Evaluation of the protective effect of oestradiol against toxicity induced by 6-hydroxydopamine and 1-methyl-4-phenylpyridinium ion (Mpp+) towards dopaminergic mesencephalic neurones in primary culture

Recent findings suggest that gonadal steroid hormones are neuroprotective and may provide clinical benefits in delaying the development of Parkinson's disease. In this report we investigated the ability of oestradiol to protect mesencephalic dopaminergic neurones cultured in serum-free or serum-supplemented medium from toxicity induced by 6-hydroxydopamine or 1-methyl-4-phenylpyridinium ion (MPP+). The efficiency of both toxins and oestradiol was evaluated by tyrosine hydroxylase (TH) immunocytochemistry, [3H]dopamine ([3H]DA) uptake, length of dopaminergic processes and lactate dehydrogenase (LDH) release measurement. In cultures grown in serum-supplemented medium, a 2-h pre-treatment with high concentrations (10-100 microM) of 17beta-oestradiol or 17alpha-oestradiol, the stereoisomer with weak oestrogenic activity, protected both dopaminergic and non-dopaminergic neurones from toxicity induced by 6-hydroxydopamine (6-OHDA; 40 or 100 microM) and by the high MPP+ concentrations (50 microM) necessary to obtain significant neuronal death under those culture conditions. At these concentrations, MPP+ was no longer selective for dopaminergic neurones but affected all cells present in the culture. In contrast, the hormonal treatments did not protect against selective degeneration of dopaminergic neurones induced by lower MPP+ concentrations (below 10 microM), related to inhibition of complex I of respiratory chain. In cultures grown in serum-free medium, oestradiol concentrations higher than 1 microM induced neuronal degeneration and no protection against 6-OHDA or MPP+ toxicity was observed at lower concentrations of the steroid. The neuroprotective effects of 17alpha- or 17beta-oestradiol evidenced in this model might be due to the antioxidant properties of these compounds. However, other non-genomic effects of the steroids cannot be excluded.     

Pramipexole protects against MPTP toxicity in non-human primates

The neurotoxin MPTP induces nigral dopaminergic cell death in primates and produces a partial model of Parkinson's disease (PD). Pramipexole is a D2/D3 dopamine receptor agonist used in the symptomatic treatment of PD, and which also protects neuronal cells against dopaminergic toxins in vitro. We now demonstrate that pramipexole partially prevents MPTP toxicity in vivo in a primate species. Common marmosets were repeatedly treated with pramipexole either before, coincidentally with, or after low-dose MPTP treatment designed to induce a partial lesion of the substantia nigra. Animals pretreated with pramipexole had a significantly greater number of surviving tyrosine hydroxylase (TH) positive neurones in the pars compacta of the substantia nigra. Pramipexole pretreatment also prevented degeneration of striatal dopamine terminals. Treatment with pramipexole concurrently with MPTP or following MPTP did not prevent TH-positive cell loss. Pramipexole pretreatment appears to induce adaptive changes that protect against dopaminergic cell loss in primates.     

Alterations in bioenergetic function induced by Parkinson's disease mimetic compounds: lack of correlation with superoxide generation

J. Neurochem. (2012) 122, 941-951. ABSTRACT: In vitro and in vivo models of Parkinson's disease (PD) suggest that increased oxidant production leads to mitochondrial dysfunction in dopaminergic neurons and subsequent cell death. However, it remains unclear if cell death in these models is caused by inhibition of mitochondrial function or oxidant production. The objective of this study was to determine the relationship between mitochondrial dysfunction and oxidant production in response to multiple PD neurotoxicant mimetics. MPP(+) caused a dose-dependent decrease in the basal oxygen consumption rate in dopaminergic N27 cells, indicating a loss of mitochondrial function. In parallel, we found that MPP(+) only modestly increased oxidation of hydroethidine as a diagnostic marker of superoxide production in these cells. Similar results were found using rotenone as a mitochondrial inhibitor, or 6-hydroxydopamine (6-OHDA) as a mechanistically distinct PD neurotoxicant, but not with exposure to paraquat. In addition, the extracellular acidification rate, used as a marker of glycolysis, was stimulated to compensate for oxygen consumption rate inhibition after exposure to MPP(+) , rotenone, or 6-OHDA, but not paraquat. Together these data indicate that MPP(+) , rotenone, and 6-OHDA dramatically shift bioenergetic function away from the mitochondria and towards glycolysis in N27 cells.     

