Increased MPTP Neurotoxicity in Vesicular Monoamine Transporter 2 Heterozygote Knockout Mice

Abstract: The neurotoxic action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been proposed to be attenuated by sequestration into intracellular vesicles by the vesicular monoamine transporter (VMAT2). The purpose of this study was to determine if mice with genetically reduced levels of VMAT2 (heterozygote knockout; VMAT2 +/−) were more vulnerable to MPTP. Striatal dopamine (DA) content, the levels of DA transporter (DAT) protein, and the expression of glial fibrillary acidic protein (GFAP) mRNA, a marker of gliosis, were assessed as markers of MPTP neurotoxicity. In all parameters measured VMAT2 +/− mice were more sensitive than their wild-type littermates (VMAT2 +/+). Administration of MPTP (7.5, 15, or 30 mg/kg, b.i.d.) resulted in dose-dependent reductions in striatal DA levels in both VMAT2 +/− and VMAT2 +/+ animals, but the neurotoxic potency of MPTP was approximately doubled in the VMAT2 +/− mice: 59 versus 23% DA loss 7 days after 7.5 mg/kg dose for VMAT2 +/− and VMAT2 +/+ mice, respectively. Dopaminergic nerve terminal integrity, as assessed by DAT protein expression, also revealed more drastic reductions in the VMAT2 +/− mice: 59 versus 35% loss at 7.5 mg/kg and 95 versus 58% loss at 15 mg/kg for VMAT2 +/− and VMAT2 +/+ mice, respectively. Expression of GFAP mRNA 2 days after MPTP was higher in the VMAT2 +/− mice than in the wild-type: 15.8- versus 7.8-fold increase at 7.5 mg/kg and 20.1- versus 9.6-fold at 15 mg/kg for VMAT2 +/− and VMAT2 +/+ mice, respectively. These observations clearly demonstrate that VMAT2 +/− mice are more susceptible to the neurotoxic effects of MPTP, suggesting that VMAT2-mediated sequestration of the neurotoxin into vesicles may play an important role in attenuating MPTP toxicity in vivo.     

Genetic ablation of tumor necrosis factor-alpha (TNF-alpha) and pharmacological inhibition of TNF-synthesis attenuates MPTP toxicity in mouse striatum

The impact of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) in the pathology of Parkinson's disease (PD) and in MPTP neurotoxicity remains unclear. Here, male TNF-alpha (-/-) deficient mice and C57bL/6 mice were treated with MPTP (4 x 15 mg/kg, 24 h intervals) and in one series, thalidomide was administered to inhibit TNF-alpha synthesis. Real-time RT-PCR revealed that the striatal mRNA levels of TNF-alpha, of the astrocytic marker glial fibrillary acidic protein (GFAP) and of the marker for activated microglia, macrophage antigen complex-1 (MAC-1), were significantly enhanced after MPTP administration. Thalidomide (50 mg/kg, p.o.) partly protected against the MPTP-induced dopamine (DA) depletion, and TNF-alpha (-/-) mice showed a significant attenuation of striatal DA and DA metabolite loss as well as striatal tyrosine hydroxylase (TH) fiber density, but no difference in nigral TH and DA transporter immunoreactivity. TNF-alpha deficient mice suffered a lower mortality (10%) compared to the high mortality (75%) seen in wild-type mice after acute MPTP treatment (4 x 20 mg/kg, 2 h interval). HPLC measurement of MPP(+) levels revealed no differences in TNF-alpha (-/-), wild-type and thalidomide treated mice. This study demonstrates that TNF-alpha is involved in MPTP toxicity and that inhibition of TNF-alpha response may be a promising target for extending beyond symptomatic treatment and developing anti-parkinsonian drugs for the treatment of the inflammatory processes in PD.     

