Guanosine exerts neuroprotective effects in the central nervous system. Apoptosis, a morphological form of programmed cell death, is implicated in the pathophysiology of Parkinson’s disease (PD). MPP+, a dopaminergic neurotoxin, produces in vivo and in vitro cellular changes characteristic of PD, such as cytotoxicity, resulting in apoptosis. Undifferentiated human SH-SY5Y neuroblastoma cells had been used as an in vitro model of Parkinson’s disease. We investigated if extracellular guanosine affected MPP+-induced cytotoxicity and examined the molecular mechanisms mediating its effects. Exposure of neuroblastoma cells to MPP+ (10 μM–5 mM for 24–72 h) induced DNA fragmentation in a time-dependent manner (p < 0.05). Administration of guanosine (100 μM) before, concomitantly with or, importantly, after the addition of MPP+ abolished MPP+-induced DNA fragmentation. Addition of MPP+ (500 μM) to cells increased caspase-3 activity over 72 h (p < 0.05), and this was abolished by pre- or co-treatment with guanosine. Exposure of cells to pertussis toxin prior to MPP+ eliminated the anti-apoptotic effect of guanosine, indicating that this effect is dependent on a Gi protein-coupled receptor, most likely the putative guanosine receptor. The protection by guanosine was also abolished by the selective inhibitor of the enzyme PI-3-K/Akt/PKB (LY294002), confirming that this pathway plays a decisive role in this effect of guanosine. Neither MPP+ nor guanosine had any significant effect on α-synuclein expression. Thus, guanosine antagonizes and reverses MPP+-induced cytotoxicity of neuroblastoma cells via activation of the cell survival pathway, PI-3-K/Akt/PKB. Our results suggest that guanosine may be an effective pharmacological intervention in PD.
Apoptosis; Caspase-3; Cell survival; Cytotoxicity; DNA fragmentation; Guanosine; 1-methyl–4-phenyl-pyridinium (MPP+); Parkinson’s disease; SH-SY5Y human neuroblastoma cells
Accumulating evidence suggests that endogenous dopamine may act as a neurotoxin and thereby participate in the pathophysiology of Parkinson’s disease (PD). Cyclooxygenase-2 (COX-2) has been implicated in the pathogenesis of PD due to its ability to generate reactive oxygen species (ROS). Inhibition of COX-2 leads to neuroprotection by preventing the formation of dopamine-quinone. In this study, we examined whether dopamine mediates 1-methyl-4-phenylpyridinium (MPP+)-induced toxicity in primary ventral mesencephalic (VM) neurons, an in vitro model of PD, and if so, whether the protective effects of COX-2 inhibitors on dopamine mediated MPP+-induced VM neurotoxicity and VM dopaminergic cell apoptosis result from the reduction of ROS. Reserpine, a dopamine-depleting agent, significantly reduced VM neurotoxicity induced by MPP+, whereas dopamine had an additive effect on MPP+-induced VM neurotoxicity and VM dopaminergic cell apoptosis. However, inhibition of COX-2 by a selective COX-2 inhibitor (DFU) or ibuprofen significantly attenuated MPP+-induced VM cell toxicity and VM dopaminergic cell apoptosis, which was accompanied by a decrease in ROS production in VM dopaminergic neurons. These results suggest that dopamine itself mediates MPP+-induced VM neurotoxicity and VM dopaminergic cell apoptosis in the presence of COX-2.
Parkinson’s disease; Ventral mesencephalon; MPP+; Apoptosis; Neuroinflammation
Gastrodia elata (GE) Blume is one of the most important traditional plants in Oriental countries and has been used for centuries to improve various conditions. The phenolic glucoside gastrodin is an active constituent of GE. The aim of this study was to investigate the neuroprotective role of gastrodin in 1-methyl-4-phenylpyridinium (MPP+)/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- (MPTP) induced human dopaminergic SH-SY5Y cells and mouse model of Parkinson's disease (PD), respectively. Gastrodin significantly and dose dependently protected dopaminergic neurons against neurotoxicity through regulating free radicals, Bax/Bcl-2 mRNA, caspase-3, and cleaved poly(ADP-ribose) polymerase (PARP) in SH-SY5Y cells stressed with MPP+. Gastrodin also showed neuroprotective effects in the subchronic MPTP mouse PD model by ameliorating bradykinesia and motor impairment in the pole and rotarod tests, respectively. Consistent with this finding, gastrodin prevented dopamine depletion and reduced reactive astrogliosis caused by MPTP as assessed by immunohistochemistry and immunoblotting in the substantiae nigrae and striatata of mice. Moreover, gastrodin was also effective in preventing neuronal apoptosis by attenuating antioxidant and antiapoptotic activities in these brain areas. These results strongly suggest that gastrodin has protective effects in experimental PD models and that it may be developed as a clinical candidate to ameliorate PD symptoms.
Oxidative stress and apoptosis are frequently cited to explain neuronal cell damage in various neurodegenerative disorders, such as Parkinson' s disease. Brassinosteroids (BRs) are phytosterols recognized to promote stress tolerance of vegetables via modulation of the antioxidative enzyme cascade. However, their antioxidative effects on mammalian neuronal cells have never been examined so far.
