Oxidative modification of peroxiredoxin is associated with drug-induced apoptotic signaling in experimental models of Parkinson disease

The aim of this study was to investigate changes in protein profiles during the early phase of dopaminergic neuronal death using two-dimensional gel electrophoresis in conjunction with mass spectrometry. Several protein spots were identified whose expression was significantly altered following treatment of MN9D dopaminergic neuronal cells with 6-hydroxydopamine (6-OHDA). In particular, we detected oxidative modification of thioredoxin-dependent peroxidases (peroxiredoxins; PRX) in treated MN9D cells. Oxidative modification of PRX induced by 6-OHDA was blocked in the presence of N-acetylcysteine, suggesting that reactive oxygen species (ROS) generated by 6-OHDA induce oxidation of PRX. These findings were confirmed in primary cultures of mesencephalic neurons and in rat brain injected stereotaxically. Overexpression of PRX1 in MN9D cells (MN9D/PRX1) exerted neuroprotective effects against death induced by 6-OHDA through scavenging of ROS. Consequently, generation of both superoxide anion and hydrogen peroxide following 6-OHDA treatment was decreased in MN9D/PRX1. Furthermore, overexpression of PRX1 protected cells against 6-OHDA-induced activation of p38 MAPK and subsequent activation of caspase-3. In contrast, 6-OHDA-induced apoptotic death signals were enhanced by RNA interference-targeted reduction of PRX1 in MN9D cells. Taken together, our data suggest that the redox state of PRX may be intimately involved in 6-OHDA-induced dopaminergic neuronal cell death and also provide a molecular mechanism by which PRX1 exerts a protective role in experimental models of Parkinson disease.     

Leptin induces apoptosis via ERK/cPLA2/cytochrome c pathway in human bone marrow stromal cells

Leptin, the Ob gene product, has emerged recently as a key regulator of bone mass. However, the mechanism mediating leptin effect remains controversial. Because the action of leptin is dependent on its receptors, we analyzed their expression in osteoblast-lineage primary human bone marrow stromal cells (hBMSC). Both the short and long forms of leptin receptors were detected in hBMSC. Leptin significantly decreased the viability of hBMSC. This cytotoxic effect was prevented by Z-Val-Ala-Asp-fluoromethylketone, a pan-caspase inhibitor, implicating that leptin-induced hBMSC death was caspase-dependent. Further investigation demonstrated that leptin activated caspase-3 and caspase-9, but not caspase-8, and increased the cleavage of poly-(ADP-ribose) polymerase and cytochrome c release into cytosol. Leptin activated ERK, but not p38 and JNK, and up-regulated cPLA2 activity; the latter was abolished by pre-treatment of cells with the MEK inhibitor (PD98059 or U0126) or cPLA2 inhibitor (AACOCF3). PD98059, U0126, and AACOCF3 also diminished the leptin-induced cytochrome c release into cytosol, cell death, and caspase-3 activation. These data indicated that leptin induced hBMSC apoptosis via ERK/cPLA2/cytochrome c pathway with activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase. To our knowledge, this is the first study demonstrating the direct detrimental effect of leptin on bone cells.     

Regulation of HIF-1α activity by overexpression of thioredoxin is independent of thioredoxin reductase status

Berberine (BBR) is one of the major alkaloids and has been reported to have a variety of pharmacologic effects, including inhibition of cell cycle progression. Here, we investigated the mechanisms of BBR protection of neuronal cells from cell death induced by the Parkinson’s disease-related neurotoxin 6-hydroxydopamine (6-OHDA). Pretreatment of SH-SY5Y cells with BBR significantly reduced 6-OHDAinduced generation of reactive oxygen species (ROS), caspase-3 activation, and subsequent cell death. BBR also upregulated heme oxygenase-1 (HO-1) expression, which conferred protection against 6-OHDA-induced dopaminergic neuron injury and besides, effect of BBR on HO-1 was reversed by siRNA-Nrf2. Furthermore, BBR induced PI3K/Akt and p38 activation, which are involved in the induction of Nrf2 expression and neuroprotection. These results suggest that BBR may be useful as a therapeutic agent for the treatment of dopaminergic neuronal diseases.     

