ApoE Mice Research Articles

NCMP-06. GENOTYPE AND SEX IMPACT MEMORY FUNCTION AFTER BRAIN IRRADIATION

Abstract PURPOSE/OBJECTIVE Cognitive changes are a serious complication that can occur after brain radiotherapy. There is a need to better understand which individuals are more susceptible to develop strategies to preserve their function. The apolipoprotein E type 4 allele (APOEe4) APOE4 is the most prevalent genetic risk factor for Alzheimer Disease. We hypothesized that APOE genotype influences memory function after a subtherapeutic dose of whole brain irradiation in male and female mice. MATERIALS/METHODS Age-matched adult female and male mice engineered with either human APOE3 or APOE4 gene knocked-in were randomized to no treatment or whole brain irradiation (0 Gy or 5 Gy x 2), then evaluated with longitudinal neurobehavioral tests of learning, memory, pain threshold and anxiety. We performed immunohistochemical analyses of mouse brain sections at 1 month and at 4 months post-radiation to quantify extracellular amyloid beta plaques and other neuropathologic hallmarks associated with neurodegeneration. RESULTS Neurobehavioral testing identified significant sex and genotype-dependent changes in memory and anxiety measures developing longitudinally after brain irradiation. At baseline (unirradiated), APOE4 mice (male and female) performed worse on multiple memory measures. However after irradiation, sex and genotype each independently impacted multiple neurobehavioral function tests and also interacted with each other to significantly influence the timing and magnitude of changes in memory function. ApoE4 significantly impacted the toxicity of radiation in a sex-dependent manner. Neuropathologic analyses including amyloid beta plaque deposition and ApoE levels are ongoing. CONCLUSIONS After brain irradiation of relatively moderate dose, neurobehavioral performance including memory are impacted in a genotype and sex-dependent manner and dynamically over time. In addition to suggesting important sex-dependent differences in memory vulnerability, these data point to new opportunities for personalizing care for patients receiving brain irradiation.

Teriparatide administration is osteoanabolic but does not impact atherosclerotic plaque calcification and progression in a mouse model of menopause

Menopause exacerbates osteoporosis and increases the risk of atherosclerotic plaque rupture, leading to cardiovascular mortality. Osteoporotic women are increasingly treated with teriparatide (TPTD, 1–34 parathyroid hormone), one of the few treatments that stimulate bone formation. Despite the fact that atherosclerotic plaque calcification is a hallmark of plaque development, the impact of TPTD administration on plaque calcification remain unclear. In this context, we sought to determine the effects of TPTD administration on atherosclerosis in ovariectomized (OVX) apolipoprotein E deficient mice (ApoE−/−), as a model of postmenopausal osteoporosis. OVX ApoE−/− mice, fed a high fat, high cholesterol diet to induce atherosclerosis, received either vehicle or TPTD daily injections (40 μg/kg/d) for 4 or 10 weeks, at which points plaques are respectively weakly and heavily calcified. After sacrifice, bone remodeling was evaluated by serum markers and bone histomorphometry. Bone architectural parameters were measured by μCT. Aortic plaques were analyzed histologically, and their calcification with von Kossa staining and the calcium tracer Osteosense. Plaque inflammation and calcification markers were measured by RT-qPCR. Intermittent TPTD increased bone volume in OVX mice, due to a higher stimulation of bone formation relatively to bone resorption. These effects were not accompanied by changes in serum levels of cholesterol, triglycerides, glucose or insulin. TPTD neither significantly affected aortic plaque size, inflammation, and calcification, even if it slightly increased vascular smooth muscle cell transdifferentiation into calcifying cells. In conclusion, TPTD exhibits osteoanabolic effects in OVX ApoE−/− mice, without significantly influencing atherosclerotic plaque progression or calcification in the short term.

Microglia depletion reduces human neuronal APOE4-related pathologies in a chimeric Alzheimer's disease model.

Despite strong evidence supporting the important roles of both apolipoprotein E4 (APOE4) and microglia in Alzheimer's disease (AD) pathogenesis, the effects of microglia on neuronal APOE4-related AD pathogenesis remain elusive. To examine such effects, we utilized microglial depletion in a chimeric model with induced pluripotent stem cell (iPSC)-derived human neurons in mouse hippocampus. Specifically, we transplanted homozygous APOE4, isogenic APOE3, and APOE-knockout (APOE-KO) iPSC-derived human neurons into the hippocampus of human APOE3 or APOE4 knockin mice and then depleted microglia in half of the chimeric mice. We found that both neuronal APOE and microglial presence were important for the formation of Aβ and tau pathologies in an APOE isoform-dependent manner (APOE4>APOE3). Single-cell RNA sequencing analysis identified two pro-inflammatory microglial subtypes with elevated MHC-II gene expression enriched in chimeric mice with human APOE4 neuron transplants. These findings highlight the concerted roles of neuronal APOE, especially APOE4, and microglia in AD pathogenesis.

