Tat­aldose reductase prevents dopaminergic neuronal cell death by inhibiting oxidative stress and MAPK activation.

Aldose reductase (AR) is known to detoxify aldehydes and prevent oxidative stress. Although AR exerts antioxidant effects, the role of AR in Parkinson's disease (PD) remains unclear. The objective of the present study was to investigate the protective effects of AR protein against 1­methyl­4­phenylpyridinium (MPP+)­induced SH­SY5Y cell death and 1­methyl­4­phenyl­1,2,3,6­tetrahydropyridine (MPTP)­induced PD in a mouse model using the cell permeable Tat­AR fusion protein. The results revealed that when Tat­AR protein was transduced into SH­SY5Y cells, it markedly protected the cells against MPP+­induced death and DNA fragmentation. It also reduced the activation of mitogen-activated protein kinase (MAPKs) and regulated the expression levels of Bcl­2, Bax and caspase­3. Immunohistochemical analysis revealed that when Tat­AR protein was transduced into the substantia nigra (SN) of mice with PD, it markedly inhibited dopaminergic neuronal cell death. Therefore, Tat­AR may be useful as a therapeutic protein for PD.

Neutral sphingomyelinase increases and delocalizes in the absence of Toll-Like Receptor 4: A new insight for MPTP neurotoxicity.

Both sphingomyelinase and Toll-Like Receptor 4 (TLR4) are implicated in neurodegenerative diseases. However, the relationship between the two molecules remains unclear. In this study, using WT and TLR4-deficient mice, treated or not with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we aimed to investigate the relation between TLR4 and neutral sphingomyelinase (nSMase) in the midbrain. We found that the lack of TLR4 caused increase in nSMase protein expression and enzyme activity in the midbrain, as well as a marked delocalization from the cell membranes. This provoked a decrease in sphingomyelin (SM) species and an increase in ceramide levels. We found that exposure of TLR4-deficient mice to MPTP reduces unsaturated SM species by increasing saturated/unsaturated SM ratio. Saturated fatty acid make SM more rigid and could contribute to reducing neural plasticity. In this study we showed that the absence of TLR4 also induced reduction of both heavy neurofilaments and glial fibrillary acidic protein (GFAP) and mice exhibited higher sensitivity to MPTP administration. We speculated about the possible association between nSMase-TLR4 complex and MPTP midbrain damage. Taken together, our findings provide for the first time indications about the role of TLR4 in change of SM metabolism in MPTP neurotoxicity.

Drug-induced Parkinson's disease modulates protein kinase A and Olfactory Marker Protein in the mouse olfactory bulb.

BACKGROUND: Olfaction is often affected in parkinsonian patients, but dopaminergic cells in the olfactory bulb are not affected by some Parkinson-inducing drugs. We investigated whether the drug MPTP produces the olfactory deficits typical of Parkinson and affects the olfactory bulb in mice. FINDINGS: Lesioned and control mice were tested for olfactory search, for motor and exploratory behavior. Brains and olfactory mucosa were investigated via immunohistochemistry for thyrosine hydroxylase, Olfactory Marker Protein and cyclic AMP-dependent protein kinase as an intracellular pathway involved in dopaminergic neurotransmission. MPTP induced motor impairment, but no deficit in olfactory search. Thyrosine hydroxylase did not differ in olfactory bulb, while a strong decrease was detected in substantia nigra and tegmentum of MPTP mice. Olfactory Marker Protein decreased in the olfactory bulb of MPTP mice, while a cyclic AMP-dependent protein kinase increased in the inner granular layer of MPTP mice. CONCLUSIONS: MPTP mice do not present behavioural deficits in olfactory search, yet immunoreactivity reveals modifications in the olfactory bulb, and suggests changes in intracellular signal processing, possibly linked to neuron survival after MPTP.

Neutral Sphingomyelinase Behaviour in Hippocampus Neuroinflammation of MPTP-Induced Mouse Model of Parkinson's Disease and in Embryonic Hippocampal Cells.

Neutral sphingomyelinase is known to be implicated in growth arrest, differentiation, proliferation, and apoptosis. Although previous studies have reported the involvement of neutral sphingomyelinase in hippocampus physiopathology, its behavior in the hippocampus during Parkinson's disease remains undetected. In this study, we show an upregulation of inducible nitric oxide synthase and a downregulation of neutral sphingomyelinase in the hippocampus of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- (MPTP-) induced mouse model of Parkinson's disease. Moreover, the stimulation of neutral sphingomyelinase activity with vitamin 1,25-dihydroxyvitamin D3 reduces specifically saturated fatty acid sphingomyelin by making sphingomyelin a less rigid molecule that might influence neurite plasticity. The possible biological relevance of the increase of neutral sphingomyelinase in Parkinson's disease is discussed.