Effect of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) on monoamine neurotransmitters in mouse brain & heart

The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was studied on dopamine (DA), norepinephrine (NE), serotonin (5HT) and γ-aminobutyric acid (GABA) neurons in mouse brain and on NE neurons of mouse heart. MPTP (45 mg/kg) was administered s.c. to mice twice daily for 2 consecutive days. This dosage regimen produced a decrease in the forebrain concentrations of DA and NE at 7 and 20 days after injection. In contrast, the forebrain concentrations of 5HT and GABA were not significantly decreased at either time. MPTP administration also produced a marked decrease in the uptake of ³H-DA into striatal slices and ³H-NE into cerebral cortical slices. In contrast, the uptake of ³H-NE into hypothalamic slices and the uptake of ³H-5HT into slices from several brain regions were not altered. MPTP initially reduced the concentration of NE in the heart, but unlike the persistent decreases in the forebrain concentrations of NE and DA, the NE concentration in the heart returned to control levels at approximately 20 days after MPTP administration. These results, showing that MPTP can produce a long lasting and selective decrease in the forebrain concentrations of NE and DA and in the uptake of radioactive DA and NE into brain slices, suggest that MPTP can cause the destruction of catecholamine neurons in mouse brain. In contrast, MPTP administration does not appear to produce long term changes in either 5HT or GABA neurons.     

Immunity of Fetal Mice to Prenatal Administration of the Dopaminergic Neurotoxin 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine

Abstract: Subcutaneous injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) HC1 (25 mg/kg) in pregnant female mice at the 17th day of gestation markedly depleted striatal dopamine (DA) concentrations in the mothers 24 h later and at 24 h and 28 days after delivery. By contrast, in the offspring of the female mice exposed to MPTP during pregnancy, fetal brain DA concentrations at 24 h after injection and at 24 h after birth and striatal DA levels at 14 and 28 days postnatally were unaffected and identical to those in age-matched controls. The postnatal ontogenesis of striatal DA levels was identical in offspring of control vehicle- and MPTP-treated pregnant mice. Also, prenatal challenge with MPTP did not make nigrostriatal DA neurons more vulnerable to a second postnatal treatment with the toxin. Striatal DA depletions were identical in 6-week-old mice given MPTP, whether they were exposed to MPTP or to vehicle in utero. Monoamine oxidase (EC 1.4.3.4; MAO) type B activity was extremely low in the fetal brain and, relatively, much lower than that of MAO-A. Prenatal MPTP administration reduced maternal striatal and also embryonal brain MAO-B activity at 24 h post treatment but did not alter the normal postnatal development of striatal MAO-A and -B activities in the offspring. Study suggests that resistance of fetal DA neurons to the DA-depleting effect of MPTP may be due, at least in part, to an absence in the embryonal brain of adequately developed MAO-B activity required for the conversion of MPTP to its toxic metabolite, 1-methyl-4-phenylpyridinium ion.     

MPTP对小鼠帕金森病造模条件探讨

目的观察不同剂量1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)对小鼠行为学及脑黑质酪氨酸羟化酶、纹状体多巴胺含量的影响,探讨MPTP致帕金森病(Parkinson′s disease,PD)样小鼠模型的最佳条件。方法C57BL小鼠分别给与MPTP不同剂量处理,测定各组小鼠爬竿时间检测动物运动协调性,应用免疫组化方法和高效液相法观察不同模型组多巴胺能神经元的变化。结果模型组各组均出现不同程度爬竿时间延长,酪氨酸羟化酶阳性细胞数减少和多巴胺含量减少。结论MPTP处理可造成小鼠的帕金森病样症状,在此种动物模型中,应根据科研目的选择MPTP的应用剂量和给药途径。     

Neurotoxicity,drugs of abuse,and the CuZn-superoxide dismutase transgenic mice

Administration of methamphetamine (METH) to animals causes loss of DA terminals in the brain. The manner by which METH causes these changes in neurotoxicity is not known. We have tested the effects of this drug in copper/zinc (CuZn)-superoxide dismutase transgenic (SOD Tg) mice, which express the human CuZnSOD gene. In nontransgenic (non-Tg) mice, acute METH administration causes significant decreases in DA and dihydroxyphenylacetic acid (DOPAC) in the striata of non-Tg mice. In contrast, there were no significant decreases in striatal DA in the SOD Tg mice. The effects of METH on DOPAC were also attenuated in SOD Tg mice. Chronic METH administration caused decreases in striatal DA and DOPAC in the non-Tg mice, but not in the SOD-Tg mice. Similar studies were carried out with 1-methyl-1,2,3,6-tetrahydropyridine (MPTP), which also causes striatal DA and DOPAC depletion. As in the case of METH, MPTP causes marked depletion of DA and DOPAC in the non-Tg mice, but not in the SOD Tg mice. These results suggest that the mechanisms of toxicity of both METH and MPTP involve superoxide radical formation.     

