EFAD Mice Research Articles

The Effect of FTY720 on Sphingolipid Imbalance and Cognitive Decline in Aged EFAD Mice.

During Alzheimer's disease (AD) progression, there is a decline in the bioactive sphingolipid sphingosine-1-phosphate (S1P). Previous research showed that FTY720, an S1P mimetic, prevented cognitive decline and reduced ceramide levels in transgenic mice with familial AD carrying the human APOE4 gene (E4FAD) at 6-7 months of age. The objective of this study is to explore the protective effects of FTY720 at late-stage AD. Male mice aged 9.5 to 10.5 months were orally administered FTY720 (0.1 mg/kg) via oral gavage for 6 weeks. A pre-test of water maze was used for evaluating the pathological status. After 4 weeks of administration, memory, locomotion, and anxiety were assessed. Cortex samples were analyzed for amyloid-β (Aβ) and sphingolipid levels. Compared with APOE3 mice, APOE4, E3FAD and E4FAD mice exhibited significant memory deficits. After 6 weeks administration, FTY720 did not alleviate memory deficits in EFAD mice. Lipid analysis revealed that S1P was significantly reduced in EFAD mice (E3FAD or E4FAD) compared to controls (APOE3 and APOE4). Ceramide level alterations were predominantly dependent on APOE isoforms rather than AD transgenes. Interestingly, Cer (d18 : 1/22 : 1) was elevated in APOE4 mice compared to APOE3, and FTY720 reduced it. E4FAD and APOE4 mice exhibited significant spatial memory deficits and higher ceramide concentrations compared to APOE3 mice. FTY720 did not reverse memory deficits in E4FAD and APOE4 mice but reduced specific ceramide species. This study provides insights into the association between sphingolipids and APOE4 in advanced AD stages, exploring potential therapeutic targeting of sphingolipid metabolism.

Apolipoprotein E polymorphisms and female fertility in a transgenic mouse model of Alzheimer’s disease

Apolipoprotein E (APOE) is a major cholesterol carrier responsible for lipid transport and injury repair in the brain. The human APOE gene (h-APOE) has 3 naturally occurring alleles: ε3, the common allele; ε4, which increases Alzheimer’s disease (AD) risk up to 15-fold; and ε2, the rare allele which protects against AD. Although APOE4 has negative effects on neurocognition in old age, its persistence in the population suggests a survival advantage. We investigated the relationship between APOE genotypes and fertility in EFAD mice, a transgenic mouse model expressing h-APOE. We show that APOE4 transgenic mice had the highest level of reproductive performance, followed by APOE3 and APOE2. Intriguingly, APOE3 pregnancies had more fetal resorptions and reduced fetal weights relative to APOE4 pregnancies. In conclusion, APOE genotypes impact fertility and pregnancy outcomes in female mice, in concordance with findings in human populations. These mouse models may help elucidate how h-APOE4 promotes reproductive fitness at the cost of AD in later life.

MTOR inhibition enhances synaptic and mitochondrial function in Alzheimer’s disease in an APOE genotype-dependent manner

Apolipoprotein ε4 (APOE4) carriers develop brain metabolic dysfunctions decades before the onset of Alzheimer’s disease (AD). A goal of the study is to identify if rapamycin, an inhibitor for the mammalian target of rapamycin (mTOR) inhibitor, would enhance synaptic and mitochondrial function in asymptomatic mice with human APOE4 gene (E4FAD) before they showed metabolic deficits. A second goal is to determine whether there may be genetic-dependent responses to rapamycin when compared to mice with human APOE3 alleles (E3FAD), a neutral AD genetic risk factor. We fed asymptomatic E4FAD and E3FAD mice with control or rapamycin diets for 16 weeks from starting from 3 months of age. Neuronal mitochondrial oxidative metabolism and excitatory neurotransmission rates were measured using in vivo 1H-[13C] proton-observed carbon-edited magnetic resonance spectroscopy, and isolated mitochondrial bioenergetic measurements using Seahorse. We found that rapamycin enhanced neuronal mitochondrial function, glutamate-glutamine cycling, and TCA cycle rates in the asymptomatic E4FAD mice. In contrast, rapamycin enhances glycolysis, non-neuronal activities, and inhibitory neurotransmission of the E3FAD mice. These findings indicate that rapamycin might be able to mitigate the risk for AD by enhancing brain metabolic functions for cognitively intact APOE4 carriers, and the responses to rapamycin are varied by APOE genotypes. Consideration of precision medicine may be needed for future rapamycin therapeutics.

