Small extracellular vesicles in plasma reveal molecular effects of modified Mediterranean-ketogenic diet in participants with mild cognitive impairment

Abstract

Extracellular vesicles (EV) have emerged as a less-invasive nano-tool for discovering biomarkers of Alzheimer’s disease and related dementia. Here, we analyzed different neuron-enriched EV from plasma to predict response and molecular mechanisms of ketogenic diet’s efficacy in mild cognitive impairment participants. The study was a randomized crossover design in which cognitively normal and mild cognitive impairment participants consumed a modified Mediterranean-ketogenic diet (MMKD) or American Heart Association diet (AHAD) for six weeks, followed by other diet after washout. L1 cell adhesion molecule (L1CAM), synaptophysin, and neural cell adhesion molecule (NCAM) surface markers were used to enrich for neuron-secreted small EV (sEVL1CAM, sEVSYP, and sEVNCAM). For the first time, we have presented multiple evidences, including immunogold labeling/Transmission electron microscopy, CD63 (clusters of differentiation 63)-ELISA based assay, confocal microscopy fluorescent images, and flow cytometry data confirming the presence of L1CAM on the surface of sEVL1CAM, validating purity and relative abundance of sEVL1CAM in the plasma. Cargo analysis of sEVL1CAM showed that MMKD intervention reduces amyloid beta 1-42 (50.3%, p = 0.011), p181-tau (34.9%, p = 0.033) and neurofilament light (54.2%, p = 0.020) in mild cognitive impairment participants. Moreover, sEVL1CAM showed better sensitivity compared to CSF in analyzing increased glutamate (6 folds, p < 0.0001) from mild cognitive impairment participants following MMKD intervention. sEVL1CAM characterization also suggested that MMKD differentially targets the expression of various glutamate receptors - glutamate receptor ionotropic NMDA1 (GRIN1), glutamate receptor ionotropic NMDA2A (GRIN2A), glutamate receptor ionotropic NMDA2B (GRIN2B) and glutamate receptor ionotropic AMPA type subunit 1 (GRIA1). Importantly, these sEVL1CAM measures strongly correlated with corresponding clinical CSF biomarkers (neurogranin, amyloid beta 1-42, neurofilament light, and tau). Furthermore, sEVL1CAM were loaded with less advanced-glycation endproducts and exhibited anti-inflammatory activity following MMKD intervention. Most importantly, the expression of monocarboxylate transporter 2 on the surface of sEVL1CAM predicted the amyloid beta 1-42 response to MMKD intervention (Area under the curve = 0.87, p = 0.0044) and offered a novel screening tool to identify participants responsive to this dietary intervention. Finally, sEVL1CAM, sEVSYP, and sEVNCAM showed significantly high concordance in analyzing amyloid beta 1-42 (Pearson correlation coefficient ≥ 0.63, p < 0.01) and neurofilament light (Pearson correlation coefficient ≥ 0.49, p < 0.05). Together, sEV in plasma offers promise in assessing the efficacy of dietary/therapeutic intervention against mild cognitive impairment/Alzheimer’s disease.

A Liquid Biopsy-Based Approach to Isolate and Characterize Adipose Tissue-Derived Extracellular Vesicles from Blood

“…sEV AT in the Blood Reflect Molecular Changes in the Adipose Tissue. Since sEVs are emerging as the "snapshot" of the parent cells and have been implicated in identifying the pathophysiological and molecular status of the host cells, 21,23 we assessed whether the most significantly altered pathway (amino acid metabolism) identified between sEV AT-Lean and sEV AT-DIO could reflect similar changes in mice AT. We extracted RNA from VAT of the lean and DIO mice and explored the regulation of the amino acid metabolism pathway using a PCR array.…”

“…Flow cytometry analysis was performed to characterize the isolated sEV and to confirm their purity from AT and plasma. 23 sEV were labeled with membrane-labeling dye CellBrite 488 or 647 (Biotium, California, USA) with and without the fluorescently labeled antibodies. sEV without dye were used as a control to set the gate for positively labeled sEV.…”

“…These methods were performed as reported recently. , Briefly, 200 mesh copper grids (with carbon-coated Formvar film) were activated with 100% ethanol for 20 min. Thirty μL sEV samples were fixed with an equal volume of 4% paraformaldehyde for 10 min at RT and then incubated with grids for 1 h at RT.…”

“…In the past decade, numerous studies have shown the feasibility of isolating cell/tissue-specific sEV from the biofluids, exploiting the specific markers expressed on the surface using immunoprecipitation methods. Neuron-derived sEV isolated from plasma have been shown to be valuable biomarkers for various neurodegenerative diseases and assessment of responses to treatment. − In recent studies, we have reported the usefulness of brain cells derived sEV from the plasma toward accessing the expression of various neurodegenerative biomarkers , as well as determining the molecular effects of specific dietary intervention against mild-cognitive impairment . Only limited attempts have been made to isolate and characterize adipose tissue-derived sEV (sEV AT ) from plasma.…”

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

“…Flow cytometry: Flow cytometry analysis was performed to evaluate the percentage of typical EV tetraspanin markers following methods reported recently (53). TE were labeled with membrane labeling dye CellBrite 488 (Biotium, California, USA) with and without the CD63-APC and CD81-PE (both from BioLegend, CA, USA) antibodies.…”