Neuroprotection by pramipexole against dopamine- and levodopa-induced cytotoxicity

Pramipexole, a novel non-ergoline dopamine (DA) agonist, has been applied successfully for treatment of Parkinson's disease (PD). We report here that pramipexole can protect dopaminergic cell line Mes23.5 against dopamine- and levodopa-induced cytotoxicity possibly through a mechanism related to antioxidant activity. In the MES 23.5 cultures, DA and L-DOPA induce a dose- and time-dependent cytotoxicity, as determined by tetrazolium salt and trypan blue assays. Furthermore, an in situ terminal deoxynucleotidyl transferase assay demonstrates that DA-induced cell death is apoptotic. Pretreatment with pramipexole in a concentration range (4-100 microM) significantly attenuates DA- or L-DOPA-induced cytotoxicity and apoptosis, an action which is not blocked by D3 antagonist U-99194 A or D2 antagonist raclopride. Pramipexole also protects MES 23.5 cells from hydrogen peroxide-induced cytotoxicity in a dose-dependent manner. In cell-free system, pramipexole can effectively inhibit the formation of melanin, an end product resulting from DA or L-DOPA oxidation. These results indicate that pramipexole exerts its neuroprotective effect possibly through a mechanism, which is independent of DA receptors but related to antioxidation or scavenging of free radicals (e.g. hydrogen peroxide). As a direct DA agonist and potentially neuroprotective agent, pramipexole remains attractive in the treatment of PD.     

Distinct Effects of Rotenone, 1-methyl-4-phenylpyridinium and 6-hydroxydopamine on Cellular Bioenergetics and Cell Death

Parkinson's disease is characterized by dopaminergic neurodegeneration and is associated with mitochondrial dysfunction. The bioenergetic susceptibility of dopaminergic neurons to toxins which induce Parkinson's like syndromes in animal models is then of particular interest. For example, rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP(+)), and 6-hydroxydopamine (6-OHDA), have been shown to induce dopaminergic cell death in vivo and in vitro. Exposure of animals to these compounds induce a range of responses characteristics of Parkinson's disease, including dopaminergic cell death, and Reactive Oxygen Species (ROS) production. Here we test the hypothesis that cellular bioenergetic dysfunction caused by these compounds correlates with induction of cell death in differentiated dopaminergic neuroblastoma SH-SY5Y cells. At increasing doses, rotenone induced significant cell death accompanied with caspase 3 activation. At these concentrations, rotenone had an immediate inhibition of mitochondrial basal oxygen consumption rate (OCR) concomitant with a decrease of ATP-linked OCR and reserve capacity, as well as a stimulation of glycolysis. MPP(+) exhibited a different behavior with less pronounced cell death at doses that nearly eliminated basal and ATP-linked OCR. Interestingly, MPP(+), unlike rotenone, stimulated bioenergetic reserve capacity. The effects of 6-OHDA on bioenergetic function was markedly less than the effects of rotenone or MPP(+) at cytotoxic doses, suggesting a mechanism largely independent of bioenergetic dysfunction. These studies suggest that these dopaminergic neurotoxins induce cell death through distinct mechanisms and differential effects on cellular bioenergetics.     

