ApoE Mice Research Articles

PS-B12-8: ANGIOTENSIN II TYPE I RECEPTOR SIGNAL REGULATES MITOCHONDRIAL DYNAMICS, CAUSING MUSCULAR MITOCHONDRIAL DYSFUNCTION AND SARCOPENIA UNDER METABOLIC STRESS

Introduction: Metabolic stress, such as oxidized low density lipoprotein (ox-LDL) and advanced glycation end products (AGE) cause atherosclerotic cardiovascular diseases through dysfunction of mitochondria, which undergo quality control by mitochondrial dynamics, fission and fusion. Mitochondria also plays a crucial role in regulating muscular function, and its dysfunction leads to sarcopenia. This study aims to clarify whether and how mitochondrial dynamics are involved in the development of sarcopenia under metabolic stress. Methods: We used human skeletal muscle cells (HSMC) in in vitro experiments and C57BL6 (WT), apolipoprotein E (ApoE) deficient and the double knockout of ApoE and AT1R (DKO) mice in in vivo experiments. Results: Administration of either ox-LDL or AGE to HSMC induced mitochondrial fission through activation of fission factor, Drp1, decreased mitochondrial membrane potential assessed by JC-1 staining and increased reactive oxygen species assessed by Mito-sox Red staining and apoptosis, which were restored by Drp1-specific inhibitor, mdivi-1. Inhibition of angiotensin II type 1 receptor (AT1R) by AT1R blocker also ameliorated excessive mitochondrial fission, its function and oxidative stress induced by either ox-LDL or AGE in HSMC. These results suggest that metabolic stress induces mitochondrial fission and its dysfunction, which may cause sarcopenia. Muscular strength and the ratio of muscle weight to tibia length were lower in ApoE than those in WT. The number of fused mitochondria and mitochondrial function were lower and oxidative stress and the number of apoptotic cell were higher in ApoE KO than those of WT. Muscle strength, the ratio of muscle weight to tibia length, the number of fused mitochondria and its function were higher and the number of apoptotic cell were lower in DKO than those in ApoE KO mice. Taken together, these results suggest that either ox-LDL or AGE causes sarcopenia through AT1R signal-dependent mitochondrial fission and subsequent mitochondrial dysfunction. Conclusion: Either ox-LDL or AGE induces mitochondrial dysfunction through AT1R signal-regulating mitochondrial fission, which causes sarcopenia. Inhibition of AT1R signal may be a potential therapeutic strategy to avoid development of sarcopenia in patients with life style related diseases, such as dyslipidemia and diabetics.

Disruption in Myelination in the Brain by APOE4

How the brain is affected by APOE4 -one of the most important genetic risk factors in patients with Alzheimer's disease (AD), is still not clear. This missing piece is a substantial barrier to understanding the risk factors of AD and their use as therapeutic targets. Blanchard and colleagues performed a study to delineate the function of APOE4 by post-mortem transcriptomic analysis of the brains of non-carriers and carriers of APOE4. The data from their results point to the fact that APOE4 influences a host of expression profiles and signaling mechanisms in all of the studied cell types. The most significant effects were observed in the cholesterol homeostasis and transport mechanisms. It was observed that the oligodendrocytes were subjected to abnormal cholesterol metabolism, which subsequently affected the electrical activity of neurons. These results were affirmed using induced pluripotent stem cells, post-mortem human brains, and targeted replacement mice. It was also seen that altered deposition of cholesterol in APOE4 carrier brains reduced myelination. Pharmacological improvement in axonal myelination of APOE4 mice demonstrated better memory and learning. By providing a single-cell atlas of the partaking signaling events, the authors concisely represented the transcriptomic effects of APOE4. A working relationship between cholesterol transport, myelination, and subsequent effects on learning and memory with APOE4 was also provided, thus unveiling potential targets for treating AD. Nature (2022) DOI: 10.1038/s41586-022-05439-w.

INVESTIGATING THE NEURAL AFFECTS OF 17 ALPHA-ESTRADIOL (17Α-E2) ON APOE4 PHENOTYPES

Abstract Aging and APOE4 genotype are the primary risk factors for late onset Alzheimer’s disease (AD). Previous drugs trials for AD have focused on amyloid beta which has yielded minimal effects; hence a drug targeting aging could prove more efficacious. 17 alpha-estradiol (17α-E2) is an enantiomer of 17 beta-estradiol that has been shown to extend lifespan in male mice. We hypothesize that 17α-E2 will improve aging related phenotypes caused by the APOE4 genotype. We enrolled 10-month-old APOE3 or APOE4 targeted replacement mice and randomized them to either control or 17α-E2 diet for 20 weeks. At weeks 15 to 17, mice underwent various behavior assays and tissues were collected at the end of the 20 weeks. We observed behavioral improvement in APOE4 mice treated with 17α-E2 and asked whether this effect is associated with: (1) change in activity or anxiety, (2) change in neural stem cells, or (3) change in microglial activation. Activity levels but not neurogenesis of microglial burden were affected by APOE genotype and 17α-E2 treatment, suggesting a potential contribution to observed memory improvement in APOE4 mice treated with 17α-E2. Continued research on the neural effects of 17α-E2 pose potential benefits to mitigate the effects of aging.

