Apoprotein Levels Research Articles

Absence of Apolipoprotein E is associated with exacerbation of prion pathology and promotes microglial neurodegenerative phenotype

Prion diseases or prionoses are a group of rapidly progressing and invariably fatal neurodegenerative diseases. The pathogenesis of prionoses is associated with self-replication and connectomal spread of PrPSc, a disease specific conformer of the prion protein. Microglia undergo activation early in the course of prion pathogenesis and exert opposing roles in PrPSc mediated neurodegeneration. While clearance of PrPSc and apoptotic neurons have disease-limiting effect, microglia-driven neuroinflammation bears deleterious consequences to neuronal networks. Apolipoprotein (apo) E is a lipid transporting protein with pleiotropic functions, which include controlling of the phagocytic and inflammatory characteristics of activated microglia in neurodegenerative diseases. Despite the significance of microglia in prion pathogenesis, the role of apoE in prionoses has not been established. We showed here that infection of wild type mice with 22L mouse adapted scrapie strain is associated with significant increase in the total brain apoE protein and mRNA levels and also with a conspicuous cell-type shift in the apoE expression. There is reduced expression of apoE in activated astrocytes and marked upregulation of apoE expression by activated microglia. We also showed apoE ablation exaggerates PrPSc mediated neurodegeneration. Apoe−/− mice have shorter disease incubation period, increased load of spongiform lesion, pronounced neuronal loss, and exaggerated astro and microgliosis. Astrocytes of Apoe−/− mice display salient upregulation of transcriptomic markers defining A1 neurotoxic astrocytes while microglia show upregulation of transcriptomic markers characteristic for microglial neurodegenerative phenotype. There is impaired clearance of PrPSc and dying neurons by microglia in Apoe−/− mice along with increased level of proinflammatory cytokines. Our work indicates that apoE absence renders clearance of PrPSc and dying neurons by microglia inefficient, while the excess of neuronal debris promotes microglial neurodegenerative phenotype aggravating the vicious cycle of neuronal death and neuroinflammation.

Apolipoprotein E Promotes Immune Suppression in Pancreatic Cancer through NF-κB-Mediated Production of CXCL1.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with few effective therapeutic options. PDAC is characterized by an extensive fibroinflammatory stroma that includes abundant infiltrating immune cells. Tumor-associated macrophages (TAM) are prevalent within the stroma and are key drivers of immunosuppression. TAMs in human and murine PDAC are characterized by elevated expression of apolipoprotein E (ApoE), an apolipoprotein that mediates cholesterol metabolism and has known roles in cardiovascular and Alzheimer's disease but no known role in PDAC. We report here that ApoE is also elevated in peripheral blood monocytes in PDAC patients, and plasma ApoE protein levels stratify patient survival. Orthotopic implantation of mouse PDAC cells into syngeneic wild-type or in ApoE-/- mice showed reduced tumor growth in ApoE-/- mice. Histologic and mass cytometric (CyTOF) analysis of these tumors showed an increase in CD8+ T cells in tumors in ApoE-/- mice. Mechanistically, ApoE induced pancreatic tumor cell expression of Cxcl1 and Cxcl5, known immunosuppressive factors, through LDL receptor and NF-κB signaling. Taken together, this study reveals a novel immunosuppressive role of ApoE in the PDAC microenvironment. SIGNIFICANCE: This study shows that elevated apolipoprotein E in PDAC mediates immune suppression and high serum apolipoprotein E levels correlate with poor patient survival.See related commentary by Sherman, p. 4186.

Superior Frontal Gyrus TOMM40-APOE Locus DNA Methylation in Alzheimer's Disease.

