Apolipoprotein E Deficiency Research Articles

NAD+ Protects against Hyperlipidemia-induced Kidney Injury in Apolipoprotein E-deficient Mice.

<p>Background: Hyperlipidemia is an independent risk factor for kidney injury. Several studies have shown that nicotinamide adenine dinucleotide (NAD+) is an important coenzyme involved in normal body metabolism. Therefore, this study aimed to investigate the possible protective effects of NAD+ against hyperlipidemia-induced kidney injury in apolipoprotein Edeficient (ApoE-/-) mice. <p> Methods: Twenty-five eight-week-old male ApoE-/- mice were randomly assigned into four groups: normal diet (ND), ND supplemented with NAD+ (ND+NAD+), high-fat diet (HFD), and HFD supplemented with NAD+ (HFD+NAD+). The mice were subjected to their respective diets for a duration of 16 weeks. Blood samples were obtained from the inferior vena cava, collected in serum tubes, and stored at -80°C until use. Kidney tissues was fixed in 10% formalin and then embedded in paraffin for histological evaluation. The remainder of the kidney tissues was snapfrozen in liquid nitrogen for Western blot analysis. <p> Results: Metabolic parameters (total cholesterol, triglycerides, low-density lipoprotein-cholesterol, creatinine, and blood urea nitrogen) were significantly higher in the HFD group compared to the other groups. Histological analysis revealed prominent pathological manifestations in the kidneys of the HFD group. The HFD+NAD+ group showed increased levels of oxidative stress markers (NRF2 and SOD2) and decreased levels of NOX4 compared to the HFD group. Furthermore, the HFD group exhibited higher levels of TGF-β, Smad3, Collagen I, Collagen III, Bax, and Bak compared to the other groups. NAD+ supplementation in the HFD+NAD+ group significantly increased the levels of SIRT3, HO-1, Bcl-2, and Bcl-xL compared to the HFD group. Additionally, NF-κB protein expression was higher in the HFD group than in the HFD+NAD+ group. <p> Conclusion: These findings demonstrated that NAD+ may hold potential as a clinical treatment for kidney injury caused by hyperlipidemia.</p>.

Apolipoprotein-E deficiency leads to brain network alteration characterized by diffusion MRI and graph theory

Apolipoprotein-E deficiency leads to brain network alteration characterized by diffusion MRI and graph theory

APOE deficiency impacts neural differentiation and cholesterol biosynthesis in human iPSC-derived cerebral organoids

BackgroundThe apolipoprotein E (APOE) gene is the strongest genetic risk factor for Alzheimer’s disease (AD); however, how it modulates brain homeostasis is not clear. The apoE protein is a major lipid carrier in the brain transporting lipids such as cholesterol among different brain cell types.MethodsWe generated three-dimensional (3-D) cerebral organoids from human parental iPSC lines and its isogenic APOE-deficient (APOE−/−) iPSC line. To elucidate the cell-type-specific effects of APOE deficiency in the cerebral organoids, we performed scRNA-seq in the parental and APOE−/− cerebral organoids at Day 90.ResultsWe show that APOE deficiency in human iPSC-derived cerebral organoids impacts brain lipid homeostasis by modulating multiple cellular and molecular pathways. Molecular profiling through single-cell RNA sequencing revealed that APOE deficiency leads to changes in cellular composition of isogenic cerebral organoids likely by modulating the eukaryotic initiation factor 2 (EIF2) signaling pathway as these events were alleviated by the treatment of an integrated stress response inhibitor (ISRIB). APOE deletion also leads to activation of the Wnt/β-catenin signaling pathway with concomitant decrease of secreted frizzled-related protein 1 (SFRP1) expression in glia cells. Importantly, the critical role of apoE in cell-type-specific lipid homeostasis was observed upon APOE deletion in cerebral organoids with a specific upregulation of cholesterol biosynthesis in excitatory neurons and excessive lipid accumulation in astrocytes. Relevant to human AD, APOE4 cerebral organoids show altered neurogenesis and cholesterol metabolism compared to those with APOE3.ConclusionsOur work demonstrates critical roles of apoE in brain homeostasis and offers critical insights into the APOE4-related pathogenic mechanisms.