Effect of the pituitary adenylate cyclase-activating polypeptide on the autophagic activation observed in in vitro and in vivo models of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder that leads to destruction of the midbrain dopaminergic (DA) neurons. This phenomenon is related to apoptosis and its activation can be blocked by the pituitary adenylate cyclase-activating polypeptide (PACAP). Growing evidence indicates that autophagy, a self-degradation activity that cleans up the cell, is induced during the course of neurodegenerative diseases. However, the role of autophagy in the pathogenesis of neuronal disorders is yet poorly understood and the potential ability of PACAP to modulate the related autophagic activation has never been significantly investigated. Hence, we explored the putative autophagy-modulating properties of PACAP in in vitro and in vivo models of PD, using the neurotoxic agents 1-methyl-4-phenylpyridinium (MPP(+)) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), respectively, to trigger alterations of DA neurons. In both models, following the toxin exposure, PACAP reduced the autophagic activity as evaluated by the production of LC3 II, the modulation of the p62 protein levels, and the formation of autophagic vacuoles. The ability of PACAP to inhibit autophagy was also observed in an in vitro cell assay by the blocking of the p62-sequestration activity produced with the autophagy inducer rapamycin. Thus, the results demonstrated that autophagy is induced in PD experimental models and that PACAP exhibits not only anti-apoptotic but also anti-autophagic properties.

1-[2-(4-Benzyloxyphenoxy)Ethyl]Imidazole inhibits monoamine oxidase B and protects against neuronal loss and behavioral impairment in rodent models of Parkinson's disease.

Monoamine oxidase B (MAO-B) is well known as a therapeutic target for Parkinson's disease (PD). MAO-B inhibitors retain antiparkinsonism abilities to improve motor function and prevent neuronal loss by decreasing dopamine metabolism and oxidative stress in the brain. From the study to find novel antiparkinsonism drugs that can inhibit MAO-B activity, neuronal loss, and behavioral deficits in the mouse model of PD, we identified that 1-[2-(4-benzyloxyphenoxy)ethyl]imidazole (BPEI) or safinamide strongly and selectively inhibited MAO-B activities in a dose-dependent manner (IC50 of BPEI and safinamide for MAO-B were 0.016 and 0.0021 µM and for MAO-A were 70.0 and 370 µM, respectively). In ex vivo studies after an administration (30 mg/kg, i.p.) of BPEI or safinamide to normal mice, the MAO-B activity in the brain was reduced by up to 90.6% or 82.4% at 1.0 hr. BPEI (20 mg/kg, i.p.) or safinamide (20 mg/kg, i.p.) significantly reversed the behavioral impairments, dopamine levels in the striatum, and neuronal loss in the substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice compared with the MPTP-alone-treated group. In the 6-hydroxydopamine-induced PD rat model, behavioral improvement by levodopa sparing activity was observed in the BPEI- or safinamide-treated (20 mg/kg, i.p.) rats. Moreover, BPEI revealed additional curative activities for nonmotor symptoms of PD such as pain, anxiety, epilepsy, and depression in rodent disease models. Therefore, BPEI has broad therapeutic potential for treating motor symptoms via strong and selective inhibitory effects on MAO-B, with additional benefits for comorbid symptoms in PD.

STEP61 is a substrate of the E3 ligase parkin and is upregulated in Parkinson's disease.

