Vernice Jackson-Lewis

Blockade of Microglial Activation Is Neuroprotective in the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Mouse Model of Parkinson Disease

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) damages the nigrostriatal dopaminergic pathway as seen in Parkinsons disease (PD), a common neurodegenerative disorder with no effective protective treatment. Consistent with a role of glial cells in PD neurodegeneration, here we show that minocycline, an approved tetracycline derivative that inhibits microglial activation independently of its antimicrobial properties, mitigates both the demise of nigrostriatal dopaminergic neurons and the formation of nitrotyrosine produced by MPTP. In addition, we show that minocycline not only prevents MPTP-induced activation of microglia but also the formation of mature interleukin-1β and the activation of NADPH–oxidase and inducible nitric oxide synthase (iNOS), three key microglial-derived cytotoxic mediators. Previously, we demonstrated that ablation of iNOS attenuates MPTP-induced neurotoxicity. Now, we demonstrate that iNOS is not the only microglial-related culprit implicated in MPTP-induced toxicity because mutant iNOS-deficient mice treated with minocycline are more resistant to this neurotoxin than iNOS-deficient mice not treated with minocycline. This study demonstrates that microglial-related inflammatory events play a significant role in the MPTP neurotoxic process and suggests that minocycline may be a valuable neuroprotective agent for the treatment of PD.

Inducible nitric oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease.

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) damages dopaminergic neurons as seen in Parkinson disease. Here we show that after administration of MPTP to mice, there was a robust gliosis in the substantia nigra pars compacta associated with significant upregulation of inducible nitric oxide synthase (iNOS). These changes preceded or paralleled MPTP-induced dopaminergic neurodegeneration. We also show that mutant mice lacking the iNOS gene were significantly more resistant to MPTP than their wild-type littermates. This study demonstrates that iNOS is important in the MPTP neurotoxic process and indicates that inhibitors of iNOS may provide protective benefit in the treatment of Parkinson disease.

Cyclooxygenase-2 is instrumental in Parkinson's disease neurodegeneration

Parkinsons disease (PD) is a neurodegenerative disorder of uncertain pathogenesis characterized by the loss of the nigrostriatal dopaminergic neurons, which can be modeled by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Increased expression of cyclooxygenase type 2 (COX-2) and production of prostaglandin E2 have been implicated in neurodegeneration in several pathological settings. Here we show that COX-2, the rate-limiting enzyme in prostaglandin E2 synthesis, is up-regulated in brain dopaminergic neurons of both PD and MPTP mice. COX-2 induction occurs through a JNK/c-Jun-dependent mechanism after MPTP administration. We demonstrate that targeting COX-2 does not protect against MPTP-induced dopaminergic neurodegeneration by mitigating inflammation. Instead, we provide evidence that COX-2 inhibition prevents the formation of the oxidant species dopamine-quinone, which has been implicated in the pathogenesis of PD. This study supports a critical role for COX-2 in both the pathogenesis and selectivity of the PD neurodegenerative process. Because of the safety record of the COX-2 inhibitors, and their ability to penetrate the blood–brain barrier, these drugs may be therapies for PD.

Time course and morphology of dopaminergic neuronal death caused by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

Mechanisms responsible for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopamine (DA) neuronal death remain unknown and in mice it is even unclear whether neuronal death does occur. In vitro studies suggest that 1-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of MPTP, kills neurons by apoptosis. Herein, we investigated whether MPTP induces DA neuronal death in vivo in mice and whether the mechanism is that of apoptosis. C57/bl Mice received different doses of MPTP administered in four intraperitoneal injections every 2 hours and were sacrificed at different time points for analyses of tyrosine hydroxylase (TH) immunohistochemistry, silver staining, and Nissl staining within the mesencephalon. We found that MPTP induces neuronal destruction in the substantia nigra pars compacta (SNpc) and the ventral tegmental area (VTA). The active phase of degeneration began at 12 h postinjection and continued up to 4 days. During this period, there was a greater decrease in TH-defined neurons than in Nissl-stained neurons suggesting that MPTP can cause a loss in TH without necessarily destroying the neuron. Thereafter, neuronal counts by both techniques equalized and there was no further loss of DA neurons. Dying neurons showed shrunken eosinophilic cytoplasm and shrunken darkly stained nuclei. Double staining revealed degenerating neurons solely among TH positive neurons of SNpc and VTA. At no time point and at no dose of MPTP was apoptosis observed. In addition, in situ labelling revealed no evidence of DNA fragmentation. This study demonstrates that the MPTP mouse model replicates several key features of neurodegeneration of DA neurons in PD and provides no in vivo evidence that, using this specific paradigm of injection, MPTP kills DA neurons by apoptosis.

