomarker and therapeutic target for the vascular contributions of Alzheimer's disease. RECENT FINDINGS: Many epidemiological studies have observed that circulating HDL levels associate with decreased Alzheimer's disease risk. However, it is now understood that the functions of HDL may be more informative than levels of HDL cholesterol (HDL-C). Animal model studies demonstrate that HDL protects against memory deficits, neuroinflammation, and cerebral amyloid angiopathy (CAA). In-vitro studies using state-of-the-art 3D models of the human blood-brain barrier (BBB) confirm that HDL reduces vascular Aß accumulation and attenuates Aß-induced endothelial inflammation. Although HDL-based therapeutics have not been tested in clinical trials for Alzheimer's disease , several HDL formulations are in advanced phase clinical trials for coronary artery disease and atherosclerosis and could be leveraged toward Alzheimer's disease . SUMMARY: Evidence from human studies, animal models, and bioengineered arteries supports the hypothesis that HDL protects against cerebrovascular dysfunction in Alzheimer's disease. Assays of HDL functions relevant to Alzheimer's disease may be desirable biomarkers of cerebrovascular health. HDL-based therapeutics may also be of interest for Alzheimer's disease, using stand-alone or combination therapy approaches.
Assuntos
Doença de Alzheimer/metabolismo , Lipoproteínas HDL/metabolismo , Doença de Alzheimer/epidemiologia , Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Animais , Comorbidade , Humanos , Resistência VascularRESUMO
apoE is the primary lipid carrier within the CNS and the strongest genetic risk factor for late onset Alzheimer's disease (AD). apoE is primarily lipidated via ABCA1, and both are under transcriptional regulation by the nuclear liver X receptor (LXR). Considerable evidence from genetic (using ABCA1 overexpression) and pharmacological (using synthetic LXR agonists) studies in AD mouse models suggests that increased levels of lipidated apoE can improve cognitive performance and, in some strains, can reduce amyloid burden. However, direct synthetic LXR ligands have hepatotoxic side effects that limit their clinical use. Here, we describe a set of small molecules, previously annotated as antagonists of the purinergic receptor, P2X7, which enhance ABCA1 expression and activity as well as apoE secretion, and are not direct LXR ligands. Furthermore, P2X7 is not required for these molecules to induce ABCA1 upregulation and apoE secretion, demonstrating that the ABCA1 and apoE effects are mechanistically independent of P2X7 inhibition. Hence, we have identified novel dual activity compounds that upregulate ABCA1 across multiple CNS cell types, including human astrocytes, pericytes, and microglia, through an indirect LXR mechanism and that also independently inhibit P2X7 receptor activity.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/agonistas , Apolipoproteínas E/agonistas , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X7/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Adamantano/análogos & derivados , Adamantano/química , Adamantano/farmacologia , Animais , Apolipoproteínas E/metabolismo , Aziridinas/química , Aziridinas/farmacologia , Benzamidas/química , Benzamidas/farmacologia , Células Cultivadas , Humanos , Camundongos , Camundongos Knockout , Estrutura Molecular , Naftoquinonas/química , Naftoquinonas/farmacologia , Antagonistas do Receptor Purinérgico P2X/química , Receptores Purinérgicos P2X7/deficiência , Bibliotecas de Moléculas Pequenas/química , Sulfonamidas/química , Sulfonamidas/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
The apolipoprotein E (APOE) gene is the most highly associated susceptibility locus for late onset Alzheimer's Disease (AD), and augmenting the beneficial physiological functions of apoE is a proposed therapeutic strategy. In a high throughput phenotypic screen for small molecules that enhance apoE secretion from human CCF-STTG1 astrocytoma cells, we show the chrysanthemic ester 82879 robustly increases expressed apoE up to 9.4-fold and secreted apoE up to 6-fold and is associated with increased total cholesterol in conditioned media. Compound 82879 is unique as structural analogues, including pyrethroid esters, show no effect on apoE expression or secretion. 82879 also stimulates liver x receptor (LXR) target genes including ATP binding cassette A1 (ABCA1), LXRα and inducible degrader of low density lipoprotein receptor (IDOL) at both mRNA and protein levels. In particular, the lipid transporter ABCA1 was increased by up to 10.6-fold upon 82879 treatment. The findings from CCF-STTG1 cells were confirmed in primary human astrocytes from three donors, where increased apoE and ABCA1 was observed along with elevated secretion of high-density lipoprotein (HDL)-like apoE particles. Nuclear receptor transactivation assays revealed modest direct LXR agonism by compound 82879, yet 10 µM of 82879 significantly upregulated apoE mRNA in mouse embryonic fibroblasts (MEFs) depleted of both LXRα and LXRß, demonstrating that 82879 can also induce apoE expression independent of LXR transactivation. By contrast, deletion of LXRs in MEFs completely blocked mRNA changes in ABCA1 even at 10 µM of 82879, indicating the ability of 82879 to stimulate ABCA1 expression is entirely dependent on LXR transactivation. Taken together, compound 82879 is a novel chrysanthemic ester capable of modulating apoE secretion as well as apoE-associated lipid metabolic pathways in astrocytes, which is structurally and mechanistically distinct from known LXR agonists.