Apolipoprotein E Deficiency Aggravates Neuronal Injury by Enhancing Neuroinflammation via the JNK/c-Jun Pathway in the Early Phase of Experimental Subarachnoid Hemorrhage in Mice

Neuronal injury is the primary cause of poor outcome after subarachnoid hemorrhage (SAH). The apolipoprotein E (APOE) gene has been suggested to be involved in the prognosis of SAH patients. However, the role of APOE in neuronal injury after SAH has not been well studied. In this study, SAH was induced in APOE-knockout (APOE−/−) and wild-type (WT) mice to investigate the impact of APOE deficiency on neuronal injury in the early phase of SAH. The experiments of this study were performed in murine SAH models in vivo and primary cultured microglia and neurons in vitro. The SAH model was induced by endovascular perforation in APOE−/− and APOE WT mice. The mortality rate, weight loss, and neurological deficits were recorded within 72 h after SAH. The neuronal injury was assessed by detecting the neuronal apoptosis and axonal injury. The activation of microglia was assessed by immunofluorescent staining of Iba-1, and clodronate liposomes were used for inhibiting microglial activation. The expression of JNK/c-Jun was evaluated by immunofluorescent staining or western blotting. The expression of TNF-α, IL-1β, and IL-6 was evaluated by ELISA. Primary cultured microglia were treated with hemoglobin (Hb) in vitro for simulating the pathological process of SAH. SP600125, a JNK inhibitor, was used for evaluating the role of JNK in neuroinflammation. Nitrite production was detected for microglial activation, and flow cytometry was performed to detect apoptosis in vitro. The results suggested that SAH induced early neuronal injury and neurological deficits in mice. APOE deficiency resulted in more severe neurological deficits after SAH in mice. The neurological deficits were associated with exacerbation of neuronal injury, including neuronal apoptosis and axonal injury. Moreover, APOE deficiency enhanced microglial activation and related inflammatory injury on neurons. Inhibition of microglia attenuated neuronal injury in mice, whereas inhibition of JNK inhibited microglia-mediated inflammatory response in vitro. Taken together, JNK/c-Jun was involved in the enhancement of microglia-mediated inflammatory injury in APOE−/− mice. APOE deficiency aggravates neuronal injury which may account for the poor neurological outcomes of APOE−/− mice. The possible protective role of APOE against EBI via the modulation of inflammatory response indicates its potential treatment for SAH.

Elimination of adrenocortical apolipoprotein E production does not impact glucocorticoid output in wild-type mice

Apolipoprotein E (APOE) deficient mice exhibit unexplained hypercorticosteronemia. Given that APOE is also produced locally within the adrenals, we evaluated the effect of adrenal-specific APOE deficiency on the glucocorticoid function. Hereto, one adrenal containing or lacking APOE was transplanted into adrenalectomized wild-type mice. Adrenal APOE deficiency did not impact adrenal total cholesterol levels. Importantly, the ability of the two adrenal types to produce glucocorticoids was also not different as judged from the similar plasma corticosterone levels. Adrenal mRNA expression levels of HMG-CoA reductase and the LDL receptor were decreased by respectively 72% (p < 0.01) and 65% (p = 0.07), suggesting that cholesterol acquisition pathways were inhibited to possibly compensate the lack of APOE. In support, a parallel increase in the expression level of the cholesterol accumulation-associated ER stress marker CHOP was detected (+117%; p < 0.05).In conclusion, our studies show that elimination of adrenocortical APOE production does not impact glucocorticoid output in wild-type mice.

Western-type diet differentially modulates osteoblast, osteoclast, and lipoblast differentiation and activation in a background of APOE deficiency