Accumulation of amyloid-β in the brain of mouse models of Alzheimer's disease is modified by altered gene expression in the presence of human apoE isoforms during aging

Apolipoprotein E4 (apoE4) is a risk factor for Alzheimer's disease (AD). Here, we investigated brain amyloid-β (Aβ) accumulation throughout the aging process in an amyloid precursor protein (APP) knock-in (KI) mouse model of AD that expresses human APPNL-G-F with or without human apoE4 or apoE3. Brain Aβ42 levels were significantly lower in 9-month-old mice that express human isoforms of apoE than in age-matched APP-KI control mice. Linear accumulation of Aβ42 began in 5-month-old apoE4 mice, and a strong increase in Aβ42 levels was observed in 21-month-old apoE3 mice. Aβ42 levels in cerebroventricular fluid were higher in apoE3 than in apoE4 mice at 6–7 months of age, suggesting that apoE3 is more efficient at clearing Aβ42 than apoE4 at these ages. However, apoE3 protein levels were lower than apoE4 protein levels in the brains of 21-month-old apoE3 and apoE4 mice, respectively, which may explain the rapid increase in brain Aβ42 burden in apoE3 mice. We identified genes that were downregulated in a human apoE-dependent (apoE4 > apoE3) and age-dependent (apoE3 = apoE4) manner, which may regulate brain Aβ burden and/or AD progression. Analysis of gene expression in AD mouse models helps identify molecular mechanisms of pleiotropy by the human APOE gene during aging.

APOE4 mice rely more on the plasma DHA than APOE3 mice, especially in glia morphology and cognitive performance.

AbstractBackgroundThe metabolism of docosahexaenoic acid (DHA), an omega‐3 fatty acid, is different in APOE4 carriers. Brain relies on the plasma DHA pool for its need but the plasma‐liver‐brain axis in relation to cognition remains obscure. We hypothesized that this relation is compromised in APOE4 mice considering the differences in fatty acid metabolism between APOE3 and APOE4 mice.MethodMale and female APOE3 and APOE4 mice were fed either a diet enriched with DHA (0.7 g DHA/100 g diet) or a control diet for 8 months. Barnes maze and object recognition tests were performed in mice. Immunofluorescence was eprformed on parafin embeded brain slides with GFAP and IBA‐1 and multiplex cytokines were measured in hippocampus protein extracts.ResultIn the cortex, a genotype effect was found where APOE4 mice had higher concentration of DHA than APOE3 mice fed the control diet. APOE4 mice had 20‐30% lower plasma DHA and arachidonic acid (AA) concentrations, adipose tissue AA concentrations than APOE3 mice, independent of diet. In APOE4 mice, there was a significant correlation between plasma DHA and scores on the Barnes maze and with better recognition index. In addition, APOE4 mice fed the DHA diet had lower hippocampal concentrations of interleukin‐7, lipopolysaccharide‐induced CXC chemokine and monocyte chemoattractant protein 1, and higher concentration of interferon‐gamma compared to APOE4 mice fed the control diet. We also found that a diet rich in DHA enhances reactive astrogliosis and ramified microglia morphology in APOE4 mice.ConclusionAltogether, our data support that APOE4 mice rely more on the plasma DHA than APOE3 mice, especially in cognitive performance and astrocytes and microglia morphology. Any disturbance in plasma DHA metabolism might have a greater impact on cognition in APOE4 carriers.

The longevity‐promoting intervention 17α‐Estradiol protects against aging phenotypes in APOE4 mice

AbstractBackgroundThe two primary risk factors for late onset Alzheimer’s Disease (AD) are aging and APOE4 genotype. While the causal relationship between aging and AD is not well defined, shared phenotypes, such as decreased metabolic function and increased inflammation, are strong leads. APOE genotype may be linked to AD phenotype through the regulation of aging processes. The NIA Interventions Testing Program found that 17α‐estradiol (17αE2) treatment can increase male mouse lifespan. Since 17αE2 has been shown to act upon systemic and neural pathways that have also been associated with AD pathology, we propose that 17αE2 may constitute a pleiotropic intervention strategy. Further, because APOE4 is associated with impaired healthspan and lifespan, 17αE2 may preferentially improve outcomes in the context of APOE4 genotype.MethodTo examine the effects at the earliest phases of AD development, we used 10‐month‐old APOE3 or APOE4 targeted replacement male mice maintained for 20 weeks on normal chow in the absence or presence of 14.4 ppm 17αE2. Mice were evaluated on various metabolic, behavioral, and molecular measures.ResultOur initial results indicate genotype differences in the impact of 17αE2 across multiple outcomes. APOE4 mice were impaired across metabolic measures including body weight, plasma leptin, and hepatic steatosis, and 17αE2 treatment significantly attenuated these metabolic phenotypes. APOE4 mice exhibited an aged phenotype compared to APOE3, notably with higher frailty indices; however, 17αE2 treatment reduced the frailty index more strongly in APOE4 mice compared to APOE3. Interestingly, transcriptomic analysis of isolated microglia from both APOE3 and APOE4 17αE2 treated mice displays a downregulation of genes relating to inflammation and upregulation of those in metabolic pathways.ConclusionThese data confirm and extend prior findings that APOE4 is linked to progeroid effects both peripherally and neurally, outcomes associated with AD risk. Importantly, although 17αE2 significantly improved a range of measure across genotypes, it showed the strongest effects in the APOE4 genotype.This research was funded by the Cure Alzheimer’s Fund.

