DYSFUNCTION OF THE CREB SIGNALING PATHWAY DURING 6-HYDROXYDOPAMINE NEUROTOXICITY

Disruption in important cell survival signaling pathways may represent a central mechanism in neurodegenerative disease processes. 6-Hydroxydopamine (6-OHDA) is an oxidative neurotoxin that is commonly used to injure catecholaminergic cells of the central and peripheral nervous systems, and has been used extensively to model Parkinson's Disease. Although it has been documented that 6-OHDA elicits phosphorylation of several kinases, downstream transcriptional effects that influence neuronal cell death are not well defined. The cAMP response element (CRE) is present in the promoter sequences of several important neuronal survival factors. Treatment of catecholaminergic neuronal cell lines (B65 and SH-SY5Y) with 6-OHDA resulted in repression of basal CRE transactivation, and message levels of CRE-mediated genes such as brain derived neurotrophic factor and the survival factor Bcl-2 were decreased in 6-OHDA-treated cells. Message levels of genes lacking CRE sequences were not affected. Interestingly, repression of CRE could be reversed by delayed treatment with cAMP several hours after initiation of 6-OHDA injury. Furthermore, this restoration of CRE-driven transcription, even up to 2 hours post 6-OHDA treatment, was associated with significant neuroprotection. In contrast to observations in other model systems, the mechanism of CRE repression did not involve decreased phosphorylation of its binding protein CREB. Instead, increased phospho-CREB was observed in 6-hydroxydopamine-treated cells, as both total CREB and phospho-CREB were markedly increased in the cytoplasm after treatment. Nuclear expression of p-CREB showed a different pattern, and was decreased in the nucleus of 6-hydroxydopamine-treated cells. 6-OHDA also decreased nuclear phospho-CREB in dopaminergic neurons of primary mouse midbrain cultures. Co-treatment with cAMP promoted/restored nuclear localization of phospho-CREB in both immortalized and primary culture systems, a trend that was associated with protection in both B65 and SY5Y cell lines. Additionally, when human Parkinson's/Lewy body brain tissue was examined, an intense clumped or granular distribution of cytoplasmic phospho-CREB was observed in degenerating substantia nigra neurons, with little to no cytoplasmic staining seen in age-matched controls. Overall, these studies suggest a common theme of impaired nuclear-cytoplasmic trafficking during oxidative neuronal injury processes, with disruption in CREB sub-cellular localization being a recurring trend. It is interesting to note that cytoplasmic accumulation of upstream CREB kinases has previously been observed in both 6-hydroxydopamine-treated cells and in degenerating Parkinson's disease neurons, further supporting a potential role for impaired nuclear import of phosphorylated signaling proteins in neuronal injury processes. These studies present important insight into oxidant-mediated modulation of survival signaling pathways in neuronal cells, may offer potential relevance to the pathogenic mechanisms underlying the progression of neurodegenerative disease