Nitration of soluble proteins in organotypic culture models of Parkinson's disease

Protein nitration due to oxidative and nitrative stress has been linked to the pathogenesis of Parkinson's disease (PD), but its relationship to the loss of dopamine (DA) or tyrosine hydroxylase (TH) activity is not clear. Here we quantified protein-bound 3-nitrotyrosine (3-NT) by a novel gas chromatography/negative chemical ionization tandem mass spectrometry technique and DA and 3,4-dihydroxyphenylalanine (DOPA) by HPLC in tissues or medium of organotypic, mouse mesencephalon cultures after acute or chronic treatments with the peroxynitrite donor 3-morpholino-sydnonimine (SIN-1), the dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP(+)) or the lipophilic complex I inhibitor rotenone. Incubation with SIN-1 (24 h) or MPP(+) treatments (48 h) caused dose-dependent protein nitration reaching a maximum of eightfold increase by 10 mM SIN-1 or twofold by 10 microM MPP(+), but significant DA depletions occurred at much lower concentrations of MPP(+) (1 microM). Chronic MPP(+) or rotenone treatments (3 weeks) caused maximum protein nitration by 1 microM (twofold) or 10nM (fourfold), respectively. Co-treatment with the nitric oxide synthase inhibitor l-NAME (300 microM) prevented protein nitration by MPP(+), but did not protect against MPP(+)-induced DA depletion or inhibition of TH activity. Acute incubation with 100 microM SIN-1 inhibited TH activity, which could be blocked by co-treatment with the tetrahydrobiopterin precursor l-sepiapterin, but tissue DA depletions required higher doses of SIN-1 (>1 mM, 24 h) and longer survival. In conclusion, protein nitration and TH activity or DA depletion are not directly related in these models.     

Matrix metalloproteinase-3 is activated by HtrA2/Omi in dopaminergic cells: relevance to Parkinson's disease

Dopaminergic neurons in the substantia nigra are particularly vulnerable, and their degeneration leads to Parkinson's disease. We have previously reported that matrix metalloproteinase-3 (MMP-3) activity is involved in dopaminergic neurodegeneration by multiple mechanisms and that this requires activation of MMP-3 from proMMP-3 by an intracellular serine protease. HtrA2/Omi is a mitochondrial serine protease that has been shown in non-dopaminergic cells to translocate into the cytosol where it triggers apoptosis. In the present study we sought to determine whether HtrA2/Omi might cause activation of MMP-3 in dopaminergic neuronal cells using CATH.a cell line. Mitochondrial stress induced by rotenone led to MMP-3 activation and HtrA2/Omi translocation into the cytosol. The MMP-3 activation involved HtrA2/Omi, because both pharmacological inhibition and siRNA-induced knockdown of HtrA2/Omi attenuated the activation induced by rotenone or MPP+. Overexpression of mature HtrA2/Omi, but not mutant HtrA2/Omi, resulted in MMP-3 activity increase and cell death. Addition of recombinant and catalytically active HtrA2/Omi to lysate of untreated cells led to activation of the endogenous MMP-3, and incubation of the HtrA2/Omi with recombinant proMMP-3 caused cleavage of proMMP-3 to a 48kD protein, corresponding to the active form, which was accompanied by an increase in MMP-3 activity. Taken together, the data indicate that HtrA2/Omi, which normally exists in the mitochondria, can cause MMP-3 activation in the cytosol under a cell stress condition, which can ultimately lead to demise of dopaminergic neuronal cells.     