Low concentrations of methamphetamine can protect dopaminergic cells against a larger oxidative stress injury: mechanistic study

Mild stress can protect against a larger insult, a phenomenon termed preconditioning or tolerance. To determine if a low intensity stressor could also protect cells against intense oxidative stress in a model of dopamine deficiency associated with Parkinson disease, we used methamphetamine to provide a mild, preconditioning stress, 6-hydroxydopamine (6-OHDA) as a source of potentially toxic oxidative stress, and MN9D cells as a model of dopamine neurons. We observed that prior exposure to subtoxic concentrations of methamphetamine protected these cells against 6-OHDA toxicity, whereas higher concentrations of methamphetamine exacerbated it. The protection by methamphetamine was accompanied by decreased uptake of both [(3)H] dopamine and 6-OHDA into the cells, which may have accounted for some of the apparent protection. However, a number of other effects of methamphetamine exposure suggest that the drug also affected basic cellular survival mechanisms. First, although methamphetamine preconditioning decreased basal pERK1/2 and pAkt levels, it enhanced the 6-OHDA-induced increase in these phosphokinases. Second, the apparent increase in pERK1/2 activity was accompanied by increased pMEK1/2 levels and decreased activity of protein phosphatase 2. Third, methamphetamine upregulated the pro-survival protein Bcl-2. Our results suggest that exposure to low concentrations of methamphetamine cause a number of changes in dopamine cells, some of which result in a decrease in their vulnerability to subsequent oxidative stress. These observations may provide insights into the development of new therapies for prevention or treatment of PD.     

Age-related toxicity of amyloid-beta associated with increased pERK and pCREB in primary hippocampal neurons: reversal by blueberry extract

Further clarification is needed to address the paradox that memory formation, aging and neurodegeneration all involve calcium influx, oxyradical production (ROS) and activation of certain signaling pathways. In aged rats and in APP/PS-1 mice, cognitive and hippocampal Ca²⁺ dysregulation was reversed by food supplementation with a high antioxidant blueberry extract. Here, we studied whether neurons were an important target of blueberry extract and whether the mechanism involved altered ROS signaling through MAP kinase and cyclic-AMP response element binding protein (CREB), pathways known to be activated in response to amyloid-beta (Aβ). Primary hippocampal neurons were isolated and cultured from embryonic, middle-age or old-age (24 months) rats. Blueberry extract was found to be equally neuroprotective against Aβ neurotoxicity at all ages. Increases in Aβ toxicity with age were associated with age-related increases in immunoreactivity of neurons to pERK and an age-independent increase in pCREB. Treatment with blueberry extract strongly inhibited these increases in parallel with neuroprotection. Simultaneous labeling for ROS and for glutathione with dichlorofluorescein and monochlorobimane showed a mechanism of action of blueberry extract to involve transient ROS generation with an increase in the redox buffer glutathione. We conclude that the increased age-related susceptibility of old-age neurons to Aβ toxicity may be due to higher levels of activation of pERK and pCREB pathways that can be protected by blueberry extract through inhibition of both these pathways through an ROS stress response. These results suggest that the beneficial effects of blueberry extract may involve transient stress signaling and ROS protection that may translate into improved cognition in aging rats and APP/PS1 mice given blueberry extract.     

