Role of glial cell derived neurotrophic factor in the protective effect of smilagenin on rat mesencephalic dopaminergic neurons damaged by MPP+

Tyrosine hydroxylase immunohistochemical analysis revealed that in cultured mesencephalic dopaminergic neurons smilagenin (SMI), added prior to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPP+), protected against the drop of neuron number and neurite outgrowth length caused by MPP+. Addition of anti-GDNF and/or anti-GFR alpha 1 functional antibodies to the medium prior to SMI, eliminated mostly, though incompletely, the action of SMI. The expression of glial cell derived neurotrophic factor (GDNF) mRNA, but not GDNF receptor alpha1 (GFR alpha 1) or receptor tyrosine kinase mRNA in MPP+ intoxicated neurons was markedly elevated as early as 2h after the addition of SMI with a peak at 24-48 h. Therefore, an important route of the protective action of SMI on dopaminergic neurons is to stimulate intrinsic GDNF expression.     

Epidermal Growth Factor Enhances Striatal Dopaminergic Parameters in the 1-Methyl-4-Phenyl-l, 2, 3, 6-Tetrahydropyridine-Treated Mouse

Intracerebroventricular infusion of epidermal growth factor (EGF) into mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced degeneration of dopaminergic nigrostriatal neurons partially enhanced the content of dopamine (DA) and 3,4-dihydroxyphenylacetic acid as well as the activity of tyrosine hydroxylase in the striatum. EGF also enhanced these parameters in control, unlesioned animals. Neurotrophic activity also was observed in embryonic mesencephalic cultures, where EGF enhanced DA uptake after a lesion with the neurotoxic metabolite of MPTP, 1-methyl-4-phenylpyridinium ion. Our in vivo and in vitro studies suggest that EGF may be a neurotrophic factor for dopaminergic neurons, or may act indirectly by inducing the release of a dopaminergic trophic factor from other cells.     

Selective Destruction of Cultured Dopaminergic Neurons from Fetal Rat Mesencephalon by 1-Methyl-4-Phenylpyridinium: Cytochemical and Morphological Evidence

Dopaminergic neurons in cultures of dissociated cells from fetal rat mesencephalon were exposed to the principal metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenyl-pyridinium ion (MPP+), and several of its structural analogues. At concentrations between 0.01 and 0.1 microM, MPP+ inhibited catecholamine accumulation as visualized by cytofluorescence. Between 0.1 and 10.0 microM, MPP+ resulted in disappearance of tyrosine hydroxylase immunoreactivity without affecting other cells in the cultures. At concentrations higher than 10 microM, MPP+ was toxic to all cells present in the cultures. The effect of low concentrations of MPP+ on catecholamine cytofluorescence of the dopaminergic neurons was partially reversible. The intermediate concentrations produced irreversible structural changes of tyrosine hydroxylase-positive cells, resulting in complete disappearance of these neurons. The morphological changes were specific to the dopaminergic neurons and were not evident in other cells viewed with phase contrast microscopy. Of the structural analogues tested, the 1-ethyl analogue of MPP+ was effective in selectively destroying dopaminergic neurons in our culture system. The antioxidants L-acetyl-carnitine, beta-carotene, and alpha-tocopherol failed to protect against MPP+ neurotoxicity when co-incubated with the toxin.     

Dopamine Synthesis as a Mechanism of Brain Plasticity in Nigrostriatal System Pathology

Using an experimental Parkinson’s disease model (symptoms develop in mice after the injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), we studied the characteristics of the synthesis of dopamine as a possible compensatory mechanism aimed at maintaining the dopamine level in the dopaminergic neurons that survived in this pathology. We found no correlation between the content and activity of tyrosine hydroxylase in the nigrostriatal system. The enzyme activity and the dopamine content showed unidirectional changes in the substantia nigra, but not in the striatum, which is apparently due to triggering other compensatory mechanisms.     

Reduced Nurrl Expression Increases the Vulnerability of Mesencephalic Dopamine Neurons to MPTP-Induced Injury

Mutation in the Nurr1 gene, a member of the nuclear receptor superfamily, causes selective agenesis of dopaminergic neurons in the midbrain of null mice. Homozygous Nurr1 knockout mice (Nurr1-/-) die 1 day after birth, but heterozygous mice (Nurr1 +/-) survive postnatally without obvious locomotor deficits. Although adult Nurr1 +/- mice show significantly reduced Nurr1 protein levels in the substantia nigra (SN), they display a normal range of tyrosine hydroxylase-positive neuron numbers in the SN and normal levels of dopamine in the striatum. The reduction in Nurr1 expression in Nurr1 +/- mice, however, confers increased vulnerability to the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) compared with wild-type (Nurr1 +/+) mice. This study suggests that Nurr1 may play an important role in maintaining mature mesencephalic dopaminergic neuron function and that a defect in Nurr1 may increase susceptibility to SN injury.     