Enteric Neurodegeneration is Mediated Through Independent Neuritic and Somal Mechanisms in Rotenone and MPP+ Toxicity

Gut motility malfunction and pathological changes in the enteric nervous system (ENS) are observed in the early stages of Parkinson’s disease (PD). In many cases disturbances in the autonomous functions such as gut motility precedes the observed loss of central motor functions in PD. However, the mechanism by which ENS degeneration occurs in PD is unknown. We show that parkinsonian mimetics rotenone and MPP+ induce neurite degeneration that precedes cell death in primary enteric neurons cultured in vitro. If the neuronal death signals originate from degenerating neurites, neuronal death should be prevented by inhibiting neurite degeneration. Our data demonstrate that overexpression of cytNmnat1, an axon protector, maintains healthy neurites in enteric neurons treated with either of the parkinsonian mimetics, but cannot protect the soma. We also demonstrate that neurite protection via cytNmnat1 is independent of mitochondrial dynamics or ATP levels. Overexpression of Bcl-xl, an anti-apoptotic factor, protects both the neuronal cell body and the neurites in both rotenone and MPP+ treated enteric neurons. Our data reveals that Bcl-xl and cytNmnat1 act through separate mechanisms to protect enteric neurites. Our findings suggest that neurite protection alone is not sufficient to inhibit enteric neuronal degeneration in rotenone or MPP+ toxicity, and enteric neurodegeneration in PD may be occurring through independent somatic and neuritic mechanisms. Thus, therapies targeting both axonal and somal protection can be important in finding interventions for enteric symptoms in PD.     

MPP+ inhibits proliferation of PC12 cells by a p21(WAF1/Cip1)-dependent pathway and induces cell death in cells lacking p21(WAF1/Cip1).

The molecular and biochemical mode of cell death of dopaminergic neurons in Parkinson's disease (PD) is uncertain. In an attempt at further clarification we studied the effects of 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), on dopaminergic PC12 cells. In humans and nonhuman primates MPTP/MPP+ causes a syndrome closely resembling PD. MPP+ toxicity is thought to be mediated by the block of complex I of the mitochondrial electron transport chain. Treatment of undifferentiated PC12 cells with MPP+ primarily inhibited proliferation of PC12 cells and secondarily led to cell death after the depletion of all energy substrates by glycolysis. This cell death showed no morphological characteristics of apoptosis and was not blocked by treatment with caspase inhibitors. The inhibition of cell growth was not dependent on an inhibition of complex I activity since MPP+ also inhibited cell proliferation in SH-SY5Y cells lacking mitochondrial DNA and complex I activity (p0 cells). As shown by flow cytometric analysis, MPP+ induced a block in the G0/G1 to S phase transition that correlated with increased expression of the cyclin-dependent kinase inhibitor p21(WAF1/Cip1) and growth arrest. Since treatment with 1 microM MPP+ caused apoptotic cell death in p21(WAF1/Cip1)-deficient (p21(-/-)) but not in parental (p21(+/+)) mouse embryo fibroblasts, our data suggest that in an early phase MPP+-induced p21(WAF1/Cip1) expression leads to growth arrest and prevents apoptosis until energy depletion finally leads to a nonapoptotic cell death.     

Inhibitory Effects of Lysophosphatidylcholine on the Dopaminergic System

Lysophosphatidylcholine (lyso-PTC) is formed by phospholipase A2 (PLA2) from phosphatidylcholine (PTC), that is produced through phosphatidylethanolamine (PTE) methylation. 1-Methyl-4-phenyl-pyridinium (MPP+), a Parkinson's disease (PD) inducing agent, and S-adenosylmethionine (SAM), a biological methyl donor, increase lyso-PTC formation and both induce PD-like changes in animal models. In the current study, we investigated the effect of lyso-PTC on the dopaminergic system to determine the modulating role of lyso-PTC in dopaminergic neurotransmission. The results of these experiments show that lyso-PTC has a remarkable inhibitory effect on dopamine D1 and D2 receptor binding activities in the striatal membrane prepared from Sprague-Dawley rats. Lyso-PTC decreased the Bmax values of both D1 and D2 receptor binding activities. The Kd values for D1 and D2 receptors were not changed, but lyso-PTC also inhibited dopamine transporter and decreased striatal dopamine turnover rate. MPP+ showed similar, but less potent effects. The current studies suggest that lyso-PTC significantly impair the dopaminergic system and might play a role in MPP+ and SAM induced PD-like changes through its inhibitory effects on dopaminergic neurotransmission.     

