apoE Expression Research Articles

The normalizing effects of the CYP46A1 activator efavirenz on retinal sterol levels and risk factors for glaucoma in Apoj−/− mice

Apolipoprotein J (APOJ) is a multifunctional protein with genetic evidence suggesting an association between APOJ polymorphisms and Alzheimer’s disease as well as exfoliation glaucoma. Herein we conducted ocular characterizations of Apoj−/− mice and found that their retinal cholesterol levels were decreased and that this genotype had several risk factors for glaucoma: increased intraocular pressure and cup-to-disk ratio and impaired retinal ganglion cell (RGC) function. The latter was not due to RGC degeneration or activation of retinal Muller cells and microglia/macrophages. There was also a decrease in retinal levels of 24-hydroxycholesterol, a suggested neuroprotectant under glaucomatous conditions and a positive allosteric modulator of N-methyl-d-aspartate receptors mediating the light-evoked response of the RGC. Therefore, Apoj−/− mice were treated with low-dose efavirenz, an allosteric activator of CYP46A1 which converts cholesterol into 24-hydroxycholesterol. Efavirenz treatment increased retinal cholesterol and 24-hydroxycholesterol levels, normalized intraocular pressure and cup-to-disk ratio, and rescued in part RGC function. Retinal expression of Abcg1 (a cholesterol efflux transporter), Apoa1 (a constituent of lipoprotein particles), and Scarb1 (a lipoprotein particle receptor) was increased in EVF-treated Apoj−/− mice, indicating increased retinal cholesterol transport on lipoprotein particles. Ocular characterizations of Cyp46a1−/− mice supported the beneficial efavirenz treatment effects via CYP46A1 activation. The data obtained demonstrate an important APOJ role in retinal cholesterol homeostasis and link this apolipoprotein to the glaucoma risk factors and retinal 24-hydroxycholesterol production by CYP46A1. As the CYP46A1 activator efavirenz is an FDA-approved anti-HIV drug, our studies suggest a new therapeutic approach for treatment of glaucomatous conditions.

APOE4 expression confers a mild, persistent reduction in neurovascular function in the visual cortex and hippocampus of awake mice

Vascular factors are known to be early and important players in Alzheimer’s disease (AD) development, however the role of the ε4 allele of the Apolipoprotein (APOE) gene (a risk factor for developing AD) remains unclear. APOE4 genotype is associated with early and severe neocortical vascular deficits in anaesthetised mice, but in humans, vascular and cognitive dysfunction are focused on the hippocampal formation and appear later. How APOE4 might interact with the vasculature to confer AD risk during the preclinical phase represents a gap in existing knowledge. To avoid potential confounds of anaesthesia and to explore regions most relevant for human disease, we studied the visual cortex and hippocampus of awake APOE3 and APOE4-TR mice using 2-photon microscopy of neurons and blood vessels. We found mild vascular deficits: vascular density and functional hyperaemia were unaffected in APOE4 mice, and neuronal or vascular function did not decrease up to late middle-age. Instead, vascular responsiveness was lower, arteriole vasomotion was reduced and neuronal calcium signals during visual stimulation were increased. This suggests that, alone, APOE4 expression is not catastrophic but stably alters neurovascular physiology. We suggest this state makes APOE4 carriers more sensitive to subsequent insults such as injury or beta amyloid accumulation.

Reelin Plasma Levels Identify Cognitive Decline in Alcohol Use Disorder Patients During Early Abstinence: The Influence of APOE4 Expression.

Apolipoprotein E (APOE)-4 isoform, reelin, and clusterin share very-low-density liporeceptor and apolipoprotein E receptor 2 receptors and are related to cognition in neuropsychiatric disorders. These proteins are expressed in plasma and brain, but studies involving plasma expression and cognition are scarce. We studied the peripheral expression (plasma and peripheral blood mononuclear cells) of these proteins in 24 middle-aged patients with alcohol use disorder (AUD) diagnosed at 4 to 12 weeks of abstinence (t = 0) and 34 controls. Cognition was assessed using the Test of Detection of Cognitive Impairment in Alcoholism. In a follow-up study (t = 1), we measured reelin levels and evaluated cognitive improvement at 6 months of abstinence. APOE4 isoform was present in 37.5% and 58.8% of patients and controls, respectively, reaching similar plasma levels in ε4 carriers regardless of whether they were patients with AUD or controls. Plasma reelin and clusterin were higher in the AUD group, and reelin levels peaked in patients expressing APOE4 (P < .05, η2 = 0.09), who showed reduced very-low-density liporeceptor and apolipoprotein E receptor 2 expression in peripheral blood mononuclear cells. APOE4 had a negative effect on memory/learning mainly in the AUD group (P < .01, η2 = 0.15). Multivariate logistic regression analyses identified plasma reelin as a good indicator of AUD cognitive impairment at t = 0. At t = 1, patients with AUD showed lower reelin levels vs controls along with some cognitive improvement. Reelin plasma levels are elevated during early abstinence in patients with AUD who express the APOE4 isoform, identifying cognitive deterioration to a great extent, and it may participate as a homeostatic signal for cognitive recovery in the long term.

