Treatment Of Mesencephalic Cultures Research Articles

Urocortin modulates dopaminergic neuronal survival via inhibition of glycogen synthase kinase-3β and histone deacetylase

Urocortin (UCN) is a member of the corticotropin-releasing hormone (CRH) family of neuropeptides that regulates stress responses. Although UCN is principally expressed in dopaminergic neurons in rat substantia nigra (SN), the function of UCN in modulating dopaminergic neuronal survival remains unclear. Using primary mesencephalic cultures, we demonstrated that dopaminergic neurons underwent spontaneous cell death when their age increased in culture. Treatment of mesencephalic cultures with UCN markedly prolonged the survival of dopaminergic neurons, whereas neutralization of UCN with anti-UCN antibody accelerated dopaminergic neurons degeneration. UCN increased intracellular cAMP levels followed by phosphorylating glycogen synthase kinase-3β (GSK-3β) on Ser9. Moreover, UCN directly inhibited the histone deacetylase (HDAC) activity and induced a robust increase in histone H3 acetylation levels. Using pharmacological approaches, we further demonstrated that inhibition of GSK-3β and HDAC contributes to UCN-mediated neuroprotection. These results suggest that dopaminergic neuron-derived UCN might be involved in an autocrine protective signaling mechanism.

Transient Receptor Potential Vanilloid Subtype 1 Mediates Cell Death of Mesencephalic Dopaminergic NeuronsIn VivoandIn Vitro

Intranigral injection of the transient receptor potential vanilloid subtype 1 (TRPV1; also known as VR1) agonist capsaicin (CAP) into the rat brain, or treatment of rat mesencephalic cultures with CAP, resulted in cell death of dopaminergic (DA) neurons, as visualized by immunocytochemistry. This in vivo and in vitro effect was ameliorated by the TRPV1 antagonist capsazepine (CZP) or iodo-resiniferatoxin, suggesting the direct involvement of TRPV1 in neurotoxicity. In cultures, both CAP and anandamide (AEA), an endogenous ligand for both TRPV1 and cannabinoid type 1 (CB1) receptors, induced degeneration of DA neurons, increases in intracellular Ca2+ ([Ca2+]i), and mitochondrial damage, which were inhibited by CZP, the CB1 antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) or the intracellular Ca2+ chelator BAPTA/AM. We also found that CAP or AEA increased mitochondrial cytochrome c release as well as immunoreactivity to cleaved caspase-3 and that the caspase-3 inhibitor z-Asp-Glu-Val-Asp-fmk protected DA neurons from CAP- or AEA-induced neurotoxicity. Additional studies demonstrated that treatment of mesencephalic cultures with CB1 receptor agonist (6aR)-trans 3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d] pyran-9-methanol (HU210) also produced degeneration of DA neurons and increases in [Ca2+]i, which were inhibited by AM251 and BAPTA/AM. The CAP-, AEA-, or HU210-induced increases in [Ca2+]i were dependent on extracellular Ca2+, with significantly different patterns of Ca2+ influx. Surprisingly, CZP and AM251 reversed HU210- or CAP-induced neurotoxicity by inhibiting Ca2+ influx, respectively, suggesting the existence of functional cross talk between TRPV1 and CB1 receptors. To our knowledge, this study is the first to demonstrate that the activation of TRPV1 and/or CB1 receptors mediates cell death of DA neurons. Our findings suggest that these two types of receptors, TRPV1 and CB1, may contribute to neurodegeneration in response to endogenous ligands such as AEA.

Nuclear translocation of nuclear transcription factor-kappa B by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors leads to transcription of p53 and cell death in dopaminergic neurons.

We describe a new molecular mechanism of cell death by excitotoxicity mediated through nuclear transcription factor kappa B (NF kappa B) in rat embryonic cultures of dopaminergic neurons. Treatment of mesencephalic cultures with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) resulted in a number of changes that occurred selectively in dopaminergic neurons, including persistent elevation in intracellular Ca(2+) monitored with Fura-2, and a significant increase in intramitochondrial oxidation of dihydrorhodamine 123, probably associated with transient increase of mitochondrial permeability, cytochrome c release, nuclear translocation of NF kappa B, and transcriptional activation of the oncogene p53. Interruption of any of these steps by specific antagonists prevented neurite pruning and programmed cell death. In contrast, cell death was not prevented by caspase antagonists and only partly prevented by nitric-oxide synthase inhibitors. This signal transduction pathway might be a contributing mechanism in ongoing neuronal death in Parkinson disease.

