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The role of estrogen receptors in autophagy in cellular model of Tauopathy

Grant number: 18/16719-8
Support type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): September 01, 2018
Effective date (End): October 31, 2021
Field of knowledge:Biological Sciences - Pharmacology - Neuropsychopharmacology
Principal Investigator:Rodrigo Portes Ureshino
Grantee:Michelle Sayuri Nishino
Home 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

In neurodegenerative diseases which has dementia as clinical condition, such as Alzheimer´s disease and Tauopathies, there are aggregations of intracellular neurofibrillary tangles composed mainly by tau protein. The search for therapies that reduce protein aggregates is important to develop strategies to stabilize the neurodegeneration progression. Some studies show that autophagy modulation is one of the most promising strategies for the treatment of diseases associated to protein aggregates. In the same way, many clinical trials demonstrate that estrogens may act as neuroprotective such condition as neurodegenerative processes, like Alzheimer's Disease. Therefore, the aim of this project is to investigate the involvement of estrogen receptors in autophagy modulation, as well as the molecular activation pathway in a cellular model of Tauopathy, with focus in autophagy as neuroprotective strategy. For this, we will use a neuroblastoma cell line (SH-SY5Y) which overexpresses the human tau protein (isoform 0N4R or mutated P301L) to test the estrogen receptor agonists and antagonists drugs, in order to evaluate if those compounds are able to induce autophagy. For this, the autophagy pathway proteins will be quantified by Western Blotting (LC3 II, p62, Beclina1, p-p70, p-ULK, pAMPK) and the cells will be transfected with GFP-mCherry-LC3 and GFP-p62 proteins vectors, to visualize autophagy flux in fluorescence microscopy. Furthermore, the cells will be first characterized by its estrogens receptors distribution (using immunofluorescence and western blotting assay) and lately the estrogen receptor genes (ER±, ER² and GPER) will be knocked out (using CRISPR-Cas9 assay). The involvement of estrogen receptors in autophagy will also be evaluated and validated in ER-knocked out cells. Then, based on autophagy gene expression activated by estrogen receptors, we will select genes that preferentially modulates autophagy by estrogenic signaling, to evaluate their contribution in this catabolic process and in neuroprotection. Therefore, the investigation of the neuroprotective role of estrogen receptors in cellular model of Tauopathy can contribute to the understanding of the impact of this hormone in neuroprotection and in neurodegenerative diseases, such as in Alzheimer's Disease. (AU)