Abstract Background We have proposed that cognitive resilience (CR) counteracts brain damage from Alzheimer’s disease (AD) or AD-related dementias such that older individuals who harbor neurodegenerative disease burden sufficient to cause dementia remain cognitively normal. However, CR traditionally is considered a binary trait, capturing only the most extreme examples, and is often inconsistently defined. Methods This study addressed existing discrepancies and shortcomings of the current CR definition by proposing a framework for defining CR as a continuous variable for each neuropsychological test. The linear equations clarified CR’s relationship to closely related terms, including cognitive function, reserve, compensation, and damage. Primarily, resilience is defined as a function of cognitive performance and damage from neuropathologic damage. As such, the study utilized data from 844 individuals (age = 79 ± 12, 44% female) in the National Alzheimer’s Coordinating Center cohort that met our inclusion criteria of comprehensive lesion rankings for 17 neuropathologic features and complete neuropsychological test results. Machine learning models and GWAS then were used to identify medical and genetic factors that are associated with CR. Results CR varied across five cognitive assessments and was greater in female participants, associated with longer survival, and weakly associated with educational attainment or APOE ε4 allele. In contrast, damage was strongly associated with APOE ε4 allele ( P value < 0.0001). Major predictors of CR were cardiovascular health and social interactions, as well as the absence of behavioral symptoms. Conclusions Our framework explicitly decoupled the effects of CR from neuropathologic damage. Characterizations and genetic association study of these two components suggest that the underlying CR mechanism has minimal overlap with the disease mechanism. Moreover, the identified medical features associated with CR suggest modifiable features to counteract clinical expression of damage and maintain cognitive function in older individuals.
Studying proteomics data of the human brain could offer numerous insights into unraveling the signature of resilience to Alzheimer’s disease. In our previous study with rigorous cohort selection criteria that excluded 4 common comorbidities, we harnessed multiple brain regions from 43 research participants with 12 of them displaying cognitive resilience to Alzheimer’s disease. Based on the previous findings, this work focuses on 6 proteins out of the 33 differentially expressed proteins associated with resilience to Alzheimer’s disease. These proteins are used to construct a decision tree classifier, enabling the differentiation of 3 groups: (i) healthy control, (ii) resilience to Alzheimer’s disease, and (iii) Alzheimer’s disease with dementia. Our analysis unveiled 2 important regional proteomic markers: Aβ peptides in the hippocampus and PA1B3 in the inferior parietal lobule. These findings underscore the potential of using distinct regional proteomic markers as signatures in characterizing the resilience to Alzheimer’s disease.
Epidemiologic and animal model data support a role for the prostaglandin pathway in AD pathogenesis. However, unexpected toxicity from protracted use of some nonsteroidal anti-inflammatory drugs (NSAIDs) compels investigation of therapeutic targets in this pathway other than COX inhibitors. Previously, we have shown that mice lacking one specific receptor for PGE2, EP2 (EP2-/-), are protected from the indirect neurotoxic effects of cerebral innate immune response mediated by CD14-dependent activation. Here we review data showing that EP2-/- microglia have a highly desirable combination of features: ablated indirect neurotoxicity following exposure to Abeta(1-42) coupled with enhanced phagocytosis of Abeta peptides, both synthetic and those deposited in human brain. These data point to microglial EP2 as a more focused target within the PG pathway for therapy in AD.
