Axon-derived PACSIN1 binds to the Schwann cell survival receptor, LRP1, and transactivates TrkC to promote gliatrophic activities.

Schwann cells (SCs) undergo phenotypic transformation and then orchestrate nerve repair following PNS injury. The ligands and receptors that activate and sustain SC transformation remain incompletely understood. Proteins released by injured axons represent important candidates for activating the SC Repair Program. The low-density lipoprotein receptor-related protein-1 (LRP1) is acutely up-regulated in SCs in response to injury, activating c-Jun, and promoting SC survival. To identify novel LRP1 ligands released in PNS injury, we applied a discovery-based approach in which extracellular proteins in the injured nerve were captured using Fc-fusion proteins containing the ligand-binding motifs of LRP1 (CCR2 and CCR4). An intracellular neuron-specific protein, Protein Kinase C and Casein Kinase Substrate in Neurons (PACSIN1) was identified and validated as an LRP1 ligand. Recombinant PACSIN1 activated c-Jun and ERK1/2 in cultured SCs. Silencing Lrp1 or inhibiting the LRP1 cell-signaling co-receptor, the NMDA-R, blocked the effects of PACSIN1 on c-Jun and ERK1/2 phosphorylation. Intraneural injection of PACSIN1 into crush-injured sciatic nerves activated c-Jun in wild-type mice, but not in mice in which Lrp1 is conditionally deleted in SCs. Transcriptome profiling of SCs revealed that PACSIN1 mediates gene expression events consistent with transformation to the repair phenotype. PACSIN1 promoted SC migration and viability following the TNFα challenge. When Src family kinases were pharmacologically inhibited or the receptor tyrosine kinase, TrkC, was genetically silenced or pharmacologically inhibited, PACSIN1 failed to induce cell signaling and prevent SC death. Collectively, these studies demonstrate that PACSIN1 is a novel axon-derived LRP1 ligand that activates SC repair signaling by transactivating TrkC.

Tumor necrosis factor receptor-1 is selectively sequestered into Schwann cell extracellular vesicles where it functions as a TNFα decoy.

Schwann cells (SCs) are known to produce extracellular vesicles (EV) that participate in cell-cell communication by transferring cargo to target cells, including mRNAs, microRNAs, and biologically active proteins. Herein, we report a novel mechanism whereby SC EVs may regulate PNS physiology, especially in injury, by controlling the activity of TNFα. SCs actively sequester tumor necrosis factor receptor-1 (TNFR1) into EVs at high density, accounting for about 2% of the total protein in SC EVs (~1000 copies TNFR1/EV). Although TNFR2 was robustly expressed by SCs in culture, TNFR2 was excluded from SC EVs. SC EV TNFR1 bound TNFα, decreasing the concentration of free TNFα available to bind to cells and thus served as a TNFα decoy. SC EV TNFR1 significantly inhibited TNFα-induced p38 MAPK phosphorylation in cultured SCs. When TNFR1 was proteolytically removed from SC EVs using tumor necrosis factor-α converting enzyme (TACE) or neutralized with antibody, the ability of TNFα to activate p38 MAPK in the presence of these EVs was restored. As further evidence of its decoy activity, SC EV TNFR1 modified TNFα activities in vitro including: (1) regulation of expression of other cytokines; (2) effects on SC morphology; and (3) effects on SC viability. SC EVs also modified the effects of TNFα on sciatic nerve morphology and neuropathic pain-related behavior in vivo. By sequestering TNFR1 in EVs, SCs may buffer against the potentially toxic effects of TNFα. SC EVs provide a novel mechanism for the spatial and temporal regulation of neuro-inflammation.

Milk exosomes and miRNA cross the placenta and promote embryo survival in mice.

