Lance A. Johnson

Apolipoprotein E knock-out and knock-in mice: atherosclerosis, metabolic syndrome, and beyond

Given the multiple differences between mice and men, it was once thought that mice could not be used to model atherosclerosis, principally a human disease. Apolipoprotein E-deficient (apoEKO) mice have convincingly changed this view, and the ability to model human-like plaques in these mice has provided scientists a platform to study multiple facets of atherogenesis and to explore potential therapeutic interventions. In addition to its well-established role in lipoprotein metabolism, recent observations of reduced adiposity and improved glucose homeostasis in apoEKO mice suggest that apoE may also play a key role in energy metabolism in peripheral organs, including adipose tissue. Finally, along with apoEKO mice, knockin mice expressing human apoE isoforms in place of endogenous mouse apoE have provided insights into how quantitative and qualitative genetic alterations interact with the environment in the pathogenesis of complex human diseases.

Differential modulation of diet-induced obesity and adipocyte functionality by human apolipoprotein E3 and E4 in mice

Objective:Apolipoprotein E (apoE), a key protein in lipid metabolism, is highly expressed in adipose tissues. Studies have shown that human APOE*4 is associated with a lower body mass index but with a greater risk of coronary heart disease compared with other APOE alleles. To define the isoform-specific role of apoE in regulating the expandability and functionality of adipose tissues, we investigated the effects of diet-induced obesity in mice whose endogenous Apoe gene has been replaced by either the human APOE*3 or APOE*4 allele.Results:After 8 weeks on a Western-type high-fat diet, male APOE4 mice displayed impaired tolerance to glucose and fat overload compared with APOE3 mice. Subcutaneous fat tissues in APOE4 and APOE3 mice after high fat feeding were not different. In contrast, although epididymal fat tissues in APOE4 mice gained 30% less weight during the high fat feeding than in APOE3 mice, they showed impaired insulin-stimulated glucose uptake ex vivo. Epididymal APOE4 adipocytes were larger in size than APOE3 adipocytes, and expressed reduced levels of mRNA for peroxisome proliferator-activated receptor γ2 and adiponectin, important markers of adipocyte functionality. Adenoviral expression of apoE3 in apoE-null culture adipocytes induced adiponectin mRNA in a dose-dependent manner, but the induction was significantly blunted in cells overexpressing apoE4. However, in contrast to the apoE3-expressing cells, Glut1, but not Glut4, expression levels were positively correlated with increased apoE4 mRNA, suggesting that apoE4 expression in adipocyte interferes in insulin-sensing pathways.Conclusion:Dysfunctional epididymal adipose tissues contribute to the accelerated impairment of glucose tolerance in APOE4 mice fed a Western-type diet. Our results underscore the importance of functionality of individual fat depots rather than total fat mass as a determinant for metabolic disturbance during diet-induced obesity.

Early Effects of Whole-Body 56Fe Irradiation on Hippocampal Function in C57BL/6J Mice

Relatively little is known about early irradiation effects on hippocampal function in wild-type mice. In this study, the effects of 56Fe irradiation on hippocampal function were assessed starting 2 weeks after whole-body irradiation. Compared to sham irradiation, radiation impaired novel object recognition in female and male C57BL/6J wild-type mice. There were no effects of irradiation on contextual fear conditioning or spatial memory retention in the water maze. It is possible that oxidative damage might contribute to radiation-induced cognitive changes. Therefore, hippocampal and cortical levels of 3-nitrotyrosine (3NT) and lipid peroxidation, measures of oxidative damage were assessed. There were no effects of irradiation on these measures of oxidative damage. As 56Fe irradiation can increase reactive oxygen species (ROS) levels, which may contribute to the impairments in novel object recognition, the effects of the antioxidant alpha-lipoic acid (ALA) on cognition following sham irradiation and irradiation were also assessed. ALA did not prevent radiation-induced impairments in novel object recognition and impaired spatial memory retention of sham-irradiated and irradiated mice in the probe trial after the first day of hidden platform training in the water maze. Thus, the novel object recognition test is particularly sensitive to detect early cognitive effects of 56Fe irradiation through a mechanism unlikely involving ROS or oxidative damage.

Enhanced hippocampus-dependent memory and reduced anxiety in mice over-expressing human catalase in mitochondria

Oxidative stress (OS) and reactive oxygen species (ROS) play a modulatory role in synaptic plasticity and signaling pathways. Mitochondria (MT), a major source of ROS because of their involvement in energy metabolism, are important for brain function. MT‐generated ROS are proposed to be responsible for a significant proportion of OS and are associated with developmental abnormalities and aspects of cellular aging. The role of ROS and MT function in cognition of healthy individuals is relatively understudied. In this study, we characterized behavioral and cognitive performance of 5‐ to 6‐month‐old mice over‐expressing mitochondrial catalase (MCAT). MCAT mice showed enhancements in hippocampus‐dependent spatial learning and memory in the water maze and contextual fear conditioning, and reduced measures of anxiety in the elevated zero maze. Catalase activity was elevated in MCAT mice in all brain regions examined. Measures of oxidative stress (glutathione, protein carbonyl content, lipid peroxidation, and 8‐hydroxyguanine) did not significantly differ between the groups. The lack of differences in these markers of oxidative stress suggests that the differences observed in this study may be due to altered redox signaling. Catalase over‐expression might be sufficient to enhance cognition and reduce measures of anxiety even in the absence of alteration in levels of OS.

