Human Apo Research Articles

An optimized method for PCR-based genotyping to detect human APOE polymorphisms

BackgroundApolipoprotein E (APOE) is one of the most polymorphic genes at two single nucleotides (rs429358 and rs7412). The various isoforms of APOE have been associated with a variety of diseases, including neurodegenerative, type 2 diabetes, etc. Hence, predicting the APOE genotyping is critical for disease risk evaluation. The purpose of this study was to optimize the tetra amplification refractory mutation system (Tetra-ARMS) PCR method for the detection of APOE mutations. Material and methodsHere, in our optimized Tetra-ARMS PCR method, different factors like cycle conditions, using HiFidelity enzyme instead of Taq polymerase and setting its best concentration, and the lack of using dimethylsulfoxide (DMSO) for amplifying the GC-regions were set up for all primer pairs. The sensitivity and accuracy were tested. For validation of the assay, the results were compared with known genotypes for the APOE gene that were previously obtained by two independent methods, RFLP and Chip-typing. ResultsSuccessful Tetra-ARMS PCR and genotyping are influenced by multiple factors. Our developed method enabled us to amplify the DNA fragment by 25 cycles without adding any hazardous reagent, like DMSO. Our findings showed 100 % accuracy and sensitivity of the optimized Tetra-ARMS PCR while both criteria were 95 % for RFLP and 100 % for the chip-typing method. In addition, our results showed 91 % and 100 % consistency with RFLP and chip typing methods, respectively. ConclusionsOur current method is a simple and accurate approach for detecting APOE polymorphisms within a large sample size in a short time and can be performed even in low-tech laboratories.

Microarray analysis identifies human apoA-IMilano and apoA-II as determinants of the liver gene expression related to lipid and energy metabolism

The phenotype of individuals carrying the apolipoprotein A-IMilano (apoA-IM), the mutant form of human apoA-I (apoA-I), is characterized by very low concentrations of HDL and apoA-I, and hypertriglyceridemia. Paradoxically, these subjects are not found to be at increased risk of premature cardiovascular disease compared to controls. Besides, various in vitro and in vivo studies have demonstrated that apoA-IM possesses greater anti-atherosclerotic activity compared to apoA-I. The molecular mechanisms explaining the apoA-IM carrier's phenotype and the apoA-IM higher efficacy are still not fully elucidated. To investigate such mechanisms, we crossed previously generated apoA-I (A-I k-in) or apoA-IM knock-in mice (A-IM k-in) with transgenic mice expressing human apoA-II but lacking murine apoA-I (hA-II) to generate hA-II/A-I k-in, and hA-II/A-IM k-in, respectively. These genetically modified mice completely reproduced the apoA-IM carrier's phenotype, including hypoalphalipoproteinemia and hypertriglyceridemia. Furthermore, by using the microarray methodology, we investigated the intrinsic differences in hepatic gene expression among these k-in mouse lines. The expression of 871, 1,018, 1129 and 764 genes was significantly altered between 1) hA-II/A-I and hA-II/A-IM k-in; 2) A-IM and hA-II/A-IM k-in; 3) A-I and A-IM; 4) A-I and hA-II/A-I k-in liver samples, respectively. Bioinformatics analysis highlighted that the hepatic expression of two genes, Elovl6 and Gatm, related to fatty acid/lipid and energy metabolism, respectively, is influenced by the presence of the apoA-IM natural variant and/or apoA-II.

