Introduction
Degeneration of nigral dopaminergic (DA) neurons is a major hallmark of Parkinson’s disease (PD), resulting in striatal dopamine loss and inappropriate fine tuning of relevant motor circuitry. About 5% of PD patients develop the disease due to familial mutations while the vast majority of cases are sporadic. No singular pathomechanism initiating PD has been identified so far. Current hypotheses encompass oxidative stress through mitochondrial dysfunction, accumulation of transition metals, dysfunction of protein folding quality control, inflammation, and depletion of neurotrophic support or of anti-oxidant defence mechanisms [
34]. Depletion of the major cellular anti-oxidant glutathione (GSH) has been described in several independent studies to take place specifically in the substantia nigra of PD patients [
27,
28,
30]. GSH depletion is not detected in patients with multiple system atrophy or progressive supranuclear palsy, despite the degeneration of dopaminergic neurons [
29,
33]. In incidental Lewy body disease, which may be considered as a pre-symptomatic form of PD [
16] and is present in up to 10% of individuals over the age of 60, GSH depletion is already present in the substantia nigra [
18], and thus appears to precede complex I dysfunctions, iron accumulation and striatal dopamine loss [
38]. These findings imply decreased GSH levels being an early event in PD aetiology, possibly initiating or facilitating a cascade of further oxidative stress lesions. This view is supported by in vitro and in vivo studies demonstrating that GSH depletion induces oxidative damage to complex I proteins [
3,
4].
GSH is a tri-peptide consisting of the amino acids glutamate, cysteine and glycine, the reactive thiol group of the cysteine residue serving as the cells most effective anti-oxidant. The rate-limiting step in GSH synthesis is carried out by glutamate–cysteine ligase (GCL), a dimeric protein composed of a catalytic (GCLc) and a modulatory subunit (GCLm). The modulatory subunit serves to increase the affinity of the catalytic subunit for its substrate glutamate, and renders the holo-enzyme less sensitive to feedback inhibition by GSH. Astrocytes synthesize the vast majority of brain GSH and secrete the peptide, but neurons can take up only the precursors derived from extra-cellular catabolism and must synthesize their own GSH. In non-enzymatic reactions GSH detoxifies NO
2 and peroxynitrite, and enzymatically reduces other anti-oxidants as dehydroascorbate and, indirectly, tocopherol and through glutaredoxin maintains sulfhydryl groups of proteins in reduced states. GSH peroxidases use GSH for enzymatic reduction of hydrogen peroxide. Oxidized GSH (GSSG) is recycled into the reduced state by GSH-reductase (GSR) [
25].
Whether GSH depletion can be a selective trigger specifically for DA neuron degeneration has not yet been demonstrated experimentally. We thus down-regulated GSH synthesis by viral vector-mediated RNA interference in DA and in non-DA neurons of the adult rat brain. We could demonstrate that depletion of anti-oxidative defence serves as an initiator for progressive DA neuron degeneration, including protein aggregation and functional motor deficits, while non-DA neurons were significantly less vulnerable to GSH depletion-induced degeneration. Dopaminergic neuron degeneration was partially prevented by α-synuclein, a synaptic vesicle-associated protein which forms the major protein component of Lewy bodies, the aggregation entities found in postmortem brains of PD patients [
12]. Unexpectedly, over-expression of either of the two subunits of GCL also induced significant degeneration of DA neurons, emphasizing that GSH levels must be maintained within a tightly controlled range and suggesting that GSH substitution as a neuroprotective strategy for PD may pose severe risk.
Methods
shRNAs
ShRNAs were selected using the DEQOR algorithm [
13] to target (1) rat glutamate–cysteine ligase catalytic subunit (GCLc, E.C. 6.3.2.2); (2) rat glutamate–cysteine ligase modulatory subunit (GCLm); and (3) rat glutathione reductase (GSR, E.C. 1.8.1.7). Control shRNAs were: EGFP-shRNA, Luciferase-shRNA and scrambled shRNA (Dharmacon-shRNA). Detailed sequences are given in Supplemental Methods. All shRNAs were tested for efficiency of target downregulation in a transient co-transfection approach as described [
23] and resulted in at least 80% of target mRNA downregulation.
