EFAD Mice Research Articles

MMP9 modulation improves specific neurobehavioral deficits in a mouse model of Alzheimer\u2019s disease

BackgroundMatrix metallopeptidase 9 (MMP9) has been implicated in a variety of neurological disorders, including Alzheimer’s disease (AD), where MMP9 levels are elevated in the brain and cerebrovasculature. Previously our group demonstrated apolipoprotein E4 (apoE4) was less efficient in regulating MMP9 activity in the brain than other apoE isoforms, and that MMP9 inhibition facilitated beta-amyloid (Aβ) elimination across the blood–brain barrier (BBB)MethodsIn the current studies, we evaluated the impact of MMP9 modulation on Aβ disposition and neurobehavior in AD using two approaches, (1) pharmacological inhibition of MMP9 with SB-3CT in apoE4 x AD (E4FAD) mice, and (2) gene deletion of MMP9 in AD mice (MMP9KO/5xFAD)ResultsTreatment with the MMP9 inhibitor SB-3CT in E4FAD mice led to reduced anxiety compared to placebo using the elevated plus maze. Deletion of the MMP9 gene in 5xFAD mice also reduced anxiety using the open field test, in addition to improving sociability and social recognition memory, particularly in male mice, as assessed through the three-chamber task, indicating certain behavioral alterations in AD may be mediated by MMP9. However, neither pharmacological inhibition of MMP9 or gene deletion of MMP9 affected spatial learning or memory in the AD animals, as determined through the radial arm water maze. Moreover, the effect of MMP9 modulation on AD neurobehavior was not due to changes in Aβ disposition, as both brain and plasma Aβ levels were unchanged in the SB-3CT-treated E4FAD animals and MMP9KO/AD mice compared to their respective controls.ConclusionsIn total, while MMP9 inhibition did improve specific neurobehavioral deficits associated with AD, such as anxiety and social recognition memory, modulation of MMP9 did not alter spatial learning and memory or Aβ tissue levels in AD animals. While targeting MMP9 may represent a therapeutic strategy to mitigate aspects of neurobehavioral decline in AD, further work is necessary to understand the nature of the relationship between MMP9 activity and neurological dysfunction.

APOE4 Promotes Tonic-Clonic Seizures, an Effect Modified by Familial Alzheimer's Disease Mutations.

Seizures are emerging as a common symptom in Alzheimer’s disease (AD) patients, often attributed to high levels of amyloid β (Aβ). However, the extent that AD disease risk factors modulate seizure activity in aging and AD-relevant contexts is unclear. APOE4 is the greatest genetic risk factor for AD and has been linked to seizures independent of AD and Aβ. The goal of the present study was to evaluate the role of APOE genotype in modulating seizures in the absence and presence of high Aβ levels in vivo. To achieve this goal, we utilized EFAD mice, which express human APOE3 or APOE4 in the absence (EFAD−) or presence (EFAD+) of familial AD mutations that result in Aβ overproduction. When quantified during cage change day, we found that unlike APOE3, APOE4 is associated with tonic-clonic seizures. Interestingly, there were lower tonic-clonic seizures in E4FAD+ mice compared to E4FAD− mice. Restraint handing and auditory stimuli failed to recapitulate the tonic-clonic phenotype in EFAD mice that express APOE4. However, after chemical-induction with pentylenetetrazole, there was a higher incidence of tonic-clonic seizures with APOE4 compared to APOE3. Interestingly, the distribution of seizures to the tonic-clonic phenotype was higher with FAD mutations. These data support that APOE4 is associated with higher tonic-clonic seizures in vivo, and that FAD mutations impact tonic-clonic seizures in a paradigm dependent manner.

Apolipoprotein E genotype-dependent nutrigenetic effects to prebiotic inulin for modulating systemic metabolism and neuroprotection in mice via gut-brain axis

