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

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

The glutamate antagonist, MK-801, does not prevent dopaminergic cell death induced by the 1-methyl-4-phenylpyridinium ion (MPP) in rat dissociated mesencephalic cultures

The neuroprotective effects of MK-801, a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor/channel, were assessed in a culture model which reproduces in vitro the selective degeneration of mesencephalic dopaminergic neurons seen in parkinsonian brains. Dissociated mesencephalic cells derived from rat embryonic brains were subjected for 24 h to intoxication by the 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPP+ at 3 and 10 microM produced selective and dose-dependent damages to dopaminergic neurons as quantified by the loss of the number of TH immunoreactive cells and the loss of [3H]DA uptake whereas other cell types remained unaffected. MK-801 at 3 and 10 microM failed to rescue degenerating dopaminergic neurons in presence of MPP+. At 50 microM, i.e. the highest concentration that is not toxic by itself in this culture system, MK-801 was also found ineffective. Furthermore, degree of dopaminergic cell damage was not reduced when repeated additions of the glutamate antagonist (10 microM/6 h for 24 h) were performed during exposure to MPP+ or when mesencephalic cultures were left after intoxication for up to 2 days in a culture medium still supplemented with MK-801 but free of toxin. In accordance with these results, MK-801 did not affect significantly the uptake of [3H]DA in control cultures, thereby suggesting that this compound cannot prevent intracellular accumulation of MPP+ within dopaminergic neurons. At higher concentrations of MPP+ (100 microM) tested, toxic effects were seen toward dopaminergic neurons and non-dopaminergic cells as quantified by Trypan blue dye accumulation and loss of [3H]GABA uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apparent opposite effects of tetrabenazine and reserpine on the toxic effects of 1-methyl-4-phenylpyridinium or 6-hydroxydopamine on nigro-striatal dopaminergic neurons

It is well documented that VMAT2 protects nigrostriatal DA neurons against MPP(+) by sequestering it inside vesicles away from its mitochondrial site of neurotoxic action. However, the implication of the VMAT2 in the mechanism of action exerted by 6-OHDA has received little attention. Therefore, the aim of the present study was to determine whether the vesicular sequestration of 6-OHDA would protect dopaminergic neurons from its toxicity similarly to what is observed with MPP(+). We injected mice with 6-OHDA 90 min after TBZ treatment. Since, unexpectedly, TBZ pretreatment prevented 6-OHDA neurotoxicity, we performed a similar experience replacing 6-OHDA with MPP(+) in order to check our experimental protocol. TBZ pretreatment similarly prevented MPP(+) neurotoxicity. This discrepancy with what is commonly describe in the literature, led us to use reserpine. Indeed, the long lasting VMAT2 inhibition induced by reserpine allowed us to inject neurotoxins while mice no longer presented hypothermia. Contrary to TBZ pretreatment, reserpine pretreatment potentiated both 6-OHDA and MPP(+) toxicity on dopaminergic neurons. Hypothermia elicited by TBZ appeared to be responsible, at least in part, for the neuroprotective effect observed. To verify this hypothesis, we investigated the influence of hypothermia on the toxic activity of both neurotoxins. A hypothermia similar to that induced by TBZ was obtained by a forced swimming test of putting mice into cool water (23 degrees C). The hypothermia prevented both 6-OHDA and MPP(+)-induced neurotoxicity. We finally reported that VMAT2 inhibition potentiates both MPP(+) and 6-OHDA neurotoxicity.     

Enhancement of glutamate uptake in 1-methyl-4-phenylpyridinium-treated astrocytes by trichostatin A

Histone deacetylases (HDAC) inhibitors have been emerging as neuroprotective agents by acting on neurons and microglia. In this study, we found trichostatin A (TSA), a HDAC inhibitor, could inhibit the elevation of glutamate in 150 microM 1-methyl-4-phenylpyridinium (MPP+)-treated primary cultured astrocytes medium when its concentration reached 132 nM. TSA of 132 nM or more could promote the uptake of [3H]-D, L-glutamate by astrocytes. Further study showed the downregulation of glutamate transporter 1 and glutamate/aspartate transporter induced by MPP+ were prevented by TSA. Therefore, these findings suggested TSA could alleviate MPP+-induced impairment of astrocytic glutamate uptake, which might be a novel mechanism contributing to neuroprotection by HDAC inhibitors.     