Cholesterol, Amyloid Beta, Fructose, and LPS Influence ROS and ATP Concentrations and the Phagocytic Capacity of HMC3 Human Microglia Cell Line.

Research has found that genes specific to microglia are among the strongest risk factors for Alzheimer's disease (AD) and that microglia are critically involved in the etiology of AD. Thus, microglia are an important therapeutic target for novel approaches to the treatment of AD. High-throughput in vitro models to screen molecules for their effectiveness in reversing the pathogenic, pro-inflammatory microglia phenotype are needed. In this study, we used a multi-stimulant approach to test the usefulness of the human microglia cell 3 (HMC3) cell line, immortalized from a human fetal brain-derived primary microglia culture, in duplicating critical aspects of the dysfunctional microglia phenotype. HMC3 microglia were treated with cholesterol (Chol), amyloid beta oligomers (AβO), lipopolysaccharide (LPS), and fructose individually and in combination. HMC3 microglia demonstrated changes in morphology consistent with activation when treated with the combination of Chol + AβO + fructose + LPS. Multiple treatments increased the cellular content of Chol and cholesteryl esters (CE), but only the combination treatment of Chol + AβO + fructose + LPS increased mitochondrial Chol content. Microglia treated with combinations containing Chol + AβO had lower apolipoprotein E (ApoE) secretion, with the combination of Chol + AβO + fructose + LPS having the strongest effect. Combination treatment with Chol + AβO + fructose + LPS also induced APOE and TNF-α expression, reduced ATP production, increased reactive oxygen species (ROS) concentration, and reduced phagocytosis events. These findings suggest that HMC3 microglia treated with the combination of Chol + AβO + fructose + LPS may be a useful high-throughput screening model amenable to testing on 96-well plates to test potential therapeutics to improve microglial function in the context of AD.

Characterization of lipoproteins in human placenta and fetal circulation as well as gestational changes in lipoprotein assembly and secretion in human and mouse placentas

In the maternal circulation, apoB-containing low-density lipoproteins (LDL) and apoA1-containing high-density lipoproteins (HDL) transport lipids. The production of lipoproteins in the placenta has been suggested, but the directionality of release has not been resolved. We compared apolipoprotein concentrations and size-exclusion chromatography elution profiles of lipoproteins in maternal/fetal circulations, and in umbilical arteries/veins; identified placental lipoprotein-producing cells; and studied temporal induction of lipoprotein-synthesizing machinery during pregnancy. We observed that maternal and fetal lipoproteins are different with respect to concentrations and elution profiles. Surprisingly, concentrations and elution profiles of lipoproteins in umbilical arteries and veins were similar indicating their homeostatic control. Human placental cultures synthesized apoB100-containing LDL-sized and apoA1-containing HDL-sized particles. Immunolocalization techniques revealed that ApoA1 was present mainly in syncytiotrophoblasts. MTP, a critical protein for lipoprotein assembly, was in these trophoblasts. ApoB was in the placental stroma indicating that trophoblasts secrete apoB-containing lipoproteins into the stroma. ApoB and MTP expressions increased in placentas from the 2nd trimester to term, whereas apoA1 expression was unchanged. Thus, our studies provide new information regarding the timing of lipoprotein gene induction during gestation, the cells involved in lipoprotein assembly and the gel filtration profiles of human placental lipoproteins.Next, we observed that mouse placenta produces MTP, apoB100, apoB48 and apoA1. The expression of genes gradually increased and peaked in late gestation. This information may be useful in identifying transcription factors regulating the induction of these genes in gestation and the importance of placental lipoprotein assembly in fetal development.

