Co-aggregation with Apolipoprotein E modulates the function of Amyloid-ß in Alzheimer's disease.

Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimer's Disease (AD), but the mechanism underlying this link is poorly understood. In particular, the relevance of direct interactions between apoE and amyloid-ß (Aß) remains controversial. Here, single-molecule imaging shows that all isoforms of apoE associate with Aß in the early stages of aggregation and then fall away as fibrillation happens. ApoE-Aß co-aggregates account for ~50% of the mass of diffusible Aß aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. We show how dynamic interactions between apoE and Aß tune disease-related functions of Aß aggregates throughout the course of aggregation. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how, in an isoform- and lipidation-specific way, apoE modulates the aggregation, clearance and toxicity of Aß. Selectively removing non-lipidated apoE4-Aß co-aggregates enhances clearance of toxic Aß by glial cells, and reduces secretion of inflammatory markers and membrane damage, demonstrating a clear path to AD therapeutics.

Neuromodulatory subcortical nucleus integrity is associated with white matter microstructure, tauopathy and APOE status.

The neuromodulatory subcortical nuclei within the isodendritic core (IdC) are the earliest sites of tauopathy in Alzheimer's disease (AD). They project broadly throughout the brain's white matter. We investigated the relationship between IdC microstructure and whole-brain white matter microstructure to better understand early neuropathological changes in AD. Using multiparametric quantitative magnetic resonance imaging we observed two covariance patterns between IdC and white matter microstructure in 133 cognitively unimpaired older adults (age 67.9 ± 5.3 years) with familial risk for AD. IdC integrity related to 1) whole-brain neurite density, and 2) neurite orientation dispersion in white matter tracts known to be affected early in AD. Pattern 2 was associated with CSF concentration of phosphorylated-tau, indicating AD specificity. Apolipoprotein-E4 carriers expressed both patterns more strongly than non-carriers. IdC microstructure variation is reflected in white matter, particularly in AD-affected tracts, highlighting an early mechanism of pathological development.

Apolipoprotein O modulates cholesterol metabolism via NRF2/CYB5R3 independent of LDL receptor.

Apolipoprotein O (APOO) plays a critical intracellular role in regulating lipid metabolism. Here, we investigated the roles of APOO in metabolism and atherogenesis in mice. Hepatic APOO expression was increased in response to hyperlipidemia but was inhibited after simvastatin treatment. Using a novel APOO global knockout (Apoo-/-) model, it was found that APOO depletion aggravated diet-induced obesity and elevated plasma cholesterol levels. Upon crossing with low-density lipoprotein receptor (LDLR) and apolipoprotein E (APOE) knockout hyperlipidemic mouse models, Apoo-/- Apoe-/- and Apoo-/- Ldlr-/- mice exhibited elevated plasma cholesterol levels, with more severe atherosclerotic lesions than littermate controls. This indicated the effects of APOO on cholesterol metabolism independent of LDLR and APOE. Moreover, APOO deficiency reduced cholesterol excretion through bile and feces while decreasing phospholipid unsaturation by inhibiting NRF2 and CYB5R3. Restoration of CYB5R3 expression in vivo by adeno-associated virus (AAV) injection reversed the reduced degree of phospholipid unsaturation while decreasing blood cholesterol levels. This represents the first in vivo experimental validation of the role of APOO in plasma cholesterol metabolism independent of LDLR and elucidates a previously unrecognized cholesterol metabolism pathway involving NRF2/CYB5R3. APOO may be a metabolic regulator of total-body cholesterol homeostasis and a target for atherosclerosis management. Apolipoprotein O (APOO) regulates plasma cholesterol levels and atherosclerosis through a pathway involving CYB5R3 that regulates biliary and fecal cholesterol excretion, independently of the LDL receptor. In addition, down-regulation of APOO may lead to impaired mitochondrial function, which in turn aggravates diet-induced obesity and fat accumulation.

Decreased mRNA expression of NR1H3 and ABCA1 in pulmonary tuberculosis patients from population of Punjab, India.

