Abstract
Impaired survival signaling may represent a central mechanism in neurodegeneration. 6-Hydroxydopamine (6-OHDA) is an oxidative neurotoxin used to injure catecholaminergic cells of the central and peripheral nervous systems. Although 6-OHDA elicits phosphorylation of several kinases, downstream transcriptional effects that influence neuronal cell death are less 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. Message levels of CRE-driven genes such as brain-derived neurotrophic factor and the survival factor Bcl-2 were decreased in 6-OHDA-treated cells, but message levels of genes lacking CRE sequences were not affected. Repression of CRE could be reversed by delayed treatment with cAMP several hours after initiation of 6-OHDA injury. Furthermore, restoration of CRE-driven transcription 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, total CREB and phospho-CREB (pCREB) were increased in the cytoplasm and decreased in the nucleus of 6-OHDA-treated cells. 6-OHDA also decreased nuclear pCREB in dopaminergic neurons of primary mouse midbrain cultures. Co-treatment with cAMP promoted/restored nuclear localization of pCREB in both immortalized and primary culture systems. Increased cytoplasmic pCREB was observed in degenerating human Parkinson/Lewy body disease substantia nigra neurons but not in age-matched controls. Notably, cytoplasmic accumulation of activated upstream CREB kinases has been observed previously in both 6-OHDA-treated cells and degenerating human neurons, supporting a potential role for impaired nuclear import of phosphorylated signaling proteins.
Highlights
Models [5,6,7,8,9]
2 The abbreviations used are: 6-OHDA, 6-hydroxydopamine; BDNF, brain-derived neurotrophic factor; CRE, cAMP response element; Cyclic AMP response element-binding protein (CREB), CRE-binding protein; CREB-binding protein (CBP), CREBbinding protein; extracellular signal regulated protein kinases (ERK), extracellular signal-regulated protein kinase; ribosomal S6 kinase (RSK), ribosomal S-6 kinase; pCREB, activated CREB phosphorylated at Ser-133; pCRE-luc, reporter plasmid in which luciferase expression is regulated by CRE sequences; pERK, activated ERK phosphorylated at Thr-202/Tyr-204; PKA, protein kinase A; PBS, phosphate-buffered saline; LDH, lactate dehydrogenase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; TH, tyrosine hydroxylase; RT, reverse transcription; Z, benzyloxycarbonyl; AFC, N-acetyl-S-farnesyl-L-cysteine; Electrophoretic Mobility Shift Assay (EMSA), electrophoretic mobility shift assay; ANOVA, analysis of variance; Bt2cAMP, dibutyryl cAMP
Cytoplasmic Accumulation of pCREB Is Observed in Dopaminergic Neurons of Human Parkinson/Lewy Body Disease Patients—We previously found that kinases capable of functioning upstream of CREB are involved in granular or clumped cytoplasmic aggregates in Parkinson disease and the closely related diffuse Lewy body dementia and that this alteration is observed in early, presymptomatic patients [36]
Summary
6-OHDA, 6-hydroxydopamine; BDNF, brain-derived neurotrophic factor; CRE, cAMP response element; CREB, CRE-binding protein; CBP, CREBbinding protein; ERK, extracellular signal-regulated protein kinase; RSK, ribosomal S-6 kinase; pCREB, activated CREB phosphorylated at Ser-133; pCRE-luc, reporter plasmid in which luciferase expression is regulated by CRE sequences; pERK, activated ERK phosphorylated at Thr-202/Tyr-204; PKA, protein kinase A; PBS, phosphate-buffered saline; LDH, lactate dehydrogenase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; TH, tyrosine hydroxylase; RT, reverse transcription; Z, benzyloxycarbonyl; AFC, N-acetyl-S-farnesyl-L-cysteine; EMSA, electrophoretic mobility shift assay; ANOVA, analysis of variance; Bt2cAMP, dibutyryl cAMP. Repression of CRE in 6-OHDA Neurotoxicity phosphorylation of CREB. Phospho-CREB accumulates in the cytoplasm and is decreased in the nucleus of 6-OHDA-treated cells, and CRE-controlled genes such as BCL-2 and BDNF are down-regulated. Reversal of CRE repression by cAMP treatment confers significant protection from 6-OHDA toxicity, even when the cAMP is added 4 h after initiation of injury. These results demonstrate a transcriptional mechanism for oxidative stress-induced neurotoxicity with potential relevance to neurodegenerative mechanisms
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