Parkinson's disease (PD) is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). The loss of SNc dopaminergic neurons affects the plasticity of striatal neurons and leads to significant motor and cognitive disabilities during the progression of the disease. PARK2 encodes for the E3 ubiquitin ligase parkin and is implicated in genetic and sporadic PD. Mutations in PARK2 are a major contributing factor in the early onset of autosomal-recessive juvenile parkinsonism (AR-JP), although the mechanisms by which a disruption in parkin function contributes to the pathophysiology of PD remain unclear. Here we demonstrate that parkin is an E3 ligase for STEP61 (striatal-enriched protein tyrosine phosphatase), a protein tyrosine phosphatase implicated in several neuropsychiatric disorders. In cellular models, parkin ubiquitinates STEP61 and thereby regulates its level through the proteasome system, whereas clinically relevant parkin mutants fail to do so. STEP61 protein levels are elevated on acute down-regulation of parkin or in PARK2 KO rat striatum. Relevant to PD, STEP61 accumulates in the striatum of human sporadic PD and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mice. The increase in STEP61 is associated with a decrease in the phosphorylation of its substrate ERK1/2 and the downstream target of ERK1/2, pCREB [phospho-CREB (cAMP response element-binding protein)]. These results indicate that STEP61 is a novel substrate of parkin, although further studies are necessary to determine whether elevated STEP61 levels directly contribute to the pathophysiology of PD.

Neuroprotective effects of aldehyde dehydrogenase 2 activation in rotenone-induced cellular and animal models of parkinsonism.

Many studies have shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) functions as a cellular protector against oxidative stress by detoxification of cytotoxic aldehydes. Within dopaminergic neurons, dopamine is metabolized by monoamine oxidase to yield 3,4-dihydroxyphenylacetaldehyde (DOPAL) then converts to a less toxic acid product by ALDH. The highly toxic and reactive DOPAL has been hypothesized to contribute to the selective neurodegeneration in Parkinson's disease (PD). In this study, we investigated the neuroprotective mechanism and therapeutic effect of ALDH2 in rotenone models for parkinsonism. Overexpression of wild-type ALDH2 gene, but not the enzymatically deficient mutant ALDH2*2 (E504K), reduced rotenone-induced cell death. Application of a potent activator of ALDH2, Alda-1, was effective in protecting against rotenone-induced apoptotic cell death in both SH-SY5Y cells and primary cultured substantia nigra (SN) dopaminergic neurons. In addition, intraperitoneal administration of Alda-1 significantly reduced rotenone- or MPTP-induced death of SN tyrosine hydroxylase (TH)-positive dopaminergic neurons. The attenuation of rotenone-induced apoptosis by Alda-1 resulted from decreasing ROS accumulation, reversal of mitochondrial membrane potential depolarization, and inhibition of activation of proteins related to mitochondrial apoptotic pathway. The present study demonstrates that ALDH2 plays a crucial role in maintaining normal mitochondrial function to protect against neurotoxicity and that Alda-1 is effective in ameliorating mitochondrial dysfunction and inhibiting mitochondria-mediated apoptotic pathway. These results indicate that ALDH2 activation could be a neuroprotective therapy for PD.

In vivo evidence of increased nNOS activity in acute MPTP neurotoxicity: a functional pharmacological MRI study.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin commonly used to produce an animal model of Parkinson's disease. Previous studies have suggested a critical role for neuronal nitric oxide (NO) synthase- (nNOS-) derived NO in the pathogenesis of MPTP. However, NO activity is difficult to assess in vivo due to its extremely short biological half-life, and so in vivo evidence of NO involvement in MPTP neurotoxicity remains scarce. In the present study, we utilized flow-sensitive alternating inversion recovery sequences, in vivo localized proton magnetic resonance spectroscopy, and diffusion-weighted imaging to, respectively, assess the hemodynamics, metabolism, and cytotoxicity induced by MPTP. The role of NO in MPTP toxicity was clarified further by administering a selective nNOS inhibitor, 7-nitroindazole (7-NI), intraperitoneally to some of the experimental animals prior to MPTP challenge. The transient increase in cerebral blood flow (CBF) in the cortex and striatum induced by systemic injection of MPTP was completely prevented by pretreatment with 7-NI. We provide the first in vivo evidence of increased nNOS activity in acute MPTP-induced neurotoxicity. Although the observed CBF change may be independent of the toxicogenesis of MPTP, this transient hyperperfusion state may serve as an early indicator of neuroinflammation.

Neuroprotective changes of thalamic degeneration-related gene expression by acupuncture in an MPTP mouse model of parkinsonism: microarray analysis.