Background:The APOE ɛ4 allele is the strongest known genetic risk factor for sporadic Alzheimer’s disease (AD). The neighboring TOMM40 gene has also been implicated in AD due to its close proximity to APOE.Objective:Here we tested whether methylation of the TOMM40-APOE locus may influence ApoE protein levels and AD pathology.Methods:DNA methylation levels across the TOMM40-APOE locus and ApoE levels were measured in superior frontal gyrus tissues of 62 human brains genotyped for APOE and scored for AD neuropathology.Results:Methylation levels within the TOMM40 CpG island in the promoter or APOE CpG island in Exon 4 did not differ between APOE ɛ4 carriers versus non-carriers. However, APOE ɛ4 carriers had significantly higher methylation the APOE promoter compared with non-carriers. Although DNA methylation at TOMM40, APOE promoter region, or APOE did not differ between AD pathological groups, there was a negative association between TOMM40 methylation and CERAD scores. ApoE protein concentrations did not significantly different between APOE ɛ4 carriers and non-carriers, or between AD pathological groups. Finally, there was no correlation between ApoE protein concentrations and DNA methylation levels.Conclusion:APOE gene methylation may not be affected by genotype, relate to AD pathology or ApoE protein levels in the superior frontal gyrus, though, DNA methylation at the ApoE promoter differed between genotype. DNA methylation at TOMM40 associated with amyloid-β plaques and longitudinal fluid intelligence. In sum, these results suggest a complicated regulation of the TOMM40-APOE locus in the brain in controlling ApoE protein levels and AD neuropathology.

Impact of Ramadan fasting on lipid profile and cardiovascular risk factors in patients with stable coronary artery disease

BackgroundThe effects of Ramadan fasting (RF) on cardiometabolic risk factors in patients with stable ischemic heart disease are not well known. AimThe aim of this study was to evaluate the impact of RF on lipid profile and cardiovascular risk factors in patients with a stable coronary heart disease. MethodsA prospective observational study carried out in the Cardiology department of Charles Nicolle Hospital (Tunisia). Eighty-four patients with a stable ischemic heart disease who intended to fast were enrolled during May 2020. Detailed clinical and biochemical assessments were performed before and after the holy month. Parameters of glycemic control, lipid profile, ultrasensitive C-reactive protein concentration (us-CRP) and homocysteine were performed before- and after- Ramadan (BR and AR, respectively). ResultsEighty-four patients including 79 males and 5 females, with a mean age of 57±7 years completed the study. Levels of cholesterol, triglycerides, low-density lipoprotein-cholesterol and apoprotein A were significantly improved AR fasting in comparison with their BR values. There was a significant decrease in blood fasting glucose, insulin level, Homeostasis model assessment of insulin resistance index and in us-CRP level. ConclusionIn patients with stable ischemic heart disease, RF may be accompanied by an improvement of lipid profile and glycemic parameters without increase in coronary events.

Identification and Enzymatic Activity Evaluation of a Novel CYP2C9 Allelic Variant Discovered in a Patient.

Warfarin is a widely prescribed anticoagulant but the doses required to attain the optimum therapeutic effect exhibit dramatic inter-individual variability. Pharmacogenomics-guided warfarin dosing has been recommended to improve safety and effectiveness. We analyzed the cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) genes among 120 patients taking warfarin. A new coding variant was identified by sequencing CYP2C9. The novel A > G mutation at nucleotide position 14,277 led to an amino acid substitution of isoleucine with valine at position 213 (I213V). The functional consequence of the variant was subsequently evaluated in vitro. cDNA of the novel variant was constructed by site-directed mutagenesis and the recombinant protein was expressed in vitro using a baculovirus–insect cell expression system. The recombinant protein expression was quantified at apoprotein and holoprotein levels. Its enzymatic activities toward tolbutamide, warfarin and losartan were then assessed. It exhibited changed apparent Km values and increases of 148%, 84% and 67% in the intrinsic clearance of tolbutamide, warfarin and losartan, respectively, compared to wild-type CYP2C9*1, indicating dramatically enhanced in vitro enzymatic activity. Our study suggests that the amino acid at position 213 in wild-type CYP2C9*1 may be important for the enzymatic activity of CYP2C9 toward tolbutamide, warfarin and losartan. In summary, a patient taking high-dose warfarin (6.0 mg/day) in order to achieve the target international normalized ratio was found to have a mutation in the CYP2C9 gene.