Apolipoprotein E4 interferes with lipid metabolism to exacerbate depression-like behaviors in 5xFAD mice.

Apolipoprotein E4 (ApoE4) allele is the strongest genetic risk factor for late-onset Alzheimer's disease, and it can aggravate depressive symptoms in non-AD patients. However, the impact of ApoE4 on AD-associated depression-like behaviors and its underlying pathogenic mechanisms remain unclear. This study developed a 5xFAD mouse model overexpressing human ApoE4 (E4FAD). Behavioral assessments and synaptic function tests were conducted to explore the effects of ApoE4 on cognition and depression in 5xFAD mice. Changes in peripheral and central lipid metabolism, as well as the levels of serotonin (5-HT) and γ-aminobutyric acid (GABA) neurotransmitters in the prefrontal cortex, were examined. In addition, the protein levels of 24-dehydrocholesterol reductase/glycogen synthase kinase-3 beta/mammalian target of rapamycin (DHCR24/GSK3β/mTOR) and postsynaptic density protein 95/calmodulin-dependent protein kinase II/brain-derived neurotrophic factor (PSD95/CaMK-II/BDNF) were measured to investigate the molecular mechanism underlying the effects of ApoE4 on AD mice. Compared with 5xFAD mice, E4FAD mice exhibited more severe depression-like behaviors and cognitive impairments. These mice also exhibited increased amyloid-beta deposition in the hippocampus, increased astrocyte numbers, and decreased expression of depression-related neurotransmitters 5-HT and GABA in the prefrontal cortex. Furthermore, lipid metabolism disorders were observed in E4FAD, manifesting as elevated low-density lipoprotein cholesterol and reduced high-density lipoprotein cholesterol in peripheral blood, decreased cholesterol level in the prefrontal cortex, and reduced expression of key enzymes and proteins related to cholesterol synthesis and homeostasis. Abnormal expression of proteins related to the DHCR24/GSK3β/mTOR and PSD95/CaMK-II/BDNF pathways was also observed. This study found that ApoE4 overexpression exacerbates depression-like behaviors in 5xFAD mice and confirmed that ApoE4 reduces cognitive function in these mice. The mechanism may involve the induction of central and peripheral lipid metabolism disorders. Therefore, modulating ApoE expression or function to restore cellular lipid homeostasis may be a promising therapeutic target for AD comorbid with depression. This study also provided a better animal model for studying AD comorbid with depression.

Estradiol improves behavior in FAD transgenic mice that express APOE3 but not APOE4 after ovariectomy.

Increasing evidence suggests that female individuals have a higher Alzheimer's disease (AD) risk associated with post-menopausal loss of circulating estradiol (E2). However, clinical data are conflicting on whether E2 lowers AD risk. One potential contributing factor is APOE. The greatest genetic risk factor for AD is APOE4, a factor that is pronounced in female individuals post-menopause. Clinical data suggests that APOE impacts the response of AD patients to E2 replacement therapy. However, whether APOE4 prevents, is neutral, or promotes any positive effects of E2 is unclear. Therefore, our goal was to determine whether APOE modulates the impact of E2 on behavior and AD pathology in vivo. To that end, mice that express human APOE3 (E3FAD) or APOE4 (E4FAD) and overproduce Aβ42 were ovariectomized at either 4 months (early) or 8 months (late) and treated with vehicle or E2 for 4 months. In E3FAD mice, we found that E2 mitigated the detrimental effect of ovariectomy on memory, with no effect on Aβ in the early paradigm and only improved learning in the late paradigm. Although E2 lowered Aβ in E4FAD mice in the early paradigm, there was no impact on learning or memory, possibly due to higher Aβ pathology compared to E3FAD mice. In the late paradigm, there was no effect on learning/memory and Aβ pathology in E4FAD mice. Collectively, these data support the idea that, in the presence of Aβ pathology, APOE impacts the response to E2 supplementation post-menopause.