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/genética , Apolipoproteínas E/genética , Astrócitos/efeitos dos fármacos , Receptores X do Fígado/genética , Piretrinas/farmacologia , Receptores de LDL/genética , Transportador 1 de Cassete de Ligação de ATP/agonistas , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Apolipoproteínas E/agonistas , Apolipoproteínas E/metabolismo , Astrócitos/citologia , Astrócitos/metabolismo , Linhagem Celular Tumoral , Ésteres , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Receptores X do Fígado/agonistas , Receptores X do Fígado/metabolismo , Camundongos , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , Cultura Primária de Células , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de LDL/agonistas , Receptores de LDL/metabolismo , Transdução de SinaisRESUMO
Many lines of evidence suggest a protective role for high-density lipoprotein (HDL) and its major apolipoprotein (apo)A-I in Alzheimer's Disease (AD). HDL/apoA-I particles are produced by the liver and intestine and, in addition to removing excess cholesterol from the body, are increasingly recognized to have vasoprotective functions. Here we tested the ability of reconstituted HDL (rHDL) consisting of human apoA-I reconstituted with soy phosphatidylcholine for its ability to lower amyloid beta (Aß) levels in symptomatic APP/PS1 mice, a well-characterized preclinical model of amyloidosis. Animals were treated intravenously either with four weekly doses (chronic study) or a single dose of 60mg/kg of rHDL (acute study). The major finding of our acute study is that soluble brain Aß40 and Aß42 levels were significantly reduced within 24h of a single dose of rHDL. By contrast, no changes were observed in our chronic study with respect to soluble or deposited Aß levels in animals assessed 7days after the final weekly dose of rHDL, suggesting that beneficial effects diminish as rHDL is cleared from the body. Further, rHDL-treated animals showed no change in amyloid burden, cerebrospinal fluid (CSF) Aß levels, neuroinflammation, or endothelial activation in the chronic study, suggesting that the pathology-modifying effects of rHDL may indeed be acute and may be specific to the soluble Aß pool. That systemic administration of rHDL can acutely modify brain Aß levels provides support for further investigation of the therapeutic potential of apoA-I-based agents for AD. This article is part of a Special Issue entitled: Vascular Contributions to Cognitive Impairment and Dementia edited by M. Paul Murphy, Roderick A. Corriveau and Donna M. Wilcock.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Amiloidose/terapia , Apolipoproteína A-I/uso terapêutico , Encéfalo/metabolismo , Lipoproteínas HDL/uso terapêutico , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/sangue , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/sangue , Amiloidose/sangue , Amiloidose/metabolismo , Amiloidose/patologia , Animais , Apolipoproteína A-I/administração & dosagem , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Humanos , Lipoproteínas HDL/administração & dosagem , Masculino , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/sangueRESUMO
BACKGROUND: Traumatic brain injury (TBI) is a major health care concern that currently lacks any effective treatment. Despite promising outcomes from many preclinical studies, clinical evaluations have failed to identify effective pharmacological therapies, suggesting that the translational potential of preclinical models may require improvement. Rodents continue to be the most widely used species for preclinical TBI research. As most human TBIs result from impact to an intact skull, closed head injury (CHI) models are highly relevant, however, traditional CHI models suffer from extensive experimental variability that may be due to poor control over biomechanical inputs. Here we describe a novel CHI model called CHIMERA (Closed-Head Impact Model of Engineered Rotational Acceleration) that fully integrates biomechanical, behavioral, and neuropathological analyses. CHIMERA is distinct from existing neurotrauma model systems in that it uses a completely non-surgical procedure to precisely deliver impacts of prescribed dynamic characteristics to a closed skull while enabling kinematic analysis of unconstrained head movement. In this study, we characterized head kinematics as well as functional, neuropathological, and biochemical outcomes up to 14d following repeated TBI (rTBI) in adult C57BL/6 mice using CHIMERA. RESULTS: Head kinematic analysis showed excellent repeatability over two closed head impacts separated at 24h. Injured mice showed significantly prolonged loss of righting reflex and displayed neurological, motor, and cognitive deficits along with anxiety-like behavior. Repeated TBI led to diffuse axonal injury with extensive microgliosis in white matter from 2-14d post-rTBI. Injured mouse brains also showed significantly increased levels of TNF-α and IL-1ß and increased endogenous tau phosphorylation. CONCLUSIONS: Repeated TBI using CHIMERA mimics many of the functional and pathological characteristics of human TBI with a reliable biomechanical response of the head. This makes CHIMERA well suited to investigate the pathophysiology of TBI and for drug development programs.