Nei-like 1 inhibition results in motor dysfunction and promotes inflammation in Parkinson’s disease mice model

Parkinson’s disease (PD) is well known as a neurodegenerative disorder with progressive loss of dopaminergic (DA) neurons. Nei-like 1 (NEIL1) is one of four mammalian DNA glycosylases involved in the progression of various diseases, including neuroinflammation. However, it is still unknown if the expression changes of NEIL1 could contribute to PD progression. In the present study, we established mouse model with PD using 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to explore the effects of NEIL1 on PD development. Here, we found that NEIL1 deletion significantly promoted the motor dysfunction in the wild type mice treated with 6-OHDA. Furthermore, DA neuronal loss was further accelerated by NEIL1 deletion in 6-OHDA-injected mice, as evidenced by the significantly reduced expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT). Furthermore, in PD mice induced by MPTP, remarkably reduced expression of NEIL1 was observed in nigra and striatum of mice. A strong positive correlation was detected in the expression of NEIL1 and the survival rate of DA neurons. Also, NEIL1 ablation further elevated the DA neuronal loss in MPTP-treated mice, accompanied with higher glial activation, as evidenced by the obvious up-regulation of glial fibrillary acidic protein (GFAP) and Ionized calcium-Binding Adapter molecule 1 (Iba1). Moreover, MPTP-triggered inflammation was highly aggravated by the loss of NEIL1 through inducing the expression of pro-inflammatory cytokines and chemokines. In contrast, promoting NEIL1 expression effectively reversedPD progression induced by MPTP in mice. Together, these results demonstrated that NEIL1 insufficiency might be a contributing factor for the progression of PD, which therefore could be considered as a novel candidate to develop effective treatments against PD progression.     

Transgenic neuronal overexpression reveals that stringently regulated p23 expression is critical for coordinated movement in mice

Background

Previous studies indicate a role of P2X₇ receptors in processes that lead to neuronal death. The main objective of our study was to examine whether genetic deletion or pharmacological blockade of P2X₇ receptors influenced dopaminergic cell death in various models of Parkinson's disease (PD).

Results

mRNA encoding P2X₇ and P2X₄ receptors was up-regulated after treatment of PC12 cells with 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP). P2X₇ antagonists protected against MPTP and rotenone induced toxicity in the LDH assay, but failed to protect after rotenone treatment in the MTT assay in PC12 cells and in primary midbrain culture. In vivo MPTP and in vitro rotenone pretreatments increased the mRNA expression of P2X₇ receptors in the striatum and substantia nigra of wild-type mice. Basal mRNA expression of P2X₄ receptors was higher in P2X₇ knockout mice and was further up-regulated by MPTP treatment. Genetic deletion or pharmacological inhibition of P2X₇ receptors did not change survival rate or depletion of striatal endogenous dopamine (DA) content after in vivo MPTP or in vitro rotenone treatment. However, depletion of norepinephrine was significant after MPTP treatment only in P2X₇ knockout mice. The basal ATP content was higher in the substantia nigra of wild-type mice, but the ADP level was lower. Rotenone treatment elicited a similar reduction in ATP content in the substantia nigra of both genotypes, whereas reduction of ATP was more pronounced after rotenone treatment in striatal slices of P2X₇ deficient mice. Although the endogenous amino acid content remained unchanged, the level of the endocannabinoid, 2-AG, was elevated by rotenone in the striatum of wild-type mice, an effect that was absent in mice deficient in P2X₇ receptors.

Conclusions

We conclude that P2X₇ receptor deficiency or inhibition does not support the survival of dopaminergic neurons in an in vivo or in vitro models of PD.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice is enhanced by ethanol or acetaldehyde

Persistent neurochemical changes consistent with parkinsonism have been reported in brains of mice treated with repeated high doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We now report that ethanol or acetaldehyde potentiate MPTP-induced damage to mouse striatum. One hour after the combined treatments (ethanol and MPTP or acetaldehyde and MPTP), the animals exhibited a marked and long-lasting catatonic posture and then returned gradually to apparently normal locomotion. Seven days after MPTP administration, depletion of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in mouse striatum were further potentiated in the group of animals treated with ethanol. This effect was more evident when the treatment was repeated twice and was dose-dependent. Acetaldehyde was more potent than ethanol in enhancing MPTP neurotoxicity. A single exposure to acetaldehyde before and during MPTP treatment produced a very consistent fall of DA, DOPAC and HVA but not serotonin (5HT) or 5-hydroxyindoleacetic acid (5HIAA) in the striatum. This suggests that ethanol effects on MPTP neurotoxicity might be related to acetaldehyde formation.     