Decreased lipidated ApoE-receptor interactions confer protection against pathogenicity of ApoE and its lipid cargoes in lysosomes.

While apolipoprotein E (APOE) is the strongest genetic modifier for late-onset Alzheimer's disease (LOAD), the molecular mechanisms underlying isoform-dependent risk and the relevance of ApoE-associated lipids remain elusive. Here, we report that impaired low-density lipoprotein (LDL) receptor (LDLR) binding of lipidated ApoE2 (lipApoE2) avoids LDLR recycling defects observed with lipApoE3/E4 and decreases the uptake of cholesteryl esters (CEs), which are lipids linked to neurodegeneration. In human neurons, the addition of ApoE carrying polyunsaturated fatty acids (PUFAs)-CE revealed an allelic series (ApoE4>ApoE3>ApoE2) associated with lipofuscinosis, an age-related lysosomal pathology resulting from lipid peroxidation. Lipofuscin increased lysosomal accumulation of tau fibrils and was elevated in the APOE4 mouse brain with exacerbation by tau pathology. Intrahippocampal injection of PUFA-CE-lipApoE4 was sufficient to induce lipofuscinosis in wild-type mice. Finally, the protective Christchurch mutation also reduced LDLR binding and phenocopied ApoE2. Collectively, our data strongly suggest decreased lipApoE-LDLR interactions minimize LOAD risk by reducing the deleterious effects of endolysosomal targeting of ApoE and associated pathogenic lipids.

Endothelial cell-specific progerin expression does not cause cardiovascular alterations and premature death.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder caused by a mutation in the LMNA gene that provokes the synthesis of progerin, a mutant version of the nuclear protein lamin A that accelerates aging and precipitates death. The most clinically relevant feature of HGPS is the development of cardiac anomalies and severe vascular alterations, including massive loss of vascular smooth muscle cells, increased fibrosis, and generalized atherosclerosis. However, it is unclear if progerin expression in endothelial cells (ECs) causes the cardiovascular manifestations of HGPS. To tackle this question, we generated atherosclerosis-free mice (LmnaLCS/LCSCdh5-CreERT2) and atheroprone mice (Apoe-/-LmnaLCS/LCSCdh5-CreERT2) with EC-specific progerin expression. Like progerin-free controls, LmnaLCS/LCSCdh5-CreERT2 mice did not develop heart fibrosis or cardiac electrical and functional alterations, and had normal vascular structure, body weight, and lifespan. Similarly, atheroprone Apoe-/-LmnaLCS/LCSCdh5-CreERT2 mice showed no alteration in body weight or lifespan versus Apoe-/-LmnaLCS/LCS controls and did not develop vascular alterations or aggravated atherosclerosis. Our results indicate that progerin expression in ECs is not sufficient to cause the cardiovascular phenotype and premature death associated with progeria.

Deletion of the asialyloglycoprotein receptor-1 (ASGR1) causes atheroprotective effects in vitro and in vivo

Abstract Introduction The asialoglycoprotein receptor 1 (ASGR1) is a hepatic receptor that clears desialylated glycoproteins from the circulation. A loss-of-function mutation in ASGR1 was reported to associate with a 34% reduction in coronary artery disease (CAD) risk, suggesting a role for ASGR1 in CAD. Aim To determine the role of ASGR1 in atherosclerosis and whether deletion of ASGR1 elicits atheroprotective effects. Methods Western blotting, mass spectrometry and flow cytometry assessed ASGR1 expression in cultured macrophages or immune cells collected from the blood and aortas of wildtype mice. Bone marrow-derived macrophages (BMDMs) from wildtype and Asgr1-/- mice were subjected to cholesterol efflux and oxidised low-density lipoprotein (oxLDL) uptake in vitro functionalassays. ELISA and qPCR measured changes in lipid regulators in BMDMs. Asgr1-/- mice were crossed with atherosclerosis-prone apolipoprotein (Apo)e-/- mice (Apoe-/-Asgr1-/-). Stable plaque area was assessed histologically in the aortic sinuses of mice fed a high-cholesterol diet for 8 weeks (n=16/group). Using the tandem stenosis surgical model, unstable plaque was assessed in carotid arteries (n=10/group). Human ASGR1 was measured by ELISA in peripheral blood mononuclear cells (PBMCs) isolated from patient samples (n=18). Results Western blotting and mass spectrometry discovered ASGR1 is expressed in macrophages. Immunofluorescence identified the presence of ASGR1 in aortic sinus plaque. Flow cytometry revealed ASGR1 is expressed in both inflammatory and patrolling monocytes, neutrophils, macrophages and dendritic cells of blood and aortas. Asgr1-/- BMDMs elicited greater cholesterol efflux (+39%, P<0.05) and decreased oxLDL uptake (-15%, P<0.05), compared to wildtype BMDMs. Moreover, Asgr1-/- BMDMs had significantly higher LDL receptor protein levels (+2-fold), and Lxra (+2-fold) and Pparg (+1.5-fold) mRNA levels than wildtype BMDMs, P<0.001. Plasma from Asgr1-/- mice had lower total (-25%, P<0.05) and LDL (-36%, P<0.05) cholesterol concentrations than wildtype mice. Apoe-/-Asgr1-/- mice developed less stable plaque in aortic sinuses (-37%, P<0.001) than Apoe-/- controls, as well as smaller unstable plaques in carotid arteries (-49%, P<0.05). Finally, in a human population, ASGR1 protein levels were higher in PBMCs from patients with coronary plaque (+61%, P<0.05), than those without plaque, determined from coronary angiograms. Conclusion ASGR1 is expressed on monocytes, macrophages and in plaques, and human PBMC ASGR1 associates with coronary plaque. Deletion of ASGR1 causes atheroprotective functions in macrophages in vitro and reduced plaque burden in vivo. Our findings demonstrate ASGR1 is important in atherosclerosis with implications for ASGR1 as a therapeutic target.

