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Glia-to-neuron transfer of miRNAs via extracellular vesicles: a new mechanism underlying inflammation-induced synaptic alterations.

ABSTRACT: Recent evidence indicates synaptic dysfunction as an early mechanism affected in neuroinflammatory diseases, such as multiple sclerosis, which are characterized by chronic microglia activation. However, the mode(s) of action of reactive microglia in causing synaptic defects are not fully understood. In this study, we show that inflammatory microglia produce extracellular vesicles (EVs) which are enriched in a set of miRNAs that regulate the expression of key synaptic proteins. Among them, miR-146a-5p, a microglia-specific miRNA not present in hippocampal neurons, controls the expression of presynaptic synaptotagmin1 (Syt1) and postsynaptic neuroligin1 (Nlg1), an adhesion protein which play a crucial role in dendritic spine formation and synaptic stability. Using a Renilla-based sensor, we provide formal proof that inflammatory EVs transfer their miR-146a-5p cargo to neuron. By western blot and immunofluorescence analysis we show that vesicular miR-146a-5p suppresses Syt1 and Nlg1 expression in receiving neurons. Microglia-to-neuron miR-146a-5p transfer and Syt1 and Nlg1 downregulation do not occur when EV-neuron contact is inhibited by cloaking vesicular phosphatidylserine residues and when neurons are exposed to EVs either depleted of miR-146a-5p, produced by pro-regenerative microglia, or storing inactive miR-146a-5p, produced by cells transfected with an anti-miR-146a-5p. Morphological analysis reveals that prolonged exposure to inflammatory EVs leads to significant decrease in dendritic spine density in hippocampal neurons in vivo and in primary culture, which is rescued in vitro by transfection of a miR-insensitive Nlg1 form. Dendritic spine loss is accompanied by a decrease in the density and strength of excitatory synapses, as indicated by reduced mEPSC frequency and amplitude. These findings link inflammatory microglia and enhanced EV production to loss of excitatory synapses, uncovering a previously unrecognized role for microglia-enriched miRNAs, released in association to EVs, in silencing of key synaptic genes.

SUBMITTER: Prada I

PROVIDER: S-EPMC5978931 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: There is a compelling evidence from animal models that early exposure to clinically relevant general anesthetics (GAs) interferes with brain development, resulting in long-lasting cognitive impairments. Human studies have been inconclusive and are challenging due to numerous confounding factors. Here, we employed primary human neural cells to analyze ketamine neurotoxic effects focusing on the role of glial cells and their activation state. We also explored the roles of astrocyte-derived extracellular vesicles (EVs) and different components of the brain-derived neurotrophic factor (BDNF) pathway. Methods: Ketamine effects on cell death were analyzed using live/dead assay, caspase 3 activity and PARP-1 cleavage. Astrocytic and microglial cell differentiation was determined using RT-PCR, ELISA and phagocytosis assay. The impact of the neuron-glial cell interactions in the neurotoxic effects of ketamine was analyzed using transwell cultures. In addition, the role of isolated and secreted EVs in this cross-talk were studied. The expression and function of different components of the BDNF pathway were analyzed using ELISA, RT-PCR and gene silencing. Results: Ketamine induced neuronal and oligodendrocytic cell apoptosis and promoted pro-inflammatory astrocyte (A1) and microglia (M1) phenotypes. Astrocytes and microglia enhanced the neurotoxic effects of ketamine on neuronal cells, whereas neurons increased oligodendrocyte cell death. Ketamine modulated different components in the BDNF pathway: decreasing BDNF secretion in neurons and astrocytes while increasing the expression of p75 in neurons and that of BDNF-AS and pro-BDNF secretion in both neurons and astrocytes. We demonstrated an important role of EVs secreted by ketamine-treated astrocytes in neuronal cell death and a role for EV-associated BDNF-AS in this effect. Conclusions: Ketamine exerted a neurotoxic effect on neural cells by impacting both neuronal and non-neuronal cells. The BDNF pathway and astrocyte-derived EVs represent important mediators of ketamine effects. These results contribute to a better understanding of ketamine neurotoxic effects in humans and to the development of potential approaches to decrease its neurodevelopmental impact.