Inhibition of Phosphatidylinositol 3-Kinase Activity Blocks Cellular Differentiation Mediated by Glial Cell Line-Derived Neurotrophic Factor in Dopaminergic Neurons

Abstract: Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for midbrain dopaminergic neurons. To begin to understand the intracellular signaling pathways used by GDNF, we investigated the role of phosphatidylinositol 3-kinase activity in GDNF-stimulated cellular function and differentiation of dopaminergic neurons. We found that treatment of dopaminergic neuron cultures with 10 ng/ml GDNF induced maximal levels of Ret phosphorylation and produced a profound increase in phosphatidylinositol 3-kinase activity, as measured by western blot analysis and lipid kinase assays. Treatment with 1 µ M 2-(4-morpholinyl)-8-phenylchromone (LY294002) or 100 n M wortmannin, two distinct and potent inhibitors of phosphatidylinositol 3-kinase activity, completely inhibited GDNF-induced phosphatidylinositol 3-kinase activation, but did not affect Ret phosphorylation. Furthermore, we examined specific biological functions of dopaminergic neurons: dopamine uptake activity and morphological differentiation of tyrosine hydroxylase-immunoreactive neurons. GDNF significantly increased dopamine uptake activity and promoted robust morphological differentiation. Treatment with LY294002 completely abolished the GDNF-induced increases of dopamine uptake and morphological differentiation of tyrosine hydroxylase-immunoreactive neurons. Our findings show that GDNF-induced differentiation of dopaminergic neurons requires phosphatidylinositol 3-kinase activation.     

Quinolinic acid but not MK-801 protects the dopaminergic system from 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine-induced toxicity in goldfish retina

The toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, intravitreally injected in goldfish eye, involves interplexiform retinal neurons and depletes tyrosine hydroxylase immunoreactivity and dopamine levels. This induced neurotoxicity was prevented by the concomitant administration in nontoxic doses (10 μg) of quinolinic acid, an endogenous structural analogue of N-methyl -aspartate with excitotoxic properties. Quinolinic acid is ineffective on the retinal degeneration induced by 1-methyl-4- phenylpyridinium ion. This fact suggests that quinolinic acid inhibits the MAO-B oxidation of 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine. MK-801, a noncompetitive antagonist of glutamate NMDA-receptors, exerts partial protective effects on MPTP-induced delayed toxicity in mammals. In the goldfish eye, MK-801, injected in low concentration, and in conjunction with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or 1-methyl-4-phenylpyridinium ion, did not prevent retinal neurodegeneration. Ten μg of MK-801 alone did not affect retinal neurons, while a higher concentration (20 μg) causes the chromatolysis of some photoreceptor nuclei.     

Inhibition of Glial Cell Line-Derived Neurotrophic Factor Induced Intracellular Activity by K-252b on Dopaminergic Neurons

Abstract: The c- ret protooncogene encodes Ret, the functional tyrosine kinase receptor for glial cell line-derived neurotrophic factor (GDNF). K-252b, a known protein tyrosine kinase inhibitor, has been shown earlier to inhibit the trophic activity of brain-derived neurotrophic factor on dopaminergic (DAergic) neurons and nerve growth factor on basal forebrain cholinergic neurons while potentiating neurotrophin-3 activity on central cholinergic and peripheral sensory neurons and PC12 cells. We tested whether K-252b would modulate GDNF-induced differentiation in DAergic neuron cultures. Exposure to 1 ng/ml GDNF increased dopamine (DA) uptake 80% above control, whereas treatment with 5 µ M K-252b decreased the efficacy of GDNF by 60%. Concentrations of GDNF of <100 pg/ml were completely inhibited, whereas concentrations of >100 pg/ml were moderately active, between 10 and 20% above control. In addition, K-252b shifted the ED₅₀ from 20 to 200 pg/ml. GDNF treatment increased soma size and neurite outgrowth in tyrosine hydroxylase-immunoreactive neurons. K-252b inhibited differentiation of these morphological parameters induced by GDNF. Furthermore, GDNF stimulated Ret autophosphorylation at maximal levels, whereas the inhibition of DA uptake and morphological differentiation by K-252b correlated with a significantly decreased level of Ret autophosphorylation. Therefore, K-252b is able to inhibit intracellular activities induced by GDNF on mesencephalic DAergic neurons.     