Acupuncture stimulations at GB34 and LR3 inhibit the reduction of tyrosine hydroxylase in the nigrostriatal dopaminergic neurons in the parkinsonism animal models. Especially, behavioral tests showed that acupuncture stimulations improved the motor dysfunction in a previous study by almost 87.7%. The thalamus is a crucial area for the motor circuit and has been identified as one of the most markedly damaged areas in Parkinson's disease (PD), so acupuncture stimulations might also have an effect on the thalamic damage. In this study, gene expression changes following acupuncture at the acupoints were investigated in the thalamus of a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism model using a whole transcript array. It was confirmed that acupuncture at these acupoints could inhibit the decrease of tyrosine hydroxylase in the thalamic regions of the MPTP model, while acupuncture at the non-acupoints could not suppress this decrease by its level shown in the acupoints. GeneChip gene array analysis showed that 18 (5 annotated genes: Dnase1l2, Dusp4, Mafg, Ndph and Pgm5) of the probes down-regulated in MPTP, as compared to the control, were exclusively up-regulated by acupuncture at the acupoints, but not at the non-acupoints. In addition, 14 (3 annotated genes; Serinc2, Sp2 and Ucp2) of the probes up-regulated in MPTP, as compared to the control, were exclusively down-regulated by acupuncture at the acupoints, but not at the non-acupoints. The expression levels of the representative genes in the microarray were validated by real-time RT-PCR. These results suggest that the 32 probes (8 annotated genes) which are affected by MPTP and acupuncture may be responsible for exerting the inhibitory effect of acupuncture in the thalamus which can be damaged by MPTP intoxication.

Neuronal or inducible nitric oxide synthase (NOS) expression level is not involved in the different susceptibility to nigro-striatal dopaminergic neurotoxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) between C57BL/6 and BALB/c mice.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces severe degeneration of dopaminergic (DA-ergic) neurons when administrated to C57BL/6 mice, but such lesions are not observed in BALB/c mice. To clarify the factors which influence such marked strain differences in the susceptibility to MPTP, the involvement of neuronal NOS (nNOS) and inducible NOS (iNOS) was investigated. MPTP was intraperitoneally (ip) administrated to adult C57BL/6 (highly sensitive) and BALB/c (resistant) mice. Immunohistochemical analysis using an antibody to tyrosine hydroxylase (TH) showed a significant decrease in TH-immunopositive areas in the striatum and TH-positive cells in the substantia nigra pars compacta (SNpc) of MPTP-treated C57BL/6 mice at 1 and 7 days (d) after administration, compared to control C57BL/6 mice. On the other hand, MPTP-treated BALB/c mice showed no significant changes. By Western blot analysis, TH, MAO-B, DAT, nNOS and iNOS protein expression levels were examined in intact and MPTP-treated mice. Intact BALB/c mice showed higher DAT protein expression in the striatum and TH protein expression in the midbrain than intact C57BL/6 mice. In addition, MPTP-treated BALB/c mice showed a more significant increase of MAO-B expression than MPTP-treated C57BL/6 mice at 12 h. The increase of nNOS and iNOS protein expressions in MPTP-treated BALB/c mice was more pronounced in the striatum and midbrain than in MPTP-treated C57BL/6 mice at 12 h and 2 d. These results indicate that MAO-B, DAT, nNOS or iNOS expression levels do not influence the different strain susceptibility to MPTP.

Resistance of the golden hamster to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-neurotoxicity is not only related with low levels of cerebral monoamine oxidase-B.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been proved to be a potent neurotoxin on dopaminergic neurons inducing most of the symptoms and cerebral lesions observed in the idiopathic Parkinson's disease (PD). Although there is a substantial body of theory and researches about the effects of MPTP on susceptible mice and nonhuman primates, there are only few studies in resistant animals, such as golden hamsters (GH). The low levels of cerebral monoamine oxidase-B (MAO-B) enzyme have been proposed as the cause of the GH insensitivity to MPTP. The aim of this study was to elucidate whether MAO-B is the only factor which confer GH resistance to MPTP. Neither loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) nor cell death in the subventricular zone (SVZ) were found in female GH in response to an acute intraperitoneal (ip) MPTP treatment. To prove the role of MAO-B in the MPTP-resistance, female and male GH was intracerebroventricularly (icv) injected with either MPTP or 1-methyl-4-phenylpyridinum (MPP(+)). Neither depletion in the number of dopaminergic neurons, nor astrogliosis, cell death in the SVZ of female and male GH were registered after an icv treatment with MPTP or MPP(+). Furthermore, we demonstrated that MAO-B is located predominantly within the endothelial cells in the blood brain barrier (BBB), but not in the astroglia. The present results raise the possibility that, in GH, other mechanisms, apart from the low levels of regional MAO-B, confer resistance to MPTP and its metabolites.