ABSTRACT Objective The goal of the study was to identify the potential nutrigenetic effects to inulin, a prebiotic fiber, in mice with different human apolipoprotein E (APOE) genetic variants. Specifically, we compared responses to inulin for the potential modulation of the systemic metabolism and neuroprotection via gut-brain axis in mice with human APOE ϵ3 and ϵ4 alleles. Method We performed experiments with young mice expressing the human APOE3 (E3FAD mice and APOE4 gene (E4FAD mice). We fed mice with either inulin or control diet for 16 weeks starting from 3 months of age. We determined gut microbiome diversity and composition using16s rRNA sequencing, systemic metabolism using in vivo MRI and metabolomics, and blood–brain barrier (BBB) tight junction expression using Western blot. Results In both E3FAD and E4FAD mice, inulin altered the alpha and beta diversity of the gut microbiome, increased beneficial taxa of bacteria and elevated cecal short chain fatty acid and hippocampal scyllo-inositol. E3FAD mice had altered metabolism related to tryptophan and tyrosine, while E4FAD mice had changes in the tricarboxylic acid cycle, pentose phosphate pathway, and bile acids. Differences were found in levels of brain metabolites related to oxidative stress, and levels of Claudin-1 and Claudin-5 BBB tight junction expression. Discussion We found that inulin had many similar beneficial effects in the gut and brain for both E3FAD and E4FAD mice, which may be protective for brain functions and reduce risk for neurodegeneration. . E3FAD and E4FAD mice also had distinct responses in several metabolic pathways, suggesting an APOE-dependent nutrigenetic effects in modulating systemic metabolism and neuroprotection.

Age, sex, and cerebral microbleeds in EFAD Alzheimer disease mice

Cerebral microbleeds (MBs) increase at later ages in association with increased cognitive decline and Alzheimer Disease (AD). MB prevalence is also increased by APOE4 and hypertension. In EFAD mice (5XFAD+/−/human APOE+/+), cerebral cortex MBs are most prevalent in E4 females at 6 months, paralleling plaque amyloid. We evaluated MBs at 2, 4, and 6 months in relation to amyloid in plaques and cerebral amyloid angiopathy (CAA) by age, sex, APOE allele, and blood pressure. At 2 mo, MBs were 50% more numerous than plaques, followed by decreased ratio of MBs:Aβ plaques with female excess to 6 mo. The stable size of MBs suggests MBs arise as single events of extravasation, which may “seed” plaque formation. Blood pressure was normal from 2 to 6 months, minimizing a role of hypertension. Memory, assessed by fear conditioning, decreased with age in correlation with MBs and amyloid. Cortical layer analysis showed prevalent MBs and plaque in layers 4 and 5. Contrarily, CAA was prevalent in layers 1 and 2, discounting its contribution to MBs.

APOE genotype and sex influence memory and dendritic spine density in an EFAD mouse model of Alzheimer’s disease

AbstractBackgroundWomen carriers of the APOE4 genotype, which is the leading genetic risk factor for late‐onset Alzheimer’s disease (AD), are more likely than women carriers of other APOE genotypes and men of any APOE genotype to develop AD. However, factors underlying the interaction between APOE genotype and sex are not well characterized. The goal of this study was to examine the effects of sex and APOE genotype on memory function and dendritic spine density in a mouse model of AD.MethodsSix month‐old gonadally‐intact male and female transgenic (Tg) mice expressing 5 familial AD mutations (5xFAD+/‐) and human APOE3 +/+ (E3FAD) or APOE4 +/+ (E4FAD) were trained on object recognition (OR) and object placement (OP) tasks designed to test object recognition and spatial memory formation. Memory for previously seen objects or locations was tested 24 or 4 hours later, respectively. Two weeks after the conclusion of behavioral testing, mice were retrained with novel objects, and brains were collected for Golgi staining. Following staining, apical and basal dendritic spines in the dorsal hippocampus (DH) and medial prefrontal cortex (mPFC) were counted and classified by morphological type.ResultsMale E3FAD mice exhibited intact memory for previously seen objects or locations, whereas male E4FADs and female E3FAD or E4FAD mice did not. The density of total, mushroom, and stubby spines on basal CA1 and mPFC dendrites was lower for E4FAD mice of both sexes compared to E3FAD mice. Preliminary data suggest a similar pattern for mPFC apical spine density.ConclusionsThese data suggest that APOE genotype and sex influence object recognition and spatial memory formation, and that APOE4 genotype is associated with reduced density of mature and immature spines in CA1 and the mPFC.