AAV vectors
Recombinant vector backbones were constructed so that expression of all shRNAs was driven by the human H1 polymerase III promoter, while expression of the EGFP reporter or of α-synuclein was driven by the human synapsin 1 gene promoter [
20], both expression cassettes being in a head-to-head arrangement. The EGFP expression cassette also contains a WPRE for enhanced mRNA stability and a human bovine growth hormone polyadenylation site. While EGFP expression was entirely neuron-restricted by the hSYN promoter, the H1 promoter is putatively active in all cell types. We ruled out that recombinant AAV-2 vectors would result in astrocyte transduction and concomitant expression of shRNAs in these cells, potentially leading to diminished supply of GSH precursors from astrocytes to neurons by constructing an AAV-2 vector driving EGFP expression from an astrocyte-specific GFAP promoter. Injection of this virus into the substantia nigra resulted in EGFP expression in no more than 5–10 astrocytes per whole midbrain, thus confirming the neuron-specific targeting approach (not shown).
Recombinant vectors were prepared as described [
24], purified by iodixanol step gradient, heparin sulphate affinity chromatography and dialysis before stored frozen at −80°C in single-use aliquots. Vector genomes were titrated by quantitative PCR. The purity of the preparations was confirmed by SDS gel electrophoresis and silver staining.
Animal procedures
All experimental animal procedures were conducted according to approved experimental animal licenses issued by the responsible animal welfare authority (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit) and controlled by the local animal welfare committee of the University Medical Center Goettingen. Intracerebral stereotaxic injections into the substantia nigra (coordinates relative to bregma = AP, −5.3; ML, +2.2; DV, −7.7 mm) and striatum (coordinates AP, +1.2; ML, +2.3; DV, −5.0 mm) of 2.5-month-old female Wistar rats, maintenance of animals and killing and perfusion were performed essentially as described [
32]. Two microliter of AAV in PBS containing 4 × 10
9 vector genomes were injected at a speed of 500 nl/min. This application resulted in transduction of 70–75% of nigral DA neurons as judged by EGFP/TH co-labelling (not shown). For numbers of animals used for each individual group please see Table 1 in Supplemental Information.
Functional/behavioural analysis of nigro-striatal projection
Apomorphine-induced circling behaviour was analysed essentially as described for 6-OHDA lesioned rats [
31]. Specifically, animals were placed into cylindrical, flat-bottomed chambers of 40 cm diameter, injected i.p. with 0.4 mg apomorphine/kg body weight and their turning behaviour recorded with a video camera for offline analysis. Only complete turns of 360°C were counted.
Quantification of GSH
The glutathione quantification assay is based on the 96-well microplate assay previously reported by Dringen [
10]. Details of the procedure are given in Supplemental Methods.
Cell viability assay
Cell viability was measured in 96-well plates using the WST-1 assay according to the protocol provided by the manufacturer (Roche Diagnostics, Germany).
Immunohistochemistry
Details are given in Supplemental Methods.
Thioflavin-S staining
EGFP fluorescence present in the tissue was eliminated by microwave boiling of mounted sections (5 min in 10 mM Na-Citrate, pH 6.0, 0.05% Tween 20; cooled to RT slowly in ca. 1 h) in order to avoid interference with emitted Thio-S fluorescence. Sections were washed in PBS (10 min), in water (5 min), incubated in 0.05% Thio-S in 50% EtOH (filtered through 0.22 μm) for 20 min, washed 3 × 5 min in 80% EtOH, then 3 × 10 min and 1 × 30 min in PBS followed by immunohistochemistry as outlined in Supplemental Methods. Due to the broad emission spectrum of Thio-S we used only Cy 5-coupled secondary antibodies in order to avoid any possible overlap of emission spectra.
