Synucleins as modulators of gene expression.

Abstract

Abstract: : Purpose: One of the features of ocular diseases is an alteration of gene expression pattern. Recently we found that synucleins act as transcriptional regulators of natrix metalloproteinases (MMP) genes and their modulation is mediated by AP–1 binding site. Synucleins belong to a family of proteins implicated in several neurodegenerative diseases and some forms of cancer. All three members of the family are expressed in the retina and optic nerve and the pattern of their expression and localization is significantly altered in eye pathologies. Here we examined the effect of alpha– and gamma–synuclein separately and together on gene expression in neuronal culture. Methods: Synucleins were expressed in HT22 neurons using a bicistronic vector pIRES. This bicistronic mammalian expression vector allows the expression of either one or two proteins. The cDNAs inserted in the A–site are expressed at a higher efficiency compared to the B–site. We confirmed the level of synuclein expression by Western blotting before proceeding to their effect on gene expression. RNA was isolated from overexpressing clones and used the Affymetrix U74A gene chip microarray. Results:The results of gene array demonstrated that the expression level of the majority of genes is not altered. However, some of the genes are considerably up– and down– regulated by alpha–synuclein and gamma–synuclein when they are expressed either separately or together. gamma–Synuclein is a stronger up–regulator, whereas a–synuclein is a stronger repressor of transcription. The expression level of several proteins involved in cell adhesion, transcription and cytoskeletal/extracellular matrix regulation is changed very significantly. The gene array data confirmed that matrix metalloproteinase MMP–9 implicated both in neurodegeneration and tumor progression is significantly upregulated by both alpha– and gamma–synuclein. The gene array showed 9 fold upregulation of MMP–9 by gamma–synuclein and 16–fold by alpha–synuclein. The expression of some other genes important for normal eye function and diseases is significantly changed, e.g. dopamine transporter, NRL and HSP70 were at least 3–fold downregulated, while NF–kappaB was 3 fold or more upregulated. Conclusion: The question of whether the influence of synucleins on gene expression is due to a direct effect on promoters of responsive genes or is exerted indirectly requires further investigation.

Keywords: gene microarray • gene/expression • protein structure/function