Leptin increases axonal growth cone size in developing mouse cortical neurons by convergent signals inactivating glycogen synthase kinase-3beta

We examined the effects of the adipose hormone leptin on the development of mouse cortical neurons. Treatment of neonatal and adult mice with intraperitoneal leptin (5 mg/kg) induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in pyriform and entorhinal cortex neurons. Stimulation of cultured embryonic cortical neurons with leptin evoked Janus kinase 2 and ERK1/2 phosphorylation and activated the downstream effector 90-kDa ribosomal protein S6 kinase. Moreover, leptin elicited the phosphorylation of the phosphatidylinositol 3-kinase effector Akt and evoked Ser-9 phosphorylation of glycogen synthase kinase-3beta (GSK3beta), an event inactivating this kinase. Leptin-mediated GSK3beta phosphorylation was prevented by the MEK/ERK inhibitor PD98059, the phosphatidylinositol 3-kinase inhibitor LY294002, or the protein kinase C inhibitor GF109203X. Exposure of cortical neurons to leptin also induced Ser-41 phosphorylation of the neuronal growth-associated protein GAP-43, an effect prevented by LY294002 and GF109203X but not by PD98059. Ser-41-GAP-43 phosphorylation is usually high in expanding axonal growth cones. Neurons exposed to 100 ng/ml leptin for 72 h displayed reduced rate of growth cone collapse, a shift of growth cone size distribution toward higher values, and a 4-fold increase in mean growth cone surface area compared with control cultures. The leptin-induced growth cone spreading was hampered in cortical neurons from Lepr(db/db) mice lacking functional leptin receptors; it was associated with localized Ser-9-GSK3beta phosphorylation and mimicked by the GSK3beta inhibitor SB216763. At concentrations preventing GSK3beta phosphorylation, PD98059, LY294002, or GF109203X reversed the leptin-induced growth cone surface enlargement. We concluded that the leptin-mediated regulation of growth cone morphogenesis in cortical neurons relies on upstream regulators of GSK3beta activity.     

Agmatine Protects Against 6-OHDA-Induced Apoptosis,and ERK and Akt/GSK Disruption in SH-SY5Y Cells

6-Hydroxydopamine (6-OHDA), a metabolite of dopamine is known to induce dopaminergic cell toxicity which makes that a suitable agent inducing an experimental model of Parkinson’s disease (PD). Agmatine has been shown to protect against some cellular and animal PD models. This study was aimed to assess whether agmatine prevents 6-OHDA-induced SH-SY5Y cell death and if yes, then how it affects Akt/glycogen synthesis kinase-3β (GSK-3β) and extracellular signal-regulated kinases (ERK) signals. The cells were treated with different drugs, and their viability was examined via MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay and morphological observation. Western blot studies were done to assess cleaved caspase-3, Akt/GSK-3β, and ERK proteins. 6-OHDA-induced cell death and caspase-3 cleavage, while agmatine prevented those changes. 6-OHDA also decreased the amount of phosphorylated Akt (pAkt)/Akt while increased GSK-3β activity which was prevented by agmatine. Additionally, this toxin increased pERK/ERK ratio which was averted again by agmatine. The PI3/Akt inhibitor, LY294002, impeded the changes induced by agmatine, while ERK inhibitor (PD98059) did not disturb the effects of agmatine, and by itself, it preserved the cells against 6-OHDA toxicity. This study revealed that agmatine is protective in 6-OHDA model of PD and affects Akt/GSK-3β and ERK pathways.     

The Potential Neuroprotection Mechanism of GDNF in the 6-OHDA-Induced Cellular Models of Parkinson’s Disease

The glial cell line-derived neurotrophic factor (GDNF) potential as a therapeutic agent for the treatment of Parkinson’s Disease (PD) has been extensively explored. However, the mechanism of the GDNF neuroprotective effects is still unclear. In this study, the neuroprotective mechanism of the GDNF in the PD cellular models, which was obtained by the 6-hydroxydopamine (6-OHDA)-induced dopaminergic (DA) cell line MN9D damage was investigated by microarray. Interestingly, 54 constitutively increased or decreased genes were detected, 17 of which have not been reported previously. The expression of 5 up-regulated and 5 down-regulated genes which displayed the most obvious changes compared to the no GDNF treatment cells and was previously proven to be related to cell survival was validated by real-time PCR and western blot. Moreover, the up-regulated gene Ager and down-regulated gene Ccnl2 which were related to the PI-3K/Akt signaling pathway, but not researched in the neuron-cells, were investigated by overexpression and RNA interference. Overexpression of Ager or knockdown the expression of Ccnl2 decreased the damage to MN9D cells caused by 6-OHDA and reduced their apoptosis. All these results suggested that the protective effects of the GDNF on the 6-OHDA damaged MN9D cells could be understood by enhancing the expression of the apoptosis inhibiting genes and decreasing the expression of the apoptosis promoting genes. Thus, this study might provide a number of specific candidates and potential targets to investigate the protective mechanism of GDNF in DA neurons.     