BACKGROUND: Mycobacterium tuberculosis (MTB) is the causative organism of tuberculosis. Cholesterol is a crucial carbon source required for the survival of MTB in host cells. Transcription factor NR1H3 along with its important target genes ABCA1 and ApoE play important role in removal of extra cholesterol from cells. Changes in the gene expression of NR1H3, ABCA1 and ApoE can affect cholesterol homeostasis and thus the survival of MTB in host cells.Therefore, the present study was designed to analyze the mRNA expression of NR1H3, ABCA1 and ApoE in pulmonary TB (PTB) patients from the population of Punjab, India. METHODS AND RESULTS: In this study, mRNA expression of the transcription factor NR1H3 and its target genes ABCA1 and ApoE was analyzed in 89 subjects, including 41 PTB patients and 48 healthy controls (HCs) by real-time quantitative PCR. It was found that the mRNA expression of both NR1H3 and ABCA1 genes was significantly lower in TB patients than in HCs (p < 0.001). Even after sex-wise stratification of the subjects, mRNA expression of NR1H3 and ABCA1 was found to be down-regulated in both male and female TB patients. No significant difference was observed in expression of ApoE (p = 0.98). CONCLUSIONS: The present study found that the mRNA expression of NR1H3 and ABCA1 is down-regulated in TB patients from Punjab state of India.

Computerized decision support is an effective approach to select memory clinic patients for amyloid-PET.

BACKGROUND: The use of amyloid-PET in dementia workup is upcoming. At the same time, amyloid-PET is costly and limitedly available. While the appropriate use criteria (AUC) aim for optimal use of amyloid-PET, their limited sensitivity hinders the translation to clinical practice. Therefore, there is a need for tools that guide selection of patients for whom amyloid-PET has the most clinical utility. We aimed to develop a computerized decision support approach to select patients for amyloid-PET. METHODS: We included 286 subjects (135 controls, 108 Alzheimer's disease dementia, 33 frontotemporal lobe dementia, and 10 vascular dementia) from the Amsterdam Dementia Cohort, with available neuropsychology, APOE, MRI and [18F]florbetaben amyloid-PET. In our computerized decision support approach, using supervised machine learning based on the DSI classifier, we first classified the subjects using only neuropsychology, APOE, and quantified MRI. Then, for subjects with uncertain classification (probability of correct class (PCC) < 0.75) we enriched classification by adding (hypothetical) amyloid positive (AD-like) and negative (normal) PET visual read results and assessed whether the diagnosis became more certain in at least one scenario (PPC≥0.75). If this was the case, the actual visual read result was used in the final classification. We compared the proportion of PET scans and patients diagnosed with sufficient certainty in the computerized approach with three scenarios: 1) without amyloid-PET, 2) amyloid-PET according to the AUC, and 3) amyloid-PET for all patients. RESULTS: The computerized approach advised PET in n = 60(21%) patients, leading to a diagnosis with sufficient certainty in n = 188(66%) patients. This approach was more efficient than the other three scenarios: 1) without amyloid-PET, diagnostic classification was obtained in n = 155(54%), 2) applying the AUC resulted in amyloid-PET in n = 113(40%) and diagnostic classification in n = 156(55%), and 3) performing amyloid-PET in all resulted in diagnostic classification in n = 154(54%). CONCLUSION: Our computerized data-driven approach selected 21% of memory clinic patients for amyloid-PET, without compromising diagnostic performance. Our work contributes to a cost-effective implementation and could support clinicians in making a balanced decision in ordering additional amyloid PET during the dementia workup.

Buyang Huanwu Decoction Alleviates Atherosclerosis by Regulating gut Microbiome and Metabolites in Apolipoprotein E-deficient Mice fed with High-fat Diet.