Platelet-activating factor receptor knockout mice are protected from MPTP-induced dopaminergic degeneration

Platelet-activating factor (PAF), a potent mediator of inflammatory and immune responses, plays various roles in neuronal functions. However, little is known about the role of PAF/platelet-activating factor receptor (PAF-R) in Parkinson’s disease. Treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) resulted in significant increases in PAF species in the striatum of wild-type mice. These increases paralleled PAF-R gene expression in wild-type mice. Although nuclear factor kappa B (NF-κB) DNA-binding activity was increased significantly in MPTP-treated wild-type mice, this increase was not significant in PAF-R antagonist ginkgolide B (GB)-treated mice or PAF-R knockout (PAF-R^−/−) mice. Pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor, significantly ameliorated the dopaminergic deficits induced by MPTP in wild-type mice. MPTP treatment significantly increased oxidative damage, the immunoreactivity of ionized calcium binding adaptor molecule 1 (Iba-1)-positive microglial cells, and microglial differentiation of the M1 type in the striatum of wild-type mice. Consistently, PDTC significantly attenuated MPTP-induced behavioral impairments in wild-type mice. However, dopaminergic deficits, oxidative damage, reactive microglial cells, and behavioral impairments induced by MPTP were not significantly observed in GB-treated mice or PAF-R^−/− mice. PDTC did not significantly alter the attenuations evident in MPTP-treated PAF-R^−/− mice, indicating that NF-κB is a critical target for neurotoxic modulation of PAF-R. We propose for the first time that PAF/PAF-R can mediate dopaminergic degeneration via an NF-κB-dependent signaling process.     

Modulatory Effects of Testosterone on 1-Methyl-4-Phenyl- 1, 2, 3, 6-Tetrahydropyridine-Induced Neurotoxicity

Abstract: In this experiment, we examined the modulatory effects of testosterone on the parkinsonism-inducing drug 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) in two strains of mice. Orchidectomized male CD-1 and C57/BI mice were implanted with either empty Silastic capsules or capsules containing testosterone and subsequently treated with MPTP. A small area of the corpus striatum was removed for determination of dopamine (DA) content, whereas the remainder was superfused and used to measure L-DOPA (5 μ M )-evoked DA release. In animals treated with MPTP, L-DOPA-evoked DA release was reduced significantly in CD-1 mice, but not in C57/BI mice, treated with testosterone. No differences in L-DOPA-stimulated DA release between MPTP- versus vehicle-treated mice was observed in either the CD-1 or C57/BI mice receiving empty Silastic capsules. Corpus striatum DA contents were more severely depleted in the MPTP-sensitive C57/BI versus the CD-1 mouse strain irrespective of hormone treatment. These results confirm previous results demonstrating differences in these two mouse strains in response to the neurotoxic effects of MPTP upon corpus striatum DA content. More interestingly, they show an important differential modulatory effect of testosterone upon L-DOPA-evoked DA release as a function of MPTP treatment and indicate that testosterone significantly alters the neurotoxic effects of MPTP in the CD-1 mouse.     

Resistance of Golden Hamster to l-Methyl-4-Phenyl-1,2,3,6 Tetrahydropyridine: Relationship with Low Levels of Regional Monoamine Oxidase B

