“Role of nuclear factor erythroid 2-related factor 2 (Nrf2) in the amelioration of amyotrophic lateral sclerosis (ALS)”

Christian Rosen¹, Bo Huang², Athanassios Fragoulis³, Thomas Pufe⁴, Christoph Jan Wruck⁵

1 Institute of Anatomy and Cell Biology, RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany

2 Institute of Anatomy and Cell Biology, RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany

3 Institute of Anatomy and Cell Biology, RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany

4 Institute of Anatomy and Cell Biology, RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany

5 Institute of Anatomy and Cell Biology, RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany

Abstract

INTRODUCTION: Mutations in Cu/Zn superoxide dismutase (SOD1) are a cause of motoneuron death in about 20 % of cases of familial amyotrophic lateral sclerosis (ALS). Although the molecular mechanism of which these mutations induce motoneuron cell death is to a large extent unknown. There is significant evidence that oxidative stress makes a major contribution to the selective death of motoneurons in this disease. Nuclear factor erythroid 2-related factor 2 (Nrf2) is known as the major regulator of a battery of genes encoding detoxifying and antioxidative enzymes via binding to the antioxidant response element (ARE). We and others had shown that activation of Nrf2 protects against a large variety of insults, particularly in neuronal cells.

The aim of the current in vitro study was to elucidate the protective role of the Nrf2/ARE-System against oxidative stress in the motoneuron-like cell line NSC34, transfected with human SOD1^G93A.

METHODS: The Nrf2/ARE-System in the motoneuron-like cell line NSC34, transfected with hSOD1^G93A, was activated via the stable/transient transfection of shRNA against the Nrf2 inhibitor Keap1. ARE activation was measured in motoneuron-like cell line NSC34, transfected with hSOD1^G93A, in a dose response assay utilising a Promega Luminometer. The potential of Keap1-RNAi knockdown NSC34 cells transfected with hSOD1^G93A was measured via cell viability assays. The expression of Nrf2 target genes and the effect of the gene silencing by RNAi were monitored by RT-PCR.

RESULTS: WST and MTT assays show a higher viability of NSC34 cells, transfected with shKeap1, compared to untransfected NSC34 cells. Correspondingly RT-PCR shows a higher expression of Nrf2 target genes compared to untransfected NSC34 cells.

CONCLUSIONS: We demonstrate that the activation of the Nrf2/ARE-System in the motor neuron-like cell line NSC34, transfected with hSOD1^G93A, leeds to a lower vulnerability against oxidative stress.

DOI^®: 10.3288/contoo.paper.1630