Effect of lithium chloride in 6-OHDA Parkinsons Disease model.

Author(s)

Ana Flavia Tostes da Silva

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2021-09-15

Examining board members

Luiz Roberto Giorgetti de Britto; Roberto de Pasquale; Lívia Clemente Motta Teixeira; Angela Cristina do Valle

• Advisor: Luiz Roberto Giorgetti de Britto

Abstract

Introduction: Parkinsons Disease (PD) is a pathological alteration which consists mainly in the loss of dopaminergic neurons in the substantia nigra (SN) and its projections to the striatum. Besides this, PD has been winning attention recently due to being considered the neurological disorder with the fastest growth in the world. In this context, a concern in testing drugs that may present potential neuroprotective effects, like lithium chloride (LiCl), raises. The objective of this study was to evaluate the effect of LiCl in dopaminergic neurons and investigate the influence of the treatment with this drug in the enzymatic expression of GSK-3&beta; in the SN and striatum in a PD model. Methodology: To induce the PD model, 6-OHDA was administered in a 2 &mu;g/&mu;l concentration, dissolved in a 0,9% NaCl and 0,2% ascorbic acid solution. The 6-OHDA injection was made in two distinct striatum points at the following coordinates: Point 1: A (anterior): +1.0 mm; ML (mediolateral): 2.0 mm in relation to bregma and DV (dorsoventral): 2.9 mm in relation to the dura mater. Point 2: P (posterior): -0.3 mm; ML: 2.3 mm in relation to bregma and DV: 2.9 mm in relation to the dura mater. The injected volume of the drug was 2 &mu;l. To the LiCl treatment, a 4 mMol/kg/day dose (capable of inhibit the GSK-3&beta; enzyme in mice models) was used, vehiculated by intraperitoneal injection for 14 days. Following, samples were collected and the enzymatic activity of the GSK-3&beta; in the SN and striatum was evaluated by immunofluorescence, RT-PCR and Immunoblotting methodologies. Results: The results found indicate that 6-OHDA promoted dopaminergic neuronal death in the SN region, evidenced by the tyrosine hydroxylase (TH) enzymatic expression reduction in the animals subjected to this neurotoxin injection, when compared to control animals. Besides this, animals that received 6-OHDA presented an increase in the Caspase-3 genic expression. Although here we relate that the PD induction was not suggestive that 6-OHDA exerts a significative effect in GSK-3&beta; mRNA nor in the active conformation of the GSK-3&beta; (Y216) enzyme in the regions of the SN and striatum at the 15th day after the lesion. About the LiCl treatment, we had a reduction in the AKT mRNA and GSK-3&beta; mRNA in animals treated with this compound. As a counterpoint, we had an increase in the GSK-3&beta; (Y216) expression that may be related to the TH mRNA reduction at the SN of animals treated with LiCl. These results are suggestive of a new mechanism of how LiCl neurotoxicity occurs. Statistical analysis: two-way ANOVA followed by a Bonferroni post-hoc test, made using GraphPad Prism 8.2.1. p-values<0.05 were considered statistically significative. Results were shown as mean ± standard error of the mean. Conclusion: Our data points that GSK-3&beta; (Y216) is present in the cell bodies of dopaminergic neurons of the SN and that, in this region, the increase of GSK-3&beta; (Y216) shows a correlation with the TH mRNA only in animals treated with LiCl and not with the PD model. These results are suggestive that the long-term LiCL treatment presents an antagonistic deleterious GSK-3 &beta; fosforilation-dependent effect to dopaminergic neurons in the SN, challenging LiCl use as a therapeutic strategy to PD. (AU)

• FAPESP's process: 18/13015-0 - Evaluation of contribution of GSK-3 in 6-OHDA model in Parkinson's Disease
• Grantee: Ana Flavia Tostes da Silva
• Support Opportunities: Scholarships in Brazil - Master