Particle size matters: Discrepancies in the health risks posed by traditional cigarettes and e-cigarettes in mice and humans

Electronic cigarettes (ECs), considered a healthier alternative to traditional cigarettes (TCs), vaporize e-liquid, which may produce harmful by-products due to thermal decomposition and metal transfer. These by-products' deposition in the respiratory tract is largely determined by particle size distribution (PSD). We employ the Multiple-Path Particle Dosimetry (MPPD) model to assess particle deposition within the human and mouse respiratory tracts. Leveraging the known connection between TC smoke inhalation and atherosclerosis, we used human aortic endothelial cells (HAECs) and ApoE-/- mice to explore the potential effects of EC aerosol inhalation on atherosclerosis. Our findings reveal that TCs exhibit a highly variable PSD, with mean diameters of approximately 300 nm for mainstream (MS) smoke and 120 nm for side stream (SS) smoke. Conversely, ECs demonstrate a more stable PSD. Combined with MPPD, the deposition fraction in the human respiratory system and mice is mainly deposited in the pulmonary region and head airway. For the Apoe-/- mice exposure experiment, preliminary findings suggest a potential impact on atherosclerosis, although not statistically significant, likely due to the limited sample size and exposure duration. This study highlights the importance of considering PSD, exposure dosage, and species-specific differences in risk assessments of EC aerosols.

Apolipoprotein A1 Inhibits Adipogenesis Progression of Human Adipose-Derived Mesenchymal Stem Cells.

According to the reports, the most vital characteristic of obesity is an aberrant accumulation of triglycerides (TG) in the adipocyte. On the other hand, circulating concentrations of apolipoprotein A1 (apoA1) have been demonstrated to be strongly correlated with the prevalence and the pathological development of obesity. Nevertheless, the underlying mechanisms whereby apoA1 modulates the pathogenesis of obesity is still not fully elucidated. Adipose-derived mesenchymal stem cells (AMSCs, isolated from the hospitalized patients were combined with 15 μg/ml recombined human apoA1 protein. The effects of apoA1 on modulating the intracellular levels of TG and the expression contents of adipogenic related cytokines were also analyzed. Furthermore, whether apoA1 modulated the adipogenesis progression via sortilin was also explored in the current research. During the adipogenesis progression, apoA1 could significantly lower the quantity of intracellular lipid droplets (LDs). Meanwhile, apoA1 could decrease the intracellular levels of TG and down-regulate the expression contents of several vital adipogenic related cytokines, such as CCAAT enhancer-binding proteins α/β (C/EBPα/β), fatty acid synthetase (FAS), and fatty acid-binding protein 4 (FABP4). Moreover, the inhibitory effect of apoA1 was further verified to be induced through upregulating the SORT1 gene expression which subsequently increased sortilin protein. Consistent with these findings, silencing the SORT1 gene expression could induce the loss-of-function (LOF) of apoA1 in modulating the adipogenesis progression of AMSCs. In conclusion, apoA1 could suppress the adipogenesis progression of human AMSCs through, at least partly, up-regulating the SORT1 gene expression which subsequently increases the sortilin protein content. Thereby, the present research sheds light on a novel pathogenic mechanism by which apoA1 regulates adipogenesis progression and proposes that apoA1 embraces the function to treat obesity in clinical practice.

Astrocytic APOE4 genotype-mediated negative impacts on synaptic architecture in human pluripotent stem cell model.

The APOE4 genotype is the strongest risk factor for the pathogenesis of sporadic Alzheimer's disease (AD), but the detailed molecular mechanism of APOE4-mediated synaptic impairment remains to be determined. In this study, we generated a human astrocyte model carrying the APOE3 or APOE4 genotype using human induced pluripotent stem cells (iPSCs) in which isogenic APOE4 iPSCs were genome edited from healthy control APOE3 iPSCs. Next, we demonstrated that the astrocytic APOE4 genotype negatively affects dendritic spine dynamics in a co-culture system with primary neurons. Transcriptome analysis revealed an increase of EDIL3, an extracellular matrix glycoprotein, in human APOE4 astrocytes, which could underlie dendritic spine reduction in neuronal cultures. Accordingly, postmortem AD brains carrying the APOE4 allele have elevated levels of EDIL3 protein deposits within amyloid plaques. Together, these results demonstrate the novel deleterious effect of human APOE4 astrocytes on synaptic architecture and may help to elucidate the mechanism of APOE4-linked AD pathogenesis.

