The use of genetic tools to understand the interaction between the endocannabinoid-nitrergic systems in PTSD-like behaviors

• Grant number: 23/02029-8
• Support Opportunities: Scholarships abroad - Research Internship - Scientific Initiation
• Start date: April 01, 2023
• End date: July 31, 2023
• Field of knowledge: Biological Sciences - Pharmacology - Neuropsychopharmacology
• Principal Investigator: Sabrina Francesca de Souza Lisboa
• Grantee: Anna Paula Amorim Marques
• Supervisor: Florian Freudenberg
• Host Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Institution abroad: Goethe University Frankfurt, Germany
• Associated to the scholarship: 22/09921-0 - Investigation of cannabinoid mechanisms involved in extinction deficits in iNOS KO animals, a genetic model of PTSD, BP.IC

Abstract

Post-Traumatic Stress Disorder (PTSD) is a psychiatric disorder that can be developed by vulnerable individuals after being exposed to intense traumatic events. Treatment for PTSD normally is done with selective serotonin reuptake inhibitors (SSRI), tricyclic antidepressants, and psychotherapy. However, since there are still several unclear mechanisms to understand this disorder, remission failure is very high. Several molecules and important neurotransmitters in the brain can be involved in mechanisms altered in PTSD, including fear processing, such as Nitric Oxide (NO). NO is a gaseous neurotransmitter involved in synaptic plasticity and neuromodulation and polymorphisms in NO system genes are associated with PTSD. Alterations in NO signaling can be triggered by NO-producing enzymes, especially, the enzymes inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS). These enzymes, when overactivated in response to stress, for example, could imbalance the homeostasis; stress exposure exert a crucial role in PTSD. In addition, PTSD patients also show alterations in the Endocannabinoid system (ECBs), which is essential for stress and fear responses. The Contextual Fear Conditioning (CFC) paradigm, which is very often used to study PTSD , is based on the formation and consolidation of new memories. Knockout mice for iNOS (iNOS KO) have deficits in conditioned fear extinction due to an overcompensation by the nNOS enzyme, supporting their use as a genetic model of PTSD. In the original project, which is associated with the current proposal, the candidate administered Arachidonoyl Serotonin (AA-5HT) - a selective antagonist of transient receptor potential vanilloid type 1 (TRPV1) and inhibitor of the Fatty Acid Amide Hydrolase enzyme (FAAH) that degradates Anandamide (AEA) - to iNOS KO mice to understand the relevance of these components to the observed deficits in conditioned fear extinction in the CFC paradigm. AA-5HT treatment, but not the sole administration of the same dose of SB366791 (SB), selective antagonist of TRPV1 receptor, or URB957 (a FAAH inhibitor), facilitated fear extinction acquisition and its evoke in iNOS KO mice, suggesting interference with both mechanisms is necessary to promote these processes. Based on this evidence and the necessary tools to further test the idea that the ECB system is modulating PTSD-like behaviors, the present project proposes to use an Adeno-Associated Virus (AAV) based vector to promote CB1 receptor overexpression or a shRNA (Small Hairpin RNAs) to promote TRPV1 or FAAH downregulation. To validate these tools, we propose to apply the AAV or shRNAs on neuronal cell cultures and quantify the expression of ECBs related-components (CB1, FAAH, TRPV1) . The host supervisor (Dr. Florian Freudenberg's laboratory at Goethe University in Frankfurt am Main) has extraordinary experience with the production of viral tools, along with great knowledge on behavioral analysis. Furthermore, this project will decisively impact the ongoing career of the student, not only given the importance of the technique to be learned, but also the opportunity to have an important partnership with a very renowned laboratory. Finally, to the best of our knowledge, there are no research groups at our University with the expertise, knowledge and technology to design, package, and amplify these viral tools, and validate them, making this experience unique. (AU)