apoE Knock-in Research Articles

Little genomic support for Cyclophilin A-matrix metalloproteinase-9 pathway as a therapeutic target for cognitive impairment in APOE4 carriers

Therapeutic targets for halting the progression of Alzheimer’s disease pathology are lacking. Recent evidence suggests that APOE4, but not APOE3, activates the Cyclophilin-A matrix metalloproteinase-9 (CypA-MMP9) pathway, leading to an accelerated breakdown of the blood–brain barrier (BBB) and thereby causing neuronal and synaptic dysfunction. Furthermore, blockade of the CypA-MMP9 pathway in APOE4 knock-in mice restores BBB integrity and subsequently normalizes neuronal and synaptic function. Thus, CypA has been suggested as a potential target for treating APOE4 mediated neurovascular injury and the resulting neuronal dysfunction and degeneration. The odds of drug targets passing through clinical trials are greatly increased if they are supported by genomic evidence. We found little evidence to suggest that CypA or MMP9 affects the risk of Alzheimer’s disease or cognitive impairment using two-sample Mendelian randomization and polygenic risk score analysis in humans. This casts doubt on whether they are likely to represent effective drug targets for cognitive impairment in human APOE4 carriers.

Impact of APOE Genotype on Diet-induced Mitochondrial Adaptations in Mouse Skeletal Muscle

Lifestyle interventions based on behaviour change principles may provide a useful mechanism in reducing dementia risk amongst older adults, however intervention acceptability remains relatively unexplored. We assessed the feasibility and acceptability of BRAIN BOOTCAMP, an Australian initiative aiming to improve dementia literary and reduce dementia risk by delivering a brain health box addressing multiple lifestyle factors through education, physical prompts and an individualised brain health profile. Semi-structured phone interviews were conducted with participants (N=94) at completion of the program (3-months) using a theoretical sampling approach to select a range of participants with varying brain health scores, age, gender, education and locality. Interview topics included participants’ overall experience and suggestions for program improvement. Interviews were transcribed and analysed using thematic analysis. Participants were mostly female (79%), with a mean age of 72.6 years (SD=5.4), from an English-speaking background (89.4%) and resided in metropolitan areas (76.6%). Participants positively perceived the program, resulting in high usability and acceptability. Valuable aspects included building dementia awareness in an innovative way, and having re-assessments which identified areas for personal improvement. Participants further discussed how the program prompted lifestyle change, including setting goals (e.g., physical activity) and facilitated a general awareness of their brain health. Suggested improvements included shorter surveys, regular check-ins, and specific tailoring of the program to be more inclusive for older adults with varying levels of health. Our study demonstrated that a simple, innovative program could be a promising medium for delivering comprehensive educational resources and induce lifestyle change for older adults.

Impact of APOE Genotype on Diet-induced Mitochondrial Adaptations in Mouse Skeletal Muscle

The forecasted adoption of autonomous vehicles (AVs) will lead to major changes to the job of truck driving. These changes may be particularly challenging for drivers, as the population of truck drivers skews much older than that of other occupations. In this study we sought to understand truck drivers’ attitudes towards AVs and the longevity of their job. We conducted focus groups with truck drivers, their supervisors, and upper-level managers of trucking companies. We relate supervisors’ and managers’ experiences working with drivers through the rollout of new technologies to further understand drivers’ initial reactions to automation and how their attitudes may develop. Based on qualitative open coding our analysis uncovered two overarching themes. The first theme is the unknown. With AVs, companies expect that experience will be less important, so they can hire younger workers. In response, drivers have expressed fear of being displaced and anxiety over the uncertainty of not knowing how their jobs will be affected. The second theme is adaptability, and desire to adapt. Older drivers have expressed resistance to adapting to AVs and to their job changing. Concerningly however, managers envision the need for a driving workforce that has experience working with technology and is adaptable. Our study identifies key challenges concerning older workers’ reactions and career decisions in response to automation. Accounting for driver reactions to AVs is necessary not only to build theory and understanding on worker reactions to automation, but also for workforce planning and to support employees, particularly older workers.

