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STUDY OF KEY IRS/PI3K/AKT PATHWAY GENES IN NEUROGENESIS AND CELL SURVIVAL PROCESSES UNDER OXIDATIVE AND NEUROTOXIC STRESS IN HUMAN NEURAL MODELS

Grant number: 23/10039-3
Support Opportunities:Regular Research Grants
Duration: February 01, 2024 - January 31, 2026
Field of knowledge:Biological Sciences - Genetics - Mutagenesis
Principal Investigator:Elza Tiemi Sakamoto Hojo
Grantee:Elza Tiemi Sakamoto Hojo
Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

Alzheimer's disease (AD) is a chronic and complex disease, characterized by progressive and irreversible degeneration of neurons and the dysregulation of multiple cellular pathways. There are still no effective treatments for patients with this disease, with large gaps regarding the molecular mechanisms involved in its development, which require elucidation. Due to the central role of the IRS/PI3K/AKT pathway in several physiological functions of the brain, it has aroused a growing interest regarding the study of its influence in neurodegenerative diseases, particularly for its involvement in cell survival, control of the antioxidant system, neuronal differentiation and also in the negative regulation of cell death pathways. Understanding how the functional status of the IRS/PI3K/AKT pathway can be modulated by the various alterations related to the development and progression of AD (insulin resistance, accumulation of A² peptides and neurofibrillary tangles, exposure to oxidative stress) has stimulated great interest, aiming at elucidating the pathophysiology of AD, as well as the development of therapeutic strategies based on molecular targets. Among these targets, PTEN gene has been investigated as an important negative regulator of the PI3K/AKT pathway. There are also downstream targets of the PI3K/AKT pathway, such as the GSK3² protein, one of the main kinases responsible for tau protein hyperphosphorylation, the MST kinase, which, by preventing the activation of the hippo pathway, linked to the induction of apoptosis, promotes neuroprotection, as well as the KEAP1 protein, the main negative regulator of NRF2, resulting in the activation of the antioxidant system. Thus, the present project to be carried out in neuronal models is based on the hypothesis that the exposure of neurons to several neurotoxic stimuli (related to typical alterations in AD) will promote alterations in the status of the PI3K/AKT pathway. Furthermore, it is expected that the modulation of the upstream and downstream genes of this pathway will promote an improvement in cellular responses, mainly regarding the induction of neuroprotection in response to treatments with neurotoxic agents, as well as neuronal differentiation. Thus, the general objective of the project is to analyze the status of the PI3K/AKT pathway after exposure to various neurotoxic stimuli, i.e., under conditions of insulin resistance, oxidative stress, toxicity of A² peptides and neurofibrillary tangles. The project also aims to investigate the impact of the inhibition of key genes (PTEN, GSK3², MST and KEAP1) on the responses of NPCs (Neural Progenitor Cells) derived from healthy elderly individuals, compared to NPCs from patients with AD, in monolayer cultures or in 3D models (neurospheres and brain organoids), in addition to the neuronal model derived from the SH-SY5Y cell line. Through various types of assays, cellular responses will be analyzed, in the presence or absence of exposure to neurotoxic agents, as a result of the lack of functionality of each of the silenced genes, using siRNA and CRISPR-Cas9 methods, in addition to chemical inhibitors. The methodologies to be applied involve analyzes of transcriptional and protein expression of genes/proteins belonging to the IRS/PI3K/AKT pathway, as well as analyzes related to neuronal differentiation and neuritogenesis, in addition to several cellular assays, such as cell viability, DNA damage, cell cycle kinetics, evaluation of cell death and analysis of mitochondrial changes. It is expected that the results to be obtained can provide unprecedented and valuable information on neurons (human model), especially in the condition of exposure to treatments with neurotoxic agents (as occurs throughout the lives of individuals and patients with AD). The research may have an impact on the search for potential relevant molecular targets for the development of therapies for patients with AD and other neurodegenerative diseases. (AU)

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