Loss of apolipoprotein E exacerbates microglial and astrocyte reactivity to sustained systemic glucocerebrosidase inhibition

AbstractBackgroundIncreased lipid storage is observed within the brains of patients with Alzheimer’s disease (AD). Genetic risk factors for AD involved in lipid processing include APOE, ABCA7 and TREM2. Lipid transport between neurons and glia is induced by mitochondrial stress and NMDA‐induced excitotoxicity in vitro and is dependent on glial apolipoprotein E (ApoE) expression. Therefore, this suggests that lipid transport between neurons and glia of the brain by ApoE and intracellular lipid processing are critical cellular adaptations to stress. This in turn suggests that insufficient ApoE activity may underpin the elevated lipid storage associated with neurodegenerative diseases.MethodTo determine whether ApoE function is required for cellular adaptation to increased lipid load relevant to dementia, 3‐month‐old WT and ApoE‐KO mice received daily intraperitoneal injections of the glucocerebrosidase inhibitor, CBE, for 18 d. Animals were sacrificed 1 d after the final dose and perfused with paraformaldehyde for immunohistochemistry, or were perfused with saline and dissected to isolate the cortex, hippocampus and substantia nigra for biochemical analysis by Western blot. Microglial and astrocyte reactivity to CBE were assessed by Iba1 and GFAP immunohistochemistry respectively.ResultMicroglial reactivity was greater in the dentate gyrus, substantia nigra and cortical layer V in ApoE‐KO mice after 18 d CBE, relative to WT. Loss of ApoE also elevated astrocyte reactivity in the dentate gyrus, substantia nigra and cortical layer V after 18 d CBE. Ongoing analyses into the molecular and cellular consequences of prolonged CBE include changes in lipid transport, tau phosphorylation, lysosome biogenesis, autophagy and proteostasis, to determine how loss of ApoE function may impact cellular adaptation to intracellular lipid formation resulting from sustained glucocerebrosidase inhibition.ConclusionThe exacerbated glial activation in ApoE‐KO animals receiving CBE suggests that ApoE expression and function may, in part, determine the capacity of neuron‐glia networks to overcome damaging stimuli. When ApoE function is insufficient, failed resolution of lipids may then lead to persistent damage and degeneration. Therefore, characterizing the consequences of limited lipid clearance on the adaptation to rising lipid load is highly relevant to understanding how failure to overcome increased lipid storage contributes to neurodegenerative processes in AD.

Imipramine treatment reduces Alzheimer’s disease neuropathologies in cerebral organoid models of Alzheimer’s disease and Down syndrome

AbstractBackgroundFamilial Alzheimer’s disease (fAD) is a autosomal dominant, early‐onset form of AD caused by genetic mutations such as duplication of the amyloid precursor protein gene (APP) on one chromosome (APPdup) or point mutations in APP such as the London mutation (APPV717I), both of which cause overproduction of the amyloid‐beta peptide (Aβ). People with Down syndrome (DS) also have a form of fAD due to triplication of APP which resides on chromosome 21. By age 40, all people with DS develop AD brain pathology, including Aβ plaques and neurofibrillary tangles of phosphorylated tau (pTau), and most go on to develop early‐onset dementia. We previously identified imipramine by high‐throughput screening for novel inhibitors of apolipoprotein E (apoE), which is the strongest risk factor for AD other than age itself. Here, we developed and characterized new cerebral organoid (CO) models of fAD and DS and used them to study common pathological mechanisms and to evaluate imipramine as a novel AD treatment.MethodWe obtained human induced pluripotent stem cells (hiPSCs) from multiple donors with fAD (APPdup or APPV717I), DS, or healthy controls (HC), all with the APOE3/3 genotype, and generated COs using validated techniques. We treated COs beginning at 45 days in vitro with 1 or 10 μM imipramine in the culture medium, and we collected treated and untreated organoids after 120 days for analysis by immunoblotting, immunohistochemistry, and ELISA.ResultCOs from fAD or DS donors were significantly reduced in size compared to those from HC donors. Increased expression of APP, Aβ, pTau, and apoE were observed in fAD and DS COs relative to HC COs. Levels of Aβ and apoE were positively correlated in all COs. Imipramine treatment significantly reduced the levels of some neuropathologies in AD COs, but did not affect the size or cellular composition of HC COs.ConclusionWe characterized several new CO models of fAD and DS, which will be useful for identifying commonalities in disease mechanisms as well as for dissecting the roles of chromosome 21 genes other than APP. Our results indicate that imipramine and other therapeutics targeting the role of apoE are promising approaches to treating AD.