Apo A1/Apo B ratio and acute coronary syndrome among peritoneal dialysis patients

Background Acute coronary syndrome (ACS) is prevalent in continuous ambulatory peritoneal dialysis (CAPD) patients. However, the association between the apoprotein profile and ACS is not well known. Therefore, we aimed to investigate the relationship between apoproteins and ACS in CAPD patients. Methods Eighty-one CAPD patients were included in this retrospective study. The primary endpoint was ACS. Predictors were baseline apoprotein levels, particularly the ratio of apoprotein A1 (Apo A1)/apoprotein B (Apo B). Cox regression was used to determine the relationship between Apo A1/Apo B and ACS. Results During follow-up, 34 (41.98%) CAPD patients experienced an ACS. ACS patients had higher levels of total cholesterol (p = 0.03), low-density lipoprotein cholesterol (LDL-C) (p = 0.04), C-reactive protein (p = 0.01), and Apo B (p < 0.01). However, hemoglobin (p = 0.01) and Apo A1/Apo B (p < 0.01) were lower in the ACS group than the non-ACS group. Patients with Apo A1/Apo B ≥ 1.105 experienced fewer ACS compared with those with Apo A1/Apo B < 1.105 (33.33% vs. 75.56%, p = 0.03). In Cox regression, Apo A1/Apo B (RR, 0.06; 95% CI, 0.00-0.77; p = 0.03) was independently associated with ACS. Conclusions Apo A1/Apo B was strongly associated with ACS and may be considered as a predictor of future ACS in CAPD patients.

Association of Apolipoprotein E in Lipoprotein Subspecies With Risk of Dementia

The ε4 allele of the apolipoprotein E (APOE) gene and lower apolipoprotein E (apoE) protein levels in plasma are risk factors for Alzheimer disease, but the underlying biological mechanisms are not fully understood. Half of plasma apoE circulates on high-density lipoproteins (HDLs). Higher apoE levels in plasma HDL were previously found to be associated with lower coronary heart disease risk, but the coexistence of another apolipoprotein, apoC3, modified this lower risk. To investigate associations between the presence of apoE in different lipoproteins with cognitive function, particularly the risk of dementia. This prospective case-cohort study embedded in the Ginkgo Evaluation of Memory Study (2000-2008) analyzed data from 1351 community-dwelling participants 74 years and older. Of this group, 995 participants were free of dementia at baseline (recruited from September 2000 to June 2002) and 521 participants were diagnosed with incident dementia during follow-up until 2008. Data analysis was performed from January 2018 to December 2019. Enzyme-linked immunosorbent assay-measured concentration of apoE in whole plasma, HDL-depleted plasma (non-HDL), HDL, and HDL subspecies that contain or lack apoC3 or apoJ. Adjusted hazard ratios for risk of dementia and Alzheimer disease during follow-up and adjusted differences (β coefficients) in Alzheimer Disease Assessment-Cognitive Subscale (ADAS-cog) and Modified Mini-Mental State Examination scores at baseline. Among 1351 participants, the median (interquartile range) age was 78 (76-81) years; 639 (47.3%) were women. The median (interquartile range) follow-up time was 5.9 (3.7-6.5) years. Higher whole plasma apoE levels and higher apoE levels in HDL were associated with better cognitive function assessed by ADAS-cog (whole plasma, β coefficient, -0.15; 95% CI, -0.24 to -0.06; HDL, β coefficient, -0.20; 95% CI, -0.30 to -0.10) but were unassociated with dementia or Alzheimer disease risk. When separated by apoC3, a higher apoE level in HDL that lacks apoC3 was associated with better cognitive function (ADAS-cog per SD: β coefficient, 0.17; 95% CI, -0.27 to -0.07; Modified Mini-Mental State Examination score per SD: β coefficient, 0.25; 95% CI, 0.07 to 0.42) and lower risk of dementia (hazard ratio per SD, 0.86; 95% CI, 0.76 to 0.99). In contrast, apoE levels in HDL that contains apoC3 were unassociated with any of these outcomes. In a prospective cohort of older adults with rigorous follow-up of dementia, the apoE level in HDL that lacked apoC3 was associated with better cognitive function and lower dementia risk. This finding suggests that the cardioprotective associations of this novel lipoprotein extend to dementia.

Coenzyme Q10 alleviates sevoflurane‑induced neuroinflammation by regulating the levels of apolipoprotein E and phosphorylated tau protein in mouse hippocampal neurons.