We analyzed the ability of 24-epibrassinolide (24-Epi), a natural BR, to protect neuronal PC12 cells from 1-methyl-4-phenylpyridinium- (MPP+-) induced oxidative stress and consequent apoptosis in dopaminergic neurons. Our results demonstrate that 24-Epi reduces the levels of intracellular reactive oxygen species and modulates superoxide dismutase, catalase, and glutathione peroxidase activities. Finally, we determined that the antioxidative properties of 24-Epi lead to the inhibition of MPP+-induced apoptosis by reducing DNA fragmentation as well as the Bax/Bcl-2 protein ratio and cleaved caspase-3. This is the first time that the potent antioxidant and neuroprotective role of 24-Epi has been shown in a mammalian neuronal cell line.
Selective degeneration of nigrostriatal dopaminergic neurons in Parkinson disease (PD) can be modeled by the administration of the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Since abnormal mitochondrial dynamics are increasingly implicated in the pathogenesis of PD, in this study, we investigated the effect of MPP+ on mitochondrial dynamics and assessed temporal and causal relationship with other toxic effects induced by MPP+ in neuronal cells. In SH-SY5Y cells, MPP+ causes a rapid increase in mitochondrial fragmentation followed by a second wave of increase in mitochondrial fragmentation, along with increased DLP1 expression and mitochondrial translocation. Genetic inactivation of DLP1 completely blocks MPP+-induced mitochondrial fragmentation. Notably, this approach partially rescues MPP+-induced decline in ATP levels and ATP/ADP ratio and increased [Ca2+]i and almost completely prevents increased reactive oxygen species production, loss of mitochondrial membrane potential, enhanced autophagy and cell death, suggesting that mitochondria fragmentation is an upstream event that mediates MPP+-induced toxicity. On the other hand, thiol antioxidant NAC or glutamate receptor antagonist D-AP5 also partially alleviate MPP+-induced mitochondrial fragmentation, suggesting a vicious spiral of events contributes to MPP+-induced toxicity. We further validated our findings in primary rat midbrain dopaminergic neurons that 0.5 μM MPP+ induced mitochondrial fragmentation only in TH-positive dopaminergic neurons in a similar pattern to that in SH-SY5Y cells but had no effects on these mitochondrial parameters in TH-negative neurons. Overall, these findings suggest that DLP1-dependent mitochondrial fragmentation plays a crucial role in mediating MPP+-induced mitochondria abnormalities and cellular dysfunction and may represent a novel therapeutic target for PD.
MPP+; mitochondrial dynamics; Parkinson disease; DLP1/Drp1; mitochondrial fragmentation; neurotoxicity
Parkinson’s disease is a debilitating neurodegenerative disease characterized by loss of midbrain dopaminergic neurons. These neurons are particularly sensitive to the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which causes parkinsonian syndromes in humans, monkeys and rodents. Although apoptotic cell death has been implicated in MPTP/MPP+ toxicity, several recent studies have challenged the role of caspase-dependent apoptosis in dopaminergic neurons. Using the midbrain-derived MN9D dopaminergic cell line, we found that MPP+ treatment resulted in an active form of cell death that could not be prevented by caspase inhibitors or over-expression of a dominant negative inhibitor of apoptotic protease activating factor 1/caspase-9. Apoptosis inducing factor (AIF) is a mitochondrial protein that may mediate caspase-independent forms of regulated cell death following its translocation to the nucleus. We found that MPP+ treatment elicited nuclear translocation of AIF accompanied by large-scale DNA fragmentation. To establish the role of AIF in MPP+ toxicity, we constructed a DNA vector encoding a short hairpin sequence targeted against AIF. Reduction of AIF expression by RNA interference inhibited large-scale DNA fragmentation and conferred significant protection against MPP+ toxicity. Studies of primary mouse midbrain cultures further supported a role for AIF in caspase-independent cell death in MPP+-treated dopaminergic neurons.
dopaminergic cells; mitochondria; neuronal cell death; Parkinson’s disease; primary midbrain neurons; RNA interference
SU5416 was originally designed as a potent and selective inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) for cancer therapy. In this study, we have found for the first time that SU5416 unexpectedly prevented 1-methyl-4-phenylpyridinium ion (MPP+)-induced neuronal apoptosis in cerebellar granule neurons, and decreased 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced loss of dopaminergic neurons and impairment of swimming behavior in zebrafish in a concentration-dependent manner. However, VEGFR-2 kinase inhibitor II, another specific VEGFR-2 inhibitor, failed to reverse neurotoxicity at the concentration exhibiting anti-angiogenic activity, strongly suggesting that the neuroprotective effect of SU5416 is independent from its anti-angiogenic action. SU5416 potently reversed MPP+-increased intracellular nitric oxide level with an efficacy similar to 7-nitroindazole, a specific neuronal nitric oxide synthase (nNOS) inhibitor. Western blotting analysis showed that SU5416 reduced the elevation of nNOS protein expression induced by MPP+. Furthermore, SU5416 directly inhibited the enzyme activity of rat cerebellum nNOS with an IC50 value of 22.7 µM. In addition, knock-down of nNOS expression using short hairpin RNA (shRNA) abolished the neuroprotective effects of SU5416 against MPP+-induced neuronal loss. Our results strongly demonstrate that SU5416 might exert its unexpected neuroprotective effects by concurrently reducing nNOS protein expression and directly inhibiting nNOS enzyme activity. In view of the capability of SU5416 to cross the blood-brain barrier and the safety for human use, our findings further indicate that SU5416 might be a novel drug candidate for neurodegenerative disorders, particularly those associated with NO-mediated neurotoxicity.