1-Methyl-4-phenylpyridinium accumulates in cerebellar granule neurons via organic cation transporter 3

1-Methyl-4-phenylpyridinium (MPP+), the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, induces apoptosis in cerebellar granule neurons (CGNs). We have tested the hypothesis that organic cation transporter (OCT) 3 mediates the accumulation and, hence, the toxicity of MPP+ in CGNs. CGNs in primary culture express OCT3 but do not express mRNA for OCT1, OCT2 or the dopamine transporter. Cerebellar astrocytes are negative for OCT3 protein by immunocytochemistry. [3H]MPP+ accumulation by CGNs exhibits first-order kinetics, and a Kt value of 5.3 +/- 1.2 micro m and a Tmax of 0.32 +/- 0.02 pmol per min per 106 cells. [3H]MPP+ accumulation is inhibited by corticosterone, beta-estradiol and decynium 22 with Ki values of 0.25 micro m, 0.17 micro m and 4.0 nm respectively. [3H]MPP+ accumulation is also inhibited by desipramine, dopamine, serotonin and norepinephrine, but is not affected by carnitine (10 mm), mazindol (9 micro m) or GBR 12909 (1 micro m). MPP+-induced caspase-3-like activation and cell death are prevented by pretreatment with 5 micro mbeta-estradiol. In contrast, the neurotoxic effects of rotenone are unaffected by beta-estradiol. Interestingly, GBR 12909 protects CGNs from both MPP+ and rotenone toxicity. In summary, CGNs accumulate MPP+ in manner that is consistent with uptake via OCT3 and the presence of this protein in CGNs explains their sensitivity to MPP+ toxicity.     

Bioenergetic and oxidative effects of free 3-nitrotyrosine in culture: selective vulnerability of dopaminergic neurons and increased sensitivity of non-dopaminergic neurons to dopamine oxidation

Protein bound and free 3-nitrotyrosine (3NT) levels are elevated in neurodegenerative diseases and have been used as evidence for peroxynitrite generation. Intrastriatal injection of free 3NT causes dopaminergic neuron injury and represents a new mouse model of Parkinson's disease (PD). We are investigating the nature of free 3NT neurotoxicity. In primary ventral midbrain cultures, free 3NT damaged dopaminergic neurons, while adjacent non-dopaminergic neurons were unaffected. Combined treatment with free 3NT and subtoxic amounts of dopamine caused extensive death of non-dopaminergic forebrain neurons in culture. Free 3NT alone directly inhibited mitochondrial complex I, decreased ATP, sensitized neurons to mitochondrial depolarization, and increased superoxide production. Subtoxic concentrations of rotenone (instead of free 3NT) caused similar results. Additionally, free 3NT and dopamine combined increased extraneuronal hydrogen peroxide and decreased intraneuronal glutathione levels more than dopamine alone. Oxidative and bioenergetic processes have been proposed to contribute to neurodegeneration in PD. As free 3NT is a compound that is increased in PD, damages dopamine neurons in vivo and in vitro and has detrimental effects on neuronal bioenergetics, it is possible that free 3NT is an endogenous contributing factor to neuronal loss, in addition to being a marker of oxidative and nitrative processes.     

Dietary folate deficiency and elevated homocysteine levels endanger dopaminergic neurons in models of Parkinson's disease.