Sevoflurane may exert neurotoxic effects on the developing brain. Coenzyme Q10 (CoQ10) has been reported to reduce sevoflurane anesthesia-induced cognitive deficiency in 6-day-old mice. However, its specific mechanisms remain unknown. Apolipoprotein E (ApoE) has been reported to lead to the initiation of neurodegeneration in patients with Alzheimer's disease (AD) and may serve an important role in anesthesia-induced neurotoxicity. The present study aimed to reveal the role of ApoE in the pathogenesis of tau protein hyperphosphorylation and neuroinflammation enhancement caused by sevoflurane anesthesia, as well as the protective mechanism of CoQ10 in an anesthetic sevoflurane treatment model of primary mouse hippocampal neurons. For that purpose, the neurons were randomly assigned to the following groups: i) Control; ii) sevoflurane; iii) control+corn oil; iv) sevoflurane+corn oil; v) control+CoQ10; and vi) control+CoQ10. CoQ10 or corn oil alone was added to the medium on day 4 of neuron culture. The neurons in the sevoflurane group were treated with 21% O2, 5% CO2 and 4.1% sevoflurane for 4 h, whereas the control group only with 21% O2 and 5% CO2 on day 5. Samples were collected immediately after anesthesia or control treatment. ATP, superoxidase dismutase (SOD)1, ApoE mRNA, total ApoE, full-length ApoE, ApoE fragments, Tau5, Tau-PS202/PT205 (AT8), Tau-PSer396/404 (PHF1), tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β levels were measured with ELISA, quantitative PCR, western blotting and immunocytochemistry. The results of the present study indicated that sevoflurane anesthesia significantly decreased the ATP and SOD levels, but increased ApoE mRNA, total ApoE protein, full-length ApoE, ApoE fragments, phosphorylated tau (AT8 and PHF1) and neuroinflammatory factor (TNF-α, IL-6 and IL-1β) expression levels compared with those in the control group. The use of CoQ10 reversed the expression of these factors. These results suggested that sevoflurane treatment damaged mouse hippocampal neurons, which may be associated with the expression of ApoE and its toxic fragments. CoQ10 improved energy replenishment and inhibited oxidative stress, which may lead to a decrease in ApoE and phosphorylated tau protein expression, thus mitigating the sevoflurane-induced neuroinflammation in mouse hippocampal neurons.

Effect ofDietary Doses of Quercetin on Hepatic Drug Metabolizing Enzymes in Spontaneously Hypertensive Rats.

Administration of quercetin (QR) has shown several health benefits in clinical and pre-clinical studies. This study investigates the effect of dietary doses of QR on hepatic drug metabolizing enzymes in spontaneously hypertensiverats in order to investigate the potential for herb-drug interactions. The activity and/or protein expression of selected cytochrome P450 (CYP) enzymes and microsomal epoxide hydrolase were measured in hepatic microsomes using specific probe substrates and/or polyclonal antibodies. Cytosolic fraction was utilized to measure protein level and activity of major antioxidant systems. The doses employed in our study did not cause any significant alterations in the activity and/or protein level of CYP1A1, CYP2A6, CYP2E, and glutathione (GSH). While the activity and apoprotein levels of CYP1A2 and CYP2B1/2 were significantly reduced by the medium and high doses of QR, the activity and/or protein level of microsomal CYP3A and cytosolic GSH-S-transferase, GSH reductase, and GSH peroxidase were significantly enhanced. Activity and protein level of CYP2C9 were significantly inhibited by all doses. Only the high-dose QR resulted in significant inhibition of both microsomal and soluble epoxide hydrolase as well as induction of the antioxidant enzymes, catalase and superoxide dismutase. This study demonstrates that dietary doses of QR may offer chemoprevention through stimulation of the endogenous antioxidant systems and inhibition of CYP enzymes involved in bioactivation of procarcinogens. However, modulation of drug metabolizing enzymes by QR could have potential for herb-drug interactions with the possibility of serious complications.

Effects of Microglial Cytokines on Alzheimer's Disease-Related Phenomena.