Comparison between survival of lazaroid-treated embryonic nigral neurons in cell suspensions,cultures and transplants

Death of transplanted dopaminergic neurons is induced both during preparation of donor tissue and after intrastriatal grafting. Oxidative stress is thought to be partly responsible for this cell death. In the present study we compared the effects of three lipid peroxidation inhibitors, the lazaroids Tirilazad mesylate, U-83836E and U-101033, on survival of embryonic mesencephalic neurons in different paradigms. The lazaroids were equally potent in preventing serum deprivation-induced death of cultured dopaminergic neurons. In a second set of experiments, mesencephalic suspensions were pretreated with lazaroids and cell survival was analyzed immediately after dissociation, after 2 or 24 h in culture or after intrastriatal transplantation. Lazaroid pretreatment failed to protect mesencephalic neurons in the in vitro paradigms and U-101033E did not protect grafted dopaminergic neurons in contrast to the neuroprotective effects previously reported for U-83836E and Tirilazad. Pretreatment with the iron chelator deferoxamine mesylate did not protect cultured or grafted dopaminergic neurons, nor did it improve neuronal survival in the serum deprivation model. U-83836E and U-101033E, but not Tirilazad, prevented cell death induced by the pro-oxidant tert-butyl hydroperoxide in suspensions. In a final experiment, we found that systemic treatment of the graft recipient rat with Tirilazad mesylate (before and during the first 3 days after grafting) improved survival of transplanted dopaminergic neurons to 180% of control values. Our results show that systemic treatment with a lipid peroxidation inhibitor for 3 days can promote graft survival, but also highlights the poor correlation between neuroprotective effect of pharmacological compounds in vitro and in grafts.     

Neuroimmunophilin ligands exert neuroregeneration and neuroprotection in midbrain dopaminergic neurons

Immunosuppressant drugs, like FK506, and nonimmunosuppressant compounds like, GPI1046 and L685818, are immunophilin ligands that specifically bind to immunophilins, like FK506 binding protein 12 (FKBP12). Several lines of evidence show that these ligands exert neurotrophic properties in neural injury models and in PC12 cells. However, the mechanism of the neurotrophic function of the immunophilin ligands is poorly known. In the present study, we use MPP+ and 6-OHDA toxicity models to examine both neuroprotective and neuroregenerative effects of immunophilin ligands on primary cultures of midbrain dopaminergic neurons. We find that FK506, GPI1046 and L685818 at concentrations from 0.01 to 1 microM partially, but significantly, protect dopaminergic neurons against both MPP+ and 6-OHDA toxicity. By Western blot analysis, we also find that all three compounds prevent tyrosine hydroxylase (TH) loss induced by MPP+ and 6-OHDA treatments. Morphologic analysis of dopaminergic neurons, by immunocytochemistry, shows that MPP+ and 6-OHDA cause the retraction and loss of neuronal processes, while FK506, GPI1046 and L685818 promote regeneration of these processes as indicated by increases in process number and length. To examine if FKBP12 is required for neurotrophic effects of immunophilin ligands, we cultured dopaminergic neurons from FKBP12 knockout mice and find that FK506 still protects dopaminergic neurons against MPP+ toxicity. These results suggest that FKBP12 is not essential for the neurotrophic properties of immunophilin ligands, and immunophilin ligands are a new class of neuroprotective and neuroregenerative agents that may have therapeutic potential in a variety of neurological disorders.     