A novel apoE-mimetic increases brain apoE levels, reduces Aβ pathology and improves memory when treated before onset of pathology in male mice that express APOE3

BackgroundAlzheimer’s disease (AD) is characterized by cognitive dysfunction and amyloid plaques composed of the amyloid-beta peptide (Aβ). APOE is the greatest genetic risk for AD with APOE4 increasing risk up to ~ 15-fold compared to APOE3. Evidence suggests that levels and lipidation of the apoE protein could regulate AD progression. In glia, apoE is lipidated via cholesterol efflux from intracellular pools, primarily by the ATP-binding cassette transporter A1 (ABCA1). Therefore, increasing ABCA1 activity is suggested to be a therapeutic approach for AD. CS-6253 (CS) is a novel apoE mimetic peptide that was developed to bind and stabilize ABCA1 and maintain its localization into the plasma membrane therefore promoting cholesterol efflux. The goal of this study was to determine whether CS could modulate apoE levels and lipidation, Aβ pathology, and behavior in a model that expresses human APOE and overproduce Aβ.MethodsIn vitro, APOE3-glia or APOE4-glia were treated with CS. In vivo, male and female, E3FAD (5xFAD+/−/APOE3+/+) and E4FAD (5xFAD+/−/APOE4+/+) mice were treated with CS via intraperitoneal injection at early (from 4 to 8 months of age) and late ages (from 8 to 10 months of age). ApoE levels, ABCA1 levels and, apoE lipidation were measured by western blot and ELISA. Aβ and amyloid levels were assessed by histochemistry and ELISA. Learning and memory were tested by Morris Water Maze and synaptic proteins were measured by Western blot.ResultsCS treatment increased apoE levels and cholesterol efflux in primary glial cultures. In young male E3FAD mice, CS treatment increased soluble apoE and lipid-associated apoE, reduced soluble oAβ and insoluble Aβ levels as well as Aβ and amyloid deposition, and improved memory and synaptic protein levels. CS treatment did not induce any therapeutic benefits in young female E3FAD and E4FAD mice or in any groups when treatment was started at later ages.ConclusionsCS treatment reduced Aβ pathology and improved memory only in young male E3FAD, the cohort with the least AD pathology. Therefore, the degree of Aβ pathology or Aβ overproduction may impact the ability of targeting ABCA1 to be an effective AD therapeutic. This suggests that ABCA1-stabilizing treatment by CS-6253 works best in conditions of modest Aβ levels.

APOE genotype and sex modulate Alzheimer's disease pathology in aged EFAD transgenic mice.

Increasing evidence supports that age, APOE and sex interact to modulate Alzheimer's disease (AD) risk, however the underlying pathways are unclear. One way that AD risk factors may modulate cognition is by impacting amyloid beta (Aβ) accumulation as plaques, and/or neuroinflammation Therefore, the goal of the present study was to evaluate the extent to which age, APOE and sex modulate Aβ pathology, neuroinflammation and behavior in vivo. To achieve this goal, we utilized the EFAD mice, which express human APOE3 or APOE4 and have five familial AD mutations (FAD) that result in Aβ42 overproduction. We assessed Aβ levels, reactive glia and Morris water maze performance in 6-, 10-, 14-, and 18-month-old EFAD mice. Female APOE4 mice had the highest Aβ deposition, fibrillar amyloid deposits and neuroinflammation as well as earlier behavior deficits. Interestingly, we found that female APOE3 mice and male APOE4 mice had similar levels of pathology. Collectively our data support that the combination of APOE4 and female sex is the most detrimental combination for AD, and that at older ages, female sex may be equivalent to APOE4 genotype.

A small-molecule TLR4 antagonist reduced neuroinflammation in female E4FAD mice

BackgroundAPOE genotype is the greatest genetic risk factor for sporadic Alzheimer’s disease (AD). APOE4 increases AD risk up to 12-fold compared to APOE3, an effect that is greater in females. Evidence suggests that one-way APOE could modulate AD risk and progression through neuroinflammation. Indeed, APOE4 is associated with higher glial activation and cytokine levels in AD patients and mice. Therefore, identifying pathways that contribute to APOE4-associated neuroinflammation is an important approach for understanding and treating AD. Human and in vivo evidence suggests that TLR4, one of the key receptors involved in the innate immune system, could be involved in APOE-modulated neuroinflammation. Consistent with that idea, we previously demonstrated that the TLR4 antagonist IAXO-101 can reduce LPS- and Aβ-induced cytokine secretion in APOE4 glial cultures. Therefore, the goal of this study was to advance these findings and determine whether IAXO-101 can modulate neuroinflammation, Aβ pathology, and behavior in mice that express APOE4.MethodsWe used mice that express five familial AD mutations and human APOE3 (E3FAD) or APOE4 (E4FAD). Female and male E4FAD mice and female E3FAD mice were treated with vehicle or IAXO-101 in two treatment paradigms: prevention from 4 to 6 months of age or reversal from 6 to 7 months of age. Learning and memory were assessed by modified Morris water maze. Aβ deposition, fibrillar amyloid deposition, astrogliosis, and microgliosis were assessed by immunohistochemistry. Soluble levels of Aβ and apoE, insoluble levels of apoE and Aβ, and IL-1β were measured by ELISA.ResultsIAXO-101 treatment resulted in lower Iba-1 coverage, lower number of reactive microglia, and improved memory in female E4FAD mice in both prevention and reversal paradigms. IAXO-101-treated male E4FAD mice also had lower Iba-1 coverage and reactivity in the RVS paradigm, but there was no effect on behavior. There was also no effect of IAXO-101 treatment on neuroinflammation and behavior in female E3FAD mice.ConclusionOur data supports that TLR4 is a potential mechanistic therapeutic target for modulating neuroinflammation and cognition in APOE4 females.