Recent association studies indicate several genes highly expressed by microglia influence Alzheimer's disease (AD) risk, which suggests microglial function contributes to this disease. Here, we evaluated how one component of microglial function, cytokine release, affects AD-related phenomena. First, we used a 3-hour lipopolysaccharide (LPS) treatment to activate mouse BV2 microglial cells. Next, we removed the LPS-containing medium, added LPS-free medium, and after 6 hours collected the medium conditioned by the activated BV2 microglial cells. We then exposed human neuronal SH-SY5Y cells to the conditioned medium for 24 hours. At the end of the 24-hour exposure, we assessed amyloid-β protein precursor (AβPP), tau, apolipoprotein E (ApoE), and lipid status. The amount of AβPP was unaffected, although a slight decrease in soluble AβPPα suggested a subtle reduction in AβPP non-amyloidogenic processing occurred. Tau mRNA increased, but total and phosphorylated tau levels were unchanged. ApoE mRNA increased, while ApoE protein levels were lower. Per cell lipid droplet number decreased and lipid oxidation increased. These results show cytokine release by activated microglial cells can influence specific AD-relevant physiologies and pathologies.

APOE hypermethylation is significantly associated with coronary heart disease in males

APOE is involved in exogenous and endogenous lipid metabolism and is a candidate gene for coronary heart disease (CHD). Using the SYBR-Green quantitative methylation-specific PCR method, we measured APOE methylation levels in 640 cases of CHD and 436 controls. Meanwhile, we used the Sequenom MassARRAY platform to genotype APOE rs7412 and rs7259620. In men, we found that CHD patients had significantly lower levels of APOE methylation than non-CHD controls (P = 0.044). In addition, rs7412-T and rs7259620-A were protective factors for CHD in males (rs7412-T: OR = 0.527, allele P = 0.004; rs7259620-A: OR = 0.743, allele P = 0.029). In the CHD group and the non-CHD control group, APOE methylation levels were inversely correlated with age (total patients: r = −0.015, P = 0.009, total controls: r = −0.185, P = 1E−04). In the female control group, the APOE methylation levels were inversely correlated with plasma APOA1 protein levels (female controls: r = −0.188, P = 0.008). In the total controls and female controls, APOE methylation levels were inversely correlated with plasma APOE protein levels (total controls: r = −0.110, P = 0.031, female controls: r = −0.188, P = 0.008). Our GDMR interaction analysis showed that there was an interaction between APOE methylation and rs7412 (P = 0.011). In addition, there was a significant interaction between APOE methylation, rs7259620, gender, smoking, LDL, TC, and APOE protein levels in the risk of CHD (P = 0.011). In summary, our research showed that the methylation of APOE was important for the risk of CHD in men.

GJA1 (connexin43) is a key regulator of Alzheimer\u2019s disease pathogenesis

GJA1 (connexin43) has been predicted as the top key driver of an astrocyte enriched subnetwork associated with Alzheimer’s disease (AD). In this study, we comprehensively examined GJA1 expression across 29 transcriptomic and proteomic datasets from post-mortem AD and normal control brains. We demonstrated that GJA1 was strongly associated with AD amyloid and tau pathologies and cognitive functions. RNA sequencing analysis of Gja1−/− astrocytes validated that Gja1 regulated the subnetwork identified in AD, and many genes involved in Aβ metabolism. Astrocytes lacking Gja1 showed reduced Apoe protein levels as well as impaired Aβ phagocytosis. Consistent with this, wildtype neurons co-cultured with Gja1−/− astrocytes contained higher levels of Aβ species than those with wildtype astrocytes. Moreover, Gja1−/− astrocytes was more neuroprotective under Aβ stress. Our results underscore the importance of GJA1 in AD pathogenesis and its potential for further investigation as a promising pharmacological target in AD.