Reduction of lipid peroxidase levels in EFAD mouse model

AbstractBackgroundCerebral microbleeds (MBs) are more prevalent in APOE4 carriers and associated with Alzheimer Disease (AD). Loss of cerebrovascular integrity, blood‐brain barrier breakdown, and reduced Aβ clearance are also shown by APOE4 carrier mice. In AD, increased brain amyloid coupled with increased oxidative stress may synergistically contribute to MBs prevalence. EFAD mice carrying familial dominant AD genes in combination with human apoE alleles are a model for sex differences in AD, with greater amyloid accumulation and microbleeds in female E4FAD mice.Independently, Aβ and heme from microbleeds contribute to increased oxidative stress. New data suggest increased lipid peroxidation in E4 mice at baseline as measured by 4‐HNE. Peroxiredoxin 6 (Prdx6) and glutathione peroxidase 4 (GPx4) are the only known antioxidant enzymes capable of detoxifying oxidized phospholipids.MethodPrdx6 and GPx4 were studied by Western blot in cortical lysates of 6mo old EFAD mice. To evaluate Aβ contribution, we analyzed Prdx6 and GPx4 in 2mo old EFAD mice, which show 2 to 10‐fold lower Aβ compared to 6mo EFAD mice, and 6mo old EFAD non carrier siblings (no AD‐transgene).ResultPrdx6 was decreased in E4FAD mice (M: ‐30%; F: ‐50%) compared to E3FAD mice. GPx4 was decreased only n E4FAD females (M: no change; F: ‐50%). Younger mice did not show differences in either lipid peroxidases at 2mo. Interestingly, Prdx6 but not GPx4 was reduced in 6mo old female E4FAD non carrier siblings.ConclusionOur data suggest that the decreased levels of Prdx6 in 6mo old female E4 carrier mice in absence of Aβ might impair their ability to reduce oxidized lipids and predispose them to membrane alteration. Moreover, aging and increase of Aβ load together might contribute to the reduction of GPx4 levels in APOE4 carriers and the accumulation of MBs. Additional studies are required to clarify the underlying interaction sex‐apoE allele in MBs onset and its contribution to the pathogenesis of AD.

The Novel Omega-6 Fatty Acid Docosapentaenoic Acid Positively Modulates Brain Innate Immune Response for Resolving Neuroinflammation at Early and Late Stages of Humanized APOE-Based Alzheimer's Disease Models.

Neuroinflammation plays a crucial role in the development and progression of Alzheimer's disease (AD), in which activated microglia are found to be associated with neurodegeneration. However, there is limited evidence showing how neuroinflammation and activated microglia are directly linked to neurodegeneration in vivo. Besides, there are currently no effective anti-inflammatory drugs for AD. In this study, we report on an effective anti-inflammatory lipid, linoleic acid (LA) metabolite docosapentaenoic acid (DPAn-6) treatment of aged humanized EFAD mice with advanced AD pathology. We also report the associations of neuroinflammatory and/or activated microglial markers with neurodegeneration in vivo. First, we found that dietary LA reduced proinflammatory cytokines of IL1-β, IL-6, as well as mRNA expression of COX2 toward resolving neuroinflammation with an increase of IL-10 in adult AD models E3FAD and E4FAD mice. Brain fatty acid assays showed a five to six-fold increase in DPAn-6 by dietary LA, especially more in E4FAD mice, when compared to standard diet. Thus, we tested DPAn-6 in aged E4FAD mice. After DPAn-6 was administered to the E4FAD mice by oral gavage for three weeks, we found that DPAn-6 reduced microgliosis and mRNA expressions of inflammatory, microglial, and caspase markers. Further, DPAn-6 increased mRNA expressions of ADCYAP1, VGF, and neuronal pentraxin 2 in parallel, all of which were inversely correlated with inflammatory and microglial markers. Finally, both LA and DPAn-6 directly reduced mRNA expression of COX2 in amyloid-beta42 oligomer-challenged BV2 microglial cells. Together, these data indicated that DPAn-6 modulated neuroinflammatory responses toward resolution and improvement of neurodegeneration in the late stages of AD models.

Apolipoprotein E Genotype-Dependent Nutrigenetic Effects to Prebiotic Inulin for Reducing Risk for Alzheimer’s Disease in a Mouse Model