Higher Neuronal Facilitation and Potentiation with APOE4 Suppressed by Angiotensin II.

Progressive hippocampal degeneration is a key component of Alzheimer's disease (AD) progression. Therefore, identifying how hippocampal neuronal function is modulated early in AD is an important approach to eventually prevent degeneration. AD-risk factors and signaling molecules likely modulate neuronal function, including APOE genotype and angiotensin II. Compared to APOE3, APOE4 increases AD risk up to 12-fold, and high levels of angiotensin II are hypothesized to disrupt neuronal function in AD. However, the extent that APOE and angiotensin II modulates the hippocampal neuronal phenotype in AD-relevant models is unknown. To address this issue, we used electrophysiological techniques to assess the impact of APOE genotype and angiotensin II on basal synaptic transmission, presynaptic, and post-synaptic activity in mice that express human APOE3 (E3FAD) or APOE4 (E4FAD) and overproduce Aβ. We found that compared to E3FAD mice, E4FAD mice have lower synaptic activity, but higher levels of paired-pulse facilitation (PPF) and long-term potentiation (LTP) in the Schaffer Collateral Commissural Pathway (SCCP) of the hippocampus. We also found that exogenous angiotensin II has a profound inhibitory effect on hippocampal LTP in both E3FAD and E4FAD mice. Collectively, our data suggests that APOE4 and Aβ are associated with a hippocampal phenotype comprised of lower basal activity and higher responses to high-frequency stimulation, the latter of which is suppressed by angiotensin II. These novel data suggest a potential mechanistic link between hippocampal activity, APOE4 genotype, and angiotensin II in AD.

Sex specific molecular networks and key drivers of Alzheimer’s disease

BackgroundAlzheimer’s disease (AD) is a progressive and age-associated neurodegenerative disorder that affects women disproportionally. However, the underlying mechanisms are poorly characterized. Moreover, while the interplay between sex and ApoE genotype in AD has been investigated, multi-omics studies to understand this interaction are limited. Therefore, we applied systems biology approaches to investigate sex-specific molecular networks of AD.MethodsWe integrated large-scale human postmortem brain transcriptomic data of AD from two cohorts (MSBB and ROSMAP) via multiscale network analysis and identified key drivers with sexually dimorphic expression patterns and/or different responses to APOE genotypes between sexes. The expression patterns and functional relevance of the top sex-specific network driver of AD were further investigated using postmortem human brain samples and gene perturbation experiments in AD mouse models.ResultsGene expression changes in AD versus control were identified for each sex. Gene co-expression networks were constructed for each sex to identify AD-associated co-expressed gene modules shared by males and females or specific to each sex. Key network regulators were further identified as potential drivers of sex differences in AD development. LRP10 was identified as a top driver of the sex differences in AD pathogenesis and manifestation. Changes of LRP10 expression at the mRNA and protein levels were further validated in human AD brain samples. Gene perturbation experiments in EFAD mouse models demonstrated that LRP10 differentially affected cognitive function and AD pathology in sex- and APOE genotype-specific manners. A comprehensive mapping of brain cells in LRP10 over-expressed (OE) female E4FAD mice suggested neurons and microglia as the most affected cell populations. The female-specific targets of LRP10 identified from the single cell RNA-sequencing (scRNA-seq) data of the LRP10 OE E4FAD mouse brains were significantly enriched in the LRP10-centered subnetworks in female AD subjects, validating LRP10 as a key network regulator of AD in females. Eight LRP10 binding partners were identified by the yeast two-hybrid system screening, and LRP10 over-expression reduced the association of LRP10 with one binding partner CD34.ConclusionsThese findings provide insights into key mechanisms mediating sex differences in AD pathogenesis and will facilitate the development of sex- and APOE genotype-specific therapies for AD.