Although the cause of Parkinson's disease (PD) is unknown, data suggest roles for environmental factors that may sensitize dopaminergic neurons to age-related dysfunction and death. Based upon epidemiological data suggesting roles for dietary factors in PD and other age-related neurodegenerative disorders, we tested the hypothesis that dietary folate can modify vulnerability of dopaminergic neurons to dysfunction and death in a mouse model of PD. We report that dietary folate deficiency sensitizes mice to MPTP-induced PD-like pathology and motor dysfunction. Mice on a folate-deficient diet exhibit elevated levels of plasma homocysteine. When infused directly into either the substantia nigra or striatum, homocysteine exacerbates MPTP-induced dopamine depletion, neuronal degeneration and motor dysfunction. Homocysteine exacerbates oxidative stress, mitochondrial dysfunction and apoptosis in human dopaminergic cells exposed to the pesticide rotenone or the pro-oxidant Fe(2+). The adverse effects of homocysteine on dopaminergic cells is ameliorated by administration of the antioxidant uric acid and by an inhibitor of poly (ADP-ribose) polymerase. The ability of folate deficiency and elevated homocysteine levels to sensitize dopaminergic neurons to environmental toxins suggests a mechanism whereby dietary folate may influence risk for PD.     

Selective vulnerability of dopaminergic neurons to microtubule depolymerization

Parkinson disease (PD) is characterized by the specific degeneration of dopaminergic (DA) neurons in substantia nigra and has been linked to a variety of environmental and genetic factors. Rotenone, an environmental PD toxin, exhibited much greater toxicity to DA neurons in midbrain neuronal cultures than to non-DA neurons. The effect was significantly decreased by the microtubule-stabilizing drug taxol and mimicked by microtubule-depolymerizing agents such as colchicine or nocodazole. Microtubule depolymerization disrupted vesicular transport along microtubules and caused the accumulation of dopamine vesicles in the soma. This led to increased oxidative stress due to oxidation of cytosolic dopamine leaked from vesicles. Inhibition of dopamine metabolism significantly reduced rotenone toxicity. Thus, our results suggest that microtubule depolymerization induced by PD toxins such as rotenone plays a key role in the selective death of dopaminergic neurons.     

Pramipexole Reduces Reactive Oxygen Species Production In Vivo and In Vitro and Inhibits the Mitochondrial Permeability Transition Produced by the Parkinsonian Neurotoxin Methylpyridinium Ion

Abstract: Sporadic Parkinson's disease is associated with a defect in the activity of complex I of the mitochondrial electron transport chain. This electron transport chain defect is transmitted through mitochondrial DNA, and when expressed in host cells leads to increased oxygen free radical production, increased antioxidant enzyme activities, and increased susceptibility to programmed cell death. Pramipexole, a chemically novel dopamine agonist used for the treatment of Parkinson's disease symptoms, possesses antioxidant activity and is neuroprotective toward substantia nigral dopamine neurons in hypoxic-ischemic and methamphetamine models. We found that pramipexole reduced the levels of oxygen radicals produced by methylpyridinium ion (MPP⁺) both when incubated with SH-SY5Y cells and when perfused into rat striatum. Pramipexole also exhibited a concentration-dependent inhibition of opening of the mitochondrial transition pore induced by calcium and phosphate or MPP⁺. These results suggest that pramipexole may be neuroprotective in Parkinson's disease by attenuating intracellular processes such as oxygen radical generation and the mitochondrial transition pore opening, which are associated with programmed cell death.     

Alpha-synuclein protects naive but not dbcAMP-treated dopaminergic cell types from 1-methyl-4-phenylpyridinium toxicity

The pre-synaptic protein, alpha-synuclein, has been associated with the pathogenesis of Parkinson's disease. The present study indicates that alpha-synuclein, but not its mutants (A53T, A30P), can protect CNS dopaminergic cells from the parkinsonism-inducing drug 1-methyl-4-phenylpyridinium (MPP+), whereas it cannot protect from the dopaminergic toxin, 6-hydroxydopamine, hydrogen-peroxide, or the beta-amyloid peptide, A-beta. Protection from MPP+ was directly correlated with the preservation of mitochondrial function. Specifically, alpha-synuclein rescued cells from MPP+ mediated decreases in mitochondrial dehydrogenase activity and loss of ATP levels by utilizing ketosis. It also prevented toxin-induced activation of the creatine kinase/creatine phosphate system. Similarly, alpha-synuclein protected cells from the complex I inhibitor rotenone and 3-nitroproprionic acid, a complex II inhibitor. Wild-type alpha-synuclein-mediated neuroprotection and subsequent alterations in energy were not found in dbcAMP-differentiated cells. These results suggest that the normal physiological role for alpha-synuclein may change during development.     