ABSTRACT: The traditional Chinese herbal prescription Buyang Huanwu decoction (BHD), effectively treats atherosclerosis. However, the mechanism of BHD in atherosclerosis remains unclear. We aimed to determine whether BHD could alleviate atherosclerosis by altering the microbiome-associated metabolic changes in atherosclerotic mice. An atherosclerotic model was established in apolipoprotein E-deficient mice fed high-fat diet, and BHD was administered through gavage for 12 weeks at 8.4 g/kg/d and 16.8 g/kg/d. The atherosclerotic plaque size, composition, serum lipid profile, and inflammatory cytokines, were assessed. Mechanistically, metabolomic and microbiota profiles were analyzed by liquid chromatography-mass spectrometry and 16S rRNA gene sequencing, respectively. Furthermore, intestinal microbiota and atherosclerosis-related metabolic parameters were correlated using Spearman analysis. Atherosclerotic mice treated with BHD exhibited reduced plaque area, aortic lumen occlusion, and lipid accumulation in the aortic root. Nine perturbed serum metabolites were significantly restored along with the relative abundance of microbiota at the family and genus levels but not at the phylum level. Gut microbiome improvement was strongly negatively correlated with improved metabolite levels. BHD treatment effectively slows the progression of atherosclerosis by regulating altered intestinal microbiota and perturbed metabolites.

Probing Protein Structural Changes in Alzheimer's Disease via Quantitative Cross-linking Mass Spectrometry.

Alzheimer's disease (AD) is a progressive neurological disorder featuring abnormal protein aggregation in the brain, including the pathological hallmarks of amyloid plaques and hyperphosphorylated tau. Despite extensive research efforts, understanding the molecular intricacies driving AD development remains a formidable challenge. This study focuses on identifying key protein conformational changes associated with the progression of AD. To achieve this, we employed quantitative cross-linking mass spectrometry (XL-MS) to elucidate conformational changes in the protein networks in cerebrospinal fluid (CSF). By using isotopically labeled cross-linkers BS3d0 and BS3d4, we reveal a dynamic shift in protein interaction networks during AD progression. Our comprehensive analysis highlights distinct alterations in protein-protein interactions within mild cognitive impairment (MCI) states. This study accentuates the potential of cross-linked peptides as indicators of AD-related conformational changes, including previously unreported site-specific binding between α-1-antitrypsin (A1AT) and complement component 3 (CO3). Furthermore, this work enables detailed structural characterization of apolipoprotein E (ApoE) and reveals modifications within its helical domains, suggesting their involvement in MCI pathogenesis. The quantitative approach provides insights into site-specific interactions and changes in the abundance of cross-linked peptides, offering an improved understanding of the intricate protein-protein interactions underlying AD progression. These findings lay a foundation for the development of potential diagnostic or therapeutic strategies aimed at mitigating the negative impact of AD.

Unveiling the oral-gut connection: chronic apical periodontitis accelerates atherosclerosis via gut microbiota dysbiosis and altered metabolites in apoE-/- Mice on a high-fat diet.

The aim of this study was to explore the impact of chronic apical periodontitis (CAP) on atherosclerosis in apoE-/- mice fed high-fat diet (HFD). This investigation focused on the gut microbiota, metabolites, and intestinal barrier function to uncover potential links between oral health and cardiovascular disease (CVD). In this study, CAP was shown to exacerbate atherosclerosis in HFD-fed apoE-/- mice, as evidenced by the increase in plaque size and volume in the aortic walls observed via Oil Red O staining. 16S rRNA sequencing revealed significant alterations in the gut microbiota, with harmful bacterial species thriving while beneficial species declining. Metabolomic profiling indicated disruptions in lipid metabolism and primary bile acid synthesis, leading to elevated levels of taurochenodeoxycholic acid (TCDCA), taurocholic acid (TCA), and tauroursodeoxycholic acid (TDCA). These metabolic shifts may contribute to atherosclerosis development. Furthermore, impaired intestinal barrier function, characterized by reduced mucin expression and disrupted tight junction proteins, was observed. The increased intestinal permeability observed was positively correlated with the severity of atherosclerotic lesions, highlighting the importance of the intestinal barrier in cardiovascular health. In conclusion, this research underscores the intricate interplay among oral health, gut microbiota composition, metabolite profiles, and CVD incidence. These findings emphasize the importance of maintaining good oral hygiene as a potential preventive measure against cardiovascular issues, as well as the need for further investigations into the intricate mechanisms linking oral health, gut microbiota, and metabolic pathways in CVD development.