Inhibition of cPLA2 ameliorates memory deficits and reduces neuroinflammation in ApoE4‐TR mice

AbstractBackgroundThe activation of calcium‐dependent cytosolic phospholipase A2 (cPLA2) is involved in inflammatory signaling by promoting the conversion of arachidonic acid (AA) into eicosanoids, while catabolizing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). We previously found higher cPLA2 activation correlated with increased pro‐inflammatory lipid metabolites in APOE4 brains. However, it is not known whether the inhibition cPLA2 will decrease neuroinflammation ApoE4‐mice.MethodFirst, ApoE4 targeted replacement mice (ApoE4‐TR, n = 3, male, 4 months old) were treated daily with vehicle or the specific cPLA2 inhibitor ASB14780 (10 mg/kg, intraperitoneal) for three days. The brains were collected and analyzed using LC‐MS based lipidomics. Second, ApoE2‐TR (n = 14, 8M/6F), ApoE3‐TR (n = 12, 5M/7F) and ApoE4‐TR (n = 17, 8M/9F) mice were fed a low‐DHA diet starting at 6 months old. ApoE4 mice were treated daily for three weeks with vehicle (n = 8, 4M/4F) or 10mg/kg/day ASB14780 IP (n = 9, 5M/4F) starting at 14 months old. Behavioral tests were subsequently conducted on the mice, after which the brains were collected for lipidomic and biochemical analyses.ResultIP ASB14780 was detected in brain tissue corresponding with decreases in pro‐inflammatory metabolites of AA after only 3 days treatment in ApoE4‐TR mice. In addition, long term ASB14780 treatment decreased brain cPLA2 phosphorylation levels and significantly increased brain EPA and DHA levels (p<0.01 for both) but did not change brain AA levels in ApoE4‐TR mice. Compared to ApoE3‐TR mice, untreated ApoE4‐TR mice had higher levels of inflammatory eicosanoids (e.g., prostaglandin E2, PGE2) resulting from AA metabolism. However, ASB14780 treatment decreased inflammatory metabolites in ApoE4‐TR mice. Additionally, ASB14780‐treatment was able to reduce microglia activation in ApoE4‐TR mice (p = 0.04 for hippocampus and p<0.01 for cortex). For behavioral experiments, ApoE4‐TR mice had significantly lower discrimination index compared to ApoE3‐TR mice at 15 months using the novel object recognition test (p = 0.029). Treatment with ASB14780 ameliorated the recognition deficits of ApoE4‐TR mice but the effects did not reach statistical significance (p = 0.15).ConclusionOur findings reveal inhibition of cPLA2 with ASB14780 could ameliorate eicosanoid‐driven neuroinflammation in ApoE4 mice, restoring synaptic dysfunction, and preventing behavioral deficits. cPLA2 represents a potential drug target to mitigate increased neuroinflammation with APOE4 and AD.

Translational potential of JAX hAPOE AD mouse model: Phenotypic characterization of cognitive function and amyloid beta

AbstractBackgroundAPOEe4 is the greatest genetic risk factor for late onset Alzheimer’s Disease (LOAD). The product of this gene, apolipoprotein E isoform ε4, has been characterized as a lipid transporter and many of its receptors and mechanisms have been identified. Studies have shown that APOEe4 exacerbates accumulation of Aβ peptides and correlates with a decline in cognitive function in humans. We investigated the translational feasibility of the humanized APOE3 (JAX #029018) and APOE4 (JAX #027894) mouse model on two hallmark pathologies of LOAD: cognitive decline and b‐amyloid (Aβ) generation in brain.MethodNovel object recognition (NOR), open field (OF), and CatWalk analyses were conducted in 18 month old transgenic mice with humanized APOEe3 homozygous (B6(SJL)‐ApoEtm1.1(APOE*3)Adiuj/J), APOEe3/4, or APOEe4 homozygous genes (B6(SJL)‐ApoEtm1.1(APOE*4)Adiuj/J). NOR analyses included discrimination index, OF center zone time, whereas CatWalk included mobility measures including cadence and speed. Aβ38, Aβ40, and Aβ42 analyses were assessed in brain and plasma.ResultNo differences were detected in NOR discrimination index, NOR combined object exploration time, or OF center zone time across sex or genotype. OF mean speed was lower in APOE3/4 mice compared to APOE4 mice. Aβ38 was not detected in any group. Hippocampal Aβ42 was significantly lower in APOE3/4 mice compared to APOE4 and APOE3 mice. No differences were detected in hippocampal Aβ40 or Aβ42/40 ratios. Plasma Aβ40, Aβ42, and Aβ42/40 were lower in females relative to males. Analyses based on sex and genotype indicated that plasma Aβ40 and Aβ42 were lower in APOE3 females vs APOE3 males, and Aβ42/40 was lower in APOE3 females vs every other group.ConclusionAPOE3/4 heterozygosity may confer a protective effect against Aβ42 deposition in the hippocampus, and female APOE mice consistently exhibit lower plasma Aβ. Translational applicability to humans on measures of cognitive decline and hippocampal Aβ42 deposition at 18 months (human age equivalent ∼56 years) for APOE mice is limited.Acknowledgements: Research supported by NIA R01AG57931 and RF1AG059093

