Subtoxic concentration of manganese synergistically potentiates 1-methyl-4-phenylpyridinium-induced neurotoxicity in PC12 cells

Endogenous or exogenous substances that are toxic to dopaminergic cells have been proposed as possible cause of idiopathic Parkinson's disease (PD). 1-Methyl-4-phenylpyridinium (MPP(+)) and manganese are dopaminergic neurotoxins causing a parkinsonism-like syndrome. Here, we studied the possible synergistic reaction between these two neurotoxins using rat PC12 pheochromocytoma cells. MPP(+) induced a delayed neurotoxicity in PC12 cells. Although low concentration of manganese did not cause cell damage, it markedly enhanced MPP(+)-induced neurotoxicity with characteristics of apoptosis, such as DNA laddering and activation of caspase-3. To understand the mechanism of enhancement of subtoxic concentration of manganese on MPP(+)-induced neurotoxicity, we investigated the reactive oxygen species (ROS) generation using a molecular probe, 2',7'-dichlorofluorescein diacetate. Although subtoxic concentration of manganese alone did not induce ROS increase, it significantly enhanced the ROS generation induced by MPP(+). We also determined the intracellular MPP(+) content. A time- and concentration-dependent increase of MPP(+) levels was found in PC12 cells treated with MPP(+). The accumulation of MPP(+) by PC12 cells was not affected by manganese. Taken together, these studies suggest that co-treatment with MPP(+) and manganese may induce synergistic neurotoxicity in PC12 cells and that subtoxic concentration of manganese may potentiate the effect of MPP(+) by an ROS-dependent pathway.     

Stromal cell-derived factor 1-mediated CXCR4 signaling in rat and human cortical neural progenitor cells

Stromal cell-derived factor 1 (SDF-1) and the chemokine receptor CXCR4 are highly expressed in the nervous system. Knockout studies have suggested that both SDF-1 and CXCR4 play essential roles in cerebellar, hippocampal, and neocortical neural cell migration during embryogenesis. To extend these observations, CXCR4 signaling events in rat and human neural progenitor cells (NPCs) were examined. Our results show that CXCR4 is expressed in abundance on rat and human NPCs. Moreover, SDF-1alpha induced increased NPCs levels of inositol 1,4,5-triphosphate, extracellular signal-regulated kinases 1/2, Akt, c-Jun N-terminal kinase, and intracellular calcium whereas it diminished cyclic adenosine monophosphate. Finally, SDF-1alpha can induce human NPC chemotaxis in vitro, suggesting that CXCR4 plays a functional role in NPC migration. Both T140, a CXCR4 antagonist, and pertussis toxin (PTX), an inactivator of G protein-coupled receptors, abrogated these events. Ultimately, this study suggested that SDF-1alpha can influence NPC function through CXCR4 and that CXCR4 is functional on NPC.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity in non-human primates is antagonized by pretreatment with nimodipine at the nigral, but not at the striatal level

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to induce parkinsonism in man and non-human primates. Hypotheses concerning the mechanism of action of MPTP have been related to the pathogenesis of nigral cell death in Parkinson's disease. For instance, alterations of calcium influxes have been reported to be implicated in both MPTP-induced parkinsonism and Parkinson's disease. Recently, we reported that nimodipine, a blocker of L-type calcium channels, prevents dopaminergic MPTP-induced neurotoxicity in C57B1/6 black mice. The present study extended these rodent findings to the non-human primate model of Parkinson's disease and assessed the effects of nimodipine, continuously applied by pellet for 18 days, on behavioural, biochemical and histological parameters, following systemic application of MPTP in common marmosets (Callithrix jacchus). The experimental design involved five groups of common marmosets and a total of 24 animals. Monkeys assigned to group I (n = 4) received subcutaneously implanted vehicle pellets 7 days prior to subcutaneous saline injections (control). Monkeys of group II (n = 4) were treated with nimodipine pellets (80 mg) and saline injections. Marmosets in group III (n = 8) were treated with vehicle pellets and received 4 times MPTP (MPTP-HCI, 2 mg/kg body weight subcutaneously, separated by an interval of 24 h for a total of 4 days). Monkeys in group IV (n = 4) and V (n = 4) were treated as group-III animals except for the implantation of nimodipine pellets (80 mg and 120 mg, respectively) 7 days prior to toxin exposure. In common marmosets MPTP induced severe parkinsonian symptoms, a pronounced dopamine depletion in the caudate-putamen (more than 99% of control) and a loss of tyrosine hydroxylase immunoreactive cells in the substantia nigra (50% percent of control) 7 days after MPTP-administration. Pretreatment with nimodipine (120 mg pellets) did neither attenuate the behavioural impairments in MPTP-treated animals nor antagonize the striatal neurotoxin-induced dopamine depletion, but almost completely prevented (in a dose-dependent manner) the MPTP-induced decrease of nigral tyrosine hydroxylase immunoreactive cells. These data suggest that application of nimodipine, during the observation period of 7 days, protects against MPTP-induced neurotoxicity in common marmosets at the cellular nigral level, but not at the synaptic striatal level, implicating differential mechanisms of actions of MPTP-induced neurotoxicity at the nigral versus the striatal level.     