Cadmium exposure modulates the gut-liver axis in an Alzheimer\u2019s disease mouse model

The human Apolipoprotein E4 (ApoE4) variant is the strongest known genetic risk factor for Alzheimer’s disease (AD). Cadmium (Cd) has been shown to impair learning and memory at a greater extent in humanized ApoE4 knock-in (ApoE4-KI) mice as compared to ApoE3 (common allele)-KI mice. Here, we determined how cadmium interacts with ApoE4 gene variants to modify the gut-liver axis. Large intestinal content bacterial 16S rDNA sequencing, serum lipid metabolomics, and hepatic transcriptomics were analyzed in ApoE3- and ApoE4-KI mice orally exposed to vehicle, a low dose, or a high dose of Cd in drinking water. ApoE4-KI males had the most prominent changes in their gut microbiota, as well as a predicted down-regulation of many essential microbial pathways involved in nutrient and energy homeostasis. In the host liver, cadmium-exposed ApoE4-KI males had the most differentially regulated pathways; specifically, there was enrichment in several pathways involved in platelet activation and drug metabolism. In conclusion, Cd exposure profoundly modified the gut-liver axis in the most susceptible mouse strain to neurological damage namely the ApoE4-KI males, evidenced by an increase in microbial AD biomarkers, reduction in energy supply-related pathways in gut and blood, and an increase in hepatic pathways involved in inflammation and xenobiotic biotransformation.

Neurobiological role of Alzheimer's disease genetic risk factor ApoE on early synaptic changes in Alzheimer-like models.

The presence of an apolipoprotein E4 (ApoE4) genotype is the major genetic risk factor for Alzheimer's disease (AD). Astrocytes are the main source of ApoE in the brain, however ApoE can also be produced by neurons and microglia. ApoE plays a role in many cell types and processes related to AD, however, it remains unclear which mechanism(s) and cellular source of ApoE are most critical for AD. One of the earliest changes in AD are early cellular changes such as endosomal and synaptic alterations. ApoE4 has been associated with impaired endosomal trafficking and dysregulated synaptic plasticity. Neuronal hyperexcitability has been reported in mice expressing human ApoE4, a dysregulation that is also seen in AD transgenic mice. Although ApoE seems to play a crucial role in neuronal changes linked to early AD, ApoE's synaptic localization and mechanisms remain poorly understood. In this study, the aim is to determine the role of ApoE, in particular ApoE4, on synaptic alterations in AD models. Mouse neurons and astrocytes are derived from wild-type, ApoE knock-out (KO), humanized ApoE3 and ApoE4 knock-in mice. Astrocyte-conditioned medium from mouse astrocytes expressing human ApoE and recombinant ApoE are used as a source for human ApoE to treat ApoE KO, wild-type and AD transgenic APP/PS1 neurons. Additionally, ApoE KO and humanized ApoE neurons are treated with synthetic Aβ or vehicle control. Analysis is performed using immunofluorescence, confocal and live cell imaging. Exogenously added and endogenously produced human ApoE is shown to be present at neurites and synaptic terminals of cultured neurons. Differences in neuronal activity are observed among different ApoE conditions using Ca2+ live cell microscopy, both in the presence and absence of elevated human Aβ. Added recombinant and endogenous ApoE appear to be present in the endosome-lysosome system of neurons. ApoE appears to localize at synapses and endosomes, sites associated with early cellular changes in AD, and seems to play a role in neuronal excitability. Determining the neurobiology of ApoE, in particular in connection with cellular sites vulnerable to early changes in AD, can contribute to a better understanding of the role of ApoE in AD.

Evidence that the gut microbiota regulates progression of neurodegeneration in a mouse model of tauopathy, in a sex‐ and ApoE isoform‐dependent manner