Abstract Objectives Apolipoprotein E ε4 (APOE4) allele is the strongest genetic risk factor for Alzheimer's disease (AD). Cognitively normal APOE4 carriers show an early decline in brain metabolic functions and gut microbiome dysbiosis before the onset of AD compared to APOE3 carriers. Our laboratory previously found that inulin, a prebiotic, is effective to restore metabolic functions and gut microbiome balance, and thus reduce risk for AD in an ApoE4 mouse model. However, whether the responses to the inulin are APOE allele-dependent remains unknown. Therefore, our objective was to identify whether inulin would differently contribute to metabolic and gut microbiome changes due to APOE genotypes. Methods We fed 3-month-old asymptomatic ApoE3 (E3FAD) and ApoE4 (E4FAD) mice prebiotic inulin or cellulose as a control for 4 months (N = 60; Male: Female = 1:1). After 4 months, we used magnetic resonance spectroscopy to measure in vivo brain scyllo-inositol level, a compound that has been demonstrated to inhibit amyloid β aggregation. We collected the fecal samples for gut microbiome sequencing, and cecal and blood samples for metabolomic profiling. Results Inulin induced scyllo-inositol in hippocampus of the brain with a higher level in E4FAD mice. Inulin increased blood metabolites in tryptophan and tyrosine metabolic pathways that enhance nervous system in E3FAD mice. It increased blood metabolites in pentose phosphate pathway and citric acid cycle that support nucleotides and nucleic acids biosynthesis and energy production in E4FAD mice. These alterations in hippocampus and blood showed inulin's impacts on systemic metabolism depending on mouse ApoE genotypes. Inulin enhanced short chain fatty acids in cecum with a greater increase in E3FAD mice. E3FAD mice showed more distinct gut microbiome patterns between inulin and control groups compared to E4FAD mice. Conclusions Inulin-induced systemic metabolism and gut microbiome changes are APOE genotype-dependent. Future studies can design human interventions that may facilitate the guide of personalized nutrition in AD prevention. Funding Sources This research was supported by grants from NIH, NIH/CTSA, and American Federation for Aging Research to A-LL and NIH/NIDDK to JDH and LMY. The 7T ClinScan small animal MRI scanner was funded by the S10 NIH Shared Instrumentation Program Grant.

HYPERTENSION DOES NOT CONTRIBUTE TO MICROBLEEDS ONSET IN FEMALE EFAD MICE

Cerebral microbleeds (MBs) contribute to pre-clinical cognitive decline and are an additional clinical burden in Alzheimer Disease (AD). Hypertension is associated with MBs, with nearly 2-fold higher likelihood for MBs per SD increment in blood pressure (BP). We investigated the possible role of age-related hypertension in the EFAD mouse model (transgenic for carrying familial AD mutations and targeted replacement of human APO-E3 or -E4). MBs were detected by Prussian Blue histochemistry. We extended prior findings with observations that MBs arise early in life, by 2 months, and confirmed female excess for ApoE3 and-E4 carriers. Wildtype C57BL/6J mice also accumulated MBs, and a 10-fold lower level and more slowly up to 21mo of age. BP was measured by the tail-cuff method. All mice had BP in the normotensive range, <150 mm Hg, systolic. Longitudinal measurements of blood pressure at ages 2, 4, and 6 months showed few age changes, except for E3FAD females at 6 months (systolic, +20%, p<0.05; diastolic, +33%, p<0.05). A possible decrease in blood pressure was observed in EFAD mice (-33%, p<0.01) compared to C57BL/6J mice. A not statistical trend of increase was observed in older C57BL/6J mice up to 18 mo of age, consistent with previous reports. Older ages are required for complete negation of role of hypertension in the MB model. Ongoing studies will examine mice older than 6 months for potential relations of blood pressure and MB, and in relation to brain amyloid deposits which surrounded MBs in our prior study.

TRANSCRIPTOMIC PROFILING OF MICROGLIA FROM AN ALZHEIMER'S DISEASE MOUSE MODEL AND FROM HUMAN INDUCED PLURIPOTENT STEM CELLS REVEALS EFFECTS OF THE APOE4 GENOTYPE

A number of risk factors for sporadic Alzheimer's disease (AD) have been identified, with the strongest genetic factor being the E4 allele of apolipoprotein E (APOE4). There are a several pathways through which APOE4 can act to increase AD risk, including via its actions on neuroinflammation and glial function. Microglia, one of the major immune cell types of the brain, are known to have a role in AD pathology and have previously been shown to have altered phenotypes in the context of APOE4. Several recent reports have demonstrated that microglia display unique transcriptional profiles in the context of AD and other neurodegenerative conditions. However, whether and how APOE4 may alter microglial transcriptomic signatures has thus far not been examined. We assessed the effects of APOE4 on microglial transcription profiles using both mouse models of AD and human induced pluripotent stem cells (iPSCs). CD11b+ microglia were isolated from EFAD mice, which carry either human APOE3 or APOE4 and the 5xFAD transgenes, and were analyzed by RNAseq. Additionally, iPSCs from APOE3 and APOE4 carriers were differentiated into microglia and their transcriptional profiles were assessed by RNAseq, both at baseline and after inflammatory insults. APOE4 is associated with transcriptional changes in a number of microglial genes, including an up-regulation of a number of inflammatory pathways and altered macrophage-associated networks. Determining how APOE4 affects microglial transcription will be critical in understanding how this AD risk factor alters the function of microglia and contributes to disease. We have identified microglial networks that are altered in the context of APOE4, enabling us to examine how these pathways may be impacting AD risk and progression.