Objective: To investigate astaxanthin (ATX) neuroprotection, and its mechanism, on a 1-methyl-4-phenyl-pyridine ion (MPP+)-induced cell model of Parkinson’s disease. Methods: Mature, differentiated PC12 cells treated with MPP+ were used as an in vitro cell model. The MTT assay was used to investigate cell viability after ATX treatment, and western blot analysis was used to observe Sp1 (activated transcription factor 1) and NR1 (NMDA receptor subunit 1) protein expression, real-time PCR was used to monitor Sp1 and NR1 mRNA, and cell immunofluorescence was used to determine the location of Sp1 and NR1 protein and the nuclear translocation of Sp1. Results: PC12 cell viability was significantly reduced by MPP+ treatment. The expression of Sp1 and NR1 mRNA and protein were increased compared with the control (p < 0.01). Following co-treatment with ATX and MPP+, cell viability was significantly increased, and Sp1 and NR1 mRNA and protein were decreased, compared with the MPP+ groups (p < 0.01). In addition, mithracycin A protected PC12 cells from oxidative stress caused by MPP+ by specifically inhibiting the expression of Sp1. Moreover, cell immunofluorescence revealed that ATX could suppress Sp1 nuclear transfer. Conclusion: ATX inhibited oxidative stress induced by MPP+ in PC12 cells, via the SP1/NR1 signaling pathway.
astaxanthin; Parkinson’s disease; MPP+; PC12 cells; Sp1; NR1
Estrogens exert many of their behavioral effects by binding to nuclear estrogen receptor (ER) proteins, ERα and ERβ. Recent studies involving ER knockout mice and selective ER agonists suggest that estradiol’s anorexigenic effect is mediated via activation of ERα. To investigate this hypothesis, we examined whether the presumptive ERα antagonist, MPP, could block estradiol’s anorexigenic effect. In the first series of experiments, the effects of MPP on food intake and uterine weight were monitored in ovariectomized (OVX) rats treated with either a physiological dose of estradiol benzoate (EB) or a selective ERα agonist (PPT). In the final experiment, food intake was monitored following acute administration of MPP in ovarian-intact (cycling) female rats. Contrary to our hypothesis, MPP failed to attenuate either EB’s or PPT’s ability to decrease food intake and increase uterine weight in OVX rats. However, in ovarian-intact rats, a similar regimen of MPP treatment attenuated the phasic decrease in food intake that is associated with estrus. We conclude that MPP may be a useful tool to investigate the behavioral actions of endogenous estradiol, but may have limited utility in studying the behavioral effects of exogenous estradiol in OVX rats.
SERM; Estradiol; Estrogen Receptor; MPP; PPT
Clavulanic acid is a psychoactive compound that has been shown to modulate central nervous system activity. Importantly, in neurotoxin-induced animal models, clavulanic acid has been shown to improve motor function (Huh et al., 2010) suggesting that it can be neuroprotective; however, the mechanism as how clavulanic acid can induce neuroprotection is not known. We demonstrate here that clavulanic acid abrogates the effects of the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) which mimics Parkinson’s disease (PD) by inducing neurodegeneration. To further establish the mechanism we identified that clavulanic acid inhibits neurotoxin-induced loss of mitochondrial membrane potential and ROS production. Consistent with these results, neurotoxin-induced increase in Bax levels was also decreased in clavulanic acid treated cells. Importantly, neurotoxin-induced release of cytochrome c levels as well as caspase activation was also inhibited in clavulanic acid treated cells. In addition, Bcl-xl levels were also restored and the Bcl-xl/Bax ratio that is critical for inducing apoptosis was increased in clavulanic acid treated cells. Overall, these results suggest that clavulanic acid is intimately involved in inhibiting neurotoxin-induced loss of mitochondrial function and induction of apoptosis that contributes towards neuronal survival.