Exosomes facilitate cell-to-cell communication by transferring regulatory molecules such as miRNA from donor to recipient cells, for example, miR-21-5p and miR-30d promote placentation. Exosomes and their miRNA cargos are not exclusively obtained from endogenous synthesis but may also be absorbed from dietary sources, such as milk. This study assessed the effects of milk exosomes and miRNA cargos on embryo development and fertility in C57BL/6 mice. Fluorophore-labeled milk exosomes, miR-21-5p and miR-30d accumulated in murine placenta and embryos following oral gavage. Seventeen mRNAs, miR-21-5p and miR-30d were differentially expressed in placentas of pregnant mice fed a milk exosome and RNA-depleted (ERD) diet or a milk exosome and RNA-sufficient (ERS) diet. Eight of these mRNAs encode proteins implicated in the synthesis of extracellular matrix components, cell adhesion and migration. Changes in mRNA expression were associated with corresponding changes in protein expression, for example, collagen type I. The size of litters born to dams fed ERD was 25-50% smaller than those born to ERS controls. This study implicates dietary exosomes and miRNA in placenta development and embryo survival.

Dietary bovine milk exosomes elicit changes in bacterial 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 nonbovine 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 3 wk, and cecum content was collected at ages 7, 15, and 47 wk. Microbial communities were identified by 16S rRNA gene sequencing. Milk exosomes altered bacterial communities in the murine cecum. The abundance of three phyla, seven families, and 52 operational taxonomic units (OTUs) was different in the ceca from mice fed ERD and ERS (P < 0.05). For example, at the phylum level, Tenericutes had more than threefold abundance in ERS mice at ages 15 and 47 wk compared with ERD mice (P < 0.05). At the family level, Verrucomicrobiaceae were much less abundant in ERS mice compared with ERD mice age 47 wk (P < 0.05). At the OTU level, four OTUs from the family of Lachnospiraceae were more than two times more abundant in ERS mice compared with ERD at age 7 and 47 wk (P < 0.05). We conclude that exosomes in bovine milk alter microbial communities in nonbovine species, suggesting that exosomes and their cargos participate in the crosstalk between bacterial and animal kingdoms.NEW & NOTEWORTHY This is the first report that exosomes from bovine milk alter microbial communities in mice. This report suggests that the gut microbiome facilitates cell-to-cell communication by milk exosomes across species boundaries, and milk exosomes facilitate communication across animal and bacteria kingdoms.

RNase H2-Dependent Polymerase Chain Reaction and Elimination of Confounders in Sample Collection, Storage, and Analysis Strengthen Evidence That microRNAs in Bovine Milk Are Bioavailable in Humans.

Background: Evidence suggests that dietary microRNAs (miRs) are bioavailable and regulate gene expression across species boundaries. Concerns were raised that the detection of dietary miRs in plasma might have been due to sample contamination or lack of assay specificity. Objectives: The objectives of this study were to assess potential confounders of plasma miR analysis and to detect miRs from bovine milk in human plasma. Methods: Potential confounders of plasma miR analysis (circadian rhythm, sample collection and storage, calibration, and erythrocyte hemolysis) were assessed by quantitative reverse transcriptase polymerase chain reaction (PCR) by using blood from healthy adults (7 men, 6 women; aged 23-57 y). Bovine miRs were analyzed by RNase H2-dependent PCR (rhPCR) in plasma collected from a subcohort of 11 participants before and 6 h after consumption of 1.0 L of 1%-fat bovine milk. Results: The use of heparin tubes for blood collection resulted in a complete loss of miRs. Circadian variations did not affect the concentrations of 8 select miRs. Erythrocyte hemolysis caused artifacts for some miRs if plasma absorbance at 414 nm was >0.300. The stability of plasma miRs depended greatly on the matrix in which the miRs were stored and whether the plasma was frozen before analysis. Purified miR-16, miR-200c, and cel-miR-39 were stable for ≤24 h at room temperature, whereas losses equaled ≤80% if plasma was frozen, thawed, and stored at room temperature for as little as 4 h. rhPCR distinguished between bovine and human miRs with small variations in the nucleotide sequence; plasma concentrations of Bos taurus (bta)-miR-21-5p and bta-miR-30a-5p were >100% higher 6 h after milk consumption than before milk consumption. Conclusions: Confounders in plasma miR analysis include the use of heparin tubes, erythrocyte hemolysis, and storage of thawed plasma at room temperature. rhPCR is a useful tool to detect dietary miRs.

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.

Tsg101 knockout in the mammary gland leads to a decrease in small extracellular vesicles in milk from C57BL/6J dams and contributes to leakiness of the gut mucosa and reduced postnatal weight gain in suckling pups.