The Prebiotic Inulin Beneficially Alters the Gut Microbiome and Associated Metabolites in an APOE4 Mouse Model (P08-133-19)

ObjectivesThe APOE4 allele is a genetic risk factor for certain diseases, due in part to alterations in lipid and glucose metabolism. The gut microbiota is also known to impact metabolic and can be beneficially modulated by prebiotics. Prebiotics are fermented into metabolites by the gut microbiota. These metabolites act as gut-brain axis components. However, the interaction of the APOE4 allele, gut microbiota, and prebiotics are unknown. The goal of the study was to use prebiotic diet to restore the gut microbiome of mice with human APOE4 (E4FAD) genes. We hypothesized that the microbial compositions of E4 mice fed inulin, compared to control fed, will correlate to metabolites being produced by the microbiome that confer benefit to host metabolism. MethodsAt 3 months of age the E4FAD mice were fed for 4 months with either control or inulin diet. We used 16S rRNA sequencing to determine gut microbiota diversity and species variations; non-targeted UPLC-MS/MS and GC-MS analysis was used to determine metabolic profiles of blood. ResultsThe inulin fed mice showed a more beneficial microbial taxa profile than those mice that were control fed. Control mice showed higher levels of dimethylglycine, choline, creatine and the polyamine spermine. Higher levels of spermine, specifically, correlate to higher levels of the Proteobacteria which has been implicated in GI disorders. E4 inulin fed mice showed higher levels of bile acids, short chain fatty acids and metabolites involved in energy, increased levels of tryptophan metabolites and robust increases in sphingomyelins. Specifically in E4 inulin fed mice we saw increases in certain genera of bacteria, all of which have been implicated in being beneficial to the composition of the microbiome and producing one or more of the above mentioned metabolites. ConclusionsWe believe the disparities of microbial metabolite production between E4 inulin fed mice and E4 control fed mice can be attributed to differences in certain taxa that produce these metabolites, and that higher levels of these taxa are due to the dietary intervention of inulin. Despite the APOE4 allele increasing one’s risk for certain diseases, we believe that beneficially modulating the gut microbiota may be one way to enhance host metabolism and decrease disease risk over time. Funding SourcesNIH/NIDDK T323048107792, NIH/NIA R01AG054459, NIEHS/NIH P42ES007380. Supporting Tables, Images and/or Graphs▪▪

APOE genotype and sex affect microglial interactions with plaques in Alzheimer\u2019s disease mice

Microglia affect Alzheimer’s disease (AD) pathogenesis in opposing manners, by protecting against amyloid accumulation in early phases of the disease and promoting neuropathology in advanced stages. Recent research has identified specific microglial interactions with amyloid plaques that exert important protective functions including attenuation of early pathology. It is unknown how these protective microglial interactions with plaques are affected by apolipoprotein E (APOE) genotype and sex, two well-established AD risk factors that modulate microglial function. We investigated this question using quantitative confocal microscopy to compare microglial interactions with amyloid plaques in male and female EFAD mice across APOE3 and APOE4 genotypes at 6 months of age. We observed that microglial coverage of plaques is highest in male APOE3 mice with significant reductions in coverage observed with both APOE4 genotype and female sex. Plaque compaction, a beneficial consequence of microglial interactions with plaques, showed a similar pattern in which APOE4 genotype and female sex were associated with significantly lower values. Within the plaque environment, microglial expression of triggering receptor expressed on myeloid cells 2 (TREM2), a known regulator of microglial plaque coverage, was highest in male APOE3 mice and reduced by APOE4 genotype and female sex. These differences in plaque interactions were unrelated to the number of microglial processes in the plaque environment across groups. Interestingly, the pattern of amyloid burden across groups was opposite to that of microglial plaque coverage, with APOE4 genotype and female sex showing the highest amyloid levels. These findings suggest a possible mechanism by which microglia may contribute to the increased AD risk associated with APOE4 genotype and female sex.

