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SummaryDifferentiation of astrocytes from human stem cells has significant potential for analysis of their role in normal brain function and disease, but existing protocols generate only immature astrocytes. Using early neuralization, we generated spinal cord astrocytes from mouse or human embryonic or induced pluripotent stem cells with high efficiency. Remarkably, short exposure to fibroblast growth factor 1 (FGF1) or FGF2 was sufficient to direct these astrocytes selectively toward a mature quiescent phenotype, as judged by both marker expression and functional analysis. In contrast, tumor necrosis factor alpha and interleukin-1β, but not FGFs, induced multiple elements of a reactive inflammatory phenotype but did not affect maturation. These phenotypically defined, scalable populations of spinal cord astrocytes will be important both for studying normal astrocyte function and for modeling human pathological processes in vitro.

Toxic organic cations can damage nigrostriatal dopaminergic pathways as seen in most parkinsonian syndromes and in some cases of illicit drug exposure. Here, we show that the organic cation transporter 3 (Oct3) is expressed in nondopaminergic cells adjacent to both the soma and terminals of midbrain dopaminergic neurons. We hypothesized that Oct3 contributes to the dopaminergic damage by bidirectionally regulating the local bioavailability of toxic species. Consistent with this view, Oct3 deletion and pharmacological inhibition hampers the release of the toxic organic cation 1-methyl-4-phenylpyridinium from astrocytes and protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration in mice. Furthermore, Oct3 deletion impairs the removal of the excess extracellular dopamine induced by methamphetamine and enhances striatal dopaminergic terminal damage caused by this psychostimulant. These results may have far-reaching implications for our understanding of the mechanism of cell death in a wide range of neurodegenerative diseases and may open new avenues for neuroprotective intervention.

The molecules underlying neuron loss in Parkinson's disease (PD) are essentially unknown, and current therapies focus on diminishing symptoms rather than preventing neuron death. We identified RTP801 as a gene whose transcripts were highly induced in a cellular model of PD in which death of neuronal catecholaminergic PC12 cells was triggered by the PD mimetic 6-OHDA. Here, we find that RTP801 protein is also induced in this and additional cellular and animal PD models. To assess the relevance of these observations to PD, we used immunohistochemistry to compare RTP801 expression in postmortem brains from PD and control patients. For all PD brains examined, expression was highly elevated within neuromelanin-containing neurons of the substantia nigra but not in cerebellar neurons. Evaluation of the potential role of RTP801 induction in our cellular model revealed that RTP801 overexpression is sufficient to promote death but does not further elevate death caused by 6-OHDA. Furthermore, RTP801 induction is requisite for death in our cellular PD models and in 6-OHDA-treated cultured sympathetic neurons in that its knockdown by short hairpin RNAs (shRNAs) is protective. The mechanism by which 6-OHDA and RTP801 induce neuron death appears to involve repression of mammalian target of rapamycin (mTOR) kinase activity, and such death is inhibited by shRNAs targeting TSC2 (tuberous sclerosis complex), a protein with which RTP801 interacts to block mTOR activation. Our findings thus suggest that the elevation of RTP801 we detect in PD substantia nigral neurons may mediate their degeneration and death and that RTP801 and its signaling cascade may be novel potential therapeutic targets for the disease.

Dysfunction of mitochondrial complex I is a feature of human neurodegenerative diseases such as Leber hereditary optic neuropathy and Parkinson's disease. This mitochondrial defect is associated with a recruitment of the mitochondrial-dependent apoptotic pathway in vivo . However, in isolated brain mitochondria, complex I dysfunction caused by either pharmacological or genetic means fails to directly activate this cell death pathway. Instead, deficits of complex I stimulate intramitochondrial oxidative stress, which, in turn, increase the releasable soluble pool of cytochrome c within the mitochondrial intermembrane space. Upon mitochondrial permeabilization by the cell death agonist Bax, more cytochrome c is released to the cytosol from brain mitochondria with impaired complex I activity. Given these results, we propose a model in which defects of complex I lower the threshold for activation of mitochondrial-dependent apoptosis by Bax, thereby rendering compromised neurons more prone to degenerate. This molecular scenario may have far-reaching implications for the development of effective neuroprotective therapies for these incurable illnesses.

The biochemical and cellular changes that occur following administration of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) are remarkably similar to that seen in idiopathic Parkinson's disease (PD). In this review, we detail the molecular activities of this compound from peripheral intoxication through its various biotransformations. In addition, we detail the interplay that occurs between the different cellular compartments (neurons and glia) that eventually consort to kill substantia nigra pars compacta (SNpc) neurons.