During the past few years, considerable evidence has uncovered a strong relationship between fat and bone metabolism. Consequently, alterations in plasma lipid metabolic pathways strongly affect bone mass and quality. We recently showed that the deficiency of apolipoprotein A-1 (APOA1), a central regulator of high-density lipoprotein cholesterol (HDL-C) metabolism, results in reduced bone mass in C57BL/6 mice. It is documented that apolipoprotein E (APOE), a lipoprotein know for its atheroprotective functions and de novo biogenesis of HDL-C, is associated with the accumulation of fat in the liver and other organs and regulates bone mass in mice. We further studied the mechanism of APOE in bone metabolism using well-characterized APOE knockout mice. We found that bone mass was remarkably reduced in APOE deficient mice fed Western-type diet (WTD) compared to wild type counterparts. Static (microCT-based) and dynamic histomorphometry showed that the reduced bone mass in APOΕ−/− mice is attributed to both decreased osteoblastic bone synthesis and elevated osteoclastic bone resorption. Interestingly, histologic analysis of femoral sections revealed a significant reduction in the number of bone marrow lipoblasts in APOΕ−/− compared to wild type mice under WTD. Analyses of whole bone marrow cells obtained from femora of both animal groups showed that APOE null mice had significantly reduced levels of the osteoblastic (RUNX2 and Osterix) and lipoblastic (PPARγ and CEBPα) cardinal regulators. Additionally, the modulators of bone remodeling RANK, RANKL, and cathepsin K were greatly increased, while OPG and the OPG/RANKL ratio were remarkably decreased in APOΕ−/− mice fed WTD, compared to their wild-type counterparts. These findings suggest that APOE deficiency challenged with WTD reduces osteoblastic and lipoblastic differentiation and activity, whereas it enhances osteoclastic function, ultimately resulting in reduced bone mass, in mice.Applying histological/histomorphometrical analyses on femora and/or lumbar vertebrae, as well as spectrometric and molecular methodologies on bone-marrow mesenchymal stem cells form APOE knock out and wild-type mice, the authors demonstrate that APOE deficient mice fed a Western-type diet have remarkably augmented bone-marrow adiposity and significantly reduced bone mass due to enhanced osteoclastic and impaired osteoblastic function.

The Role of Apolipoprotein E and Ethanol Exposure in Age-Related Changes in Choline Acetyltransferase and Brain-Derived Neurotrophic Factor Expression in the Mouse Hippocampus.

Disruption of apolipoprotein E (APOE) is responsible for age-dependent neurodegeneration and cognitive impairment. Elderly individuals are more sensitive than young individuals to the effects of ethanol (EtOH), particularly those affecting cognition. We investigated the role of APOE deficiency and EtOH exposure on age-dependent alterations in choline acetyltransferase (ChAT) and brain-derived neurotrophic factor (BDNF) mRNA and protein expression in the mouse hippocampus. Three-month-old (young) and 12-month-old (aged) ApoE-knockout (ApoE-KO) and wild-type (WT) mice were treated with saline or 2g/kg EtOH, and the bilateral hippocampus was collected after 60min for real-time PCR and western blotting analyses. ChAT (P < 0.01) and BDNF (P < 0.01) expression were significantly decreased in both young and aged saline- and EtOH-treated ApoE-KO mice versus young and aged saline- and EtOH-treated WT mice. Aged saline- and EtOH-treated ApoE-KO mice exhibited greater differences in ChAT and BDNF expression (P < 0.01) than young saline- and EtOH-treated ApoE-KO mice. Aged EtOH-treated WT mice also exhibited larger decreases in BDNF expression (P < 0.01)-but not in ChAT expression-than young EtOH-treated WT mice. EtOH decreased ChAT and BDNF expression in both young (P < 0.01) and aged (P < 0.01) ApoE-KO mice versus EtOH-free ApoE-KO mice of the same age. EtOH also decreased BDNF expression in aged (P < 0.01) WT mice versus EtOH-free aged WT mice. In summary, these results suggest that APOE deficiency and EtOH exposure cause age-dependent decreases in ChAT and BDNF in the hippocampus. Importantly, the decreases in ChAT and BDNF were greater in aged EtOH-treated mice, particularly those lacking APOE, raising the possibility that APOE-deficient individuals who consume alcohol may be at greater risk of memory deficit.

