Células estaminais neurais como alvos terapêuticos na depressão: estudo da sinergia entre receptores canabinóides de tipo 2 e exercício físico

The primary aim of the project is to identify potential targets for treating MDD by exploring the antidepressant and procognitive effects achieved by the combined treatment with CB2R inhibition together with PE. We have devised 4 objectives: Objective 1) Determine whether the CB2R inhibition and PE-mediated effects are mediated by CB2R expressed in aNSC or in microglia in a pre-clinical depression model. 1.1) To investigate the intrinsic capacity of CB2R+ aNSCs and microglia in regulating depressive-like behavior, cell-specific inducible deletion of CB2Rs in aNSCs and microglia will be performed using Nestin-Cnr2-iKO and Tmem119-2A-Cnr2-iKO (inducible knockout) animals, respectively. We will generate these transgenic animals by crossing Cnr2-floxed with Nes-cre/ERT2 and Tmem119-2A-CreERT2 animals. These models will be subjected to uCMS to determine the specific contributions of CB2R+ aNSCs and microglia to the modulation of depressive-like behavior (Fig.4A, B1). Objective 2) Evaluate the role of AHN in mediating the antidepressant and procognitive effects resulting from CB2R inhibition together with PE in a pre-clinical depression model. 2.1) To fully characterize and evaluate AHN dependency on CB2R and PE-mediated antidepressant effects, AHN will be ablated through X-ray irradiation to target dividing aNSCs in the dentate gyrus (DG) hippocampal region in mice subjected to uCMS (Fig.4A, B2). Objective 3) Study the neuromodulatory factors induced by CB2R modulation and PE in a pre-clinical depression model. 3.1) To identify potential neuromodulatory factors induced by the co-treatment, we will perform cell sorting using Nestin-GFP, Tmem119-2A-EGFPF, and Dcx-mRFP mice to isolate aNSC, microglia, and neuroblasts/young neurons, respectively, from the dentate gyrus. Then, we will perform a proteomic analysis by tandem mass tag mass spectrometry (TMT-MS) approach. The proteins whose expression changes with the co-treatment will be analyzed for their association with biological function. 3.2) To determine which cell types are producing the putative neuromodulators in situ hybridization-RNAscope will be performed. This technique allows the detection of target RNA within intact cells giving the information regarding the cell types in which the target RNA is being expressed. Objective 4) Investigate the relation between eCBs, neurotrophic factors and AHN in MDD in a clinical setting. 4.1) A clinical study involving control individuals and patients with MDD will be conducted to search for new biomarkers for early diagnostics, and for better prognosis and patient stratification. Given that recent evidence suggests that dysregulations in the ECS are likely associated with hippocampal dysfunctions, as well as depressive-like behavior, we propose to evaluate serum levels of components of the ECS, namely endocannabinoids and related fatty acids, and whether these are correlated with distinct neurotrophic factor levels, known regulators of AHN and key players in PE. 4.2) Neuroimaging analysis will be performed to assess the potential correlation between MDD severity and AHN. Changes in metabolism, hippocampal volume, fractional anisotropy, and the default mode network at resting state will be measured. 4.3) Genetic screening for CNR2 polymorphisms will be performed to correlate with MDD severity and stress susceptibility.