Different susceptibility to 1-methyl-4-phenylpyridium (MPP(+))-induced nigro-striatal dopaminergic cell loss between C57BL/6 and BALB/c mice is not related to the difference of monoamine oxidase-B (MAO-B).

Subcutaneous and intraperitoneal administrations of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induce selective dopaminergic (DA-ergic) neuronal death in many animal species. After passing through the blood-brain barrier (BBB), MPTP is converted to 1-methy-4-phenylpiridinium (MPP(+)) by astrocytic monoamine oxidase-B (MAO-B). MPP(+) then induces the dopaminergic neuronal death. In mice, marked strain differences in the susceptibility to MPTP-injection have been reported. To clarify which factor(s) cause the strain differences, MPTP or MPP(+) was intracerebroventricularly (icv) injected into adult C57BL/6 (highly susceptible to MPTP) and BALB/c (resistant to MPTP) mice. The brain tissues including the striatum and substantia nigra pars compacta (SNpc) were examined immunohistochemically using an antibody to tyrosine hydrocyrase (TH). MPP(+)-injected C57BL/6 mice showed a significant decrease in TH-immunopositive areas in the striatum at Day 3 post injection (p<0.01), and TH-positive cells in the SNpc at Days 1 and 3 (p<0.01), respectively, compared to saline-injected control mice. In addition, MPP(+)-injected BALB/c mice showed a significant decrease in TH-positive areas in the striatum at Days 1 and 3, and SNpc TH-positive cells in the SNpc at Day 3, respectively (p<0.05). However, the decrease rates in the BALB/c mice were lower than that in C57BL/6 mice. MPTP-injected C57BL/6 mice, however, showed no lesions in the striatum and SNpc at Days 1 and 7 after icv injection. All the present findings indicate that factors other than MAO-B can influence the strain susceptibility between C57BL/6 and BALB/c mice after the conversion from MPTP to MPP(+).

[Changes of heme oxygenase-1 expression in the nigrostriatal system of MPTP-treated SAMP8 mouse].

AIM: To explore the relationship between the expression of hemeoxygenase-1 (HO-1) and the dopaminergic system impairment in MPTP-treated SAMP8 mice. METHODS: 6-month-old male SAMP8 mice received MPTP (20 mg/kg) subcutaneous injection at 2-h intervals for 4 times, and the control group was treated with an equal volume of normal saline. Mice were sacrificed at 6 h, 24 h, 3 d and 8 d after the first injection for the detection of the changes of tyrosine hydroxylase (TH) and HO-1 in the nigrostriatal system by immunohistochemistry and Western blotting. RESULTS: TH-positive neuronal loss was visible at 6 h (14.23%, P<0.05), 24 h (23.85%, P<0.01), 3 d (36.77%, P<0.001), and 8 d (45.90%, P<0.001), and the significant progression of dopaminergic neuronal loss occurred most prominently in the MPTP group from 24 h to 3 d (24 h vs 3 d, P<0.05). There was a significant decrease of striatal TH immunoreactive cells in the MPTP group (P<0.05). Additionally, HO-1 positive cells were detected in striatum just only at 3 d, with the increase of HO-1 protein expression in MPTP groups. Western blot analysis showed no change of HO-1 protein levels in the midbrain after MPTP treatment compared to those of the normal saline group. CONCLUSION: MPTP caused the loss of dopaminergic neuron number and the decrease of TH protein levels in SAMP8 mice. The up-regulation of HO-1 was ephemeral, and its effects related with Parkinson's disease was limited in this study.

Inhibition of rho kinase enhances survival of dopaminergic neurons and attenuates axonal loss in a mouse model of Parkinson's disease.

Axonal degeneration is one of the earliest features of Parkinson's disease pathology, which is followed by neuronal death in the substantia nigra and other parts of the brain. Inhibition of axonal degeneration combined with cellular neuroprotection therefore seem key to targeting an early stage in Parkinson's disease progression. Based on our previous studies in traumatic and neurodegenerative disease models, we have identified rho kinase as a molecular target that can be manipulated to disinhibit axonal regeneration and improve survival of lesioned central nervous system neurons. In this study, we examined the neuroprotective potential of pharmacological rho kinase inhibition mediated by fasudil in the in vitro 1-methyl-4-phenylpyridinium cell culture model and in the subchronic in vivo 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease. Application of fasudil resulted in a significant attenuation of dopaminergic cell loss in both paradigms. Furthermore, dopaminergic terminals were preserved as demonstrated by analysis of neurite network in vitro, striatal fibre density and by neurochemical analysis of the levels of dopamine and its metabolites in the striatum. Behavioural tests demonstrated a clear improvement in motor performance after fasudil treatment. The Akt survival pathway was identified as an important molecular mediator for neuroprotective effects of rho kinase inhibition in our paradigm. We conclude that inhibition of rho kinase using the clinically approved small molecule inhibitor fasudil may be a promising new therapeutic strategy for Parkinson's disease.