Alzheimer's disease (AD) is a fatal progressive neurodegenerative disease. Mounting evidence supports that an unbalanced gut microbiota (GM) is linked to amyloid-β deposition potentially by disrupting neuroinflammation and metabolic homeostasis. However, the contribution of the GM to tau-mediated neurodegeneration is poorly characterized. Furthermore, recent studies have reported that the configuration of the GM is also affected by ApoE isoforms, which is the strongest genetic risk factor for late-onset AD. Here, we explore the hypothesis that the GM regulates tau-mediated neurodegeneration in a sex- and ApoE isoform- dependent manner, using a mouse model of tauopathy.Male and female P301S tau transgenic mice were crossed with human ApoE knock-in mice (ApoE3 or ApoE4) or ApoE knockout mice (EKO) to generate P301S::ApoE3/E3, ApoE4/E4, or P301S/EKO mice, termed TE3F, TE4F, and TEKO, respectively. The GM in each group was perturbed by gastric gavage of a combination of antibiotics (ABX) from post-natal day 16-22; controls were gavaged with water (H2O). Mice were housed in a specific pathogen-free facility and fed a standard mouse chow diet ad libitum until they were euthanized at 9 months of age. In addition, a separate group of male and female TE4F mice were housed under germ-free conditions. Collected brains were sectioned, stained with 0.1% Sudan black and used to measure volumes of regions of interest.In males, TE3F and TE4F mice treated with H2O showed significant hippocampal atrophy compared to P301S mice lacking a functional ApoE gene (TEKO). However, TE3F mice treated with a short-term ABX showed significantly milder hippocampal atrophy compared to the water-treated group (p < 0.001, t-test). ABX treatment of TE4F animals was also associated with milder atrophy, although the effect did not reach statistical significance (p = 0.09, t-test). There were no effects in TEKO mice treated with ABX. Remarkably, these phenotypic effects of ABX treatment were not observed in females. Moreover, germ-free TE4F mice showed a marked decrease in brain atrophy compared to conventionally-raised animals; this was true for both sexes.Our results indicate that tau-mediated neurodegeneration occurs in a ApoE- dependent manner and is influenced by the gut microbiota and gender.

APOE variant in the receptor binding domain confers cognitive resilience to familial Alzheimer's mutations and cell-type specific gene expression changes in the hippocampus.

Cognitive resilience to familial Alzheimer's Disease (FAD) is a phenomenon whereby cognitive functioning is better than predicted based on highly penetrant mutations in APP and PS1. This resilience is likely mediated by unidentified genetic factors, and we hypothesize that these factors provide key targets for treatment and prevention of AD. We previously discovered that the most commonly used background strain in AD model research (C57BL/6J, B6) is resilient to AD-related cognitive decline. Using genetic mapping, we determined that cognitive resilience is associated with a variant in the Apoe receptor binding domain and utilized CRISPR to generate a novel Apoe knockin mouse (Apoe KIB6/E163D ). Contextual fear conditioning (CFC), tau pathology, and single-nuclear RNAseq hippocampi analyses were performed in male and female Apoe KIB6/E163D x 5XFAD progeny that differed by only a single variant in Apoe leading to the E163D base pair change in the protein. We found that, indeed, cognitive resilience to the 5XFAD transgene was conferred in ApoeB6/B6 mice compared to Apoe KIB6/E163D mice tested on CFC. Although minimal differences in hyperphosphorylated tau abundance were observed between 5XFAD-ApoeWT/WT mice compared to 5XFAD-Apoe KIB6/E163D , we uncovered robust differences in the transcriptomes of hippocampal excitatory and inhibitory neurons and microglia using single-nuclear sequencing. Gene enrichment and pathway analyses of differentially expressed genes suggest that the 5XFAD-ApoeB6/B6 genotype promotes cation transport and suppression of amyloid fibril formation in excitatory neurons, promotes genes involved in ribosome assembly and protein translation in inhibitory neurons, and enhances gene expression associated with stress response in microglia. Our results suggest that 5XFAD-ApoeB6/B6 homozygotes exhibit cognitive resilience to causal FAD mutations that involve dynamic regulation of gene networks across multiple cell types, emphasizing the need for well-aligned cell culture models of cognitive resilience to validate targets and screen resilience-based interventions. Future work leveraging in vivo electrophysiology and imaging will establish mechanisms whereby Apoe variants alter neural and synaptic function. Understanding the relationship between protective genetic factors and cognitive resilience are crucial for determining mechanisms of resilience and uncovering novel targets for intervention.

Effect of APOE alleles on the glial transcriptome in normal aging and Alzheimer's disease.

