From understanding to action: Exploring molecular connections of Down syndrome to Alzheimer's disease for targeted therapeutic approach.

Down syndrome (DS) is caused by a third copy of chromosome 21. Alzheimer's disease (AD) is a neurodegenerative condition characterized by the deposition of amyloid-beta (Aß) plaques and neurofibrillary tangles in the brain. Both disorders have elevated Aß, tau, dysregulated immune response, and inflammation. In people with DS, Hsa21 genes like APP and DYRK1A are overexpressed, causing an accumulation of amyloid and neurofibrillary tangles, and potentially contributing to an increased risk of AD. As a result, people with DS are a key demographic for research into AD therapeutics and prevention. The molecular links between DS and AD shed insights into the underlying causes of both diseases and highlight potential therapeutic targets. Also, using biomarkers for early diagnosis and treatment monitoring is an active area of research, and genetic screening for high-risk individuals may enable earlier intervention. Finally, the fundamental mechanistic parallels between DS and AD emphasize the necessity for continued research into effective treatments and prevention measures for DS patients at risk for AD. Genetic screening with customized therapy approaches may help the DS population in current clinical studies and future biomarkers.

The absorption of bovine milk small extracellular vesicles largely depends on galectin 3 and galactose ligands in human intestinal cells and C57BL/6J mice.

Small extracellular vesicles in milk (sMEVs) have attracted attention in drug delivery and as bioactive food compounds. Previous studies implicate galactose residues on the sMEV surface in sMEV transport across intestinal and endothelial barriers in humans, but details of glycoprotein-dependent transport are unknown. We used a combination of cell biology and genetics protocols to identify glycoproteins on the sMEV surface that facilitate sMEV absorption. We identified 256 proteins on the bovine sMEVs surface by using LC-MS/MS, and bioinformatics analysis suggested that 42, 13, and 13 surface proteins were N-, O-, and 13 C-glycosylated, respectively. Lectin blots confirmed the presence of mannose, galactose, N-acetyl galactose, fucose, and neuraminate. When surface proteins were removed by various treatment with various proteases, sMEV uptake decreased by up to 58% and 67% in FHs-74 Int and Caco-2 cells, respectively, compared with controls (P < 0.05). When glycans were removed by treatment with various glycosidases, sMEV uptake decreased by up to 54% and 74% in FHs-74 Int and Caco-2 cells, respectively (P < 0.05). When galactose and N-acetyl galactosamine residues were blocked with agglutinins, sMEV uptake decreased by more than 50% in FHs-74 Int cells (P < 0.05). When bovine sMEVs were administered to Galectin-3 knockout mice by oral gavage, hepatic sMEV accumulation decreased by 56% compared with wild-type mice (P < 0.05), consistent with a role of ß-galactoside glycan structures in the absorption of sMEVs. We conclude that sMEVs are decorated with glycoproteins, and Galectin-3 and its galactose ligands are particularly important for sMEV absorption.NEW & NOTEWORTHY This is the first paper to assess the role of unique glycans and their Galectin-3 receptor in the transport and distribution of small extracellular vesicles ("exosomes") from milk in mammals. The research assessed milk exosome transport and distribution by using multiple approaches and platforms including cell cultures, various exosome labels, knockout and mutant mice, enzymatic removal of surface proteins and glycans, and lectin blocking of glycans.

Plant-food-derived Bioactives in Managing Hypertension: From Current Findings to Upcoming Effective Pharmacotherapies.