Endometrial immune cells at the time of implantation in mares – preliminary results

Implantation in mares occurs approximately between days 35 when the chorionic girdle starts to invade endometrium and day 45 when the allantochorion is more firmly attached to endometrium. Immune cells are known to be present in the endometrium over this period but their phenotype and role in implantation in the mare is poorly understood. The aim of this study was to phenotypically characterize subpopulations of immune cells in the endometrium around the time of implantation using single cell RNAseq. Clinically healthy mares were monitored via transrectal ultrasonography. Ovulation was induced with hCG when the follicle diameter was ≥35mm and uterine edema was present. Mares were inseminated with cooled semen. Morphologically normal embryos were flushed by non-surgical lavage at day 33 (n=2) and day 42 (n=2) post ovulation, endometrial biopsies obtained, and serum samples collected for progesterone assays. Biopsies were digested to obtain single cell suspensions, PBMC enriched over Ficoll and then 10 000 cells per sample were processed for scRNA-Seq on the 10X Genomics Chromium platform (Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1; Dual Index). Sequencing was performed at the initial depth of ∼20 000 reads/cell. Data was analyzed with Seurat R package v4.0 and the EquCab 3.0 as the reference. Clusters were annotated using established markers of immune cells . (Patel et al. BMC biology. 2021;19:1-13).Unsupervised graph-based clustering of 53909 cells detected 17 clusters of cells that expressed 17667 genes. Cluster 3 was annotated as NK-like cells based on expression of LY49F, LY49C, GNLY, GZMA, CTSW and CD96. Cluster 4 was annotated as heterogenous T cell populations based on the expression of CD3 genes, CCL5, CD2 and CD5. Cluster 7 was assigned to macrophages due to expression of C1Q transcripts, CD68, APOE, DQ and DR transcripts, CXCL10 and CCL8. Both cluster 12 and 16 showed transcripts characteristic for neutrophils including CXCL8, IL1RW, IL1B, IL1BP, TG, RGS2, CCRL2 and PLEK. Cells in cluster 15 expressed genes characteristic both for γδT cells like CCL3, IL2RB, GLNY, GZMA, CTSW, but also Tregs as IL2R and CD80. Cells in cluster 17 expressed markers characteristic for plasma cells and B cells such as ENSECAG00000039599, TP53INP1, CD79A, MEF2C, PRDX4. Some clusters could not be annotated (cluster 1, 2, 5, 6, 8, 9, 10, 11) and are currently being screened for markers for endometrial cells and fibroblasts. In conclusion, these results indicate scRNAseq is a viable approach to simultaneously identify a large number of immune populations present in the endometrium. Deeper sequencing is needed to further differentiate and annotate subpopulations of cells within the clusters and to compare these subpopulations prior to and after chorionic girdle invasion.

Chicken cecal microbiota reduces abdominal fat deposition by regulating fat metabolism

Cecal microbiota plays an essential role in chicken health. However, its contribution to fat metabolism, particularly in abdominal fat deposition, which is a severe problem in the poultry industry, is still unclear. Here, chickens at 1, 4, and 12 months of age with significantly (p < 0.05) higher and lower abdominal fat deposition were selected to elucidate fat metabolism. A significantly (p < 0.05) higher mRNA expression of fat anabolism genes (ACSL1, FADS1, CYP2C45, ACC, and FAS), a significantly (p < 0.05) lower mRNA expression of fat catabolism genes (CPT-1 and PPARα) and fat transport gene APOAI in liver/abdominal fat of high abdominal fat deposition chickens indicated that an unbalanced fat metabolism leads to excessive abdominal fat deposition. Parabacteroides, Parasutterella, Oscillibacter, and Anaerofustis were found significantly (p < 0.05) higher in high abdominal fat deposition chickens, while Sphaerochaeta was higher in low abdominal fat deposition chickens. Further, Spearman correlation analysis indicated that the relative abundance of cecal Parabacteroides, Parasutterella, Oscillibacter, and Anaerofustis was positively correlated with abdominal fat deposition, yet cecal Sphaerochaeta was negatively correlated with fat deposition. Interestingly, transferring fecal microbiota from adult chickens with low abdominal fat deposition into one-day-old chicks significantly (p < 0.05) decreased Parabacteroides and fat anabolism genes, while markedly increased Sphaerochaeta (p < 0.05) and fat catabolism genes (p < 0.05). Our findings might help to assess the potential mechanism of cecal microbiota regulating fat deposition in chicken production.