Dementia with Lewy bodies (DLB) is the second most common form of dementia and shows more severely impaired performance on tests of executive functions compared to Alzheimer disease. Here the authors demonstrate selective spinodendritic degeneration of medium spiny neurons in regions of the caudate nucleus that subserve executive functions and propose that this may underlie, at least in part, the heightened executive dysfunction observed in patients with DLB.
Parkinson disease (PD) is an already prevalent neurodegenerative disease that is poised to at least double over the next 25 years. Although best known for its characteristic movement disorder, PD is now appreciated commonly to cause cognitive impairment, including dementia, and behavioral changes. Dementia in patients with PD is consequential and has been associated with reduced quality of life, shortened survival, and increased caregiver distress. Here we review clinical presentation and progression, pathological bases, identification of genetic risk factors, development of small molecule biomarkers, and emerging treatments for cognitive impairment in patients with PD.
Recent studies underline the potential relevance of microglial innate immune activation in Alzheimer disease. Primary mouse microglia that lack prostaglandin E2 receptor subtype 2 (EP2) show decreased innate immune-mediated neurotoxicity and increased amyloid β (Aβ) peptide phagocytosis, features that were replicated in vivo. Here, we tested the hypothesis that scavenger receptor CD36 is an effector of EP2-regulated Aβ phagocytosis. CD36 expression was 143-fold greater in mouse primary microglia than in primary astrocytes. Three different means of suppressing EP2 signaling increased and an agonist of EP2 decreased CD36 expression in primary wild-type microglia. Activation of Toll-like receptor (TLR) 3, TLR4, and TLR7, but not TLR2 or TLR9, reduced primary microglial CD36 transcription and cell surface CD36 protein and reduced Aβ42 phagocytosis as well. At each step, the effects of innate immune activation on CD36 were reversed by at least 50% by an EP2 antagonist, and this partial rescue of microglia Aβ42 phagocytosis was largely mediated by CD36 activity. Finally, we showed in hippocampus of wild-type mice that innate immune activation suppressed CD36 expression by an EP2-dependent mechanism. Taken together with results of others that found brain clearance of Aβ peptides and behavioral improvements mediated by CD36 in mice, regulation of CD36-mediated Aβ phagocytosis by suppression of EP2 signaling may provide a new approach to suppressing some aspects of Alzheimer disease pathogenesis. Recent studies underline the potential relevance of microglial innate immune activation in Alzheimer disease. Primary mouse microglia that lack prostaglandin E2 receptor subtype 2 (EP2) show decreased innate immune-mediated neurotoxicity and increased amyloid β (Aβ) peptide phagocytosis, features that were replicated in vivo. Here, we tested the hypothesis that scavenger receptor CD36 is an effector of EP2-regulated Aβ phagocytosis. CD36 expression was 143-fold greater in mouse primary microglia than in primary astrocytes. Three different means of suppressing EP2 signaling increased and an agonist of EP2 decreased CD36 expression in primary wild-type microglia. Activation of Toll-like receptor (TLR) 3, TLR4, and TLR7, but not TLR2 or TLR9, reduced primary microglial CD36 transcription and cell surface CD36 protein and reduced Aβ42 phagocytosis as well. At each step, the effects of innate immune activation on CD36 were reversed by at least 50% by an EP2 antagonist, and this partial rescue of microglia Aβ42 phagocytosis was largely mediated by CD36 activity. Finally, we showed in hippocampus of wild-type mice that innate immune activation suppressed CD36 expression by an EP2-dependent mechanism. Taken