Small RNA sequencing of brain-derived extracellular vesicles from post-mortem brain tissue of controls and Alzheimer's disease patients with different APOE genotypes
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE159541 | GEO | 2020/10/20
REPOSITORIES: GEO
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Project description:Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque. Effects of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with macaque, suggesting opportunities for optimization. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.
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Project description:Extracellular vesicles (EVs) have gained significant attention as potential diagnostic and therapeutic tools due to their role in intercellular communication and their ability to encapsulate various biomolecules. However, the isolation of EVs from tissue remains challenging, often requiring enzymatic digestion steps that may introduce unwanted biases and affect EV integrity. In this study, we present an efficient non-enzymatic approach for the isolation of brain-derived extracellular vesicles (BDEVs). Our method employs sequential ultracentrifugation and a density-based gradient to achieve a high yield and purity of BDEVs from brain tissue without any enzymatic digestion. Characterization of the isolated EVs revealed their typical morphology and size distribution, as confirmed by transmission electron microscopy and nanoparticle tracking analysis. We show by western blot that known BDEV markers, such as Flotlin-1, CD81, Alix, and the prion protein (PrP) are present without proteolytic cuts introduced by the enzymatic digestion of the tissue. We also show that the absence ofGM130 ,widely accepted as a negative marker to assess BDEV’s purity, is due to enzymatic digestion rather than lack of this protein in the sample. Moreover, we find no differences in mRNA content between the non-enzymatically and the enzymatically isolated BDEVs, showing that the intraluminal part of the BDEVs is not affected by enzymatic isolation. Similarly, our proteomic analysis shows unaltered protein expression in BDEVs isolated with both methods. However, other proteins, potentially in the BDEVs corona and membrane, are differentially expressed, highlighting the consequences of the enzymatically-based isolation protocol.
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Project description:The apolipoprotein E4 (APOE4) gene is the strongest genetic risk factor for late-onset Alzheimer’s disease (AD). ApoE is a lipid carrier abundantly expressed in both brain and periphery. As peripheral apoE is separated from brain apoE by the blood-brain barrier, it remains unclear whether peripheral apoE impacts brain functions and AD pathogenesis. To investigate this, we developed novel mouse models that conditionally express human APOE3 or APOE4 only in the liver with no detectable apoE in the brain. We found that liver-expressed apoE4 compromised synaptic plasticity and cognitive behaviors likely by impairing cerebrovascular functions. Remarkably, liver-expressed apoE4 exacerbated brain amyloid pathology, whereas apoE3 reduced it. Our findings demonstrate a pathogenic effect of peripheral apoE4, providing strong rationale for targeting peripheral apoE to treat AD.
2022-06-15 | GSE125501 | GEO
Project description:Alzheimer's disease (AD) is the most common neurodegenerative disorder. Extracellular vesicles (EVs) are carriers of nucleic acids, lipids and proteins, and are known to play a significant role in neurodegenerative pathogenesis. The goals of this study are to compare long RNA (exLR) profiling of EVs in normal human brains with which in AD human brain. EVs were isolated from frontal cortex of normal control (n=10) and AD (n=8) donors.
2022-12-31 | GSE197505 | GEO
Project description:Alzheimer’s disease (AD) is the most common cause of late-life dementia characterized by progressive neurodegeneration and brain deposition of amyloid-β (Aβ) and phosphorylated tau. The APOE ε2 encoding apolipoprotein E (APOE2) is a protective allele against AD among the three genotypes (APOE ε2, ε3, ε4), while APOE4 is the strongest genetic factor substantially increasing AD risk. APOE regulates brain lipid homeostasis and maintaining synaptic plasticity and neuronal function, where APOE2 has a superior function compared to APOE3 and APOE4. Gene therapy that increases APOE2 levels in the brain is, therefore, a promising therapeutic strategy for AD treatment. We previously reported that PEGylated liposomes conjugated with transferrin and a cell-penetrating peptide Penetratin sufficiently deliver chitosan-APOE2 cDNA plasmid complex into the brain of wild-type mice. Here, we investigated how brain-targeting liposome-based APOE2 gene delivery influences Aβ)-related pathologies in amyloid model AppNL-G-F knockin mice at 12-month-old. We found a trend of reductions of insoluble Aβ levels in the mouse cortices 1 month after APOE2 gene therapy. Furthermore, in the AppNL-G-F knockin mice that received the APOE2 gene therapy, brain transcriptome analysis through RNA-sequencing identified the upregulation of genes/pathways related to neuronal development. This was supported by increases of Dlg4 and Syp mRNAs coding synaptic proteins in the experimental group. On the other hand, we found that APOE2 gene delivery increased soluble Aβ levels, including oligomers, as well as exacerbated neurite dystrophy and decreased synaptophysin. Together, our results suggest that brain-targeting liposome-based APOE2 gene therapy is potentially beneficial for synaptic formation at the transcriptional level. Forced APOE2 expressions, however, may exacerbate Aβ toxicity by increasing the dissociation of Aβ oligomers from aggregates in the presence of considerable amyloid burden.