Efficient systemic CNS delivery of a therapeutic antisense oligonucleotide with a blood-brain barrier-penetrating ApoE-derived peptide.

Antisense oligonucleotide (ASO) has emerged as a promising therapeutic approach for treating central nervous system (CNS) disorders by modulating gene expression with high selectivity and specificity. However, the poor permeability of ASO across the blood-brain barrier (BBB) diminishes its therapeutic success. Here, we designed and synthesized a series of BBB-penetrating peptides (BPP) derived from either the receptor-binding domain of apolipoprotein E (ApoE) or a transferrin receptor-binding peptide (THR). The BPPs were conjugated to phosphorodiamidate morpholino oligomers (PMO) that are chemically analogous to the 2'-O-(2-methoxyethyl) (MOE)-modified ASO approved by the FDA for treating spinal muscular atrophy (SMA). The BPP-PMO conjugates significantly increased the level of full-length SMN2 in the patient-derived SMA fibroblasts in a concentration-dependent manner with minimal to no toxicity. Furthermore, the systemic administration of the most potent BPP-PMO conjugates significantly increased the expression of full-length SMN2 in the brain and spinal cord of SMN2 transgenic adult mice. Notably, BPP8-PMO conjugate showed a 1.25-fold increase in the expression of full-length functional SMN2 in the brain. Fluorescence imaging studies confirmed that 78% of the fluorescently (Cy7)-labelled BPP8-PMO reached brain parenchyma, with 11% uptake in neuronal cells. Additionally, the BPP-PMO conjugates containing retro-inverso (RI) D-BPPs were found to possess extended half-lives compared to their L-counterparts, indicating increased stability against protease degradation while preserving the bioactivity. This delivery platform based on BPP enhances the CNS bioavailability of PMO targeting the SMN2 gene, paving the way for the development of systemically administered neurotherapeutics for CNS disorders.

Quercetin Inhibits Neuronal Pyroptosis and Ferroptosis by Modulating Microglial M1/M2 Polarization in Atherosclerosis.

Atherosclerosis (AS) with iron and lipid overload and systemic inflammation is a risk factor for Alzheimer's disease. M1 macrophage/microglia participate in neuronal pyroptosis and recently have been reported to be the ferroptosis-resistant phenotype. Quercetin plays a prominent role in preventing and treating neuroinflammation, but the protective mechanism against neurodegeneration caused by iron deposition is poorly understood. ApoE-/- mice were fed a high-fat diet with or without quercetin treatment. The Morris water maze and novel object recognition tests were conducted to assess spatial learning and memory, and nonspatial recognition memory, respectively. Prussian blue and immunofluorescence staining were performed to assess the iron levels in the whole brain and in microglia, microglia polarization, and the degree of microglia/neuron ferroptosis. In vitro, we further explored the molecular biological alterations associated with microglial polarization, neuronal pyroptosis, and ferroptosis via Western blot, flow cytometry, CCK8, LDH, propidium iodide, and coculture system. We found that quercetin improved brain lesions and spatial learning and memory in AS mice. Iron deposition in the whole brain or microglia was reversed by the quercetin treatment. In the AS group, the colocalization of iNOS with Iba1 was increased, which was reversed by quercetin. However, the colocalization of iNOS with PTGS2/TfR was not increased in the AS group, suggesting a character resisting ferroptosis. Quercetin induced the expression of Arg-1 and decreased the colocalizations of Arg-1 with PTGS2/TfR. In vitro, ox-LDL combined with ferric ammonium citrate treatment (OF) significantly shifted the microglial M1/M2 phenotype balance and increased the levels of free iron, ROS, and lipid peroxides, which was reversed by quercetin. M1 phenotype induced by OF caused neuronal pyroptosis and was promoted to ferroptosis by L-NIL treatment, which contributed to neuronal ferroptosis as well. However, quercetin induced the M1 to M2 phenotype and inhibited M2 macrophages/microglia and neuron pyroptosis or ferroptosis. In summary, quercetin alleviated neuroinflammation by inducing the M1 to M2 phenotype to inhibit neuronal pyroptosis and protected neurons from ferroptosis, which may provide a new idea for neuroinflammation prevention and treatment.