Evaluation of the neurotoxicity of N-methyl-1-(4-methoxyphenyl)-2-aminopropane (para-methoxymethamphetamine, PMMA)

These studies assessed the neurotoxic potential of N-methyl-1-(4-methoxyphenyl)-2-aminopropane (para-methoxymethamphetamine; PMMA), an amphetamine analog that has surfaced in the illicit drug market. Repeated subcutaneous injections of PMMA caused lasting, dose-related reductions in regional brain concentrations of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), and in the density of [³H]paroxetine-labelled 5-HT uptake sites. Comparison of the neurotoxic potential of PMMA to that of para-methoxyamphetamine (PMA) and 3,4-methyl-enedioxymethamphetamine (MDMA) showed that equivalent doses of PMMA and PMA (80 mg/kg) produced comparable depletions of 5-HT, but that these depletions were not as pronounced as those induced by a lower dose of MDMA (20 mg/kg). Striatal DA was not affected on a long-term basis by any of the ring-substituted amphetamines evaluated in this study. These data suggest that PMMA, like PMA and MDMA, produces long-term (possibly neurotoxic) effects on brain serotonin neurons, but that PMMA is less potent than MDMA as a 5-HT neurotoxin. Further, they raise concern over the illicit use of PMMA since humans could be more sensitive than rodents to the 5-HT neurotoxic effects of PMMA and related drugs.     

Mechanisms of MPTP (1-methyl-4-phenyl-l, 2, 3, 6-tetrahydropyridine) neurotoxicity to striatal dopamine neurons in mice

1. MPTP given to mice in 4 daily doses (20 mg/kg s.c.) resulted in 56–70% depletion of striatal dopamine 1 week after the last dose.

2. Pretreatment with deprenyl or MD 240928, selective inhibitors of monoamine oxidase type B, or with amfonelic acid or nomifensine, selective inhibitors of dopamine uptake, prevented the depletion of striatal dopamine. In contrast, pretreatment with -methyltyrosine, Ro 4-1284 or haloperidol did not prevent the depletion of striatal dopamine by MPTP.

3. The results are compatible with the view that dopamine itself is not involved in the neurotoxic effect of MPTP but that MPP+, a metabolite of MPTP formed by MAO-B and accumulated by the dopamine uptake carrier, is responsible for the neurotoxicity.     

In vivo protection of striatal dopaminergic system against 1-methyl-4-phenylpyridinium neurotoxicity by phenobarbital

We have studied the effect of a semichronic and acute treatment of phenobarbital on in vivo 1-methyl-4-phenylpyridinium ion- (MPP+)-induced neurotoxicity. A group of rats were intraperitoneally injected for 12 days with phenobarbital (80 mg/Kg of body weight, semichronic treatment) in order to induce cytochrome P450 levels in brain. At day 10 of treatment, rats received unilateral left striatal injections of 1 or 2 μg of MPP+. Two days after the injection of the toxin a dose-dependent loss of dopamine uptake along with a concomitant decrease of dopamine levels and its metabolites was produced in control rats. In phenobarbital treated animals striatal injection of 1 μg of MPP+ did not produce any effect on dopaminergic parameters but injection of 2 μg of MPP+ caused losses of dopamine levels and dopamine transporter although smaller than in control rats. TH immunohistochemistry in semichronic phenobarbital treated rats also demonstrated the protective effect of this drug against MPP+ toxicity. Dopamine uptake in synaptosomes from semichronic phenobarbital treated rats did not change with respect to the controls, thereby diminished MPP+ toxicity in phenobarbital treated rats is not due to an alterated uptake of the toxin. Neuroprotection found by intraperitoneal injection of phenobarbital 30 min before MPP+ intrastriatal injection (acute treatment) could discard the induction of cytochrome P450 as responsible for this suppressed neurotoxicity of MPP+. The neuroprotective effect of phenobarbital could be produced by its action as an excitatory amino acid antagonist or as a GABA agonist. J. Neurosci. Res. 49:301–308, 1997. © 1997 Wiley-Liss, Inc.     

