Small Extracellular Vesicles in Milk Cross the Blood-Brain Barrier in Murine Cerebral Cortex Endothelial Cells and Promote Dendritic Complexity in the Hippocampus and Brain Function in C57BL/6J Mice

Abstract

Human milk contains large amounts of small extracellular vesicles (sEVs) and their microRNA cargos, whereas infant formulas contain only trace amounts of sEVs and microRNAs. We assessed the transport of sEVs across the blood-brain barrier (BBB) and sEV accumulation in distinct regions of the brain in brain endothelial cells and suckling mice. We further assessed sEV-dependent gene expression profiles and effects on the dendritic complexity of hippocampal granule cells and phenotypes of EV depletion in neonate, juvenile and adult mice. The transfer of sEVs across the BBB was assessed by using fluorophore-labeled bovine sEVs in brain endothelial bEnd.3 monolayers and dual chamber systems, and in wild-type newborn pups fostered to sEV and cargo tracking (ECT) dams that express sEVs labeled with a CD63-eGFP fusion protein for subsequent analysis by serial two-photon tomography and staining with anti-eGFP antibodies. Effects of EVs on gene expression and dendritic architecture of granule cells was analyzed in hippocampi from juvenile mice fed sEV and RNA-depleted (ERD) and sEV and RNA-sufficient (ERS) diets by using RNA-sequencing analysis and Golgi-Cox staining followed by integrated neuronal tracing and morphological analysis of neuronal dendrites, respectively. Spatial learning and severity of kainic acid-induced seizures were assessed in mice fed ERD and ERS diets. bEnd.3 cells internalized sEVs by using a saturable transport mechanism and secreted miR-34a across the basal membrane. sEVs penetrated the entire brain in fostering experiments; major regions of accumulation included the hippocampus, cortex and cerebellum. Two hundred ninety-five genes were differentially expressed in hippocampi from mice fed ERD and ERS diets; high-confidence gene networks included pathways implicated in axon guidance and calcium signaling. Juvenile pups fed the ERD diet had reduced dendritic complexity of dentate granule cells in the hippocampus, scored nine-fold lower in the Barnes maze test of spatial learning and memory, and the severity of seizures was 5-fold higher following kainic acid administration in adult mice fed the ERD diet compared to mice fed the ERS diet. We conclude that sEVs cross the BBB and contribute toward optimal neuronal development, spatial learning and memory, and resistance to kainic acid-induced seizures in mice.

Human Milk Extracellular Vesicles: A Biological System with Clinical Implications

“…After the infants ingests the milk, their intestinal mucosa may be a primary target, but appreciable amounts of hMEVs are also absorbed into the infant circulation. The membrane contains adhesion molecules that can guide the vesicle to specific types of cells, both in the gut lining, and the distant organ systems after some of the hMEVs pass into the blood circulation [ 83 , 86 , 147 , 189 , 190 , 191 ]. The cargo of the hMEVs include both biologically active proteins and miRNAs that are capable of programing the recipient cells to induce physiological responses in breastfed infants.…”

Emerging role of exosomes in vascular diseases

“…243) used the thiol-maleimide crosslinking reaction to generate E3 aptamer-PEG-cholesterol adducts to modify the exosome surface. In addition, labeling could be achieved by generating CD63EGFP fusion proteins, such as by cotransfecting stable cell lines using the PCL6-CD63EGFP plasmid, which resulted in CD63-EGFP fusion protein-tagged exosomes, were then analyzed by serial two-photon tomography (STP) and anti-EGFP antibody staining (244,245). Among the bioluminescent markers, exosomal surface-expressed reporter proteins such as Gaussia luciferase, firefly luciferase or kidneyworm luciferase produced bioluminescence when their substrates, such as cecropin (CTZ), were added (246).…”

Milk exosomal microRNAs: friend or foe?—a narrative review

“…After oral gavage of bovine MEX to mice, MEX accumulated in the brain (32). In mice, MEX cross the blood-brain-barrier and promote dendritic complexity in the hippocampus, whereas dietary depletion of bovine MEX impaired sensorimotor gating and cognitive performance (147). In early postnatal life, developmental processes are critical for establishing proper neuronal connectivity.…”

“…Adipocytes of obese mice secreted MALAT1-containing exosomes, which increased appetite and weight when administered to lean mice (329). It is thus conceivable that MEX, which reach the brain (32,147), may also have an impact on hypothalamic appetite and satiety control. One of the gut hormones sending satiety signals to the hypothalamus is cholecystokinin (CCK), which is secreted from intestinal mucosa cells when the duodenum is filled with food (330).…”

“…Notably, α-synuclein promotes IAPP fibril formation in vitro and enhanced β-cell amyloid formation in vivo, whereas β-cell amyloid formation was reduced in mice on a SNCA −/− background (465). As bovine MEX are able to cross the blood-brain barrier and reach the brain (32,147), MEX miR-148a may stimulate neuronal α-synuclein in the substantia nigra, whereas MEX miR-21 may suppress lysosome-associated membrane protein type 2A (LAMP2A) (466,467), a critical effector protein of chaperone-mediated autophagy clearing neurotoxic α-synuclein (349).…”