Generation of three isogenic, gene-edited iPSC lines carrying the APOE-Christchurch mutation into the three common APOE variants: APOE2Ch, APOE3Ch and APOE4Ch.

Late-onset Alzheimer's disease (AD) has become the paradigm of a non-mendelian complex neurodegenerative disease, for which a major genetic determinant is known, the APOE locus. A rare APOE variant named Christchurch (APOEch) yielding a missense mutation from Arginine to Serine at amino acid 136, has been suggested to exert a protective effect in an individual carrying the most penetrant form of Familial AD (Paisa mutation in PSEN1 gene, E280A). We describe here a new set of induced pluripotent stem cell (iPSC) lines, where the Christchurch mutation (Ch) has been introduced by gene editing into the APOE locus of three isogenic iPSC lines carrying the more common APOE variants (APOE 2/2, APOE 3/3, and an APOE 4/4) in homozygosity. Brain cells derived from these iPSC lines will enable a better understanding of APOE biology in general and facilitate the study of how the Christchurch variant affects the function of each APOE genotype. This set of iPSC lines are globally available via the European Bank of iPSCs, EBiSC.org.

Apolipoprotein E is a prognostic factor for pancreatic cancer and associates with immune infiltration.

BACKGROUND: The expression level of apolipoprotein E (APOE) in pancreatic ductal adenocarcinoma (PDAC) and its effect on the prognosis of PDAC patients are not clear. The effect of APOE on the immune status of patients with PDAC has not been elucidated. METHODS: We obtained pancreatic cancer data from the TCGA and GETx databases. Patients with PDAC who underwent pancreatic surgery at the Second Affiliated Hospital of Jiaxing University between 2012 and 2021 were included. Clinical pathological data were recorded, plasma APOE levels were measured, and tissue samples were collected. A tissue microarray was generated using the collected tissue samples. APOE and CD4 staining was performed to determine immunoreactive scores (IRSs). The expression of APOE in the plasma and tumour tissues of pancreatic cancer patients was analysed and compared. The correlations between plasma APOE levels, tissue APOE levels and clinicopathological characteristics were analysed. Survival prognosis was analysed using Kaplan-Meier survival analysis and Cox multivariate regression analysis. The correlations between APOE expression levels and immune biomarkers and immune cells were further analysed. Single-cell analysis of APOE distribution in various cells was performed on the TISCH website. RESULTS: APOE was highly expressed in the tumour tissue of pancreatic cancer patients, and high plasma APOE levels were associated with poor prognosis. Females, patients with high-grade disease and patients with pancreatic head carcinoma had high plasma APOE levels. High APOE expression in tumour tissues was associated with good prognosis. Mononuclear macrophages in the pancreatic cancer microenvironment primarily expressed APOE. APOE levels positively correlated with immune biomarkers, such as CD8A, PDCD1, GZMA, CXCL10, and CXCL9, in the tumour microenvironment. APOE promoted CD4 + T cell or dendritic cell infiltration in the tumour microenvironment. CONCLUSIONS: APOE may affect the occurrence and development of pancreatic cancer by regulating the infiltration of immune cells in the tumour microenvironment.

The low-density lipoprotein receptor and apolipoprotein E associated with CCHFV particles mediate CCHFV entry into cells.

The Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging pathogen of the Orthonairovirus genus that can cause severe and often lethal hemorrhagic diseases in humans. CCHFV has a broad tropism and can infect a variety of species and tissues. Here, by using gene silencing, blocking antibodies or soluble receptor fragments, we identify the low-density lipoprotein receptor (LDL-R) as a CCHFV entry factor. The LDL-R facilitates binding of CCHFV particles but does not allow entry of Hazara virus (HAZV), another member of the genus. In addition, we show that apolipoprotein E (apoE), an exchangeable protein that mediates LDL/LDL-R interaction, is incorporated on CCHFV particles, though not on HAZV particles, and enhances their specific infectivity by promoting an LDL-R dependent entry. Finally, we show that molecules that decrease LDL-R from the surface of target cells could inhibit CCHFV infection. Our study highlights that CCHFV takes advantage of a lipoprotein receptor and recruits its natural ligand to promote entry into cells.

Simultaneous quantification of six proteins related to liver injury using nano liquid chromatography-tandem mass spectrometry.

RATIONALE: In clinical diagnosis of liver injury, which is an important health concern, serum aminotransferase assays have been the go-to method used worldwide. However, the measurement of serum enzyme activity has limitations, including inadequate disease specificity and enzyme specificity. METHODS: With the high selectivity and specificity provided by nano liquid chromatography-tandem mass spectrometry (LC/MS/MS), this work describes a method for the simultaneous determination of six proteins in liver that can be potentially used as biomarkers for liver injury: glutamic-pyruvic transaminase 1 (GPT1), glutamic oxaloacetic transaminase 1 (GOT1), methionine adenosyl transferase 1A (MAT1A), glutathione peroxidase 1 (GPX1), cytokeratin 18 (KRT18) and apolipoprotein E (APOE). RESULTS: In validation, the method was shown to have good selectivity and sensitivity (limits of detection at pg/mL level). The analytical method revealed that, compared with normal mice, in carbon tetrachloride-induced acute liver injury mice, liver MAT1A and GPX1 were significantly lower (p < 0.01 and p < 0.05, respectively), KRT18 was significantly higher (p < 0.05) and APOE and GPT1 were marginally significantly lower (p between 0.05 and 0.1). This is the first work reporting the absolute contents of GPT1, GOT1, MAT1A, GPX1 and KRT18 proteins based on LC/MS. CONCLUSIONS: The proposed method provides a basis for establishing more specific diagnostic indicators of liver injury.

Quinic acid regulated TMA/TMAO-related lipid metabolism and vascular endothelial function through gut microbiota to inhibit atherosclerotic.

BACKGROUND: Quinic acid (QA) and its derivatives have good lipid-lowering and hepatoprotective functions, but their role in atherosclerosis remains unknown. This study attempted to investigate the mechanism of QA on atherogenesis in Apoe-/- mice induced by HFD. METHODS: HE staining and oil red O staining were used to observe the pathology. The PCSK9, Mac-3 and SM22a expressions were detected by IHC. Cholesterol, HMGB1, TIMP-1 and CXCL13 levels were measured by biochemical and ELISA. Lipid metabolism and the HMGB1-SREBP2-SR-BI pathway were detected by PCR and WB. 16 S and metabolomics were used to detect gut microbiota and serum metabolites. RESULTS: QA or low-frequency ABX inhibited weight gain and aortic tissue atherogenesis in HFD-induced Apoe-/- mice. QA inhibited the increase of cholesterol, TMA, TMAO, CXCL13, TIMP-1 and HMGB1 levels in peripheral blood of Apoe-/- mice induced by HFD. Meanwhile, QA or low-frequency ABX treatment inhibited the expression of CAV-1, ABCA1, Mac-3 and SM22α, and promoted the expression of SREBP-1 and LXR in the vascular tissues of HFD-induced Apoe-/- mice. QA reduced Streptococcus_danieliae abundance, and promoted Lactobacillus_intestinalis and Ileibacterium_valens abundance in HFD-induced Apoe-/- mice. QA altered serum galactose metabolism, promoted SREBP-2 and LDLR, inhibited IDOL, FMO3 and PCSK9 expression in liver of HFD-induced Apoe-/- mice. The combined treatment of QA and low-frequency ABX regulated microbe-related Glycoursodeoxycholic acid and GLYCOCHENODEOXYCHOLATE metabolism in HFD-induced Apoe-/- mice. QA inhibited TMAO or LDL-induced HCAECs damage and HMGB1/SREBP2 axis dysfunction, which was reversed by HMGB1 overexpression. CONCLUSIONS: QA regulated the gut-liver lipid metabolism and chronic vascular inflammation of TMA/TMAO through gut microbiota to inhibit the atherogenesis in Apoe-/- mice, and the mechanism may be related to the HMGB1/SREBP2 pathway.