Ascorbate prevents cell death from prolonged exposure to glutamate in an in vitro model of human dopaminergic neurons

Ascorbate (vitamin C) is a nonenzymatic antioxidant highly concentrated in the brain. In addition to mediating redox balance, ascorbate is linked to glutamate neurotransmission in the striatum, where it renders neuroprotection against excessive glutamate stimulation. Oxidative stress and glutamatergic overactivity are key biochemical features accompanying the loss of dopaminergic neurons in the substantia nigra that characterizes Parkinson's disease (PD). At present, it is not clear whether antiglutamate agents and ascorbate might be neuroprotective agents for PD. Thus, we tested whether ascorbate can prevent cell death from prolonged exposure to glutamate using dopaminergic neurons of human origin. To this purpose, dopamine‐like neurons were obtained by differentiation of SH‐SY5Y cells and then cultured for 4 days without antioxidant (antiaging) protection to evaluate glutamate toxicity and ascorbate protection as a model system of potential factors contributing to dopaminergic neuron death in PD. Glutamate dose dependently induced toxicity in dopaminergic cells largely by the stimulation of AMPA and metabotropic receptors and to a lesser extent by N‐methyl‐D‐aspartate and kainate receptors. At relatively physiological levels of extracellular concentration, ascorbate protected cells against glutamate excitotoxicity. This neuroprotection apparently relies on the inhibition of oxidative stress, because ascorbate prevented the pro‐oxidant action of the scavenging molecule quercetin, which occurred over the course of prolonged exposure, as is also seen with glutamate. Our findings show the relevance of ascorbate as a neuroprotective agent and emphasize an often underappreciated role of oxidative stress in glutamate excitotoxicity. Occurrence of a glutamate–ascorbate link in dopaminergic neurons may explain previous contradictions regarding their putative role in PD. © 2013 Wiley Periodicals, Inc.     

Ginsenosides Rb1 and Rg1 effects on mesencephalic dopaminergic cells stressed with glutamate

Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), is a well known and popular herbal medicine used worldwide. Among more than 30 ginsenosides, the active ingredients of ginseng, ginsenosides Rb1 and Rg1 are regarded as the main compounds responsible for many pharmaceutical actions of ginseng. In our study, primary cultures from embryonic mouse mesencephala were exposed to neurotoxic glutamate concentration and potential protective effects of these two ginsenosides on survival and neuritic growth of dopaminergic cells were tested. Treatment of primary mesencephalic culture with 500 microM glutamate for 15 min on the 10th day in vitro (DIV) increased the release of lactate dehydrogenase (LDH) into the culture medium, the propidium iodide (PI) uptake by cultured cells and the total number of nuclei with condensed and fragmented chromatin (apoptotic features) as evaluated with Hoechst 33342. Moreover, it extensively decreased the number of tyrosine hydroxylase immunopositive (TH+) cells and adversely affected the length and number of their neuronal processes. The toxic effect of glutamate was primarily mediated by over-activation of N-methyl-D-aspartate receptor (NMDA) as treatment of cultured cells with (+)-MK 801, an NMDA receptor antagonist, nearly abolished dopaminergic cells loss and LDH release induced by glutamate. When either ginsenoside was added alone for six consecutive days (at final concentrations 0.1, 1, 10, 20 microM), ginsenoside Rb1 (at 10 microM) significantly enhanced the survival of dopaminergic neurons compared to untreated controls. In these cultures, neurite lengths and numbers were not affected by both ginsenosides. Against glutamate exposure, ginsenosides Rb1 and Rg1 could not prevent cell death. However when pre-treating for 4 days or post-treating for 2 days following glutamate exposure, they significantly increased the numbers and lengths of neurites of surviving dopaminergic cells. Thus our study indicates that ginsenosides Rb1 and Rg1 have a partial neurotrophic and neuroprotective role in dopaminergic cell culture.     