A novel splicing mutation in the ABCA1 gene, causing Tangier disease and familial HDL deficiency in a large family

Tangier disease is a rare disorder of lipoprotein metabolism that presents with extremely low levels of HDL cholesterol and apoprotein A-I. It is caused by mutations in the ATP-binding cassette transporter A1 (ABCA1) gene. Clinical heterogeneity and mutational pattern of Tangier disease are poorly characterized. Moreover, also familial HDL deficiency may be caused by mutations in ABCA1 gene.ATP-binding cassette transporter A1 (ABCA1) gene mutations in a patient with Tangier disease, who presented an uncommon clinical history, and in his family were found and characterized. He was found to be compound heterozygous for two intronic mutations of ABCA1 gene, causing abnormal pre-mRNAs splicing. The novel c.1510-1G > A mutation was located in intron 12 and caused the activation of a cryptic splice site in exon 13, which determined the loss of 22 amino acids of exon 13 with the introduction of a premature stop codon. Five heterozygous carriers of this mutation were also found in proband's family, all presenting reduced HDL cholesterol and ApoAI (0.86 ± 0.16 mmol/L and 92.2 ± 10.9 mg/dL respectively), but not the typical features of Tangier disease, a phenotype compatible with the diagnosis of familial HDL deficiency. The other known mutation c.1195-27G > A was confirmed to cause aberrant retention of 25 nucleotides of intron 10 leading to the insertion of a stop codon after 20 amino acids of exon 11. Heterozygous carriers of this mutation also showed the clinical phenotype of familial HDL deficiency.Our study extends the catalog of pathogenic intronic mutations affecting ABCA1 pre-mRNA splicing. In a large family, a clear demonstration that the same mutations may cause Tangier disease (if in compound heterozygosis) or familial HDL deficiency (if in heterozygosis) is provided.

Apolipoprotein E DNA methylation and posttraumatic stress disorder are associated with plasma ApoE level: A preliminary study

Mild traumatic brain injury (mTBI) occurred in 15–30% of Veterans returning from Iraq and Afghanistan. We examined whether DNA methylation of the apolipoprotein E (APOE) gene promoter region or plasma ApoE protein levels are altered in mTBI. APOE promoter region DNA methylation, APOE genotype, and plasma ApoE concentration were determined in 87 Veterans with or without mTBI who were recruited from 2010-2014. Plasma ApoE concentration was found to be associated with Posttraumatic Stress Disorder (PTSD) symptom severity ratings by hierarchical linear regression (p = .013) and ANCOVA (p = .007). Hierarchical linear regression revealed that plasma ApoE concentration was associated with APOE-ε4 genotype status (p=.022). Higher ApoE plasma levels were found in ε3/ε3 Veterans than in APOE-ε4 carriers (p = .031). Furthermore, plasma ApoE concentration was associated experiment-wise with DNA methylation at CpG sites -877 (p = .021), and -775 (p = .014). The interaction between APOE-ε4 genotype and having a PTSD diagnosis was associated with DNA methylation at CpG site -675 (p = .009).

EPICARDIAL OBESITY AS ONE OF THE BASIC CRITERIA FOR METABOLICALLY UNHEALTHY OBESITY PHENOTYPE AND THE PREDICTOR OF SUBCLINICAL ATHEROSCLEROSIS

Aim. To assess the effect of epicardial obesity on the development of dyslipidemia and polyvascular subclinical atherosclerosis, and to assess the prognostic significance of various obesity criteria in the development of cardiovascular risk. Methods. Male patients with arterial hypertension and without clinical symptoms of atherosclerosis of any localization and type 2 diabetes were included in the study. All the patients were enrolled into two groups, depending on the metabolic phenotypes of obesity assessed by epicardial fat thickness and body mass index. Both groups were comparable in waist circumference. All patients in both groups had abdominal obesity (AO≥94 cm). The study groups underwent a comparative assessment of parameters of lipid metabolism and levels of Apo-proteins. Coronary atherosclerosis was measured with multispiral computed tomography. Atherosclerotic lesions of brachiocephalic arteries were assessed by duplex scanning was performed. Epicardial fat thickness was measured by echocardiography. Results. Significant relationships between epicardial obesity and the development of dyslipidemia and polyvascular subclinical atherosclerosis have been determined. Waist circumference and body mass index have low prognostic significance for developing cardiovascular risk. Conclusion. Epicardial obesity significantly affects the development of subclinical atherosclerosis of the coronary and brachiocephalic arteries. The assessment of cardiovascular risk in patients with arterial hypertension and without other diagnosed cardiovascular diseases reported that it is necessary to measure epicardial fat thickness along with traditional risk factors. Epicardial fat thickness is considered to be a criterion for visceral obesity, contributing to the development of cardiometabolic disorders.