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.

Exercise-induced upregulation of APOE expression is attenuated by dietary supplementation with resveratrol

The ε4 variant of the Apolipoprotein E gene (APOE4) is the most prevalent genetic risk factor for Alzheimer’s disease (AD). Approximately 25% of people carry at least one copy of the variant which increases risk of AD two-to-three-fold relative to those who carry the ε3 variant. Low ApoE levels are typically reported in Alzheimer’s patients and animal studies have reveal lower levels of ApoE in ε4 carriers relative to ε3 carriers. Therefore, reduced ApoE levels has been associated with increased risk for dementia. Physical exercise and active lifestyles are well-accepted therapeutic strategies for the prevention or delay of Alzheimer’s disease and other dementias, Additionally, there is growing evidence that the consumption of antioxidant nutritional supplements may also be therapeutic and slow progression of AD. But the effect of physical activity or antioxidant intake on APOE expression is not well-studied.The objective of this study is to determine the impact of exercise and resveratrol intake on APOE expression. We hypothesized that both resveratrol intake and exercise would increase APOE expression in brain with an additive effect when combined. Targeted replacement mice, homozygous for either APOE3 or APOE4 were randomized into four groups based on activity and resveratrol dietary supplementation: sedentary standard, sedentary resveratrol, exercise standard, and exercise resveratrol. Exercised groups were subjected to daily treadmill running for 8 weeks. Resveratrol treated groups were fed a chow supplemented with resveratrol. Results showed that exercise induced a significant increase in ApoE protein in brain cortex of both APOE3 and APOE4 mice. Resveratrol treatment alone had no significant effect on brain APOE expression, but in combination with exercise, attenuated the exercise-induced increase. While biochemical variations between APOE3 and APOE4 mice likely contribute to the differential risk for AD, the exercise-induced increase in ApoE levels in both genotypes supports a role for APOE in the therapeutic mechanisms of exercise. The timing of antioxidant supplementation in conjunction with exercise interventions should be critically considered to ensure best therapeutic efficacy of exercise. This research was supported by NIH grants R25AG047843 and 2U54MD007597-31 This is the full abstract presented at the American Physiology Summit 2023 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.

Sleep deprivation modulates APOE and LDL receptor-related protein 1 through thyroid hormone T4 and impairs Aβ clearance in hippocampus of rats

Alzheimer's disease is the most common form of dementia. One of its pathological hallmarks is Aβ accumulation, which is influenced by APOE genotype and expression, as well as by sleep homeostasis. However, conflicting mechanisms for APOE roles in Aβ clearance have been reported, and the relationship between APOE and sleep also remains unclear. In this study, we aimed to investigate how hormonal alteration caused by sleep deprivation affects APOE and its receptors in rats, and to evaluate the role of different cell types in Aβ clearance. Paradoxical sleep deprivation for 96 h increased Aβ level in hippocampus with concomitant reduction of APOE and LRP1 at the time point within the resting period. Sleep deprivation also significantly reduced T4 levels in both active and resting times. To evaluate the effect of T4 variation, C6 glial cells and primary brain endothelial cells were treated with T4. High T4 level (300 ng/mL) increased APOE, but reduced LRP1 and LDL-R in C6 cells, while in primary endothelial cells, LDL-R levels were increased. Treatment of C6 cells with exogenous APOE reduced LRP1 and Aβ uptake. These results suggest that T4 modulates LRP1 and LDL-R in both cell types, but in the opposite manner, thus, sleep deprivation might modify the ratio of the receptors in blood-brain barrier and glial cells by altering T4 levels. Considering that LRP1 and LDL-R are important for Aβ clearance, sleep deprivation might also affect the degree of participation of glia in Aβ clearance, and consequently, turnover of Aβ in the brain.