Stimulation of the regrowth of MPP + -damaged dopaminergic fibers by the treatment of mesencephalic cultures with basigin

Basigin (Bsg) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily and widely expressed in the central nervous system. To elucidate functional role of Bsg in the central nervous system, the effects of its glutathione-S-transferase (GST) fusion protein on the number and neurite outgrowth of cultured rat mesencephalic dopaminergic neurons were measured. The fusion protein was not able to promote the survival and neurite outgrowth of tyrosine hydroxylase (TH)-positive neurons under serum-free condition. However, the treatment of 1-methyl-4-phenylpyridinium (MPP+)-exposed cultures with the fusion protein resulted in stimulation of the regrowth of damaged TH-positive fibers. Basic fibroblast growth factor (bFGF) also stimulated the regrowth of neurites in damaged neurons. These results indicate that Bsg may play an important role in the regrowth of damaged dopaminergic fibers.

Hydrogen peroxide removal and glutathione mixed disulfide formation during metabolic inhibition in mesencephalic cultures.

Compromised mitochondrial energy metabolism and oxidative stress have been associated with the pathophysiology of Parkinson's disease. Our previous experiments exemplified the importance of GSH in the protection of neurons exposed to malonate, a reversible inhibitor of mitochondrial succinate dehydrogenase/complex II. This study further defines the role of oxidative stress during energy inhibition and begins to unravel the mechanisms by which GSH and other antioxidants may contribute to cell survival. Treatment of mesencephalic cultures with 10 microM buthionine sulfoximine for 24 h depleted total GSH by 60%, whereas 3 h exposure to 5 mM 3-amino-1,2,4-triazole irreversibly inactivated catalase activity by 90%. Treatment of GSH-depleted cells with malonate (40 mM) for 6, 12 or 24 h both potentiated and accelerated the time course of malonate toxicity, however, inhibition of catalase had no effect. In contrast, concomitant treatment with buthionine sulfoximine plus 3-amino-1,2,4-triazole in the presence of malonate significantly potentiated toxicity over that observed with malonate plus either inhibitor alone. Consistent with these findings, GSH depletion enhanced malonate-induced reactive oxygen species generation prior to the onset of toxicity. These findings demonstrate that early generation of reactive oxygen species during mitochondrial inhibition contributes to cell damage and that GSH serves as a first line of defense in its removal. Pre-treatment of cultures with 400 microM ascorbate protected completely against malonate toxicity (50 mM, 12 h), whereas treatment with 1 mM Trolox provided partial protection. Protein-GSH mixed disulfide formation during oxidative stress has been suggested to either protect vulnerable protein thiols or conversely to contribute to toxicity. Malonate exposure (50 mM) for 12 h resulted in a modest increase in mixed disulfide formation. However, exposure to the protective combination of ascorbate plus malonate increased membrane bound protein-GSH mixed disulfides three-fold. Mixed disulfide levels returned to baseline by 72 h of recovery indicating the reversible nature of this formation. These results demonstrate an early role for oxidative events during mitochondrial impairment and stress the importance of the glutathione system for removal of reactive oxygen species. Catalase may serve as a secondary defense as the glutathione system becomes limiting. These findings also suggest that protein-GSH mixed disulfide formation under these circumstances may play a protective role.

Energy stress-induced dopamine loss in glutathione peroxidase-overexpressing transgenic mice and in glutathione-depleted mesencephalic cultures.

The role of the glutathione system in protecting dopamine neurons from a mild impairment of energy metabolism imposed by the competitive succinate dehydrogenase inhibitor, malonate, was investigated in vitro and in vivo. Treatment of mesencephalic cultures with 10 microM buthionine sulfoxamine for 24 h reduced total glutathione levels in the cultures by 68%. Reduction of cellular glutathione per se was not toxic to the dopamine population, but potentiated toxicity when the cultures were exposed to malonate. In contrast, transgenic mice overexpressing glutathione peroxidase (hGPE) that received an intrastriatal infusion of malonate (3 mumol) into the left side had significantly less loss of striatal dopamine than their hGPE-negative littermates when assayed 1 week following infusion. These studies demonstrate that manipulation of the glutathione system influences susceptibility of dopamine neurons to damage due to energy impairment. The findings may provide insight into the loss of dopamine neurons in Parkinson's disease in which defects in both energy metabolism and the glutathione system have been identified.