Reduction of zinc-positive terminal fields in striatum of mouse after 1-methyl-4-phenylpyridinium neurotoxicity

Zinc is an essential trace element in the central nervous system and is located in three distinct pools: free zinc, vesicular zinc and protein-bound zinc. Zinc may serve as an endogenous neuromodulator and has been associated with neuropathologies. This study was undertaken to determine whether levels of vesicular zinc in neuronal terminals would decrease in response to the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+). Adult male C-57 black mice were injected with MPP+ (0.72 mg/kg) into their right lateral ventricle. All animals were killed at 1, 2, 24 h and 7 days after MPP+ or saline administration. The brains were stained for zinc sulfides and the density of zinc-positive terminal fields was evaluated after MPP+ administration. The relative optical density analysis of zinc-positive terminal fields showed significant decreases in the striatum at 1, 2 and 24 h (24, 18 and 14%, respectively, versus control) and ventricular epithelium (1, 2, 24 h and 7 days). The hippocampus showed increase in the stratum oriens and stratum radiatum at different times. MPP+ administration reduced dopamine levels at 24h and 7 days (36 and 40%, respectively, versus control) as a result of the neurotoxic action of MPP+. The decrease of zinc-positive neuronal terminal fields in the striatum after MPP+ administration is most likely due to a neuronal release of vesicular zinc in response to its dopaminergic neurotoxicity.     

NEDD4-1 shRNA对U251胶质瘤细胞增殖和凋亡的影响

目的探讨NEDD4-1 shRNA对U251胶质瘤细胞增殖及凋亡的影响。方法构建NEDD4-1 shRNA表达载体。以不同比例质粒和脂质体转染U251细胞,通过荧光显微镜观察细胞存活情况,优化转染效率。转染48 h后采用Western blot、RT-PCR分别检测NEDD4-1蛋白及mRNA的表达,流式细胞术检测细胞周期,MTT法检测细胞增殖,DAPI荧光染色检测细胞凋亡。结果 NEDD4-1 shRNA表达载体构建成功。脂质体与质粒的最佳转染效率比例为2.5μl∶1μg,48 h转染效率在60%～70%。与对照组比较,转染NEDD4-1 shRNA后,U251胶质瘤细胞NEDD4-1的蛋白及mRNA表达水平均下降(均P<0.05),细胞大多停滞在G0-G1期(均P<0.05),增殖率明显降低(均P<0.05),凋亡率显著增加(均P<0.05)。结论 NEDD4-1作为PTEN泛素连接酶,为胶质瘤基因治疗提供靶点。     

1-Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol) is toxic to dopaminergic neuroblastoma SH-SY5Y cells via impairment of cellular energy metabolism

The endogenous neurotoxin 1-methyl-6,7-dihydroxy-1,2,3, 4-tetrahydroisoquinoline (salsolinol), which is structurally similar to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been reported to inhibit mitochondrial complex I (NADH-Q reductase) activity as does the MPTP metabolite 1-methyl-4-phenylpyridinium ion (MPP(+)). However, the mechanism of salsolinol leading to neuronal cell death is still unknown. Thus, we correlated indices of cellular energy production and cell viability in human dopaminergic neuroblastoma SH-SY5Y cells after exposure to salsolinol and compared these results with data obtained with MPP(+). Both toxins induce time and dose-dependent decrease in cell survival with IC(50) values of 34 microM and 94 microM after 72 h for salsolinol and MPP(+), respectively. Furthermore, salsolinol and MPP(+) produce a decrease of intracellular net ATP content with IC(50) values of 62 microM and 66 microM after 48 h, respectively. In contrast to MPP(+), salsolinol does not induce an increase of intracellular net NADH content. In addition, enhancing glycolysis by adding D-glucose to the culture medium protects the cells against MPP(+) but not salsolinol induced cellular ATP depletion and cytotoxicity. These results suggest that cell death induced by salsolinol is due to impairment of cellular energy supply, caused in particular by inhibition of mitochondrial complex II (succinate-Q reductase), but not complex I.     

Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in goldfish cerebellum: neurochemical and immunocytochemical analysis

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administered in goldfish for 3 consecutive days (10 mg kg⁻¹ i.p.), caused cerebellar disappearance of dopamine-hydroxylase (DBH) immunoreactive fibres, whereas the noradrenergic cell bodies located in the medulla oblongata appeared intact. This effect was coupled with marked decreases in cerebellar noradrenaline (NA) and dopamine (DA) levels. An increase of immunostaining for glial fibrillary acidic protein (GFAP) was also observed. In the cerebellum of MPTP-treated fish, the contents of glutamate and GABA were significantly reduced, whereas glutamine was strongly increased. These modifications were concomitant with a significant increase of glutamine synthetase (GS) activity, whereas glutamic acid decarboxylase (GAD) activity was decreased. No changes in choline acetyltransferase (ChAT) and ornithine decarboxylase (ODC) activities were observed. High affinity uptake of glutamate and GABA was strongly reduced. Pretreatment of fish with either the monoamine oxidase inhibitor pargyline or the catecholamine (CA) uptake blocker mazindol largely prevented such modifications. The NMDA-sensitive glutamate receptor uncompetitive antagonist, dizocilpine maleate (MK-801), failed to protect against MPTP-induced damage. In conclusion, the neurotoxic effects of MPTP in goldfish cerebellum appear to be not specific against catecholaminergic terminals and could promote astrocytic reactions.     

Benzamide, an inhibitor of poly(ADP-ribose) polymerase, attenuates methamphetamine-induced dopamine neurotoxicity in the C57B1/6N mouse

Previous studies have indicated that the activation of poly(ADP-ribose) polymerase (PARP), an enzyme involved in DNA plasticity-related phenomena, is an early event occurring in glutamate-induced neurotoxicity in vitro, and that inhibitors of PARP, including benzamide, are protective against both glutamate- and methamphetamine (METH)-induced neurotoxicity in vitro. To evaluate a central neuroprotective potential of benzamide in vivo, the present study examined the effect of benzamide on the nigrostriatal dopamine toxicity (i.e. long-lasting striatal dopamine depletion) induced by METH in the C57B1/6N mouse. Intraperitoneal injection of METH at 2-h intervals (4 injections of 5 mg/kg, 4 injections of 10 mg/kg, or 2 injections of 20 mg/kg) dose-dependently reduced the levels of striatal dopamine in male C57B1/6N mice by up to 53% at 7 days post-treatment. Administration of benzamide (2 injections of 160 mg/kg spaced by a 4 h interval) during the different METH treatment protocols partially and significantly attenuated the METH-induced dopamine depletions. Benzamide (160 mg/kg i.p.) by itself had no acute effect on striatal dopamine metabolism and did not reduce body temperature. The concentrations of benzamide measured in the striatum at different times following this same dose of drug were in a range (0.09–0.64 mM) reported in in vitro studies to be both neuroprotective and effective in inhibiting PARP activity. These results indicate a neuroprotective potential of benzamide in vivo and suggest a role of PARP in METH neurotoxicity.     

Effect of serotonergic, corticostriatal and kainic acid lesions on the dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,5,6- tetrahydropyridine (MPTP) in mice

In mice, prior destruction of striatal 5-hydroxytryptamine (5-HT) neurons by intrastriatal or intraventricular injections of 5,7-dihydroxytryptamine did not abolish or attenuate DA depletions produced in striatum by 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). This suggests that although they contain monoamine oxidase type B, the oxidative conversion of MPTP to 1-methyl-4-phenyl-pyridinium ion (MPP+) does not take place in 5-HT neurons. Likewise, decortication and kainic acid lesions did not prevent or enhance striatal MPTP-induced DA decrements suggesting that corticostriatal projections and striatal neurons are not involved in the mechanisms of MPTP neurotoxicity.     

