Toxicity of beta-amyloid peptide aggregates on the SH-SY5Y human neuroblastoma cell line

Author(s)

Nathalia Reges Pinheiro

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2023-03-06

Examining board members

Adriano Silva Sebollela; Vitor Marcel Faça; Cilene Lino de Oliveira

• Advisor: Adriano Silva Sebollela

Abstract

Aggregates of the ß-amyloid peptide (Aß) are the main neurotoxins associated with synaptoxicity and neurodegeneration in Alzheimer\'s Disease (AD). These aggregates accumulate in the brains of AD patients. Amongst these aggregates, soluble species termed Aß oligomers (AßOs) are considered the most neurotoxic ones. AßOs are metastable entities formed by a mechanism not yet completely understood. The various species of AßOs described present different conformations and toxic activities. Therefore, elucidating the biochemical characteristics of the most relevant AßOs species for AD has been a critical bottleneck in the search for efficient treatments for AD. Conformation-sensitive antibodies selective to AßO have been developed as a tool for the structural elucidation of toxic species and as potential therapeutic agents for rescuing cognitive impairment in AD. Previously, our group has selected an scFv-type antibody NUsc1 showing a remarkable ability to distinguish AßOs from Aß monomers and fibrils, in addition to having as a preferential target a subpopulation of high molecular weight AßOs present in the brain of individuals with AD. Supported by these findings, here we carried out a study to identify and isolate a new high molecular weight AßO subpopulation recognized by NUsc1 under non-denaturing conditions. Size and shape of the isolated AßO species were analyzed by gel filtration chromatography and transmission electron microscopy. Furthermore, we investigated the toxicity induced by this AßO subpopulation on SH-SY5Y human neuroblastoma cells differentiated into mature neurons and evaluated the ability of NUsc1 to neutralize this toxicity. Interestingly, NUsc1 partially neutralized the toxicity induced by this subpopulation of high molecular weight AßOs, in contrast to the capacity of this antibody to completely neutralize the toxicity caused by the standard AßOs population against which NUsc1 was selected. In parallel, motivated by the diversity of protocols and wide use of SH-SY5Y cells in studies on the neurotoxicity of Aß aggregates, we decided to study the parameters of differentiation of SH-SY5Y cells and challenge with the Aß peptide that influence the size effect of toxicity elicited by this treatment. We conducted a comprehensive systematic review of the literature through the search in reference bases and the selection of articles according to pre-established criteria. Based on the quantitative synthesis of experimental data extracted from selected articles, we identified the most commonly used methods for the assessment of Aß toxicity on SH-SY5Y cells. Subsequently, through metaanalysis of the extracted data, we determined that the duration of differentiation of SH-SY5Y cells, and the duration of exposure to Aß peptide, are predictors of larger reduction in the viability of SH-SY5Y cells by exposure to Aß aggregates. Thus, we determined the critical experimental parameters for the use of SH-SY5Y cells in studies of Aß neurotoxicity, as well as for the selection and characterization of novel neuroprotective agents with potential for the treatment of AD. (AU)

• FAPESP's process: 18/10721-0 - Neutralizing beta-amyloid peptide oligomers neurotoxicity by the controlled neuronal expression of the conformational single chain antibody NUsc1
• Grantee: Nathalia Reges Pinheiro
• Support Opportunities: Scholarships in Brazil - Doctorate