Assuntos
Lesões Encefálicas/metabolismo , Lesões Encefálicas/fisiopatologia , Modelos Animais de Doenças , Animais , Fenômenos Biomecânicos , Humanos , Interleucina-1beta/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Brain lipoprotein metabolism is dependent on lipoprotein particles that resemble plasma high-density lipoproteins but that contain apolipoprotein (apo) E rather than apoA-I as their primary protein component. Astrocytes and microglia secrete apoE but not apoA-I; however, apoA-I is detectable in both cerebrospinal fluid and brain tissue lysates. The route by which plasma apoA-I enters the central nervous system is unknown. METHODS AND RESULTS: Steady-state levels of murine apoA-I in cerebrospinal fluid and interstitial fluid are 0.664 and 0.120 µg/mL, respectively, whereas brain tissue apoA-I is ≈10% to 15% of its levels in liver. Recombinant, fluorescently tagged human apoA-I injected intravenously into mice localizes to the choroid plexus within 30 minutes and accumulates in a saturable, dose-dependent manner in the brain. Recombinant, fluorescently tagged human apoA-I accumulates in the brain for 2 hours, after which it is eliminated with a half-life of 10.3 hours. In vitro, human apoA-I is specifically bound, internalized, and transported across confluent monolayers of primary human choroid plexus epithelial cells and brain microvascular endothelial cells. CONCLUSIONS: Following intravenous injection, recombinant human apoA-I rapidly localizes predominantly to the choroid plexus. Because apoA-I mRNA is undetectable in murine brain, our results suggest that plasma apoA-I, which is secreted from the liver and intestine, gains access to the central nervous system primarily by crossing the blood-cerebrospinal fluid barrier via specific cellular mediated transport, although transport across the blood-brain barrier may also contribute to a lesser extent.
Assuntos
Apolipoproteína A-I/administração & dosagem , Apolipoproteína A-I/farmacocinética , Barreira Hematoencefálica/metabolismo , Plexo Corióideo/metabolismo , Animais , Apolipoproteína A-I/sangue , Apolipoproteína A-I/líquido cefalorraquidiano , Apolipoproteína A-I/genética , Transporte Biológico , Permeabilidade Capilar , Células Cultivadas , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Feminino , Meia-Vida , Humanos , Injeções Intravenosas , Taxa de Depuração Metabólica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacocinética , Distribuição TecidualRESUMO
Apolipoprotein E (apoE) is the major lipid carrier in the central nervous system. As apoE plays a major role in the pathogenesis of Alzheimer disease (AD) and also mediates repair pathways after several forms of acute brain injury, modulating the expression, secretion, or function of apoE may provide potential therapeutic approaches for several neurological disorders. Here we show that progesterone and a synthetic progestin, lynestrenol, significantly induce apoE secretion from human CCF-STTG1 astrocytoma cells, whereas estrogens and the progesterone metabolite allopregnanolone have negligible effects. Intriguingly, lynestrenol also increases expression of the cholesterol transporter ABCA1 in CCF-STTG1 astrocytoma cells, primary murine glia, and immortalized murine astrocytes that express human apoE3. The progesterone receptor inhibitor RU486 attenuates the effect of progestins on apoE expression in CCF-STTG1 astrocytoma cells but has no effect on ABCA1 expression in all glial cell models tested, suggesting that the progesterone receptor (PR) may participate in apoE but does not affect ABCA1 regulation. These results suggest that selective reproductive steroid hormones have the potential to influence glial lipid homeostasis through liver X receptor-dependent and progesterone receptor-dependent pathways.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Apolipoproteínas E/metabolismo , Hormônios/farmacologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Apolipoproteína A-I/metabolismo , Apolipoproteína E3/metabolismo , Apolipoproteínas E/genética , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Colesterol/metabolismo , Estrogênios/farmacologia , Homeostase/efeitos dos fármacos , Humanos , Receptores X do Fígado , Linestrenol/farmacologia , Camundongos , Receptores Nucleares Órfãos/metabolismo , Progesterona/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Progesterona/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Regulação para Cima/efeitos dos fármacosRESUMO
