Novel method for collecting hippocampal interstitial fluid extracellular vesicles (EV-ISF) reveals sex-dependent changes in microglial EV proteome in response to Aβ pathology

Abstract

Brain-derived extracellular vesicles (EVs) play an active role in Alzheimer's disease (AD), relaying important physiological information about their host tissues. Circulating EVs are protected from degradation, making them attractive AD biomarkers. However, it is unclear how circulating EVs relate to EVs isolated from disease-vulnerable brain regions. We developed a novel method for collecting EVs from the hippocampal interstitial fluid (ISF) of live mice. EVs (EVISF) were isolated via ultracentrifugation and characterized by nanoparticle tracking analysis, immunogold labeling, and flow cytometry. Mass spectrometry and proteomic analyses were performed on EVISF cargo. EVISF were 40-150 nm in size and expressed CD63, CD9, and CD81. Using a model of cerebral amyloidosis (e.g. APPswe,PSEN1dE9 mice), we found protein concentration increased but protein diversity decreased with A deposition. Genotype, age, and Aβ deposition modulated proteostasis- and immunometabolic-related pathways. Changes in the microglial EVISF proteome were sexually dimorphic and associated with a differential response of plaque associated microglia. We found that female APP/PS1 mice have more amyloid plaques, less plaque associated microglia, and a less robust- and diverse- EVISF microglial proteome. Thus, in vivo microdialysis is a novel technique for collecting EVISF and offers a unique opportunity to explore the role of EVs in AD.

Extracellular Vesicles Slow Down Aβ(1–42) Aggregation by Interfering with the Amyloid Fibril Elongation Step

“…ThT fluorescence was used to monitor the kinetics of Aβ(1–42) amyloid fibril formation in the absence and presence of the SH-SY5Y- and HEK293-T-derived EVs. We used size exclusion chromatography-purified monomeric peptide solutions (2 μM) as the starting material to obtain reproducible kinetics. , Figure a,b shows the kinetic curves for Aβ(1–42) amyloid fibril formation across a range of different EV particle concentrations that are in line with reported abundances of EVs in cerebrospinal or interstitial brain fluid . The data show that both EV types slow the aggregation rate of Aβ(1–42) in a concentration-dependent manner.…”

“…12,43 Figure 2a,b shows the kinetic curves for Aβ(1−42) amyloid fibril formation across a range of different EV particle concentrations that are in line with reported abundances of EVs in cerebrospinal 44 or interstitial brain fluid. 45 The data show that both EV types slow the aggregation rate of Aβ(1−42) in a concentration-dependent manner. This was accompanied by a decrease in end-point ThT fluorescence (taken as the mean ThT signal over the final 3 h of the plateau phase for each sample, especially for the HEK293-T-derived EVs Figure 2c).…”

“…However, similar extremes in substoichiometric inhibition have been observed with linoleic acid and the amyloid-forming peptide NACore, and the effect is ascribed to the co-assembly of fatty acid aggregates and the amyloid species at the early stages of aggregation. 71 Moreover, whereas the EV concentrations used in this work were within the physiological range, 44,45 total Aβ(1−42) concentrations in brain fluids are generally lower 72 even though significant concentration variation likely exists. 73 This suggests that Aβ(1−42):EV ratios in the brain may be lower than those assessed here and that elongation inhibitory mechanisms could potentially be even more effective in vivo.…”

Extracellular vesicles from GABAergic but not glutamatergic neurons protect against neurological dysfunction following cranial irradiation

“…This may promote beneficial GABAergic signaling to normalize the excitatory/inhibitory balance in the irradiated brain. The use of stem cell derived EV to treat various neurological orders has been reviewed 9 – 11 , and a recent study isolating EV from hippocampal interstitial fluid found the microglial EV proteome sensitive to sex, age, and Alzheimer’s pathology, opening the door to using brain derived EV as biomarkers to diagnose and stage neurodegenerative disease progression 39 . Further, cargo analysis and omics level studies will be required to provide more precise mechanistic context for our findings.…”

Efficient enzyme-free isolation of brain-derived extracellular vesicles

“…Given the growing interest in and the relevance of isolating EVs from complex tissues such as the brain, the number of protocols is increasing (Vella et al, 2017;Hurwitz et al, 2018;Brenna et al, 2020;Huang et al, 2020;Su et al, 2021;D'Acunzo et al, 2021;Gomes et al, 2023;Huang et al, 2023;Pait et al, 2024). Brain EVs isolation is challenging since BDEVs need to be released from the extracellular matrix (ECM), which consists of an intricate meshwork of (glyco-)proteins, large proteoglycans, and other molecules.…”