Comparative Study of Cutaneous Squamous Cell Carcinogenesis in Different Hairless Murine Models

Background: In recent decades, a significant global increase in the incidence of non-melanoma skin cancer has been observed. To explore the pathogenesis of and potential therapeutic approaches for squamous cell carcinoma, various in vivo studies using mouse models have been conducted. However, investigations comparing different hairless mouse models, with or without melanin, as well as models with hypercholesterolemia and immunosuppression, in terms of their ability to induce squamous cell carcinoma have yet to be undertaken. Methods: Four mouse strains, namely SKH-hr1, SKH-hr2, SKH-hr2+ApoE, and immunodeficient Nude (Foxn1 knockout), were exposed to UVA and UVB radiation three times per week, initially to 1 Minimal Erythemal Dose (MED), incrementally increased weekly to a maximum dose of 3 MED. Clinical evaluation, photodocumentation, and biophysical parameters were monitored, along with proteasome protein activity and histopathological assessments. Results: The SKH-hr1 model primarily developed actinic keratosis without significant progression to invasive squamous cell carcinoma (SCC), while the SKH-hr2 and SKH-hr2+ApoE models exhibited a higher likelihood and intensity of papilloma and aggressive SCC formation, with the latter showing upregulated proteasome activity. Histopathological analysis confirmed the presence of poorly differentiated, invasive SCCs in the SKH-hr2 and SKH-hr2+ApoE models, contrasting with the less aggressive SCCs in the Nude mice and the mixed lesions observed in the SKH-hr1 mice. Conclusions: The SKH-hr2+ApoE and SKH-hr2 mice were identified as the most suitable for further exploration of squamous cell carcinogenesis. In contrast, the SKH-hr1 mice were found to be the least suitable, even though they are albino. Notably, proteasome analysis revealed a potential role of proteasome activity in squamous cell carcinogenesis.

Local Shear Stress and Dyslipidemia Interfere with Actin Cyto-Skeleton and Lysosomal Organization Contributing to Vascular Fragility

Shear stress is one of the major hemodynamic forces acting on the endothelium. However, it is not well known how endothelial cells (EC) respond mechanically to these stimuli in vivo. Here we investigated whether changes in biomechanics properties and shear stress could increase cell susceptibility to injury, contributing to vascular fragility. We surgically implanted a shear stress modifier device on the carotid artery of ApoE-knockout mice (ApoE−/−), which, due to its shape, causes a gradual stenosis in the vessel, resulting in distinct shear stress patterns. Our data show actin fibers accumulation in areas with higher lipid deposition in ApoE−/−, indicating that dyslipidemia might interfere with EC actin cytoskeleton organization. We also showed that both shear stress and dyslipidemia were important for EC susceptibility to injury. Furthermore, lysosomal distribution, an important organelle for plasma membrane repair, was altered in ApoE−/−, which could compromise EC’s ability to repair from damage. Therefore, dyslipidemia and variations in shear stress patterns not only affect cellular mechanics by compromising the actin cytoskeleton organization, but also enhance cell susceptibility to injury and alter vesicle trafficking in vascular cells. This may likely contribute to vascular fragility and thus to the initial steps of atherosclerosis development.

A cell-autonomous role for border-associated macrophages in ApoE4 neurovascular dysfunction and susceptibility to white matter injury.