The roles of APOEε4 and APOEε2-the strongest genetic risk and protective factors for Alzheimer's disease-in glial responses remain elusive. We tested the hypothesis that APOE alleles differentially impact glial responses by investigating their effects on the glial transcriptome from elderly control brains with no neuritic amyloid plaques. We identified a cluster of microglial genes that are upregulated in APOEε4 and downregulated in APOEε2 carriers relative to APOEε3 homozygotes. This microglia-APOE cluster is enriched in phagocytosis-including TREM2 and TYROBP-and proinflammatory genes, and is also detectable in brains with frequent neuritic plaques. Next, we tested these findings in APOE knock-in mice exposed to acute (lipopolysaccharide challenge) and chronic (cerebral β-amyloidosis) insults and found that these mice partially recapitulate human APOE-linked expression patterns. Thus, the APOEε4 allele might prime microglia towards a phagocytic and proinflammatory state through an APOE-TREM2-TYROBP axis in normal aging as well as in Alzheimer's disease.

High-fat diet increases gliosis and immediate early gene expression in APOE3 mice, but not APOE4 mice

BackgroundAPOE4 is the strongest genetic risk factor for Alzheimer’s disease (AD), and obesity is a strong environmental risk factor for AD. These factors result in multiple central nervous system (CNS) disturbances and significantly increase chances of AD. Since over 20% of the US population carry the APOE4 allele and over 40% are obese, it is important to understand how these risk factors interact to affect neurons and glia in the CNS.MethodsWe fed male and female APOE3 and APOE4 knock-in mice a high-fat diet (HFD-45% kcal fat) or a "control" diet (CD-10% kcal fat) for 12 weeks beginning at 6 months of age. At the end of the 12 weeks, brains were collected and analyzed for gliosis, neuroinflammatory genes, and neuronal integrity.ResultsAPOE3 mice on HFD, but not APOE4 mice, experienced increases in gliosis as measured by GFAP and Iba1 immunostaining. APOE4 mice on HFD showed a stronger increase in the expression of Adora2a than APOE3 mice. Finally, APOE3 mice on HFD, but not APOE4 mice, also showed increased neuronal expression of immediate early genes cFos and Arc.ConclusionsThese findings demonstrate that APOE genotype and obesity interact in their effects on important processes particularly related to inflammation and neuronal plasticity in the CNS. During the early stages of obesity, the APOE3 genotype modulates a response to HFD while the APOE4 genotype does not. This supports a model where early dysregulation of inflammation in APOE4 brains could predispose to CNS damages from various insults and later result in the increased CNS damage normally associated with the APOE4 genotype.

Aggregate Trends of Apolipoprotein E on Cognition in Transgenic Alzheimer's Disease Mice.

Background:Apolipoprotein E (APOE) genotypes typically increase risk of amyloid-β deposition and onset of clinical Alzheimer’s disease (AD). However, cognitive assessments in APOE transgenic AD mice have resulted in discord.Objective:Analysis of 31 peer-reviewed AD APOE mouse publications (n = 3,045 mice) uncovered aggregate trends between age, APOE genotype, gender, modulatory treatments, and cognition.Methods:T-tests with Bonferroni correction (significance = p < 0.002) compared age-normalized Morris water maze (MWM) escape latencies in wild type (WT), APOE2 knock-in (KI2), APOE3 knock-in (KI3), APOE4 knock-in (KI4), and APOE knock-out (KO) mice. Positive treatments (t+) to favorably modulate APOE to improve cognition, negative treatments (t–) to perturb etiology and diminish cognition, and untreated (t0) mice were compared. Machine learning with random forest modeling predicted MWM escape latency performance based on 12 features: mouse genotype (WT, KI2, KI3, KI4, KO), modulatory treatment (t+, t–, t0), mouse age, and mouse gender (male = g_m; female = g_f, mixed gender = g_mi).Results:KI3 mice performed significantly better in MWM, but KI4 and KO performed significantly worse than WT. KI2 performed similarly to WT. KI4 performed significantly worse compared to every other genotype. Positive treatments significantly improved cognition in WT, KI4, and KO compared to untreated. Interestingly, negative treatments in KI4 also significantly improved mean MWM escape latency. Random forest modeling resulted in the following feature importance for predicting superior MWM performance: [KI3, age, g_m, KI4, t0, t+, KO, WT, g_mi, t–, g_f, KI2] = [0.270, 0.094, 0.092, 0.088, 0.077, 0.074, 0.069, 0.061, 0.058, 0.054, 0.038, 0.023].Conclusion:APOE3, age, and male gender was most important for predicting superior mouse cognitive performance.