Endothelial Cell APOE3 Regulates Neurovascular, Neuronal, and Behavioral Function.

Specialized brain endothelial cells and human APOE3 are independently important for neurovascular function, yet whether APOE3 expression by endothelial cells contributes to brain function is currently unknown. In the present study, we determined whether the loss of endothelial cell APOE3 impacts brain vascular and neural function. We developed APOE3fl/fl/Cdh5(PAC)-CreERT2+/- (APOE3Cre+/-) and APOE3fl/fl/Cdh5(PAC)-CreERT2-/- (APOE3Cre-/-, control) mice and induced endothelial cell APOE3 knockdown with tamoxifen at ≈4 to 5 weeks of age. Neurovascular and neuronal function were evaluated by biochemistry, immunohistochemistry, behavioral testing, and electrophysiology at 9 months of age. We found that the loss of endothelial APOE3 expression was sufficient to cause neurovascular dysfunction including higher permeability and lower vessel coverage in tandem with deficits in spatial memory and fear memory extinction and a disruption of cortical excitatory/inhibitory balance. Our data collectively support the novel concept that endothelial APOE3 plays a critical role in the regulation of the neurovasculature, neural circuit function, and behavior.

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

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

The APOE-TOMM40 Humanized Mouse Model: Characterization of Age, Sex, and PolyT Variant Effects on Gene Expression.

The human chromosome 19q13.32 is a gene rich region and has been associated with multiple phenotypes, including late onset Alzheimer's disease (LOAD) and other age-related conditions. Here we developed the first humanized mouse model that contains the entire TOMM40 and APOE genes with all intronic and intergenic sequences including the upstream and downstream regions. Thus, the mouse model carries the human TOMM40 and APOE genes and their intact regulatory sequences. We generated the APOE-TOMM40 humanized mouse model in which the entire mouse region was replaced with the human (h)APOE-TOMM40 loci including their upstream and downstream flanking regulatory sequences using recombineering technologies. We then measured the expression of the human TOMM40 and APOE genes in the mice brain, liver, and spleen tissues using TaqMan based mRNA expression assays. We investigated the effects of the '523' polyT genotype (S/S or VL/VL), sex, and age on the human TOMM40- and APOE-mRNAs expression levels using our new humanized mouse model. The analysis revealed tissue specific and shared effects of the '523' polyT genotype, sex, and age on the regulation of the human TOMM40 and APOE genes. Noteworthy, the regulatory effect of the '523' polyT genotype was observed for all studied organs. The model offers new opportunities for basic science, translational, and preclinical drug discovery studies focused on the APOE genomic region in relation to LOAD and other conditions in adulthood.

Carboxyl truncation of α-synuclein occurs early and is influenced by human APOE genotype in transgenic mouse models of α-synuclein pathogenesis

Post-translational modifications to the carboxyl (C) terminus domain of α-synuclein can play an important role in promoting the pathologic aggregation of α-synuclein. Various cleavages that diminish this highly charged, proline-rich region can result in exposure of hydrophobic, aggregation-prone regions, thereby accelerating the aggregation kinetics of α-synuclein into misfolded, pathologic forms. C-terminally truncated forms of α-synuclein are abundant in human diseased brains compared to controls, suggesting a role in disease pathogenesis. Factors that alter the homeostatic proteolytic processing of α-synuclein may ultimately tip the balance towards a progressive disease state. Apolipoprotein E (APOE) has been implicated in the acceleration of cognitive impairment in patients with Lewy body diseases. The APOE4 isoform has been found to cause dysregulation in the endosomal–lysosomal pathway, which could result in altered α-synuclein degradation as a potential mechanism for promoting its pathologic misfolding. Herein, we investigate the spatiotemporal accumulation of C-terminally truncated α-synuclein in a seeded and progressive mouse model of synucleinopathy. Furthermore, we study how this process is influenced in the context of mice that are altered to express either the human APOE3 or APOE4 isoforms. We found that specific C-terminal truncation of α-synuclein occurs at early stages of pathogenesis. We also found that proteolytic processing of this domain differs across various brain regions and is influenced by the presence of different human APOE isoforms. Our data demonstrate an early pathogenic role for C-terminally truncated α-synuclein, and highlight the influence of APOE isoforms in modulating its impact.

