Multi-Omics Analysis of Microglial Extracellular Vesicles From Human Alzheimer’s Disease Brain Tissue Reveals Disease-Associated Signatures

Abstract

Alzheimer’s disease (AD) is the most common cause of dementia, yet there is no cure or diagnostics available prior to the onset of clinical symptoms. Extracellular vesicles (EVs) are lipid bilayer-delimited particles that are released from almost all types of cell. Genome-wide association studies have linked multiple AD genetic risk factors to microglia-specific pathways. It is plausible that microglia-derived EVs may play a role in the progression of AD by contributing to the dissemination of insoluble pathogenic proteins, such as tau and Aβ. Despite the potential utility of EVs as a diagnostic tool, our knowledge of human brain EV subpopulations is limited. Here we present a method for isolating microglial CD11b-positive small EVs from cryopreserved human brain tissue, as well as an integrated multiomics analysis of microglial EVs enriched from the parietal cortex of four late-stage AD (Braak V-VI) and three age-matched normal/low pathology (NL) cases. This integrated analysis revealed 1,000 proteins, 594 lipids, and 105 miRNAs using shotgun proteomics, targeted lipidomics, and NanoString nCounter technology, respectively. The results showed a significant reduction in the abundance of homeostatic microglia markers P2RY12 and TMEM119, and increased levels of disease-associated microglia markers FTH1 and TREM2, in CD11b-positive EVs from AD brain compared to NL cases. Tau abundance was significantly higher in AD brain-derived microglial EVs. These changes were accompanied by the upregulation of synaptic and neuron-specific proteins in the AD group. Levels of free cholesterol were elevated in microglial EVs from the AD brain. Lipidomic analysis also revealed a proinflammatory lipid profile, endolysosomal dysfunction, and a significant AD-associated decrease in levels of docosahexaenoic acid (DHA)-containing polyunsaturated lipids, suggesting a potential defect in acyl-chain remodeling. Additionally, four miRNAs associated with immune and cellular senescence signaling pathways were significantly upregulated in the AD group. Our data suggest that loss of the homeostatic microglia signature in late AD stages may be accompanied by endolysosomal impairment and the release of undigested neuronal and myelin debris, including tau, through extracellular vesicles. We suggest that the analysis of microglia-derived EVs has merit for identifying novel EV-associated biomarkers and providing a framework for future larger-scale multiomics studies on patient-derived cell-type-specific EVs.

Differential Effects of APOE Genotype on MicroRNA Cargo of Cerebrospinal Fluid Extracellular Vesicles in Females With Alzheimer’s Disease Compared to Males

“…After loading 0.1 µg of CSF SEC Fx protein lysate, we detected an enrichment of GLAST, which is a marker for astrocyte-derived EVs ( Figure 5B ) ( You et al, 2020 ). However, we were unable to detect NCAM-1 and SYP, which are markers for neuronal-derived EVs; as well as TMEM119, which is a microglia-derived EV marker ( Figure 5B ) ( Togashi et al, 2009 ; Satoh et al, 2016 ; Sathe et al, 2019 ; Vukojevic et al, 2020 ; Cohn et al, 2021 ; Crews et al, 2021 ; Ruan et al, 2021 ; You et al, 2022 ). We also performed an immunoblot assay using 0.1 µg protein for CD11b, which is another marker for microglia-derived EVs ( Gu et al, 2021 ), and detected faint bands in Fxs 6–9, 10–13, and 14–17 ( Figure 5B ).…”

Small but Mighty—Exosomes, Novel Intercellular Messengers in Neurodegeneration

“…The lipidomic analyses revealed a proinflammatory phenotype and a potential defect in acyl-chain remodeling. Lastly, miRNAs associated with immune and cellular senescence signaling pathways were increased in AD brain-derived microglial exosomes [192]. These data suggest that a significant change in the molecular composition of exosomes reflects changes in microglia consistent with a diseased state.…”

“…To better understand possible dynamic interactions between proteins, lipids and RNA in Alzheimer's disease, high-throughput techniques have recently been applied. Cohn and colleagues took an 'omics' integrative approach to analyze microglial exosomes [192]. In this study, the authors isolated microglial exosomes from the parietal cortex of late-stage Alzheimer's disease patients.…”

An Essential Role for Alzheimer’s-Linked Amyloid Beta Oligomers in Neurodevelopment: Transient Expression of Multiple Proteoforms during Retina Histogenesis

“…The possibility that AβOs might be secreted from their sites of assembly and diffuse to distal sites is consistent with our findings that AβOs in retina extracts could associate with cultured retina neurons (not shown). Current thinking favors the idea that pathological AβOs and pTau in Alzheimer's disease are spread extracellularly by exosomes [96,97]. Retina exosomes have been isolated [98], including from embryonic avian retina [99], where they are developmentally regulated [45].…”