Abstract: Effects of acute and chronic administration of 1 -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were investigated for dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetic acid, in nucleus caudatus putamen (NCP), limbic system, and substantia nigra (SN) of golden hamster and BALB/c and C57/BL mice to obtain a clue for the variance of MPTP toxicity between the strains and species. Regional differences in the levels of monoamine oxidase (MAO) and the in vitro effects of MAO inhibitors were also determined and correlated with MPTP neurotoxicity. Concentrations of MPTP in the brains of mice and golden hamster at 10 min were comparable. Golden hamster was found to be resistant to the administration of MPTP as indicated by a lack of any alteration from the normal content of DA in NCP, limbic system, and SN. Both strains of mice exhibited >50% and >75% depletion of DA (C57/BL and BALB/c, respectively). The metabolites-to-DA ratios were decreased and increased in golden hamster and mouse strains, respectively, after acute or chronic treatment. Whereas the content of total MAO in golden hamster was one-third to one-sixth of any nuclei or mitochondria of both strains of mice, the ratio of MAO A to B was significantly higher in the former species. A possible involvement of discrete regional MAO activity in determining the extent of susceptibility of a species to MPTP toxicity is indicated from the study because (1) susceptibility as evidenced by DA depletion of a species coincided with high levels of MAO activity in SN and NCP, and (2) a highly positive correlation existed with total MAO and MAO B activity, there was a lack of correlation with MAO A activity, and a negative correlation existed with MAO A-to-B ratio and DA depletion. Hence, we propose that the resistance of a species to MPTP toxicity may depend on the content as well as the ratios of the two forms of MAO in NCP and SN. In other words, a higher MAO activity and a relative dominance of MAO B in these nuclei are critical in determining the susceptibility of a species to MPTP neurotoxicity.     

Prevention of the Nigrostriatal Toxicity of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine by Inhibitors of 3,4-Dihydroxyphenylethylamine Transport

The 3,4-dihydroxyphenylethylamine (DA, dopamine) uptake inhibitors GBR 13,069, amfonelic acid, WIN-35,065-2, WIN-35,428, nomifensine, mazindol, cocaine, McN-5908, McN-5847, and McN-5292 were effective in preventing [3H]DA and [3H]1-methyl-4-phenylpyridinium (MPP+) uptake in rat and mouse neostriatal tissue slices. These DA uptake inhibitors also were effective in attenuating the MPP+-induced release of [3H]DA in vitro. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration to mice (6 X 25 mg/kg i.p.) resulted in a large (70-80%) decrement in neostriatal DA. WIN-35,428 (5 mg/kg), GBR 13,069 (10 mg/kg), McN-5292 (5 mg/kg), McN-5908 (2 mg/kg), and amfonelic acid (2 mg/kg), when administered intraperitoneally 30 min prior to each MPTP injection, fully protected against MPTP-induced neostriatal damage. Other DA uptake inhibitors showed partial protection in vivo at the doses selected. Desmethylimipramine did not prevent [3H]MPP+ uptake or MPP+-induced release of [3H]DA in vitro, and did not protect against MPTP neurotoxicity in vivo. These results support the hypothesis put forth previously by others that the active uptake of MPP+ by dopaminergic neurons is necessary for toxicity.     

Involvement of cytochrome P450 2E1 in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of Parkinson's disease

Elucidation of the biochemical steps leading to the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced degeneration of the nigrostriatal dopamine (DA) pathway has provided new clues to the pathophysiology of Parkinson's disease. In line with the enhancement of MPTP toxicity by diethyldithiocarbamate (DDC), here we demonstrate how other cytochrome P450 (CYP) 2E1 inhibitors, such as diallyl sulphide (DAS) and phenylethylisothiocyanate (PIC), also potentiate the selective DA neurone degeneration in C57/bl mice. In addition, we show that CYP 2E1 is present in the brain and in the basal ganglia of this mouse strain, as measured by RT-PCR, western blot analysis and immunohistochemistry. A kinetic analysis of MPTP and its metabolites, by means of the microdialysis technique in the striatum, indicates that no detoxification metabolic pathway is affected by any of these inhibitors. This does not rule out, however, that an undetected detoxification pathway involving CYP 2E1 is operating. In order to provide direct evidence for this isozyme involvement, CYP 2E1 knockout mice were challenged with MPTP or the combined treatment. Here we show that these transgenic mice have a low sensitivity to MPTP alone, similar to their wild-type counterparts, suggesting that it is likely that transgenic mice compensate for the missing enzyme. However, DDC pretreatment completely fails to enhance MPTP toxicity in CYP 2E1 knockout mice, whereas this enhancement is regularly present in wild-type animals. This study indicates that the occurrence of CYP 2E1 in C57/bl mouse brain is relevant to MPTP toxicity, and suggests that this isozyme may have a detoxificant role related to the efflux transporter of the toxin.     