Human milk contains 2.2 ± 1.5×1011 small extracellular vesicles (sEVs) per milliliter and human infants consume 1.7×1014 milk sEVs (sMEVs) daily in 800 mL milk. Infant formula contains trace amounts of sMEVs. To date, eight adverse effects of milk depletion and five beneficial effects of sMEV supplementation have been reported including studies in infants and neonate mice. Formula-fed infants do not realize the benefits of sMEVs. Most of the phenotyping studies reported to date have the limitation that sMEV depletion and supplementation were initiated after mice were weaned. Here, we used a genetics approach for assessing effects of sMEV depletion on the development of suckling mice. Newborn C57BL/6J pups were fostered to Tumor Susceptibility Gene 101 (Tsg101) mammary-specific knockout (KO) dams or C57BL/6J dams (controls) in synchronized pregnancies. Tsg101 KO was associated with an 80% decrease of sMEVs. Postnatal weight gain and gut health (histology, morphology, and barrier function) were assessed until weaning at age three weeks. We observed a significant decrease in weight gain, length of small intestine, villi height, crypt depth, and intestinal barrier function in male and female pups fostered to Tsg101 dams compared to pups fostered to control dams. The effect size varied between 11 and 32 percent. Maternal Tsg101 KO did not affect the dams' health, content of macronutrients and dry mass of milk and had no effect on the amount of milk consumed by pups. We conclude that sMEVs are important for growth and gut health in neonate mice.

Holocarboxylase synthetase knockout is embryonic lethal in mice.

Holocarboxylase synthetase (HLCS) catalyzes the biotinylation of five distinct biotin-dependent carboxylases and perhaps chromatin proteins. HLCS deficiency causes multiple carboxylase deficiency which results in fatal consequences unless patients are diagnosed early and treated with pharmacological doses of biotin. The objective of this study was to develop an HLCS conditional knockout (KO) mouse and assess effects of HLCS knockout on embryo survival. In the mouse, exon 8 is flanked by LoxP sites, thereby removing a catalytically important region upon recombination by Cre. HLCS conditional KO mice were backcrossed for 14 generations with C57BL/6J mice to yield Hlcstm1Jze. Fertility and weight gain were normal and no frank disease phenotypes and abnormal feeding behavior were observed in the absence of Cre. HLCS knockout was embryonic lethal when dams homozygous for both the floxed Hlcs gene and tamoxifen-inducible Cre recombinase (denoted Hlcstm1.1Jze) were injected with tamoxifen on gestational days 2.5 and 10.5. This is the first report of an HLCS conditional KO mouse, which enables studies of the roles of HLCS and biotin in intermediary metabolism.

Cloning and expression of NS3 helicase fragment of hepatitis C virus and the study of its immunoreactivity in HCV infected patients.

OBJECTIVES: Hepatitis C is a major cause of liver failure worldwide. Current therapies applied for this disease are not fully effective and produce side effects in most cases. Non-structural protein 3 helicase (NS3) of HCV is one of the key enzymes in viral replication and infection. Therefore, this region is a promising target to design new drugs and therapies against HCV infection. The aim of this study was cloning and expression of HCV NS3 helicase fragment in Escherichia coli BL21 (DE3) using pET102/D-TOPO expression vector and studying immunoreactivity of the expressed antigen in Iranian infected with hepatitis C. MATERIALS AND METHODS: The viral RNA was extracted from the serum of HCV infected patient. The NS3 helicase region was amplified by RT-PCR. The PCR product was directionally cloned into the expression vector pET102/D-TOPO and transformed into the BL21 strain of E. coli (DE3). The transformed bacteria were then induced by adding 1mM isopropyl-ß-D-thiogalactopyranoside (IPTG) into the culture medium to enhance the protein expression. SDS-PAGE and western blotting were carried out to identify the protein under investigation, and finally purified recombinant fusion protein was used as the antigen for ELISA method. RESULTS: The insertion of the DNA fragment of the NS3 region into the expression vector was further confirmed by PCR and sequencing. SDS-PAGE analysis showed the successful expression of the recombinant protein of interest. Furthermore, immunoreactivity of fusion NS3 helicase was confirmed by ELISA and western blotting. CONCLUSION: It seems that this recombinant protein could be a useful source of antigen for future studies on HCV diagnosis and therapy.