ATP-binding cassette transporter A1 (ABCA1) mediates cellular cholesterol efflux in the brain and influences whole brain cholesterol homeostasis. Activation of liver X receptors (LXRs), transcription factors that increase the expression of cholesterol transport genes including ABCA1, reduces neuroinflammation and pathology in neurodegenerative animal models suggesting that in addition to its involvement in cholesterol transport, ABCA1 may play a role in modulating the inflammatory response in the brain. We investigated the cell-type specific role of ABCA1 in neuroinflammation in vivo using mice specifically lacking brain ABCA1 (ABCA1(-B/-B)) as well as mice lacking neuronal (ABCA1(-N/-N)) and astrocytic (ABCA1(-Ast/-Ast)) ABCA1. ABCA1(-B/-B) mice exhibit cortical astrogliosis, increased inflammatory gene expression as well as activation of mitogen-activated protein kinases (MAPKs) following acute lipopolysaccharide (LPS) administration. Microglia cultured from ABCA1(-B/-B) mice exhibit augmented LPS-induced secretion of tumor necrosis factor α (TNFα) and decreased phagocytic activity, indicating an increase in a pro-inflammatory response. ABCA1(-N/-N) mice develop astrogliosis but show no change in inflammatory gene expression. Intriguingly, ABCA1(-Ast/-Ast) mice show neither astrogliosis nor elevated expression of inflammatory markers. Cortical apolipoprotein E (apoE) levels are reduced in ABCA1(-Ast/-Ast) but not in ABCA1(-N/-N) mice, providing in vivo evidence for the specific role of astrocyte ABCA1 in regulating brain apoE levels. Interestingly, cortical neuronal death is increased in 17month-old ABCA1(-B/-B) mice but not in ABCA1(-N/-N) or ABCA1(-Ast/-Ast) mice. Our findings suggest that coordinated ABCA1 activity across neurons and glial cells influences neuroinflammation and neurodegeneration.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Encéfalo/metabolismo , Inflamação/metabolismo , Degeneração Neural/metabolismo , Neurônios/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/imunologia , Animais , Encéfalo/imunologia , Encéfalo/patologia , Morte Celular , Imunofluorescência , Immunoblotting , Imuno-Histoquímica , Inflamação/genética , Inflamação/imunologia , Camundongos , Camundongos Knockout , Degeneração Neural/genética , Degeneração Neural/imunologia , Neuroglia/imunologia , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Traumatic brain injury (TBI) increases Alzheimer's disease (AD) risk and leads to the deposition of neurofibrillary tangles and amyloid deposits similar to those found in AD. Agonists of Liver X receptors (LXRs), which regulate the expression of many genes involved in lipid homeostasis and inflammation, improve cognition and reduce neuropathology in AD mice. One pathway by which LXR agonists exert their beneficial effects is through ATP-binding cassette transporter A1 (ABCA1)-mediated lipid transport onto apolipoprotein E (apoE). To test the therapeutic utility of this pathway for TBI, we subjected male wild-type (WT) and apoE-/- mice to mild repetitive traumatic brain injury (mrTBI) followed by treatment with vehicle or the LXR agonist GW3965 at 15 mg/kg/day. GW3965 treatment restored impaired novel object recognition memory in WT but not apoE-/- mice. GW3965 did not significantly enhance the spontaneous recovery of motor deficits observed in all groups. Total soluble Aß(40) and Aß(42) levels were significantly elevated in WT and apoE-/- mice after injury, a response that was suppressed by GW3965 in both genotypes. WT mice showed mild but significant axonal damage at 2 d post-mrTBI, which was suppressed by GW3965. In contrast, apoE-/- mice showed severe axonal damage from 2 to 14 d after mrTBI that was unresponsive to GW3965. Because our mrTBI model does not produce significant inflammation, the beneficial effects of GW3965 we observed are unlikely to be related to reduced inflammation. Rather, our results suggest that both apoE-dependent and apoE-independent pathways contribute to the ability of GW3965 to promote recovery from mrTBI.
