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Alzheimer stage proteomics study

ABSTRACT: This project aims to study the pathological role of Extracellular vesicles (EVs) in human brain and the correlation between EVs secretion with whole brain proteome. EVs are extracted from human brain tissue of Alzheimer dementia (AD) patients, VaD patients and age matched healthy controls using PROSPR method. Alzheimer patients includes stage3 (AD3), stage4 (AD4), stage5 (AD5) and stage6 (AD6). Whole brain proteome are performed simultaneously. Proteins associated with pre-symptomatic AD, AD progression and other pathological ways are analysed. The potential targets are further validated with MRM approach

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain

DISEASE(S): Vascular Dementia

SUBMITTER: Xue Guo

LAB HEAD: Sze Siu Kwan

PROVIDER: PXD015578 | Pride | 2020-05-04

REPOSITORIES: Pride

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Project description:Background: Acute coronary syndromes (ACS) are associated with aberrant gene expression and epigenetic mechanisms. In particular, de novo DNA methylation is typically linked to gene silencing, but its role in heart disease remains not fully understood. Extracellular vesicles (EVs) are active components in cellular communication for their ability to carry a plethora of signalling biomolecules, thus representing a promising new diagnostic/therapeutic approach in cardiovascular diseases (CVDs). Indeed, there is the need of novel biomarkers for ACS prediction and timely detection. Purpose: We hypothesized that specific epigenetic signals can be carried by EVs. In this regard, we isolated and characterized circulating EVs from ACS patients and evaluated their potential role to influence DNA methylation in target cells. Methods: Circulating EVs were recovered, by ultracentrifugation, from plasma samples of 19 ACS patients and 50 healthy subjects (HS). Nanoparticle tracking analysis (NTA) and western blot (WB) were used to confirm the EVs integrity and purity. Peripheral blood mononuclear cells (PBMCs) of volunteer donors were treated with both ACS and HS derived EVs and genomic DNA was extracted to perform epigenome wide analysis through Reduced Representation Bisulfite Sequencing. ShinyGO, PANTHER, and STRING tools were interrogated to perform GO and PPI network analyses. Flow Cytometry, qRT-PCR, and WB analysis were also performed to evaluate and validate both intra-vesicular and intra-cellular signals. Results: Plasma ACS-derived EVs showed a significant up-regulation of DNA methyltransferases (DNMTs) gene expression levels as compared to HS (P<0.001). Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B were significantly increased in plasma ACS-EVs. DNA methylation analysis of PBMCs from volunteer donors treated with HS- and ACS-derived EVs showed that RNF166 and CCND3 genes resulted the most hyper- and hypo-methylated, respectively, after by ACS-EV treatment. In addition, PPI network analysis specifically evidenced the subnetwork with SRC, as a hub gene, connecting it to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, known as important genes already involved in the onset of CVDs. Surprising, other novel genes, BAIAP2, SYP, CHL1, and SHB, which were hypomethylated, were found significantly overexpressed in PBMCs (P<0.005), underlying the fundamental modulating properties of EV cargo in atherosclerosis. Conclusions: These findings support the significant role of ACS plasma-derived EVs, inducing de novo DNA methylation signals, and modulating specific signaling pathways in target cells.

Project description:Alzheimer’s Disease (AD) is the most common cause of dementia in the elderly population without a cure or early diagnostics currently available. Despite the demonstrated ability of extracellular vesicles (EVs) to spread tau and Aβ pathology in AD models and their potential utility as a diagnostic and treatment-monitoring tool, our knowledge of human brain EV subpopulations, their molecular composition and roles in disease progression is very limited. Genome-wide association studies linked multiple AD genetic risk factors to microglia-specific pathways suggesting a potential role of microglia-derived EVs in AD progression. Here we are presenting a method for isolation of microglial CD11b-positive small EVs from cryopreserved human brain tissue and multi-omics analysis of the EVs from the parietal cortex of 4 late-stage AD (Braak V-VI) and 3 age-matched normal/low pathology (NL) cases. We identified a total of 1000 proteins by quantitative proteomics, analyzed 594 individual lipid species by targeted lipidomics, and 105 miRNAs using a NanoString miRNA expression panel. We found significant reduction in abundance of homeostatic microglia markers, P2RY12 and TMEM119, and increased levels of disease associated microglia markers, FTH1 and TREM2, in microglial EVs from AD brain compared to NL cases. Protein tau abundance was also significantly higher in AD brain-derived microglial EVs. These changes were accompanied by upregulation of synaptic and neuron-specific proteins in the AD group. Levels of free-cholesterol were elevated in microglial EVs from AD brain. Lipidomic analysis also revealed a pro-inflammatory lipid profile, endolysosomal dysfunction and significant AD-associated decrease in levels of docosahexaenoic acid (DHA)-containing polyunsaturated lipids of different classes, suggesting a potential defect in acyl-chain remodeling. Several immune pathways and senescence were among the top pathways controlled by 4 miRNAs significantly upregulated in the AD group. Our data suggest that loss of the homeostatic microglia signature in late AD stages may accompany endolysosomal impairment and release of undigested neuronal and myelin debris, including tau, through extracellular vesicles. These data validate a method of isolation of small cell-type specific EVs from human brain tissue, suggests new potential EV-associated markers and disease-related pathways, and provides a framework for future large-scale multi-omics study.

			Action	DRS
	170413_EVsB_C2_A.DAT	Other
	170413_EVsB_C2_A01.raw	Raw
	170413_EVsB_C2_A02.raw	Raw
	170413_EVsB_C2_A03.raw	Raw
	170413_EVsB_C2_A04.raw	Raw

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Alzheimer stage proteomics study

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