Altered L‐carnitine and acylcarnitine profiles in APOE e4 carriers with Alzheimer’s disease reflect impaired fatty acid oxidation.

AbstractBackgroundAbnormal lipid metabolism is now considered one of the key biological features of Alzheimer’s disease (AD). The L‐carnitine system plays an important role in lipid metabolism, particularly fatty acid (FA) transport and metabolism in mitochondria. Blood and tissue acylcarnitine accumulation reflects incomplete fatty acid beta‐oxidation (FAO), corresponding with insulin resistance, oxidative stress and inflammation. We hypothesized that changes in blood and brain markers of the L‐carnitine system would indicate dysfunctional FAO in aging e4 carriers, thereby influencing AD pathogenesis.MethodPlasma and brain L‐carnitine, L‐carnitine metabolites and acylcarnitine were analyzed in controls and patients with early or moderate AD stratified by APOE genotypes. The L‐carnitine system was examined over a range of ages (10 to 50 weeks) in plasma and brains from transgenic mice expressing different human APOE isoforms and EFAD mice with human APOE and 5 x AD mutations.ResultAmong e4 carriers, plasma L‐carnitine decreased, but its metabolite, Trimethylamine N‐oxide, increased in patients with early or moderate AD. Among non‐e4 carriers these changes are detected only in moderate AD patients. Among e4 carriers, plasma long chain acylcarnitines (LCA) and very‐LCA (VLCA) were lowest in moderate AD patients. In e4 carriers, plasma acylcarnitines correlated with cerebrovascular, amyloid and tau pathologies. Whereas in e2 carriers, both brain and plasma acylcarnitines were correlated with amyloid, tau and cerebrovascular pathologies. In APOE4‐TR mice, medium chain acylcarnitines (MCA) and LCA decreased with age within the cerebrovasculature. Parenchymal acylcarnitines increased at an earlier age (25 weeks) among APOE4‐TR mice compared to mice with other APOE genotypes (50 weeks).ConclusionThese data suggest that among e4 carriers, the L‐carnitine system‐mediated FAO is dysregulated early in the disease process of AD. Age‐dependent decreases in MCA and LCA in the cerebrovasculature in APOE4‐TR compared to APOE3‐TR mice indicate FAO deficits at an earlier age in APOE4‐TR. Understanding the role of APOE4 in impaired FAO with age in relation to the L‐carnitine system is required for developing novel preventative/therapeutic strategies for AD.

APOE e4 dependent deficits in brain DHA phospholipids and mfsd2a in Alzheimer’s disease patients with severe cerebral amyloid angiopathy

AbstractBackgroundThe apolipoprotein E e4 allele (APOE4) is a major risk factor, contributing to vascular pathologies in AD which include CAA, BBB dysfunction and reduced cerebral vascular integrity. The diminished capacity of APOE4 to transport docosahexaenoic acid (DHA), an essential fatty acid required for the structural and functional maintenance and vascular integrity of the brain, could also contribute to AD pathogenesis. However, it remains undetermined if changes in the brain DHA content of phospholipids (PL) and DHA transporters exist in relation to APOE4, AD diagnosis and CAA pathology.MethodWe performed liquid chromatography/mass spectrometry‐based PL analysis of the cerebrovascular and parenchymal fractions from autopsied human brain of pathologically confirmed AD cases and controls. We performed an antibody‐based examination of the major facilitator superfamily domain containing 2A (mfsd2a) protein in the cerebrovasculature from these subjects and brain homogenates from transgenic mice with human APOE (APOE‐TR) and APOE‐TR crossed with mice with five AD mutations (EFAD). We performed proteomic analyses of the cerebrovascular and parenchymal fractions of the brains from 50‐week‐old APOE‐TR.ResultIn the cerebrovasculature and parenchyma, DHA containing PL species were lower in ε4 carriers with AD compared to control ε4 carriers. We observed an ε4‐dependent decrease in mfsd2a level in the cerebrovasculature of ε4 carriers compared to non‐carriers. Stratification of DHA containing PL by CAA showed that these PL levels were reduced in ε4 positive AD patients with severe CAA. In addition, brain levels of DHA containing PC species and mfsd2a were lower in APOE4‐TR and E4FAD mice compared to other genotypes in each mouse model. Furthermore, protein levels of glucose transporter 1 (Glut1) and a metabolite transporter, monocarboxylate transporter 1 (MCT1), were lower in the cerebrovasculature of APOE4‐TR relative to APOE3‐TR and APOE2‐TR mice.ConclusionThese findings demonstrate that brain DHA deficiencies in ε4 carriers may be due to reduced mfsd2a expression and partly associated with severe CAA. Thus, targeting this transport mechanism may improve the bioavailability of DHA into the brains of ε4 carriers who are at risk of developing AD.