Yolk precursor synthesis and deposition in hierarchical follicles and effect on egg production performance of hens

Egg production of hens is related to ovarian follicles development. The hierarchical follicle development accompanies the deposition of a large amount of yolk precursor. The aim of this study was to illustrate the effects of strain and age on yolk deposition and egg production. The experiment compared yolk synthesis, transport, and deposition in 3 groups of hens: one of a high-yield commercial hybrid laying breed (Jinghong No.1) in 2 stages (35 wk and 75 wk; JH35, JH75) and one of Chinese native breed (Lueyang Black-Boned chicken) at 35 wk (LY35). The results showed that the number of hierarchical follicles in JH35 and JH75 was significantly more than in LY35. At the same time, the yolk weight of the LY35 and JH75 was significantly higher than that of JH35. The expression of apolipoprotein A1 and apolipoprotein B genes in the liver of JH35 was higher than that of JH75. The expression of the very low-density lipoprotein receptor gene in the JH75 ovary was higher than that of the other 2 groups. The plasma concentrations of very low-density lipoprotein and vitellogenin were no significant difference among groups. The yolk deposition in hierarchical follicles based on the fat-soluble dyes measurement meant that the rate of yolk deposition of LY35 was lower than the other 2 groups. In most cases, the yolk deposition of JH75 was higher than that of the other groups, but the process showed greater fluctuation over time. These results meant that the rate and stability of yolk deposition played an essential role in affecting egg performance. In summary, both strain and age were related to egg production, but the 2 factors might impact yolk deposition and egg-laying performance differently. The egg performance may be affected by both yolk precursor synthesis and deposition for different strains, but it may be affected by yolk precursor deposition for the old laying hens.

Potential Molecular Mechanisms of Alzheimer's Disease from Genetic Studies.

The devastating effects of Alzheimer's disease (AD) are yet to be ameliorated due to the absence of curative treatment options. AD is an aging-related disease that affects cognition, and molecular imbalance is one of its hallmarks. There is a need to identify common causes of molecular imbalance in AD and their potential mechanisms for continuing research. A narrative synthesis of molecular mechanisms in AD from primary studies that employed single-cell sequencing (scRNA-seq) or spatial genomics was conducted using Embase and PubMed databases. We found that differences in molecular mechanisms in AD could be grouped into four key categories: sex-specific features, early-onset features, aging, and immune system pathways. The reported causes of molecular imbalance were alterations in bile acid (BA) synthesis, PITRM1, TREM2, olfactory mucosa (OM) cells, cholesterol catabolism, NFkB, double-strand break (DSB) neuronal damage, P65KD silencing, tau and APOE expression. What changed from previous findings in contrast to results obtained were explored to find potential factors for AD-modifying investigations.

Overexpression of salusin‑α upregulates AdipoR2 and activates the PPARα/ApoA5/SREBP‑1c pathway to inhibit lipid synthesis in HepG2 cells.

Salusin‑α and adiponectin, are vasoactive peptides with numerous similar biological effects related to lipid metabolism. Adiponectin has been shown to reduce fatty acid oxidation and to inhibit lipid synthesis of liver cells through its receptor, adiponectin receptor2 (AdipoR2), but whether salusin‑α is able to interact with AdipoR2, was not previously reported. To investigate this, invitro experiments were carried out. The overexpression and interference recombinant plasmids were constructed with salusin‑α. The lentiviral expression systems of salusin‑α overexpression and interference were respectively synthesized in 293Tcells, and 293Tcells were infected with the lentivirus. Finally, the association between salusin‑α and AdipoR2 was analyzed by semi‑quantitative PCR. Subsequently, HepG2 cells were also infected with these viruses. The expression levels of AdipoR2, peroxisome proliferator‑activated receptor‑α (PPARα), apolipoproteinA5 (ApoA5) and sterol regulatory element‑binding transcription factor1 (SREBP‑1c) were detected by western blotting, and AdipoR2 inhibitor (thapsigargin) and agonist [4‑phenyl butyric acid (PBA)] were used to observe the resultant changes in the aforementioned molecules. The results obtained revealed that the overexpression of salusin‑α increased the level of AdipoR2 in 293TandHepG2cells, led to an upregulation of the levels of PPARα and ApoA5, and inhibited the expression of SREBP‑1c, whereas the salusin‑α interference lentivirus exerted the opposite effects. Notably, thapsigargin inhibited the expression of AdipoR2, PPARα and ApoA5 in HepG2 cells of pHAGE‑Salusin‑α group, and caused an increase in the level of SREBP‑1c, whereas the opposite effects were observed in pLKO.1‑shSalusin‑α#1 group upon treatment with PBA. Taken together, these data demonstrated that overexpression of salusin‑α upregulated AdipoR2, which in turn activated the PPARα/ApoA5/SREBP‑1c signaling pathway to inhibit lipid synthesis in HepG2 cells, thereby providing theoretical data on which to base the clinical application of salusin‑α as a novel peptide for molecular intervention in fatty liver disease.