Assuntos
Apolipoproteínas E/metabolismo , Benzoatos/farmacologia , Benzilaminas/farmacologia , Lesões Encefálicas/fisiopatologia , Receptores Nucleares Órfãos/agonistas , Recuperação de Função Fisiológica/efeitos dos fármacos , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Cognição/efeitos dos fármacos , Citocinas/metabolismo , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Fragmentos de Peptídeos/metabolismoRESUMO
Lipid transport in the brain is coordinated by glial-derived lipoproteins that contain apolipoprotein E (apoE) as their primary protein. Here we show that apoE is secreted from wild-type (WT) primary murine mixed glia as nascent lipoprotein subspecies ranging from 7.5 to 17 nm in diameter. Negative-staining electron microscropy (EM) revealed rouleaux, suggesting a discoidal structure. Potassium bromide (KBr) density gradient ultracentrifugation showed that all subspecies, except an 8.1 nm particle, were lipidated. Glia lacking the cholesterol transporter ABCA1 secreted only 8.1 nm particles, which were poorly lipidated and nondiscoidal but could accept lipids to form the full repertoire of WT apoE particles. Receptor-associated-protein (RAP)-mediated inhibition of apoE receptor function blocked appearance of the 8.1 nm species, suggesting that this particle may arise through apoE recycling. Selective deletion of the LDL receptor (LDLR) reduced the level of 8.1 nm particle production by approximately 90%, suggesting that apoE is preferentially recycled through the LDLR. Finally, apoA-I stimulated secretion of 8.1 nm particles in a dose-dependent manner. These results suggest that nascent glial apoE lipoproteins are secreted through multiple pathways and that a greater understanding of these mechanisms may be relevant to several neurological disorders.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apolipoproteínas E/química , Apolipoproteínas E/metabolismo , Metabolismo dos Lipídeos , Neuroglia/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Apolipoproteínas E/genética , Encéfalo/citologia , Encéfalo/metabolismo , Células Cultivadas , Colesterol/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Neuroglia/química , Neuroglia/citologia , Tamanho da Partícula , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMO
Brain cholesterol, which is synthesized locally, is a major component of myelin and cell membranes and participates in neuronal functions, such as membrane trafficking, signal transduction, neurotransmitter release, and synaptogenesis. Here we show that brain cholesterol biosynthesis is reduced in multiple transgenic and knock-in Huntington's disease (HD) rodent models, arguably dependent on deficits in mutant astrocytes. Mice carrying a progressively increased number of CAG repeats show a more evident reduction in cholesterol biosynthesis. In postnatal life, the cholesterol-dependent activities of neurons mainly rely on the transport of cholesterol from astrocytes on ApoE-containing particles. Our data show that mRNA levels of cholesterol biosynthesis and efflux genes are severely reduced in primary HD astrocytes, along with impaired cellular production and secretion of ApoE. Consistently, in CSF of HD mice, ApoE is mostly associated with smaller lipoproteins, indicating reduced cholesterol transport on ApoE-containing lipoproteins circulating in the HD brain. These findings indicate that cholesterol defect is robustly marked in HD animals, implying that strategies aimed at selectively modulating brain cholesterol metabolism might be of therapeutic significance.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Colesterol/metabolismo , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Análise de Variância , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Apolipoproteínas E/líquido cefalorraquidiano , Encéfalo/patologia , Células Cultivadas , Colesterol/biossíntese , Modelos Animais de Doenças , Feminino , Doença de Huntington/líquido cefalorraquidiano , Doença de Huntington/genética , Masculino , Camundongos , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Ratos , Esteróis/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Sinaptossomos/metabolismo , Sinaptossomos/patologia , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