Apolipoprotein E4 (ApoE4), the strongest genetic risk factor for sporadic Alzheimer's disease, is also a risk factor for microvascular pathologies leading to cognitive impairment, particularly subcortical white matter injury. These effects have been attributed to alterations in the regulation of the brain blood supply, but the cellular source of ApoE4 and the underlying mechanisms remain unclear. In mice expressing human ApoE3 or ApoE4, we report that border-associated macrophages (BAMs), myeloid cells closely apposed to neocortical microvessels, are both sources and effectors of ApoE4 mediating the neurovascular dysfunction through reactive oxygen species. ApoE4 in BAMs is solely responsible for the increased susceptibility to oligemic white matter damage in ApoE4 mice and is sufficient to enhance damage in ApoE3 mice. The data unveil a new aspect of BAM pathobiology and highlight a previously unrecognized cell-autonomous role of BAM in the neurovascular dysfunction of ApoE4 with potential therapeutic implications.

CCN4 (WISP-1) reduces apoptosis and atherosclerotic plaque burden in an ApoE mouse model

Background and aimsCCN4/WISP-1 regulates various cell behaviours that contribute to atherosclerosis progression, including cell adhesion, migration, proliferation and survival. We therefore hypothesised that CCN4 regulates the development and progression of atherosclerotic plaques. MethodsWe used a high fat fed ApoE−/− mouse model to study atherosclerotic plaque progression in the brachiocephalic artery and aortic root. In protocol 1, male ApoE−/− mice with established plaques were given a CCN4 helper-dependent adenovirus to see the effect of treatment with CCN4, while in protocol 2 male CCN4−/−ApoE−/− were compared to CCN4+/+ApoE−/− mice to assess the effect of CCN4 deletion on plaque progression. ResultsCCN4 overexpression resulted in reduced occlusion of the brachiocephalic artery with less apoptosis, fewer macrophages, and attenuated lipid core size. The amount of plaque found on the aortic root was also reduced. CCN4 deficiency resulted in increased apoptosis and occlusion of the brachiocephalic artery as well as increased plaque in the aortic root. Additionally, in vitro cells from CCN4−/−ApoE−/− mice had higher apoptotic levels. CCN4 deficiency did not significantly affect blood cholesterol levels or circulating myeloid cell populations. ConclusionsWe conclude that in an atherosclerosis model the most important action of CCN4 is the effect on cell apoptosis. CCN4 provides pro-survival signals and leads to reduced cell death, lower macrophage number, smaller lipid core size and reduced atherosclerotic plaque burden. As such, the pro-survival effect of CCN4 is worthy of further investigation, in a bid to find a therapeutic for atherosclerosis.

Quantitative and Kinetic Proteomics Reveal ApoE Isoform-dependent Proteostasis Adaptations in Mouse Brain.

Apolipoprotein E (ApoE) polymorphisms modify the risk of neurodegenerative disease with the ApoE4 isoform increasing and ApoE2 isoform decreasing risk relative to the 'wild-type control' ApoE3 isoform. To elucidate how ApoE isoforms alter the proteome, we measured relative protein abundance and turnover in transgenic mice expressing a human ApoE gene (isoform 2, 3, or 4). This data provides insight into how ApoE isoforms affect the in vivo synthesis and degradation of a wide variety of proteins. We identified 4849 proteins and tested for ApoE isoform-dependent changes in the homeostatic regulation of ~2700 ontologies. In the brain, we found that ApoE4 and ApoE2 both lead to modified regulation of mitochondrial membrane proteins relative to the wild-type control ApoE3. In ApoE4 mice, this regulation is not cohesive suggesting that aerobic respiration is impacted by proteasomal and autophagic dysregulation. ApoE2 mice exhibited a matching change in mitochondrial matrix proteins and the membrane which suggests coordinated maintenance of the entire organelle. In the liver, we did not observe these changes suggesting that the ApoE-effect on proteostasis is amplified in the brain relative to other tissues. Our findings underscore the utility of combining protein abundance and turnover rates to decipher proteome regulatory mechanisms and their potential role in biology.

APOE Christchurch enhances a disease-associated microglial response to plaque but suppresses response to tau pathology.

Apolipoprotein E ε4 (APOE4) is the strongest genetic risk factor for late-onset Alzheimer's disease (LOAD). A recent case report identified a rare variant in APOE, APOE3-R136S (Christchurch), proposed to confer resistance to autosomal dominant Alzheimer's Disease (AD). However, it remains unclear whether and how this variant exerts its protective effects. We introduced the R136S variant into mouse Apoe (ApoeCh) and investigated its effect on the development of AD-related pathology using the 5xFAD model of amyloidosis and the PS19 model of tauopathy. We used immunohistochemical and biochemical analysis along with single-cell spatial transcriptomics and proteomics to explore the impact of the ApoeCh variant on AD pathological development and the brain's response to plaques and tau. In 5xFAD mice, ApoeCh enhances a Disease-Associated Microglia (DAM) phenotype in microglia surrounding plaques, and reduces plaque load, dystrophic neurites, and plasma neurofilament light chain. By contrast, in PS19 mice, ApoeCh suppresses the microglial and astrocytic responses to tau-laden neurons and does not reduce tau accumulation or phosphorylation, but partially rescues tau-induced synaptic and myelin loss. We compared how microglia responses differ between the two mouse models to elucidate the distinct DAM signatures induced by ApoeCh. We identified upregulation of antigen presentation-related genes in the DAM response in a PS19 compared to a 5xFAD background, suggesting a differential response to amyloid versus tau pathology that is modulated by the presence of ApoeCh. These findings highlight the ability of the ApoeCh variant to modulate microglial responses based on the type of pathology, enhancing DAM reactivity in amyloid models and dampening neuroinflammation to promote protection in tau models. This suggests that the Christchurch variant's protective effects likely involve multiple mechanisms, including changes in receptor binding and microglial programming.