The effects of sex and APOE genotype on the gut microbiome in EFAD transgenic mice

While the gut microbiome (GM), the collective genome of bacteria in the gastrointestinal tract, is primarily studied in metabolism and immune defense, the GM may also serve as a diagnostic tool and therapeutic target in neurological disorders, including Alzheimer's disease (AD). While age is the greatest risk factor for AD, the e4allele of APOE is the greatest genetic risk factor, increasing risk up to 15‐fold compared to APOE3, the common genotype. APOE4 is associated with the accelerated accumulation of the peptide amyloid‐b(Ab), which can aggregate to form both amyloid plaques and small soluble aggregates or oligomeric Ab (oAb), the latter considered a proximal neurotoxin. Importantly, among APOE4 carriers, females have an increased AD risk, rate of cognitive loss and accumulation of Ab42 compared to males. Although the GM of individuals with AD exhibit specific microbial differences compared to healthy controls, a greater understanding of how age, APOE genotype and sex contribute to the altered GM in AD may help determine the diagnostic potential of the GM composition. To study the interactions among these AD risk factors, we developed the EFAD‐transgenic mice, which express the human APOE genotypes and over‐express human Ab42. Thus, our hypothesis is that APOE genotype and sex interact to modulate the GM composition in EFAD mice. Fecal samples from 4 month (M) ♂ and ♀ E3FAD and E4FAD mice were sequenced and compared. The Shannon H index, a measure of species richness and diversity, identified a negative correlation between diversity and severity of AD pathology in the 4M EFAD cohorts of mice: ♂E4FAD &gt; ♀E4FAD and ♀E3FAD &gt; ♀E4FAD. Analysis of similarities (ANOSIM) demonstrated significant differences in the GM in E3FAD vs E4FAD across different taxonomic levels (operational taxonomic unit/OTU, Genus, and Family). Comparison across sex within APOE genotype revealed a significant difference between ♂E3FAD vs ♂E4FAD and ♀E3FAD vs ♀E4FAD at all taxonomic levels. Additionally, there is a significant effect for ♂E4FAD vs ♀E4FAD, though only at the OTU level. Further, heatmap analysis demonstrates clustering of ♀E4FAD samples based on 29 OTUs. Therefore, the synergistic effect of APOE4 and female sex on AD risk is exhibited by the GM, observed by the stratification of the EFAD cohorts. This study is the first step in defining the composition of the GM as a function APOE genotype and sex modulation of AD pathology.Support or Funding InformationLaDu lab funding includes institutional funds from the University of Illinois College of Medicine, the Bridge to the Doctorate Fellowship, and the Honor's College Undergraduate Research Award at the University of Illinois at Chicago.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

APOE genotype affects metabolic and Alzheimer-related outcomes induced by Western diet in female EFAD mice.

Development of Alzheimer's disease (AD) is regulated by interactive effects of genetic and environmental risk factors. The most significant genetic risk factor for AD is the ε4 allele of apolipoprotein E ( APOE4), which has been shown to exert greater AD risk in women. An important modifiable AD risk factor is obesity and its associated metabolic dysfunctions. Whether APOE genotype might interact with obesity in females to regulate AD pathogenesis is unclear. To investigate this issue, we studied the effects of Western diet (WD) on female EFAD mice, a transgenic mouse model of AD that includes human APOE alleles ε3 (E3FAD) and ε4 (E4FAD). EFAD mice were fed either control (10% fat, 7% sugar) or WD (45% fat, 17% sugar), and both metabolic and neuropathologic outcomes were determined. Although E4FAD mice generally exhibited poorer metabolic status at baseline, E3FAD mice showed greater diet-induced metabolic impairments. Similarly, E4FAD mice exhibited higher levels of AD-related pathology overall, but only E3FAD showed significant increases on select measures of β-amyloid pathology after exposure to WD. These data demonstrate a gene-environment interaction between APOE and obesogenic diets in females. Understanding how AD-promoting effects of obesity are modulated by genetic factors will foster the identification of at-risk populations and development of preventive interventions.-Christensen, A., Pike, C. J. APOE genotype affects metabolic and Alzheimer-related outcomes induced by Western diet in female EFAD mice.

NORMALIZATION OF HIPPOCAMPAL SIRTUIN 1 LEVELS IN A MURINE APOLIPOPROTEIN E4-5XFAD MODEL OF AD RESTORES COGNITIVE FUNCTION

