Cell Death Caused by the Synergistic Effects of Zn2+ and Dopamine is Mediated by a Stress Sensor Gene Gadd45b - Implication in the Pathogenesis of Parkinson’s Disease

We have previously found that Zn2+ and DA synergistically increase the apoptosis of PC12 cells and also deplete striatal dopamine content (Lo et al. 2004). To study the possible molecular mechanisms involved in the synergistic effects of Zn2+ and DA, mRNA isolated from Zn2+ and DA-treated PC12 cells was subjected to microarray analysis.

Microarray analysis

Microarray analysis was performed by the National Microarray & Gene Expression Analysis Core Facility of the National Research Program for Genomic Medicine, National Yang-Ming University. Microarray data set was generated using Affymetrix Rat Genome 230 2.0 platform that used samples from three independent experiments for from each of the four treatment sets: control, DA alone, Zn2+ alone, and Zn2+/DA, as described above.

The raw dataset (CEL file) was normalized using the RMA algorithm and the ‘affy’ package of the Bioconductor (http://www.bioconductor.org) (Gentleman et al. 2004) software suite, part of the R Project for Statistical Computing (http://www.r-project.org). The default RMA settings were used for the background correction, normalization and summarization of all expression values. In addition, the expression values of each gene were normalized across samples using a range from zero to one. This step preserves the richness in the original expression values for each gene among the samples and helps us easily visualize the distribution of expression values of the significant genes.

To select candidate genes involved in the Zn2+/DA -induced synergistic effects, the following criteria were set: (1) the difference between the means of the expression values for the conditions ‘control’ and ‘DA alone’, needed to be less than 0.1; (2) the mean of the expression values for the condition ‘Zn2+ alone’ should have a value at least 0.6 higher than the value of the mean of the expression values for the condition ‘DA’; and (3) the mean of the expression values for the condition ‘Zn2+/DA’ should have a value at least 0.2 higher than the value of the mean of the expression values for the condition ‘Zn2+’. Using the above criteria, Gadd45b obtained the second highest score and was selected for this study (Table S1).

Name Treatment File Name
Ctrl-1 Serum free DMEM 3hr S0157-005-Control.zip
Ctrl-2 Serum free DMEM 3hr S0157-009-Control.zip
Ctrl-3 Serum free DMEM 3hr S0162-001-Control.zip
DA-1 Serum free DMEM 1hr + 200 μM DA 2hr S0157-007-Da.zip
DA-2 Serum free DMEM 1hr + 200 μM DA 2hr S0157-010-Da.zip
DA-3 Serum free DMEM 1hr + 200μM DA 2hr S0162-002-Da.zip
Zn-1 400μM ZnCl2 1hr + Serum free DMEM 2hr S0157-006-Zn.zip
Zn-2 400μM ZnCl2 1hr + Serum free DMEM 2hr S0157-011-Zn.zip
Zn-3 400μM ZnCl2 1hr + Serum free DMEM 2hr S0162-003-Zn.zip
ZnDA-1 400μM ZnCl2 1hr + 200μM DA 2hr S0157-008-ZnDa.zip
ZnDA-2 400μM ZnCl2 1hr + 200μM DA 2hr S0157-012-ZnDa.zip
ZnDA-3 400μM ZnCl2 1hr + 200μM DA 2hr S0162-004-ZnDa.zip
Normalized Data RMA.txt
RMA-normalized.txt