Neurovascular imaging with QUTE-CE MRI in APOE4 rats reveals early vascular abnormalities.

Cerebrovascular abnormality is linked to Alzheimer’s disease and related dementias (ADRDs). ApoE-Ɛ4 (APOE4) is known to play a critical role in neurovascular dysfunction, however current medical imaging technologies are limited in quantification. This cross-sectional study tested the feasibility of a recently established imaging modality, quantitative ultra-short time-to-echo contrast-enhanced magnetic resonance imaging (QUTE-CE MRI), to identify small vessel abnormality early in development of human APOE4 knock-in female rat (TGRA8960) animal model. At 8 months, 48.3% of the brain volume was found to have significant signal increase (75/173 anatomically segmented regions; q<0.05 for multiple comparisons). Notably, vascular abnormality was detected in the tri-synaptic circuit, cerebellum, and amygdala, all of which are known to functionally decline throughout AD pathology and have implications in learning and memory. The detected abnormality quantified with QUTE-CE MRI is likely a result of hyper-vascularization, but may also be partly, or wholly, due to contributions from blood-brain-barrier leakage. Further exploration with histological validation is warranted to verify the pathological cause. Regardless, these results indicate that QUTE-CE MRI can detect neurovascular dysfunction with high sensitivity with APOE4 and may be helpful to provide new insights into health and disease.

Region-Specific Differences in the Apoe4-dependent Response to Focal Brain Injury.

Apolipoprotein E (apoE) plays a role in various physiological functions including lipid transport, synaptic plasticity, and immune modulation. Epidemiological studies suggest that the apoE4 allele increases the risk of post-traumatic sequelae. This study was performed to investigate region-specific effects of the apoE4 isoform on post-traumatic neurodegeneration. Two focal brain injuries were introduced separately in the motor cortex and hippocampus of apoE4 knock-in, apoE3 knock-in, apoE knockout, and wild-type (WT) mice. Western blotting showed that the expression levels of pre-synaptic and post-synaptic markers at the recovery stage were lower in the hippocampal injury core in apoE4 mice, compared with apoE3 and WT mice. Fast glial activation (determined by immunohistochemistry with glial fibrillary acidic protein, ionized calcium binding adaptor molecule 1, and cluster of differentiation 45 antibodies) was characteristic of apoE4 mice with hippocampal injury penumbra. apoE4-specific changes were not observed after cortical injury. The intensity of microglial activation in the hippocampus was inversely correlated with the volume of injury reduction on sequential magnetic resonance imaging examinations, when validated using matched samples. These findings indicate that the effects of the interaction between apoE4 and focal brain damage are specific to the hippocampus. Manipulation of inflammatory cell responses could be beneficial for reducing post-traumatic hippocampal neurodegeneration in apoE4 carriers.

Features of Lipid Metabolism in Humanized ApoE Knockin Rat Models.

Apolipoprotein E (ApoE), an essential plasma apolipoprotein, has three isoforms (E2, E3, and E4) in humans. E2 is associated with type III hyperlipoproteinemia. E4 is the major susceptibility gene to Alzheimer’s disease (AD) and coronary heart disease (CHD). We investigated lipid metabolism and atherosclerotic lesions of novel humanized ApoE knockin (hApoE KI) rats in comparison to wide-type (WT) and ApoE knockout (ApoE KO) rats. The hApoE2 rats showed the lowest bodyweight and white fat mass. hApoE2 rats developed higher serum total cholesterol (TC), total triglyceride (TG), and low- and very low density lipoprotein (LDL-C&VLDL-C). ApoE KO rats also exhibited elevated TC and LDL-C&VLDL-C. Only mild atherosclerotic lesions were detected in hApoE2 and ApoE KO aortic roots. Half of the hApoE2 rats developed hepatic nodular cirrhosis. A short period of the Paigen diet (PD) treatment led to the premature death of the hApoE2 and ApoE KO rats. Severe vascular wall thickening of the coronary and pulmonary arteries was observed in 4-month PD-treated hApoE4 rats. In conclusion, hApoE2 rats develop spontaneous hyperlipidemia and might be suitable for studies of lipid metabolism-related diseases. With the PD challenge, hApoE4 KI rats could be a novel model for the analysis of vascular remodeling.