Monitoring DHA and AA Brain Uptake in ApoE4 Mice with 18F‐Fluorinated PET Tracers

AbstractBackgroundPolyunsaturated fatty acids (PUFAs) are the precursors of various lipid mediators of inflammation. Brain imaging techniques for monitoring the brain’s uptake of PUFAs would not only uncover the effects of risk factors for excessive neuroinflammation associated with Alzheimer’s disease (AD) and related dementia but also direct the development of methods to prevent or treat AD. Radiosynthesized 11C‐docosahexaenoic acid (DHA) and 11C‐arachidonic acid (AA) have already been utilized in positron emission tomography (PET) studies in humans [1, 2]. Because 18F has a longer half‐life than 11C, fluorinated PUFA derivatives would be more favorable alternatives for PET studies of brain uptake.MethodsWe prepared 18F‐fluorinated analogues of DHA (18F‐FDHA) and AA (18F‐FAA) as PUFA PET tracers. Radiosynthesis was performed via a nucleophilic fluorination scheme using the tosylate ester derivatives as precursors. We then performed bolus injection of each tracer into 16‐month‐old APOE4 knock‐in mice and scanned them by dynamic PET‐MRI over 45 min to assess region‐specific uptake. PUFA uptake kinetics in the cerebral region was evaluated using the time‐dependent activity in the lumen of the right ventricle as the image‐derived input function and using an irreversible two‐tissue compartment model to calculate the incorporation coefficient (Ki).ResultsBoth 18F‐FDHA and 18F‐FAA were successfully synthesized in high radiochemical purity (≥98%) and were found to be stable in the formulation and mouse serum for 2 hr. The mean Ki values for the cerebral region were determined to be 6.31 ± 0.98 µL mL‐1 min‐1 for 18F‐FDHA (n = 4) and 6.75 ± 0.68 µL mL‐1 min‐1 for 18F‐FAA (n = 5).ConclusionUsing an image‐based method with 18F‐fluorinated PUFA tracers, we were able to determine Ki values for both AA and DHA. The use of these PUFA PET tracers has the potential to elucidate deleterious changes in PUFA metabolism during various phases of AD as well as effects due to dietary interventions and drug treatment.

Translational potential of JAX humanized APOE mice: Peripheral biomarkers of pathology

AbstractBackgroundAlzheimer’s Disease (AD) drives the global rising incidence of neurodegenerative dementia. Disease progression includes cerebral aggregation of amyloid‐beta plaques and immune activation. Many AD animal models carry mutations that cause an early onset of these phenotypes, but the mutations are rare in human populations; most cases are idiopathic and late‐onset AD (LOAD). Another approach utilizing a transgenic APOE animal model may better model clinical populations. APOE4 is the predominant genetic risk factor for LOAD and confers increasing risk for LOAD in allelic fashion, relative to normative APOE3. This study investigates the JAX humanized APOE knock‐in mouse model of LOAD’s translational potential for peripheral biomarkers of pathology in aged, male and female mice.MethodAged mice of both sexes (23‐25 months old; 39M/37F) with APOE3/3,APOE3/4, andAPOE4/4 genotypes underwent blood plasma amyloid‐beta (Aß) measurement. A subset of these animals (30M/28F) additionally received peripheral immunophenotyping of whole blood via flow cytometry. Two‐way ANOVA and post‐hoc t‐tests were conducted to determine data significance.ResultsFemale mice had lower Aß42 plasma levels as well as a decreased Aß42/40 ratio, suggesting higher levels of the aggregate‐prone Aß42 protein in the brain, but there were no observed APOE genotype effects. Blood plasma Aß40 levels did not differ by genotype or sex. Whole blood flow cytometry showed an elevation in CD8+ memory T cells in APOE4/4 mice relative to APOE3/3 andAPOE3/4 mice. Aged female mice had a peripheral immune cell profile consistent with a more reactive and dysfunctional immune system than genotype‐matched aged males: higher levels of CD11b+ B cells, CD4+ helper T cells, and CD8+ cytotoxic T cells along with a lower percentage of naïve CD4+ and CD8+ T cells.ConclusionIn mice aged to resemble a ∼70‐year‐old human population, these results suggest increased brain levels of the aggregate‐prone Aß42 isoform and a reactive peripheral immune system in female APOE mice. This sex difference is consistent with the increased prevalence of LOAD in women. However, the specificity of APOE4 genotype effects may define the translational potential of this humanized APOE mouse model of late‐onset Alzheimer’s Disease.Research supported by T32AG061897, R01AG057931, and RF1AG059093 to RDB.