Diethyldithiocarbamate Potentiates the Neurotoxicity of In Vivo l-Methyl-4-Phenyl-1, 2, 3, 6-Tetrahydropyridine and of In Vitro 1-Methyl-4-Phenylpyridinium

Diethyldithiocarbamic acid (DDC) potentiates in vivo neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and in vitro neurotoxicity of 1-methyl-4-phenylpyridinium (MPP+). Male C57B1/6 mice were given two or five injections of MPTP (30 mg/kg i.p.) preceded 0.5 h by DDC (400 mg/kg i.p.). The mice were tested for catalepsy, akinesia, or motor activity during and after the period of dosing. Striatal and hippocampal tissues were obtained at 2 and 7 days following the last injection and evaluated for dopamine and norepinephrine levels, respectively. These same tissues were also analyzed for the levels of glial fibrillary acidic protein (GFAP), an astrocyte-localized protein known to increase in response to neural injury. Pretreatment with DDC potentiated the effect of MPTP in striatum and resulted in substantially greater dopamine depletion, as well as a more pronounced elevation in GFAP. In hippocampus, the levels of norepinephrine and GFAP were not different from controls in mice receiving only MPTP, but pretreatment with DDC resulted in a sustained depletion of norepinephrine and an elevation of GFAP, suggesting that damage was extended to this brain area by the combined treatment. Mice receiving MPTP preceded by DDC also demonstrated a more profound, but reversible, catalepsy and akinesia compared to those receiving MPTP alone. Systemically administered MPP+ decreased heart norepinephrine, but did not alter the striatal levels of dopamine or GFAP, and pretreatment with DDC did not alter these effects, but did increase lethality. DDC is known to increase brain levels of MPP+ after MPTP, but our data indicate that this is not due to a movement of peripherally generated MPP+ into CNS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of acute and sub-chronic l-dopa therapy on striatal l-dopa methylation and dopamine oxidation in an MPTP mouse model of Parkinsons disease

Aims

The molecular mechanisms for the loss of 3,4-dihydroxyphenylalanine (l-dopa) efficacy during the treatment of Parkinson's disease (PD) are unknown. Modifications related to catecholamine metabolism such as changes in l-dopa and dopamine (DA) metabolism, the modulation of catecholamine enzymes and the production of interfering metabolites are the primary concerns of this study.

Main methods

Normal (saline) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) pre-treated mice were primed with 100 mg/kg of l-dopa twice a day for 14 days, and a matching group remained l-dopa naïve. l-dopa naive and primed mice received a challenge dose of 100 mg/kg of l-dopa and were sacrificed 30 min later. Striatal catecholamine levels and the expression and activity of catechol-O-methyltransferase (COMT) were determined.

Key findings

Normal and MPTP pre-treated animals metabolize l-dopa and DA similarly during l-dopa therapy. Administration of a challenge dose of l-dopa increased l-dopa and DA metabolism in l-dopa naïve animals, and this effect was enhanced in l-dopa primed mice. The levels of 3-OMD in MPTP pre-treated animals were almost identical to those in normal mice, which we found are likely due to increased COMT activity in MPTP pre-treated mice.

Significance

The results of this comparative study provide evidence that sub-chronic administration of l-dopa decreases the ability of the striatum to accumulate l-dopa and DA, due to increased metabolism via methylation and oxidation. This data supports evidence for the metabolic adaptation of the catecholamine pathway during long-term treatment with l-dopa, which may explain the causes for the loss of l-dopa efficacy.     

The Neuroprotective Effect of Overexpression of Calbindin-D28k in an Animal Model of Parkinson’s Disease

Overexpression of calbindin-D_28k (CaBP-28 k) induces neurite outgrowth in dopaminergic neuronal cells and could provide some protection to dopaminergic neurons against the pathological process in Parkinson’s disease. Transgenic mice CaBP-28 k overexpression and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse models were generated, and the effect of midbrain dopamine neurons in ethology was also assessed. Tyrosine hydroxylase (TH)-immunoreactive neurons were counted, and the concentration of total protein and dopamine (DA) of striatum corpora was measured in four animal models. Results showed that the positive TH cells, content of DA, and ability of ethology in MPTP-induced transgenic mice were significantly higher than that in MPTP-induced wild-type mice. The findings demonstrate that overexpression of CaBP-28 k could provide protection for DA neurons from neurodegeneration. It would provide a potential strategy in the treatment of Parkinson’s diseases.     

