N-Acetylcysteine Prevents Loss of Dopaminergic Neurons in the EAAC1(-/-) Mouse

Department of Neurology, University of California, San Francisco, San Francisco Veterans Affairs Medical Center, USA.
Annals of Neurology (Impact Factor: 9.98). 03/2011; 69(3):509-20. DOI: 10.1002/ana.22162
Source: PubMed


Dopaminergic neuronal death in Parkinson's disease (PD) is accompanied by oxidative stress and preceded by glutathione depletion. The development of disease-modifying therapies for PD has been hindered by a paucity of animal models that mimic these features and demonstrate an age-related progression. The EAAC1(-/-) mouse may be useful in this regard, because EAAC1(-/-) mouse neurons have impaired neuronal cysteine uptake, resulting in reduced neuronal glutathione content and chronic oxidative stress. Here we aimed to (1) characterize the age-related changes in nigral dopaminergic neurons in the EAAC1(-/-) mouse, and (2) use the EAAC1(-/-) mouse to evaluate N-acetylcysteine, a membrane-permeable cysteine pro-drug, as a potential disease-modifying intervention for PD.
Wild-type mice, EAAC1(-/-) mice, and EAAC1(-/-) mice chronically treated with N-acetylcysteine were evaluated at serial time points for evidence of oxidative stress, dopaminergic cell death, and motor abnormalities.
EAAC1(-/-) mice showed age-dependent loss of dopaminergic neurons in the substantia nigra pars compacta, with more than 40% of these neurons lost by age 12 months. This neuronal loss was accompanied by increased nitrotyrosine formation, nitrosylated α-synuclein, and microglial activation. These changes were substantially reduced in mice that received N-acetylcysteine.
These findings suggest that the EAAC1(-/-) mouse may be a useful model of the chronic neuronal oxidative stress that occurs in PD. The salutary effects of N-acetylcysteine in this mouse model provide an impetus for clinical evaluation of glutathione repletion in PD.

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Available from: Tiina Kauppinen, Jul 17, 2014
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    • "Indeed, in EAAT3 deficient mice, the dopaminergic neurons of the substantia nigra pars compacta, a neuronal population highly susceptible to oxidative stress, show decreased levels of neuronal glutathione, increased evidence for oxidative stress and decreased neuronal survival during aging [24]. Treatment of these mice with the cysteine analogue N-acetylcysteine (NAC), which is taken up by cells in an EAAT3 independent manner, rescues the phenotype supporting the idea that EAAT3 activity is important for cellular cysteine homeostasis [4], [25]. "
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    ABSTRACT: Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1-5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1-3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools.
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    • "PDC being a substrate inhibitor of neuronal and glial EAATs, the question of which EAAT mediates PDC-triggered neurodegeneration arises. Although the contribution of glial EAATs cannot be excluded, a role of EAAC1 can be suggested since SNc DA neurons in EAAC1 −/− mice undergo age-related neurodegeneration associated with reduced GSH levels and increased oxidative stress (Berman et al., 2011). "
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    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.
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    • "Natural antioxidant phytocompound curcumin showed neuroprotective capacity in the 6-OHDA model of PD [86], [87] and also attenuated aggregation of α-synuclein in cell model of PD [88], [89]. The simpler anti-oxidant N-acetylcysteine prevents loss of DA neurons in the EAAC1-/- mouse [90]. Oral N-acetylcysteine reduced loss of dopaminergic terminals in α-synuclein overexpressing mice model [91]. "
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    ABSTRACT: Parkinson's disease (PD) is the second most common degenerative disorder of the central nervous system that impairs motor skills and cognitive function. To date, the disease has no effective therapies. The identification of new drugs that provide benefit in arresting the decline seen in PD patients is the focus of much recent study. However, the lengthy time frame for the progression of neurodegeneration in PD increases both the time and cost of examining potential therapeutic compounds in mammalian models. An alternative is to first evaluate the efficacy of compounds in Caenorhabditis elegans models, which reduces examination time from months to days. n-Butylidenephthalide is the naturally-occurring component derived from the chloroform extract of Angelica sinensis. It has been shown to have anti-tumor and anti-inflammatory properties, but no reports have yet described the effects of n-butylidenephthalide on PD. The aim of this study was to assess the potential for n-butylidenephthalide to improve PD in C. elegans models. In the current study, we employed a pharmacological strain that expresses green fluorescent protein specifically in dopaminergic neurons (BZ555) and a transgenic strain that expresses human α-synuclein in muscle cells (OW13) to investigate the antiparkinsonian activities of n-butylidenephthalide. Our results demonstrate that in PD animal models, n-butylidenephthalide significantly attenuates dopaminergic neuron degeneration induced by 6-hydroxydopamine; reduces α-synuclein accumulation; recovers lipid content, food-sensing behavior, and dopamine levels; and prolongs life-span of 6-hydroxydopamine treatment, thus revealing its potential as a possible antiparkinsonian drug. n-Butylidenephthalide may exert its effects by blocking egl-1 expression to inhibit apoptosis pathways and by raising rpn-6 expression to enhance the activity of proteasomes. n-Butylidenephthalide may be one of the effective neuroprotective agents for PD.
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