Identification of Inhibitors of CD36-Amyloid Beta Binding as Potential Agents for Alzheimer's Disease.
ABSTRACT: Neuroinflammation is one of the hallmarks of Alzheimer's disease pathology. Amyloid ? has a central role in microglia activation and the subsequent secretion of inflammatory mediators that are associated with neuronal toxicity. The recognition of amyloid ? by microglia depends on the expression of several receptors implicated in the clearance of amyloid and in cell activation. CD36 receptor expressed on microglia interacts with fibrils of amyloid inducing the release of proinflammatory cytokines and amyloid internalization. The interruption of the interaction CD36-amyloid ? compromises the activation of microglia cells. We have developed and validated a new colorimetric assay to identify potential inhibitors of the binding of amyloid ? to CD36. We have found seven molecules, structural analogues of the Trichodermamide family of natural products that interfere with the interaction CD36-amyloid ?. By combining molecular docking and dynamics simulations, we suggested the second fatty acids binding site within the large luminal hydrophobic tunnel, present in the extracellular domain of CD36, as the binding pocket of these compounds. Free energy calculations predicted the nonpolar component as the driving force for the binding of these inhibitors. These molecules also inhibited the production of TNF-?, IL-6, and IL-1? by peritoneal macrophages stimulated with fibrils of amyloid ?. This work serves as a platform for the identification of new potential anti-inflammatory agents for the treatment of Alzheimer's disease.
Project description:Particulate ligands, including cholesterol crystals and amyloid fibrils, induce production of interleukin 1? (IL-1?) dependent on the cytoplasmic sensor NLRP3 in atherosclerosis, Alzheimer's disease and diabetes. Soluble endogenous ligands, including oxidized low-density lipoprotein (LDL), amyloid-? and amylin peptides, accumulate in such diseases. Here we identify an endocytic pathway mediated by the pattern-recognition receptor CD36 that coordinated the intracellular conversion of those soluble ligands into crystals or fibrils, which resulted in lysosomal disruption and activation of the NLRP3 inflammasome. Consequently, macrophages that lacked CD36 failed to elicit IL-1? production in response to those ligands, and targeting CD36 in atherosclerotic mice resulted in lower serum concentrations of IL-1? and accumulation of cholesterol crystals in plaques. Collectively, our findings highlight the importance of CD36 in the accrual and nucleation of NLRP3 ligands from within the macrophage and position CD36 as a central regulator of inflammasome activation in sterile inflammation.
Project description:TREM2 plays a critical role in the alleviation of Alzheimer's disease by promoting A? phagocytosis by microglia, but the detailed molecular mechanism underlying TREM2-induced direct phagocytic activity of A? remains to be revealed. We found that learning and memory functions were improved in aged TREM2 TG mice, with the opposite effects in KO mice. The amount of phagocytosed A? was significantly reduced in the primary microglia of KO mice. CD36 expression in primary microglia was greater in TG than in WT mice but was substantially decreased in KO mice. The expression of C/EBP?, an upstream transcriptional activator of CD36, was also elevated in primary microglia of TG mice but decreased in KO mice. The transcription of CD36 was markedly increased by TREM2 overexpression, and this effect was suppressed by a mutation of the C/EBP? binding site on the CD36 promoter. The TREM2-induced expression of CD36 and C/EBP? was inhibited by treatment with PI3K/AKT signaling blockers, and phosphorylation of AKT was elevated in TREM2-overexpressing BV2 cells. The present study provides evidence that TREM2 is required for preventing loss of memory and learning in Alzheimer's disease by regulating C/EBP?-dependent CD36 expression and the consequent A? phagocytosis.
Project description:Oculoleptomeningeal amyloidosis (OA) is a fatal and untreatable hereditary disease characterized by the accumulation of transthyretin (TTR) amyloid within the central nervous system. The mechanisms underlying the pathogenesis of OA, and in particular how amyloid triggers neuronal damage, are still unknown. Here, we show that amyloid fibrils formed by a mutant form of TTR, A25T, activate microglia, leading to the secretion of tumor necrosis factor-? (TNF-?), interleukin-6 (IL-6) and nitric oxide. Further, we found that A25T amyloid fibrils induce the activation of Akt, culminating in the translocation of NF?B to the nucleus of microglia. While A25T fibrils were not directly toxic to neurons, the exposure of neuronal cultures to media conditioned by fibril-activated microglia caused synapse loss that culminated in extensive neuronal death via apoptosis. Finally, intracerebroventricular (i.c.v.) injection of A25T fibrils caused microgliosis, increased brain TNF-? and IL-6 levels and cognitive deficits in mice, which could be prevented by minocycline treatment. These results indicate that A25T fibrils act as pro-inflammatory agents in OA, activating microglia and causing neuronal damage.