2024-11-13 | GSE279476 | GEO
Project description:The most common genetic risk factor for late onset Alzheimer Disease (AD) is the APOE4 allele, with evidence for gain- and loss-of-function as a primary mechanism. ApoE knockout in mice abrogates AD phenotypes but causes severe atherosclerosis due to the role of liver ApoE in cholesterol homeostasis. Previous attempts to therapeutically block brain-specific ApoE in adult models of AD only modestly reduced ApoE expression and no significant impact on amyloid burden. Here, we optimized a divalent siRNA (di-siRNA) to selectively silence ApoE in brain (>2 months). By measuring transcriptomic changes upon knockdown of ApoE in the CNS in multiple mouse models of AD, we find that ApoE knockdown results in an upregulation of the innate immune response, likely through activation of microglia. Additionally, we find that the changes in the transcriptome were similar, but not identical, two weeks and two months post-treatment. We find that mild reduction of ApoE in the liver does not cause widespread transcriptomic changes, but near complete knockdown of ApoE in the liver results in the dysregulation of many lipid metabolism-related pathways.
2023-10-02 | GSE186756 | GEO
Project description:Given the nefarious role that Extracellular vesicles (EVs) can play in disease pathogenesis, there is a growing interest in examining the contents of EVs in many disorders, including depression, HIV, and HIV-Associated Neurocognitive Disorder (HAND). EVs are particularly enriched with miRNA, and the nervous system expresses approximately 70% of all miRNA. miRNA typically regulate gene expression via degrading or suppressing mRNA translation, and these small non-coding RNAs, which are thought to be shuttled between cells via EVs, have been implicated in a host of diseases and conditions, including depression and cognitive decline. To our knowledge, however, this is the first study to examine brain-derived (bd) EV miRNA content in a preclinical model of HIV-associated CI and depression. We report 4 miRNAs, including miR-429-3p, miR-200c-3p, miR-183-5p, and miR-200b-3p, that are significantly upregulated in bdEVs of EcoHIV-infected mice and normalized by novel treatment.
2021-08-28 | GSE182743 | GEO
Project description:Human APOE genotype is intricately involved in the homeostasis of the central nervous system, which is particularly evident from its association with neurodegenerative disease. Compared to APOE3, APOE4 is associated with greater cognitive decline in aging, poorer outcomes following stroke and traumatic brain injury, and is a major genetic risk factor for Alzheimer’s disease. Human APOE genotypes can differentially modulate neuronal function via both direct and indirect pathways. Of the indirect pathways, increasing evidence supports that APOE4-associated neurovascular dysfunction plays a significant role in neurodegeneration. For example, deficits in cerebral blood flow, neuronal-vascular coupling and blood-brain barrier integrity are observed with APOE4 both during aging and in Alzheimer’s disease patients; data that has been recapitulated in mouse models. Therefore, the identification of cellular mechanistic pathways that contribute to APOE-modulated vascular function is important for fundamental and disease-focused research. Highly specialized brain endothelial cells (BECs) are essential for virtually all neurovascular functions and, therefore, the influence of apoE on the cerebrovasculature likely involves alterations in brain endothelial cells function, directly or via supporting cells. ApoE synthesis has been demonstrated in multiple cell types in both the brain (e.g. astrocytes, pericytes and neurons) and periphery (e.g. hepatocytes and macrophages). Current research has largely focused on the effects of apoE produced by supporting cell types, particularly astrocytes and pericytes, on BEC function. However, less explored is whether BECs produce apoE to modulate their function. Data from limited studies on apoE production by BECs and APOE genotype-specific functional differences are conflicting. Evaluation of whether the APOE genotype of BECs modulates their function is important, as this could represent a novel mechanisms that contributes to altered cerebrovascular function in aging and AD. The goal of this study was to evaluate the role of human APOE genotype in modulating the phenotype and/or function of brain endothelial cells in vitro. To this end, we utilized primary brain endothelial cells isolated from APOE-targeted replacement mice that express human APOE3 or APOE4 under the endogenous mouse APOE promoter. BECs are highly specialized and functionally complex to maintain homeostasis of the central nervous system and APOE genotype could theoretically alter levels of any number of molecules and associated functions. Thus, without a priori knowledge we initially evaluated the extent that APOE modulated the transcriptomic profile of brain endothelial cells.
2021-03-24 | GSE160483 | GEO