Clavulanic acid; MPTP; Neuroprotection; Parkinson’s disease (PD); Mitochondria; Apoptosis
San-Huang-Xie-Xin-Tang (SHXT), composed of Coptidis rhizoma, Scutellariae radix, and Rhei rhizoma, is a traditional Chinese medicine used for complementary and alternative therapy of cardiovascular and neurodegenerative diseases via its anti-inflammatory and antioxidative effects. The aim of this study is to investigate the protective effects of SHXT in the 1–methyl–4–phenylpyridinium (MPP+)/1–methyl–4–phenyl–1,2,3,6–tetrahydropyridine (MPTP) models of Parkinson's disease. Rat primary mesencephalic neurons and mouse Parkinson disease model were used in this study. Oxidative stress was induced by MPP+ in vitro and MPTP in vivo. In MPP+-treated mesencephalic neuron cultures, SHXT significantly increased the numbers of TH-positive neurons. SHXT reduced apoptotic signals (cytochrome and caspase) and apoptotic death. MPP+-induced gp91phox activation and ROS production were attenuated by SHXT. In addition, SHXT increased the levels of GSH and SOD in MPP+-treated neurons. In MPTP animal model, SHXT markedly increased TH-positive neurons in the substantia nigra pars compacta (SNpc) and improved motor activity of mice. In conclusion, the present results reveal the evidence that SHXT possesses beneficial protection against MPTP-induced neurotoxicity in this model of Parkinson's disease via its antioxidative and antiapoptotic effects. SHXT might be a potentially alternative and complementary medicine for neuroprotection.
Apoptosis is a widely accepted component of the pathogenesis of Parkinson's disease (PD), a debilitating neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra. However, additional death programs were implicated, and current understanding of the cycle of intracellular events that leads to the demise of these neurons is limited. Gene therapy strategies proposed to inhibit apoptosis have met with relatively limited success. Here we report that the anti-apoptotic HSV-2 gene ICP10PK protects neuronally differentiated PC12 cells from death caused by 1-methyl-4-phenylpyridinium (MPP+) (in vitro PD model) through inhibition of calpain I activation and the resulting inhibition of Bax translocation to the mitochondria, AIF release and caspase-3 activation. Neuroprotection is through ICP10PK-mediated activation of the PI3-K/Akt survival pathway and up-regulation/stabilization of the anti-apoptotic protein Bcl-2 and the cytoprotective chaperone Hsp70.
ICP10PK; Parkinson's disease; MPP+; Programmed cell death; calpain; gene therapy
Lipopolysaccharide (LPS) is known to damage hepatocytes by cytokines released from activated Kupffer cells, but the ancillary role of LPS as a direct hepatotoxin is less well characterized. The aim of this study was to determine the direct effect of LPS on hepatocyte viability and the underlying signaling mechanism. Rat hepatocyte cultures treated overnight with LPS (500 ng/mL) induced apoptosis as monitored morphologically (Hoechst 33258) and biochemically (cleavage of caspase 3 and 9 and the appearance of cytochrome C in the cytoplasm). LPS-induced apoptosis was additive to that induced by glycochenodeoxycholate or Fas ligand, was associated with activation of c-Jun N-terminal kinase B (JNK) and p38 mitogen-activated protein kinases (MAPK), and inhibition of protein kinase (AKT). Inhibition of JNK by SP600125, but not of p38 MAPK by SB203580 attenuated LPS-induced apoptosis, indicating JNK dependency. CPT-2-Me-cAMP, an activator of cAMP-GEF, decreased apoptosis due to LPS alone or in combination with glycochenodeoxycholate or Fas ligand. CPT-2-Me-cAMP also prevented LPS-induced activation of JNK and inhibition of AKT. Taken together, these results suggest that LPS can induce hepatocyte apoptosis directly in vitro in a JNK-dependent manner and activation of cAMP-GEF protects against the LPS-induced apoptosis most likely by reversing the effect of LPS on JNK and AKT.
apoptosis; cAMP-GEF; AKT; exchange protein activated by cAMP (EPAC); lipopolysaccharide; JNK
Astaxanthin (AST) is a powerful antioxidant that occurs naturally in a wide variety of living organisms. We have investigated the role of AST in preventing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced apoptosis of the substantia nigra (SN) neurons in the mouse model of Parkinson’s disease (PD) and 1-methyl-4-phenylpyridinium (MPP+)-induced cytotoxicity of SH-SY5Y human neuroblastoma cells. In in vitro study, AST inhibits MPP+-induced production of intracellular reactive oxygen species (ROS) and cytotoxicity in SH-SY5Y human neuroblastoma cells. Preincubation of AST (50 μM) significantly attenuates MPP+-induced oxidative damage. Furthermore, AST is able to enhance the expression of Bcl-2 protein but reduce the expression of α-synuclein and Bax, and suppress the cleavage of caspase-3. Our results suggest that the protective effects of AST on MPP+-induced apoptosis may be due to its anti-oxidative properties and anti-apoptotic activity via induction of expression of superoxide dismutase (SOD) and catalase and regulating the expression of Bcl-2 and Bax. Pretreatment with AST (30mg /kg) markedly increases tyrosine hydroxylase (TH)-positive neurons and decreases the argyrophilic neurons compared with the MPTP model group. In summary, AST shows protection from MPP+/MPTP-induced apoptosis in the SH-SY5Y cells and PD model mouse SN neurons, and this effect may be attributable to upregulation of the expression of Bcl-2 protein, downregulation of the expression of Bax and α-synuclein, and inhibition of the activation of caspase-3. These data indicate that AST may provide a valuable therapeutic strategy for the treatment of progressive neurodegenerative disease such as Parkinson’s disease.