Clonidine Suppresses 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Reductions of Striatal Dopamine and Tyrosine Hydroxylase Activity in Mice

Abstract: Recent findings have shown that excitatory amino acid may be involved in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity. At the same time, evidence is accumulating that the endogenous nor-adrenergic system plays a protective role in MPTP-induced striatal dopamine (DA) depletion and nigral dopaminergic cell death. Recently, α₂-adrenoceptors located on glutamatergic axons have been shown to inhibit glutamate overflow. In this study, we evaluated the effects of an α₂-agonist (clonidine) and an α₂-antagonist (yohimbine) on MPTP-induced striatal DA depletion and tyrosine hydroxylase activity reduction. We show that clonidine is able to prevent the neurotoxicity of MPTP in mice. To exert this effect, clonidine (0.5 mg/kg) must be administered at least twice (30 min before and 30 min after MPTP). Administration of another α₂-agonist (detomidine, 0.3 mg/kg) attenuated the neurotoxicity induced by MPTP. We provide evidence that the protective effect obtained with clonidine was not due to decreased striatal content of 1-methyl-4-phenylpyridinium (MPP⁺). We also show that yohimbine, which is a classic α₂-adrenoceptor antagonist with low affinity for imidazoline receptors, produced by itself an enhancement of MPTP toxicity and was able to block the protective effect of clonidine. These data raise the possibility that α₂-adrenoceptor may modulate the susceptibility of the nigrostriatal dopaminergic pathway to neurotoxicity.     

IL-17 is involved in bone resorption in mouse periapical lesions

Periapical lesions are induced by bacterial infection of the dental pulp and result in destruction of the surrounding alveolar bone. Although various immunological studies concerning periapical bone resorption have been reported, the role of cytokines in the formation of periapical lesions remains unclear. In this study, the role of IL-17A in periapical lesions in mice was investigated. Normal C57BL/6, IFN-γ^−/−, TNF-α^−/−, and IL-17A^−/− mice were subjected to pulp exposure and infected with Prevotella intermedia (ATCC25611) and Porphyromonas gingivalis (ATCC33277) in the mandibular first molar. Periapical lesions were determined by μCT on day 21 after infection, and 3D visual construction was performed using 3D picture quantification software. The expression of IL-17A mRNA in periapical lesions was determined by the RT-PCR and real-time RT-PCR method. Periapical lesions developed in wild-type, IFN-γ^−/−, and TNF-α^−/− mice after infection with P. intermedia and P. gingivalis . However, periapical lesions were not observed in IL-17A^−/− mice. The expression of IL-17A mRNA was significantly induced in periapical lesions of wild-type mice after infection. These results suggest that IL-17A, but not IFN-γ or TNF-α, plays an important role in the formation of periapical lesions.     

Neurochemical and Histochemical Characterization of Neurotoxic Effects of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine on Brain Catecholamine Neurones in the Mouse

Systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) caused a rapid and long-lasting reduction of both 3,4-dihydroxyphenylalanine (dopamine, DA) and noradrenaline (NA) in mouse brain, as observed histo- and neurochemically. The depleting effects were more pronounced after repeated MPTP administration and the most marked reductions were observed after 2 X 50 mg MPTP/kg s.c., when DA in striatum and NA in frontal cortex were reduced by greater than 90% 1 week after MPTP. Mice with such catecholamine depletions were markedly sedated and almost completely immobilized. The behavioural syndrome after MPTP resembled that seen after reserpine, a monoamine-depleting drug. MPTP also caused a long-lasting reduction of catecholamine uptake in striatal DA and cortical NA nerve terminals and reduced tyrosine hydroxylase activity in these regions. There was no evidence that MPTP caused any marked DA and NA cell body death. MPTP given acutely transiently elevated serotonin levels. The results are compatible with a neurotoxic action of MPTP on both DA and NA nerve terminals. The nigro-striatal DA and the locus coeruleus NA neurone systems appeared to be most susceptible. Synthesis and utilization of residual striatal DA and cortical NA were increased, as often observed in partially denervated monoamine-innervated brain regions. Both DA and NA showed a gradual recovery, which took months to become complete and may have been related to a regrowth of catecholamine nerve terminals.