Translational potential of JAX humanized APOE mice: trajectories of resilient versus susceptible aging by sex and weight

AbstractBackgroundAging, chromosomal sex, and genetic risk factors interact to determine the trajectory of an individual’s age‐related biological changes and, in turn, may predispose individual risk or resilience to AD. To advance translational validity of the JAX humanized APOE (hAPOE) mouse model, we investigated whether a well‐documented preclinical AD symptom, inexplicable weight loss, was evident in our hAPOE mouse colony. Further we determined whether this hallmark symptom could be used to detect subpopulations of vulnerable vs resilient.MethodWe used an autoregressive hidden Markov model (AHMM) to identify trajectories of JAX humanized APOE mice based on their longitudinal weight data. We assumed 10 hidden states to estimate proper trajectories. We ran 10 AHMM models to ensure stability and each model was run with 1500 iterations to reach convergence. The AHMM with maximum trace value was selected as the main model. The key trajectories were then identified by clustering together mice ending in the same hidden state. Weight data were collected from November 2018 to September 2021 for 1196 hAPOE mice (652M, 544F) at different time points ranging from 5 to 28 months of age. Genotypes included APOEe3/3, APOEe3/4 and APOEe4/4.ResultAHMM results identified 5 main weight trajectories for mice ending in one of the 5 most probable ending states (formally named A,B,C,D,E). Of these, three states (A,B,D) captured weight loss trajectories for 426 (36%) mice, one state (C) was characterized by an increase in weight for 152 (36%) mice and 403 (13%) mice ending in a state stable (E) with no significant weight loss or increase. Weight loss was more apparent in female mice, while weight increase was more typical of male mice.ConclusionThis mouse cohort displays weight loss that may correspond to individuals exhibiting a hallmark inexplicable weight loss starting around 15/20 months (corresponding to human 50/55 years old) that can be symptomatic of the prodromal phase of AD.AcknowledgementsThis work was supported R01AG057931, RF1AG059093 and the Center for Innovation in Brain Science to RDB.

Translational potential of JAX humanized APOE mice: Peripheral interferon gamma induces systemic immuno‐metabolic dysfunction

AbstractBackgroundInflammation is a well‐documented feature of Alzheimer’s disease (AD) in human brain. Previous findings from our group demonstrated upregulated immune transcripts in the AD brain as well as sex differences in immune transcript levels during pre‐clinical midlife aging. Using our humanized APOE mouse model, we identified elevated immune transcripts in the female brain including interferon signaling genes, CD3, CD4, and MHCII. We hypothesized that activation via increased interferon gamma (IFN‐γ) during endocrine aging may disrupt the immune‐metabolic balance in the brain and therefore advance changes in grey and white matter. With this study, we test the hypothesis that increased levels of peripheral IFN‐γ induces genotype dependent immuno‐metabolic changes in the JAX humanized APOE (hAPOE) mouse model.MethodTo determine the role of IFN‐γ in female midlife aging and AD risk, pre‐menopausal and peri‐menopausal hAPOE mice were intraperitoneally treated with recombinant IFN‐γ for 9 days. Immunophenotyping using multi‐color flow cytometry was conducted microglial reactivity, phagocytosis, neutral lipid content, oxidative stress, and the presence of lymphocytes were determined. Structural and diffusion weighted analysis using MRI was conducted to evaluate grey and white matter structural integrity in the brain. ELISA assays were conducted to assess the presence of beta amyloid 40 and 42 and levels of pro‐inflammatory cytokines.ResultIFN‐γ treated animals exhibited upregulation of markers that perpetuate neuroinflammation and white matter catabolism including MHCII, CD8, pHrodo, and BODIPY. Peripheral immune effects were genotype dependent while immune signaling in brain largely exhibited treatment‐dependent effects. Fasting blood glucose levels were significantly increased by IFN‐γ consistent with an accelerated aging profile. Diffusion metrics including mean and radial kurtosis in addition to mean, axial, and radial diffusivity were greater in IFN‐treated animals.ConclusionPeripheral increase of interferon gamma signaling during female endocrine aging in hAPOE mice induced 1) dysfunction of metabolic systems and neuroinflammatory response and 2) decreases in white and grey matter microstructural integrity throughout the brain. These outcomes support the investigation of therapeutic strategies targeting interferon signaling pathways at the earliest stages of midlife aging to reduce risk of AD or delay AD onset.

Translational potential of JAX humanized APOE mice: Hippocampal volume decline in very old mice