Black carrot anthocyanins exhibit neuroprotective effects against MPP+ induced cell death and cytotoxicity via inhibition of oxidative stress mediated apoptosis

Parkinson’s disease (PD) is a common chronic neurodegenerative disease induced by the death of dopaminergic neurons. Anthocyanins are naturally found antioxidants and well-known for their preventive effects in neurodegenerative disorders. Black carrots (Daucus carota L. ssp. sativus var. atrorubens Alef.) are a rich source of anthocyanins predominantly including acylated cyanidin-based derivatives making them more stable. However, there have been no reports analysing the neuroprotective role of black carrot anthocyanins (BCA) on PD. In order to investigate the potential neuroprotective effect of BCA, human SH-SY5Y cells were treated with MPP+?(1-methyl-4-phenylpyridinium) to induce PD associated cell death and cytotoxicity. Anthocyanins were extracted from black carrots and the composition was determined by HPLC–DAD. SH-SY5Y cells were co-incubated with BCA (2.5, 5, 10, 25, 50, 100 µg/ml) and 0.5 mM MPP+?to determine the neuroprotective effect of BCA against MPP+?induced cell death and cytotoxicity. Results indicate that BCA concentrations did not have any adverse effect on cell viability. BCA revealed its cytoprotective effect, especially at higher concentrations (50, 100 µg/ml) by increasing metabolic activity and decreasing membrane damage. BCA exhibited antioxidant activity via scavenging MPP+?induced reactive oxygen species (ROS) and protecting dopaminergic neurons from ROS mediated apoptosis. These results suggest a neuroprotective effect of BCA due to its high antioxidant and antiapoptotic activity, along with the absence of cytotoxicity. The elevated stability of BCA together with potential neuroprotective effects may shed light to future studies in order to elucidate the mechanism and further neuro-therapeutic potential of BCA which is promising as a neuroprotective agent.

     

HEK-293 cells expressing the human dopamine transporter are susceptible to low concentrations of 1-methyl-4-phenylpyridine (MPP+) via impairment of energy metabolism.

Selective dopaminergic neurotoxicity induced by 1-methyl-4-phenylpyridine (MPP+) is believed to be due to the transmembrane uptake by the dopamine transporter and subsequent inhibition of mitochondrial complex I and/or production of free radicals. However, little is known about the molecular sequence of intracellular events leading to cell death induced by low concentrations of MPP+. Here we stably express the human dopamine transporter (hDAT) in human embryonic kidney HEK-293 cells to correlate cytotoxicity and indices of cellular energy metabolism after exposure to low concentrations of MPP+. The permanent ektopic expression of hDAT in HEK-293 cells confers time and dose-dependent cytotoxicity at nanomolar concentrations of MPP+ with an IC50 value of 740 nM after 48 h. MPP+ initially induces a fast increase of cellular NADH content within the first 6 h, followed by a slow reduction of intracellular ATP (IC50 value of 690 nM after 48 h) as well as reduction of intracellular ATP/ADP ratio. These changes of cellular energy metabolism precede reduction of cell viability. The toxic effects of MPP+ are blocked by the hDAT inhibitor GBR12909 with EC50 values of 110 and 60 nM for cytotoxicity and ATP depletion, respectively. Antioxidants such as D-alpha-tocopherol and ascorbic acid do not have significant protective effects against MPP+ toxicity. This study shows that HEK-293 cells expressing the hDAT gene are highly sensitive to MPP+ due to (i) transmembrane uptake of MPP+ by the dopamine transporter, (ii) cellular energy depletion, probably caused by inhibition of mitochondrial complex I activity and (iii) that the toxicity is independent from the presence of antioxidants. This cell system may serve as a screening system for endogenous and exogenous compounds with similar effects compared to MPP+ as well as protective agents.     