Chemotherapy promotes astrocytic response to Aβ deposition, but not Aβ levels, in a mouse model of amyloid and APOE

Many cancer survivors experience cancer-related cognitive impairment (CRCI), which is characterized by problems of attention, working memory, and executive function following chemotherapy and/or hormonal treatment. APOE4, the strongest genetic risk factor for Alzheimer's Disease (AD), is also a risk factor for CRCI, especially among survivors exposed to chemotherapy. We explored whether the effects of APOE genotype to chemotherapy were associated with an increase in AD pathological processes, using a mouse model of amyloid (5XFAD) along with the E3 or E4 alleles of human APOE (E3FAD and E4FAD). Six-month-old female E3FAD mice (control n = 5, treated n = 5) and E4FAD (control n = 6, treated n = 6) were treated with two doses of doxorubicin (total 10 mg/kg) or DMSO vehicle. After six weeks, mice were euthanized and brains were analyzed by immunohistochemistry and biochemical assays. Doxorubicin-treated mice had the same level of Aβ in the brain as control mice, as measured by 6E10 immunohistochemistry, Aβ40 and Aβ42 ELISAs, and plaque morphologies. Doxorubicin significantly increased the level of the astrocytic response to Aβ deposits, which was independent of APOE genotype; no effects of doxorubicin were observed on the microglial responses. These data are consistent with a model in which the effects of doxorubicin on risk of CRCI are unrelated amyloid accumulation, but possibly related to glial responses to damage.

Fasting-mimicking diet cycles reduce neuroinflammation to attenuate cognitive decline in Alzheimer's models.

The effects of fasting-mimicking diet (FMD) cycles in reducing many aging and disease risk factors indicate it could affect Alzheimer's disease (AD). Here, we show that FMD cycles reduce cognitive decline and AD pathology in E4FAD and 3xTg AD mouse models, with effects superior to those caused by protein restriction cycles. In 3xTg mice, long-term FMD cycles reduce hippocampal Aβ load and hyperphosphorylated tau, enhance genesis of neural stem cells, decrease microglia number, and reduce expression of neuroinflammatory genes, including superoxide-generating NADPH oxidase (Nox2). 3xTg mice lacking Nox2 or mice treated with the NADPH oxidase inhibitor apocynin also display improved cognition and reduced microglia activation compared with controls. Clinical data indicate that FMD cycles are feasible and generally safe in a small group of AD patients. These results indicate that FMD cycles delay cognitive decline in AD models in part by reducing neuroinflammation and/or superoxide production in the brain.

APOE4 homozygote females are resistant to the beneficial effects of 17β-estradiol on memory and CA1 dendritic spine density in the EFAD mouse model of Alzheimer's disease

Female APOE4 carriers are at greatest risk of Alzheimer's disease (AD). The potent estrogen 17β-estradiol (E2) may mediate AD risk, as the onset of memory decline coincides with the menopausal transition. Whether APOE genotype mediates E2’s effects on memory and neuronal morphology is poorly understood. We used the APOE+/+/5xFAD+/- (EFAD) mouse model to examine how APOE3 homozygote (E3FAD), APOE3/4 heterozygote (E3/4FAD), and APOE4 homozygote (E4FAD) genotypes modulate effects of E2 on object and spatial memory consolidation, dendritic spine density, and dorsal hippocampal estrogen receptor expression in 6-month-old ovariectomized EFAD mice. Dorsal hippocampal E2 infusion enhanced memory consolidation and increased CA1 apical spine density in E3FAD and E3/4FAD, but not E4FAD, mice. CA1 basal mushroom spines were also increased by E2 in E3FADs. E4FAD mice exhibited reduced CA1 and mPFC basal spine density, and increased dorsal hippocampal ERα protein, independent of E2. Overall, E2 benefitted hippocampal memory and structural plasticity in females bearing one or no APOE4 allele, whereas two APOE4 alleles impeded the memory-enhancing and spinogenic effects of E2.