Phosphatidylglycerol Acts as a Switch for Cholesterol-Dependent Membrane Binding of ApoE Signal Peptide.

The apolipoprotein E (ApoE) signal peptide is a short stretch of N-terminal amino acids that direct the ApoE protein to the endoplasmic reticulum after synthesis. Previous studies have shown that this peptide can bind to lipid membranes in a cholesterol-dependent manner; however, the mechanism of this interaction is yet to be clarified. In this study, we aimed to investigate how the composition of neighboring lipids affects the membrane-binding of the ApoE signal peptide. We found that a negatively charged lipid, such as phosphatidylglycerol, can act as a switch that reduces the binding efficiency of the peptide to cholesterol-rich membranes. Interestingly, phosphatidylethanolamine does not activate the cholesterol-dependent binding of the ApoE signal peptide yet acts synergistically to enhance the cholesterol sensitivity in phosphatidylglycerol-containing membranes. To the best of our knowledge, this is the first report of modulation of the affinity of a peptide for a membrane by a neighboring lipid rather than by the lipid-binding domain of the peptide. Our findings revealed a novel role of lipid diversity in modulating the membrane binding of the ApoE signal peptide and its potential implications in the unidirectional trafficking of a newly synthesized protein from the ribosomes to the endoplasmic reticulum.

Contactin 5 and Apolipoproteins Interplay in Alzheimer's Disease.

Background: Apolipoproteins and contactin 5 are proteins associated with Alzheimer's disease (AD) pathophysiology. Apolipoproteins act on transport and clearance of cholesterol and phospholipids during synaptic turnover and terminal proliferation. Contactin 5 is a neuronal membrane protein involved in key processes of neurodevelopment. Objective: To investigate the interactions between contactin 5 and apolipoproteins in AD, and the role of these proteins in response to neuronal damage. Methods: Apolipoproteins (measured by Luminex), contactin 5 (measured by Olink's proximity extension assay), and cholesterol (measured by liquid chromatography mass spectrometry) were assessed in the cerebrospinal fluid (CSF) and plasma of cognitively unimpaired participants (n = 93). Gene expression was measured using polymerase chain reaction in the frontal cortex of autopsied-confirmed AD (n = 57) and control subjects (n = 31) and in the hippocampi of mice following entorhinal cortex lesions. Results: Contactin 5 positively correlated with apolipoproteins B (p = 5.4×10-8), D (p = 1.86×10-4), E (p = 2.92×10-9), J (p = 2.65×10-9), and with cholesterol (p = 0.0096) in the CSF, and with cholesterol (p = 0.02), HDL (p = 0.0143), and LDL (p = 0.0121) in the plasma. Negative correlations were seen between CNTN5, APOB (p = 0.034) and APOE (p = 0.015) mRNA levels in the brains of control subjects. In the mouse model, apoe and apoj gene expression increased during the reinnervation phase (p <  0.05), while apob (p = 0.023) and apod (p = 0.006) increased in the deafferentation stage. Conclusions: Extensive interactions were observed between contactin 5 and apolipoproteins and cholesterol, possibly due to neuronal damage. The alterations in gene expression of apolipoproteins suggest a role in axonal, terminal, and synaptic remodeling in response to entorhinal cortex damage.

Global O-glycoproteome enrichment and analysis enabled by a combinatorial enzymatic workflow.