Detection of early Alzheimer's disease-like molecular alterations in a mouse model expressing human ApoE4.

The E4 allele of apolipoprotein E (ApoE4) is a key genetic risk factor for late-onset Alzheimer's disease (AD), increasing the risk of developing the disease by up to three-fold. However, the mechanisms by which ApoE4 contributes to AD pathogenesis are poorly understood. Here, we utilize a mouse model expressing either human ApoE3 or human ApoE4 to examine the effects of the E4 allele on a wide range of genetic and molecular pathways that are altered in early AD pathology. We demonstrate that ApoE4-expressing mice begin to show early differential expression of multiple genes, leading to alterations in downstream pathways related to neural cell maintenance, insulin signaling, amyloid processing and clearance, and synaptic plasticity. These alterations may result in the earlier accumulation of pathological proteins such as β-amyloid that may build up within cells, leading to the accelerated degeneration of neurons and astrocytes as observed in ApoE4-positive individuals. We also examine the metabolic effects associated with a high-fat diet (HFD) in male ApoE4-expressing mice compared with regular chow diet (RD) fed mice at different ages. We found that young ApoE4-expressing mice fed HFD developed metabolic disturbances, such as elevated weight gain, blood glucose, and plasma insulin levels that cumulatively have been observed to increase the risk of AD in humans. Taken together, our results reveal early pathways that could mediate ApoE4-related AD risk and may help identify more tractable therapeutic targets for treating ApoE4-associated AD.

Synaptojanin 1 Modulates Functional Recovery After Incomplete Spinal Cord Injury in Male Apolipoprotein E Epsilon 4 Mice.

Apolipoprotein E epsilon 4 (ApoE4) is the second most common variant of ApoE, being present in ∼14% of the population. Clinical reports identify ApoE4 as a genetic risk factor for poor outcomes after traumatic spinal cord injury (SCI) and spinal cord diseases such as cervical myelopathy. To date, there is no intervention to promote recovery of function after SCI/spinal cord diseases that is specifically targeted at ApoE4-associated impairment. Studies in the human and mouse brain link ApoE4 to elevated levels of synaptojanin 1 (synj1), a lipid phosphatase that degrades phosphoinositol 4,5-bisphosphate (PIP2) into inositol 4-monophosphate. Synj1 regulates rearrangements of the cytoskeleton as well as endocytosis and trafficking of synaptic vesicles. We report here that, as compared to ApoE3 mice, levels of synj1 messenger RNA and protein were elevated in spinal cords of healthy ApoE4 mice associated with lower PIP2 levels. Using a moderate-severity model of contusion SCI in mice, we found that genetic reduction of synj1 improved locomotor function recovery at 14 days after SCI in ApoE4 mice without altering spared white matter. Genetic reduction of synj1 did not alter locomotor recovery of ApoE3 mice after SCI. Bulk RNA sequencing revealed that at 14 days after SCI in ApoE4 mice, genetic reduction of synj1 upregulated genes involved in glutaminergic synaptic transmission just above and below the lesion. Overall, our findings provide evidence for a link between synj1 to poor outcomes after SCI in ApoE4 mice, up to 14 days post-injury, through mechanisms that may involve the function of excitatory glutaminergic neurons.