Risk factors for severe COVID-19 disease increase SARS-CoV-2 infectivity of endothelial cells and pericytes.

Coronavirus disease 2019 (COVID-19) was initially considered a primarily respiratory disease but is now known to affect other organs including the heart and brain. A major route by which COVID-19 impacts different organs is via the vascular system. We studied the impact of apolipoprotein E (APOE) genotype and inflammation on vascular infectivity by pseudo-typed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses in mouse and human cultured endothelial cells and pericytes. Possessing the APOE4 allele or having existing systemic inflammation is known to enhance the severity of COVID-19. Using targeted replacement human APOE3 and APOE4 mice and inflammation induced by bacterial lipopolysaccharide (LPS), we investigated infection by SARS-CoV-2. Here, we show that infectivity was higher in murine cerebrovascular pericytes compared to endothelial cells and higher in cultures expressing APOE4. Furthermore, increasing the inflammatory state of the cells by prior incubation with LPS increased infectivity into human and mouse pericytes and human endothelial cells. Our findings provide insights into the mechanisms underlying severe COVID-19 infection, highlighting how risk factors such as APOE4 genotype and prior inflammation may exacerbate disease severity by augmenting the virus's ability to infect vascular cells.

Low to moderate dose 137Cs (γ) radiation promotes M2 type macrophage skewing and reduces atherosclerotic plaque CD68+ cell content in ApoE(−/−) mice

The effects of low doses of ionizing radiation on atherosclerosis remain uncertain, particularly as regards the generation of pro- or anti-inflammatory responses, and the time scale at which such effects can occur following irradiation. To explore these phenomena, we exposed atheroprone ApoE(−/−) mice to a single dose of 0, 0.05, 0.5 or 1 Gy of 137Cs (γ) administered at a 10.35 mGy min−1 dose rate and evaluated short-term (1–10 days) and long-term consequences (100 days). Bone marrow-derived macrophages were derived from mice 1 day after exposure. Irradiation was associated with a significant skewing of M0 and M2 polarized macrophages towards the M2 phenotype, as demonstrated by an increased mRNA expression of Retnla, Arg1, and Chil3 in cells from mice exposed to 0.5 or 1 Gy compared with non-irradiated animals. Minimal effects were noted in M1 cells or M1 marker mRNA. Concurrently, we observed a reduced secretion of IL-1β but enhanced IL-10 release from M0 and M2 macrophages. Effects of irradiation on circulating monocytes were most marked at day 10 post-exposure, when the 1 Gy dose was associated with enhanced numbers of both Ly6CHigh and Ly6Low cells. By day 100, levels of circulating monocytes in irradiated and non-irradiated mice were equivalent, but anti-inflammatory Ly6CLow monocytes were significantly increased in the spleen of mice exposed to 0.05 or 1 Gy. Long term exposures did not affect atherosclerotic plaque size or lipid content, as determined by Oil red O staining, whatever the dose applied. Similarly, irradiation did not affect atherosclerotic plaque collagen or smooth muscle cell content. However, we found that lesion CD68+ cell content tended to decrease with rising doses of radioactivity exposure, culminating in a significant reduction of plaque macrophage content at 1 Gy. Taken together, our results show that short- and long-term exposures to low to moderate doses of ionizing radiation drive an anti-inflammatory response, skewing bone marrow-derived macrophages towards an IL-10-secreting M2 phenotype and decreasing plaque macrophage content. These results suggest a low-grade athero-protective effect of low and moderate doses of ionizing radiation.

#1957 Microbial and metabolomic remodeling by Sichuan dark tea-based formula improves obesity-induced renal lipid metabolism disorder in mice

