Synergy Between Glutathione Peroxidase-1 and Astrocytic Growth Factors Suppresses Free Radical Generation and Protects Dopaminergic Neurons against 6-Hydroxydopamine

Abstract

The degeneration of dopaminergic neurons in the course of Parkinson disease is largely blamed on oxidative damage in the brain. This study examined the potency of glutathione peroxidase-1 (GPX-1) to protect dopaminergic neurons against toxicity induced by the parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA). We generated pLV-GPX1, a recombinant lentivirus vector carrying the coding sequence for human GPX-1, into the SK-N-MC neuroblastoma cell line. The pLV-GPX1-infected neurons showed an over 3-fold increase in enzyme expression and a 2.6-fold increase in enzyme activity compared to the pLV-EGFP-infected control cells. In the pLV-GPX1-infected cells, we also detected significantly increased neuronal survival and resistance to 6-OHDAmediated toxicity compared to our controls (75 AE 4% versus 51 AE 7%, p < 0.001). To maximize this protection, the neurons were treated with conditioned medium taken from growing primary astrocytes (astro-CM). We found the treated pLV-GPX1-infected neurons even more significantly resistant to 6-OHDA toxicity compared to their untreated counterparts (86 AE 5% versus 75 AE 4%, p < 0.001). Concomitant with increased neuroprotection, copresence of overexpressed GPX-1 and astro-CM significantly increased glutathione (GSH) levels compared to when either of the two was present ( p < 0.001). Further analysis showed nearly 2.7-fold reduction, in the presence of astro-CM, of hydrogen peroxide (H 2 O 2 ) levels released from the pLV-GPX1-infected neurons compared to control groups ( p < 0.001). Finally, regression analysis between H 2 O 2 levels and cell viability showed that co-presence of GPX-1 and astro-CM reduced 33% of cell death rate ( p < 0.05). These data highlight the antioxidant properties of GPX-1 in protecting dopaminergic neurons and further emphasize the capacity of astrocytes in pumping growth-inducing factors that may synergize with GPX-1 to accelerate neuroprotection.

Hippocampal antioxidative system in mesial temporal lobe epilepsy

“…These loci were observed in GD, CA regions, and alveus, and appear to represent bundles of astrocytes with highly expressed GPx. It has been proposed that astrocytic GPx is essential for H 2 O 2 detoxification in the central nervous system and that astrocytes protect neurons from oxidative stress . It is important to point out that H 2 O 2 is less reactive and has a larger diffusion radius compared to superoxide.…”

“…It has been proposed that astrocytic GPx is essential for H 2 O 2 detoxification in the central nervous system and that astrocytes protect neurons from oxidative stress. [23][24][25] It is important to point out that H 2 O 2 is less reactive and has a larger diffusion radius compared to superoxide. It leaks from mitochondria and cells, which is facilitated by some aquaporins.…”

SLUG and SOX9 Cooperatively Regulate Tumor Initiating Niche Factors in Breast Cancer

“…Recombinant lentivirus stocks were produced and concentrated as previously reported [9], Transfer vectors pTK380-Slug, pWPXL-Sox9, pLKO.Sh-SLUG and pLKO.Sh-SOX9 (Addgene), were respectively used for the coding sequences of mouse Slug and Sox9, and human shRNAs targeting SLUG and SOX9. Infected cells were expanded and used for RT-PCR.…”

Optimized Quantities of GDNF Overexpressed by Engineered Astrocytes Are Critical for Protection of Neuroblastoma Cells Against 6-OHDA Toxicity

“…To ensure of cDNA quality and sample equality, we also used standard GAPDH primers for each cDNA sample as we described (Gardaneh et al 2011). …”

“…One solution is to use astrocytic conditioned medium that allows monitoring function of single factors and dissecting their signaling pathways (Yoshida et al 1995;Dhandapani et al 2003;Zhu et al 2006;Ding et al 2009;Arai and Lo 2010). It also allows detection of possible synergism between its components and factors expressed within neurons, as shown in our recent study (Gardaneh et al 2011). Therefore, we used the astro-CM method that allowed monitoring positive and adverse effects of GDNF on neuronal cell survival:…”