Astaxanthin; Parkinson’s disease; MPTP; Apoptosis; Substantia nigra neuron
The angiogenic factor, angiogenin, has been recently linked to both Amyotrophic Lateral Sclerosis (ALS) and Parkinson Disease (PD). We have recently shown that endogenous angiogenin levels are dramatically reduced in an alpha-synuclein mouse model of PD and that exogenous angiogenin protects against cell loss in neurotoxin-based cellular models of PD. Here, we extend our studies to examine whether activation of the prosurvival Akt pathway is required for angiogenin's neuroprotective effects against 1-methyl-4-phenylpyridinium (MPP+), as observed in ALS models, and to test the effect of virally-mediated overexpression of angiogenin in an in vivo PD model. Using a dominant negative Akt construct, we demonstrate that inhibition of the Akt pathway does not reduce the protective effect of angiogenin against MPP+ toxicity in the dopaminergic SH-SY5Y cell line. Furthermore, an ALS-associated mutant of angiogenin, K40I, which fails to induce Akt phosphorylation, was similar to wildtype angiogenin in protection against MPP+. These results confirm previous work showing neuroprotective effects of angiogenin against MPP+, and indicate that Akt is not required for this protective effect. We also investigated whether adeno-associated viral serotype 2 (AAV2)-mediated overexpression of angiogenin protects against dopaminergic neuron loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model. We found that angiogenin overexpression using this approach does not reduce the MPTP-induced degeneration of dopaminergic cells in the substantia nigra, nor limit the depletion of dopamine and its metabolites in the striatum. Together, these findings extend the evidence for protective effects of angiogenin in vitro, but also suggest that further study of in vivo models is required to translate these effects into meaningful therapies.
Aberrant mitochondrial function appears to play a central role in dopaminergic neuronal loss in Parkinson's disease (PD). 1-methyl-4-phenylpyridinium iodide (MPP+), the active metabolite of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a selective inhibitor of mitochondrial complex I and is widely used in rodent and cell models to elicit neurochemical alterations associated with PD. Recent findings suggest that Glycogen Synthase Kinase-3β (GSK-3β), a critical activator of neuronal apoptosis, is involved in the dopaminergic cell death. In this study, the role of GSK-3β in modulating MPP+-induced mitochondrial dysfunction and neuronal death was examined in vivo, and in two neuronal cell models namely primary cultured and immortalized neurons. In both cell models, MPTP/MPP+ treatment caused cell death associated with time- and concentration-dependent activation of GSK-3β, evidenced by the increased level of the active form of the kinase, i.e. GSK-3β phosphorylated at tyrosine 216 residue. Using immunocytochemistry and subcellular fractionation techniques, we showed that GSK-3β partially localized within mitochondria in both neuronal cell models. Moreover, MPP+ treatment induced a significant decrease of the specific phospho-Tyr216-GSK-3β labeling in mitochondria concomitantly with an increase into the cytosol. Using two distinct fluorescent probes, we showed that MPP+ induced cell death through the depolarization of mitochondrial membrane potential. Inhibition of GSK-3β activity using well-characterized inhibitors, LiCl and kenpaullone, and RNA interference, prevented MPP+-induced cell death by blocking mitochondrial membrane potential changes and subsequent caspase-9 and -3 activation. These results indicate that GSK-3β is a critical mediator of MPTP/MPP+-induced neurotoxicity through its ability to regulate mitochondrial functions. Inhibition of GSK-3β activity might provide protection against mitochondrial stress-induced cell death.
•We investigate the role of PPARδ in a model of Parkinson’s disease.•PPARδ is upregulated after the neurotoxin MPTP.•PPARδ antagonism enhances MPP+ toxicity which is reversible by PPARδ agonism.•PPARδ agonism protects against MPTP-toxicity.
Peroxisome proliferator-activated receptor (PPAR)-γ and PPARα have shown neuroprotective effects in models of Parkinson’s disease (PD). The role of the third, more ubiquitous isoform PPARδ has not been fully explored. This study investigated the role of PPARδ in PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to model the dopaminergic neurodegeneration of PD. In vitro administration of the PPARδ antagonist GSK0660 (1 μM) increased the detrimental effect of 1-methyl-4-phenylpyridinium iodide (MPP+) on cell viability, which was reversed by co-treatment with agonist GW0742 (1 μM). GW0742 alone did not affect MPP+ toxicity. PPARδ was expressed in the nucleus of dopaminergic neurons and in astrocytes. Striatal PPARδ levels were increased (over two-fold) immediately after MPTP treatment (30 mg/kg for 5 consecutive days) compared to saline-treated mice. PPARδ heterozygous mice were not protected against MPTP toxicity. Intra-striatal infusion of GW0742 (84 μg/day) reduced the MPTP-induced loss of dopaminergic neurons (5036 ± 195) when compared to vehicle-infused mice (3953 ± 460). These results indicate that agonism of PPARδ provides protection against MPTP toxicity, in agreement with the effects of other PPAR agonists.
