Elsevier

Neurobiology of Disease

Volume 65, May 2014, Pages 69-81
Neurobiology of Disease

Progressive Parkinsonism by acute dysfunction of excitatory amino acid transporters in the rat substantia nigra

https://doi.org/10.1016/j.nbd.2014.01.011Get rights and content

Highlights

  • Acute nigral EAAT dysfunction induces selective death of dopaminergic neurons.

  • The primary insult involves GSH depletion, oxidative stress and excitotoxicity.

  • Dopamine neuron loss is progressive, evolves bilaterally and leads to motor deficit.

  • Novel rodent model recapitulating main pathological features of Parkinsonism

  • Evolving model potentially suitable for testing disease-modifying strategies

Abstract

Parkinson's disease (PD) is characterized by the progressive degeneration of substantia nigra (SN) dopamine neurons, involving a multifactorial cascade of pathogenic events. Here we explored the hypothesis that dysfunction of excitatory amino acid transporters (EAATs) might be involved. Acutely-induced dysfunction of EAATs in the rat SN, by single unilateral injection of their substrate inhibitor l-trans-pyrrolidine-2,4-dicarboxylate (PDC), triggers a neurodegenerative process mimicking several PD features. Dopamine neurons are selectively affected, consistent with their sustained excitation by PDC measured by slice electrophysiology. The anti-oxidant N-acetylcysteine and the NMDA receptor antagonists ifenprodil and memantine provide neuroprotection. Besides oxidative stress and NMDA receptor-mediated excitotoxicity, glutathione depletion and neuroinflammation characterize the primary insult. Most interestingly, the degeneration progresses overtime with unilateral to bilateral and caudo-rostral evolution. Transient adaptive changes in dopamine function markers in SN and striatum accompany cell loss and axonal dystrophy, respectively. Motor deficits appear when neuron loss exceeds 50% in the most affected SN and striatal dopamine tone is dramatically reduced. These findings outline a functional link between EAAT dysfunction and several PD pathogenic mechanisms/pathological hallmarks, and provide a novel acutely-triggered model of progressive Parkinsonism.

Introduction

Parkinson's disease (PD) is a neurodegenerative movement disorder in which cell death mainly affects substantia nigra (SN) dopamine (DA) neurons. Neuronal loss is progressive and motor symptoms appear when 50–60% of nigral DA neurons are lost. A number of mechanisms have been implicated in such cell death, including oxidative stress, mitochondrial dysfunction, protein misfolding/aggregation, autophagy, neuroinflammation and excitotoxicity (Ahmed et al., 2012, Cheung and Ip, 2009, Olanow, 2007, Schapira and Jenner, 2011, Yacoubian and Standaert, 2009). Depletion of glutathione (GSH), the main brain antioxidant, is considered as an early and key component of the pathological process. Reduced levels of GSH are measured specifically in the SN of PD patients and incidental Lewy body disease (presumably representing presymptomatic PD), but not in other pathologies affecting DA neurons (Jenner et al., 1992, Pearce et al., 1997, Perry et al., 1982, Sian et al., 1994a, Sian et al., 1994b). Down-regulation of GSH synthesis in vivo in rodents impacts mitochondrial complex-I activity and results in nigral DA neuron degeneration associated with protein aggregation (Chinta et al., 2007, Garrido et al., 2011). Dysfunction of excitatory amino acid transporters (EAATs) might link several of these PD pathogenic mechanisms. DA neurons express the neuronal transporter EAAC1 (EAAT3) at high levels (Plaitakis and Shashidharan, 2000, Shashidharan et al., 1997), and pathological conditions leading to excessive neuronal depolarization can affect or even reverse EAAT function by altering the transport driving force (Danbolt, 2001): for example, overactive glutamatergic inputs from the subthalamic nucleus (STN) to DA neurons (Rodriguez et al., 1998), or ATP depletion and subsequent impairment of the Na+/K+-ATPase resulting from mitochondrial dysfunction. Neuroinflammation can also affect glial EAAT function (McNaught and Jenner, 2000, Tilleux and Hermans, 2007). Besides clearing extracellular glutamate, EAATs provide substrates for GSH production since they uptake not only glutamate but also cyst(e)ine (Hayes et al., 2005). EAAT dysfunction might then, in turn, sustain excitotoxicity and oxidative stress. Accordingly, oxidative stress on EAAC1 has been involved in MPTP-induced GSH depletion (Aoyama et al., 2008), and EAAC1−/− mice show an age-dependent loss of nigral DA neurons that is prevented by N-acetylcysteine (Berman et al., 2011). Interestingly, the EAAT substrate inhibitor l-trans-pyrrolidine-2,4-dicarboxylate (PDC) triggers preferential DA neuron death in vitro through a mechanism involving oxidative stress and excitotoxicity, and can induce early loss of DA neurons when injected in the SN (Nafia et al., 2008), whereas it does not induce neuronal damage in other brain regions (Massieu et al., 1995, Montiel et al., 2005).

