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