Isolation of recombinant apolipoprotein E4 N-terminal domain by foam fractionation

Apolipoprotein (apo) E functions in lipoprotein metabolism as a low density lipoprotein receptor ligand. ApoE is comprised of two structural domains, a 22 kDa N-terminal (NT) domain that adopts a helix bundle conformation and a 10 kDa C-terminal domain with strong lipid binding affinity. The NT domain is capable of transforming aqueous phospholipid dispersions into discoidal reconstituted high density lipoprotein (rHDL) particles. Given the utility of apoE-NT as a structural component of rHDL, expression studies were conducted. A plasmid construct encoding a pelB leader sequence fused to the N-terminus of human apoE4 (residues 1–183) was transformed into Escherichia coli. Upon expression, the fusion protein is directed to the periplasmic space where leader peptidase cleaves the pelB sequence, generating mature apoE4-NT. In shaker flask expression cultures, apoE4-NT escapes the bacteria and accumulates in the medium. In a bioreactor setting, however, apoE4-NT was found to combine with gas and liquid components in the culture medium to generate large quantities of foam. When this foam was collected in an external vessel and collapsed into a liquid foamate, analysis revealed that apoE4-NT was the sole major protein present. The product protein was further isolated by heparin affinity chromatography (60–80 mg/liter bacterial culture), shown to be active in rHDL formulation, and documented to serve as an acceptor of effluxed cellular cholesterol. Thus, foam fractionation provides a streamlined process to produce recombinant apoE4-NT for biotechnology applications.

Investigating the effect of human apolipoprotein E isoforms on expression level of CLS and MINO genes involved in mitochondrial function in transgenic Drosophila model

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by aberrant lipid metabolism and mitochondrial dysfunction, features related to mitochondria-associated ER membranes (MAM). ApoE is recognized as the major AD risk factor. Human ApoE has three isoforms including Apoε2, Apoε3 and Apoε4. It has revealed that ε4 allele carriers are at higher risk of developing AD than ε3 allele carriers. Given that Apoε4 is a major AD risk factor and that MAM dysfunction in lipid metabolism may affect mitochondrial function, we assessed the expression pattern of cardiolipin synthetase (CLS) and glycerol-3-phosphate acyltransferase 2 (MINO) genes in Drosophila model of AD. To the best of our knowledge, this is the first study to investigate the effect of ApoE isoforms on mitochondrial function in Drosophila model of AD. RNA extraction was performed using glazt2a.gal4.UAS.hapoe transgenic flies harboring Apoε isoforms. Following cDNA synthesis, mRNA expression levels of target (CLS and MINO) and housekeeping (eEF1α) genes were measured by qRT-PCR. Our results did not show a significant decrease in CLS and MINO expression level in Apoɛ3 and Apoɛ4 groups as compared to the control. Although this reduction was not statistically significant, the decreasing trend indicates a kind of disruption in lipid biosynthesis and may directly affect mitochondrial function. Though the impact of Apoɛ4 on AD pathogenesis has been elucidated, the exact mechanism through which Apoɛ4 mediates AD progression is still unknown. Investigating the biology of ApoE isoforms using both human and AD animal models is recommended.

Effects of long‐term oral treatment with a novel estrogen receptor beta agonist on memory, vasomotor, and anxiety outcomes in an EFAD mouse model of Alzheimer’s disease

