Evaluationof theeffectsof thecannabinoidagonistWIN55212-2in cell model of klotho deletion.

Author(s)

Isabela Ribeiro Possebom

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2024-07-29

Examining board members

Elisa Mitiko Kawamoto Iwashe; Alline Cristina de Campos; Carolina Demarchi Munhoz; Aline Camargo Ramos

• Advisor: Elisa Mitiko Kawamoto Iwashe

Abstract

The neurogenesis processs, where neurons are formed from neural progenitor cells (BCCs), is important for neural circuit maintenance and central nervous system (CNS) homeostasis. Decreased neurogenesis is associated with cognitive decline and neurodegenerative diseases. However, it is necessary to thoroughly understand its respective mechanisms and functions, for the manipulation of neurogenesis to be an applicable therapeutic strategy. Therefore, recent studies have described that Klotho protein deficiency can negatively affect the process of neurogenesis in the hippocampus. Furthermore, the cannabinoid agonist WIN55212-2 has been shown to be capable of promoting neurogenesis in NPCs. The objective of the present project was to evaluate the influence of Klotho protein hypofunction generated by the mutation of just one allele of the Klotho gene; as well as the action of the cannabinoid agonist WIN55212-2 in the process of neurogenesis. To achieve this objective, mouse embryos of the Klotho lineage were used between 14.5-16.5 days of development, of the wild (Kl+/+) and heterozygous (Kl+/-) genotypes. By dissecting the telencephalon, a culture of NPCs was generated and used in the following tests: 1) Cell viability, methyltetrazolium (MTT) and lactate dehydrogenase (LDH) to standardize the concentration of WIN55212-2; 2) Proliferation assays in the absence and presence of WIN55212-2; 3) Cellular differentiation assays in the absence and presence of WIN55212-2. Data were statistically analyzed by unpaired Student\'s t-testor2-way ANOVA. Differences were considered significant at p-value <0.05. None of the concentrations of WIN55212-2 at 48 hours (20; 10; 5; 2. 5 and 0.5 <font face = \"symbol\">mM) increased cellular cytotoxicity compared to the control, dimethylformaldehyde (DMF). In the proliferation assay, there were no significant differences between the genotypes, only a trend towards an increase in the number of kl +/- neurospheres (n=5), compared to the control group, Kl+/+(n=5). In the differentiation assay, kl+/- neurospheres (n=4) showed a decrease in the total CB2 fluorescence intensity compared to kl+/+. Treatment with WIN55212-2 did not significantly alter the parameters quantified in the proliferation and differentiation assays. Thus, we observed that the mutation of one allele of the klotho gene, although it did not change the rate of neurosphere proliferation, was able to reduce CB2 fluorescence. The project was approved by the Animal Use Ethics Committee (CEUA 4028260422). (AU)

• FAPESP's process: 22/04708-7 - Evaluation of the effects of the cannabinoid agonist win- 55212-2 in the cell model of klotho deletion.
• Grantee: Isabela Ribeiro Possebom
• Support Opportunities: Scholarships in Brazil - Master