Project description:In atherosclerosis and Alzheimer's disease, deposition of the altered self components oxidized low-density lipoprotein (LDL) and amyloid-beta triggers a protracted sterile inflammatory response. Although chronic stimulation of the innate immune system is believed to underlie the pathology of these diseases, the molecular mechanisms of activation remain unclear. Here we show that oxidized LDL and amyloid-beta trigger inflammatory signaling through a heterodimer of Toll-like receptors 4 and 6. Assembly of this newly identified heterodimer is regulated by signals from the scavenger receptor CD36, a common receptor for these disparate ligands. Our results identify CD36-TLR4-TLR6 activation as a common molecular mechanism by which atherogenic lipids and amyloid-beta stimulate sterile inflammation and suggest a new model of TLR heterodimerization triggered by coreceptor signaling events.
Project description:Deposition of amyloid-? (A?) in cerebral arteries, known as cerebral amyloid angiopathy (CAA), occurs both in the setting of Alzheimer's disease and independent of it, and can cause cerebrovascular insufficiency and cognitive deficits. The mechanisms leading to CAA have not been established, and no therapeutic targets have been identified. We investigated the role of CD36, an innate immunity receptor involved in A? trafficking, in the neurovascular dysfunction, cognitive deficits, and amyloid accumulation that occurs in mice expressing the Swedish mutation of the amyloid precursor protein (Tg2576). We found that Tg2576 mice lacking CD36 have a selective reduction in A?1-40 and CAA. This reduced vascular amyloid deposition was associated with preservation of the A? vascular clearance receptor LRP-1, and protection from the deleterious effects of A? on cerebral arterioles. These beneficial vascular effects were reflected by marked improvements in neurovascular regulation and cognitive performance. Our data suggest that CD36 promotes vascular amyloid deposition and the resulting cerebrovascular damage, leading to neurovascular dysfunction and cognitive deficits. These findings identify a previously unrecognized role of CD36 in the mechanisms of vascular amyloid deposition, and suggest that this scavenger receptor is a putative therapeutic target for CAA and related conditions.
Project description:There is extensive evidence that oxidative stress induces cellular dysfunction in the brain and plays a critical role in Alzheimer's disease (AD) pathogenesis. Hypoxia increases factors involved in oxidative stress injury and contributes to the onset and progression of AD. Nuclear factor erythroid 2-related factor 2 (NRF2), a major component regulating antioxidant response, is attenuated in the AD brain. Importantly, NRF2 directly regulates the alternative first exons of CD36, an important participant in oxidative and inflammatory processes. To explore the effects of hypoxia-induced deterioration of AD-like pathogenesis and investigate the correlation between hypoxia-induced NRF2 signal alterations and CD36 expression, we examined the NRF2 signaling, CD36, and oxidative stress events in hypoxia-treated APPswe/PSEN1dE9 (APP/PS1) mice brain.We observed that hypoxia treatment increased oxidative stress, exacerbated inflammation, and aggravated learning defects in aged APP/PS1 mice. Microglia from hypoxia-treated mice brain exhibited marked reduction in CD36 expression and inhibition of ?-amyloid (A?) degradation. Accordingly, hypoxia treatment caused a decrease in transactivation of NRF2 target genes in the aging mouse brain. Intranasal administration with a lentiviral vector encoding human NRF2 increased CD36 expression, ameliorated the weak antioxidant response triggered by hypoxia, diminished A? deposition, and improved spatial memory defects.In this study, we demonstrated for the first time that NRF2 intranasal treatment-induced increases of CD36 could enhance A? clearance in AD transgenic mouse.These results suggest that targeting NRF2-mediated CD36 expression might provide a beneficial intervention for cognitive impairment and oxidative stress in AD progression.
Project description:A pathological hallmark of Alzheimer disease (AD) is deposition of amyloid ? (A?) in the brain. A? binds to microglia via a receptor complex that includes CD36 leading to production of proinflammatory cytokines and neurotoxic reactive oxygen species and subsequent neurodegeneration. Interruption of A? binding to CD36 is a potential therapeutic strategy for AD. To identify pharmacologic inhibitors of A? binding to CD36, we developed a 384-well plate assay for binding of fluorescently labeled A? to Chinese hamster ovary cells stably expressing human CD36 (CHO-CD36) and screened an Food and Drug Administration-approved compound library. The assay was optimized based on the cells' tolerance to dimethyl sulfoxide, A? concentration, time required for A? binding, reproducibility, and signal-to-background ratio. Using this assay, we identified four compounds as potential inhibitors of A? binding to CD36. These compounds were ursolic acid, ellipticine, zoxazolamine, and homomoschatoline. Of these compounds, only ursolic acid, a naturally occurring pentacyclic triterpenoid, successfully inhibited binding of A? to CHO-CD36 cells in a dose-dependent manner. The ursolic acid effect reached a plateau at ~20 ?m, with a maximal inhibition of 64%. Ursolic acid also blocked binding of A? to microglial cells and subsequent ROS production. Our data indicate that cell-based high-content screening of small molecule libraries for their ability to block binding of A? to its receptors is a useful tool to identify novel inhibitors of receptors involved in AD pathogenesis. Our data also suggest that ursolic acid is a potential therapeutic agent for AD via its ability to block A?-CD36 interactions.