Parkinson disease (PD) is a common neurodegenerative disease of unknown origin that is characterized, mainly, by a significant reduction in the number of dopamine neurons in the substantia nigra pars compacta (SNpc) of the brain and a dramatic reduction in dopamine levels in the corpus striatum. For reasons that we do not know, the dopamine neuron seems to be more vulnerable to damage than any other neuron in the brain. Although hypotheses of damage to the dopamine neuron include oxidative stress, growth factor decline, excitotoxicity, inflammation in the SNpc and protein aggregation, oxidative stress in the nigrostriatal dopaminergic system garners a significant amount of attention. In the oxidative stress hypothesis of PD, superoxide, nitric oxide and dopamine all conspire to create an environment that can be detrimental to the dopamine neuron. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), the tool of choice for investigations into the mechanisms involved in the death of dopamine neurons in PD, has been used extensively in attempts to sort out what happens in and around the dopamine neuron. Herein, we review the roles of dopamine, superoxide and nitric oxide in the demise of the dopamine neuron in the MPTP model of PD as it relates to the death of the dopamine neuron noted in PD.

Accumulating evidence suggests that apoptotic and inflammatory factors contribute to the demise of dopaminergic neurons. In this respect, Fas, a member of the tumor necrosis factor receptor family with proapoptotic and inflammatory functions, was reported to be elevated within the striatum and substantia nigra pars compacta (SNc) of Parkinson's disease (PD) patients. Accordingly, the present investigation evaluated the function of Fas in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD. Injection of MPTP increased nigral Fas expression, and mice lacking Fas displayed attenuated MPTP-induced SNc dopaminergic loss and microglial activation. In addition, Fas induction was blocked by expression of a dominant-negative c-Jun adenovirus that also protected dopamine neurons from MPTP-induced damage. Together, these data suggest the critical nature of the c-Jun-Fas signaling pathway in MPTP-induced neuronal loss. Although critical for degeneration of the soma, Fas deficiency did not significantly prevent the reduction of dopaminergic terminal fibers within the striatum or normalize the activation of striatal microglia and elevation of the postsynaptic activity marker ΔFosB induced by denervation. Interestingly, Fas-deficient mice displayed a pre-existing reduction in striatal dopamine levels and locomotor behavior when compared with wild-type mice. Despite the reduced terminals, dopamine levels were not further suppressed by MPTP treatment in mutant mice, raising the possibility of a compensatory response in basal ganglia function in Fas-deficient mice.

Recent evidence indicates that cyclin-dependent kinases (CDKs, cdks) may be inappropriately activated in several neurodegenerative conditions. Here, we report that cdk5 expression and activity are elevated after administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a toxin that damages the nigrostriatal dopaminergic pathway. Supporting the pathogenic significance of the cdk5 alterations are the findings that the general cdk inhibitor, flavopiridol, or expression of dominant-negative cdk5, and to a lesser extent dominant-negative cdk2, attenuates the loss of dopaminergic neurons caused by MPTP. In addition, CDK inhibition strategies attenuate MPTP-induced hypolocomotion and markers of striatal function independent of striatal dopamine. We propose that cdk5 is a key regulator in the degeneration of dopaminergic neurons in Parkinson's disease.

Parkinson disease (PD) is a neurodegenerative disorder characterized by a loss of the nigrostriatal dopaminergic neurons accompanied by a deficit in mitochondrial respiration. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin that causes dopaminergic neurodegeneration and a mitochondrial deficit reminiscent of PD. Here we show that the infusion of the ketone body d-beta-hydroxybutyrate (DbetaHB) in mice confers partial protection against dopaminergic neurodegeneration and motor deficits induced by MPTP. These effects appear to be mediated by a complex II-dependent mechanism that leads to improved mitochondrial respiration and ATP production. Because of the safety record of ketone bodies in the treatment of epilepsy and their ability to penetrate the blood-brain barrier, DbetaHB may be a novel neuroprotective therapy for PD.

Mutations in alpha-synuclein cause a form of familial Parkinson's disease (PD), and wild-type alpha-synuclein is a major component of the intraneuronal inclusions called Lewy bodies, a pathological hallmark of PD. These observations suggest a pathogenic role for alpha-synuclein in PD. Thus far, however, little is known about the importance of alpha-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 alpha-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.