Uncoupling of ATP-depletion and cell death in human dopaminergic neurons

The mitochondrial inhibitor 1-methyl-4-phenylpyridinium (MPP(+)) is the toxicologically relevant metabolite of 1-methyl-4-phenyltetrahydropyridine (MPTP), which causes relatively selective degeneration of dopaminergic neurons in the substantia nigra. Dopaminergic LUHMES cells were used to investigate whether ATP-depletion can be uncoupled from cell death as a downstream event in these fully post-mitotic human neurons. Biochemical assays indicated that in the homogeneously differentiated cell cultures, MPP(+) was taken up by the dopamine transporter (DAT). MPP(+) then triggered oxidative stress and caspase activation, as well as ATP-depletion followed by cell death. Enhanced survival of the neurons in the presence of agents interfering with mitochondrial pathology, such as the fission inhibitor Mdivi-1 or a Bax channel blocker suggested a pivotal role of mitochondria in this model. However, these compounds did not prevent cellular ATP-depletion. To further investigate whether cells could be rescued despite respiratory chain inhibition by MPP(+), we have chosen a diverse set of pharmacological inhibitors well-known to interfere with MPP(+) toxicity. The antioxidant ascorbate, the iron chelator desferoxamine, the stress kinase inhibitor CEP1347, and different caspase inhibitors reduced cell death, but allowed ATP-depletion in protected cells. None of these compounds interfered with MPP(+) accumulation in the cells. These findings suggest that ATP-depletion, as the initial mitochondrial effect of MPP(+), requires further downstream processes to result in neuronal death. These processes may form self-enhancing signaling loops, that aggravate an initial energetic impairment and eventually determine cell fate.     

Neuroprotection afforded by prior citicoline administration in experimental brain ischemia: effects on glutamate transport

BACKGROUND AND PURPOSE: Cytidine-5'-diphosphocholine (citicoline or CDP-choline), an intermediate in the biosynthesis of phosphatidylcholine, has shown beneficial effects in a number of CNS injury models including cerebral ischemia. Citicoline is the only neuroprotectant that has proved efficacy in patients with moderate to severe stroke. However, the precise mechanism by which citicoline is neuroprotective is not fully known. The present study was designed to search for mechanisms of citicoline neuroprotective properties using in vivo and in vitro models of brain ischemia. METHODS: Focal brain ischemia was produced in male adult Fischer rats by occluding both the common carotid and middle cerebral arteries. Brain glutamate levels were determined at fixed intervals after occlusion. Animals were then sacrificed, and infarct volume and brain ATP levels were measured. As in vitro model of ischemia, rat cultured cortical neurones or astrocytes, isolated or in co-culture, were exposed to oxygen-glucose deprivation (OGD) either in the absence or in the presence of citicoline (1-100 microM). Viability was studied by measuring LDH release. Glutamate release and uptake, and ATP levels were also determined. RESULTS: Citicoline (0.5, 1 and 2 g/kg i.p. administered 1 h before the occlusion) produced a reduction of the infarct size measured at striatum (18, 27 and 42% inhibition, respectively, n = 8, P < 0.05 vs. ischemia), effect that correlated with the inhibition caused by citicoline on ischemia-induced increase in glutamate concentrations after the onset of the ischemia. Citicoline also inhibited ischemia-induced decrease in cortical and striatal ATP levels. Incubation of cultured rat cortical neurones with citicoline (10 and 100 microM) prevented OGD-induced LDH and glutamate release and caused a recovery in ATP levels after OGD, confirming our previous results. In addition, citicoline (100 microM) caused an increase in glutamate uptake and in EAAT2 glutamate transporter membrane expression in cultured rat astrocytes. CONCLUSIONS: Our present findings show novel mechanisms for the neuroprotective effects of citicoline, which cooperate to decrease brain glutamate release after ischemia.     