AbstractBackgroundThe prevalence and severity of Alzheimer’s disease (AD) are increased by female sex, apolipoprotein E (APOE) genotype, and age, such that postmenopausal female carriers of the Apoe4 allele are at greater risk of developing AD than age‐matched males. Although estrogen therapy shows promise in preventing and reducing menopause‐ and AD‐related symptoms, activation of estrogen receptor alpha (ERa) is associated with increased risk for health concerns, such as breast cancer. Therefore, treatments more selective for estrogen receptor beta (ERb) may provide estrogen‐related health benefits without the risks of activating ERa. Long‐term or acute treatment of wild‐type mice with the novel ERb agonist, EGX358, improves memory and reduces drug‐induced vasomotor alterations. However, efficacy of this compound in an AD model is unknown.MethodFemale APOE‐TR+/+/5xFAD+/− (EFAD) mice expressed five familial AD and were homozygous for human APOE3 (E3FAD) or heterozygous for APOE3 and APOE4 (E3/4FAD). Mice were ovariectomized at 5 months of age and then treated orally with vehicle (1% DMSO) or EGX358 (10 mg/kg/day) via hydrogel for 8 weeks. Mice were weighed weekly and tested for spatial and object memory in the object placement (OP) and object recognition (OR) tasks, respectively. Anxiety‐like behaviors were also tested in the open field and elevated plus maze, and vasomotor symptoms were assessed via tail temperature following injection of the tachykinin receptor 3 agonist, senktide.ResultEGX358 significantly enhanced OR memory in both E3FAD and E3/4FAD mice. However, treatment did not affect OP memory or anxiety in the open field or elevated plus maze. Interestingly, there were significant genotype differences in open field, vasomotor response, and body weight such that E3/4FADs spent more time in the center of open field, had a greater increase in tail temperature following senktide injection, and gained more weight compared to E3FADs.ConclusionOur findings indicate a potential for ERβ‐specific estrogen treatment to improve memory for the identity of objects. Unexpected genotype differences indicate the need to further study how APOE genotype affects anxiety and metabolic outcomes in AD.

Targeting apolipoprotein E and N-terminal amyloid β-protein precursor interaction improves cognition and reduces amyloid pathology in Alzheimer’s mice

Apolipoprotein E (apoE) interaction with amyloid β-protein precursor (APP) has garnered attention as the therapeutic target for Alzheimer's disease (AD). Having discovered the apoE antagonist (6KApoEp) that blocks apoE binding to N-terminal APP, we tested the therapeutic potential of 6KApoEp on AD-relevant phenotypes in amyloid β-protein precursor/presenilin 1 (APP/PS1) mice that express each human apoE isoform of apoE2, apoE3, or apoE4 (designated APP/PS1/E2, APP/PS1/E3, or APP/PS1/E4 mice). At 12months of age, we intraperitoneally administered 6KApoEp (250μg/kg) or vehicle once daily for 3months. At 15months of age, blockage of apoE and N-terminal APP interaction by 6KApoEp treatment improved cognitive impairment in most tests of learning and memory, including novel object recognition and maze tasks in APP/PS1/E2, APP/PS1/E3, and APP/PS1/E4 mice versus each vehicle-treated mouse line and did not alter behavior in nontransgenic littermates. Moreover, 6KApoEp therapy ameliorated brain parenchymal and cerebral vascular β-amyloid deposits and decreased abundance of amyloid β-protein (Aβ) in APP/PS1/E2, APP/PS1/E3, and APP/PS1/E4 mice versus each vehicle-treated mouse group. Notably, the highest effect in Aβ-lowering by 6KApoEp treatment was observed in APP/PS1/E4 mice versus APP/PS1/E2 or APP/PS1/E3 mice. These effects occured through shifting toward lessened amyloidogenic APP processing due to decreasing APP abundance at the plasma membrane, reducing APP transcription, and inhibiting p44/42 mitogen-activated protein kinase phosphorylation. Our findings provide the preclinical evidence that 6KApoEp therapy aimed at targeting apoE and N-terminal APP interaction is a promising strategy and may be suitable for patients with AD carrying the apoE4 isoform.

Role of white matter hyperintensity in effects of apolipoprotein E on cognitive injury.