APOE4 accelerates advanced-stage vascular and neurodegenerative disorder in old Alzheimer's mice via cyclophilin A independently of amyloid-β.

Apolipoprotein E4 (APOE4), the main susceptibility gene for Alzheimer's disease (AD), leads to vascular dysfunction, amyloid-β pathology, neurodegeneration and dementia. How these different pathologies contribute to advanced-stage AD remains unclear. Using aged APOE knock-in mice crossed with 5xFAD mice, we show that, compared to APOE3, APOE4 accelerates blood-brain barrier (BBB) breakdown, loss of cerebral blood flow, neuronal loss and behavioral deficits independently of amyloid-β. BBB breakdown was associated with activation of the cyclophilin A-matrix metalloproteinase-9 BBB-degrading pathway in pericytes. Suppression of this pathway improved BBB integrity and prevented further neuronal loss and behavioral deficits in APOE4;5FAD mice while having no effect on amyloid-β pathology. Thus, APOE4 accelerates advanced-stage BBB breakdown and neurodegeneration in Alzheimer's mice via the cyclophilin A pathway in pericytes independently of amyloid-β, which has implication for the pathogenesis and treatment of vascular and neurodegenerative disorder in AD.

Neuronal ApoE upregulates MHC-I expression to drive selective neurodegeneration in Alzheimer's disease.

Selective neurodegeneration is a critical causal factor in Alzheimer's disease (AD); however, the mechanisms that lead some neurons to perish, whereas others remain resilient, are unknown. We sought potential drivers of this selective vulnerability using single-nucleus RNA sequencing and discovered that ApoE expression level is a substantial driver of neuronal variability. Strikingly, neuronal expression of ApoE-which has a robust genetic linkage to AD-correlated strongly, on a cell-by-cell basis, with immune response pathways in neurons in the brains of wild-type mice, human ApoE knock-in mice and humans with or without AD. Elimination or over-expression of neuronal ApoE revealed a causal relationship among ApoE expression, neuronal MHC-I expression, tau pathology and neurodegeneration. Functional reduction of MHC-I ameliorated tau pathology in ApoE4-expressing primary neurons and in mouse hippocampi expressing pathological tau. These findings suggest a mechanism linking neuronal ApoE expression to MHC-I expression and, subsequently, to tau pathology and selective neurodegeneration.

Characterization of Lipid Metabolism and Atherosclerotic Phenotypes in Novel Humanized ApoE Knockin Rats

Apolipoprotein E (ApoE) is one of the essential plasma apolipoproteins involved majorly in lipids’ metabolism. Human ApoE has three isoforms (E2, E3, and E4) differing by one or two amino acids. E3 is the most common one with a population allelic frequency of 78% and considered normal. E2 (7%) is associated with genetic disorder type III hyperlipoproteinemia. E4 (15%) is the major susceptibility gene related to the onset of Alzheimer's disease (AD) and cardiovascular diseases (CVDs). Gene modified mice carrying a mutant ApoE gene or different ApoE isoforms, such as ApoE knockout (ApoE KO) mouse and humanized ApoE knockin (hApoE KI) mice, provide a series of beneficial mouse models in the studies of atherosclerosis, hyperlipidemia, and AD. However, alternative animal models are necessary to investigate the roles and underlying mechanisms of ApoE in human diseases. Recently, the novel hApoE KI rat models created by Envigo RMS, Saint Louis, MO (preciously named Horizon Discovery and SAGE Labs respectively) by replacing endogenous rat ApoE with human ApoE2, E3, or E4 allele are available. Hypothesis The hApoE KI rats could recapitulate the diversities of metabolic phenotypes in human carrying polymorphic ApoE proteins, and will be ideal animal models to study the roles of ApoE proteins. Methods We examed the body weight, serum lipid levels, the mass of fat tissue, and vascular atherosclerotic lesions of these hApoE rat models in comparison to wide-type Sprague Dawley (WT) and ApoE KO rats, as well as the metabolic changes, death rate, and development of vascular atherosclerosis of these rats with 4-months atherogenic Paigen diet (PD) (40 kcal% Fat, 1.25% Cholesterol, 0.5% Cholic Acid). Results We investigate the body weight starting from week seven till month six, and found that all the hApoE KI and ApoE KO rats had a lower body weight than WT at the corresponding age. The hApoE2 KI rats showed lowest body weight. At six months (P6m), all hApoE KI and KO rats had lower anti-atherogenic lipoprotein HDL. The total cholesterol (TC), total triacylglycerol (TG), and LDL/VLDL of hApoE2 KI rats were higher than those of WT, hApoE3 KI and hApoE4 KI rats. ApoE KO rats also exhibited elevated total cholesterol and LDL/VLDL, but not total triacylglycerol. Surprisingly, the mass of white fat was less in hApoE2 KI rats. No obvious aortic plaques were identified in the en face aortas stained by Oil Red O of all normal diet-fed hApoE KI and ApoE KO rats at P6m. A mild atherosclerotic lesion was detected in frozen section of hApoE2 KI and ApoE KO aortic roots. After challenged with the PD, hApoE2 KI and ApoE KO rats died within two and half weeks. Causes of death were different: hApoE2 KI rats might be due to fat embolism in big vessels and heart chambers; ApoE KO rats might be caused by massive pulmonary alveolar hydrops (accompanying considerable lipid-laden macrophages accumulated in the lung). Thicker walls of small vessels in lung and heart, as well as necrosis of cardiomyocytes, were demonstrated in hApoE4 KI rats after four months PD. Conclusion hApoE2 rats develop hyperlipoproteinemia but are not a suitable animal model for the study of atherosclerosis. With the assistance of a PD challenge, hApoE4 KI rats could be a good model for the analysis of CVDs.