GDNF

The identification of novel factors that promote neuronal survival could have profound effects on developing new therapeutics for neurodegenerative disorders. Glial cell line-derived neurotrophic factor (GDNF) is a novel protein purified and cloned based on its marked ability to promote dopaminergic neuronal function. GDNF, now known to be the first identified member of a family of factors, signals through the previously known receptor tyrosine kinase, Ret. Unlike most ligands for receptor tyrosine kinases, GDNF does not bind and activate Ret directly, but requires the presence of GPI-linked coreceptors. There are several coreceptors with differing affinities for the GDNF family members. The profile of coreceptors in a cell may determine which factor preferentially activates Ret. In vivo differences in localization of the GDNF family members, its coreceptors and Ret suggest this ligand/receptor interaction has extensive and multiple functions in the CNS as well as in peripheral tissues. GDNF promotes survival of several neuronal populations both in vitro and in vivo. Dopaminergic neuronal survival and function are preserved by GDNF in vivo when challenged by the toxins MPTP and 6-hydroxydopamine. Furthermore, GDNF improves the symptoms of pharmacologically induced Parkinson's disease in monkeys. Several motor neuron populations isolated in vitro are also rescued by GDNF. In vivo, GDNF protects these neurons from programmed cell death associated with development and death induced by neuronal transection. These experiments suggest that GDNF may provide significant therapeutic opportunities in several neurodegenerative disorders.     

Granulocyte-colony stimulating factor is neuroprotective in a model of Parkinson's disease

We have recently shown that the hematopoietic Granulocyte-Colony Stimulating Factor (G-CSF) is neuroprotective in rodent stroke models, and that this action appears to be mediated via a neuronal G-CSF receptor. Here, we report that the G-CSF receptor is expressed in rodent dopaminergic substantia nigra neurons, suggesting that G-CSF might be neuroprotective for dopaminergic neurons and a candidate molecule for the treatment of Parkinson's disease. Thus, we investigated protective effects of G-CSF in 1-methyl-4-phenylpyridinium (MPP+)-challenged PC12 cells and primary neuronal midbrain cultures, as well as in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. Substantial protection was found against MPP+-induced dopaminergic cell death in vitro. Moreover, subcutaneous application of G-CSF at a dose of 40 microg/Kg body weight daily over 13 days rescued dopaminergic substantia nigra neurons from MPTP-induced death in aged mice, as shown by quantification of tyrosine hydroxylase-positive substantia nigra cells. Using HPLC, a corresponding reduction in striatal dopamine depletion after MPTP application was observed in G-CSF-treated mice. Thus our data suggest that G-CSF is a novel therapeutic opportunity for the treatment of Parkinson's disease, because it is well-tolerated and already approved for the treatment of neutropenic conditions in humans.     

GDNF - a stranger in the TGF-beta superfamily?

Glial cell line-derived neurotrophic factor (GDNF) family, consisting of GDNF, neurturin, artemin and persephin are distant members of the transforming growth factor-beta (TGF-beta) superfamily. Unlike other members of the TGF-beta superfamily, which signal through the receptor serine-threonine kinases, GDNF family ligands activate intracellular signalling cascades via the receptor tyrosine kinase Ret. GDNF family ligands first bind to the glycosylphosphatidylinositol (GPI)-anchored GDNF family receptor alpha (GFRalpha) and then the GDNF family ligand-GFRalpha complex binds to and stimulates autophosphorylation of Ret. Alternatively, a preassociated complex between GFRalpha and Ret could form the binding site for the GDNF family ligand. GFRalpha1, GFRalpha2, GFRalpha3 and GFRalpha4 are the physiological coreceptors for GDNF, neurturin, artemin and persephin, respectively. Although all GDNF family ligands signal via activated Ret, GDNF can signal also via GFRalpha1 in the absence of Ret. GPI-anchored GFRalpha receptors are localized in plasma membrane to lipid rafts. GDNF binding to GFRalpha1 also recruits Ret to the lipid rafts and triggers association with Src, which is required for effective downstream signalling, leading to differentiation and neuronal survival. GDNF family ligands are potent survival factors for midbrain dopamine neurons, motoneurons, noradrenergic neurons, as well as for sympathetic, parasympathetic and sensory neurons. However, for most neuronal populations, except for motoneurons, TGF-beta is required as a cofactor for GDNF family ligand signalling. Because GDNF and neurturin can rescue dopamine neurons in the animal models of Parkinson disease, as well as motoneurons in vivo, hopes have been raised that GDNF family ligands may be new drugs for the treatment of neurodegenerative diseases. GDNF also has distinct functions outside the nervous system, promoting ureteric branching in kidney development and regulating spermatogenesis.     