DMEM, Dulbecco’s Modified Eagle Medium; DMSO, dimethyl sulfoxide; DPBS, Dulbecco’s phosphate-buffered saline; EDTA, ethylenediaminetetraacetic acid; FCS, foetal calf serum; GFAP, glial fibrillary acid protein; IL, interleukin; LDH, lactate dehydrogenase; MAC-1, macrophage antigen complex-1; MPP+, 1-methyl-4-phenylpyridinium iodide; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MTT, 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; PD, Parkinson’s disease; PFA, paraformaldehyde; PPAR, peroxisome proliferator-activated receptor; TH, tyrosine hydroxylase; TNFα, tumour necrosis factor-α; MPP+; neurodegeneration; neuroinflammation; SH-SY5Y; MPTP; PPAR delta
The role of the β2 Adrenergic Receptor (β2AR) in the regulation of chronic neurodegenerative inflammation within the CNS is poorly understood. The purpose of this study was to determine neuroprotective effects of long-acting β2AR agonists such as salmeterol in rodent models of Parkinson’s disease. Results showed salmeterol exerted potent neuroprotection against both LPS and MPTP/MPP+-induced dopaminergic neurotoxicity both in primary neuron-glia cultures (at sub-nanomolar concentrations) and in mice (1–10 μg/kg/day doses). Further studies demonstrated that salmeterol-mediated neuroprotection is not a direct effect on neurons; instead, it is mediated through the inhibition of LPS-induced microglial activation. Salmeterol significantly inhibited LPS-induced production of microglial pro-inflammatory neurotoxic mediators, such as TNFα, superoxide and nitric oxide, as well as the inhibition of TAK1-mediated phosphorylation of MAPK and p65 NF-κB. The anti-inflammatory effects of salmeterol required β2AR expression in microglia, but were not mediated through the conventional GPCR/cAMP pathway. Rather, salmeterol failed to induce microglial cAMP production, could not be reversed by either PKA inhibitors or an EPAC agonist, and was dependent on beta-arrestin2 expression. Together, our results demonstrate that administration of extremely low doses of salmeterol exhibit potent neuroprotective effects by inhibiting microglial cell activation through a β2AR/β-arrestin2-dependent but cAMP/PKA independent pathway.
Monocytes/Macrophages; Neuroimmunology; Inflammation
The present study examined the inhibitory effect of licorice compounds glycyrrhizin and a metabolite 18β-glycyrrhetinic acid on the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the mouse and on the 1-methyl-4-phenylpyridinium (MPP+)-induced cell death in differentiated PC12 cells. MPTP treatment increased the activities of total superoxide dismutase, catalase and glutathione peroxidase and the levels of malondialdehyde and carbonyls in the brain compared to control mouse brain. Co-administration of glycyrrhizin (16.8 mg/kg) attenuated the MPTP effect on the enzyme activities and formation of tissue peroxidation products. In vitro assay, licorice compounds attenuated the MPP+-induced cell death and caspase-3 activation in PC12 cells. Glycyrrhizin up to 100µM significantly attenuated the toxicity of MPP+. Meanwhile, 18β-glycyrrhetinic acid showed a maximum inhibitory effect at 10µM; beyond this concentration the inhibitory effect declined. Glycyrrhizin and 18β-glycyrrhetinic acid attenuated the hydrogen peroxide- or nitrogen species-induced cell death. Results from this study indicate that glycyrrhizin may attenuate brain tissue damage in mice treated with MPTP through inhibitory effect on oxidative tissue damage. Glycyrrhizin and 18β-glycyrrhetinic acid may reduce the MPP+ toxicity in PC12 cells by suppressing caspase-3 activation. The effect seems to be ascribed to the antioxidant effect.
Glycyrrhizin; MPTP; MPP+; Brain tissue damage; Cell death; Inhibitory effect
Peroxisome proliferator-activated receptor γ (PPARγ) agonists have been shown to provide neuroprotection in a number of neurodegenerative diseases including Parkinson's disease and Alzheimer's disease. These protective effects are primarily considered to result from the anti-inflammatory actions of PPARγ, however, there is increasing evidence that anti-oxidant mechanisms may also contribute. This study explored the impact of the PPARγ agonist rosiglitazone and the PPARγ antagonist GW9662 in the MPP+/MPTP (1-methyl-4-phenylpyridinium/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease, focussing on oxidative stress mechanisms. Rosiglitazone attenuated reactive oxygen species formation induced by MPP+ in SH-SY5Y cells concurrent with an upregulation of glutathione-S-transferase activity, but not superoxide dismutase activity. These responses were not attenuated by cotreatment with GW9662 suggesting that PPARγ activation is not required. The localisation of PPARγ in vivo to dopaminergic neurons of the substantia nigra pars compacta (SNpc) was established by immunohistochemistry and PPARγ levels were found to be upregulated 7 days after MPTP treatment. The importance of PPARγ in protecting against MPTP toxicity was confirmed by treating C57BL6 mice with GW9662. Treatment with GW9662 increased MPTP-induced neuronal loss in the SNpc whilst not affecting MPTP-induced reductions in striatal dopamine and 3,4-dihdroxyphenylacetic acid. GW9662 also caused neuronal loss in the SNpc of saline-treated mice. The evidence presented here supports the role of anti-oxidant mechanisms in the protective effects of PPARγ agonists in neurodegenerative diseases, but indicates that these effects may be independent of PPARγ activation. It also demonstrates the importance of PPARγ activity for neuronal survival within the SNpc.