雷公藤内酯可减轻MPP+诱导的大鼠多巴胺神经元损伤

目的以小胶质细胞激活为特征的神经炎性反应作为帕金森病（Parkinson’s disease,PD）一种重要的致病原因,正受到越来越多的关注。本文的目的是研究1-甲基-4-苯基-毗啶（l-methyl-4-phenylpyridinium,MPP^＋）所诱导的偏侧PD大鼠模型中小胶质细胞的激活,观察雷公藤内酯作为一种小胶质细胞抑制剂对多巴胺神经元的保护作用及其对MPP^＋所导致的大鼠行为学异常的改善效果。方法通过黑质区域显微注射MPP^＋制作偏侧PD大鼠模型。分别在基线、MPP^＋注射后第1、3、7、14、21天时通过测定黑质区域OX-42的免疫荧光强度评定小胶质细胞的激活程度,测定酪氨酸羟化酶的表达情况评定存活多巴胺神经元的数量;通过阿朴吗啡诱导的旋转行为、前肢跨步及触须引发的不对称放置试验得分测评行为学表现。结果黑质区域MPP注射导致小胶质细胞的激活、多巴胺神经元进行性死亡以及行为学缺陷的不断加重。雷公藤内酯可以显著抑制小胶质细胞的激活,部分阻止MPP^＋对多巴胺神经元的毒性,改善行为学异常。结论上述结果显示雷公藤内酯对MPP^＋诱导的偏侧PD大鼠模型多巴胺神经元具有保护作用,其机制可能与抑制小胶质细胞的激活有关,这为PD免疫抑制治疗提供了实验依据。     

1-methyl-4-phenylpyridinium neurotoxicity is attenuated by adenoviral gene transfer of human Cu/Zn superoxide dismutase

Oxidative stress has been suggested to be an important mediator of dopaminergic cell death in Parkinson's disease (PD). We investigated the neuroprotective potential of Cu/Zn superoxide dismutase (SOD1) overexpression in the rat substantia nigra (SN) following adenovirus-mediated gene transfer. Human dopaminergic SK-N-SH cells were transduced with adenoviral vectors expressing either human SOD1 (Ad-SOD1) or beta-galactosidase (Ad-betagal) before exposure to 1 mM of the 1-methyl-4-phenylpyridinium ion (MPP+). A strong neuroprotective effect of SOD1 gene transfer was observed in the SK-N-SH cells exposed to MPP+ compared with controls. Adult rats were then given unilateral injections of either Ad-SOD1 or Ad-betagal into the striatum, and MPP+ was administered 8 days later at the same location. Strong transgene expression was detected in the SN dopaminergic neurons, a consequence of retrograde axonal transport of the adenoviral particles. The amphetamine-induced rotational behavior of the rats was markedly lower in Ad-SOD1-injected rats than in control animals. Also, behavioral recovery significantly correlated with the number of tyrosine hydrolase-expressing neurons in the SN of the treated rats. These results are consistent with oxidative stress contributing to the MPP+ -induced neurodegenerative process. They also indicate that SOD1 gene transfer into the nigrostriatal system may be a potential neuroprotective strategy for treating PD.     

Neurotrophic and neuroprotective effects of tripchlorolide,an extract of Chinese herb Tripterygium wilfordii Hook F,on dopaminergic neurons

It has been reported recently that the immunosuppressant FK506 produced neurotrophic and neuroprotective effects on dopaminergic neurons in vitro and in vivo. We investigated whether tripchlorolide, an immunosuppressive extract of Chinese herb Tripterygium wilfordii Hook F, could exert similar neurotrophic and neuroprotective effects similar to those of FK506. It was found that tripchlorolide promoted axonal elongation and protected dopaminergic neurons from a neurotoxic lesion induced by 1-methyl-4-phenylpyridinium ion (MPP+) at concentrations of as low as 10(-12) to 10(-8) M. In situ hybridization study revealed that tripchlorolide stimulated brain-derived neurotrophic factor (BDNF) mRNA expression. In vivo administration of tripchlorolide (1 microg/kg, ip) for 28 days effectively attenuated the rotational behavior challenged by D-amphetamine in the model rats by transection of the medial forebrain bundle. In addition, tripchlorolide treatment (0.5 or 1 microg/kg/day for 28 days) increased the survival of dopaminergic neurons in substantia nigra pars compacta by 50 and 67%, respectively. Moreover, tripchlorolide markedly prevented the decrease in amount of dopamine in the striatum of model rats. Taken together, our data provide the first evidence that tripchlorolide acts as a neuroprotective molecule that rescues MPP+ or axotomy-induced degeneration of dopaminergic neurons, which may imply its therapeutic potential for Parkinson's disease. The underlying mechanism may be relevant to its neurotrophic effect and its efficacy in stimulating the expression of BDNF.     

