Cocaine‐Specific Effects on Exosome Biogenesis in Microglial Cells

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.

Human Adenovirus Serotype 3 Infection Modulates the Biogenesis and Composition of Lung Cell-Derived Extracellular Vesicles

“…Approximately 500 μ L of purified EVs was collected below the meniscus of the centrifuge tube. Collected EVs were quantified using the Bradford–Lowry quantitation method [ 24 , 42 , 44 ].…”

“…After 15 seconds (s), samples were observed under TEM (Tecnai) 120 kV (FEI, Hillsboro, USA) at 80 kV within 24 h as compared to the negatively stained grids. Digital images were captured with a BioSprint 29 CCD Camera (AMT, Woburn, MA, USA) [ 44 ].…”

“…To test the effect of HAdV3 on A549 cellderived EVs, we infected cells in exosome-free complete DMEM/F12. Medium only was used as a control; EVs were isolated and purified from cell supernatant using a standard ultracentrifugation procedure [25,[42][43][44]. Isolated EV size and particles per mL were evaluated via TEM and NTA, respectively.…”

Ethanol Induces Extracellular Vesicle Secretion by Altering Lipid Metabolism through the Mitochondria-Associated ER Membranes and Sphingomyelinases

“…Studies have shown that microglia increase EV secretion after a stimulus [3,4], and the content of microglia-derived EVs differs depending on the nature of the stimuli, triggering either pro-or anti-inflammatory responses [5][6][7]. Interestingly, drugs of addiction such as cocaine [8] or ethanol [9] have also been shown to alter microglial EV secretion. Specifically, ethanol was reported to increase the content of pro-inflammatory cytokines and chemokines in microglia-derived EVs in a dose-dependent manner [9].…”

Extracellular Vesicles: Progress and Challenges in the Study of Human Immunodeficiency Virus and Cocaine-associated Pulmonary Arterial Hypertension

“…Also, no perfect EV purification method exists, and it is difficult to obtain pure, uncontaminated, and unselected populations of EVs, especially from complex biological fluids like plasma ( 13 ). Lastly, administration of cocaine can itself have a strong effect on the rate of EV production as well as the type of cargo that they deliver to target cells ( 18 ), adding another layer of complexity to the study of this system. By using the HIV-transgenic rat model, validating their principal findings across strata of viremia, varying EV purification techniques, and using complementary cell culture–based experiments, Krishnamachary and colleagues have attenuated, but not eliminated, some of these potential confounders.…”