A sedentary lifestyle has evoked a high risk of cardiovascular (CV) disease, diabetes, and obesity, all of them with high morbimortality rates and with a common denominator, hypertension. Numerous pharmacological drugs have been used for the treatment of hypertension. However, the side effects associated with the use of existing pharmacological therapies have triggered a demand for plant-based medications. In this connection, the aim of this review was to provide an in-depth analysis of the use of plant-derived bioactives for the effective management of hypertension. Phytoconstituents from leaves, bark, stem, roots, seeds, and fruits of medicinal plants grown in our different regions of the globe have been highly searched. Among them, polyphenols (e.g., flavonoids as quercetin, anthocyanins as cyanidin, tannins as ellagic acid, stilbenes as resveratrol, lignans as honokiol and others as hydroxytyrosol or curcumin), organosulfur compounds (e.g. s-allyl cysteine and allicin), fatty acids (e.g. α-lipoic acid, DHA and oleic acid), alkaloids (e.g. berberine or tetrandrine) and some terpenes have been intensively investigated for the management of hypertension, with effective ability being stated in controlling high blood pressure and related health problems both in vivo and in vitro studies. Some of the activities presented by these bioactive compounds are reducing oxidative stress, renin-angiotensin system control, SIRT1 activation, regulating platelet aggregation and COX activity, anti-atherogenic effects, anti-inflammatory properties, vasorelaxation and other results that translate into the prevention or control of hypertension. The knowledge of these bioactive compounds is important in developing countries where traditional medicine is the majority, but it can also give rise to new approaches in hypertension therapy.

Evaluation of blood-based, extracellular vesicles as biomarkers for aging-related TDP-43 pathology.

Introduction: Limbic predominant age related TDP-43 encephalopathy neuropathological change (LATE-NC) is a recently characterized brain disease that mimics Alzheimer's disease (AD) clinically. To date, LATE-NC is difficult to diagnose antemortem using clinical information or biomarkers. Recent studies suggest concentrations of extracellular vesicle (EVs) protein cargo derived from neuronal and glial cells may serve as useful diagnostic biomarkers for AD and other neurodegenerative diseases. Methods: TDP-43 was evaluated in neuronal (NDEVs), astrocyte (ADEVs), and microglial derived extracellular vesicles (MDEVs). EV preparations were isolated from the plasma of research subjects with autopsy-confirmed diagnoses, including many with LATE (n = 22). Quantified TDP-43 concentrations were compared to the cohort that included healthy controls, mild cognitively impairment (MCI), and AD dementia with diagnoses other than LATE-NC (n = 42). Results: TDP-43 was significantly elevated in plasma ADEVs derived from autopsy confirmed LATE-NC subjects, with or without comorbid AD pathology. Measurable levels of TDP-43 were also detected in EV-depleted plasma; however, TDP-43 levels were not significantly different between persons with and without eventual autopsy confirmed LATE-NC. No correlation was observed between EV TDP-43 levels with cognition-based variables, sex, and APOE carrier status. Discussion: Blood-based EVs, specifically measuring TDP-43 accumulation in ADEVs, may serve as a potential diagnostic tool to rapidly identify subjects who are currently living with LATE-NC.

Quantitation of Exosomes and Their MicroRNA Cargos in Frozen Human Milk.

We assessed feasibility of analyzing exosomes and microRNA cargos in frozen human milk as a pre-requisite for epidemiological studies of milk exosomes. We collected milk from five mother-preterm infant dyads at 3 time points during postnatal hospital care for storage at -80°C. We purified exosomes by ultracentrifugation, probed marker proteins using immunoblots, assessed size and counts with a nanoparticle tracker, and quantified three microRNAs with quantitative PCR. Positive exosome marker proteins were detectable; ß-casein was the only detectable contaminant. Exosome count and size trended to decrease from early to late samples (count: 2.3×109 ± 3.8×109 to 5.6×108 ± 9.7×108 exosomes/mL; size: 117 ± 25 to 92 ± 16 nm). Two microRNAs were detectable in early samples only; cycle threshold values equaled 28.7 ± 0.7 for miR-30d-5p and miR-125a-5p; miR-423-5p was not detectable. We conclude that the analysis of exosomes and quantification of microRNAs is feasible in human milk previously stored at -80°C.

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.

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.

Ultrasonication of Milk Decreases the Content of Exosomes and MicroRNAs in an Exosome-Defined Rodent Diet.