MPP(+) injection into rat substantia nigra causes secondary glial activation but not cell death in the ipsilateral striatum

Injection of MPP(+) into the substantia nigra causes extensive necrosis and anterograde degeneration of pars compacta dopaminergic neurons. We studied secondary effects in the ipsilateral striatum by examining dopaminergic terminals, signs of neuronal damage, and glial reactivity at 1, 2, 3, and 7 days after injection of MPP(+) into the substantia nigra. Dopaminergic terminals and uptake sites were evaluated with [(3)H]GBR-12935 binding and tyrosine hydroxylase immunoreactivity. Glial reaction was examined with markers of astrocytes and microglia. Stereology was used to evaluate any changes in neuronal density. Tyrosine hydroxylase immunoreactivity and [(3)H]GBR-12935 binding markedly decreased (74%) from days 2 to 7. Loss of dopaminergic terminals in the ipsilateral striatum was accompanied by an intense astroglial and, to a lesser extent, microglial reaction. However, no signs of cell damage, neuronal loss, or disruption of the blood-brain barrier were found in the striatum. Resident astroglial and microglial cells showed a morphological shift and notable changes in protein expression typical of glial reactivity, yet the presence of macrophage-like cells was not detected. This study shows that injection of MPP(+) in the substantia nigra causes a secondary reaction within the ipsilateral striatum involving the transformation of quiescent glia to reactive glia. It is suggested that stimuli derived from damaged dopaminergic terminals within the striatum are able to activate resident glia and that this glial transformation may promote repair and regeneration.     

Transient receptor potential vanilloid subtype 1 contributes to mesencephalic dopaminergic neuronal survival by inhibiting microglia-originated oxidative stress

The present study examined whether capsaicin (CAP), an agonist of transient receptor potential vanilloid subtype 1 (TRPV1) can prevent 1-methyl-4-phenylpyridinium (MPP(+))-induced dopaminergic (DA) neuronal death in the substantia nigra (SN). Unilateral injection of MPP(+) into the median forebrain bundle of rat brain resulted in a significant loss of nigral DA neurons, assessed by tyrosine hydroxylase (TH) immunostaining. In parallel, activation of microglia, visualized by OX-42 and OX-6 immunostaining were also observed in the SN, where degeneration of nigral neurons was found. By contrast, MPP(+) neurotoxicity was partially inhibited by co-treatment with MPP(+) and CAP. Interestingly, CAP significantly decreased not only immunoreactivity of OX-42 and OX-6 but also production of microglia-derived reactive oxygen species (ROS) in the SN of MPP(+)-treated rats. In experiments designed to further verify effectiveness of CAP against microglia-derived neurotoxicity, CAP inhibited ROS production and blocked MPP(+)-induced death of DA neurons in co-cultures of mesencephalic neurons and microglia, but not in microglia-free, neuron-enriched mesencephalic cultures. This beneficial effect was reversed by capsazepine, an antagonist of TRPV1, expressed in microglia, indicating TRPV1 involvement. Our data demonstrate for the first time that CAP may inhibit microglial activation-mediated oxidative stress via TRPV1, suggesting that CAP and its analogs may have therapeutic value by inhibiting microglial activation and/or ROS generation that occurs in Parkinson's disease.     