The cholesterol transporter ATP-binding cassette transporter A1 (ABCA1) moves lipids onto apolipoproteins including apolipoprotein E (apoE), which is the major cholesterol carrier in the brain and an established genetic risk factor for late-onset Alzheimer disease (AD). In amyloid mouse models of AD, ABCA1 deficiency exacerbates amyloidogenesis, whereas ABCA1 overexpression ameliorates amyloid load, suggesting a role for ABCA1 in Aß metabolism. Agonists of liver X receptors (LXR), including GW3965, induce transcription of several genes including ABCA1 and apoE, and reduce Aß levels and improve cognition in AD mice. However, the specific role of ABCA1 in mediating beneficial responses to LXR agonists in AD mice is unknown. We evaluated behavioral and neuropathogical outcomes in GW3965-treated female APP/PS1 mice with and without ABCA1. Treatment of APP/PS1 mice with GW3965 increased ABCA1 and apoE protein levels. ABCA1 was required to observe significantly elevated apoE levels in brain tissue and cerebrospinal fluid upon therapeutic (33 mg/kg/day) GW3965 treatment. At 33 mg/kg/day, GW3965 was also associated with a trend toward redistribution of Aß to the carbonate-soluble pool independent of ABCA1. APP/PS1 mice treated with either 2.5 or 33 mg/kg/day of GW3965 showed a clear trend toward reduced amyloid burden in hippocampus and whole brain, whereas APP/PS1-treated mice lacking ABCA1 failed to display reduced amyloid load in the whole brain and showed trends toward increased hippocampal amyloid. Treatment of APP/PS1 mice with either dose of GW3965 completely restored novel object recognition memory to wild-type levels, which required ABCA1. These results suggest that ABCA1 contributes to several beneficial effects of the LXR agonist GW3965 in APP/PS1 mice.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Amiloide/química , Benzoatos/farmacologia , Benzilaminas/farmacologia , Memória , Receptores Nucleares Órfãos/química , Reconhecimento Visual de Modelos , Presenilina-1/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Animais , Apolipoproteínas E/metabolismo , Feminino , Hipocampo/metabolismo , Humanos , Receptores X do Fígado , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Although apolipoprotein E (apoE) is the most common genetic risk factor for Alzheimer's Disease (AD), how apoE participates in AD pathogenesis remains incompletely understood. ApoE is also the major carrier of lipids in the brain. Here, we review studies showing that the lipidation status of apoE influences the metabolism of Abeta peptides, which accumulate as amyloid deposits in the neural parenchyma and cerebrovasculature. One effect of apoE is to inhibit the transport of Abeta across the blood-brain-barrier (BBB), particularly when apoE is lipidated. A second effect is to facilitate the proteolytic degradation of Abeta by neprilysin and insulin degrading enzyme (IDE), which is enhanced when apoE is lipidated. We also describe how apoE becomes lipidated and how this impacts Abeta metabolism. Specifically, genetic loss of the cholesterol transporter ABCA1 impairs apoE lipidation and promotes amyloid deposition in AD mouse models. ABCA1 catalyses the ATP-dependent transport of cholesterol and phospholipids from the plasma membrane to lipid-free apolipoproteins including apoE. Conversely, selective overexpression of ABCA1 increases apoE lipidation in the central nervous system (CNS) and eliminates the formation of amyloid plaques in vivo. Deficiency of Liver-X-Receptors (LXRs), transcription factors that stimulate ABCA1 and apoE expression, exacerbates AD pathogenesis in vivo, whereas treatment of AD mice with synthetic LXR agonists reduces amyloid load and improves cognitive performance. These studies provide new insights into the mechanisms by which apoE affects Abeta metabolism, and offer opportunities to develop novel therapeutic approaches to reduce the leading cause of dementia in the elderly.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/fisiologia , Apolipoproteínas E/metabolismo , Apolipoproteínas E/fisiologia , Lipídeos/fisiologia , Animais , Humanos , Metabolismo dos Lipídeos/fisiologia , Modelos BiológicosRESUMO