together with results of others that found brain clearance of Aβ peptides and behavioral improvements mediated by CD36 in mice, regulation of CD36-mediated Aβ phagocytosis by suppression of EP2 signaling may provide a new approach to suppressing some aspects of Alzheimer disease pathogenesis. Observational studies in patients and experimental studies that use a variety of model systems have concluded that affected regions of the brain in Alzheimer disease (AD) experience a proinflammatory, pro-oxidative state.1Galasko D. Montine T.J. Biomarkers of oxidative damage and inflammation in Alzheimer's disease.Biomark Med. 2010; 4: 27-36Crossref PubMed Scopus (165) Google Scholar Indeed, recent studies have highlighted several genes involved in the innate immune response whose variants are associated with increased risk of AD, especially the TREM2 gene.2Lambert J.C. Ibrahim-Verbaas C.A. Harold D. Naj A.C. Sims R. 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Variant of TREM2 associated with the risk of Alzheimer's disease.N Engl J Med. 2013; 368: 107-116Crossref PubMed Scopus (1558) Google Scholar TREM2 encodes the triggering receptor expressed on myeloid cells-2, a protein expressed by myeloid lineage cells, including microglia in brain, which can function as a receptor for phagocytosis,5Hsieh C.L. Koike M. Spusta S.C. Niemi E.C. Yenari M. Nakamura M.C. Seaman W.E. A role for TREM2 ligands in the phagocytosis of apoptotic neuronal cells by microglia.J Neurochem. 2009; 109: 1144-1156Crossref PubMed Scopus (290) Google Scholar modulate the innate immune response in brain,6Forabosco P. Ramasamy A. Trabzuni D. Walker R. Smith C. Bras J. Levine A.P. Hardy J. Pocock J.M. Guerreiro R. Weale M.E. Ryten M. Insights into TREM2 biology by network analysis of human brain gene expression data.Neurobiol Aging. 2013; 34: 2699-2714Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 7Sieber M.W. Jaenisch N. Brehm M. Guenther M. 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TREM-2 promotes host resistance against Pseudomonas aeruginosa infection by suppressing corneal inflammation via a PI3K/Akt signaling pathway.Invest Ophthalmol Vis Sci. 2013; 54: 3451-3462Crossref PubMed Scopus (47) Google Scholar The prostaglandin (PG) pathway, a cascade initiated by enzyme-catalyzed synthesis of PGH2 from arachidonic acid by the cyclooxygenases (COXs), COX-1 or COX-2, is an essential component of the innate immune response. Multiple studies have reproducibly shown that nonselective COX or COX-2–selective inhibition by nonsteroidal anti-inflammatory drugs (NSAIDs) can suppress amyloid β (Aβ) accumulation and associated behavioral changes in transgenic mouse models of AD10Lim G.P. Yang F. Chu T. Chen P. Beech W. Teter B. Tran T. Ubeda O. Ashe K.H. Frautschy S.A. Cole G.M. Ibuprofen suppresses plaque pathology and inflammation in a mouse model for Alzheimer's disease.J Neurosci. 2000; 20: 5709-5714Crossref PubMed Google Scholar and can reduce the risk of AD dementia in people by approximately 60%.11Szekely C.A. Zandi P.P. Non-steroidal anti-inflammatory drugs and Alzheimer's disease: the epidemiological evidence.CNS Neurol Disord Drug Targets. 2010; 9: 132-139Crossref PubMed Scopus (93) Google Scholar Together these results suggest that NSAIDs may effectively suppress some aspect of what is now appreciated to be early steps in AD pathogenesis.12Sperling R.A. Aisen P.S. Beckett L.A. Bennett D.A. Craft S. Fagan A.M. Iwatsubo T. Jack Jr., C.R. Kaye J. Montine T.J. Park D.C. Reiman E.M. Rowe C.C. Siemers E. Stern Y. Yaffe K. Carrillo M.C. Thies B. Morrison-Bogorad M. Wagster M.V. Phelps C.H. Toward defining the preclinical stages of Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease.Alzheimers Dement. 