Astrocyte-specific IKK2 activation in mice is sufficient to induce neuroinflammation but does not increase susceptibility to MPTP.

A key regulator of inflammatory gene expression is the transcription factor NF-κB that is controlled by the IκB proteins. We used a transgenic mouse model expressing a constitutively active IκB-kinase-2 (IKK2-CA) in astrocytes under control of the human glial fibrillary acidic protein promotor (IKK2-mice) to investigate neuroinflammation, proinflammatory cytokine expression, microglial activation and a potential enhanced susceptibility to the neurotoxin MPTP (4×10 mg/kg). Readouts included the determination of cytokines, striatal dopamine (DA), nigral tyrosine hydroxylase (TH) positive neurons, microglial activation and motor activity. IKK2-CA expression in astrocytes conditionally induced by the tet-off system resulted in a widespread neuroinflammation indicated by the increased expression of inflammatory cytokines and the presence of activated microglia and astrogliosis. Additionally, striatal DA concentrations but not nigral TH-positive neurons were reduced in IKK2-mice by 20%. Motor activity of IKK2-mice was not affected. Surprisingly, there was a similar reduction in striatal DA concentrations and the number of nigral TH-positive neurons in IKK2 and control mice after MPTP treatment. In conclusion, although naïve IKK2-mice showed reduced striatal DA concentrations and an increase in inflammatory markers in the brain, a higher susceptibility to MPTP was not observed. This finding argues against a prominent role of astrocyte specific, IKK2-mediated neuroinflammation in MPTP-induced neurodegeneration.

Glutathione S-transferase pi mediates MPTP-induced c-Jun N-terminal kinase activation in the nigrostriatal pathway.

Parkinson's disease (PD) is a progressive movement disorder resulting from the death of dopaminergic neurons in the substantia nigra. Neurotoxin-based models of PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) recapitulate the neurological features of the disease, triggering a cascade of deleterious events through the activation of the c-Jun N-terminal kinase (JNK). The molecular mechanisms underlying the regulation of JNK activity under cellular stress conditions involve the activation of several upstream kinases along with the fine-tuning of different endogenous JNK repressors. Glutathione S-transferase pi (GSTP), a phase II detoxifying enzyme, has been shown to inhibit JNK-activated signaling by protein-protein interactions, preventing c-Jun phosphorylation and the subsequent trigger of the cell death cascade. Here, we use C57BL/6 wild-type and GSTP knockout mice treated with MPTP to evaluate the regulation of JNK signaling by GSTP in both the substantia nigra and the striatum. The results presented herein show that GSTP knockout mice are more susceptible to the neurotoxic effects of MPTP than their wild-type counterparts. Indeed, the administration of MPTP induces a progressive demise of nigral dopaminergic neurons together with the degeneration of striatal fibers at an earlier time-point in the GSTP knockout mice when compared to the wild-type mice. Also, MPTP treatment leads to increased p-JNK levels and JNK catalytic activity in both wild-type and GSTP knockout mice midbrain and striatum. Moreover, our results demonstrate that in vivo GSTP acts as an endogenous regulator of the MPTP-induced cellular stress response by controlling JNK activity through protein-protein interactions.

Recovery of hypothalamic tuberoinfundibular dopamine neurons from acute toxicant exposure is dependent upon protein synthesis and associated with an increase in parkin and ubiquitin carboxy-terminal hydrolase-L1 expression.