Decreases in the level of major longevity determinant sirtuin 1 (SirT1) have been associated with Alzheimer's Disease (AD) and expression of a major genetic risk factor for sporadic AD, apolipoprotein E4 (ApoE4) [Kumar et al. 2013 PLoS One 8(4):e61560; Theendakara et al. 2013 PNAS 110(45):18303]. We previously identified a compound - A03 - that normalized SirT1 levels in the presence of ApoE4 in vitro. Here, we determined A03’s efficacy in enhancing SirT1 in brain and improving memory in vivo in a murine model of AD wherein human targeted-replacement ApoE4 (huApoE4 TR) is expressed in the 5XFAD model of AD (E4FAD). E4FAD mice expressing huApoE4 TR and human amyloid precursor protein (huAPP) with three mutations (K670N/M671L; I716V; & V717I) and human presenilin 1 (PS1) with two mutations (M146L & L286V) received 20 mg/kg A03 BID for a total dose of 40 mg/kg/day by the oral route for 56 days. Vehicle-only E4FAD and vehicle-only aged-matched (9-12 mo) non-transgenic (NTg) groups were used as controls. Working object and spatial memory were assessed using the Novel Object Recognition (NOR) and Novel Location Recognition (NLR) testing paradigms, respectively, both pre- and end-study. Levels of sirtuin 1 and soluble amyloid precursor protein alpha (sAPPα) were determined in hippocampi and frontal cortices of all mice using custom AlphaLISAs (Perkin-Elmer), and amyloid-beta (Aβ) 1-40 and 1-42 were determined by ELISA (Thermo). A03 was found to be orally available and brain-penetrant, and 56-day oral treatment significantly increased SirT1 in the hippocampi of E4FAD mice as compared to vehicle-treated E4FAD mice. A03 treatment of E4FAD mice also improved working object memory in NOR (novelty preference) significantly as compared to E4FAD vehicle-treated controls.

THE EFFECT OF SEX AND APOE ON AD PATHOLOGY DURING AGING IN EFAD MICE

THE EFFECT OF SEX AND APOE ON AD PATHOLOGY DURING AGING IN EFAD MICE

Neuronal pentraxin 1: A synaptic-derived plasma biomarker in Alzheimer's disease

Synaptic neurodegeneration is thought to be an early event initiated by soluble β-amyloid (Aβ) aggregates that closely correlates with cognitive decline in Alzheimer disease (AD). Apolipoprotein ε4 (APOE4) is the most common genetic risk factor for both familial AD (FAD) and sporadic AD; it accelerates Aβ aggregation and selectively impairs glutamate receptor function and synaptic plasticity. However, its molecular mechanisms remain elusive and these synaptic deficits are difficult to monitor. AD- and APOE4-dependent plasma biomarkers have been proposed, but synapse-related plasma biomarkers are lacking. We evaluated neuronal pentraxin 1 (NP1), a potential CNS-derived plasma biomarker of excitatory synaptic pathology. NP1 is preferentially expressed in brain and involved in glutamate receptor internalization. NP1 is secreted presynaptically induced by Aβ oligomers, and implicated in excitatory synaptic and mitochondrial deficits. Levels of NP1 and its fragments were increased in a correlated fashion in both brain and plasma of 7–8 month-old E4FAD mice relative to E3FAD mice. NP1 was also found in exosome preparations and reduced by dietary DHA supplementation. Plasma NP1 was higher in E4FAD+ (APOE4+/+/FAD+/−) relative to E4FAD- (non-carrier; APOE4+/+/FAD−/−) mice, suggesting NP1 is modulated by Aβ expression. Finally, relative to normal elderly, plasma NP1 was also elevated in patients with mild cognitive impairment (MCI) and elevated further in the subset who progressed to early-stage AD. In those patients, there was a trend towards increased NP1 levels in APOE4 carriers relative to non-carriers. These findings indicate that NP1 may represent a potential synapse-derived plasma biomarker relevant to early alterations in excitatory synapses in MCI and early-stage AD.

EFAD transgenic mice as a human APOE relevant preclinical model of Alzheimerʼns disease

Identified in 1993, APOE4 is the greatest genetic risk factor for sporadic Alzheimer's disease (AD), increasing risk up to 15-fold compared with APOE3, with APOE2 decreasing AD risk. However, the functional effects of APOE4 on AD pathology remain unclear and, in some cases, controversial. In vivo progress to understand how the human (h)-APOE genotypes affect AD pathology has been limited by the lack of a tractable familial AD-transgenic (FAD-Tg) mouse model expressing h-APOE rather than mouse (m)-APOE. The disparity between m- and h-apoE is relevant for virtually every AD-relevant pathway, including amyloid-β (Aβ) deposition and clearance, neuroinflammation, tau pathology, neural plasticity and cerebrovascular deficits. EFAD mice were designed as a temporally useful preclinical FAD-Tg-mouse model expressing the h-APOE genotypes for identifying mechanisms underlying APOE-modulated symptoms of AD pathology. From their first description in 2012, EFAD mice have enabled critical basic and therapeutic research. Here we review insights gleaned from the EFAD mice and summarize future directions.