► We investigate the role of PPARγ in a model of Parkinson's disease. ► Rosiglitazone has PPARγ independent anti-oxidant effects. ► Rosiglitazone involves upregulation of glutathione-S-transferase activity. ► PPARγ is upregulated after the neurotoxin MPTP. ► PPARγ antagonism is toxic to neurons in the SNpc.
BSO, buthionine-sulfoximine; DCF, 2′7′-dichlorofluorescein; DCF-DA, 2′7′-dichlorofluorescein diacetate; DOPAC, 3,4-dihydrophenylacetic acid; GFAP, glial fibrillary acid protein; GST, glutathione-S-transferase; Iba1, ionized calcium-binding adaptor molecule 1; LDH, lactate dehydrogenase; MPP+, 1-methyl-4-phenylpyridinium; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MTT, 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide; PD, Parkinson's disease; PPARγ, peroxisome proliferator-activated receptor γ; ROS, reactive oxygen species; SNpc, substantia nigra pars compacta; SOD, superoxide dismutase; TH, tyrosine hydroxylase; Parkinson's disease; Peroxisome proliferator-activated receptor γ; MPTP; MPP+; Neurodegeneration
Mammalian homologues of the Drosophila canonical transient receptor potential (TRP) proteins have been implicated to function as plasma membrane Ca2+ channels. This study examined the role of TRPC1 in human neuroblastoma (SH-SY5Y) cells. SH-SY5Y cells treated with an exogenous neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP+) significantly decreased TRPC1 protein levels. Confocal microscopy on SH-SY5Y cells treatment with MPP+ showed decreased plasma membrane staining of TRPC1. Importantly, overexpression of TRPC1 reduced neurotoxicity induced by MPP+. MPP+-induced α-synuclein expression was also suppressed by TRPC1 overexpression. Protection of SH-SY5Y cells against MPP+ was significantly decreased upon the overexpression of antisense TRPC1 cDNA construct or the addition of a nonspecific transient receptor potential channel blocker lanthanum. Activation of TRPC1 by thapsigargin or carbachol decreased MPP+ neurotoxicity, which was partially dependent on external Ca2+. Staining of SH-SY5Y cells with an apoptotic marker (YO-PRO-1) showed that TRPC1 protects SH-SY5Y neuronal cells against apoptosis. Further, TRPC1 overexpression inhibited cytochrome c release and decreased Bax and Apaf-1 protein levels. Interpretation of the above data suggests that reduction in the cell surface expression of TRPC1 following MPP+ treatment may be involved in dopaminergic neurodegeneration. Furthermore, TRPC1 may inhibit degenerative apoptotic signaling to provide neuroprotection against Parkinson’s disease-inducing agents.
The regulation of mitochondrial quality has emerged as a central issue in neurodegeneration, diabetes, and cancer. We utilized repeated low-dose applications of the complex I inhibitor 1-methyl-4-phenylpyridinium (MPP+) over 2 weeks to study cellular responses to chronic mitochondrial stress. Chronic MPP+ triggered depletion of functional mitochondria resulting in diminished capacities for aerobic respiration. Inhibiting autophagy/mitophagy only partially restored mitochondrial content. In contrast, inhibiting activation of extracellular signal-regulated protein kinases conferred complete cytoprotection with full restoration of mitochondrial functional and morphological parameters, enhancing spare respiratory capacity in MPP+ co-treated cells above that of control cells. Reversal of mitochondrial injury occurred when U0126 was added 1 week after MPP+, implicating enhanced repair mechanisms. Chronic MPP+ caused a >90% decrease in complex I subunits, along with decreases in complex III and IV subunits. Decreases in respiratory complex subunits were reversed by co-treatment with U0126, ERK1/2 RNAi or transfection of dominant-negative MEK1, but only partially restored by degradation inhibitors. Chronic MPP+ also suppressed the de novo synthesis of mitochondrial DNA-encoded proteins, accompanied by decreased expression of the mitochondrial transcription factor TFAM. U0126 completely reversed each of these deficits in mitochondrial translation and protein expression. These data indicate a key, limiting role for mitochondrial biogenesis in determining the outcome of injuries associated with elevated mitophagy.