Dopamine measurement
Animals were killed by decapitation and their brains quickly removed. Both striata were prepared individually and quick-frozen on dry ice. 143 μl perchloric acid 70% was added per 50 mg of sample and the mixture homogenized by mechanical beads (Precellys 24 homogenizer) and centrifuged twice. 20 μl of the resulting supernatant was then analysed by HPLC with electrochemical detection as described [
19].
Statistics
For statistics, the respective groups were evaluated by one-way ANOVA with Bonferroni’s multiple comparison test using GraphPad Prism. Differences between groups were considered statistically significant if p < 0.05. Significances are indicated with *p < 0.05, **p < 0.01, ***p < 0.001. All data are presented as mean ± standard deviation (SD) unless otherwise indicated.
Discussion
It has long been debated whether GSH depletion as found in PD patients’ substantia nigra is a cause or a consequence of DA neuron degeneration, and if DA neurons would indeed be specifically vulnerable to GSH depletion. Targeting of the catalytic subunit of the rate-limiting enzyme in GSH synthesis, glutamate–cysteine ligase (GCLc) has been the traditional approach to elucidate this issue. Pharmacological inhibition by infusion of the GCLc inhibitor buthionine sulfoxamine (BSO) into the adult rat brain did not induce degeneration of DA neurons [
35]. While constitutive knock-out of GCLc was embryonically lethal [
7], a conditionally GCLc anti-sense RNA-expressing mouse demonstrated oxidative damage of mitochondrial complex I as a consequence of GSH depletion and showed a minor loss of DA neurons in aged animals [
4]. However, given the possible off-target effects of expression of the anti-sense RNA and the lack of any control for such effects [
1], this model must be considered with caution. Furthermore, no effects of GSH depletion in non-DA neurons could be assessed, and no behavioural readout could be obtained. We now demonstrate that by an RNAi-mediated approach we could unequivocally induce progressive DA neuron degeneration by targeting the expression of GCLc or GCLm. While nigral DA neurons degenerated progressively over time in a pace depending on potency of the respective shRNA to decrease GSH levels as determined in vitro, extra-nigral non-DA neurons and striatal neurons were apparently less vulnerable. This finding contradicts data obtained from cultured dopaminergic neurons, which were more resistant to BSO application than non-dopaminergic midbrain neurons [
26]. In AAV-GCLc-shRNA#2 transduced striatal neurons we found an almost tenfold reduction of fluorescence of the EGFP reporter compared to controls, which might be explained by the strong quenching of the EGFP fluorophore by reactive oxygen species like singlet oxygen derived from lipid peroxidation [
11] under conditions of depleted anti-oxidant defence capabilities. This quenching of the EGFP reporter co-expressed with the GCLc-targeting shRNA demonstrates substantial impact on striatal neuron physiology, which, however, degenerated to a much lesser extent than nigral DA neurons. These results for the first time suggest a selective vulnerability of DA neurons towards depletion of anti-oxidant defence, which might be an explanation for their preferential death in PD patients.
GSH depletion induced an apparent continuum of formation of Thio-S positive aggregate structures within affected DA neurons. The morphology of these aggregates resembled the so-called pale bodies (PB) which are thought to be a precursor stage of Lewy bodies (LB), and the obvious morphogenesis of these structures recapitulates the human pathology revealed by immunohistochemical methods in postmortem tissue [
21]. However, since they are negative for α-synuclein and ubiquitin immunoreactivity, their composition appeared to be quite different from Lewy bodies. Thus, while GSH depletion can be a selective trigger for DA neurons to degenerate, further impacts must occur in patients to cause α-synuclein aggregation. Intriguingly, our attempts to foster GSH-induced degeneration by over-expression of the familial α-synuclein mutant A53T demonstrated that α-synuclein partially ameliorated DA neuron survival. It should be emphasized that for rodent α-synuclein T53 is the wild-type amino acid, and that AAV-2-mediated over-expression of α-synuclein-A53T in the rat nigra did not result in DA cell body loss at 9 weeks in an independent study [
5]. Thus, this protection may not be representative for other α-synuclein mutants or more enforced expression of α-synuclein. However, this finding is reminiscent of α-synuclein’s neuroprotective effect in CSP-α-deficient mice [
2], which is mediated through interaction with synaptic vesicles. Together with the early appearance of ipsilateral rotation behaviour and the prevalence of Fluoro-Jade positive neurites rather than cell bodies, protection achieved by α-synuclein suggest that GSH-depletion-induced neurodegeneration might be initiated at synaptic or axonal sites.