AbstractBackgroundWhole brain and hippocampal atrophy are the most prominent structural features of late‐onset Alzheimer’s disease (LOAD) and are accepted endpoints in many clinical trials. The APOE ε4 allele is the strongest genetic risk factor for LOAD, with the ε4/ε4 genotype associated with the greatest atrophy rates. To date, there is limited hippocampal volume reporting in preclinical APOE models. To assess the translational potential of a humanized APOE (hAPOE) mouse model, we report age, sex, and genotype effects on total brain and hippocampal volume.MethodHigh resolution ex‐vivo MRIs were obtained from two separate cohorts of male and female hAPOE mice (Cohort 1: n=53, 18.47±0.99 months; Cohort 2: n=33, 24.24±0.58 months). A validated mouse brain atlas was used for volumetric analysis. Cohorts were analyzed separately with genotype by sex analyses of variance for total brain volume (sum of all regions in atlas) as well as left and right hippocampal volume as percent of total brain volume.ResultIn Cohort 1, hAPOEε4/ε4 mice had significantly larger brains than hAPOEε3/ε3s or hAPOEε3/ε4s, with female hAPOEε4/ε4s having the largest brains (p<0.001). Regardless of genotype, females had smaller left (p=0.006) and right (p<0.001) hippocampi relative to males. In Cohort 2, there were no genotype or sex differences in total brain volume. However, left (p=0.044) and right (p=0.034) hippocampal volumes were smaller for hAPOEε4/ε4 mice compared to hAPOEε3/ε4 mice without sex effects.ConclusionOur findings capitalize on a large dataset (n=88) of high‐resolution MRIs. At 18‐20 months old (∼60 human years), hAPOEε4/ε4 mice had greater total brain volume than comparably aged hAPOEε3/ε3 or hAPOEε3/ε4 mice without hippocampal atrophy. However, hippocampal atrophy was evident when mice were aged to 24‐25 months (∼70 human years). These findings of larger brain volume and greater hippocampal atrophy in hAPOEε4/ε4 carriers are consistent with large‐scale AD imaging datasets. The timing of evident hippocampal atrophy in these mice also coincides with the global average age of clinical diagnosis. Based on these findings, the translational potential of this model requires utilizing very old mice, limiting the window for therapeutic testing.AcknowledgementsNIA R01AG057931, RF1AG059093; University of Arizona TBIR (NIH S10 OD025016)

Translational potential of JAX humanized‐APOE mice: Building the foundation for translational validity by applying human clinical trial principles

AbstractBackgroundThe 4th isoform of apolipoprotein E is the most prevalent genetic risk factor for Alzheimerʼs Disease (AD) and transgenic APOE animal models are widely used. However, APOE4’s penetrance is only ∼50‐60% and many studies do not use sufficient sample sizes to account for this.MethodTo address knowledge gaps regarding APOE penetrance and advance translatability of JAX humanized APOE (hAPOE) mouse models, we utilized a translational animal data management system recapitulating core aspects of human clinical trials: unique mouse IDs linked to all tissue samples and analytic outcomes. Transgenic C57BL/6J mice carrying hAPOE alleles (hAPOEe3/3, hAPOEe3/4 or hAPOEe4/4) were bred and longitudinally tracked for identification and analysis of susceptible and resilient subpopulations through population‐level studies on monthly weights (N = 1129 mice, N = 13262 weights), clinical observations (N = 2754 mice), and survival rates (N = 559 mice not utilized in studies).ResultColony‐wide analyses showed few differences, potentially leading to incorrect conclusions of minimal allelic effects. In this study, sub‐populations of hAPOEe4/4 mice exhibit divergent weight trajectories beginning at 15mo. Moreover, hAPOEe4/4 females displayed aberrations in fur barbering trajectories. The clinical observation of dermatitis was also noted more often in hAPOEe4/4 mice compared to hAPOEe3/3. Accordingly, hAPOEe4/4 animals, especially females, exhibit reduced survival rates, potentially due to divergent trajectories of susceptibility and resilience.ConclusionThe present study identified distinct subpopulations of hAPOE mice exhibiting clear susceptible vs. resilient phenotypes within longitudinal data. Therefore, results from cross‐sectional studies of hAPOE mice with small, average cohorts will be highly variable. Future studies selecting cohorts based on clinical observations such as weight and phenotypic indicator trajectory will advance translatability to human APOE4‐AD.Acknowledgements: Research supported by T32AG061897, R01AG057931, RF1AG059093, and Center for Innovation in Brain Science (CIBS) to RDB.

APOE genotype alters the lipid droplet proteome and modulates droplet dynamics

AbstractBackgroundThe microglial immune response is a significant contributor to Alzheimer’s disease (AD) pathophysiology and neurodegeneration. Aging microglia accumulate lipid droplets (LDs), have high levels of reactive oxygen species, secrete pro‐inflammatory cytokines, and are defective in phagocytosis. The E4 allele of Apolipoprotein E (APOE) is the strongest genetic risk factor for late‐onset AD, and is associated with heightened neuroinflammation and increased LD formation. We hypothesize E4 microglia have increased LD formation under basal conditions and a higher capacity to form LDs under stress, resulting in greater pro‐inflammatory cytokine production. We characterized LD development in microglia in the context of APOE genotype and analyzed LD surface proteins and lipid content from control and lipopolysaccharide (LPS) stimulated ApoE3 and ApoE4 mice.MethodPrimary microglia were isolated from mice expressing human ApoE3 and ApoE4. Microglia were exposed to 250uM oleic acid (OA), 10ug/mL LPS, OA+LPS, dead N2A cells, or dead N2As+LPS. ApoE3 and ApoE4 expressing mice were injected with saline (control) or LPS (5mg/kg) and perfused at 24h. Livers were extracted, the LD enriched supernatant fraction was collected after centrifugation, and proteomic and lipidomic analyses were performed.ResultPrimary microglia from ApoE4 mice accumulated more LDs at baseline, with exogenous OA, LPS stimulation, and N2As as a percentage of E3 control across multiple experiments (E3 v E4 p‐values: baseline, 0.0317; LPS, 0.0032; OA, 0.0277; N2A, 0.0192). Western blots on LD fractions confirm LD enrichment by surface protein, PLIN2, along with increased expression of PLIN2 (i.e. more LDs) in E4 LPS mice. Proteomics reveal LD fractions from E4 mice are enriched for proteins involved in innate immunity, while E3 LDs are enriched for proteins involved in lipid b‐oxidation.ConclusionE4 microglia accumulate more LDs compared to E3 microglia under all conditions tested. The proteomic profile of E4 liver LDs support the hypothesis that E4 expression increases inflammation under basal conditions, and upon stimulation, causes a more robust response. Increased LD formation is present in non‐aged, non‐diseased E4 cells, suggesting preclinical dysfunction associated with the highest risk APOE genotype. A better understanding of LD dynamics within these cells and their functional implications can provide targets to improve E4‐related outcomes.