Project description:OBJECTIVE:The scavenger receptor CD36 is injurious in acute experimental focal stroke and neurodegenerative diseases in the adult. We investigated the effects of genetic deletion of CD36 (CD36ko) on acute injury, and oxidative and inflammatory signaling after neonatal stroke. METHODS:Postnatal day 9 CD36ko and wild-type (WT) mice were subjected to a transient middle cerebral artery occlusion (MCAO). Injury, phagocytosis of dying cells, and CD36 inflammatory signaling were determined. RESULTS:While the volume of tissue at risk by diffusion-weighted magnetic resonance imaging during MCAO was similar in neonatal CD36ko and WT mice, by 24 hours after reperfusion, injury was more severe in CD36ko and was associated with increased caspase-3 cleavage and reduced engulfment of neurons expressing cleaved caspase-3 by activated microglia. No significant superoxide generation was observed in activated microglia in injured WT, whereas increased superoxide production in vessels and nuclear factor (NF)-?B activation induced by MCAO were unaffected by lack of CD36. Lyn expression was higher in injured CD36ko, and cell type-specific patterns of Lyn expression were altered; Lyn was expressed in endothelial cells and microglia in WT but predominantly in dying neurons in CD36ko. INTERPRETATION:Lack of CD36 results in poorer short-term outcome from neonatal focal stroke due to lack of attenuation of NF-?B-mediated inflammation and diminished removal of apoptotic neuronal debris. Although inhibition of CD36 does not seem to be a good therapeutic target for protection after acute neonatal stroke, as it is after adult stroke, seeking better understanding of CD36 signaling in particular cell populations may reveal important therapeutic targets for neonatal stroke.
Project description:Haplodeficiency of the microglia gene TREM2 increases risk for late-onset Alzheimer's disease (AD) but the mechanisms remain uncertain. To investigate this, we used high-resolution confocal and super-resolution (STORM) microscopy in AD-like mice and human AD tissue. We found that microglia processes, rich in TREM2, tightly surround early amyloid fibrils and plaques promoting their compaction and insulation. In Trem2- or DAP12-haplodeficient mice and in humans with R47H TREM2 mutations, microglia had a markedly reduced ability to envelop amyloid deposits. This led to an increase in less compact plaques with longer and branched amyloid fibrils resulting in greater surface exposure to adjacent neurites. This was associated with more severe neuritic tau hyperphosphorylation and axonal dystrophy around amyloid deposits. Thus, TREM2 deficiency may disrupt the formation of a neuroprotective microglia barrier that regulates amyloid compaction and insulation. Pharmacological modulation of this barrier could be a novel therapeutic strategy for AD.
Project description:Alzheimer's disease is characterized by progressive accumulation of ?-amyloid (A?)-containing amyloid plaques, and microglia play a critical role in internalization and degradation of A?. Our previous research confirmed that Nogo-66 binding to Nogo receptors (NgR) expressed on microglia inhibits cell adhesion and migration in vitro.The adhesion and migration of microglia isolated from WT and APP/PS1 mice from different ages were measured by adhesion assays and transwells. After NEP1-40 (a competitive antagonist of Nogo/NgR pathway) was intracerebroventricularly administered via mini-osmotic pumps for 2 months in APP/PS1 transgenic mice, microglial recruitment toward A? deposits and CD36 expression were determined.In this paper, we found that aging led to a reduction of microglia adhesion and migration to fA?1-42 in WT and APP/PS1 mice. The adhesion and migration of microglia to fA?1-42 were downregulated by the Nogo, which was mediated by NgR, and the increased inhibitory effects of the Nogo could be observed in aged mice. Moreover, Rho GTPases contributed to the effects of the Nogo on adhesion and migration of microglia to fA?1-42 by regulating cytoskeleton arrangement. Furthermore, blocking the Nogo/NgR pathway enhanced recruitment of microglia toward A? deposits and expression of CD36 in APP/PS1 mice.Taken together, Nogo/NgR pathway could take part in A? pathology in AD by modulating microglial adhesion and migration to A? and the Nogo/NgR pathway might be an important target for treating AD.