2011; 7: 280-292Abstract Full Text Full Text PDF PubMed Scopus (4406) Google Scholar These compelling experimental model and human data formed the rationale for multiple clinical trials. Two clinical trials targeted what is now understood to be relatively advanced AD: one with NSAIDs in AD dementia and one with NSAIDs in mild cognitive impairment, a diagnostic group enriched for prodromal AD; both failed.13Aisen P.S. Schafer K.A. Grundman M. Pfeiffer E. Sano M. Davis K.L. Farlow M.R. Jin S. Thomas R.G. Thal L.J. Alzheimer's Disease Cooperative StudyEffects of rofecoxib or naproxen vs placebo on Alzheimer disease progression: a randomized controlled trial.JAMA. 2003; 289: 2819-2826Crossref PubMed Scopus (876) Google Scholar, 14Thal L.J. Ferris S.H. Kirby L. Block G.A. Lines C.R. Yuen E. Assaid C. Nessly M.L. Norman B.A. Baranak C.C. Reines S.A. Rofecoxib Protocol 078 study groupA randomized, double-blind, study of rofecoxib in patients with mild cognitive impairment.Neuropsychopharmacology. 2005; 30: 1204-1215Crossref PubMed Scopus (341) Google Scholar A third trial targeted early-stage AD,15Martin B.K. Szekely C. Brandt J. Piantadosi S. Breitner J.C. Craft S. Evans D. Green R. Mullan M. ADAPT Research GroupCognitive function over time in the Alzheimer's Disease Anti-inflammatory Prevention Trial (ADAPT): results of a randomized, controlled trial of naproxen and celecoxib.Arch Neurol. 2008; 65: 896-905Crossref PubMed Scopus (305) Google Scholar similar to the epidemiologic cohorts, but was terminated because of toxicity, likely related to perturbation of prostacyclin and thromboxane metabolism.16Hoozemans J.J. Veerhuis R. Rozemuller J.M. Eikelenboom P. Soothing the inflamed brain: effect of non-steroidal anti-inflammatory drugs on Alzheimer's disease pathology.CNS Neurol Disord Drug Targets. 2011; 10: 57-67Crossref PubMed Scopus (36) Google Scholar, 17Jaturapatporn D. Isaac M.G. McCleery J. Tabet N. Aspirin, steroidal and non-steroidal anti-inflammatory drugs for the treatment of Alzheimer's disease.Cochrane Database Syst Rev. 2012; 2: Cd006378PubMed Google Scholar, 18Montine T.J. Sonnen J.A. Milne G. Baker L.D. Breitner J.C. Elevated ratio of urinary metabolites of thromboxane and prostacyclin is associated with adverse cardiovascular events in ADAPT.PLoS One. 2010; 5: e9340Crossref PubMed Scopus (15) Google Scholar Despite these disappointing therapeutic outcomes, some data suggest pharmacodynamic effects of NSAIDs in the central nervous system of patients with AD.19Babiloni C. Frisoni G.B. Del Percio C. Zanetti O. Bonomini C. Cassetta E. Pasqualetti P. Miniussi C. De Rosas M. Valenzano A. Cibelli G. Eusebi F. Rossini P.M. Ibuprofen treatment modifies cortical sources of EEG rhythms in mild Alzheimer's disease.Clin Neurophysiol. 2009; 120: 709-718Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 20Breitner J.C. Baker L.D. Montine T.J. Meinert C.L. Lyketsos C.G. Ashe K.H. Brandt J. Craft S. Evans D.E. Green R.C. Ismail M.S. Martin B.K. Mullan M.J. Sabbagh M. Tariot P.N. ADAPT Research GroupExtended results of the Alzheimer's disease anti-inflammatory prevention trial.Alzheimers Dement. 2011; 7: 402-411Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar We and others have sought to disentangle the potentially beneficial effects of NSAIDs in early AD pathogenesis from toxic effects by focusing on specific PG receptors distal in the signaling cascade rather than proximal COX inhibition.21Cimino P.J. Keene C.D. Breyer R.M. Montine K.S. Montine T.J. Therapeutic targets in prostaglandin E2 signaling for neurologic disease.Curr Med Chem. 2008; 15: 1863-1869Crossref PubMed Scopus (85) Google Scholar, 22Shi J. Wang Q. Johansson J.U. Liang X. Woodling N.S. Priyam P. Loui T.M. Merchant M. Breyer R.M. Montine T.J. Andreasson K. Inflammatory prostaglandin E2 signaling in a mouse model of Alzheimer disease.Ann Neurol. 2012; 72: 788-798Crossref PubMed Scopus (77) Google Scholar, 23Andreasson K. Emerging roles of PGE2 receptors in models of neurological disease.Prostaglandins Other Lipid Mediat. 