Abstract Background and Aims The theory of medicine-food homology is long-standing and has been widely applied in traditional Chinese medicine. The Sichuan dark tea-based medicated dietary formula (alternatively referred to as Qing, or clarity in Chinese) is previously used for its lipid-lowering properties in apoE mice. This study further investigated the renoprotective effects of Qing on diet-induced obesity (DIO) mice and explored the specific mechanisms involved. Method Male C57BL/6 mice were randomly assigned to three groups, a control group, a DIO group, and a Qing treatment group, or the Qing group, with 8 mice in each group. The mice in the control group were given normal maintenance feed and purified water, and the other two groups were fed a high-fat diet for 12 weeks to establish the DIO model. After that, high-fat diet continued in the DIO group, while the Qing group was given Qing at the same time for 12 weeks, during which period the weight of the mice was monitored and recorded every week. The mice were sacrificed after 12 weeks. Serum samples were collected and the levels of triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin were measured to evaluate liver function. In addition, renal lipids were extracted to determine the levels of TG and TC in the kidney and periodic acid-Schiff (PAS) and oil red O stainings were performed to evaluate kidney pathological injury. Western blot was performed to determine the phosphorylated AMPK (pAMPK)/AMPK ratio in the kidney tissue. RT-qPCR and Western blot were used to determine the expression of proteins related to fatty acid oxidation and key proteins related to lipid synthesis. 16SrRNA and metabolomics were applied to analyze the gut microbiota in the intestinal contents and its metabolites. Results Compared with those of the control group, the levels of liver mass (P=0.0003), serum ALT(P < 0.0001), AST (P=0.0001), TC (P=0.0191) and TG (P=0.0101) in the model group were significantly increased. Qing treatment can effectively reduce liver mass (P=0.0316), and improve AST (P=0.0012), ALT (P=0.0027), TC (P=0.0200), TG (P=0.0499) levels of mice compared to those of the DIO group. Qing treatment inhibited the expression of lipid synthesis related genes (SREBP-1, FASN and SCD1) and promoted the expression of fatty acid oxidation related genes (CPT1A, PGC1α and PPARγ) in kidneys (Fig. 1). Obesity-triggered dysfunction of structure and composition of gut microbiota were improved by Qing (Fig. 2), where the content of short-chain fatty acids, especially isovaleric acid and propionic acid, were also restored (Fig. 3). Conclusion Our data suggested that Sichuan dark tea-based medicated dietary formula may improve renal lipid metabolism by regulating gut microbiota and the levels of intestinal short-chain fatty acids, thereby protecting obesity-related kidney injury. Isovaleric acid and propionic acid may be the metabolites key to its regulation of gut microbiota.

Cellular senescence induced by cholesterol accumulation is mediated by lysosomal ABCA1 in APOE4 and AD.

Cellular senescence is a hallmark of aging and has been implicated in Alzheimer's disease (AD) pathogenesis. Cholesterol accumulation drives cellular senescence; however, the underlying mechanisms are unclear. ATP-binding cassette transporter A1 (ABCA1) plays an important role in cholesterol homeostasis. ABCA1 expression and its trafficking is afiltered in APOE4 and AD cellular and mouse models. However, whether ABCA1 trafficking is involved in cellular senescence in APOE4 and AD remains unknown. We examined the association between cellular senescence and ABCA1 expression in human postmortem brain samples using transcriptomic, histological, and biochemical analyses. An unbiased proteomic screening was performed to identify targets that mediate cellular ABCA1 trafficking. APOE4-TR mice, immortalized, primary and induced pluripotent stem cell (iPSC) models were used to examine the cholesterol-ABCA1-senescence pathways. Bulk and single nuclei transcriptomic profiling of the human dorsolateral prefrontal cortex from the Religious Order Study/Memory Aging Project (ROSMAP) revealed upregulation of cellular senescence transcriptome signatures in AD, which was strongly correlated with ABCA1 expression. Immunofluorescence and immunoblotting analyses confirmed increased ABCA1 expression in AD brain tissues, which was associated with lipofuscin-stained lipids and mTOR phosphorylation. Using discovery proteomics, caveolin-1, a sensor of cellular cholesterol accumulation, was identified to promote ABCA1 endolysosomal trafficking. Greater caveolin-1 expression was found in both APOE4-TR mouse models and AD human brains. Cholesterol induced mTORC1 activation was regulated by ABCA1 expression or its lysosomal trapping. Reducing cholesterol by cyclodextrin in APOE4-TR mice reduced ABCA1 lysosome trapping and increased ABCA1 recycling to efflux cholesterol to HDL particles, reducing mTORC1 activation and senescence-associated neuroinflammation. In human iPSC-derived astrocytes, the reduction of cholesterol by cyclodextrin attenuated inflammatory responses. Cholesterol accumulation in APOE4 and AD induced caveolin-1 expression, which traps ABCA1 in lysosomes to activate mTORC1 pathways and induce cellular senescence. This study provided novel insights into how cholesterol accumulation in APOE4 and AD accelerates senescence.