Long-term regulation of signalling components of adenylyl cyclase and mitogen-activated protein kinase in the pre-frontal cortex of human opiate addicts

Opiate addiction involves the development of chronic adaptive changes in micro -opioid receptors and associated pathways (e.g. cAMP signalling) which lead to neuronal plasticity in the brain. This study assessed the status of cAMP and mitogen-activated protein kinase (MAPK) pathways in brains (pre-frontal cortex) of chronic opiate addicts. In these subjects (n = 24), the immunodensities of adenylyl cyclase-I, PKA Calpha, total and phosphorylated CREB were not different from those in sex-, age- and PMD-matched controls. Moreover, the ratio pCREB/tCREB was similar in opiate addicts (0.74) and controls (0.76), further indicating that opiate addiction in humans is not associated with an upregulation of several key components of cAMP signalling in the pre-frontal cortex. In contrast, the components of MAPK cascade (Ras/c-Raf-1/MEK/ERK) were decreased in the same brains. Notably, pronounced downregulations of phosphorylated MEK (85%) and ERK1/2 (pERK1: 81%; pERK2: 80%) were quantitated in brains of opiate addicts. Chronic morphine treatment in rats (10-100 mg/kg for 5 days) was also associated with decreases of pERK1/2 (59-68%) in the cortex. In SH-SY5Y cells, morphine also stimulated the activity of pERK1/2 (2.5-fold) and the MEK inhibitor PD98059 blocked this effect (90%). The abnormalities of MAPK signalling might have important consequences in the long term development of various forms of neural plasticity associated with opiate addiction in humans.     

Phosphorylation of p38 MAPK induced by oxidative stress is linked to activation of both caspase-8- and -9-mediated apoptotic pathways in dopaminergic neurons

We evaluated the contribution of p38 mitogen-activated protein kinase and the events upstream/downstream of p38 leading to dopaminergic neuronal death. We utilized MN9D cells and primary cultures of mesencephalic neurons treated with 6-hydroxydopamine. Phosphorylation of p38 preceded apoptosis and was sustained in 6-hydroxydopamine-treated MN9D cells. Co-treatment with PD169316 (an inhibitor of p38) or expression of a dominant negative p38 was neuroprotective in death induced by 6-hydroxydopamine. The superoxide dismutase mimetic and the nitric oxide chelator blocked 6-hydroxydopamine-induced phosphorylation of p38, suggesting a role for superoxide anion and nitric oxide in eliciting a neurotoxic signal by activating p38. Following 6-hydroxydopamine treatment, inhibition of p38 prevented both caspase-8- and -9-mediated apoptotic pathways as well as generation of truncated Bid. Consequently, 6-hydroxydopamine-induced cell death was rescued by blockading activation of caspase-8 and -9. In primary cultures of mesencephalic neurons, the phosphorylation of p38 similarly appeared in tyrosine hydroxylase-positive, dopaminergic neurons after 6-hydroxydopamine treatment. This neurotoxin-induced phosphorylation of p38 was inhibited in the presence of superoxide dismutase mimetic or nitric oxide chelator. Co-treatment with PD169316 deterred 6-hydroxydopamine-induced loss of dopaminergic neurons and activation of caspase-3 in these neurons. Furthermore, inhibition of caspase-8 and -9 significantly rescued 6-hydroxydopamine-induced loss of dopaminergic neurons. Taken together, our data suggest that superoxide anion and nitric oxide induced by 6-hydroxydopamine initiate the p38 signal pathway leading to activation of both mitochondrial and extramitochondrial apoptotic pathways in our culture models of Parkinson's disease.     

