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bioRxiv2021

Milk Exosomes 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

BackgroundHuman milk contains large amounts of exosomes (MEs) and their regulatory microRNA cargos, whereas infant formulas contain only trace amounts of MEs and microRNAs. Breastfeeding has been implicated in optimal brain development but experimental evidence linking ME intake with brain development is limited. ObjectivesWe assessed the transport of MEs across the blood-brain barrier (BBB) and ME accumulation in distinct regions of the brain in brain endothelial cells and suckling mice. We further assessed BME-dependent gene expression profiles and effects on the dendritic complexity of hippocampal granule cells and phenotypes of BME depletion in neonate, juvenile and adult mice. MethodsThe transfer of MEs across the BBB was assessed by using bovine MEs labeled with FM4-64 or loaded with IRDye-labeled miR-34a in murine brain endothelial bEnd.3 cell monolayers and dual chamber systems, and in wild-type newborn pups fostered to exosome and cargo tracking (ECT) dams that express MEs endogenously labeled with a CD63-eGFP fusion protein for subsequent analysis by serial two-photon tomography and staining with anti-eGFP antibodies. Effects of MEs on gene expression and dendritic architecture of granule cells was analyzed in hippocampi from juvenile mice fed exosome and RNA-depleted (ERD) and exosome 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. ResultsbEnd.3 cells internalized MEs by using a saturable transport mechanism and secreted miR-34a across the basal membrane. MEs 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 male mice fed ERD and ERS diets; high-confidence gene networks included pathways implicated in axon guidance and calcium signaling. Only one gene was differentially expressed in females fed the experimental diets. 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 (P < 0.01), 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 (P < 0.01). ConclusionsMEs cross the BBB and contribute toward optimal neuronal development, spatial learning and memory, and resistance to kainic acid-induced seizures in mice.

neuroscience↗

bioRxiv2018

Dietary Bovine Milk Exosomes Elicit Changes in Microbial Communities in C57BL/6 Mice

Exosomes and exosome-like vesicles participate in cell-to-cell communication in animals, plant and bacteria. Dietary exosomes in bovine milk are bioavailable in non-bovine species, but a fraction of milk exosomes reaches the large intestine. We hypothesized that milk exosomes alter the composition of the gut microbiome in mice. C57BL/6 mice were fed AIN-93G diets, defined by their content of bovine milk exosomes and RNA cargos: exosome/RNA depleted (ERD) versus exosome/RNA-sufficient (ERS) diets. Feeding was initiated at age three weeks and cecum content was collected at ages 7, 15 and 47 weeks. Microbial communities were identified by 16S rRNA gene sequencing. The dietary intake of exosomes and age had significant effects on the microbial communities in the cecum. At the phylum level, the abundance of Verrucomicrobia was greater in mice fed ERD compared to ERS, and the abundance of both Firmicutes and Tenericutes was smaller in mice fed ERD compared to ERS at age 47 weeks. At the family level, the abundance of Anaeroplasmataceae was greater in mice fed ERD compared to ERS, and the abundance of Bifidobacteriaceae, Lachnospiraceae, and Dehalobacteriaceae was significantly greater in mice fed ERS than mice fed ERD at age 15 weeks. Exosome feeding significantly altered the abundance of 52 operational taxonomic units; diet effects were particularly strong in the Lachnospiraceae, Ruminococcaceae and the Verrucomicrobiaceae families. We conclude that exosomes in bovine milk alter microbial communities in non-bovine species, suggesting that exosomes and their cargos participate in the crosstalk between bacterial and animal kingdoms.\n\nIMPORTANCEVirtually all living cells, including bacteria communicate through exosomes, which can be found in all body fluids. Exosomes and the RNA cargos have been implicated in all aspects of health and disease, e.g., metastasis of cancer, neuronal signaling and embryonic development. Previously, we reported that exosomes and their microRNA cargos are not solely derived from endogenous synthesis, but may also be obtained from dietary sources such as bovine milk in non-bovine mammals. Here, we report for the first time that bovine milk exosomes communicate with the intestinal microbiome and alters microbial communities in mice. This is the first report suggesting that the gut microbiome facilitates the signaling by dietary exosomes across kingdoms: animal (cow) [->] bacteria [->] animal (mouse).

microbiology↗