The expression of the cholesterol transporter ATP-binding cassette transporter A1 (ABCA1) in the brain and its role in the lipidation of apolipoproteins indicate that ABCA1 may play a critical role in brain cholesterol metabolism. To investigate the role of ABCA1 in brain cholesterol homeostasis and trafficking, we characterized mice that specifically lacked ABCA1 in the CNS, generated using the Cre/loxP recombination system. These mice showed reduced plasma high-density lipoprotein (HDL) cholesterol levels associated with decreased brain cholesterol content and enhanced brain uptake of esterified cholesterol from plasma HDL. Increased levels of HDL receptor SR-BI in brain capillaries and apolipoprotein A-I in brain and CSF of mutant mice were evident. Cholesterol homeostasis changes were mirrored by disturbances in motor activity and sensorimotor function. Changes in synaptic ultrastructure including reduced synapse and synaptic vesicle numbers were observed. These data show that ABCA1 is a key regulator of brain cholesterol metabolism and that disturbances in cholesterol transport in the CNS are associated with structural and functional deficits in neurons. Moreover, our findings also demonstrate that specific changes in brain cholesterol metabolism can lead to alterations in cholesterol uptake from plasma to brain.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Colesterol/metabolismo , Neurônios/fisiologia , Neurônios/ultraestrutura , Regulação para Cima/genética , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/fisiologia , Animais , Comportamento/fisiologia , Colesterol/biossíntese , Homeostase/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Sinapses/fisiologiaRESUMO
Lipid trafficking in the brain is essential for the maintenance and repair of neuronal membranes, especially after neurotoxic insults. However, brain lipid metabolism is not completely understood. In plasma, LCAT catalyses the esterification of free cholesterol on circulating lipoproteins, a key step in the maturation of HDL. Brain lipoproteins are apolipoprotein E (apoE)-containing, HDL-like particles secreted initially as lipid-poor discs by glial cells. LCAT is synthesized within the brain, suggesting that it may play a key role in the maturation of these lipoproteins. Here we demonstrate that astrocytes are the primary producers of brain LCAT. This LCAT esterifies free cholesterol on nascent apoE-containing lipopoproteins secreted from glia. ApoE is the major LCAT activator in glia-conditioned media (GCM), and both the cholesterol transporter ABCA1 and apoE are required to generate glial LCAT substrate particles. LCAT deficiency leads to the appearance of abnormal approximately 8 nm particles in GCM, and exogenous LCAT restores the lipoprotein particle distribution to the wild-type (WT) pattern. In vivo, complete LCAT deficiency results in a dramatic increase in apoE-HDL and reduced apolipoprotein A-I (apoA-I)-HDL in murine cerebrospinal fluid (CSF). These data show that brain LCAT esterifies cholesterol on glial-derived apoE-lipoproteins, and influences CSF apoE and apoA-I levels.
Assuntos
Astrócitos/metabolismo , Colesterol , Ésteres/química , Lipoproteínas/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apolipoproteínas E/metabolismo , Astrócitos/citologia , Células Cultivadas , Colesterol/química , Colesterol/metabolismo , Meios de Cultivo Condicionados/química , Humanos , Lipídeos/líquido cefalorraquidiano , Lipídeos/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Neurônios/metabolismoRESUMO
OBJECTIVE: The purpose of this study was to evaluate the effects of whole body overexpression of human ABCG1 on atherosclerosis in apoE(-/-) mice. METHODS AND RESULTS: We generated BAC transgenic mice in which human ABCG1 is expressed from endogenous regulatory signals, leading to a 3- to 7-fold increase in ABCG1 protein across various tissues. Although the ABCG1 BAC transgene rescued lung lipid accumulation in ABCG1(-/-) mice, it did not affect plasma lipid levels, macrophage cholesterol efflux to HDL, atherosclerotic lesion area in apoE(-/-) mice, or levels of tissue cholesterol, cholesterol ester, phospholipids, or triglycerides. Subtle changes in sterol biosynthetic intermediate levels were observed in liver, with chow-fed ABCG1 BAC Tg mice showing a nonsignificant trend toward decreased levels of lathosterol, lanosterol, and desmosterol, and fat-fed mice exhibiting significantly elevated levels of each intermediate. These changes were insufficient to alter ABCA1 expression in liver. CONCLUSIONS: Transgenic human ABCG1 does not influence atherosclerosis in apoE(-/-) mice but may participate in the regulation of tissue cholesterol biosynthesis.