Apolipoprotein E Deficiency Increases Remnant Lipoproteins and Accelerates Progressive Atherosclerosis, But Not Xanthoma Formation, in Gene-Modified Minipigs

Deficiency of apolipoprotein E (APOE) causes familial dysbetalipoproteinemia in humans resulting in a higher risk of atherosclerotic disease. In mice, APOE deficiency results in a severe atherosclerosis phenotype, but it is unknown to what extent this is unique to mice. In this study, APOE was targeted in Yucatan minipigs. APOE-/- minipigs displayed increased plasma cholesterol and accumulation of apolipoprotein B-48-containing chylomicronremnants on low-fat diet, which was significantly accentuated upon feeding a high-fat, high-cholesterol diet. APOE-/- minipigs displayed accelerated progressive atherosclerosis but not xanthoma formation. This indicates that remnant lipoproteinemia does not induce early lesions but is atherogenic inpre-existing atherosclerosis.

Activation of the NLRP3 inflammasome in Porphyromonas gingivalis-accelerated atherosclerosis

Porphyromonas gingivalis has been shown to accelerate atherosclerotic lesion development in hyperlipidemic animals. Atherosclerosis is a disease characterized by inflammation of the arterial wall. Recent studies have suggested that the NLRP3 inflammasome plays an important role in the development of vascular inflammation and atherosclerosis. Herein, we investigated a possible association between the inflammasome in atherosclerosis and periodontal disease induced by P. gingivalis infection using apolipoprotein E-deficient, spontaneously hyperlipidemic (Apoe(shl)) mice. Oral infection with wild-type (WT) P. gingivalis significantly increased the area of aortic sinus covered with atherosclerotic plaque and alveolar bone loss, compared with KDP136 (gingipain-null mutant) or KDP150 (FimA-deficient mutant) challenge. WT challenge also increased IL-1β, IL-18 and TNF-α production in peritoneal macrophages, and gingival or aortic gene expression of Nod-like receptor family, pyrin domain containing 3 (NLRP3), pro-IL-1β, pro-IL-18 and pro-caspase-1. Porphyromonas gingivalis genomic DNA was detected more in the aorta, gingival tissue, liver and spleen of WT-challenged mice than those in KDP136- or KDP150-challenged mice. We conclude that WT P. gingivalis activates innate immune cells through the NLRP3 inflammasome compared with KDP136 or KDP150. The NLRP3 inflammasome may play a critical role in periodontal disease and atherosclerosis induced by P. gingivalis challenge through sustained inflammation.

Apolipoprotein-E deficiency predisposes to the development of osteoporosis following long-term exposure to western-type diet, in mice

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

Apolipoprotein-E deficiency prevents obesity but predisposes to the development of osteoporosis following long-term exposure to Western-type diet, in mice

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

Apolipoprotein E-Deficient Lipoproteins Induce Foam Cell Formation by Activation of PERK-EIF-2α Signaling Cascade

Transformation of macrophages into foam cells by apolipoprotein (Apo) E-deficient, ApoB48-containing (E(-)/B48) lipoproteins has been shown to be associated with increased phosphorylation of eukaryotic initiation factor-2α (eIF-2α). The present report examined the causal relationship between eIF-2α phosphorylation and lipid accumulation in macrophages induced by E(-)/B48 lipoproteins. E(-)/B48 lipoproteins increased eIF-2α phosphorylation and cholesterol ester accumulation, while lipoprotein degradation decreased and lysosomal acid lipase and cathepsin B mRNA translation was inhibited in mouse peritoneal macrophages (MPMs). These responses were overcome by overexpression of a nonphosphorylatable eIF-2α mutant in MPMs. Incubation of MPMs with E(-)/B48 lipoproteins also increased the phosphorylation of RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK), but not other eIF-2α kinases. Overexpression of a nonphosphorylatable PERK mutant inhibited PERK and eIF-2α phosphorylation, and alleviated cholesterol ester accumulation induced by E(-)/B48 lipoproteins. PERK is an eIF-2α kinase activated by endoplasmic reticulum (ER) stress. Taken together, findings from this report suggest that induction of ER stress, i.e., activation of the PERK-eIF2α signaling cascade, is a mechanism by which E(-)/B48 lipoproteins down-regulate lysosomal hydrolase synthesis, inhibit lysosomal lipoprotein degradation, and increase intracellular lipoprotein and cholesterol ester accumulation, resulting in foam cell formation.