Skeletal Muscle Mitochondrial Response to Diet Differs by Genotype and Sex in an Alzheimer’s Disease Mouse Model

Introduction: Skeletal muscle mass is reduced in the early stages of cognitive decline. Apolipoprotein ε4 ( APOE4) is the most prevalent genetic risk factor for Alzheimer’s disease (AD) and is associated with faster motor decline in cognitively healthy individuals. APOE4 also increases the risk of insulin resistance and cardiovascular disease, conditions associated with altered bioenergetic function in muscle. However, no studies have examined the role of APOE4 in modulating muscle physiology and the impact this would have on cognitive function. We sought to determine if APOE4 affects skeletal muscle mitochondrial metabolism in response to a high-fat diet (HFD) and if this is reflected in whole-body metabolism. We also examined the role of sex since APOE4 carriers who are female are more likely to develop AD. Methods: Male and female APOE3 (n=32) and APOE4 (n= 29) targeted-replacement mice on a C57BL/6 background purchased from Taconic were fed a standard chow diet until 4 months old. Mice were then switched to a low-fat diet (LFD, 10% kcal fat) or HFD (45% kcal fat) until sacrifice. Glucose tolerance was tested at 7 months by administering an intraperitoneal glucose injection of 1g/kg lean mass. Blood glucose was measured before and 15, 30, 45, 60, 90 and 120 minutes after glucose injection. Resting respiratory exchange ratio (RER) was determined using the Promethion Indirect Calorimetry system at 8 months to determine the relative contribution of carbohydrates versus lipids to whole body metabolism. Mice were sacrificed at 9 months and carbohydrate-driven oxygen consumption was measured at basal, state 3 (ADP), state 3 + glutamate, state 3S (succinate) and uncoupled respiratory states in mitochondria isolated from quadriceps tissue on the Oroboros Oxygraph-2k system. Results: Mice fed a HFD had greater glucose area under the curve (p<0.0001) and reduced RER (p= <0.001) compared to mice fed a LFD, regardless of sex or genotype. At the level of skeletal muscle, APOE4 female mice had increased state 3 (p=0.027) and state 3 + glutamate oxygen consumption (p=0.037) on a HFD compared to a LFD while no diet effect was observed in APOE4 males or any APOE3 mice. Conclusion: We provide evidence that female APOE4 mice are susceptible to diet-induced increases in carbohydrate-driven mitochondrial oxygen consumption in muscle. Future studies are needed to determine the underlying mechanism and consequences of increased muscle mitochondrial activity, which could help identify treatment targets in female APOE4 carriers. BHTP T32 AG078114, P30 AG072973 (Chelsea Johnson). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Age and APOE4 genotype alter cerebrovascular function and stiffness

Two of the greatest risk factors for late-onset Alzheimer’s disease (LOAD) are age and the APOE4 genotype. The gene encoding for apolipoprotein E ( APOE) has three isoforms in humans ( E2, E3, E4). E3 has the highest prevalence, while E4 exponentially increases the risk for AD. Additionally, it is well known that the E4 genotype is accompanied by an altered cerebral blood flow. However, the mechanisms underlying these blood flow changes in E4 individuals remain unclear and may be mediated by changes to cerebral artery structure and function. We aimed to determine if APOE genotype alters the impact of age on cerebral artery endothelial function and stiffness. We studied male and female, young E3 (n=20; ~6 months), aged E3 (n=9; ~24 months young E4 (n=22; ~6 months), and aged E4 (n=18; ~24 months) mice. We assessed endothelium-dependent and -independent vasodilation and vasoconstriction in isolated, pressurized posterior cerebral arteries (PCAs). Passive stiffness was measured in isolated PCAs after incubation in a calcium-free solution. Data are mean ± SD. Both old E3 and old E4 mice had impaired PCA endothelium-dependent vasodilation to acetylcholine compared with young mice ( E3: 29±10.6% vs 46±15.2%, p=0.02; E4: 30±10.8 vs 49±14.3%, p=0.0004). Sex comparisons found that the PCA maximal dilation to acetylcholine was 21% greater in young E3 females compared with young E3 males (52±12.5% vs 41±16%, p=0.04), and 13% greater in young E4 females compared with young E4 males (52±10% vs 45±16%, p=0.07), but there were no sex differences among the old groups. Additionally, the PCA responses to acetylcholine in the presence of nitric oxide synthase inhibitor L-NAME did not differ between groups (p>0.05). Endothelium-independent dilation, measured as the PCA response to sodium nitroprusside, also did not differ between groups (p>0.05). Aged E3 mice, compared with young E3, had a lower maximal constriction to potassium chloride (20±11% vs 59±16%, p<0.0001) and endothelin-1 (23±16% vs 53±14%, p=0.0005). In contrast, young E4 and old E4 mice had a similar maximal constriction to potassium chloride (35±13% vs 43±18%, p>0.05) and endothelin-1 (48±12% vs 47±20%, p>0.05). Lastly, we found that old mice had a greater PCA β stiffness index compared with young mice for both the E3 (15.5±4.5 vs 10.6±1.4 AU, p=0.0008) and E4 (13.4±2.9 vs 10.2±2.5 AU, p>0.0001) groups. In summary, we found that age impairs cerebral artery endothelial function in both APOE3 and APOE4 mice, and young female mice have better endothelial function than their male counterparts regardless of genotype. In addition, vasoconstrictor responsiveness appears to decline in old age in APOE3 mice, whereas vasoconstrictor responsiveness is maintained in old APOE4 mice. This maintained sensitivity to vasoconstrictors in old age may explain why APOE4 individuals have reduced cerebral blood flow and greater LOAD risk. Luvaas Family Fund, Alzheimer’s Association ALZDISCOVERY-1049110. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Allelic modulation of large (second-order) mesenteric artery mechanical properties in adult APOE3 & APOE4 Mice