APOE4 genotype or ovarian hormone loss influence open field exploration in an EFAD mouse model of Alzheimer's disease

Anxiety is a prominent and debilitating symptom in Alzheimer's disease (AD) patients. Carriers of APOE4, the greatest genetic risk factor for late-onset AD, may experience increased anxiety relative to carriers of other APOE genotypes. However, whether APOE4 genotype interacts with other AD risk factors to promote anxiety-like behaviors is less clear. Here, we used open field exploration to assess anxiety-like behavior in an EFAD mouse model of AD that expresses five familial AD mutations (5xFAD) and human APOE3 or APOE4. We first examined whether APOE4 genotype exacerbates anxiety-like exploratory behavior in the open field relative to APOE3 genotype in a sex-specific manner among six-month-old male and female E3FAD (APOE3+/+/5xFAD+/−) and E4FAD mice (APOE4+/+/5xFAD+/−). Next, we determined whether circulating ovarian hormone loss influences exploratory behavior in the open field among female E3FAD and E4FADs. APOE4 genotype was associated with decreased time in the center of the open field, particularly among female EFADs. Furthermore, ovariectomy (OVX) decreased time in the center of the open field among female E3FADs to levels similar to intact and OVXed E4FAD females. Our results suggest that APOE4 genotype increased anxiety-like behavior in the open field, and that ovarian hormones may protect against an anxiety-like phenotype in female E3FAD, but not E4FAD mice.

The detrimental effects of APOE4 on risk for Alzheimer's disease may result from altered dendritic spine density, synaptic proteins, and estrogen receptor alpha

Women carriers of APOE4, the greatest genetic risk factor for late-onset Alzheimer's disease (AD), are at highest risk of developing AD, yet factors underlying interactions between APOE4 and sex are not well characterized. Here, we examined how sex and APOE3 or APOE4 genotypes modulate object and spatial memory, dendritic spine density and branching, and protein expression in 6-month-old male and female E3FAD and E4FAD mice (APOE+/+/5xFAD+/−). APOE4 negatively impacted object recognition and spatial memory, with male E3FADs exhibiting the best memory across 2 object-based tasks. In both sexes, APOE4 reduced basal dendritic spine density in the medial prefrontal cortex and dorsal hippocampus. APOE4 reduced dorsal hippocampal levels of PDS-95, synaptophysin, and phospho-CREB, yet increased levels of ERα. E4FAD females exhibited strikingly increased GFAP levels, in addition to the lowest levels of PSD-95 and pCREB. Overall, our results suggest that APOE4 negatively impacts object memory, dendritic spine density, and levels of hippocampal synaptic proteins and ERα. However, the general lack of sex differences or sex by genotype interactions suggests that the sex-specific effects of APOE4 on AD risk may be related to factors unexplored in the present study.

APOE4 homozygotes are resistant to the mnemonic benefits of 17β-estradiol in the EFAD mouse model of Alzheimer's disease.

Loss of circulating estrogens at menopause may contribute to increased risk for Alzheimer's disease (AD) in females relative to males. APOE4, the greatest genetic risk factor for AD, acts synergistically with estrogens to increase risk in females. However, despite the relative contributions of APOE genotype and estrogen loss to sex differences in AD risk, these factors are rarely examined in concert in basic research. Hence, our knowledge of how APOE genotype may interact with estrogens to contribute to AD risk, or whether estrogens may be of benefit to particular genotypes, is limited. The present study was designed to test whether APOE genotype interacts with E2 to modulate cognition and dendritic spine density in a mouse model of AD. Effects of 17β-estradiol (E2 ) treatment on memory were examined in ovariectomized (OVXed) transgenic mice expressing 5 familial AD mutations (5XFAD) and two copies of human APOE3 (E3FAD), APOE4 (E4FAD), or one copy each of APOE3/APOE4 (E3/4FAD). OVXed female E3FAD, E3/4FAD, and E4FAD mice received bilateral dorsal hippocampal (DH) infusion of E2 immediately after training in object recognition (OR) and object placement (OP) tasks. Next, the effect of APOE genotype to modulate estrogenic effects on spines was examined. E3FAD, E3/4FAD, and E4FAD mice received DH infusion of E2 and brains were collected for Golgi impregnation and quantification of dendritic spine density 2 h later. DH infusion of E2 facilitated OR and OP memory in E3FAD and E3/4FADs, but not E4FADs. Similarly, DH infusion of E2 increased CA1 apical dendritic spine density in E3FADs and E3/4FADs, but not E4FADs. CA1 basal spine density was significantly reduced by E4FAD genotype. The significant benefit of E2 only in E3FAD and E3/4FAD mice suggests that having two copies of the APOE4 allele results in lower susceptibility to the memory-enhancing effects of E2 in the EFAD model. These findings provide novel evidence that E2 may be of clinical benefit to APOE3 and APOE3/4, but not homozygous APOE4, carriers.