Parkinson's disease; 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; autophagy; mitochondrial biogenesis; mitogen-activated protein kinases; mitochondrial transcription factor A
We have previously shown that the androgen-independent prostate cancer cells DU145, despite expressing Fas and FasL, were resistant to anti-Fas-induced apoptosis, and that this resistance could be overcome by pretreating the cells with sublethal doses of camptothecin. Here, we provide evidence that SAPK/JNK activity is required for camptothecin sensitization to anti-Fas-induced apoptosis. Camptothecin, but not Fas ligation, was shown to activate SAPK/JNK in a time-dependent manner, and to induce c-Jun expression. The effects were more prominent in cells treated with both camptothecin and anti-Fas. The expression levels of MKP-1, a phosphatase which regulates SAPK/JNK and which has been implicated in prostate cancer resistance to apoptosis, remained unchanged. Inhibition of caspases had no effect on the SAPK/JNK activation, suggesting that this activation is an upstream event in the Fas-signalling pathway, and is independent of caspase activity. Antisense oligonucleotides targeted to JNK1 and JNK2 reversed the effect of camptothecin. These results suggest that stress kinase activation can significantly influence the fate of androgen-independent prostate cancer cells following Fas receptor ligation. © 2000 Cancer Research Campaign
prostate cancer; apoptosis; Fas; camptothecin; SAPK/JNK
Neurotoxicity is involved in various neurodegenerative diseases including Parkinson’s disease (PD), which affects mesencephalic dopaminergic neurons of the substantia nigra (SN). Positive α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor modulators (PARMs, a.k.a. Ampakines, such as CX614) increase brain-derived neurotrophic factor (BDNF) protein levels in vivo and in cultured hippocampal slices. BDNF is a survival factor for various neuronal cell types including mesencephalic dopaminergic neurons. Using cultured mesencephalic and hippocampal slices, we investigated whether preincubation with CX614 could provide neuroprotection against MPP+ toxicity and whether such neuroprotection was mediated by BDNF. Various treatment protocols were tested to demonstrate CX614-induced neuroprotection against MPP+. Pretreatment with CX614 significantly reduced MPP+-induced toxicity and increased BDNF levels in both hippocampal and mesencephalic cultured slices; CX614 pretreatment for 6 h in hippocampal slices and 24 h in mesencephalic slices was sufficient to produce significant neuroprotection as assessed with lactate dehydrogenase release in slice medium and propidium iodide uptake in slices. Both a BDNF scavenger and an inhibitor of the BDNF receptor TrkB, abrogated CX614-mediated reduction of MPP+-induced toxicity. Inhibition of Ca2+-activated proteases, calpains, was also protective against MPP+-induced toxicity. However, co-application of calpain inhibitor with CX614 abolished CX614-mediated protection, suggesting a dual action of calpains in this model. We conclude that CX614 is neuroprotective against MPP+-induced toxicity, an effect mediated by increased BDNF expression and activation of BDNF-dependent signaling pathways. Our results provide support for using PARMs as a new therapy for neurodegenerative disorders, including PD.
Ampakine; BDNF; Dopamine; Hippocampus; Mesencephalon; Tyrosine hydroxylase; Calpain
The human M-Phase Phosphoprotein 1 (MPP1), previously identified through a screening of a subset of proteins specifically phosphorylated at the G2/M transition (1), is characterized as a plus-end-directed kinesin-related protein. Recombinant MPP1 exhibits in vitro microtubule-binding and microtubule-bundling properties as well as microtubules-stimulated ATPase activity. In gliding experiments using polarity-marked microtubules, MPP1 is a slow molecular motor that moves towards the microtubule plus-end at a 0.07 μm/s speed. In cycling cells, MPP1 localizes mainly to the nuclei in interphase. During mitosis, MPP1 is diffuse throughout the cytoplasm in metaphase and subsequently localizes to the midzone to further concentrate on the midbody. MPP1 suppression by RNA interference induces failure of cell division late in cytokinesis. We conclude that MPP1 is a new mitotic molecular motor required for completion of cytokinesis.
Adenosine Triphosphatases; metabolism; Amino Acid Sequence; Animals; Cell Cycle Proteins; Cell Division; physiology; Cell Line; Cell Movement; Cells; Cultured; Cloning; Molecular; Cytoplasm; metabolism; DNA; Complementary; metabolism; Electrophoresis; Polyacrylamide Gel; Epitopes; Flow Cytometry; G2 Phase; Gene Library; Genome; Green Fluorescent Proteins; Hela Cells; Humans; Immunoblotting; Insects; Kinesin; metabolism; Kinetics; Luminescent Proteins; metabolism; Metaphase; Microscopy; Fluorescence; Microtubules; metabolism; Mitosis; Molecular Sequence Data; Mutagenesis; Site-Directed; Phosphoproteins; metabolism; physiology; Phosphorylation; Protein Structure; Tertiary; RNA Interference; RNA; Small Interfering; metabolism; Recombinant Fusion Proteins; metabolism; Recombinant Proteins; metabolism; Sequence Analysis; DNA; Sequence Homology; Amino Acid; Time Factors; Tissue Distribution; Transfection