Peripheral Inflammation, Apolipoprotein E4, and Amyloid-β Interact to Induce Cognitive and Cerebrovascular Dysfunction.

Cerebrovascular dysfunction is rapidly reemerging as a major process of Alzheimer’s disease (AD). It is, therefore, crucial to delineate the roles of AD risk factors in cerebrovascular dysfunction. While apolipoprotein E4 (APOE4), Amyloid-β (Aβ), and peripheral inflammation independently induce cerebrovascular damage, their collective effects remain to be elucidated. The goal of this study was to determine the interactive effect of APOE4, Aβ, and chronic repeated peripheral inflammation on cerebrovascular and cognitive dysfunction in vivo. EFAD mice are a well-characterized mouse model that express human APOE3 (E3FAD) or APOE4 (E4FAD) and overproduce human Aβ42 via expression of 5 Familial Alzheimer’s disease (5xFAD) mutations. Here, we utilized EFAD carriers [5xFAD+/−/APOE+/+ (EFAD+)] and noncarriers [5xFAD−/−/APOE+/+ (EFAD−)] to compare the effects of peripheral inflammation in the presence or absence of human Aβ overproduction. Low-level, chronic repeated peripheral inflammation was induced in EFAD mice via systemic administration of lipopolysaccharide (LPS; 0.5 mg/kg/wk i.p.) from 4 to 6 months of age. In E4FAD+ mice, peripheral inflammation caused cognitive deficits and lowered post-synaptic protein levels. Importantly, cerebrovascular deficits were observed in LPS-challenged E4FAD+ mice, including cerebrovascular leakiness, lower vessel coverage, and cerebral amyloid angiopathy-like Aβ deposition. Thus, APOE4, Aβ, and peripheral inflammation interact to induce cerebrovascular damage and cognitive deficits.

DIETARY LINOLEIC ACID DIFFERENTIALLY INFLUENCES BRAIN FADS ACTIVITIES INCREASING AN N-6 METABOLITE THAT INHIBITS INFLAMMATION AND PROMOTES AMYLOID-β CLEARANCE

Chronic inflammation, involved in many diseases including Alzheimer's disease (AD) is tightly regulated by polyunsaturated fatty acids (PUFAs). Many studies suggest a protective role of n-3, and opposite role of n-6 PUFAs against inflammation because n-6 arachidonic acid is a precursor of pro-inflammatory eicosanoids. However, emerging evidence reveals intake of major n-6 PUFA, linoleic acid (LA) can have anti-inflammatory effects while low LA is associated with risk of cognitive decline, but the underlying mechanisms are poorly understood. Therefore, in this study, we investigated whether dietary LA has anti-inflammatory effects in AD cellular and animal models and explored the mechanisms involved. Two AD mouse models were used. First in aged Tg2576 mice, dietary LA was given from 17 to 20 months; second in more aggressive EFAD mice, LA was given from 3 to 7 months. At the end of the experiments, the brain was collected for measurement of brain fatty acids, pro-inflammatory cytokines and COX2. Aβ oligomer- or LPS-stimulated microglial BV2 cells were used as cellular models to directly evaluate effects of LA and some n-6 metabolites. We found that dietary LA and ApoE genotype differently influenced brain fatty acid desaturases (FADS1(delta-5), FADS2 (delta4/6/8)) activities, and altered brain fatty acid composition with reduction of arachidonic acid precursor DGLA (20:3n-6) and elevation of docosapentaenoic acid (DPA, 22:5n-6) that was greater in E4FAD. This was accompanied by a reduction of plaques, cyclooxygenase-2 enzyme (COX2) expression and pro-inflammatory cytokines (IL1β, IL-6 and TNFα) but elevation of inflammation-resolving cytokine IL-10 compared to standard diet animals in both AD models. DPAn-6 directly inhibited Aβ oligomer- or LPS-induced COX2 expression in cultured microglial BV2 cells and promoted Aß clearance. This is the first report that LA influences brain FADS activities, inhibiting CNS inflammation while increasing DPA that directly promotes amyloid-β phagocytosis and COX-2 reduction. Since genetic variants in FADS genes regulate desaturase enzyme activity and fatty acid composition while diet interacts with genetic variants affecting disease risk, our study suggests further in vivo tests with DPA (n-6) supplements and a possible dietary strategy with LA intake for developing clinical personalized AD interventions.