2-Aminopurine Inhibits Lipid Accumulation Induced by Apolipoprotein E-Deficient Lipoprotein in Macrophages: Potential Role of Eukaryotic Initiation Factor-2α Phosphorylation in Foam Cell Formation

We previously reported that apolipoprotein (Apo) E-deficient, ApoB48-containing (E^–/B48) lipoproteins inhibited expression of lysosomal hydrolase and transformed mouse peritoneal macrophages (MPMs) into foam cells. The present study examined the effect of 2-aminopurine (2-AP), an inhibitor of eukaryotic initiation factor (eIF)-2α phosphorylation, on E^–/B48 lipoprotein-induced changes in gene expression and foam cell formation. Our data demonstrated that E^–/B48 lipoproteins enhanced phosphorylation of eIF-2α in macrophages. Incubation of MPMs with E^–/B48 lipoproteins inhibited the translation efficiency of mRNAs encoding lysosomal acid lipase, cathepsin B, and cation-dependent mannose 6 phosphate receptor, with a parallel reduction in the level of these proteins. Addition of 2-AP to the culture media alleviated the suppressive effect of E^–/B48 lipoproteins on lysosomal hydrolase mRNA translation, increased macrophage degradation of E^–/B48 lipoproteins, and inhibited foam cell formation. Transfection of MPMs with a nonphosphorylatable eIF-2α mutant also attenuated the suppressive effect of E^–/B48 lipoproteins on expression of lysosomal acid lipase, associated with a reduced accumulation of cellular cholesterol esters. This is the first demonstration that ApoE-deficient lipoproteins inhibit lysosomal hydrolase synthesis and transform macrophages into foam cells through induction of eIF-2α phosphorylation.

Expression and activity of methionine cycle genes are altered following folate and vitamin E deficiency under oxidative challenge: Modulation by apolipoprotein E-deficiency

Folate deficiency increases neuronal oxidative damage and potentiates the deleterious effects of apolipoprotein E (ApoE) deficiency. Mice lacking ApoE (ApoE − / − mice) upregulate the expression and activity of another enzyme, glutathione synthase (GS), when deprived of folate, in an apparent attempt to compensate for increased oxidative damage. Herein, we examined the influence of ApoE and folate deficiency on expression and activity of several enzymes of the methionine cycle. Expression and activity of methylene tetrahydrofolate reductase was increased in the order ApoE+/+

A new concept in the pharmacology of neuroprotection.

Vasoactive intestinal peptide (VIP), originally discovered in the intestine as a peptide of 28 amino acids, was later found to be a major brain peptide having neuroprotective activities. To exert neuroprotective activity, VIP requires glial cells secreting neuroprotective proteins. Activity-dependent neurotrophic factor (ADNF) is a recently isolated factor secreted by glial cells under the action of VIP. This protein, isolated by sequential chromatographic methods, was named activity-dependent neurotrophic factor since it protected neurons from death associated with blockade of electrical activity. A fourteen-amino-acid fragment of ADNF (ADNF-14) and the more potent, nine-amino-acid derivative (ADNF-9), exhibit activity that surpasses that of the parent protein with regard to potency and a broader range of effective concentration. Furthermore, the peptides exhibit protective activity in Alzheimer's disease-related systems (e.g., beta-amyloid toxicity and apolipoprotein E deficiencies, genes that have been associated with Alzheimer's disease onset and progression). ADNP is another glial mediator of VIP-associated neuroprotection. NAP, an eight-amino-acid peptide derived from ADNP (sharing structural and functional similarities with ADNF-9), was identified as the most potent neuroprotectant described to-date in an animal model of apolipoprotein E-deficiency (knock-out mice). These femtomolar-acting peptides form a basis for a new concept in pharmacology: femtomolar neuroprotection.