NADPH oxidase mediates oxidative stress in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease.

Parkinsons disease (PD) is a neurodegenerative disorder of uncertain pathogenesis characterized by a loss of substantia nigra pars compacta (SNpc) dopaminergic (DA) neurons, and can be modeled by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Both inflammatory processes and oxidative stress may contribute to MPTP- and PD-related neurodegeneration. However, whether inflammation may cause oxidative damage in MPTP and PD is unknown. Here we show that NADPH-oxidase, the main reactive oxygen species (ROS)-producing enzyme during inflammation, is up-regulated in SNpc of human PD and MPTP mice. These changes coincide with the local production of ROS, microglial activation, and DA neuronal loss seen after MPTP injections. Mutant mice defective in NADPH-oxidase exhibit less SNpc DA neuronal loss and protein oxidation than their WT littermates after MPTP injections. We show that extracellular ROS are a main determinant in inflammation-mediated DA neurotoxicity in the MPTP model of PD. This study supports a critical role for NADPH-oxidase in the pathogenesis of PD and suggests that targeting this enzyme or enhancing extracellular antioxidants may provide novel therapies for PD.

Protocol for the MPTP mouse model of Parkinson's disease

This protocol describes our method of producing a reliable mouse model of Parkinsons disease (PD) using the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We discuss the particulars of the model, provide key references and outline what investigators need to know to develop the MPTP mouse model of PD safely and successfully. Completion of this protocol depends on the regimen of MPTP used and on the actual planned studies, which often range from 7 to 30 d. This protocol calls for implementation of safety measures and for the acquisition of several pieces of equipment, which are a one-time investment worth making if one elects to use this model on a regular basis.

Mutant LRRK2R1441G BAC transgenic mice recapitulate cardinal features of Parkinson's disease

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinsons disease. We created a LRRK2 transgenic mouse model that recapitulates cardinal features of the disease: an age-dependent and levodopa-responsive slowness of movement associated with diminished dopamine release and axonal pathology of nigrostriatal dopaminergic projection. These mice provide a valid model of Parkinsons disease and are a resource for the investigation of pathogenesis and therapeutics.

The parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) : A technical review of its utility and safety

Parkinsons disease (PD) is a common disabling neurodegenerative disorder the cardinal clinical features of which include tremor, rigidity and slowness of movement (Fahn and Przedborski 2000). These symptoms are attributed mainly to a profound reduction of dopamine in the striatum due to a dramatic loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) (Fahn and Przedborski 2000). Thus far, both the cause and the mechanisms of PD remain unknown. Over the years, investigators have used experimental models of PD produced by several compounds such as reserpine, 6-hydroxydopamine, methamphetamine, and 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) to provide insights into the mechanisms responsible for the demise of dopaminergic neurons in PD. To this end, MPTP has emerged unquestionably as a popular tool for inducing a model of PD in a variety of animal species including monkeys, rodents, cats, and pigs (Kopin and Markey 1988). The sensitivity to MPTP and therefore its ability to induce parkinsonism closely follows the phylogenetic tree where the species most closely related to humans are the most vulnerable to this neurotoxin. Due to the signi®cant neurotoxicity of MPTP, it is important that researchers appreciate the potential hazards of this toxin. Given this, the purpose of this review is to inform the researcher of the hazardous nature of MPTP and to provide guidance for its safe handling and use.