Peroxisome proliferator-activated receptor δ improves the features of atherosclerotic plaque vulnerability by regulating smooth muscle cell phenotypic switching.

Vascular smooth muscle cells (SMCs) undergo phenotypic switching during sustained inflammation, contributing to an unfavourable atherosclerotic plaque phenotype. PPARδ plays an important role in regulating SMC functions; however, its role in atherosclerotic plaque vulnerability remains unclear. Here, we explored the pathological roles of PPARδ in atherosclerotic plaque vulnerability in severe atherosclerosis and elucidated the underlying mechanisms. Plasma levels of PPARδ were measured in patients with acute coronary syndrome (ACS) and stable angina (SA). SMC contractile and synthetic phenotypic markers, endoplasmic reticulum (ER) stress, and features of atherosclerotic plaque vulnerability were analysed for the brachiocephalic artery of apolipoprotein E-knockout (ApoE-/- ) mice, fed a high-cholesterol diet (HCD) and treated with or without the PPARδ agonist GW501516. In vitro, the role of PPARδ was elucidated using human aortic SMCs (HASMCs). Patients with ACS had significantly lower plasma PPARδ levels than those with SA. GW501516 reduced atherosclerotic plaque vulnerability, a synthetic SMC phenotype, ER stress markers, and NLRP3 inflammasome expression in HCD-fed ApoE-/- mice. ER stress suppressed PPARδ expression in HASMCs. PPARδ activation inhibited ER stress-induced synthetic phenotype development, ER stress-NLRP3 inflammasome axis activation and matrix metalloproteinase 2 (MMP2) expression in HASMCs. PPARδ inhibited NFκB signalling and alleviated ER stress-induced SMC phenotypic switching. Low plasma PPARδ levels may be associated with atherosclerotic plaque vulnerability. Our findings provide new insights into the mechanisms underlying the protective effect of PPARδ on SMC phenotypic switching and improvement the features of atherosclerotic plaque vulnerability.

Quantitative Proteomic Analysis Reveals apoE4-Dependent Phosphorylation of the Actin-Regulating Protein VASP

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer’s disease. While neurons generally produce a minority of the apoE in the central nervous system, neuronal expression of apoE increases dramatically in response to stress and is sufficient to drive pathology. Currently, the molecular mechanisms of how apoE4 expression may regulate pathology are not fully understood. Here, we expand upon our previous studies measuring the impact of apoE4 on protein abundance to include the analysis of protein phosphorylation and ubiquitylation signaling in isogenic Neuro-2a cells expressing apoE3 or apoE4. ApoE4 expression resulted in a dramatic increase in vasodilator-stimulated phosphoprotein (VASP) S235 phosphorylation in a protein kinase A (PKA)-dependent manner. This phosphorylation disrupted VASP interactions with numerous actin cytoskeletal and microtubular proteins. Reduction of VASP S235 phosphorylation via PKA inhibition resulted in a significant increase in filopodia formation and neurite outgrowth in apoE4-expressing cells, exceeding levels observed in apoE3-expressing cells. Our results highlight the pronounced and diverse impact of apoE4 on multiple modes of protein regulation and identify protein targets to restore apoE4-related cytoskeletal defects.

Relationship of Muscle Apolipoprotein E Expression with Markers of Cellular Stress, Metabolism, and Blood Biomarkers in Cognitively Healthy and Impaired Older Adults.

