Functional Repression of cAMP Response Element in 6-Hydroxydopamine-treated Neuronal Cells

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-OHDAtreated 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.Oxidative stress has been implicated in aging and a variety of pathological processes. In particular, oxidative mechanisms have been strongly linked to the pathogenesis of age-related neurodegenerative diseases (1). Neurons may be particularly susceptible to oxidants because of high metabolic activity and relatively low levels of endogenous antioxidants in the brain. Indeed, markers for lipid peroxidation and protein nitration are increased in affected brain areas in Alzheimer disease, Parkinson disease, and amyotrophic lateral sclerosis (2-4), and antioxidants provide protection in their respective animal and culture models (5-9). However, the mechanism(s) by which reactive oxygen species influence cell survival and death decisions are incompletely defined (10, 11). Given the progressive nature of neurodegenerative diseases, a better understanding of mechanisms that could impair adaptive responses to injury would be particularly pertinent.6-Hydroxydopamine (6-OHDA) 2 is a redox active analog of catecholamine neurotransmitters, which has been used to lesion the nigrostriatal system that deg...

6-Hydroxydopamine induces mitochondrial ERK activation

“…In addition, this sustained ERK activation is associated with nuclear translocation and cellular differentiation, while transient EGF-mediated ERK activation remains predominantly cytosolic and is associated with a proliferative response [69][70][71]. In pathologic conditions, different cell fates can also be mediated by different temporal and spatial patterns of kinase activation, with nuclear localization promoting neuroprotective signaling responses not observed during cytoplasmic accumulation of activated phosphoproteins [20]. In recent years, there has been intensifying interest in the intersection between kinase signaling pathways and mitochondria [38,72], particularly with the recognition that kinases and other proteins implicated genetically in parkinsonian neurodegeneration localize to mitochondria [73,74].…”

“…In recent years, there has been intensifying interest in the intersection between kinase signaling pathways and mitochondria [38,72], particularly with the recognition that kinases and other proteins implicated genetically in parkinsonian neurodegeneration localize to mitochondria [73,74]. In addition to altered subcellular distributions of signaling proteins observed in PD neurons [20,21,29] normal mitochondrial functions for phosphoproteins traditionally thought of as nuclear regulators have begun to be elucidated [75]. Poderosos and colleagues have demonstrated the presence of mitochondrial ERK in the matrix, intermembrane space, and outer membrane of the rat CNS [39].…”

“…In experiments assessing the effect of [5,10,15,20-tetrakis(1-ethylpyridinium-2-yl)porphyrinato] manganese(III) pentachloride (AEOL 10113, gift of Incara Pharmaceuticals, Research Triangle Park, NC), manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP, Alexis Biochemicals, San Diego, CA), and beef liver catalase (Roche Molecular Biochemicals, Indianapolis, IN, 260,000 U/ml), reagents were diluted in D10 and added 30 minutes prior to the addition of 6-OHDA or at different intervals following 6-OHDA (delayed administration time course). Heat-inactivation of catalase (5 minutes, 100 degrees) resulted in >90% loss of activity as confirmed by 2 assays for catalase activity: Amplex red catalase assay kit (Molecular Probes, Eugene, OR) or by monitoring the disappearance of hydrogen peroxide at 240 nm [28].…”

“…Notably, 6-OHDA injury recapitulates several features of degenerating neurons observed in human PD tissues. These include proteasome inhibition, α-synuclein aggregation, oxidation and nitration of proteins, increased protein ubiquitination, cleaved caspase 3 expression, glutathione depletion, and cytoplasmic accumulation of activated signaling proteins [14][15][16][17][18][19][20][21]. A better understanding of 6-OHDA mediated neurotoxicity could lend important insights into injury and degeneration pathways shared among different causes of dopaminergic neuron degeneration.…”

Splice Variant Biomarkers for Parkinson's Disease

“…Not surprisingly, this group of genes is involved in neurodegeneration. For example, reduced CREB signaling appears to be an underlying mechanism for neuronal death in PD (Chalovich et al, 2006) and tauopathies (Ljungberg et al, 2012). Neuroprotective effects afforded by leptin and the liver growth factor observed in 6-OHDA models of PD involve the regulation of CREB signaling (Weng et al, 2007;Gonzalo-Gobernado et al, 2013).…”