Dose-dependent lesions of the dopaminergic nigrostriatal pathway induced by instrastriatal injection of 6-hydroxydopamine

Animal models with partial lesions of the dopaminergic nigrostriatal pathway may be useful for developing neuroprotective and neurotrophic therapies for Parkinsons disease. To develop such a model, different doses of 6-hydroxydopamine (0.0, 0.625, 1.25, 2.5 and 5.0 micrograms/microliters in 3.5 microliters of saline) were unilaterally injected into the striatum of rats. Animals that received 1.25 to 5.0 micrograms/microliters 6-hydroxydopamine displayed dose-dependent amphetamine and apomorphine-induced circling. 6-Hydroxydopamine also caused dose-dependent reductions in [3H]mazindol-labeled dopamine uptake sites in the lesioned striatum and ipsilateral substantia nigra pars compacta (up to 93% versus contralateral binding), with smaller losses in the nucleus accumbens, olfactory tubercle and ventral tegmental area. In the substantia nigra pars compacta and the ventral tegmental area, the number of Nissl-stained neurons decreases in parallel with the reduction in [3H]mazindol binding. The reduction in [3H]mazindol binding in the striatum and the nucleus accumbens, and the reduction in [3H]mazindol binding and in the number of Nissl-stained neurons in the substantia nigra pars compacta and the ventral tegmental area is stable for up to 12 weeks after the lesion. Macroscopically, forebrain coronal sections showed normal morphology, except for rats receiving 5.0 micrograms/microliters 6-hydroxydopamine in which striatal cross-sectional area was reduced, suggesting that this high dose non-specifically damages intrinsic striatal neurons. Nissl-stained sections revealed an area of neuronal loss and intense gliosis centered around the needle track, which increased in size with the dose of neurotoxin. Striatal [3H]sulpiride binding was increased by 2.5 micrograms/microliters and 5.0 micrograms/microliters 6-hydroxydopamine, suggesting up-regulation of dopamine D2 receptors. Striatal binding of [3H]CGS 21680-labeled adenosine A2a receptors, but not of [3H]SCH 23390-labeled dopamine D1 receptors, was reduced at the highest dose, suggesting preservation of the striatal intrinsic neurons with the lower doses. This study indicates that intrastriatal injection of different doses of 6-hydroxydopamine can be used to cause increasing amounts of dopamine denervation, which could model Parkinsons disease of varying degrees of severity. Injecting 3.5 microliters of 2.5 micrograms/microliters 6-hydroxydopamine appears to be particularly useful as a general model of early Parkinsons disease, since it induces a lesion characterized by robust drug-induced rotation, changes in binding consistent with approximately 70% dopamine denervation, approximately 19% dopamine D22 receptor up-regulation, negligible intrinsic striatal damage and stability for at least 12 weeks. This study outlines a technique for inducing partial lesions of the nigrostriatal dopamine pathway in rats.(ABSTRACT TRUNCATED AT 400 WORDS)

α-Synuclein Up-Regulation in Substantia Nigra Dopaminergic Neurons Following Administration of the Parkinsonian Toxin MPTP

Abstract: Mutations in α‐synuclein cause a form of familial Parkinson’s disease (PD), and wild‐type α‐synuclein is a major component of the intraneuronal inclusions called Lewy bodies, a pathological hallmark of PD. These observations suggest a pathogenic role for α‐synuclein in PD. Thus far, however, little is known about the importance of α‐synuclein in the nigral dopaminergic pathway in either normal or pathological situations. Herein, we studied this question by assessing the expression of synuclein‐1, the rodent homologue of human α‐synuclein, in both normal and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐intoxicated mice. In normal mice, detectable levels of synuclein mRNA and protein were seen in all brain regions studied and especially in ventral midbrain. In the latter, there was a dense synuclein‐positive nerve fiber network, which predominated over the substantia nigra, and only few scattered synuclein‐positive neurons. After a regimen of MPTP that kills dopaminergic neurons by apoptosis, synuclein mRNA and protein levels were increased significantly in midbrain extracts; the time course of these changes paralleled that of MPTP‐induced dopaminergic neurodegeneration. In these MPTP‐injected mice, there was also a dramatic increase in the number of synuclein‐immunoreactive neurons exclusively in the substantia nigra pars compacta; all synuclein‐positive neurons were tyrosine hydroxylase‐positive, but none coexpressed apoptotic features. These data indicate that synuclein is highly expressed in the nigrostriatal pathway of normal mice and that it is up‐regulated following MPTP‐induced injury. In light of the synuclein alterations, it can be suggested that, by targeting this protein, one may modulate MPTP neurotoxicity and, consequently, open new therapeutic avenues for PD.