While it has been demonstrated in vitro and in vivo that depletion of GSH leads to nitric oxide-mediated oxidative damage of mitochondrial complex I components [
4,
8], the mechanism of neuronal cell loss is less clear. Cultured cortical neurons which were depleted by GCLc or GCLm demonstrated features of apoptotic cell death [
8], but in concordance with results obtained in postmortem tissue [
22], we did not detect significant activation of caspases in nigral DA neurons and no fragmented nuclei by GSH depletion. Activation of astrocytes was moderate, while staining for activated microglia showed more robust increase, as seen in postmortem PD brains [
14]. However, microglia morphology remained ramified and no cells with spherical macrophage-like shape appeared, indicating that classical necrosis might not be a prominent feature of DA neuronal death after GSH depletion.
GSH depletion induced a progressive decline in motor control, allowing us to follow the degeneration of the nigro-striatal system by simple behavioural assessment. Thus, this animal model is well suited for testing drugs aimed at protecting DA neurons from degeneration in functional assays, with the option to be transferred to brains of non-human primates. As it is based on a patho-physiological principle detected in brains of sporadic PD patients, it may be a valuable alternative to toxin-based models of PD currently employed, since compounds tested successfully in MPTP and 6-OHDA models have met with very limited benefit in clinical trials [
17].
Increase of GSH production by GCLc over-expression was protective against oxidative stress in cultured cortical neurons [
8]. The obvious strategy to increase GSH production in DA neurons as a means to protect them from degeneration, however, led to significant cell death in our in vivo study, presumably due to formation of GSH adducts on cellular proteins as detected by a GSH-adduct-specific antibody. In recent years, it has become evident that GSH has important functions besides its reducing activity. In general, thiolation or dethiolation of cellular proteins by GSH may either increase or decrease their levels of activity or binding to other cellular structures [
6,
36,
37]. Specifically within the CNS, GSH has been demonstrated to be able to modify NMDA receptor activity and leukotriene metabolism, and thus is directly involved in neuromodulatory events [
9,
15]. Due to the overriding GSH content of astrocytes it is not possible to reliably quantify the extent to which GSH was reduced or elevated in individual neurons in vivo, but in cultured neurons over-expressing either the regulatory or the catalytic subunit of GCL we measured only moderate increases in GSH levels, in the range of 1.5- to 2.5-fold to controls. Thus, our results suggest that even relatively moderate increases in GSH can initiate degeneration of DA neurons, arguing against delivery of GCLc or GCLm to the brain, e.g. by gene therapeutic attempts, as a potential treatment strategy for PD.
In conclusion, our results demonstrate the distinct dependence of nigral DA neurons on tight control of GSH levels, suggesting that disturbed GSH homeostasis can be a trigger in the initiation of DA neuron degeneration in PD.
Acknowledgments
We are grateful to Ulrike Schöll for excellent technical assistance with primary neuron preparations, Erin Butler and Anja Drinkut for help with HPLC, and Cathy Ludwig for critically reading the manuscript. This work was supported by the European Community’s Seventh Framework Programme FP7/2007-2013 under grant agreement no. HEALTH-F5-2008-222925, by the German Research Council-funded Center of Molecular Physiology of the Brain (CMPB) and by the European Research Training Network 2004–2007 under contract no. MRTN-CT-2003-504636.