Evaluation of the Biological Activity of Several Analogs of the Dopaminergic Neurotoxin 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine

Several analogs of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were synthesized and screened for their capacity to be oxidized by monoamine oxidase (MAO-A or MAO-B) and their capacity to produce nigrostriatal dopaminergic neurotoxicity in mice. All of the compounds were relatively weak substrates for MAO-A but many of the compounds were found to be good substrates for MAO-B. Only three of the compounds, in addition to MPTP itself, were found to be neurotoxic. These were 1-methyl-4-cyclohexyl-1,2,3,6-tetrahydropyridine, 1-methyl-4-(2'-methylphenyl)-1,2,3,6-tetrahydropyridine and 1-methyl-4-(3'-methoxyphenyl)-1,2,3,6-tetrahydropyridine. All three of these neurotoxic compounds were found to be substrates for MAO-B; in contrast no compound was found to be neurotoxic that was not oxidized by MAO-B. The capacity of the compounds studied to be oxidized by MAO-B appears to be an important aspect of the neurotoxic process.     

Metabotropic glutamate receptors in neurodegeneration/neuroprotection: Still a hot topic?

Moving from early studies, we here review the most recent evidence linking metabotropic glutamate (mGlu) receptors to processes of neurodegeneration/neuroprotection. The use of knockout mice and subtype-selective drugs has increased our knowledge of the precise role played by individual mGlu receptor subtypes in these processes. Activation of mGlu1 and mGlu5 receptors may either amplify or reduce neuronal damage depending on the context and the nature of the toxic insults. In contrast, mGlu1 and mGlu5 receptors antagonists are consistently protective in in vitro and in vivo models of neuronal death. A series of studies suggest that mGlu1 receptor antagonists or negative allosteric modulators (NAMs) are promising candidates for the treatment of ischemic brain damage, whereas mGlu5 receptor NAMs, which have been clinically developed for the treatment of Parkinson's disease (PD) and l-DOPA-induced dyskinesias, protect nigro-striatal dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity in mice and monkeys. Activation of glial mGlu3 receptors promotes the formation of various neurotrophic factors, such as transforming growth factor-β (TGF-β), glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF). Hence, selective mGlu3 receptor agonists or positive allosteric modulators (PAMs) (not yet available) are potentially helpful in the treatment of chronic neurodegenerative disorders such as PD, Alzheimer's disease (AD), and amyotrophic lateral sclerosis. Selective mGlu2 receptor PAMs should be used with caution in AD patients because these drugs are shown to amplify β-amyloid neurotoxicity. Finally, mGlu4 receptor agonists/PAMs share with mGlu5 receptor NAMs the ability to improve motor symptoms associated with PD and attenuate nigro-striatal degeneration at the same time. No data are yet available on the role of mGlu7 and mGlu8 receptors in neurodegeneration/neuroprotection.     

Identification of the key amino acids of glial cell line-derived neurotrophic factor family receptor alpha1 involved in its biological function

Glial cell line-derived neurotrophic factor (GDNF) plays a critical role in neurodevelopment and survival of midbrain dopaminergic and spinal motor neurons in vitro and in vivo. The biological actions of GDNF are mediated by a two-receptor complex consisting of a glycosylphosphatidylinositol-linked cell surface molecule, the GDNF family receptor alpha1 (GFRalpha1), and receptor protein tyrosine kinase Ret. Although structural analysis of GDNF has been extensively examined, less is known about the structural basis of GFRalpha1 function. In this study, based on evolutionary trace method and relative solvent accessibility prediction of residues, a set of trace residues that are solvent-accessible was selected for site-directed mutagenesis. A series of GFRalpha1 mutations was made, and PC12 cell lines stably expressing different GFRalpha1 mutants were generated. According to the survival and differentiation responses of these stable PC12 cells upon GDNF stimulation and the GDNF-GFRalpha1-Ret interaction assay, residues 152NN153, Arg259, and 316SNS318 in the GFRalpha1 central region were found to be critical for GFRalpha1 binding to GDNF and eliciting downstream signal transduction. The single mutation R259A in the GFRalpha1 molecule simultaneously lost its binding ability to GDNF and Ret. However N152A/N153A or S316A/N317A/S318A mutation in the GFRalpha1 molecule still retained the ability to bind with Ret. These findings suggest that distinct structural elements in GFRalpha1 may be involved in binding to GDNF and Ret.     