Protection from 1-methyl-4-phenylpyridinium (MPP +) toxicity and stimulation of regrowth of MPP +-damaged dopaminergic fibers by treatment of mesencephalic cultures with EGF and basic FGF

Several peptide growth factors can maintain survival or promote recovery of injured central neurons. In the present study, the effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the toxicity produced by the dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium (MPP +), were investigated in rat mesencephalic dopaminergic neurons in culture. High affinity [ 3H]DA uptake and morphometric analyses of tyrosine hydroxylase immunostained neurons were used to assess the extent of MPP + toxicity, dopaminergic neuronal survival and growth of neurites. Consistent with previous reports, EGF and bFGF treatments stimulated neuritic outgrowth in dopaminergic neurons, increased DA uptake and enhanced their long-term survival in vitro. These growth factors also stimulated proliferation of astrocytes. The time course of EGF and bFGF effects on dopaminergic neurons coincided with the increase in glial cell density, suggesting that proliferation of glia mediates their trophic effects. Several findings from our study support this possibility. When MPP + was applied to cultures at 4 days in vitro, before glial cells had proliferated, the damage to dopaminergic neurons was not affected by EGF or bFGF pretreatments. However, when cultures maintained in the presence of the growth factors for 10 days were exposed to MPP +, after they had become confluent with dividing glial cells, the MPP +-induced decreases in DA uptake and cell survival were significantly attenuated. Furthermore, when glial cell proliferation was inhibited by 5-fluoro-2′-deoxyuridine, the protective effects of EGF and bFGF against MPP + toxicity were abolished. Continuous treatment of MPP +-exposed cultures with EGF or bFGF resulted in the stimulation of process regrowth of damaged dopaminergic neurons with concomitant recovery of DA uptake, suggesting that the injured neurons are able to respond to the trophic effects of EGF and bFGF. In summary, our study shows that the trophic effects of EGF and bFGF on mesencephalic dopaminergic neurons include protection from the toxicity produced by MPP + and promotion of recovery of MPP +-damaged neurons. Stimulation of glial cell proliferation is necessary for these effects.

Erratum for Engele et al., The Neurotrophic Effects of Fibroblast Growth Factors on Dopaminergic Neuronsin vitroAre Mediated by Mesencephalic Glia

The authors of “The Neurotrophic Effects of Fibroblast Growth Factors on Dopaminergic Neurons in vitro Are Mediated by Mesencephalic Glia” (J. Engele and M.C. Bohn,J Neurosci11:3070–3078) wish to correct an error in that paper. Treatment of mesencephalic cultures with aFGF or bFGF does not increase the survival of tyrosine hydroxylase-immunoreactive neurons by 190–210%, as reported in line 16 of the abstract, but by 90–110%. The authors regret this error.

Epidermal growth factor exerts neuronotrophic effects on dopaminergic and GABAergic CNS neurons: Comparison with basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) have been described to exert neuronotrophic effects on central nervous system neurons in culture. To study the selectivity of trophic actions of these growth factors, neurotransmitter-identified populations of embryonic rat mesencephalon were used. At 20 days in vitro, EGF (3 ng/ml) promoted survival and neurite outgrowth from these neurons. The neuritogenic effect of bFGF (3 ng/ml) was, however, more robust. Quantitative analysis with the neurofilament monoclonal antibody RR97 and ELISA confirmed the differential response, bFGF being 2-2.5 times more effective at all concentrations tested (ED100: 3-10 ng/ml for both EGF and bFGF). At 10 days in vitro, EGF displayed no trophic activity--even at 30 ng/ml. Treatment of mesencephalic cultures with EGF (3 ng/ml) for 20 days stimulated [3H]dopamine and [14C]GABA uptakes about 4-fold. While bFGF (3 ng/ml) also stimulated GABA uptake some 4-fold, dopamine uptake was increased almost 20-fold. Thus, EGF is also capable of enhancing the transmitter traits of selected central neuronal populations; however, the actions of bFGF appear to preferentially address dopaminergic cells.

Regeneration of monoaminergic and cholinergic neurons in the mammalian central nervous system

Regeneration of monoaminergic and cholinergic neurons in the mammalian central nervous system