Surgery Performed Under Propofol Anesthesia Induces Cognitive Impairment and Amyloid Pathology in ApoE4 Knock-In Mouse Model.

Background: Postoperative cognitive dysfunction (POCD) following anesthesia and surgery is a common and severe complication, especially in elderly patients. A pre-existing cognitive impairment may impart susceptibility to further cognitive dysfunction; the mechanism remains unclear. We hypothesized that the specific impacts of anesthesia and surgery on individuals with preclinical Alzheimer’s disease (AD) may render them more susceptible to an increase in the risk of cognitive impairment. The aim of this study was to compare the cognitive impairment between normal adult mice and those with preclinical AD after propofol anesthesia and surgery.Methods: We performed abdominal surgery in cognitively pre-symptomatic, 5-month-old male mice with sporadic AD (apolipoprotein E4 allele, ApoE4-KI) and age-matched (C57BL/6J) controls. Propofol anesthesia (170 mg/kg) was induced via retro-orbital injection over 2 h. Morris water maze (MWM) and Y-maze tests were conducted 2 days before and 2, 4, and 7 days after surgery. The mean escape latencies and spontaneous alternation percentages were the major outcomes. Neuronal apoptosis in hippocampal sections was evaluated using the terminal dUTP nick-end labeling (TUNEL) assay. Hippocampal amyloid beta (Aβ) levels were assessed via quantitative immunohistochemistry (IHC).Results: The control mice exhibited increased mean escape latencies of MWM at postoperative 2 and 4, but not at day 7; ApoE4-KI mice exhibited such increases at postoperative days 2, 4 and 7. Significant differences between ApoE4-KI and control mice in terms of the mean escape latencies were evident at days 2 and 7 (both P < 0.05). However, performance on a non-hippocampal memory tasks (Y-maze test) did not differ. More TUNEL-positive neurons were evident in the hippocampal CA3 region of ApoE4-KI mice at postoperative days 2 and 4, but not at day 7 compared to the control group (both P < 0.05). IHC revealed significantly elevated Aβ deposition in the hippocampal CA3 region of ApoE4-KI mice at postoperative days 4 and 7 compared to control mice (both P < 0.05).Conclusions: Propofol anesthesia followed by surgery induced persistent changes in cognition, and pathological hippocampal changes in pre-symptomatic, but vulnerable AD mice. It would be appropriate to explore whether preclinical AD patients are more vulnerable to POCD development.