Tinuvin 123, a compound used in the manufacture of plastics, has recently been suggested to possibly cause Parkinson's disease (PD). Herein, we revisited this issue by assessing the effect of Tinuvin 123 on dopaminergic neurons of the substantia nigra following its stereotaxic injection in the rat. Twenty-one days post unilateral stereotaxic injection of Tinuvin 123, systemic injection of both apomorphine and amphetamine caused rotations toward the side of the lesion in these rats. Tinuvin 123 produced a small to moderate dose-dependent reduction in striatal levels of dopamine and metabolites on the side of the lesion. This compound also produced dramatic cell loss in the substantia nigra on the side of the lesion. However, the loss of cells lacked the phenotypic specificity for tyrosine hydroxylase (TH)-positive neurons that is expected with a dopaminergic neurotoxin. Indeed, aside from a robust glial reaction, both TH-positive and glutamic acid dehydrogenase (GAD)-positive neurons were destroyed, and near the site of the injection, there was complete tissue destruction. This study indicates that, using this mode of injection, Tinuvin 123 exerts a dramatic tissue toxicity without any evidence of specificity for dopaminergic neurons. Thus, our data argues against a role for Tinuvin 123 as an environmental toxin causing a clinical condition characterized by the selective loss of dopaminergic neurons as seen in PD.

Mitochondrial complex I deficits have long been associated with Parkinson disease (PD). However, it remains unknown whether such defects represent a primary event in dopaminergic neurodegeneration.Apoptosis-inducing factor (AIF) is a mitochondrial protein that, independently of its proapoptotic properties, plays an essential physiologic role in maintaining a fully functional complex I. We used AIF-deficient harlequin (Hq) mice, which exhibit structural deficits in assembled complex I, to determine whether primary complex I defects linked to AIF depletion may cause dopaminergic neurodegeneration.Despite marked reductions in mitochondrial complex I protein levels, Hq mice did not display apparent alterations in the dopaminergic nigrostriatal system. However, these animals were much more susceptible to exogenous parkinsonian complex I inhibitors, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Subtoxic doses of MPTP, unable to cause damage to wild-type animals, produced marked nigrostriatal dopaminergic degeneration in Hq mice. This effect was associated with exacerbated complex I inhibition and increased production of mitochondrial-derived reactive oxygen species (ROS) in Hq brain mitochondria. The antioxidant superoxide dismutase-mimetic compound tempol was able to reverse the increased susceptibility of Hq mice to MPTP. Supporting an instrumental role for mitochondrial-derived ROS in PD-related neurodegeneration, transgenic mice overexpressing mitochondrially targeted catalase exhibited an attenuation of MPTP-induced mitochondrial ROS and dopaminergic cell death.Structural complex I alterations linked to AIF deficiency do not cause dopaminergic neurodegeneration but increase the susceptibility of dopaminergic neurons to exogenous parkinsonian neurotoxins, reinforcing the concept that genetic and environmental factors may interact in a common molecular pathway to trigger PD.

We report that rapamycin, an allosteric inhibitor of certain but not all actions of the key cellular kinase mammalian target of rapamycin (mTOR), protects neurons from death in both cellular and animal toxin models of Parkinson's disease (PD). This protective action appears to be attributable to blocked translation of RTP801/REDD1/Ddit4, a protein that is induced in cell and animal models of PD and in affected neurons of PD patients and that causes neuron death by leading to dephosphorylation of the survival kinase Akt. In support of this mechanism, in PD models, rapamycin spares phosphorylation of Akt at a site critical for maintenance of its survival-promoting activity. The capacity of rapamycin to provide neuroprotection in PD models appears to arise from its selective suppression of some but not all actions of mTOR, as indicated by the contrasting finding that Torin1, a full catalytic mTOR inhibitor, is not protective and induces Akt dephosphorylation and neuron death.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's 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 Parkinson's disease and are a resource for the investigation of pathogenesis and therapeutics.

There is increasing evidence that neuron death in neurode-generative diseases, such as Parkinson's disease, is due to the activation of programmed cell death. However, the upstream mediators of cell death remain largely unknown. One approach to the identification of upstream mediators is to perform gene expression analysis in disease models. Such analyses, performed in tissue culture models induced by neurotoxins, have identified up-regulation of CHOP/GADD153, a transcription factor implicated in apoptosis due to endoplasmic reticulum stress or oxidative injury. To evaluate the disease-related significance of these findings, we have examined the expression of CHOP/GADD153 in neurotoxin models of parkinsonism in living animals. Nuclear expression of CHOP protein is observed in developmental and adult models of dopamine neuron death induced by intrastriatal injection of 6-hydroxydopamine (6OHDA) and in models induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). CHOP is a mediator of neuron death in the adult 60HDA model because a null mutation results in a reduction in apoptosis. In the chronic MPTP model, however, while CHOP is robustly expressed, the null mutation does not protect from the loss of neurons. We conclude that the role of CHOP depends on the nature of the toxic stimulus. For 6OHDA, an oxidative metabolite of dopamine, it is a mediator of apoptotic death.

