Effects of B-micrustoxin, phospholipase A2 isolated from Micrurus lemniscatus venom, on the cytoskeleton of cultured astrocytes and glioblastomas U138.

• Grant number: 22/15518-4
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: April 01, 2023
• End date: December 31, 2023
• Field of knowledge: Biological Sciences - Pharmacology - Toxicology
• Principal Investigator: Solange Castro Afeche
• Grantee: Caio Henrique Tida Oliveira
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

Toxins with phospholipase A2 (FLA2) activity are found in snake venoms of the Elapidae and Viperidae families and are characterized by their neurotoxic activity, causing neuromuscular junction blockade due to their presynaptic action. Although the action of these toxins occurs at the neuromuscular junction, several studies have used central neuronal cells to characterize their mechanisms of action, and these proved to be a good model. Little is known, however, about the activities performed by these enzymes in glial cells, such as astrocytes, and this cell type plays important roles in both the central and peripheral nervous systems. Work from our laboratory showed that an FLA2 toxin isolated from the venom of the snake Micrurus lemniscatus, B-micrustoxin (formerly Mlx-9), induced cell death in hippocampal neurons and reduced astrocyte cell viability and proliferation, with a predominance of the phase G2/M of the cell cycle. Furthermore, this toxin induced an increase in cell cycle regulatory proteins p53, p21 and p27 in astrocytes. In glioblastomas, U138 and U251, B-micrustoxin reduced cell viability. Studies in the literature have shown the activation of p53 after disruptions in the cytoskeleton, for example in the formation of the mitotic spindle and centrosome. Thus, the objective of this study will be to characterize the actions of the toxin FLA2 B-micrustoxin, isolated from the venom of the snake Micrurus lemniscatus, on astrocytes and glioblastomas in culture, regarding the integrity of the cytoskeleton. Therefore, astrocytes from Wistar rats will be obtained by enzymatic dissociation and maintained in culture in DMEM medium + 10% FBS, at 37°C, 5% CO2. Human glioblastomas U138, with astrocytic origin, will be purchased from the ATCC and maintained in culture in DMEM medium + 10% FBS. The cells will be treated with B-micrustoxin (2 and 20nM) for 24h and the cytoskeleton evaluated in confocal microscopy using the fluorophores SiR-actin and Sir-tubulin. Cell proliferation of U138 glioblastomas will also be analyzed. This study intends to contribute to a better understanding of the cellular events that lead to toxicity induced by B-micrustoxin, from the venom of the snake Micrurus leminiscatus, in astrocytes and glioblastomas and opens perspectives of new targets to be explored aiming at a possible therapeutic use.