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      Cocaine‐Specific Effects on Exosome Biogenesis in Microglial Cells

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          Abstract

          Cocaine is a highly addictive stimulant and a well-known drug, with multiple effects on physiology. Cocaine can have direct effects on all cell types in the brain, including microglia. Microglia can be activated by other conditions, such as infection, inflammation, or injury. However, how cocaine regulates microglia and the influence of cocaine on microglial-derived exosomes remains unknown. Exosomes are nanovesicles that are responsible for intercellular communications, signaling, and trafficking necessary cargo for cell homeostasis. In this study, we hypothesized that cocaine affects exosome biogenesis and composition in BV2 microglial cells. BV2 microglial cells were cultured in exosome-depleted RPMI-1640 media and were treated according to the experimental designs. We observed that cell viability decreased by 11% at 100 µM cocaine treatment but was unaffected at other concentrations. After treatments, the exosomes were isolated from the condition media. Purified exosomes were characterized and quantified using transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA). By NTA, there was a significant decrease in particles/mL after cocaine treatment. There was a 39.5%, 58.1%, 32.3% and 28.1% decrease in particles/mL at 100 nM, 1 μM, 10 μM and 100 μM cocaine, respectively. The characterization of exosomes and exosomal protein was performed by western/dot blot analyses. Tetraspanins CD11b, CD18 and CD63 were relatively unchanged after cocaine treatment. The heat shock proteins (Hsps), Hsp70 and Hsp90, were both significantly increased at 10 μM and 100 μM, but only hsp70 was significantly increased at 10 nM. The Rab proteins were assessed to investigate their role in cocaine-mediated exosomal decrease. Rab11 was significantly decreased at 10 nM, 100 nM, 1 μM, 10 μM and 100 μM by 15%, 28%, 25%, 38% and 22%, respectively. Rab27 was decreased at all concentrations but only significantly decreased at 100 nM, 1 μM and 100 μM cocaine by 21%, 24% and 23%, respectively. Rab35 had no significant changes noted when compared to control. Rab7 increased at all cocaine concentrations but only a significant increase in expression at 100 nM and 10 μM by 1.32-fold and 1.4-fold increase. Cocaine was found to alter exosome biogenesis and composition in BV2 microglial cells. Western and dot blot analyses verified the identities of purified exosomes, and the specific protein compositions of exosomes were found to change in the presence of cocaine. Furthermore, cocaine exposure modulated the expression of exosomal proteins, such as Hsps and Rab GTPases, suggesting the protein composition and formation of microglial-derived exosomes were regulated by cocaine.

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          The online version of this article (doi:10.1007/s11064-021-03231-2) contains supplementary material, which is available to authorized users.

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          Most cited references76

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          Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.

          In the 1980s, exosomes were described as vesicles of endosomal origin secreted from reticulocytes. Interest increased around these extracellular vesicles, as they appeared to participate in several cellular processes. Exosomes bear proteins, lipids, and RNAs, mediating intercellular communication between different cell types in the body, and thus affecting normal and pathological conditions. Only recently, scientists acknowledged the difficulty of separating exosomes from other types of extracellular vesicles, which precludes a clear attribution of a particular function to the different types of secreted vesicles. To shed light into this complex but expanding field of science, this review focuses on the definition of exosomes and other secreted extracellular vesicles. Their biogenesis, their secretion, and their subsequent fate are discussed, as their functions rely on these important processes.
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            Exosomes: a common pathway for a specialized function.

            Exosomes are membrane vesicles that are released by cells upon fusion of multivesicular bodies with the plasma membrane. Their molecular composition reflects their origin in endosomes as intraluminal vesicles. In addition to a common set of membrane and cytosolic molecules, exosomes harbor unique subsets of proteins linked to cell type-associated functions. Exosome secretion participates in the eradication of obsolete proteins but several findings, essentially in the immune system, indicate that exosomes constitute a potential mode of intercellular communication. Release of exosomes by tumor cells and their implication in the propagation of unconventional pathogens such as prions suggests their participation in pathological situations. These findings open up new therapeutic and diagnostic strategies.
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              Temporal Tracking of Microglia Activation in Neurodegeneration at Single-Cell Resolution

              SUMMARY Microglia, the tissue-resident macrophages in the brain, are damage sensors that react to nearly any perturbation, including neurodegenerative diseases such as Alzheimer’s disease (AD). Here, using single-cell RNA sequencing, we determined the transcriptome of more than 1,600 individual microglia cells isolated from the hippocampus of a mouse model of severe neurodegeneration with AD-like phenotypes and of control mice at multiple time points during progression of neurodegeneration. In this neurodegeneration model, we discovered two molecularly distinct reactive microglia phenotypes that are typified by modules of co-regulated type I and type II interferon response genes, respectively. Furthermore, our work identified previously unobserved heterogeneity in the response of microglia to neurodegeneration, discovered disease stage-specific microglia cell states, revealed the trajectory of cellular reprogramming of microglia in response to neurodegeneration, and uncovered the underlying transcriptional programs.
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                Author and article information

                Contributors
                skumar@peds.uab.edu
                Bjcrenshaw0320@gmail.com
                swilliams@vt.edu
                courtneerbell@yahoo.com
                qmatthews@alasu.edu
                bsims@peds.uab.edu
                Journal
                Neurochem Res
                Neurochem Res
                Neurochemical Research
                Springer US (New York )
                0364-3190
                1573-6903
                8 February 2021
                8 February 2021
                2021
                : 46
                : 4
                : 1006-1018
                Affiliations
                [1 ]GRID grid.265892.2, ISNI 0000000106344187, Department of Pediatrics/Division of Neonatology and Center of Glial Biology in Medicine at the University of Alabama School of Medicine, UAB Women and Infant Center, , University of Alabama, ; 1700 6th Ave South, Birmingham, AL 35294 USA
                [2 ]GRID grid.251976.e, ISNI 0000 0000 9485 5579, Microbiology Program, Department of Biological Sciences, College of Science, Technology, Engineering and Mathematics, , Alabama State University, ; Montgomery, AL 36104 USA
                Article
                3231
                10.1007/s11064-021-03231-2
                7946671
                33559104
                4c4295b3-d97e-4ffe-9fe2-c152e83a829e
                © The Author(s) 2021

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 26 May 2020
                : 18 November 2020
                : 4 January 2021
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: 1R15DA045564-01
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000001, National Science Foundation;
                Award ID: 1900377
                Award Recipient :
                Categories
                Original Paper
                Custom metadata
                © Springer Science+Business Media, LLC, part of Springer Nature 2021

                Neurosciences
                cocaine,exosomes,bv2 microglia,heat shock proteins,lipids,rab gtpases
                Neurosciences
                cocaine, exosomes, bv2 microglia, heat shock proteins, lipids, rab gtpases

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