Magnetic Resonance Imaging (MRI) T2-weighted white matter hyperintensity (WMH) is a marker of small vessel cerebrovascular pathology and is of ischemic origin. The prevalence and severity of WMH is associated with cardiovascular risk factors, aging, and cognitive injury in mild cognitive impairment (MCI), vascular dementia, and Alzheimer's disease (AD). WMH especially affects executive function, with additional effects on memory and global cognition. Apolipoprotein E (apoE) plays a role in cholesterol metabolism and neuronal repair after injury. Human and animal studies support a role for apoE in maintaining white matter integrity. In humans, there are three major human apoE isoforms, E2, E3, and E4. Human apoE isoforms differ in risk to develop AD and in association with WMH. In this Mini Review, we propose an increased focus on the role of WMH in cognitive health and cognitive injury and the likely role of apoE and apoE isoform in modulating these effects. We hypothesize that apoE and apoE isoforms play a role in modulating WMH via apoE isoform-dependent effects on oxylipins and 7-ketocholesterol, as well as amyloid related vascular injury, as seen in cerebral amyloid angiopathy.

APOEε4 and risk of Alzheimer's disease - time to move forward.

The inheritance of Apolipoprotein E4 (APOEε4) brings the highest genetic risk of Alzheimer's disease (AD), arguably the highest genetic risk in human pathology. Since the discovery of the association, APOE protein isoforms have been at the center of tens of thousands of studies and reports. While, without a doubt, our knowledge about the normal physiological function of APOE isoforms in the brain has increased tremendously, the questions of how the inheritance of the APOEε4 allele translates into a risk of AD, and the risk is materialized, remain unanswered. Moreover, the knowledge about the risk associated with APOEε4 has not helped design a meaningful preventative or therapeutic strategy. Animal models with targeted replacement of Apoe have been generated and, thanks to the recent NIH/NIA/Alzheimer's disease Association initiative, are now freely available to AD researchers. While helpful in many aspects, none of the available models recapitulates normal physiological transcriptional regulation of the human APOE gene cluster. Changes in epigenetic regulation of APOE alleles in animal models in response to external insults have rarely been if ever, addressed. However, these animal models provide a useful tool to handle questions and investigate protein-protein interactions with proteins expressed by other recently discovered genes and gene variants considered genetic risk factors of AD, like Triggering Receptor expressed on Myeloid cells 2 (TREM2). In this review, we discuss genetic and epigenetic regulatory mechanisms controlling and influencing APOE expression and focus on interactions of APOE and TREM2 in the context of microglia and astrocytes' role in AD-like pathology in animal models.

Inhibition of ACAT as a Therapeutic Target for Alzheimer's Disease Is Independent of ApoE4 Lipidation.

APOE4, encoding apolipoprotein E4 (apoE4), is the greatest genetic risk factor for Alzheimer's disease (AD), compared to the common APOE3. While the mechanism(s) underlying APOE4-induced AD risk remains unclear, increasing the lipidation of apoE4 is an important therapeutic target as apoE4-lipoproteins are poorly lipidated compared to apoE3-lipoproteins. ACAT (acyl-CoA: cholesterol-acyltransferase) catalyzes the formation of intracellular cholesteryl-ester droplets, reducing the intracellular free cholesterol (FC) pool. Thus, inhibiting ACAT increases the FC pool and facilitates lipid secretion to extracellular apoE-containing lipoproteins. Previous studies using commercial ACAT inhibitors, including avasimibe (AVAS), as well as ACAT-knock out (KO) mice, exhibit reduced AD-like pathology and amyloid precursor protein (APP) processing in familial AD (FAD)-transgenic (Tg) mice. However, the effects of AVAS with human apoE4 remain unknown. In vitro, AVASinduced apoE efflux at concentrations of AVASmeasured in the brains of treated mice. AVAS treatment of male E4FAD-Tg mice (5xFAD+/-APOE4+/+) at6-8months had no effect on plasma cholesterol levels or distribution, the original mechanism forAVAS treatment of CVD. In the CNS, AVAS reduced intracellular lipid droplets, indirectly demonstrating target engagement. Surrogate efficacy was demonstrated by an increase in Morris water maze measures of memory and postsynaptic protein levels. Amyloid-beta peptide (Aβ) solubility/deposition and neuroinflammation were reduced, critical components of APOE4-modulated pathology. However, there was no increase in apoE4 levels or apoE4 lipidation, while amyloidogenic and non-amyloidogenic processing of APP were significantly reduced. This suggests that the AVAS-induced reduction in Aβ via reduced APP processing was sufficient to reduce AD pathology, as apoE4-lipoproteins remained poorly lipidated.