Serum apolipoprotein E may be a novel biomarker of migraine.

Migraine attacks alter various molecules that might be related to the pathophysiology of migraine, such as serotonin, calcitonin gene-related peptide, and nitric oxide. The underlying pathophysiology of migraine is as yet unclear. We explored key proteins related to the pathogenesis of migraine here. Serum was collected from two patients with migraine with aura (MA) and seven patients with migraine without aura (MO) during attack-free periods and migraine attacks. Samples were analyzed using 2-dimensional gel electrophoresis. Nineteen protein spots were altered between the attack-free versus migraine attack periods. Mass spectrometric analysis was performed to identify the proteins within each of the 19 altered spots. Thirty-six proteins were significantly altered in samples collected during attack-free periods versus migraine attacks. The protein with the statistically most significant MASCOT/Mowse score (268±112) among lipoproteins was apolipoprotein (ApoE). In the MA and MO groups, ApoE protein levels were significantly higher during migraine attack than during the attack-free period (p<0.05). ApoE protein levels were also significantly increased in the MA group during the attack-free period compared to healthy controls and patients with tension type headaches (p<0.01). Migraine alters ApoE levels, especially in MA. ApoE might play an important role in the pathophysiology of migraine, and may act as a diagnostic biomarker of migraine.

Diallyl disulphide inhibits apolipoprotein(a) expression in HepG2 cells through the MEK1-ERK1/2-ELK-1 pathway

BackgroundLipoprotein(a) [LP(a)] is implicated as a common and independent risk factor for cardiovascular diseases. The therapeutic options currently available for reducing plasma LP(a) concentrations are limited. Diallyl disulphide (DADS), the main component of garlic, regulates lipid metabolism in hepatocytes and adipocytes through ERK1/2 signalling. This study aimed to assess the effect of DADS on apolipoprotein(a) [apo(a)] in HepG2 cells. We also determined the effects of DADS on apo(a) expression and secretion in HepG2 cells as well as the underlying mechanisms.MethodsWe examined the role of DADS on apo(a) expression in HepG2 cells by treating cell with different concentrations of DADS (10, 20, 40 and 80 μg/mL) for 24 h or treating cells with 40 μg/mL DADS for 0, 6, 12, 24 and 48 h. Then we used quantitative real-time PCR to analysis apo(a) mRNA levels, used Western blot to analysis apo(a) protein levels and used enzyme-linked immunosorbent assay to test apo(a) secreted levels. To farther determined the role of DADS, we applied Transfection of small interfering RNA to knockdown ELK-1levels and applied PD98059, a specific inhibitor of ERK1/2, to block ERK1/2 signal.ResultsThe results show DADS inhibited apo(a) at both the mRNA and protein levels in HepG2 cells in a dose-dependent manner. DADS-mediated inhibition of apoa(a) expression in HepG2 cells was attenuated when the cells were cultured in medium containing PD98059 (ERK1/2 inhibitor) or were transfected with siRNAs against MEK1 or ELK-1. Overexpression of apo(a) yielded similar results.ConclusionsThis study reveals that DADS can downregulate apo(a) expression in a dose-dependent manner via the MEK-ERK12-ELK-1 pathway.