Glutamine Antagonist JHU-083 Normalizes Aberrant Hippocampal Glutaminase Activity and Improves Cognition in APOE4 Mice

Given the emergent aging population, the identification of effective treatments for Alzheimer's disease (AD) is critical. We investigated the therapeutic efficacy of JHU-083, a brain-penetrable glutamine antagonist, in treating AD using the humanized APOE4 knock-in mouse model. Cell culture studies were performed using BV2 cells and primary microglia isolated from hippocampi of adult APOE4 knock-in mice to evaluate the effect of JHU-083 treatment on LPS-induced glutaminase (GLS) activity and inflammatory markers. Six-month-old APOE4 knock-in mice were administered JHU-083 or vehicle via oral gavage 3x/week for 4-5 months and cognitive performance was assessed using the Barnes maze. Target engagement in the brain was confirmed using a radiolabeled GLS enzymatic activity assay, and electrophysiology, gastrointestinal histology, blood chemistry, and CBC analyses were conducted to evaluate the tolerability of JHU-083. JHU-083 inhibited the LPS-mediated increases in GLS activity, nitic oxide release, and pro-inflammatory cytokine production in cultured BV2 cells and primary microglia isolated from APOE4 knock-in AD mice. Chronic treatment with JHU-083 in APOE4 mice improved hippocampal-dependent Barnes maze performance. Consistent with the cell culture findings,postmortem analyses of APOE4 mice showed increased GLS activity in hippocampal CD11b+ enriched cells versus age-matched controls, which was completely normalized by JHU-083 treatment. JHU-083 was well-tolerated, showing no weight loss effect or overt behavioral changes. Peripheral nerve function, gastrointestinal histopathology, and CBC/clinical chemistry parameters were all unaffected by chronic JHU-083 treatment. These results suggest that the attenuation of upregulated hippocampal glutaminase by JHU-083 represents a new therapeutic strategy for AD.

In Vivo Chimeric Alzheimer’s Disease Modeling of Apolipoprotein E4 Toxicity in Human Neurons

Despite its clear impact on Alzheimer's disease (AD) risk, apolipoprotein (apo) E4's contributions to AD etiology remain poorly understood. Progress in answering this and other questions in AD research has been limited by an inability to model human-specific phenotypes in an invivo environment. Here we transplant human induced pluripotent stem cell (hiPSC)-derived neurons carrying normal apoE3 or pathogenic apoE4 into human apoE3 or apoE4 knockin mouse hippocampi, enabling us to disentangle the effects of apoE4 produced in human neurons and in the brain environment. Using single-nucleus RNA sequencing (snRNA-seq), we identify key transcriptional changes specific to human neuron subtypes in response to endogenous or exogenous apoE4. We also find that Aβ from transplanted human neurons forms plaque-like aggregates, with differences in localization and interaction with microglia depending on the transplant and host apoE genotype. These findings highlight the power of invivo chimeric disease modeling for studying AD.

A mouse model of chemotherapy-related cognitive impairments integrating the risk factors of aging and APOE4 genotype

Some cancer survivors experience marked cognitive impairment, referred to as cancer-related cognitive impairment (CRCI). CRCI has been linked to the genetic factor APOE4, the strongest genetic risk factor for Alzheimer’s disease (AD). We used APOE knock-in mice to test whether the relationship between APOE4 and CRCI can be demonstrated in a mouse model, to identify associations of chemotherapy with behavioural and structural correlates of cognition, and to test whether chemotherapy affects markers of AD. Twelve-month old C57BL/6 J female APOE3 (n = 30) and APOE4 (n = 31) knock-in mice were randomized to treatment with either doxorubicin (10 mg/kg) or saline. Behavioural assays at 2–21 weeks-post exposure included open field maze, elevated zero maze, pre-pulse inhibition, Barnes maze, and fear conditioning. Ex-vivo magnetic resonance imaging was used to determine regional volume differences at 31–35 weeks-post exposure, and tissue sections were analyzed for markers of AD pathogenesis. Minimal toxicities were observed in the aged mice after doxorubicin exposure. In the Barnes maze assay, APOE3 mice did not exhibit impairment in spatial learning after doxorubicin treatment, but APOE4 mice demonstrated significant impairments in both the initial identification of the escape hole and the latency to full escape at 6 weeks post-exposure. Both APOE3 and APOE4 mice treated with doxorubicin showed impairment of spatial memory. Grey matter volume in the frontal cortex decreased in APOE4 mice treated with doxorubicin vs. APOE3 mice. This study demonstrates cognitive impairments in aged APOE4 knock-in mice after doxorubicin treatment and establishes this system as a novel and powerful model of CRCI.