Cleavage of Bax is mediated by caspase-dependent or -independent calpain activation in dopaminergic neuronal cells: protective role of Bcl-2.

Two cysteine protease families, caspase and calpain, are known to participate in cell death. We investigated whether a stress-specific protease activation pathway exists, and to what extent Bcl-2 plays a role in preventing drug-induced protease activity and cell death in a dopaminergic neuronal cell line, MN9D. Staurosporine (STS) induced caspase-dependent apoptosis while a dopaminergic neurotoxin, MPP(+) largely induced caspase-independent necrotic cell death as determined by morphological and biochemical criteria including cytochrome c release and fluorogenic caspase cleavage assay. At the late stage of both STS- and MPP(+)-induced cell death, Bax was cleaved into an 18-kDa fragment. This 18-kDa fragment appeared only in the mitochondria-enriched heavy membrane fraction of STS-treated cells, whereas it was detected exclusively in the cytosolic fraction of MPP(+)-treated cells. This proteolytic cleavage of Bax appeared to be mediated by calpain as determined by incubation with [(35)S]methionine-labelled Bax. Thus, cotreatment of cells with calpain inhibitor blocked both MPP(+)- and STS-induced Bax cleavage. Intriguingly, overexpression of baculovirus-derived inhibiting protein of caspase, p35 or cotreatment of cells with caspase inhibitor blocked STS- but not MPP(+)-induced Bax cleavage. This appears to indicate that calpain activation may be either dependent or independent of caspase activation within the same cells. However, cotreatment with calpain inhibitor rescued cells from MPP(+)-induced but not from STS-induced neuronal cell death. In these paradigms of dopaminergic cell death, overexpression of Bcl-2 prevented both STS- and MPP(+)-induced cell death and its associated cleavage of Bax. Thus, our results suggest that Bcl-2 may play a protective role by primarily blocking drug-induced caspase or calpain activity in dopaminergic neuronal cells.     

Dopaminergic cell death induced by MPP(+), oxidant and specific neurotoxicants shares the common molecular mechanism

Recent etiological study in twins (Tanner et al. 1999) strongly suggests that environmental factors play an important role in typical, non-familial Parkinson's disease (PD), beginning after age 50. Epidemiological risk factor analyses of typical PD cases have identified several neurotoxicants, including MPP(+) (the active metabolite of MPTP), paraquat, dieldrin, manganese and salsolinol. Here, we tested the hypothesis that these neurotoxic agents might induce cell death in our nigral dopaminergic cell line, SN4741 (Son et al. 1999) through a common molecular mechanism. Our initial experiments revealed that treatment with both MPP(+) and the other PD-related neurotoxicants induced apoptotic cell death in SN4741 cells, following initial increases of H(2)O(2)-related ROS activity and subsequent activation of JNK1/2 MAP kinases. Moreover, we have demonstrated that during dopaminergic cell death cascades, MPP(+), the neurotoxicants and an oxidant, H(2)O(2) equally induce the ROS-dependent events. Remarkably, the oxidant treatment alone induced similar sequential molecular events: ROS increase, activation of JNK MAP kinases, activation of the PITSLRE kinase, p110, by both Caspase-1 and Caspase-3-like activities and apoptotic cell death. Pharmacological intervention using the combination of the antioxidant Trolox and a pan-caspase inhibitor Boc-(Asp)-fmk (BAF) exerted significant neuroprotection against ROS-induced dopaminergic cell death. Finally, the high throughput cDNA microarray screening using the current model identified downstream response genes, such as heme oxygenase-1, a constituent of Lewy bodies, that can be the useful biomarkers to monitor the pathological conditions of dopaminergic neurons under neurotoxic insult.