Evaluation of estrogen-mediated neuroprotection in cellular model of the tauopathy

Abstract

Alzheimer's disease is clinically characterized by decreased cognitive ability and dementia. The main risk factor for this disease is the aging process, and it is observed a higher incidence in women. Many studies have pointed out that estrogens have neuroprotective effects in neurodegenerative processes. It is well known that the formation of neurofibrillary tangles, consisting mainly by hyperphosphorylated tau protein, contributes to the pathogenesis of this disease. Therefore, therapeutic strategies that aim to remove these protein aggregates are desirable, for example, by the modulation of autophagy. Therefore, the objective of this study is to develop and characterize a neuroblastoma cell line that overexpresses human tau protein, seeking for agonists/antagonists of estrogen receptors that may lead to a reduction in tau protein. For this purpose, SH-SY5Y cells will be transfected with plasmid DNA encoding the human 0N4R tau protein (isoform with 4 microtubule-binding domain repeats, which is more prone to hyperphosphorylate) or the mutated tau P301L (isoform with 4 microtubule-binding domain repeats, which is linked to the familiar forms of tauopathies). After verifying the integration of the tau gene to cell DNA, it will be evaluated the ability of this protein to form aggregates (neurofibrillary tangles) by quantifying the tau protein by Western blot (total and phosphorylated tau), and will be also analyzed its cytoplasmic distribution by fluorescence microscopy. Toxicity tests will be performed to verify the sensitivity to cell death, detected by flow cytometry (Annexin V-PI kit, caspase 3). Finally, a screening of a nuclear receptors compound library, especially composed by the steroid hormones and analogues, will be performed in search of substances that are able to reduce the protein aggregates, and which possibly activate the autophagy. Therefore, the evaluation of the regulation of estrogen receptors in cytoprotection is important to delineate new pharmacological therapies for dementias, such as in Alzheimer's disease. (AU)