BACKGROUND: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids, and microRNAs. Consumption of an exosome- and RNA-depleted (ERD) diet elicited phenotypes compared with controls fed an exosome- and RNA-sufficient (ERS) diet in mice. All other ingredients were identical in the diets. ERD and ERS diets were prepared by substituting ultrasonicated and nonultrasonicated milk, respectively, for casein in the AIN-93G formulation. OBJECTIVES: The objective of this study was to assess the effect of ultrasonication of milk on exosome content and bioavailability, and cargo content. METHODS: Bovine milk was ultrasonicated and exosomes were isolated by ultracentrifugation [ultrasonicated exosomes (USEs)]; controls were not ultrasonicated [nonultrasonicated exosomes (NSEs)]. Exosome count, size, and morphology were assessed using a nanoparticle tracker and electron microscopy. RNAs, lipids, and proteins were analyzed by RNA sequencing and MS. Intestinal transport, bioavailability, and distribution were measured by using fluorophore-labeled USEs and NSEs in Caco-2 cells, FHs 74 Int cells, and C57BL/6J mice (n = 3; age: 6-8 wk). RESULTS: The exosome count was 76% ± 22% lower in USEs than in NSEs (P < 0.05). Ultrasonication caused a degradation of ≤100% of microRNAs. USEs and NSEs contained 145 and 332 unique lipid signatures, respectively (P < 0.05). We detected a total of 525 and 484 proteins in USEs and NSEs, respectively. The uptake of USEs decreased by 46% ± 30% and 40% ± 27% compared with NSEs in Caco-2 and FHs 74 Int cells, respectively (P < 0.05). The hepatic accumulation of USEs was 48% ± 28% lower than the accumulation of NSEs in mice (P < 0.05). CONCLUSIONS: Ultrasonication of milk depletes bioavailable BMEs in studies of Caco-2 cells, FHs 74 Int cells, and C57BL/6J mice and causes a near-complete degradation of microRNA cargos.

Isolation of extracellular vesicles from byproducts of cheesemaking by tangential flow filtration yields heterogeneous fractions of nanoparticles.

Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as bioactive food compounds and for their use in drug delivery. The utility of small EV in milk (sMEV) as an animal feed additive and in drug delivery would be enhanced by cost-effective large-scale protocols for the enrichment of sMEV from byproducts in dairy plants. Here, we tested the hypothesis that sMEV may be enriched from byproducts of cheesemaking by tangential flow filtration (EV-FF) and that the sMEV have properties similar to sMEV prepared by ultracentrifugation (sMEV-UC). Three fractions of EV were purified from the whey fraction of cottage cheese making by using EV-FF that passed through a membrane with a 50-kDa cutoff (50 penetrate; 50P), and subfractions of 50P that were retained (100 retentate; 100R) or passed through (100 penetrate; 100P) a membrane with a 100-kDa cutoff; sMEV-UC controls were prepared by serial ultracentrifugation. The abundance of sMEV (<200 nm) was less than 0.3% in EV-FF compared with sMEV-UC (1012/mL of milk). Despite the low EV count, the protein content (mg/mL) of 100R (63 ± 0.02; ± standard deviation) was higher than that of 50P (0.75 ± 0.10), 100P (0.65 ± 0.40), and sMEV-UC (27 ± 0.02). There were 17, 14, 35, and 75 distinct proteins detected by nontargeted mass spectrometry analysis in 50P, 100R, 100P, and sMEV-UC, respectively. Exosome markers CD9, CD63, CD81, HSP-70, PDCD6IP, and TSG101 were detected in control sMEV-UC but not in EV-FF by using targeted mass spectrometry and immunoblot analyses. Negative exosome markers, APOB, ß-integrin, and histone H3 were below the limit of detection in EV-FF and control sMEV-UC analyzed by immunoblotting. The abundance of the major milk fat globule protein butyrophilin showed the following pattern: 100R ≫ 100P = 50P > sMEV-UC. More than 100 mature microRNA were detected in sMEV-UC by using sequencing analysis, compared with 36 to 60 microRNA in EV-FF. Only 100R and sMEV-UC yielded mRNA in quantities and qualities sufficient for sequencing analysis; an average of 276,000 and 838,000 reads were mapped to approximately 14,600 and 18,500 genes in 100R and sMEV-UC, respectively. In principal component analysis, microRNA, mRNA, and protein in EV-FF preparations clustered separately from control sMEV-UC. We conclude that under the conditions used here, flow filtration yields a heterogeneous population of milk EV.

Milk-Derived Exosomes and Metabolic Regulation.