Impact of dietary choline on lipid metabolism and atherosclerosis development in apoEKO mice deficient or overexpressing apolipoprotein A-I: Selected Abstract - XVI SITeCS Congress

TMAO, a metabolite of dietary choline, is considered a pro-atherogenic molecule for its ability to interfere with the reverse cholesterol transport, in which apolipoprotein A-I and HDL play a key role. In the present work it was evaluated how TMAO impacts on the development of atherosclerosis in mice with different levels of apoA-I/HDL. Mice deficient in both murine apoA-I and apoE (DKO) and DKO mice overexpressing human apoA-I (DKO/hA-I), characterized by extremely low or high plasma HDL levels respectively, were fed for 16 weeks two standard rodent diets, differing only in their choline content (0.09% or 1.2%). At the end of the dietary treatment, atherosclerosis development was quantified at the aortic sinus, targeted plasma metabolomics was performed, and gene expression was evaluated in liver, duodenum, jejunum and ileum. With both diets, DKO mice developed much larger plaques than DKO/hA-I mice. High-choline diet increased plasma TMAO levels in both genotypes. Interestingly, a worsening of plaque development by high choline diet occurred in DKO/hA-I mice only (0.057±0.048 mm2 vs 0.0988±0.064 mm2, p<0.01). Plasma metabolomics indicated that choline supplementation, only in the presence of HDL, significantly increased the concentration of some ceramide species in addition to several markers of impaired kidney function. High-choline diet increased the hepatic gene expression of Fmo1 and Fmo2 in DKO/hA-I, whereas the expression of Scarb1 was lower in DKO/hA-I compared to DKO mice, regardless of the dietary treatment. Intestinal expression of genes involved in inflammatory response and in lipid metabolism was comparable between genotypes and was not modified by choline supplementation. In conclusion, high choline diet increased plasma TMAO concentration in both genotypes, but affected atherosclerosis development, plasma metabolome and hepatic gene expression only in high HDL mice. Intestinal gene expression was not affected neither by genotype nor by dietary choline content.

LRP1 in vascular mural cells modulates cerebrovascular integrity and function in the presence of APOE4.

Cerebrovasculature is critical in maintaining brain homeostasis; its dysregulation often leads to vascular cognitive impairment and dementia (VCID) during aging. VCID is the second most prevalent cause of dementia in the elderly, after Alzheimer's disease (AD), with frequent cooccurrence of VCID and AD. While multiple factors are involved in the pathogenesis of AD and VCID, APOE4 increases the risk for both diseases. A major apolipoprotein E (apoE) receptor, the low-density lipoprotein receptor-related protein 1 (LRP1), is abundantly expressed in vascular mural cells (pericytes and smooth muscle cells). Here, we investigated how deficiency of vascular mural cell LRP1 affects the cerebrovascular system and cognitive performance using vascular mural cell-specific Lrp1-KO mice (smLrp1-/-) in a human APOE3 or APOE4 background. We found that spatial memory was impaired in the 13- to 16-month-old APOE4 smLrp1-/- mice but not in the APOE3 smLrp1-/- mice, compared with their respective littermate control mice. These disruptions in the APOE4 smLrp1-/- mice were accompanied with excess paravascular glial activation and reduced cerebrovascular collagen IV. In addition, blood-brain barrier (BBB) integrity was disrupted in the APOE4 smLrp1-/- mice. Together, our results suggest that vascular mural cell LRP1 modulates cerebrovasculature integrity and function in an APOE genotype-dependent manner.