Sex differences in activation of the hypothalamic-pituitary-adrenal axis by methamphetamine.

Dysregulation of hypothalamic–pituitary–adrenal (HPA) axis activation is associated with changes in addiction‐related behaviors. In this study, we tested whether sex differences in the acute effects of methamphetamine (MA) exposure involve differential activation of the HPA axis. Male and female mice were injected with MA (1 mg/kg) or saline for comparison of plasma corticosterone and analysis of the immediate early gene c‐Fos in brain. There was a prolonged elevation in corticosterone levels in female compared to male mice. C‐Fos was elevated in both sexes following MA in HPA axis‐associated regions, including the hypothalamic paraventricular nucleus (PVN), central amygdala, cingulate, and CA3 hippocampal region. MA increased the number of c‐Fos and c‐Fos/glucocorticoid receptor (GR) dual‐labeled cells to a greater extent in males than females in the cingulate and CA3 regions. MA also increased the number of c‐fos/vasopressin dual‐labeled cells in the PVN as well as the number and percentage of c‐Fos/GR dual‐labeled cells in the PVN and central amygdala, although no sex differences in dual labeling were found in these regions. Thus, sex differences in MA‐induced plasma corticosterone levels and activation of distinct brain regions and proteins involved in HPA axis regulation may contribute to sex differences in acute effects of MA on the brain.

Apolipoprotein E4 in Macrophages Enhances Atherogenesis in a Low Density Lipoprotein Receptor-dependent Manner

Apolipoprotein E (apoE) and the low density lipoprotein receptor (LDLr) are well recognized determinants of atherosclerosis. In addition to hepatocytes, where both are highly expressed and contribute to plasma lipoprotein clearance, they are expressed in vascular cells and macrophages. In this study, we examined the effects of human apoE isoforms and LDLr levels in atherogenic pathways in primary macrophages ex vivo and atherosclerosis development after bone marrow transfer in vivo using mice expressing human apoE isoforms and different levels of LDLr expression. Increases in LDLr expression significantly increased cholesterol delivery into macrophages in culture, and the effects were more prominent with lipoproteins containing apoE4 than those containing apoE3. Conversely, increased LDLr expression reduced cholesterol efflux in macrophages expressing apoE4 but not in macrophages expressing apoE3. Furthermore, apoE3 protected VLDL from oxidation in vitro more than did apoE4. In LDLr-deficient mice expressing the human apoE4 isoform, Apoe4/4 Ldlr–/–, the replacement of bone marrow cells with those expressing LDLr increased atherosclerotic lesions in a dose-dependent manner compared with mice transplanted with cells having no LDLr. In contrast, atherosclerosis in Apoe3/3 Ldlr–/– mice, expressing the human apoE3 isoform, did not differ by the levels of macrophage LDLr expression. Our results demonstrate that apoE4, but not apoE3, in macrophages enhances atherosclerotic plaque development in mice in an LDLr-dependent manner and suggests that this interaction may contribute to the association of apoE4 with an increased cardiovascular risk in humans.

Human LDL Receptor Enhances Sequestration of ApoE4 and VLDL Remnants on the Surface of Hepatocytes but Not Their Internalization in Mice

Objective—In humans, apolipoprotein (apo) E4 is associated with elevated plasma cholesterol levels and a high risk of developing atherosclerosis, whereas apoE2 is protective. Here we investigate the mechanism by which mice expressing human apoE isoforms recapitulate this association when they also express high levels of human low-density lipoprotein receptor (LDLR). Methods and Results—Primary hepatocytes from apoE4 mice secreted less apoE into the medium than hepatocytes from apoE2 mice. Increased LDLR expression decreased this secretion and increased degradation of apoE4. An apoE4-GFP fusion protein expressed in the liver of apoE-deficient mice accumulated on the hepatocyte surface bordering the space of Disse in an LDLR-dependent manner. Fluorescence-labeled very low–density lipoprotein (VLDL) remnants accumulated on the hepatocyte surface in apoE4 mice with high LDLR, but they were internalized poorly. In contrast, apoE2-GFP did not accumulate on the hepatocyte surface even when the LDLR expression was high, but apoE2 mice with high LDLR internalized the remnants avidly without sequestering them on the hepatocyte surface. Conclusions—The high affinity of apoE4 to the LDLR enhances VLDL sequestration on the hepatocyte surface but delays their internalization. This delay likely increases VLDL conversion to cholesterol-enriched remnants in apoE4 mice with high LDLR, and probably to LDL in humans with apoE4.