GM1 ganglioside enhances Ret signaling in striatum

It has been proposed that GM1 ganglioside promotes neuronal growth, phenotypic expression, and survival by modulating tyrosine kinase receptors for neurotrophic factors. Our studies tested the hypothesis that GM1 exerts its neurotrophic action on dopaminergic neurons, in part, by interacting with the GDNF (glia cell‐derived neurotrophic factor) receptor complex, Ret tyrosine kinase and GFRα1 co‐receptor. GM1 addition to striatal slices in situ increased Ret activity in a concentration‐ and time‐dependent manner. GM1‐induced Ret activation required the whole GM1 molecule and was inhibited by the kinase inhibitors PP2 and PP1. Ret activation was followed by Tyr1062 phosphorylation and PI3 kinase/Akt recruitment. The Src kinase was associated with Ret and GM1 enhanced its phosphorylation. GM1 responses required the presence of GFRα1, and there was a GM1 concentration‐dependent increase in the binding of endogenous GDNF which paralleled that of Ret activation. Neutralization of the released GDNF did not influence the Ret response to GM1, and GM1 had no effect on GDNF release. Our in situ studies suggest that GM1 via GFRα1 modulates Ret activation and phosphorylation in the striatum and provide a putative mechanism for its effects on dopaminergic neurons. Indeed, chronic GM1 treatment enhanced Ret activity and phosphorylation in the striatum of the MPTP‐mouse and kinase activation was associated with recovery of dopamine and DOPAC deficits.

     

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.     

Ret is essential to mediate GDNF's neuroprotective and neuroregenerative effect in a Parkinson disease mouse model