A comprehensive analysis of site-specific protein O-glycosylation is hindered by the absence of a consensus O-glycosylation motif, the diversity of O-glycan structures, and the lack of a universal enzyme that cleaves attached O-glycans. Here, we report the development of a robust O-glycoproteomic workflow for analyzing complex biological samples by combining four different strategies: removal of N-glycans, complementary digestion using O-glycoprotease (IMPa) with/without another protease, glycopeptide enrichment, and mass spectrometry with fragmentation of glycopeptides using stepped collision energy. Using this workflow, we cataloged 474 O-glycopeptides on 189 O-glycosites derived from 79 O-glycoproteins from human plasma. These data revealed O-glycosylation of several abundant proteins that have not been previously reported. Because many of the proteins that contained unannotated O-glycosylation sites have been extensively studied, we wished to confirm glycosylation at these sites in a targeted fashion. Thus, we analyzed selected purified proteins (kininogen-1, fetuin-A, fibrinogen, apolipoprotein E, and plasminogen) in independent experiments and validated the previously unknown O-glycosites.

Effect of moxibustion on the SIRT1/FOXO3a signaling pathway in ApoE-/- mice with atherosclerosis. / 艾灸对ApoE-/-åŠ¨è„‰ç²¥æ ·ç¡¬åŒ–å°é¼ SIRT1/FOXO3a信号通路的影响.

OBJECTIVES: To observe the effects of moxibustion on blood lipid metabolism, pathological morphology of thoracic aorta, and the expression of silent information regulator 1 (SIRT1) and forkhead box transcription factor O3a (FOXO3a) in ApoE-/- atherosclerosis (AS) mice, so as to explore the potential mechanism of moxibustion in preventing and treating AS. METHODS: Ten C57BL/6J mice were fed a normal diet as the control group, and 30 ApoE-/- mice were fed a high-fat diet to establish the AS model, which were randomly divided into the model group, simvastatin group, and moxibustion group, with 10 mice in each group. From the first day of modeling, mice in the moxibustion group received mild moxibustion treatment at "Shenque"(CV8), "Yinlingquan"(SP9), bilateral "Neiguan"(PC6) and "Xuehai"(SP10) for 30 min per time；the mice in the simvastatin group were given simvastatin orally (2.5 mg·kg-1·d-1), with both treatments given once daily, 5 times a week, with a total intervention period of 12 weeks. The body weight and general condition of the mice were observed and recorded during the intervention period. After the intervention, the contents of serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using an automated biochemistry analyzer. Hematoxylin eosin (HE) staining was used to observe the pathological morphology of the thoracic aorta. ELISA was used to measure the contents of serum oxidized low-density lipoprotein (ox-LDL) and superoxide dismutase (SOD) activity. Western blot and real-time fluorescent quantitative PCR analysis were used to detect the expression levels of SIRT1 and FOXO3a protein and mRNA in the thoracic aorta. RESULTS: Compared with the control group, body weight at the 8th and 12th week, serum TC, TG, LDL-C, and ox-LDL contents of the model group mice were significantly increased(P<0.05, P<0.01), while the HDL-C contents, SOD activity, and the expression levels of SIRT1 protein and mRNA in the thoracic aorta were significantly decreased(P<0.05, P<0.01). HE staining showed thickening of the aortic intima, endothelial cell degeneration, swelling, and shedding. Compared with the model group, body weight at the 8th and 12th week, serum TC, TG, LDL-C, and ox-LDL contents of mice in the simvastatin group and moxibustion group were significantly decreased(P<0.01), while the serum SOD activity, expression levels of SIRT1 protein and mRNA in the thoracic aorta were significantly increased(P<0.01). The HDL-C contents were significantly increased in the simvastatin group(P<0.05). The thoracic aortic structure was more intact in both groups, with a more regular lumen and orderly arrangement of the elastic membrane in the media, and a slight amount of endothelial cell degeneration and swelling in the intima. There was no significant difference in the evaluated indexes between the moxibustion group and the simvastatin group and the pathological changes in the thoracic aorta were similar between the two groups. CONCLUSIONS: Moxibustion can reduce the body weight of AS model mice, regulate lipid levels, repair vascular intima, and alleviate endothelial damage. Its mechanism of action may be related to the regulation of the SIRT1/FOXO3a signaling pathway to improve oxidative damage.