Hypothalamic tuberoinfundibular dopamine (TIDA) neurons remain unaffected in Parkinson disease (PD) while there is significant degeneration of midbrain nigrostriatal dopamine (NSDA) neurons. A similar pattern of susceptibility is observed in acute and chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse and rotenone rat models of degeneration. It is not known if the resistance of TIDA neurons is a constitutive or induced cell-autonomous phenotype for this unique subset of DA neurons. In the present study, treatment with a single injection of MPTP (20 mg/kg; s.c.) was employed to examine the response of TIDA versus NSDA neurons to acute injury. An acute single dose of MPTP caused an initial loss of DA from axon terminals of both TIDA and NSDA neurons, with recovery occurring solely in TIDA neurons by 16 h post-treatment. Initial loss of DA from axon terminals was dependent on a functional dopamine transporter (DAT) in NSDA neurons but DAT-independent in TIDA neurons. The active metabolite of MPTP, 1-methyl, 4-phenylpyradinium (MPP+), reached higher concentration and was eliminated slower in TIDA compared to NSDA neurons, which indicates that impaired toxicant bioactivation or distribution is an unlikely explanation for the observed resistance of TIDA neurons to MPTP exposure. Inhibition of protein synthesis prevented TIDA neuron recovery, suggesting that the ability to recover from injury was dependent on an induced, rather than a constitutive cellular mechanism. Further, there were no changes in total tyrosine hydroxylase (TH) expression following MPTP, indicating that up-regulation of the rate-limiting enzyme in DA synthesis does not account for TIDA neuronal recovery. Differential candidate gene expression analysis revealed a time-dependent increase in parkin and ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1) expression (mRNA and protein) in TIDA neurons during recovery from injury. Parkin expression was also found to increase with incremental doses of MPTP. The increase in parkin expression occurred specifically within TIDA neurons, suggesting that these neurons have an intrinsic ability to up-regulate parkin in response to MPTP-induced injury. These data suggest that TIDA neurons have a compensatory mechanism to deal with toxicant exposure and increased oxidative stress, and this unique TIDA neuron phenotype provides a platform for dissecting the mechanisms involved in the natural resistance of central DA neurons following toxic insult.

Apoptosis signal-regulating kinase 1 mediates MPTP toxicity and regulates glial activation.

Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase 3 family, is activated by oxidative stress. The death-signaling pathway mediated by ASK1 is inhibited by DJ-1, which is linked to recessively inherited Parkinson's disease (PD). Considering that DJ-1 deficiency exacerbates the toxicity of the mitochondrial complex I inhibitor 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we sought to investigate the direct role and mechanism of ASK1 in MPTP-induced dopamine neuron toxicity. In the present study, we found that MPTP administration to wild-type mice activates ASK1 in the midbrain. In ASK1 null mice, MPTP-induced motor impairment was less profound, and striatal dopamine content and nigral dopamine neuron counts were relatively preserved compared to wild-type littermates. Further, microglia and astrocyte activation seen in wild-type mice challenged with MPTP was markedly attenuated in ASK⁻/⁻ mice. These data suggest that ASK1 is a key player in MPTP-induced glial activation linking oxidative stress with neuroinflammation, two well recognized pathogenetic factors in PD. These findings demonstrate that ASK1 is an important effector of MPTP-induced toxicity and suggest that inhibiting this kinase is a plausible therapeutic strategy for protecting dopamine neurons in PD.

Protective effect of panaxatriol saponins extracted from Panax notoginseng against MPTP-induced neurotoxicity in vivo.

AIM OF THE STUDY: Panaxatriol saponins (PTS), the main constituents extracted from Panax notoginseng, a Chinese herbal medicine, has been shown to be an effective agent on various diseases. Our previous study has demonstrated that PTS is an inducer of thioredoxin-1 (Trx-1) and has a possible potential as a therapeutic agent for Parkinson's disease (PD). However, the effect of PTS on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in vivo is unknown. MATERIALS AND METHODS: Using locomotor activity test and traction test, we detected the effect of PTS on MPTP-induced behavioral impairment. Tyrosine hydroxylase, Trx-1, cyclooxygenase-2, pro-caspase-9, pro-caspase-12 and caspase-3 expressions in the anatomical region of substantia nigra pars compacta (SNc) were tested by Western blot. RESULTS: PTS provided neuroprotection against the loss of dopaminergic neurons and behavioral impairment caused by MPTP. MPTP-induced neuronal death in the SNc was suppressed by PTS through increasing Trx-1 expression, suppressing cyclooxygenase-2 over-expression and inhibiting mitochondria-mediated apoptosis. CONCLUSIONS: PTS, an inducer of Trx-1, has pluripharmacological properties in the protection against PD including enhancing antioxidant activity, acting as neurotrophic factor, modulating inflammation and inhibiting mitochondria-mediated apoptosis.