EFFECT OF INCREASING APOLIPOPROTEIN E ON INFLAMMATION IN THE BRAIN

Apolipoprotein E (apoE) is a key lipid transport protein in brain and plasma. Humans have three apoE isoforms; namely, apoE2, apoE3 and apoE4. Of the three isoforms, apoE4 is strongly associated with late-onset and sporadic forms of Alzheimer's disease (AD). In addition to its role in neuronal repair, synaptogenesis, and clearance of toxic Aß fragments from brain, apoE has been suggested to play a critical role in inflammation in the brain. Using pan LXR agonists as a tool compound for increasing brain and astrocytic apoE levels, we sought to investigate the effect of increasing astrocytic apoE on brain inflammation induced by lipopolysaccharide (LPS) both in vitro and in vivo. Using primary mouse microglia and astrocytes and C57/bl6 wild type mice as our model system, we evaluated the effect of increasing apoE protein levels using T0901317 (pan LXR agonist) on LPS induced inflammation. We used RT-qPCR to evaluate the mRNA levels of apoE and apoE lipidating genes and MSD assays to evaluate cytokine protein levels in cell media and brain homogenates. TO901317 treatment led to an increase in brain apoE levels in vivo in wild type mice and in vitro, in both primary murine astrocytes and microglia. This increase in apoE was accompanied by an increase in apoE lipidating genes (Abca1 and Abcg1). Treatment with LPS led to a significant inflammatory response as seen by an increase in pro-inflammatory cytokines, TNFα and IL6. However, TO901317 treatment led to a significant reduction in the levels of the pro-inflammatory cytokines both in vitro (astrocytes and microglia) and in vivo in the brain. Our results indicate that increasing brain and gial apoE using a pan LXR agonist attenuates LPS induced inflammation. Our data suggests that apoE could play an important role in promoting anti- inflammatory effects in the brain. Given the strong association of apoE with AD, increasing lipidated apoE in the brain could have multi-prong benefits including anti-inflammatory effects.

Abstract 181: Insulin Receptor Signaling Regulates ApoA-I Secretion from Hepatocytes

The aim of the study was to assess the effect of insulin receptor (IR) signaling on hepatic apoA-I metabolism. The experimental approach was to compare hepatic apoA-I expression, secretion and cellular localization in IRfl/fl mice and LIRKO mice in which hepatic insulin receptors were specifically deleted by AAV delivered Cre-recombinase. Results showed that IR mRNA and protein levels were markedly reduced in the livers of LIRKO mice compared to control IRfl/fl mice. As expected, LIRKO mice exhibited decreased glucose tolerance and reduced hepatic insulin signaling. Knockdown of hepatic IR decreased plasma HDL cholesterol and apoA-I levels. Whereas apoA-I mRNA levels were similar in LIRKO and control hepatocytes, apoA-I protein levels were increased in both the liver and primary hepatocytes isolated from LIRKO mice. In contrast to apoA-I, apoE and apoB protein levels in the liver and in cultured hepatocytes, as well as in the plasma, were similar in LIRKO and control mice. ApoA-I accumulation in LIRKO hepatocytes was associated with a decreased rate of apoA-I secretion. Immunofluorescence staining demonstrated that apoA-I accumulated in LIRKO hepatocytes in membrane bound inclusions. These inclusions shared markers characteristic of early, late and recycling endosomes, and of lysosomes. We conclude that IR-mediated insulin signaling plays an important role in hepatic apoA-I secretion and consequent nascent HDL formation. Reduced apoA-I secretion from liver into the circulation may contribute to the lower HDL levels typically associated with insulin resistance.

Daily rhythms of cognition-related factors are modified in an experimental model of Alzheimer’s disease

The accumulation of amyloid-β (Aβ) peptides in the brain of Alzheimer disease patients is associated to cognitive deficit, increased oxidative stress, and alterations in the circadian rhythms. Brain-derived neurotrophic factor (BDNF) and Neurogranin (RC3), play an important role in the synaptic plasticity underlying memory and learning. Previously, we observed BDNF and RC3 expression follow a daily rhythmic pattern in the hippocampus of young rats. The objective of this study was to investigate the effects of an intracerebroventricular (i.c.v) injection of aggregated Aβ peptide (1–42) on temporal patterns of ApoE protein, Bdnf and Rc3 mRNA, lipid peroxidation (LPO) and reduced glutathione (GSH) levels, in the rat hippocampus. We observed an i.c.v. injection of Aβ aggregates phase shifts daily BDNF and RC3 expression as well as LPO and decreased the mesor of GSH rhythms. ApoE protein levels vary rhythmically throughout the day. ApoE levels increase at ZT 03:39±00:22 in the hippocampus of control rats and at ZT 06:30±00:28 in the treated animals. Thus, elevated levels of Aβ aggregates, characteristic of AD, altered temporal patterns of cognition related-factors, probably, as a consequence of changes in the daily variation of ApoE-mediated Aβ aggregates clearance as well as in the 24h rhythms of the cellular redox state.