Translational potential of JAX humanized‐APOE mice: sex differences in bioenergetic function in brain

AbstractBackgroundThe preclinical phase of late onset Alzheimer’s disease (LOAD), known as the preclinical phase, can start 10 to 20 years prior to onset of clinical symptoms. Hypometabolism and impaired mitochondrial bioenergetics can emerge during prodromal phase as early indicators of AD risk. APOEe4, the greatest genetic risk factor for LOAD, is well documented to induce lipid metabolic dysregulation. We therefore sought to determine the impact of APOEe4. The following study analyzed body composition and mitochondrial functions to elucidate how the presence of APOEe4 in a mouse model contributes to bioenergetic changes, and whether they affect females and males equally.Method18mo old transgenic mouse models (JAX #029018 and #027894) carrying humanized APOE alleles (hAPOE3/3, hAPOE3/4 or hAPOE4/4) were assessed for fat and lean mass content via EchoMRI. A week later, fasting blood glucose levels were assessed prior to sacrifice, and brain tissues were collected to determine mitochondria function by Complex I and IV activity.ResultBody composition analyses indicated that hAPOEe4 males had significantly lower fat percentage than male APOEe3 mice. Females, irrespective of APOE genotype, exhibited significantly lower lean weight relative to males. No sex or APOE genotype difference was detected in fasting blood glucose plasma levels nor in mitochondrial Complex I activity. In contrast, mitochondrial Complex IV activity was significantly lower in females relative to males. Lastly, Complex IV activity was lower in mice exhibiting dominance aggressive behavior, specifically barbering, irrespective of sex or genotype.ConclusionAPOE mice resembling a human population of approximately 60 years of age displayed a sex difference in body composition parameters, where total and lean weight was higher in males than females. Sex differences also emerged in bioenergetic function, with females exhibiting lower mitochondria Complex IV activity relative to males. Sex differences are consistent with human data, whereas no genotype differences were observed and thus may influence the translational potential of the humanized APOE mouse model in LOAD research. However, the translational potential might be greater when linked with phenotypic characteristics.This work was supported by NIA grants R01AG057931, and RF1AG059093

Single amino acid change in the receptor binding domain of mouse APOE results in altered protein dynamics and worsened AD‐related cognitive impairment

AbstractBackground APOE genotype is the strongest genetic modifier of late‐onset Alzheimer’s disease (AD) in humans. In genetically diverse mice, natural variation in the Apoe locus predicts AD‐related cognitive decline, with C57BL/6J (B6) mice harboring a protective variant compared to DBA/2J (D2) (Neuner 2019, Neuron). The mechanisms by which protective and detrimental Apoe/APOE variants affect cognitive decline in AD are not fully understood yet, but likely could provide an opportunity for personalized treatment and prevention strategies.MethodHere, we have used CRISPR/Cas9 gene editing to introduce the D2 Apoe variant into the B6 genome. We crossed these heterozygous APOEB6/E163D mice to B6 congenic heterozygous 5XFAD carriers in order to explore the effects of this single variant on AD‐related cognitive function, pathology, cell‐type specific gene dysregulation, and hippocampal and cortical network function. We also performed molecular dynamics modeling to ask how this variant affects APOE protein biophysics.Result5XFAD‐APOEB6/E163D mice showed exacerbated cognitive dysfunction compared to 5XFAD‐APOEB6/B6 mice, indicating that the single D2 variant in Apoe significantly contributes to AD‐related cognitive decline. We did not observe differences in amyloid pathology or phosphorylated tau across Apoe genotypes, suggesting that the effect on cognitive function is not mediated by pathological protein aggregation. However, snRNAseq revealed that excitatory neuron gene expression was substantially dysregulated in 5XFAD‐APOEB6/E163D mice compared to other cell types; we then performed in vivo electrophysiology to characterize how this translates to circuit‐level neuronal dysfunction. Finally, computational modeling of protein structure and function showed that the APOEE163D variant results in protein states most closely resembling human APOE4, particularly in the presence of lipids, suggesting that APOEE163D may show impaired lipid binding and transport function.ConclusionC57BL/6J mice harbor a protective Apoe variant which confers resiliency to AD‐related cognitive dysfunction. This protection, and–by the same measure–the detrimental effects of the DBA/2J variant of Apoe (APOEE163D), is likely ultimately mediated by altered neuronal gene expression and activity. We are currently exploring the intermediate mechanisms between APOEE163D and neuronal function; altered APOE protein states hint at impairments in lipid binding and transport, which may result in pathological lipid and protein accumulation affecting neuron function.