Degeneration of the nigrostriatal dopaminergic pathway, the hallmark of Parkinson's disease, can be recapitulated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. Herein, we demonstrate that adoptive transfer of copolymer-1 immune cells to MPTP recipient mice leads to T cell accumulation within the substantia nigra pars compacta, suppression of microglial activation, and increased local expression of astrocyte-associated glial cell line-derived neurotrophic factor. This immunization strategy resulted in significant protection of nigrostriatal neurons against MPTP-induced neurodegeneration that was abrogated by depletion of donor T cells. Such vaccine treatment strategies may provide benefit for Parkinson's disease.

The massive dopaminergic neuronal loss that occurs in Parkinson's disease shows features of apoptosis. In the current study we have characterised the neuronal death in an animal model of Parkinson's disease. 6-Hydroxydopamine infused in the striatum of adult rats induced progressive loss of dopamine neurons, identified as tyrosine hydroxylase immunoreactive profiles, in the ipsilateral substantia nigra starting at day 5 post-lesion (32%). Silver staining revealed the presence of apoptotic profiles with neuronal morphology in the substantia nigra ipsilateral to the intrastriatal 6-hydroxydopamine injection. These apoptotic nuclei were first observed at day 6 post-lesion, peaked between days 7 and 10 and then abruptly declined. The apoptotic morphology of 6-hydroxydopamine-induced neuronal death was confirmed by electron microscopic studies. These data show that intrastriatal 6-hydroxydopamine-induced dopaminergic neuronal death in the adult rat is apoptotic and supports the use of this lesion protocol as an animal model of Parkinson's disease.

Parkinson's disease (PD) is most commonly a sporadic illness, and is characterized by degeneration of substantia nigra dopamine (DA) neurons and abnormal cytoplasmic aggregates of alpha-synuclein. Rarely, PD may be caused by missense mutations in alpha-synuclein. MPTP, a neurotoxin that inhibits mitochondrial complex I, is a prototype for an environmental cause of PD because it produces a pattern of DA neurodegeneration that closely resembles the neuropathology of PD. Here we show that alpha-synuclein null mice display striking resistance to MPTP-induced degeneration of DA neurons and DA release, and this resistance appears to result from an inability of the toxin to inhibit complex I. Contrary to predictions from in vitro data, this resistance is not due to abnormalities of the DA transporter, which appears to function normally in alpha-synuclein null mice. Our results suggest that some genetic and environmental factors that increase susceptibility to PD may interact with a common molecular pathway, and represent the first demonstration that normal alpha-synuclein function may be important to DA neuron viability. ispartof: Proceedings of the National Academy of Sciences of the United States of America vol:99 issue:22 pages:14524-9 ispartof: location:United States status: published

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) damages the nigrostriatal dopaminergic pathway as seen in Parkinson's 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-1beta 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.

Parkinson's disease is a common neurodegenerative disorder characterized by the progressive loss of the dopaminergic neurons in the substantia nigra pars compacta. The loss of these neurons is associated with a glial response composed mainly of activated microglial cells and, to a lesser extent, of reactive astrocytes. This glial response may be the source of trophic factors and can protect against reactive oxygen species and glutamate. Aside from these beneficial effects, the glial response can mediate a variety of deleterious events related to the production of reactive species, and pro-inflammatory prostaglandin and cytokines. This article reviews the potential protective and deleterious effects of glial cells in the substantia nigra pars compacta of Parkinson's disease.

Dopaminergic neurons in the substantia nigra pars compacta (SNpc) undergo natural cell death during development in rats. Controversy exists as to the occurrence of this phenomenon in SNpc dopaminergic neurons in the developing mouse. Herein, by using an array of morphologic techniques, we show that many SNpc neurons fulfill the criteria for apoptosis and that the number of apoptotic neurons in the SNpc vary in a time-dependent manner from postnatal day 2 to 32. These dying neurons also show evidence of DNA fragmentation, of activated caspase-3, and of cleavage of beta-actin. Some, but not all of the SNpc apoptotic neurons still express their phenotypic marker tyrosine hydroxylase, confirming their dopaminergic nature. Consistent with the importance of target-derived trophic support in modulating developmental cell death, we demonstrate that destruction of intrinsic striatal neurons by a local injection of quinolinic acid (QA) dramatically enhances the magnitude of SNpc apoptosis and results in a lower number of adult SNpc dopaminergic neurons. Strengthening the apoptotic nature of the observed SNpc developmental cell death, we demonstrate that overexpression of the anti-apoptotic protein Bcl-2 attenuates both natural and QA-induced SNpc apoptosis. The present study provides compelling evidence that developmental neuronal death with a morphology of apoptosis does occur in the SNpc of mice and that this process plays a critical role in regulating the adult number of dopaminergic neurons in the SNpc.