Here we show that a single unilateral PDC injection in the rat SN initiates a self-sustaining degenerative process selectively affecting DA versus non-DA neurons, which progresses up to 120 days after the injection. This provides a novel animal model exhibiting a unique combination of PD hallmarks: i) presumed cell death mode (GSH depletion, oxidative stress, NMDA receptor-mediated excitotoxicity, neuroinflammation); ii) evolution pattern of DA neuron loss from unilateral to bilateral and with a caudo-rostral gradient; iii) development of compensatory mechanisms; iv) appearance of motor deficits.

Section snippets

Materials and methods

Experiments were performed on male Wistar Hannover rats in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and all efforts were made to minimize their number and sufferance. Protocols were approved by the local ethical committee and conformed to the ethical guidelines of the French Ministry of Agriculture and Forests (Animal Health and Protection Veterinary Service).

DA and non-DA SN neurons show differential in vitro electrophysiological responses to PDC

In order to study the acute effect of PDC on SN neurons and its possible action mechanisms, we first tested this drug in vitro on brain slices by means of whole-cell patch-clamp electrophysiology.

DA neurons of the SNr were identified off-line by the co-localization of both Alexa Fluor 568 fluorescence (injected via the patch-clamp micropipette) and TH immunostaining (Fig. 2A), and on-line by their localization, size and membrane properties in response to hyperpolarizing and depolarizing current

Discussion

Glutamate-mediated mechanisms have been implicated in PD pathophysiology and pathogenesis (Blandini et al., 1996, Carlsson and Carlsson, 1990, Johnson et al., 2009). EAAT function is essential for glutamate homeostasis and to provide metabolic substrates for antioxidant defenses. EAAC1 −/− mice have been reported to show age-dependent neurodegeneration involving oxidative stress, including loss of DA neurons, brain atrophy, and cognitive and motivational behavioral impairments but little motor

Conclusion

None of the available neurotoxin-based or genetic rodent models of PD recapitulates all the hallmarks of this pathology, thus capturing as many of these hallmarks in one single model remains a major challenge (Chesselet et al., 2012, Dawson et al., 2010, Jackson-Lewis et al., 2012, Magen and Chesselet, 2010, Martinez and Greenamyre, 2012). Here we generated a novel rodent model of progressive Parkinsonism of easy implementation, based on the manipulation of endogenous glutamate-mediated

Acknowledgments

This work was supported by the Centre National de la Recherche Scientifique (CNRS), Aix-Marseille University, and a France Parkinson grant (to L.H.-A.). M.A. was supported by a fellowship from the French Ministry of Education and Research. The authors are grateful to Dr. Annie Daszuta for her advice and expertise in immunocytochemistry. This work was performed using France-BioImaging infrastructure supported by the Agence Nationale de la Recherche (ANR-10-INSB-04-01, call “Investissements

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