Dopamine-related and caspase-independent apoptosis in dopaminergic neurons induced by overexpression of human wild type or mutant alpha-synuclein

Human wild type (WT) and mutant alpha-synuclein (alpha-syn) genes were overexpressed using a Tet-on expression system in stably transfected dopaminergic MN9D cells. Their overexpression induced caspase-independent and dopamine-related apoptosis not rescued by general caspase inhibitor Z-VAD-FMK. While apoptosis due to overexpression of WT alpha-syn was completely abrogated by a specific tyrosine hydroxylase (TH) inhibitor, alpha-methyl-p-tyrosine (alpha-MT), the inhibitor only partially rescued apoptosis caused by overexpression of alpha-syn mutants. In addition, overexpression of mutants enhanced the toxicity of 1-methyl-4-phenylpyridinium (MPP+) and 6-hydroxyldopamine (6-OHDA) to MN9D cells, whereas overexpression of WT protected MN9D cells against MPP+ toxicity, but not against 6-OHDA. We conclude that WT alpha-syn is beneficial to dopaminergic neurons but its overexpression in the presence of endogenous dopamine makes it a potential threat to the cells. In contrast, mutant alpha-syn not only caused the loss of WT protective function but also the gain-of-toxicity which becomes more serious in the presence of dopamine and neurotoxins.     

Isoliquiritigenin Isolated from Licorice Glycyrrhiza uralensis Prevents 6-Hydroxydopamine-Induced Apoptosis in Dopaminergic Neurons

Licorice (Glycyrrhiza uralensis) is a medicinal herb containing various bioactive components implicated in antioxidative, anti-inflammatory, antiviral, and neuroprotective effects, but the effects of licorice against Parkinson’s disease (PD)-related dopaminergic cell death have not been studied. In this study, we investigated the protective effects of isoliquiritigenin (ISL) isolated from Glycyrrhiza uralensis on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in a dopaminergic cell line, SN4741. ISL (1 μM) significantly attenuated 6-OHDA (50 μM)-induced reactive oxygen species (ROS) and nitric oxide (NO) generation and apoptotic cell death. ISL pretreatment effectively suppressed 6-OHDA-mediated upregulation of Bax, p-c-Jun N-terminal kinase (JNK), p-p38 mitogen-activated protein (MAP) kinase, cytochrome c release, and caspase 3 activation. In addition, ISL significantly attenuated 6-OHDA-induced Bcl-2, brain-derived neurotrophic factor (BDNF), and mitochondrial membrane potential (MMP) reduction. Pharmacological inhibitors of the phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) pathway reversed ISL-mediated neuroprotection against 6-OHDA toxicity in SN4741 cells. These results provide the first evidence that ISL can protect dopaminergic cells under oxidative stress conditions by regulating the apoptotic process.     

The neuronal insulin receptor in its environment

The statin atorvastatin (ATV) given as a post‐treatment has been reported beneficial in stroke, although the mechanisms involved are not well understood so far. Here, we investigated in vitro the effect of post‐treatment with ATV and its main bioactive metabolite ortho‐hydroxy ATV (o‐ATV) on neuroprotection after oxygen and glucose deprivation (OGD), and the role of the pro‐survival cAMP response element‐binding protein (CREB). Post‐OGD treatment of primary cultures of rat cortical neurons with o‐ATV, but not ATV, provided neuroprotection to a specific subset of cortical neurons that were large and positive for glutamic acid decarboxylase (large‐GAD⁽⁺⁾ neurons, GABAergic). Significantly, only these GABAergic neurons showed an increase in phosphorylated CREB (pCREB) early after neuronal cultures were treated post‐OGD with o‐ATV. We found that o‐ATV, but not ATV, increased the neuronal uptake of glutamate from the medium; this provides a rationale for the specific effect of o‐ATV on pCREB in large‐GABAergic neurons, which have a higher ratio of synaptic (pCREB‐promoting) vs extrasynaptic (pCREB‐reducing) N‐methyl‐D‐aspartate (NMDA) receptors (NMDAR) than that of small‐non‐GABAergic neurons. When we pharmacologically increased pCREB levels post‐OGD in non‐GABAergic neurons, through the selective activation of synaptic NMDAR, we observed as well long‐lasting neuronal survival. We propose that the statin metabolite o‐ATV given post‐OGD boosts the intrinsic pro‐survival factor pCREB in large‐GABAergic cortical neurons in vitro, this contributing to protect them from OGD.