Individuals with mild cognitive impairment (MCI) have reduced lipid-stimulated mitochondrial respiration in skeletal muscle. A major risk factor for Alzheimer's disease (AD), the apolipoprotein E4 (APOE4) allele, is implicated in lipid metabolism and is associated with metabolic and oxidative stress that can result from dysfunctional mitochondria. Heat shock protein 72 (Hsp72) is protective against these stressors and is elevated in the AD brain. Our goal was to characterize skeletal muscle ApoE and Hsp72 protein expression in APOE4 carriers in relationship to cognitive status, muscle mitochondrial respiration and AD biomarkers. We analyzed previously collected skeletal muscle tissue from 24 APOE4 carriers (60y+) who were cognitively healthy (CH, n = 9) or MCI (n = 15). We measured ApoE and Hsp72 protein levels in muscle and phosphorylated tau181 (pTau181) levels in plasma, and leveraged previously collected data on APOE genotype, mitochondrial respiration during lipid oxidation, and VO2 max. Muscle ApoE (p = 0.013) and plasma pTau181 levels (p < 0.001) were higher in MCI APOE4 carriers. Muscle ApoE positively correlated with plasma pTau181 in all APOE4 carriers (R2 = 0.338, p = 0.003). Hsp72 expression negatively correlated with ADP (R2 = 0.775, p = <0.001) and succinate-stimulated respiration (R2 = 0.405, p = 0.003) in skeletal muscle of MCI APOE4 carriers. Plasma pTau181 negatively tracked with VO2 max in all APOE4 carriers (R2 = 0.389, p = 0.003). Analyses were controlled for age. This work supports a relationship between cellular stress in skeletal muscle and cognitive status in APOE4 carriers.

Immune landscape and the key role of APOE+ monocytes of lupus nephritis under the single-cell and spatial transcriptional vista.

Lupus nephritis (LN) is among the most common complication of systemic lupus erythematosus (SLE) with high mortality and morbidity. The analysis of LN kidney's local immune response through single-cell and spatial transcriptome enables the study of potential therapeutic targets. By single cell sequencing and spatial transcriptome, we profile cells from LN kidney and normal kidney tissues to characterize cellular composition and elucidate the potential upstream monocyte/macrophage (Mono/MΦ) initiating the auto-immune response. After the high-throughput synergy screening, we performed the immunofluorescence to identify the specific cells in LN patients. The function experiments were finished by flow cytometry and Elisa. By immunofluorescence and spatial transcriptome, we identified differential subsets of Mono/MΦ and demonstrated that they exhibit temporal expression of TIMP1, IL1B, SPP1 and APOE. With the function experiments, we found that the APOE+ Mono may be compensatorily increased in LN, and the capacity of antigen presenting was decreased with the overexpression of APOE. Furthermore, how do the LN-specific Mono/MΦ transport in and out the glomerulus to active the local immune response remains unclear. Our results showed that lymphangiogenesis occurred in LN kidneys but not in normal kidneys, suggesting the presence of a new lymphatic vessel may serve as a 'green channel' for LN-specific Mono/MΦ. In LN, APOE+ Mono are compensatorily elevated, with decreased antigen presenting ability and reduced secretion of interferons. The lymphangiogenesis in LN prompts the trafficking of Mono/MΦ in LN kidney.

Mitochondria Profoundly Influence Apolipoprotein E Biology.

Mitochondria can trigger Alzheimer's disease (AD)-associated molecular phenomena, but how mitochondria impact apolipoprotein E (APOE; apoE) is not well known. Consider whether and how mitochondrial biology influences APOE and apoE biology. We measured APOE expression in human SH-SY5Y neuronal cells with different forms of mitochondrial dysfunction including total, chronic mitochondrial DNA (mtDNA) depletion (ρ0 cells); acute, partial mtDNA depletion; and toxin-induced mitochondrial dysfunction. We further assessed intracellular and secreted apoE protein levels in the ρ0 cells and interrogated the impact of transcription factors and stress signaling pathways known to influence APOE expression. SH-SY5Y ρ0 cells exhibited a 65-fold increase in APOE mRNA, an 8-fold increase in secreted apoE protein, and increased intracellular apoE protein. Other models of primary mitochondrial dysfunction including partial mtDNA-depletion, toxin-induced respiratory chain inhibition, and chemical-induced manipulations of the mitochondrial membrane potential similarly increased SH-SY5Y cell APOE mRNA. We explored potential mediators and found in the ρ0 cells knock-down of the C/EBPα and NFE2L2 (Nrf2) transcription factors reduced APOE mRNA. The activity of two mitogen-activated protein kinases, JNK and ERK, also strongly influenced ρ0 cell APOE mRNA levels. Primary mitochondrial dysfunction either directly or indirectly activates APOE expression in a neuronal cell model by altering transcription factors and stress signaling pathways. These studies demonstrate mitochondrial biology can influence the biology of the APOE gene and apoE protein, which are implicated in AD.