2010; 91: 104-112Crossref PubMed Scopus (148) Google Scholar PGE2 is the principal proinflammatory PG, and increased PGE2 concentrations in cerebrospinal fluid have been found in patients with AD,24Montine T.J. Sidell K.R. Crews B.C. Markesbery W.R. Marnett L.J. Roberts 2nd, L.J. Morrow J.D. Elevated CSF prostaglandin E2 levels in patients with probable AD.Neurology. 1999; 53: 1495-1498Crossref PubMed Google Scholar particularly early in the disease process.25Combrinck M. Williams J. De Berardinis M.A. Warden D. Puopolo M. Smith A.D. Minghetti L. Levels of CSF prostaglandin E2, cognitive decline, and survival in Alzheimer's disease.J Neurol Neurosurg Psychiatry. 2006; 77: 85-88Crossref PubMed Scopus (78) Google Scholar In addition, the inducible form of PGE2 synthase is increased in AD brain and is a key component in Aβ-mediated neurotoxicity in a mouse model of AD.26Akitake Y. Nakatani Y. Kamei D. Hosokawa M. Akatsu H. Uematsu S. Akira S. Kudo I. Hara S. Takahashi M. Microsomal prostaglandin E synthase-1 is induced in alzheimer's disease and its deletion mitigates alzheimer's disease-like pathology in a mouse model.J Neurosci Res. 2013; 91: 909-919Crossref PubMed Scopus (39) Google Scholar We, therefore, have focused on PGE2 and its functionally antagonistic receptor subtypes, EP1 to EP4, which are widely expressed in central nervous system cells, including microglia, astrocytes, and neurons.21Cimino P.J. Keene C.D. Breyer R.M. Montine K.S. Montine T.J. Therapeutic targets in prostaglandin E2 signaling for neurologic disease.Curr Med Chem. 2008; 15: 1863-1869Crossref PubMed Scopus (85) Google Scholar Primary microglia from mice homozygous deficient for the EP2 gene (PTGER2; EP2−/−) have the highly desirable dual phenotype of suppressed TLR-activated microglia-mediated neurotoxicity that depends on the adaptor protein DOCK2 and enhanced microglial phagocytosis of Aβ that is DOCK2 independent.27Shie F.S. Montine K.S. Breyer R.M. Montine T.J. Microglial EP2 is critical to neurotoxicity from activated cerebral innate immunity.Glia. 2005; 52: 70-77Crossref PubMed Scopus (100) Google Scholar, 28Shie F.S. Breyer R.M. Montine T.J. Microglia lacking E Prostanoid Receptor subtype 2 have enhanced Abeta phagocytosis yet lack Abeta-activated neurotoxicity.Am J Pathol. 2005; 166: 1163-1172Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 29Cimino P.J. Sokal I. Leverenz J. Fukui Y. Montine T.J. DOCK2 is a microglial specific regulator of central nervous system innate immunity found in normal and Alzheimer's disease brain.Am J Pathol. 2009; 175: 1622-1630Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar These studies show that microglial EP2 is necessary for lipopolysaccharide-induced microglia-mediated neurotoxicity in microglia–neuron cocultures through regulation of inducible nitric oxide synthase and COX-2 and subsequent neurotoxic cytokine production, including IL-1β production,27Shie F.S. Montine K.S. Breyer R.M. Montine T.J. Microglial EP2 is critical to neurotoxicity from activated cerebral innate immunity.Glia. 2005; 52: 70-77Crossref PubMed Scopus (100) Google Scholar and indicate EP2-dependent neurotoxicity in EP2−/− microglia, with reduced paracrine neurotoxicity in response to aggregated Aβ in neuron–microglia cocultures.28Shie F.S. Breyer R.M. Montine T.J. Microglia lacking E Prostanoid Receptor subtype 2 have enhanced Abeta phagocytosis yet lack Abeta-activated neurotoxicity.Am J Pathol. 2005; 166: 1163-1172Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar Recently, these findings were confirmed in mice with conditional deletion of EP2 in myeloid lineage cells and in bone marrow transplant experiments.30Keene C.D. Chang R.C. Lopez-Yglesias A.H. Shalloway B.R. Sokal I. Li X. Reed P.J. Keene L.M. Montine K.S. Breyer R.M. Rockhill J.K. Montine T.J. Suppressed accumulation of cerebral amyloid {beta} peptides in aged transgenic Alzheimer's disease mice by transplantation with wild-type or prostaglandin E2 receptor subtype 2-null bone marrow.Am J Pathol. 