Identification of novel neuroprotective genes against oxidative stress : a combined genetic and proteomic approach

Abstract

Introduction: Oxidative stress is a pathological condition caused by an imbalance between oxygen radical production and their detoxification. Oxidative stress in the brain is considered a contributing factor in neurodegenerative diseases, including Alzheimer's disease. Murine Neuro2A cells were chronically exposed to H2O2 and resistant clones (named Neuro2A-HR populations) with up to 70 fold higher tolerance to H2O2 were selected. Differential gene expression in the selected Neuro2A-HR subpopulations was analysed using microarrays, the top hits of the genes were validated by qRT-PCR and 11 of the validated genes were cloned in pIRES2-EGFP plasmid in order to be overexpressed. Aim: The general aim of this work was to identify novel neuroprotective genes and proteins involved in the mechanism of defence against oxidative stress by investigating the mechanism of resistance of the selected Neuro2A-HR cells via a genetic and a proteomic approach. Methods: Genetic approach: stable transfected single clones were generated. Single clones were overexpressing at different level five genes of interest: Glutathione Stransferase alpha 3 (Gsta3); Microsomal glutathione S-transferase 1 (Mgst1); I/I hydrolase domain-containing protein 3 (Abhd3); Cytotoxic T lymphocyteassociated protein 2 alpha (Ctla-2I) and Major facilitator superfamily domain containing 6 (Mfsd6) in Neuro2A cells. Level of gene expression was measured via qRT-PCR. The single clones were also tested for their resistance toward H2O2 exposure to evaluate their role in the protection from H2O2-induced cell death. Proteomic approach: two of the Neuro2A-HR subpopulations (E3 andE725) were used for analysing the proteome in order to evaluate differentially increased and decreased proteins and identify them by using the 2D-PAGE and LC-ESI/MS Mass Spectrometry. Results: Gene overexpression was a successful tool and Gsta3, Mgst1, Ctla-2IA and Mfsd6 genes demonstrated to be protective when overexpressed in the cells. Selected single clones showed different levels of expression and also different levels of resistance, but all the clones that had an expression of the target genes over 1.5 fold resulted in an increased resistance when compared to the parental Neuro2A. Conversely Abhd3 overexpression did not result in an increased resistance against oxidative stress. From the proteomic analysis of the two Neuro2A-HR subpopulations, 40 proteins were identified that were (A) present in only in Neuro2A-HR subpopulation (both) and only in Neuro2A parental or (B) showing a consistent increase or decrease in volume in both of the Neuro2A-HR subpopulations (E3 and E725) and (C) presenting a linear relationship inside the Neuro2A-HR subpopulation following their differential resistance to H2O2. Future work: Further investigations are necessary, especially in the validation of the targets identified from the proteomic part of the project but this project was a step forward in understanding how the oxidative stress can be fought. A common function amongst the identified genes and proteins was lipid metabolism, which seems to play an important role in cell survival and mediating protection against oxidative stress. Some of the identified targets have previously been found to be involved in inflammation and also in Alzheimer's disease, such as Apolipoprotein E, Cathepsin B and Splicing factor proline/glutamine-rich. The targets identified therefore likely represent key gene and protein changes that could protect against the oxidative stress associated with disease states.Introduction: Oxidative stress is a pathological condition caused by an imbalance between oxygen radical production and their detoxification. Oxidative stress in the brain is considered a contributing factor in neurodegenerative diseases, including Alzheimer's disease. Murine Neuro2A cells were chronically exposed to H2O2 and resistant clones (named Neuro2A-HR populations) with up to 70 fold higher tolerance to H2O2 were selected. Differential gene expression in the selected Neuro2A-HR subpopulations was analysed using microarrays, the top hits of the genes were validated by qRT-PCR and 11 of the validated genes were cloned in pIRES2-EGFP plasmid in order to be overexpressed. Aim: The general aim of this work was to identify novel neuroprotective genes and proteins involved in the mechanism of defence against oxidative stress by investigating the mechanism of resistance of the selected Neuro2A-HR cells via a genetic and a proteomic approach. Methods: Genetic approach: stable transfected single clones were generated. Single clones were overexpressing at different level five genes of interest: Glutathione Stransferase alpha 3 (Gsta3); Microsomal glutathione S-transferase 1 (Mgst1); I/I hydrolase domain-containing protein 3 (Abhd3); Cytotoxic T lymphocyteassociated protein 2 alpha (Ctla-2I) and Major facilitator superfamily domain containing 6 (Mfsd6) in Neuro2A cells. Level of gene expression was measured via qRT-PCR. The single clones were also tested for their resistance toward H2O2 exposure to evaluate their role in the protection from H2O2-induced cell death. Proteomic approach: two of the Neuro2A-HR subpopulations (E3 andE725) were used for analysing the proteome in order to evaluate differentially increased and decreased proteins and identify them by using the 2D-PAGE and LC-ESI/MS Mass Spectrometry. Results: Gene overexpression was a successful tool and Gsta3, Mgst1, Ctla-2IA and Mfsd6 genes demonstrated to be protective when overexpressed in the cells. Selected single clones showed different levels of expression and also different levels of resistance, but all the clones that had an expression of the target genes over 1.5 fold resulted in an increased resistance when compared to the parental Neuro2A. Conversely Abhd3 overexpression did not result in an increased resistance against oxidative stress. From the proteomic analysis of the two Neuro2A-HR subpopulations, 40 proteins were identified that were (A) present in only in Neuro2A-HR subpopulation (both) and only in Neuro2A parental or (B) showing a consistent increase or decrease in volume in both of the Neuro2A-HR subpopulations (E3 and E725) and (C) presenting a linear relationship inside the Neuro2A-HR subpopulation following their differential resistance to H2O2. Future work: Further investigations are necessary, especially in the validation of the targets identified from the proteomic part of the project but this project was a step forward in understanding how the oxidative stress can be fought. A common function amongst the identified genes and proteins was lipid metabolism, which seems to play an important role in cell survival and mediating protection against oxidative stress. Some of the identified targets have previously been found to be involved in inflammation and also in Alzheimer's disease, such as Apolipoprotein E, Cathepsin B and Splicing factor proline/glutamine-rich. The targets identified therefore likely represent key gene and protein changes that could protect against the oxidative stress associated with disease states.

Date of Award	2015
Original language	English