Cannabidiol mitigates the long-term cognitive effects produced by the signaling pathway mediated by exosomes derived from neural cells infected with SARS-CoV-2: standardization of a cognitive model for long COVID.

• Grant number: 23/05979-7
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: March 01, 2024
• End date: July 31, 2027
• Field of knowledge: Biological Sciences - Pharmacology - Neuropsychopharmacology
• Principal Investigator: Alline Cristina de Campos
• Grantee: Bruno Lemes Marques
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated scholarship(s): 24/18340-7 - Extracellular vesicles derived from SARS-CoV-2 infected cells induces neuroplastic changes in neuron-glia enriched cultures derived from human olfactory neuroepithelium., BE.EP.DR

Abstract

Approximately one-third of COVID-19 patients may develop psychiatric and neurological problems up to six months after infection, a phenomenon known as Long COVID. These symptoms include cognitive issues, memory loss, tingling sensations, dizziness, sensitivity to light and noise, and autonomic dysfunction, regardless of the severity of the respiratory illness. Exosomes, nanosized biovesicles, play a role in the dissemination of viral particles and can promote neuroinflammatory and neurodegenerative changes depending on their content. Recent studies show that cannabidiol (CBD), derived from Cannabis sativa, may reduce neuroinflammation and cognitive changes in animal models of neurodegenerative diseases. Considering that ongoing inflammation may underlie the neurological symptoms of Long COVID, CBD may offer neuroprotective effects and alleviate these symptoms, possibly by influencing the exosomal payload released during SARS-CoV-2 infection. In this regard, this study will investigate whether exosomes are crucial for the neuroinvasiveness and cognitive deficits observed in the neurological syndrome caused by COVID-19. It will also assess whether CBD treatment can mitigate these effects by reducing the viral and pro-inflammatory content of exosomes. First, we will examine the impact of exosomes from human neuroblastoma cells (SH-SY5Y) infected with SARS-CoV-2 on SH-SY5Y cells in vitro. The SH-SY5Y cells will be infected with the SARS-CoV2 P1 virus, and exosomes will be isolated from the supernatant via ultracentrifugation. Exosomes will be assessed through NanoTracking analysis (NTA), Transmission Electron Microscopy (TEM), and micro-RNA sequencing, where we will analyze the size, concentration, morphology, and content of these vesicles. After characterization, SH-SY cells will be incubated for 24 hours with exosomes isolated from SH cells infected with SARS-CoV-2 or MOCK. Cell proliferation and viability will be assessed through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and BrdU (5-Bromo-3-deoxyuridine) assays. In a second experiment to investigate whether exosomes from human neuroblastoma infected with SARS-CoV-2 affect cognition and whether CBD is capable of reversing such changes, we will use the Barnes Maze Test, Novel Object Recognition Test (NORT), and Object Location Test (OLT). Subsequently, these animals will be euthanized, and their brains will be removed for the analysis of neuronal degeneration using FluoroJadeC, Western Blotting (IL-1, IL-6, TNF-±, IL-10, TGF-², CB1, CB2, PKA, ERK, PI3K, AKT, GSK-3², BCL-2, Caspase 3), and ELISA for cytokines (IL-2, IL-4, IL-6, IL-10, IFN-gamma, and TNF-alpha). CBD treatment will be administered daily for seven days. Finally, to understand the role of microglial cells in the potential neuroprotective effect of CBD on cognitive deficits caused by exosomes from neuroblastomas infected with SARS-CoV-2, transgenic animals TMEM19-2A + CreERT2 x Ai39 (RCL-eNpHR3.0/EYFP) will be optogenetically manipulated, enabling the selective activation of microglial cells in the nervous system of these animals, to examine the contribution of these cells to the positive effects of CBD.