2010; 177: 346-354Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 31Johansson J.U. Pradhan S. Lokteva L.A. Woodling N.S. Ko N. Brown H.D. Wang Q. Loh C. Cekanaviciute E. Buckwalter M. Manning-Bog A.B. Andreasson K.I. Suppression of inflammation with conditional deletion of the prostaglandin E2 EP2 receptor in macrophages and brain microglia.J Neurosci. 2013; 33: 16016-16032Crossref PubMed Scopus (63) Google Scholar Microglial phagocytosis is mediated largely by a class of surface proteins called scavenger receptors. CD36, a type B scavenger receptor originally identified as a receptor for long-chain fatty acids and oxidized low-density lipoprotein, is a microglial binding site for Aβ,32Silverstein R.L. Febbraio M. CD36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior.Sci Signal. 2009; 2: re3Crossref PubMed Scopus (662) Google Scholar facilitates intracellular nucleation of Aβ peptides into fibrils,33Sheedy F.J. Grebe A. Rayner K.J. Kalantari P. Ramkhelawon B. Carpenter S.B. Becker C.E. Ediriweera H.N. Mullick A.E. Golenbock D.T. Stuart L.M. Latz E. Fitzgerald K.A. Moore K.J. CD36 coordinates NLRP3 inflammasome activation by facilitating intracellular nucleation of soluble ligands into particulate ligands in sterile inflammation.Nat Immunol. 2013; 14: 812-820Crossref PubMed Scopus (579) Google Scholar and mediates some of the biological effects of Aβ peptides, including innate immune activation and oxidative stress.34Coraci I.S. Husemann J. Berman J.W. Hulette C. Dufour J.H. Campanella G.K. Luster A.D. Silverstein S.C. El-Khoury J.B. CD36, a class B scavenger receptor, is expressed on microglia in Alzheimer's disease brains and can mediate production of reactive oxygen species in response to beta-amyloid fibrils.Am J Pathol. 2002; 160: 101-112Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 35Park L. Wang G. Zhou P. Zhou J. Pitstick R. Previti M.L. Younkin L. Younkin S.G. Van Nostrand W.E. Cho S. Anrather J. Carlson G.A. Iadecola C. Scavenger receptor CD36 is essential for the cerebrovascular oxidative stress and neurovascular dysfunction induced by amyloid-beta.Proc Natl Acad Sci U S A. 2011; 108: 5063-5068Crossref PubMed Scopus (111) Google Scholar, 36El Khoury J.B. Moore K.J. Means T.K. Leung J. Terada K. Toft M. Freeman M.W. Luster A.D. CD36 mediates the innate host response to beta-amyloid.J Exp Med. 2003; 197: 1657-1666Crossref PubMed Scopus (368) Google Scholar CD36 is expressed on microglia in AD brains.34Coraci I.S. Husemann J. Berman J.W. Hulette C. Dufour J.H. Campanella G.K. Luster A.D. Silverstein S.C. El-Khoury J.B. CD36, a class B scavenger receptor, is expressed on microglia in Alzheimer's disease brains and can mediate production of reactive oxygen species in response to beta-amyloid fibrils.Am J Pathol. 2002; 160: 101-112Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar CD36 overexpression in human brain correlates with β-amyloid deposition but not with AD; it is undetectable in healthy brains without Aβ deposition.37Ricciarelli R. D'Abramo C. Zingg J.M. Giliberto L. Markesbery W. Azzi A. Marinari U.M. Pronzato M.A. Tabaton M. CD36 overexpression in human brain correlates with beta-amyloid deposition but not with Alzheimer's disease.Free Radic Biol Med. 2004; 36: 1018-1024Crossref PubMed Scopus (47) Google Scholar We are unaware of any data to link the specific PG receptors with CD36 function. Here, we tested the hypothesis that EP2 suppression of Aβ phagocytosis is mediated, at least in part, by CD36. Dulbecco's modified Eagle's medium/F-12 medium and fetal bovine serum were purchased from Hyclone Laboratories (Logan, UT). Papain and DNase I were from Worthington Biochemical (Lakewood, NJ). Butaprost, NS-398, 17-phenyl trinor PGE2, CAY10598, ZK118182, 17-phenyl trinor PGF2α, carbaprostacyclin, U-46619, AH 6809, and CD36 blocking antibody (Clone JC63.1) were from Cayman Chemical Company (Ann Arbor, MI). Dibutyryl cAMP, forskolin, tribromoethanol (Avertin), and polyinosinic-polycytidylic acid (PIC; TLR3 ligand) were from Sigma-Aldrich (St. Louis, MO). Pam3CSK4 (TLR2 ligand), loxoribine (TLR7 ligand), and CpG (TLR9 ligand) were from Invivogen (San Diego, CA). Lipopolysaccharide (TLR4 ligand) was purchased from Calbiochem (La Jolla, CA). pHrodo fluorogenic dye was from Invitrogen (Carlsbad, CA). β Amyloid 1-42 (Aβ42) was from American Peptide Company (Sunnyvale, CA). C57BL/6 mice and CD36−/− (B6.129S1-Cd36tm1Mfe/J) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). EP2−/− mice were a gift from Dr. Richard Breyer (Vanderbilt University, Nashville, TN). The animals were maintained in a specific pathogen-free environment. All procedures were approved by the University of Washington Institutional Animal Care and Use Committee. Ten-week-old male mice were anesthetized with isoflurane inhalation and placed in a stereotactic instrument. After the skull was exposed through skin incision, a cranial window (2 to 3 mm) on the left lateral ventricle was opened by drilling with a small burr bit. Five microliter of reagent was injected over a period of 5 minutes by using a Hamilton microsyringe; injections were 200 ng of AH 6809, 50 ng of PIC, AH 6809 plus PIC, or vehicle. After injection, the mice were placed on an isothermal pad and continuously observed until recovery. Twenty-four hours after injection, the mice were sacrificed by i.p. injection of tribromoethanol and perfused with ice-cold phosphate-buffered saline, and brain tissues were rapidly harvested for analysis. Primary microglia and astrocytes were isolated from brains of newborn mice and cultured as previously described.38Li X. Cudaback E. Keene C.D. Breyer R.M. Montine T.J. Suppressed microglial E prostanoid receptor 1 signaling selectively reduces tumor necrosis factor alpha and interleukin 6 secretion from toll-like receptor 3 activation.Glia. 2011; 59: 569-576Crossref PubMed Scopus (18) Google Scholar, 39Li X. Cudaback E. Breyer R.M. Montine K.S. Keene C.D. Montine T.J. Eicosanoid receptor subtype-mediated opposing regulation of TLR-stimulated expression of astrocyte glial-derived neurotrophic factor.FASEB J. 2012; 26: 3075-3083Crossref PubMed Scopus (12) Google Scholar Medium concentrations of PGE2 were measured by ELISA exactly as described previously.38Li X. Cudaback E. Keene C.D. Breyer R.M. Montine T.J. Suppressed microglial E prostanoid receptor 1 signaling selectively reduces tumor necrosis factor alpha and interleukin 6 secretion from toll-like receptor 3 activation.Glia. 2011; 59: 569-576Crossref PubMed Scopus (18) Google Scholar Cells were incubated with the compounds described at the indicated concentrations and times. We note that, although EP antagonist AH 6809 has nearly equal affinity for EP1 and DP1 in human tissue,40Abramovitz M. Adam M. Boie Y. Carriere M. Denis D. Godbout C. Lamontagne S. Rochette C. Sawyer N. Tremblay N.M. Belley M. Gallant M. Dufresne C. Gareau Y. Ruel R. Juteau H. Labelle M. Ouimet N. Metters K.M. The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs.Biochim Biophys Acta. 2000; 1483: 285-293Crossref PubMed Scopus (468) Google Scholar it has been shown to be selective for EP2 in mouse tissue.41Kiriyama M. Ushikubi F. Kobayashi T. Hirata M. 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Reverse transcription was performed with Omniscript RT Kit (Qiagen). TaqMan probes and primers (Mm01135198_m1 for CD36, fMm99999915_g1 for GAPDH) were purchased from Applied Biosystems (Carlsbad, CA). PCR were performed on an Applied Biosystems ViiA 7 Real-Time PCR System by using the relative quantitative method. Microglia were preincubated with 10 μg/mL of anti-mouse CD16/CD32 antibody (BD Biosciences, San Jose, CA) for 5 minutes on ice to block Fc receptor–mediated binding. The cells were then incubated with 5 μg/mL of phycoerythrin-conjugated CD36 or mouse IgA control (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 30 minutes on