Oral Administration of Branched-Chain Amino Acids Attenuates Atherosclerosis by Inhibiting the Inflammatory Response and Regulating the Gut Microbiota in ApoE-Deficient Mice.

Atherosclerosis (AS) is a chronic inflammatory disease that serves as a common pathogenic underpinning for various cardiovascular diseases. Although high circulating branched-chain amino acid (BCAA) levels may represent a risk factor for AS, it is unclear whether dietary BCAA supplementation causes elevated levels of circulating BCAAs and hence influences AS, and the related mechanisms are not well understood. Here, ApoE-deficient mice (ApoE-/-) were fed a diet supplemented with or without BCAAs to investigate the effects of BCAAs on AS and determine potential related mechanisms. In this study, compared with the high-fat diet (HFD), high-fat diet supplemented with BCAAs (HFB) reduced the atherosclerotic lesion area and caused a significant decrease in serum cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels. BCAA supplementation suppressed the systemic inflammatory response by reducing macrophage infiltration; lowering serum levels of inflammatory factors, including monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6); and suppressing inflammatory related signaling pathways. Furthermore, BCAA supplementation altered the gut bacterial beta diversity and composition, especially reducing harmful bacteria and increasing probiotic bacteria, along with increasing bile acid (BA) excretion. In addition, the levels of total BAs, primary BAs, 12α-hydroxylated bile acids (12α-OH BAs) and non-12α-hydroxylated bile acids (non-12α-OH BAs) in cecal and colonic contents were increased in the HFB group of mice compared with the HFD group. Overall, these data indicate that dietary BCAA supplementation can attenuate atherosclerosis induced by HFD in ApoE-/- mice through improved dyslipidemia and inflammation, mechanisms involving the intestinal microbiota, and promotion of BA excretion.

Exercise modulates APOE expression in brain cortex of female APOE3 and APOE4 targeted replacement mice

Apolipoprotein E (ApoE) is the main cholesterol carrier of the brain and the ε4 gene variant (APOE4) is the most prevalent genetic risk factor for Alzheimer's disease (AD), increasing risk up to 15-fold. Several studies indicate that APOE4 modulates critical factors for neuronal function, including brain-derived neurotrophic factor (BDNF) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). Both proteins show exercise-induced upregulation, which is presumed to mediate many of the beneficial effects of physical activity including improved cognition; however, there is variability in results between individuals potentially in-part due to genetic variations including APOE isoform. This study aimed to determine if the two most prevalent human APOE isoforms influence adaptive responses to exercise-training. Targeted replacement mice, homozygous for either APOE3 or APOE4 were randomized into exercised and sedentary groups. Baseline locomotor function and voluntary wheel-running behavior was reduced in APOE4 mice. Exercised groups were subjected to daily treadmill running for 8 weeks. ApoE protein in brain cortex was significantly increased by exercise in both genotypes. PGC-1α mRNA levels in brain cortex were significantly lower in APOE4 mice, and only tended to increase with exercise in both genotypes. Hippocampal BDNF protein were similar between genotypes and was not significantly modulated by treadmill running. Behavioral and biochemical variations between APOE3 and APOE4 mice likely contribute to the differential risk for neurological and vascular diseases and the exercise-induced increase in ApoE levels suggests an added feature of the potential efficacy of physical activity as a preventative and therapeutic strategy for neurogenerative processes in both genotypes.

APOE4 impairs myelination via cholesterol dysregulation in oligodendrocytes.

APOE4 is the strongest genetic risk factor for Alzheimer's disease1-3. However, the effects of APOE4 on the human brain are not fully understood, limiting opportunities to develop targeted therapeutics for individuals carrying APOE4 and other risk factors for Alzheimer's disease4-8. Here, to gain more comprehensive insights into the impact of APOE4 on the human brain, we performed single-cell transcriptomics profiling of post-mortem human brains from APOE4 carriers compared with non-carriers. This revealed that APOE4 is associated with widespread gene expression changes across all cell types of the human brain. Consistent with the biological function of APOE2-6, APOE4 significantly altered signalling pathways associated with cholesterol homeostasis and transport. Confirming these findings with histological and lipidomic analysis of the post-mortem human brain, induced pluripotent stem-cell-derived cells and targeted-replacement mice, we show that cholesterol is aberrantly deposited in oligodendrocytes-myelinating cells that are responsible for insulating and promoting the electrical activity of neurons. We show that altered cholesterol localization in the APOE4 brain coincides with reduced myelination. Pharmacologically facilitating cholesterol transport increases axonal myelination and improves learning and memory in APOE4 mice. We provide a single-cell atlas describing the transcriptional effects of APOE4 on the aging human brain and establish a functional link between APOE4, cholesterol, myelination and memory, offering therapeutic opportunities for Alzheimer's disease.