Project description:In rheumatoid arthritis (RA), extracellular vesicles (EVs) are associated with both the propagation and attenuation of joint inflammation and destruction. However, the specific EV content responsible for these processes is largely unknown. Investigations into identifying EV content are confounded by the challenges in obtaining high-quality EV preparations from synovial fluid. Implementing a size exclusion chromatography-based method of EV isolation, coupled with small RNA sequencing, we accurately characterised EV miRNAs in synovial fluid obtained from RA patients and investigated the differences between joints with high- and low-grade inflammation. Synovial fluid was obtained from the joints of 12 RA patients and, based on leukocyte counts, classified as either high (n = 7)- or low (n = 5)-grade inflammation. Using size exclusion chromatography, EVs were purified and small RNA was extracted and sequenced on a NextSeq 500. Sequencing reads were aligned to miRBase v21, and differences in miRNA profiles between RA patients with high- and low-grade joint inflammation were analysed. In total, 1972 distinct miRNAs were identified from RA synovial fluid EVs. miRNAs with less than five reads in fewer than five patients were filtered out, leaving 318 miRNAs for analysis. Analysis of the most abundant miRNAs suggested that they negatively regulate multiple genes relevant to inflammation, including signal transducer and activator of transcription 3 (STAT3), which lies downstream of IL-6 and has a pro-inflammatory role in RA. Synovial fluid from joints with high-grade inflammation contained 3.5-fold more EV miRNA per mL of synovial fluid (p = 0.0017). Seventy-eight EV miRNAs were differentially expressed between RA joints with high- and low-grade inflammation, and pathway analysis revealed that their target genes were commonly involved a variety of processes, including cellular apoptosis, proliferation and migration. Of the 49 miRNAs that were elevated in joints with high-grade inflammation, pathway analysis revealed that genes involved in cytokine-mediated signalling pathways were significantly enriched targets. In contrast, genes associated with reactive oxygen species signalling were significantly enriched as targets of the 29 miRNAs elevated in joints with low-grade inflammation. Our study identified an abundance of EV miRNAs from the synovial fluid of RA patients with the potential to modulate inflammation. In doing so, we defined potential mechanisms by which synovial fluid EVs may contribute to RA pathophysiology.

Project description:BACKGROUND:Extracellular vesicles (EVs) are nano-sized particles secreted by most cells. EVs carry nucleic acids that hold promise as potential biomarkers in various diseases. Human immunodeficiency virus type 1 (HIV) infects CD4+ T cells and induces immune dysfunction, inflammation, and EV secretion, but little is known about EV small RNA cargo in relation to immune dysregulation in HIV-infected individuals. Here, we characterize small RNA carried by circulating EVs in HIV-positive subjects on antiretroviral therapy (ART) relative to uninfected controls by next-generation RNA sequencing. RESULTS:Plasma EVs isolated from HIV-positive and HIV-negative subjects in test (n?=?24) and validation (n?=?16) cohorts were characterized by electron microscopy, nanoparticle tracking analysis, and immunoblotting for exosome markers. EVs were more abundant in plasma from HIV-positive compared to HIV-negative subjects. Small RNA sequencing of plasma EVs in the test cohort identified diverse small RNA species including miRNA, piRNA, snRNA, snoRNA, tRNA, and rRNA, with miRNA being the most abundant. A total of 351 different miRNAs were detected in plasma EVs, with the top 50 miRNAs accounting for 90% of all miRNA reads. miR-26a-5p was the most abundant miRNA, followed by miR-21-5p and miR-148-3p. qRT-PCR analysis showed that six miRNAs (miR-10a-5p, -?21-5p, -27b-3p, -?122-5p, -146a-5p, -?423-5p) were significantly increased in plasma EVs from HIV-positive compared to HIV-negative subjects in the validation cohort. Furthermore, miR-21-5p, -27b-3p, -146a-5p, and?-?423-5p correlated positively with metabolite markers of oxidative stress and negatively with anti-inflammatory polyunsaturated fatty acids. Over-representation and pathway enrichment analyses of miRNAs and their target genes predicted functional association with oxidative stress responses, interferon gamma signaling, Toll-like receptor signaling, TGF beta signaling, and Notch signaling. CONCLUSIONS:HIV-positive individuals on ART have increased abundance of circulating EVs carrying diverse small RNAs, with miRNAs being the most abundant. Several miRNAs associated with inflammation and oxidative stress are increased in circulating EVs of HIV-positive individuals, representing potential biomarkers of targetable pathways that contribute to disease pathogenesis.

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Glia-to-neuron transfer of miRNAs via extracellular vesicles: a new mechanism underlying inflammation-induced synaptic alterations.

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