ice in the dark. Thereafter, microglia were washed twice with phosphate-buffered saline and resuspended in 4% paraformaldehyde for fixation. The fluorescence intensity was determined by BD FACScan flow cytometry. Microglia were incubated with 5 μmol/L pHrodo-labeled Aβ42 for 2 hours at 37°C. The nonspecific signal was determined by incubation of microglia with the same concentration of labeled Aβ42 for 2 hours at 4°C. Thereafter, microglia were washed three times in phosphate-buffered saline and fixed in 4% paraformaldehyde. The samples were analyzed by LSRII flow cytometry (BD Biosciences) with a 561-nm excitation laser. Human CD36 was expressed in Chinese hamster ovary (CHO) cells as described by others.44Wilkinson K. Boyd J.D. Glicksman M. Moore K.J. El Khoury J. A high content drug screen identifies ursolic acid as an inhibitor of amyloid beta protein interactions with its receptor CD36.J Biol Chem. 2011; 286: 34914-34922Crossref PubMed Scopus (70) Google Scholar Briefly, cells were maintained in Ham's F-12 medium that contained 10% fetal bovine serum and penicillin/streptomycin. Human full-length CD36 cDNA was obtained from GE Healthcare (Lafayette, CO) and cloned into the expression vector pIRES2-EGFP at EcoRI and XbaI sites. The recombinant vector or empty vector was transfected into CHO cells by Lipofectamine 2000 according to the manufacturer's protocol. The expression of CD36 was confirmed by quantitative PCR with Taqman expression assay. Two days after transfection, cells were collected for phagocytosis assay and inhibition with sulfosuccinimidyl oleate (SSO) as described by others.45Kuda O. Pietka T.A. Demianova Z. Kudova E. Cvacka J. Kopecky J. Abumrad N.A. Sulfo-N-succinimidyl oleate (SSO) inhibits fatty acid uptake and signaling for intracellular calcium via binding CD36 lysine 164: SSO also inhibits oxidized low density lipoprotein uptake by macrophages.J Biol Chem. 2013; 288: 15547-15555Crossref PubMed Scopus (95) Google Scholar Statistical analyses were performed with GraphPad Prism 5 (GraphPad Inc., San Diego, CA) with α set to 0.05. All P values presented in the text or figures were corrected for multiple comparisons when appropriate with the method of Bonferroni. Microglia, and less so astrocytes, are cellular effectors of innate immune response in the cerebral cortex. CD36 expression in primary wild-type (WT) cultures of microglia and astrocytes prepared from the same five mice was determined by quantitative real-time PCR with each sample normalized to endogenous control GAPDH. CD36 expression in microglia was on average 143-fold greater in microglial than in astrocytes (P < 0.0001). For this reason, subsequent experiments focused on microglia. Because many endogenous activators of innate immune response, including Aβ,46Reed-Geaghan E.G. Savage J.C. Hise A.G. Landreth G.E. CD14 and toll-like receptors 2 and 4 are required for fibrillar A{beta}-stimulated microglial activation.J Neurosci. 2009; 29: 11982-11992Crossref PubMed Scopus (403) Google Scholar, 47Tang S.C. Lathia J.D. Selvaraj P.K. Jo D.G. Mughal M.R. Cheng A. Siler D.A. Markesbery W.R. Arumugam T.V. Mattson M.P. 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TREM-2, triggering receptor expressed on myeloid cell-2, negatively regulates TLR responses in dendritic cells.Eur J Immunol. 2012; 42: 176-185Crossref PubMed Scopus (113) Google Scholar we measured primary WT mouse microglia CD36 expression after exposure for 18 hours to specific TLR activators that are known to activate microglia.38Li X. Cudaback E. Keene C.D. Breyer R.M. Montine T.J. Suppressed microglial E prostanoid receptor 1 signaling selectively reduces tumor necrosis factor alpha and interleukin 6 secretion from toll-like receptor 3 activation.Glia. 2011; 59: 569-576Crossref PubMed Scopus (18) Google Scholar Activators of TLR3, TLR4, and TLR7, but not TLR2 or TLR9, suppressed microglia expression of CD36 (Figure 1A). A seven-poi