Cholinergic systems and long-term potentiation in memory-impaired apolipoprotein E-deficient mice

Impairments in cholinergic neurotransmitter systems of the basal forebrain are a hallmark of Alzheimer's disease pathophysiology. The presence of the ε4 allele of apolipoprotein E was recently implicated as a major risk factor in both familial and sporadic Alzheimer's disease. The present study examined the integrity of cholinergic and non-cholinergic systems in apolipoprotein E-deficient, memory-impaired mice. Choline acetyltransferase activity, hippocampal acetylcholine release, nicotinic and muscarinic (M1 and M2) receptor binding sites and acetylcholinesterase cell or terminal density showed no signs of alteration in either three-month or 9.5-month-old apolipoprotein E-deficient mice compared to controls. In contrast, long-term potentiation was found to be markedly reduced in these mice, but increases in the strength of stimulation induced the same level of long-term potentiation as that observed in controls. These alterations did not appear to be the consequence of modifications in the binding properties of glutamatergic receptors ( N-methyl- d-aspartate and [ RS]-α-amino-3-hydroxy-5-methylisoxazole propionic acid) but from defective regulation of the ( RS)-α-amino-3-hydroxy-5-methylisoxazole propionic acid receptor by phospholipase A2 activity. These results support the notion that apolipoprotein E plays a fundamental role in neuronal plasticity, which could in turn affect cognitive performance through imbalances in extra- and intracellular lipid homeostasis.

Complete sequence of a novel protein containing a femtomolar-activity-dependent neuroprotective peptide.

The vulnerability of neurons and the irreversibility of loss make discoveries of neuroprotective compounds fundamentally important. Here, the complete coding sequence of a novel protein (828 amino acids, pI 5.99), derived from mouse neuroglial cells, is revealed. The sequence contained (1) a neuroprotective peptide, NAPVSIPQ, sharing structural and immunological homologies with the previously reported, activity-dependent neurotrophic factor; (2) a glutaredoxin active site; and (3) a zinc binding domain. Gene expression was enriched in the mouse hippocampus and cerebellum and augmented in the presence of the neuropeptide vasoactive intestinal peptide, in cerebral cortical astrocytes. In mixed neuron-astrocyte cultures, NAPVSIPQ provided neuroprotection at subfemtomolar concentrations against toxicity associated with tetrodotoxin (electrical blockade), the beta-amyloid peptide (the Alzheimer's disease neurotoxin), N-methyl-D-aspartate (excitotoxicity), and the human immunodeficiency virus envelope protein. Daily NAPVSIPQ injections to newborn apolipoprotein E-deficient mice accelerated the acquisition of developmental reflexes and prevented short-term memory deficits. Comparative studies suggested that NAPVSIPQ was more efficacious than other neuroprotective peptides in the apolipoprotein E-deficiency model. A potential basis for rational drug design against neurodegeneration is suggested with NAPVSIPQ as a lead compound. The relative enrichment of the novel mRNA transcripts in the brain and the increases found in the presence of vasoactive intestinal peptide, an established neuroprotective substance, imply a role for the cloned protein in neuronal function.

Apolipoprotein-E deficiency results in an altered stress responsiveness in addition to an impaired spatial memory in young mice

It has been suggested that Alzheimer's disease (AD) is associated with an altered neurotrophic function of apolipoprotein-E (ApoE) and abnormal neuroendocrine activities. In the present study we investigated stress responsiveness of ApoE-deficient mice. Firstly, two sessions of restraint were introduced, 20 min per day for two (session 1) and three (session 2) consecutive days. In session 1, there was no difference between genotypes in open-field activity in response to restraint stress. In session 2, spatial memory was assessed in a Morris Water Maze `Place Learning Set' task immediately following stress. Restraint stress caused a significant impairment of spatial memory in wild-type mice. The non-restraint ApoE-deficient mice showed a severe impairment of spatial memory similar to that of the restrained wild-type mice. Restraint stress had no obvious effect on spatial memory in ApoE-deficient mice until the third day of testing, when there was a decrease in reference memory compared with their non-restraint controls. In addition, the first session of restraint stress had an inhibitory effect on food intake in wild-type but not ApoE-deficient mice, and a longer-lasting effect on body weight in the wild-type than ApoE-deficient mice. ApoE-deficient mice showed a weaker corticosterone response to the initial restraint stress and a slower descending rate in serum corticosterone level during a 30-min post-stress period than their wild-type controls. However, higher baseline levels and stronger corticosterone responses were observed in ApoE-deficient mice than in wild-type mice when exposed to repeated restraint stress. The expression of ApoE mRNA was upregulated in the hypothalamus in wild-type mice exposed to repeated restraint stress. Taken together, these results demonstrate that ApoE deficiency causes a memory impairment and an altered stress responsiveness in mice.