Previous studies from our lab have demonstrated age-related changes in vascular structure/function in posterior cerebral arteries (PCA) and left common carotid artery (CA) in male and female mice expressing human-APOE targeted replacement of APOE3 (B6.129P2-APOEtm2(APOE*3)MaeN8) and APOE4 (B6.129P2-APOEtm3(APOE*4)MaeN8) (Taconic Labs). Therefore, we hypothesize that similar changes may be observed in the systemic circulation. We isolated second-order mesenteric arteries (MA) from adult (6-month-old) male and female mice expressing APOE3 (n=3) and APOE4 (n=4). Mesenteric artery segments (5-8 mm in length) were rapidly isolated and cannulated on an arteriograph to assess mechanical properties: lumen diameter (LD), wall thickness (WT), wall:lumen ratio (W:L), distensibility, stress, strain, and stress versus strain (SvS) in the presence of Ca2+-free Krebs + diltiazem at intraluminal pressures ranging from 10mmHg–140mmHg. Phenylephrine and potassium-induced contractions were assessed in Ca2+-containing Krebs at intraluminal pressures ranging from 10-140mmHg. Data were analyzed using two-way ANOVA, Student’s t-test, and analysis of nonlinear fit. Values were considered statistically different at p<0.05. Our preliminary data indicated APOE4 mice had a greater WT and W:L compared to APOE3. Interestingly, there were no significant differences in the remaining passive mechanical properties (LD, distensibility, stress, strain, SvS). In addition, phenylephrine and potassium-induced contractions were similar in the APOE3 and APOE4 mice. This data suggests changes in passive mechanical properties in APOE4 mice which may resemble premature aging. ABRC/ADHS18-205211 (DME, CBJ), Arizona Alzheimer’s Consortium (funded by the Arizona Department of Health Services, Contract No. CTR040636) and matching funds from Midwestern University (DME), Biomedical Sciences Program (IW, SM, DME), Biomedical Sciences Start-up Funds (DME). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Small mesenteric artery passive mechanical properties in adult APOE3 & APOE4 Mice

Previous studies from our lab have demonstrated changes in vascular structure and function in posterior cerebral arteries and left common carotid artery (CA) in adult male and female mice expressing human-ApoE targeted replacement of APOE3 (B6.129P2-Apoetm2(APOE*3)MaeN8) and APOE4 (B6.129P2-Apoetm3(APOE*4)MaeN8) (Taconic Labs). Therefore, we hypothesized that similar changes may be observed in the systemic circulation. We isolated 4thor 5th-order mesenteric arteries from adult 6-month-old male and female mice expressing hAPOE3 (n=5) or hAPOE4 (n=4). Mesenteric artery segments (5-8 mm in length) were isolated and cannulated on an arteriograph to assess vascular mechanical properties; lumen diameter, wall thickness, wall:lumen ratio, distensibility, stress versus strain (SvS), compliance, incremental modulus of elasticity (Einc), and calculated pulse wave velocity under passive conditions (Ca2+-free Krebs plus diltiazem) at intraluminal pressures ranging from 10mmHg to 140mmHg. In addition, phenylephrine-induced contraction and potassium-induced contraction were assessed using Ca2+-containing Krebs. Data was analyzed using two-way ANOVA, Student’s t-test, and analysis of nonlinear fit. Values were considered statistically different at p<0.05. In our preliminary data, lumen diameter, distensibility, compliance, strain, and calculated pulse wave velocity were similar in APOE3 and APOE4 mice. Wall thickness and wall:lumen ratio were significantly greater in APOE4 compared to APOE3 mice. Wall stress and Einc were significantly less in APOE4 compared to APOE3 mice. Interestingly, the APOE4 mice exhibited a significantly lesser stress per given amount of strain compared to the APOE3 mice. Phenylephrine- and potassium-induced contractions were similar in APOE3 and APOE4 mice. This data suggests there are changes in passive mechanical properties in small mesenteric arteries from APOE4 mice, which resemble a hypertensive phenotype. ABRC/ADHS18-205211 (DME, CBJ), Arizona Alzheimer’s Consortium (funded by the Arizona Department of Health Services, Contract No. CTR040636) and matching funds from Midwestern University (DME), Biomedical Sciences Program (SM, IW, DME), Biomedical Sciences Start-up Funds (DME). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.