Exosomes are natural nanoparticles that play an important role in cell-to-cell communication. Communication is achieved through the transfer of cargos, such as microRNAs, from donor to recipient cells and binding of exosomes to cell surface receptors. Exosomes and their cargos are also obtained from dietary sources, such as milk. Exosome and cell glycoproteins are crucial for intestinal uptake. A large fraction of milk exosomes accumulates in the brain, whereas the tissue distribution of microRNA cargos varies among distinct species of microRNA. The fraction of milk exosomes that escapes absorption elicits changes in microbial communities in the gut. Dietary depletion of exosomes and their cargos causes a loss of circulating microRNAs and elicits phenotypes such as loss of cognitive performance, increase in purine metabolites, loss of fecundity, and changes in the immune response. Milk exosomes meet the definition of bioactive food compounds.

Biological Activities of Extracellular Vesicles and Their Cargos from Bovine and Human Milk in Humans and Implications for Infants.

Extracellular vesicles (EVs) in milk harbor a variety of compounds, including lipids, proteins, noncoding RNAs, and mRNAs. Among the various classes of EVs, exosomes are of particular interest, because cargo sorting in exosomes is a regulated, nonrandom process and exosomes play essential roles in cell-to-cell communication. Encapsulation in exosomes confers protection against enzymatic and nonenzymatic degradation of cargos and provides a pathway for cellular uptake of cargos by endocytosis of exosomes. Compelling evidence suggests that exosomes in bovine milk are transported by intestinal cells, vascular endothelial cells, and macrophages in human and rodent cell cultures, and bovine-milk exosomes are delivered to peripheral tissues in mice. Evidence also suggests that cargos in bovine-milk exosomes, in particular RNAs, are delivered to circulating immune cells in humans. Some microRNAs and mRNAs in bovine-milk exosomes may regulate the expression of human genes and be translated into protein, respectively. Some exosome cargos are quantitatively minor in the diet compared with endogenous synthesis. However, noncanonical pathways have been identified through which low concentrations of dietary microRNAs may alter gene expression, such as the accumulation of exosomes in the immune cell microenvironment and the binding of microRNAs to Toll-like receptors. Phenotypes observed in infant-feeding studies include higher Mental Developmental Index, Psychomotor Development Index, and Preschool Language Scale-3 scores in breastfed infants than in those fed various formulas. In mice, supplementation with plant-derived MIR-2911 improved the antiviral response compared with controls. Porcine-milk exosomes promote the proliferation of intestinal cells in mice. This article discusses the above-mentioned advances in research concerning milk exosomes and their cargos in human nutrition. Implications for infant nutrition are emphasized, where permitted, but data in infants are limited.

Human vascular endothelial cells transport foreign exosomes from cow's milk by endocytosis.

Encapsulation of microRNAs in exosomes confers protection against degradation and a vehicle for shuttling of microRNAs between cells and tissues, and cellular uptake by endocytosis. Exosomes can be found in foods including milk. Humans absorb cow's milk exosomes and deliver the microRNA cargo to peripheral tissues, consistent with gene regulation by dietary nucleic acids across species boundaries. Here, we tested the hypothesis that human vascular endothelial cells transport milk exosomes by endocytosis, constituting a step crucial for the delivery of dietary exosomes and their cargo to peripheral tissues. We tested this hypothesis by using human umbilical vein endothelial cells and fluorophore-labeled exosomes isolated from cow's milk. Exosome uptake followed Michaelis-Menten kinetics (Vmax = 0.057 ± 0.004 ng exosome protein × 40,000 cells/h; Km = 17.97 ± 3.84 µg exosomal protein/200 µl media) and decreased by 80% when the incubation temperature was lowered from 37°C to 4°C. When exosome surface proteins were removed by treatment with proteinase K, or transport was measured in the presence of the carbohydrate competitor d-galactose or measured in the presence of excess unlabeled exosomes, transport rates decreased by 45% to 80% compared with controls. Treatment with an inhibitor of endocytosis, cytochalasin D, caused a 50% decrease in transport. When fluorophore-labeled exosomes were administered retro-orbitally, exosomes accumulated in liver, spleen, and lungs in mice. We conclude that human vascular endothelial cells transport bovine exosomes by endocytosis and propose that this is an important step in the delivery of dietary exosomes and their cargo to peripheral tissues.