     

Involvement of corticotropin-releasing factor receptor 2 beta in differentiation of dopaminergic MN9D cells

Corticotropin releasing factor (CRF) mediates various responses to stress through CRF receptors 1 and 2. CRF receptor 2 has two forms, 2alpha and 2beta each of which appears to have distinct roles. Here we used dopaminergic neuron-derived MN9D cells to investigate the function of CRF receptor 2 in dopamine neurons. We found that n-butyrate, a histone deacetylase inhibitor, induced MN9D cell differentiation and increased gene expression of all CRF receptors. CRF receptor 2beta was minimally expressed in MN9D cells; however, its expression dramatically increased during differentiation. CRF receptor 2beta expression levels appeared to correlate with neurite outgrowth, suggesting CRF receptor 2beta involvement in neuronal differentiation. To validate this statement, we made a CRF receptor 2beta-overexpressing MN9D/CRFR2 beta stable cell line. This cell line showed robust neurite outgrowth and GAP43 overexpression, together with MEK and ERK activation, suggesting MN9D cell neuronal differentiation. From these results, we conclude that CRF receptor 2beta plays an important role in MN9D cell differentiation by activating the MEK/ERK signaling pathway.     

The Expression and Release of Hsp60 in 6-OHDA Induced In Vivo and In Vitro Models of Parkinson’s Disease

In Parkinson’s disease, dopaminergic neuron damage/death causes the release of soluble substances that are selectively toxic to neighboring/additional dopaminergic neurons through the activation of microglia. Hsp60 can be released from injured cells of central nervous system to activate microglia. However, its expression and role in Parkinson’s disease has not been well understood. Here, we performed a 6-OHDA treated Parkinson’s disease model in adult rats. Western blot analysis showed a time-course expression of Hsp60, which decreased gradually and then rose back. Immunofluorescence staining showed that Hsp60 was decreased in dopaminergic neuron, and most Hsp60 located on the surface of activated microglia. Furthermore, in cellular Parkinson’s disease model, Hsp60 was obviously detected in the culture supernatants after 6-OHDA treatment, and a concomitant decrease in cell extracts. Taken together, our results suggested that Hsp60 could be released extracellularly to activate microglia in Parkinson’s disease model.     

Chronic leptin treatment stimulates lipid oxidation in immortalized and primary mouse skeletal muscle cells

Leptin administration enhances lipid oxidation in skeletal muscle. Nevertheless, direct and chronic effect of leptin has not been well characterized. Here, we measured the effect of leptin on skeletal muscles and their signaling pathways using differentiated C₂C₁₂ myotubes and primary myotube cultures. Differentiated myotubes expressed both the short and long forms of leptin receptors. Leptin increased lipid oxidation in myotubes in a concentration- and time-dependent manner, with significant induction of lipid oxidation occurring after 6 h. Actinomycin D completely blocked leptin-induced lipid oxidation. Leptin significantly increased phosphorylation of JAK2 and STAT3 in myotubes, and leptin-induced lipid oxidation was abolished by treatment with a JAK2 inhibitor or STAT3 siRNA. We then used mouse myotubes to measure these effects under physiological conditions. Leptin increased lipid oxidation, which again was blocked by a JAK2 inhibitor and STAT3 siRNA. These results suggest that the JAK2/STAT3 signaling pathway may underlie the chronic effects of leptin on lipid oxidation in skeletal muscles.