A novel key driver gene of Alzheimer's disease impacts AD-related cognitive deficit, pathology and neuro-inflammation in sex- and APOE-specific manners.

Alzheimer's Disease (AD) has complex etiologies and the impact of sex on AD varies over the course of disease development. Literature provides some evidence of sex-specific contributions to AD. However, molecular mechanisms of sex biased differences in AD remain elusive. Multi-omics data in tandem with systems biology approaches offer a new avenue to not only dissect sex-stratified molecular mechanisms of AD but also develop sex-specific diagnostic and therapeutic strategies for AD. We integrated RNA expression datasets from the para-hippocampal gyrus of the Mount Sinai Medical Center Brain Bank (MSBB) and the prefrontal cortex from the Religious Orders Study and Rush Memory and Aging Project (ROSMAP) to perform multiscale network analysis. The differentially expressed genes (DEGs) between AD and control, female and male, ApoE3 and ApoE4 genotype were rank-ordered based on their relevance to clinical dementia rating scale (CDR) and AD pathology (Braak stage, CERAD and plaque density). The trend analyses and gene co-expression network analysis were then performed to identify key drivers with sexually dimorphic expression patterns or differentially respond to APOE genotypes between sexes. In this study, we identified ldl receptor-related protein (lrp10), a receptor possibly involved in the uptake of ApoE and APP trafficking as the highest-ranked key driver gene candidate accounting for the sex differences in AD. Lrp10 mRNA was increased in female AD patients when compared to female controls or male AD patients. In contrast, LRP10 protein levels were reduced in AD when compared to controls, suggesting possible post-translational modifications of LRP10 in AD human brains. Gene perturbation studies in EFAD mouse hippocampal brain regions suggest that over-expression of lrp10 improved cognitive function, reduced Aβ42 and p-Tau, and increased microglial recruitment around amyloid plaques in female E4FAD mice. In contrast, down-regulation of lrp10 exacerbated cognitive impairment as well as increased brain Aβ42 and total tau levels in male E3FAD mice. These results strongly support our hypothesis that lrp10 is a causal regulator gene for AD, whose expression is strongly associated with cognition performance in sex- and APOE-specific manners. Our findings further implicate a role of lrp10 in regulating microglial function and neuro-inflammation in AD.

APOE genotype and sex affect astrocyte interactions with amyloid plaques in EFAD transgenic mice.

Glial interactions with plaques are thought to affect Alzheimer's disease (AD) pathogenesis in both beneficial and deleterious manners. Positive effects of microglial interactions with amyloid plaques include both plaque compaction and protection of local neurites, perhaps resulting from barrier-like microglial formations. We previously demonstrated that both APOE4 genotype and female sex are associated with impaired microglial interactions with plaques. Although it is well-established that astrocytes have bidirectional functional interactions with microglia and are localized in close proximity to amyloid plaques, the role of astrocyte-plaque interactions are not well understood. Further, the potential moderating effects of APOE genotype and biological sex have not been investigated with regards to astrocyte-plaque interactions in AD. To investigate the role of APOE genotype and biological sex on astrocyte interactions with Aβ plaques, we studied both male and female EFAD mice, which have mouse APOE replaced with human APOE and are hemizygous for the 5xFAD transgenes. Fixed brains from 6-month-old mice were stained with amyloid dyes, immunolabeled with markers of astrocytes, microglia, and dystrophic neurites and analyzed with quantitative, high-resolution confocal microscopy. We observed that astrocytic processes interact with plaques in a similar way to microglia, in which coverage was highest in male APOE3 mice. Importantly, significant reductions in astrocyte coverage of amyloid plaques was observed with both APOE4 genotype and female sex. Plaque compaction also showed a similar pattern where APOE4 genotype and female sex were associated with significantly lower values relative to male sex and APOE3 genotype. Functionally, neuritic dystrophy was significantly higher with both APOE4 genotype and female sex within the plaque environment. These findings suggest that astrocytes complement microglial plaque coverage in a manner that appears to protect the neuronal milieu from plaque-associated damage. These protective astrocytic interactions are dependent on APOE genotype and sex, with outcomes being best in males with APOE3 and poorest in females with APOE4. Collectively, these findings provide new insight into the role of glia as contributors to the relationships among sex, APOE, and AD.