Evaluation of epigenetic alterations of estrogen receptors in in vitro models of tauopathies

• Grant number: 25/05035-4
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: July 01, 2025
• End date: June 30, 2026
• Field of knowledge: Biological Sciences - Pharmacology - Neuropsychopharmacology
• Principal Investigator: Rodrigo Portes Ureshino
• Grantee: Pedro Henrique Zonaro
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated research grant: 24/04261-8 - Investigation of mitochondria alterations in in vivo and in vitro models of tauopathies: evaluation of estrogenic compounds treatment, AP.R

Abstract

Several studies have evaluated the neuroprotective role of estrogens in neurodegenerative diseases, and researchs have sought to relate the activity of estrogen receptors with the modulation of pathophysiological processes. Tauopathies are neurodegenerative diseases characterized by decreased cognitive function, clinical symptoms of dementia, and in some cases, changes in mobility. In this conformation, the tau protein leads to a cellular neurotoxic state with impaired axonal transport of organelles, modifying its physiology. Epigenetic alterations comprise a series of post-translational modifications that lead to diverse responses, even after activation of the transcription factor indicated for a given cellular function, but few studies have directly related protein alterations associated with neurodegeneration with patterns of epigenetic changes, and it is interesting to investigate the global effects that protein accumulation produces at the transcriptional and translational levels. Our group have previously developed a model of tau protein overexpression (isoform 0N4R) in SH-SY5Y cells, which presents morphological and cellular physiology alterations, such as mitochondrial alterations, but it is important to consider that isoforms such as the full-length 2N4R (more common in Alzheimer's disease) may have different alterations related to neuropathology and molecular biology. Thus, this project aims to develop an overexpression model of the EGFP-tau 2N4R protein for comparison with the 0N4R model in SH-SY5Y cells. In addition, we will seek to evaluate the DNA methylation pattern in estrogen receptors possibly correlated with changes in these cellular models, and their consequences on cellular physiology. To this end, we will clone the MAPTvEGFP-tau 2N4R gene in the expression vector already standardized for the model that expresses the 0N4R isoform, with conditional overexpression (Tet-On system), followed by its transduction into SH-SY5Y cells and will be validated. For the epigenetic study, initially, for each estrogen receptor gene (ER¿, ER¿ and GPER1) the bisulfite conversion method will be used, whose genes will subsequently be amplified by PCR and sequenced, detecting the methylation sites. Thus, target genes that may be involved in neuroprotection, such as BDNF and Bcl-2, will be evaluated, in addition to the clearance of the EGFP-tau protein after estrogen treatment by Western Blot method. This study seeks to contribute to the understanding of the relationship between tau protein and mitochondrial function, with a focus on the role of estrogenic compounds in a cellular model of AD and other dementias.