Apolipoprotein E-low density lipoprotein receptor interaction affects spatial memory retention and brain ApoE levels in an isoform-dependent manner

Human apolipoprotein E (apoE) exists in three isoforms: apoE2, apoE3 and apoE4. APOE ε4 is a major genetic risk factor for cardiovascular disease (CVD) and Alzheimers disease (AD). ApoE mediates cholesterol metabolism by binding various receptors. The low-density lipoprotein receptor (LDLR) has a high affinity for apoE, and is the only member of its receptor family to demonstrate an apoE isoform specific binding affinity (E4>E3>>E2). Evidence suggests that a functional interaction between apoE and LDLR influences the risk of CVD and AD. We hypothesize that the differential cognitive effects of the apoE isoforms are a direct result of their varying interactions with LDLR. To test this hypothesis, we have employed transgenic mice that express human apoE2, apoE3, or apoE4, and either human LDLR (hLDLR) or no LDLR (LDLR(-/-)). Our results show that plasma and brain apoE levels, cortical cholesterol, and spatial memory are all regulated by isoform-dependent interactions between apoE and LDLR. Conversely, both anxiety-like behavior and cued associative memory are strongly influenced by APOE genotype, but these processes appear to occur via an LDLR-independent mechanism. Both the lack of LDLR and the interaction between E4 and the LDLR were associated with significant impairments in the retention of long term spatial memory. Finally, levels of hippocampal apoE correlate with long term spatial memory retention in mice with human LDLR. In summary, we demonstrate that the apoE-LDLR interaction affects regional brain apoE levels, brain cholesterol, and cognitive function in an apoE isoform-dependent manner.

ApoE2 Exaggerates PTSD-Related Behavioral, Cognitive, and Neuroendocrine Alterations

Apolipoprotein E (apoE) is an essential component of lipoprotein particles in both the brain and periphery, and exists in three isoforms in the human population: E2, E3, and E4. ApoE has numerous, well-established roles in neurobiology. Most notably, E4 is associated with earlier onset and increased risk of Alzheimer’s disease (AD). Although possession of E2 is protective in the context of AD, E2 appears to confer an increased incidence and severity of posttraumatic stress disorder (PTSD). However, the biological processes underlying this link remain unclear. In this study, we began to elucidate these associations by examining the effects of apoE on PTSD severity in combat veterans, and on PTSD-like behavior in mice with human apoE. In a group of 92 veterans with PTSD, we observed significantly higher Clinician-Administered PTSD Scale and PTSD Checklist scores in E2+ individuals, as well as alterations in salivary cortisol levels. Furthermore, we measured behavioral and biological outcomes in mice expressing human apoE after a single stressful event as well as following a period of chronic variable stress, a model of combat-related trauma. Mice with E2 showed impairments in fear extinction, and behavioral, cognitive, and neuroendocrine alterations following trauma. To the best of our knowledge, these data constitute the first translational demonstration of PTSD severity in men and PTSD-like symptoms in mice with E2, and point to apoE as a novel biomarker of susceptibility, and potential therapeutic target, for PTSD.

Apolipoprotein E4 Exaggerates Diabetic Dyslipidemia and Atherosclerosis in Mice Lacking the LDL Receptor

OBJECTIVE We investigated the differential roles of apolipoprotein E (apoE) isoforms in modulating diabetic dyslipidemia—a potential cause of the increased cardiovascular disease risk of patients with diabetes. RESEARCH DESIGN AND METHODS Diabetes was induced using streptozotocin (STZ) in human apoE3 (E3) or human apoE4 (E4) mice deficient in the LDL receptor (LDLR−/−). RESULTS Diabetic E3LDLR−/− and E4LDLR−/− mice have indistinguishable levels of plasma glucose and insulin. Despite this, diabetes increased VLDL triglycerides and LDL cholesterol in E4LDLR−/− mice twice as much as in E3LDLR−/− mice. Diabetic E4LDLR−/− mice had similar lipoprotein fractional catabolic rates compared with diabetic E3LDLR−/− mice but had larger hepatic fat stores and increased VLDL secretion. Diabetic E4LDLR−/− mice demonstrated a decreased reliance on lipid as an energy source based on indirect calorimetry. Lower phosphorylated acetyl-CoA carboxylase content and higher gene expression of fatty acid synthase in the liver indicated reduced fatty acid oxidation and increased fatty acid synthesis. E4LDLR−/− primary hepatocytes cultured in high glucose accumulated more intracellular lipid than E3LDLR−/− hepatocytes concomitant with a 60% reduction in fatty acid oxidation. Finally, the exaggerated dyslipidemia in diabetic E4LDLR−/− mice was accompanied by a dramatic increase in atherosclerosis. CONCLUSIONS ApoE4 causes severe dyslipidemia and atherosclerosis independent of its interaction with LDLR in a model of STZ-induced diabetes. ApoE4-expressing livers have reduced fatty acid oxidation, which contributes to the accumulation of tissue and plasma lipids.