1-Benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ), an endogenous neurotoxin,induces dopaminergic cell death through apoptosis

Endogenous MPTP-like neurotoxins such as 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ) have been suspected in the etiology of Parkinson's disease (PD). 1BnTIQ was found in a concentration three times higher in cerebrospinal fluid of PD brains than control subjects [J. Neurochem. 65 (6) (1995) 2633]. In the present study, we have evaluated the mechanisms of 1BnTIQ toxicity in human dopaminergic SH-SY5Y cells and tested the neuroprotective action of SKF-38393, a dopamine receptor (D(1)) agonist. 1BnTIQ dose dependently decreased cell viability in dopaminergic SH-SY5Y cells and the extent of cell death was more pronounced when compared to MPP(+). Similar to MPP(+), 1BnTIQ significantly decreased [3H]dopamine uptake. 1BnTIQ significantly increased lipid peroxidation, Bax expression, and active caspase-3 formation. Furthermore, it decreased the expression of Bcl-xL, an anti-apoptotic protein, in these cells. SKF-38393, a dopamine receptor (D(1)) agonist (1 and 10 microM) completely prevented the cell death and significantly increased cell viability. These results strongly suggest that 1BnTIQ induces dopaminergic cell death by apoptosis and dopamine receptor agonists may be useful neuroprotective agents against 1BnTIQ toxicity.     

Basic fibroblast growth factor protects striatal neurons in vitro from NMDA-receptor mediated excitotoxicity.

Basic fibroblast growth factor (bFGF) promotes the survival and outgrowth of neurons. In this study the neuroprotective effects of bFGF were examined in 12-18-day-old cultured striatal neurons exposed to glutamic acid, kainic acid (KA), and quinolinic acid (QA), an N-methyl-D-aspartate (NMDA)-receptor agonist. Results showed that preincubation with bFGF (6 pM) from the day of plating significantly increased the survival of striatal neurons treated for 3 h with glutamate (3 mM) or QA (1 mM), but had little effect on KA (1 mM) induced toxicity. Moreover, maximum protection by bFGF against glutamate neurotoxicity was observed in cultures treated as little as 2 h before glutamate exposure. These results show that bFGF markedly protects striatal neurons from NMDA-receptor induced neurotoxicity.     

MPP(+) and glutamate in the degeneration of nigral dopaminergic neurons

Glutamate neurotoxicity can be an experimental oxidative stress, and we investigated glutamate toxicity against cultured rat mesencephalic neurons. Although glutamate showed similar toxicity against dopaminergic and nondopaminergic neurons, nitric oxide (NO) showed neurotoxicity restricted exclusively in nondopaminergic neurons. An inhibitor of NO synthase had no significant effect on the glutamate toxicity against dopaminergic neurons, however, it had a significant antagonistic effect on that against nondopaminergic neurons. These findings indicate the presence of two mechanisms of glutamate neurotoxicity, one being not mediated by NO, found in dopaminergic neurons, and the other being mediated via NO, found in nondopaminergic neurons. In contrast to NO, peroxynitrite (ONOO⁻), an active metabolite of NO, caused significant cytotoxicity against dopaminergic and nondopaminergic neurons, suggesting that conversion of NO to ONOO⁻ is suppressed in dopaminergic neurons. After pretreatment with small doses of methyl-4-phenylpyridium ion (MPP⁺), NO caused significant cytotoxicity against dopaminergic neurons, and glutamate toxicity was enhanced only against dopaminergic neurons. Therefore, sublethal dose of MPP⁺ enhances glutamate toxicity against dopaminergic neurons, probably by the facilitation of suppressed NO conversion to ONOO⁻ in dopaminergic neurons. Finally, to provide basic data for neuroprotective therapy in Parkinson's disease, we investigated neuroprotection against glutamate toxicity by dopamine agonists. Preincubation with the D2 type dopamine agonists provides neuroprotection against glutamate neurotoxicity and the protective effects blocked by a D2 antagonist, indicating that D2 agonists provide protection mediated not only by the inhibition of dopamine turnover, but also via D2 type dopamine receptor.     

