Down regulation of DJ-1 enhances cell death by oxidative stress, ER stress, and proteasome inhibition

doi:10.1016/j.bbrc.2003.11.056

Biochemical and Biophysical Research Communications

Volume 312, Issue 4, 26 December 2003, Pages 1342-1348

https://doi.org/10.1016/j.bbrc.2003.11.056 Get rights and content

Abstract

Mutations in DJ-1 gene have been linked to autosomal recessive early onset parkinsonism (AR-EOP). Although the mechanism of neuronal cell death due to DJ-1 mutation has not been fully elucidated, loss of DJ-1 function was considered to cause the phenotype. Here, we demonstrated that the down regulation of endogenous DJ-1 of the neuronal cell line by siRNA enhanced the cell death which was induced by oxidative stress, ER stress, and proteasome inhibition, but not by pro-apoptotic stimulus. The cell death with hydrogen peroxide was dramatically rescued by over-expression of wild-type DJ-1, but not by that of L166P mutant DJ-1. Furthermore, DJ-1 rescued the cell death caused by over-expression of Pael receptor, which was a substrate of Parkin, another gene product for autosomal recessive juvenile parkinsonism. These results suggest that loss of protective activity of DJ-1 from neuro-toxicity induced by these stresses contributes to neuronal cell death in AR-EOP with mutant DJ-1.

Section snippets

Methods

Plasmid constructs, cell culture, transfection, Western blotting. The constructions of expression plasmids of human DJ-1 were previously reported [5]. The coding region of human DJ-1 cDNA was subcloned into pEGFP-N1 (Clontech) (pEGFP-DJ-1). To make L166P mutant DJ-1 expression vector, thymin was changed to cytosine at position 497 from ORF start in of pEGFP-DJ-1 using the QuickChange site-directed mutagenesis system (Stratagene). siRNA-expressing vector was constructed by a previously reported

Results

Both siRNA vectors reduced expression level of endogenous mouse DJ-1 in Neuro2a cells or endogenous human DJ-1 in 293T cells by more than 90% on band intensity of Western blotting at 48 h after the transfection (Fig. 1A). Without stresses, down regulation of DJ-1 or over-expression of wild-type and mutant DJ-1 alone was confirmed to have no effect on cell death by LDH assay (data not shown).

After down regulation of endogenous DJ-1, Neuro2a cells were much more susceptible to the oxidative stress

Discussion

Homozygous deletion, missense mutations (L166P, M26I), and compound heterozygous mutation in DJ-1 gene have been reported to be directly pathogenic for the phenotype of AR-EOP [2], [8], [9]. A homozygous 14 kb large deletion removing exons 1–5 [2], and a compound heterozygous mutation leading to the frameshift in the exon 1 and the splice error in exon 7 [8], both are predicted to result in a loss of functional protein. Recently, L166P DJ-1 protein proved to be unstable and rapidly degraded when

Acknowledgements

This work was supported by grants from the Ministry of Education, Science and Culture of Japan, and from Ministry of Health, Labour and Welfare of Japan.

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Down regulation of DJ-1 enhances cell death by oxidative stress, ER stress, and proteasome inhibition

Abstract

Section snippets

Methods

Results

Discussion

Acknowledgements

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Cell

J. Biol. Chem.

Neuron

Mol. Cell.

Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism

Nature

Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism

Science

DJ-1 is an indicator for endogenous reactive oxygen species elicited by endotoxin

Free Radic. Res.