Study of intracellular signalling and metabolism mediated by estrogen in a neurodegeneration cell model

• Grant number: 19/20131-9
• Support Opportunities: Scholarships in Brazil - Master
• Start date: October 01, 2019
• End date: September 30, 2021
• Field of knowledge: Biological Sciences - Pharmacology - Neuropsychopharmacology
• Principal Investigator: Rodrigo Portes Ureshino
• Grantee: Bruno Gabriel Pereira
• Host Institution: Instituto de Ciências Ambientais, Químicas e Farmacêuticas (ICAQF). Universidade Federal de São Paulo (UNIFESP). Campus Diadema. Diadema , SP, Brazil
• Associated research grant: 16/20796-2 - Study of estrogen receptors mediated autophagy against tau toxicity in cell and zebrafish models, AP.JP

Abstract

Many studies have shown that estrogens play a neuroprotective role in neurodegenerative processes that are characterized by symptoms of dementia and cognitive impairment, such as in Alzheimer's disease. In neurodegenerative diseases, such as tauopathies, the accumulation of protein aggregates induces cytotoxicity and leads to neuronal loss by apoptosis. However, the intracellular mechanisms and pathways activated by these hormones still need to be further explored. This project aims to investigate the intracellular signalling pathways mediated by activation/inhibition of estrogen receptors in a tauopathy cell model. Initially, it will be studied a GPER activation-mediated signalling cascades, such as cAMP measurements. The investigation of intracellular mobilization and buffering of Ca2+ upon estrogen / antiestrogen stimulation in SH-SY5Y cells will be assessed, using fluorescence indicators and expressing to Ca2+-sensitive proteins that are directed to organelles (PERICAM), which will be analysed by fluorescence microscopy. In addition, it will performed a bioenergetic study, assessing mitochondrial O2 consumption by respiratory chain stimulation (ADP: O consumption) and complex I. We will complement this study by evaluating mitochondrial membrane potential with fluorescent marker TMRE, generation of reactive oxygen species (DCF labelling) and ATP generation (luciferase biochemical assay). By using Western blot and immunoprecipitation assays, the ERK/MAPK pathway will be studied, as well as the transcriptional factors and estrogen element response activation by estrogen receptors ER± and ER². The project will proceed with the assessment of the impact of human tau protein (overexpression of isoform 0N4R wild type or mutated P301L) on the cellular homeostasis disturbance that prevents the physiological degradation of this protein. It will be performed the same assays in SH-SY5Y cell (Ca2+ signalling, cell metabolism study, estrogen signalling pathway). Therefore, the study of cellular signalling alterations promotes by the overexpression of human tau protein could contribute to the understanding of the role of this protein on the pathophysiology of dementias, among Alzheimer's disease. (AU)