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival and regeneration-promoting factor for dopaminergic neurons in cell and animal models of Parkinson disease (PD). GDNF is currently tested in clinical trials on PD patients with so far inconclusive results. The receptor tyrosine kinase Ret is the canonical GDNF receptor, but several alternative GDNF receptors have been proposed, raising the question of which signaling receptor mediates here the beneficial GDNF effects. To address this question we overexpressed GDNF in the striatum of mice deficient for Ret in dopaminergic neurons and subsequently challenged these mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Strikingly, in this established PD mouse model, the absence of Ret completely abolished GDNF''s neuroprotective and regenerative effect on the midbrain dopaminergic system. This establishes Ret signaling as absolutely required for GDNF''s effects to prevent and compensate dopaminergic system degeneration and suggests Ret activation as the primary target of GDNF therapy in PD.Glial cell line-derived neurotrophic factor (GDNF) is the founding member of the four ligands in the GDNF family, which belong to the transforming growth factor-β superfamily.¹ GDNF was characterized as a potent survival factor for many neurons in culture such as dopaminergic, motor, sympathetic, parasympathetic, sensory and enteric neurons.^{1, 2} In addition, in dopaminergic neuron cultures GDNF stimulates neuronal differentiation, neurite outgrowth, synapse formation and dopamine release.^{1, 2}As degeneration of midbrain dopaminergic neurons in the substantia nigra pars compacta (SNpc) represents a major hallmark of Parkinson disease (PD), the most common neurodegenerative movement disorder, GDNF has raised considerable interest as a therapeutic molecule for the treatment of PD.^{3, 4, 5} PD affects >2% of individuals over the age of 60 years, but no curative treatment is available to date, mainly due to a lack of understanding disease etiology.^{6, 7, 8} Preclinical studies in the established 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) rodent and primate models of PD demonstrated a substantial neuroprotection and regeneration effect by striatal provided GDNF or its close relative neurturin.^{3, 4, 9} However, clinical phase II trials on PD patients using GDNF or neurturin did so far not convincingly recapitulate their beneficial effects on the dopaminergic system in humans most likely due to technical problems and the selection of advanced PD patients.^{10, 11, 12, 13}GDNF signaling is highly complex as this neurotrophic factor can bind to a variety of receptors, thus being able to induce pleiotropic effects. GDNF efficiently binds to the GPI-linked GDNF family receptor α1 (GFRα1).^{1, 2} It has been shown that the GDNF/GFRα1 complex can activate not only the canonical GDNF receptor Ret, a receptor tyrosine kinase which signals through the sarcoma protein (Src)/rat sarcoma (Ras)/mitogen-activated protein kinase (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt, NF-κB (nuclear factor ''kappa-light-chain-enhancer'' of activated B cells), JNK (c-Jun N-terminal kinases) and PLCγ (phospholipase γ) pathway, but also with other signaling inducing receptors.^{1, 2, 4, 5, 13} So far, at least four alternative GDNF receptors have been described which are all expressed in midbrain dopaminergic neurons, NCAM,^{14, 15} the integrins αV and βI,^{14, 16} syndecan 3¹⁷ and N-cadherin.¹⁸ Interestingly, Ret is not essential during pre- and postnatal development of the mouse dopaminergic system,^{19, 20, 21, 22, 23} but specifically required for the maintenance of SNpc dopaminergic neurons and their striatal innervation in aged mice.^{23, 24, 25} In contrast, GDNF seems most likely under physiological conditions to be dispensable during development and maintenance of midbrain dopaminergic neurons in mice, although conflicting results exist.^{26, 27, 28} Thus, Ret might be activated by a GDNF-independent mechanism to stimulate SNpc dopaminergic neuron survival. In addition, the in vivo function of the alternative GDNF receptors in the dopaminergic system under physiological and pathophysiological conditions, like PD, and their dependence on GDNF has not yet been addressed in detail. This raised the important question which GDNF receptor might be required to mediate GDNF''s reported neuroprotective and regenerative effect in the dopaminergic system in PD animal models and potentially in PD patients.^{5, 29}Previously, we showed in dopaminergic neuron-specific Ret knockout mice that Ret receptor loss does not result in a higher vulnerability of midbrain dopaminergic neurons against MPTP but to less resprouting of left over dopaminergic neuron axons in the striatum after MPTP intoxication.³⁰ In adult mice endogenous GDNF levels are rather low.^{26, 31} Therefore, we could not rule out in that study the possibility, that higher levels of GDNF—as also used in the clinical GDNF trials in PD patients—might have neuroprotective and regenerating effects even in the absence of the Ret receptor. Here we addressed now this question by viral overexpression of GDNF in MPTP-treated mice lacking expression of Ret again specifically in dopaminergic neurons.^{23, 30} We found that in the absence of Ret in dopaminergic neurons even a substantial overexpression of GDNF in the striatum does not have a neuroprotective and regenerative effect. Thus, despite the expression of alternative GDNF receptors on midbrain dopaminergic neurons, the presence of the canonical GDNF receptor Ret seems to be mandatory for mediating GDNF''s beneficial survival and axonal resprouting effect in these neurons.     

Selective Visualization of Rodent Locus Ceruleus by a Radiolabeled N-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Analog

The neurotoxic metabolite of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridinium, selectively accumulates in dopaminergic neurons via the dopamine reuptake system. Consequently, nontoxic radiolabeled MPTP analogs may be potentially useful for visualizing catecholaminergic neurons in vivo. N-Methyl-4-(4-hydroxy-3-[125I]iodobenzyl)-1,2,3,6-tetrahydropyridine [( 125I]MHTP), an analog of the nontoxic N-methyl-4-benzyl-1,2,3,6-tetrahydropyridine, has been studied in rats and mice. After intravenous administration of [125I]MHTP to rodents, the initial accumulation of radioactivity within the brain was found to be comparable to that of radiolabeled MPTP. Following intravenous administration of [125I]MHTP, in vivo autoradiographic visualization of the rodent brain revealed selective accumulation of [125I]MHTP-derived radioactivity within the locus ceruleus; there was no accumulation of the radiotracer within dopaminergic fibers and cell bodies. The accumulation of radioactivity within the locus ceruleus was blocked by pretreatment with pargyline, a result suggesting that an MHTP metabolite formed by monoamine oxidase was responsible for the localization of the radiotracer within this structure. The anatomical distribution of the radiolabel demonstrates selective accumulation of this metabolite within noradrenergic cell bodies and those fibers making up the locus ceruleus. These findings further suggest that nontoxic metabolites of MPTP may become useful for in vivo labeling of selected populations of catecholaminergic neurons.