Development of Purkinje cells in humans: an immunohistochemical study using a monoclonal antibody against the inositol 1, 4, 5-triphosphate type 1 receptor (IP3R1)

Immunohistochemical analyses were carried out on the Purkinje cells from 21 autopsied fetal and early postnatal normal cerebella using a monoclonal antibody against the inositol 1, 4, 5-triphosphate type 1 receptor (IP₃R1) as a cytochemical marker of Purkinje cells. In normal adult cerebella used as positive controls, the cell bodies, axons, and dendrites, including spiny branchlets of the Purkinje cells, were specifically stained by the antibody. In the fetal cerebella examined, the IP₃R1 immunoreactivity was first detected in the soma of multilayered cells just beneath the molecular layer at 16 weeks of gestation. The IP₃R1 immunoreactivity gradually increased in area of positive staining from soma to dendrites and spiny branchlets, and the dendritic outgrowth rapidly progressed during 6 months after birth. The Purkinje cell maturation was more advanced in the vermis than in the hemisphere, more in the posterior lobe than in the anterior lobe, and more at the bottom of the folia than at the top. Partial absence of the Purkinje cells in the cerebellar cortex was observed in three cases. Heterotopias including Purkinje cells were often noted in the cerebellar white matter in five cases. Received: 8 October 1998 / Revised, accepted: 11 January 1999     

Distribution of protein phosphatases-1 alpha and -1 gamma 1 and the D(1) dopamine receptor in primate prefrontal cortex: Evidence for discrete populations of spines.

The function of G protein-coupled receptors depends on the availability of the appropriate signal transduction proteins in close proximity to the receptor. We have examined and quantified in primate prefrontal cortex the subcellular distribution of two isoforms of protein phosphatase-1 (PP1), PP1 alpha and PP1 gamma 1, which are components of the signal transduction pathway accessed by the D(1) dopamine receptor. Both PP1 alpha- and PP1 gamma 1-labeled puncta are seen in cortex, basal ganglia, hippocampus, and thalamus. Viewed with the electron microscope, both PP1 isoforms are selectively localized to dendritic spines and are found in different percentages of spines; PP1 alpha is present in roughly 70% and PP1 gamma 1 in roughly 40% of dendritic spines. Our analysis indicates that three populations of spines are defined by the distribution of these PP1 isoforms: those that contain both PP1 alpha and PP1 gamma 1, those that contain only PP1 alpha and those that contain neither. The D(1) receptor is present in a subset of the population that contains both PP1 alpha and PP1 gamma 1. The nonhomogeneous distribution of signal transduction proteins in the spines and dendrites of cortical pyramidal cells may help to explain differences in the actions of receptors that nominally use the same signal-transduction pathway.     

Reversible serotoninergic neurotoxicity ofN-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mouse striatum studied by neurochemical and immunohistochemical approaches

Effects ofN-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on serotonin (5-HT) were studied in mouse striatum. The 5-HT level was significantly reduced at 1 week after MPTP, though returned at 4 weeks. The level of 5-hydroxyindoleacetic acid (5-HIAA) increased at both 1 and 4 weeks. The ratio of increase in 5-HT induced by pargyline administration was larger in MPTP-pretreated mice than in vehicle-pretreated controls at 1 week, although it was identical in both groups at 4 weeks. The 5-HT immunohistochemical study confirmed the biochemical change of 5-HT. These results suggest that MPTP functionally affects the 5-HT metabolism in mouse striatum.     

Involvement of oxidative stress in di-2-ethylhexyl phthalate (DEHP)-induced apoptosis of mouse NE-4C neural stem cells

Di-2-ethylhexyl phthalate (DEHP) has been widely used as a plasticizer in industry and can cause neurotoxicity; however, the underlying mechanism remains unclear. In the study, we found that DEHP significantly inhibited viability of mouse NE-4C neural stem cells and caused lactate dehydrogenase (LDH) release from the cells. DEHP dramatically increased the levels of apoptosis-related proteins such as cleaved Caspase-8, cleaved Caspase-3 and Bax, as well as decreased Bcl-2 protein level. DEHP could also significantly increase the total numbers of AnnexinV-positive/PI-negative and AnnexinV-positive/PI-positive staining cells. Hoechst 33342 staining showed that marked DNA condensation and apoptotic bodies could be found in the ZnO NPs-treated cells. These results indicated that DEHP could induce apoptosis of NE-4C cells. Meanwhile, DEHP could significantly increase malondialdehyde (MDA) level, and decrease the content of glutathione (GSH) and activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), respectively, implying that DEHP could induce oxidative stress of NE-4C cells. Furthermore, N-Acetyl-l-cysteine (NAC), an inhibitor of oxidative stress, could rescue the inhibition of cell viability and induction of apoptosis by DEHP. Taken together, our results showed that oxidative stress was involved in DEHP-induced apoptosis of mouse NE-4C cells.