Methylphenylpyridium ion (MPP+) enhances glutamate-induced cytotoxicity against dopaminergic neurons in cultured rat mesencephalon

Parkinson's disease is characterized by chronic progression of dopaminergic neuronal death, the mechanism of which is still unknown. Although methyl-4-phenylpyridium ion (MPP+) or MPP+-like substance, that can reduce mitochondrial complex I activity, is supposed to be a causative agent for Parkinson's disease, it is difficult to explain the chronic neuronal degeneration for years. It is important to identify other putative agents capable of causing chronic cell death besides MPP⁺. We hypothesized that treatment with small doses of MPP⁺, not causing severe damage to dopaminergic neurons but merely reducing the activity of mitochondrial complex I, can be a model of Parkinson's disease, and that glutamate can be a putative agent causing chronic neuronal degeneration. Using primary culture of the rat mesencephalon, we investigated glutamate-induced cytotoxicity against dopaminergic and non-dopaminergic neurons with or without the pretreatment with MPP⁺. Brief exposure to glutamate showed similar cytotoxicity against both dopaminergic and non-dopaminergic neurons. An N-methyl-D -aspartate receptor antagonist completely blocked the glutamate-induced cytotoxicity against both dopaminergic and non-dopaminergic neurons. In the dopaminergic neurons, MPP⁺ caused cytotoxicity that was not blocked by co-administration of MK-801. After pretreatment with small doses of MPP⁺, sub-lethal doses of glutamate caused severe cell damage restricted to dopaminergic neurons, suggesting that MPP⁺ potentiates the glutamate-induced cytotoxicity only against dopaminergic neurons. As glutamate is putatively capable of causing cytotoxicity against dopaminergic neurons, the present findings might be important in considering the pathogenesis of dopaminergic neuronal degeneration and a possible therapeutic application of glutamate receptor antagonists in Parkinson's disease. © 1996 Wiley-Liss, Inc.     

Estrogen modulates responses of striatal dopamine neurons to MPP(+): evaluations using in vitro and in vivo techniques

In vitro superfusion and in vivo electrochemistry were used to investigate the role of estrogen in modulating MPP(+)-induced dopamine output in the corpus striatum and nucleus accumbens of ovariectomized female rats. For in vitro superfusion experiments, dopamine and dihydroxyphenylacetic acid release were determined using HPLC with electrochemical detection from superfusion of corpus striatum fragments with Kreb's ringer phosphate buffer pulsed with MPP(+) alone or MPP(+) with estrogen. The in vivo electrochemistry experiments recorded the dopamine signal from carbon fiber microelectrodes stereotaxically passed through the corpus striatum and nucleus accumbens. Dopamine release was stimulated by pressure ejection of MPP(+) alone or in combination with estrogen through glass micropipettes fastened to the electrodes. Dopamine output from superfusion chambers which received infusion of MPP(+) with estrogen showed significantly lower output of dopamine compared with chambers which received MPP(+) alone. Outputs of dihydroxyphenylacetic acid did not increase following MPP(+) infusions. Data from the electrochemistry experiments demonstrated that estrogen significantly reduced both the amplitude and clearance rates of the MPP(+)-evoked dopamine signal in both the corpus striatum and nucleus accumbens. Results of this study demonstrate that: (1) MPP(+) evokes striatal dopamine release and this effect is significantly reduced in the presence of estrogen as determined by both in vivo electrochemistry and in vitro superfusion: (2) similar, albeit attenuated effects are observed in the nucleus accumbens as determined with in vivo electrochemistry; (3) estrogen acts to inhibit the clearance of dopamine in both the striatum and nucleus accumbens; and (4) estrogen may function as a neuroprotectant by reducing the uptake of neurotoxin into dopaminergic neurons.