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Study of possible changes in the endocannabinoid, endovaniloid, and neuroimmune systems in brain after stimulation of microglia cells: involvement of epigenetic mechanisms

Grant number: 21/05406-1
Support type:Scholarships in Brazil - Master
Effective date (Start): June 01, 2021
Effective date (End): May 31, 2023
Field of knowledge:Biological Sciences - Pharmacology - Neuropsychopharmacology
Principal researcher:Sabrina Francesca de Souza Lisboa
Grantee:Sávio Lima Bastos
Home Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Associated research grant:17/19731-6 - Identification of epigenetic mechanisms induced by stress which modulate endocannabinoid signaling and neuroimmunological mechanisms as new therapeutic targets to treat the posttraumatic stress disorder (PTSD), AP.JP


Summaries and aims: the initial aim of this subproject is to verify the content of EVs released by microglial cells after being stimulated with ATP or capsaicin, a TRPV1 receptor agonist. In addition, to verify the effect of these EVs on the synaptic expression of molecules from the endocannabinoid, endovaniloid, immune and glutamatergic systems, as well as enzymes responsible for epigenetic modifications.Work plan-Methodology:Animals: C57BL/6 newborn mice (1-3 days old) or pregnant female mice to obtain 14-day-old embryos.Drugs: Capsaicin, TRPV1 agonist (0.01-1 µM, Tocris) (36), LPS (10-100 ng/ml in culture; Invivogen); ATP (0.1-10 mM, Sigma-Aldrich), trypsin (0.25%), glutamine (200 nM), penicillin G (100 U/ml), streptomycin (100 ¼g/ml), fungisone (0.25 ¼g/ml), gentamicin (50 ¼g/ml).Primary microglia culture: Primary microglia cultures will be obtained from the total brain of newborn mice aseptically, as previously described (76). The collected cells will be added (105 cells/500 ¼l) in 24-well plates coated with poly-l-lysine. After 48 hours, the cells will be washed with serum-free DMEM and supplemented with antibiotics, containing the experimental treatments to be investigated.Obtaining extracellular vesicles (EVs): EVs will be isolated and processed as described (73). The primary microglia cultures will be exposed to serum-free culture medium for 10 min and the medium will be collected to quantify EV constitutive production. Then, the cultures to be stimulated with ATP (for activation of the NLRP3 inflammasome) will be incubated first with LPS (4-6h). All cultures will be maintained in complete medium before adding ATP or capsaicin (1h). The EVs (exosomes) will be isolated using a commercial kit (Invitrogen, Thermo). The exosomes will be subjected to the Western blotting technique to check the purity of the exosomes obtained by detecting the levels of CD63, CD9 and Tsg101 (enriched in exosomes), as described in (78). The total RNA will be purified from the exosome samples using the commercial kit Total Exosome RNA Isolation Kit (Invitrogen, Thermo). IL-1², HDAC2, DNMTs 3a and 3b, CB1, CB2, TRPV1, FAAH mRNA levels will be determined. Beta-actin, GAPDH and will be used as endogenous controls. A fraction of the EVs obtained will be frozen at -80oC for lipid extraction by the chloroform: ethanol method (2: 1, v/v) and detection of ECB levels (AEA and 2-AG) by liquid chromatography coupled to the mass spectrophotometer, as described in the literature (77).Primary culture of hippocampal and cortical neurons: It will be performed as described (59). The cells (5x105 cells/cm2) obtained will be placed in plates coated with poly-l-lysine and laminin and kept in supplemented medium until the time of use (7-12 days). Primary cultures will be incubated with EVs or only with culture medium. After the incubation period, the cells will be washed, the neurons collected and processed to obtain synaptoneurosomes. Independent cultures (1.6 x 105 cells) will be used for co-culture experiments.Obtaining synaptoneurosomes: To verify the alteration in the expression of molecules in the synaptic terminal by EVs, synaptoneurosomes will be isolated as described (77). The mRNA and proteins will be obtained in order to determine the expression of DNMTs, HDAC2, caspase-1, IL-1RI, proteins associated with IL-1RI (IL-1RacP, MyD88, IRAK), PKC, total and phosphorylated TRPV1, CB1, CB2, FAAH, NAPE-PLD and DGL5ü (36, 59, 69). To investigate the possibility of postsynaptic changes in EVs-induced glutamatergic neurotransmission, the levels of the AMPA receptor GluR1 and GluR2 subunits will be determined, as well as the NR1 and NR2 subunits of NMDA glutamate receptors. (AU)

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