Both these authors contributed equally to this study.
Protection of midbrain dopaminergic neurons by the end-product of purine metabolism uric acid: potentiation by low-level depolarization
Article first published online: 19 MAR 2009
© 2009 The Authors. Journal Compilation © 2009 International Society for Neurochemistry
Journal of Neurochemistry
Volume 109, Issue 4, pages 1118–1128, May 2009
How to Cite
Guerreiro, S., Ponceau, A., Toulorge, D., Martin, E., Alvarez-Fischer, D., Hirsch, E. C. and Michel, P. P. (2009), Protection of midbrain dopaminergic neurons by the end-product of purine metabolism uric acid: potentiation by low-level depolarization. Journal of Neurochemistry, 109: 1118–1128. doi: 10.1111/j.1471-4159.2009.06040.x
- Issue published online: 14 APR 2009
- Article first published online: 19 MAR 2009
- Received January 8, 2009; revised manuscript received March 6, 2009; accepted March 10, 2009.
- oxidative stress;
- uric acid
High plasma levels of the end product of purine metabolism uric acid (UA) predict a reduced risk of developing Parkinson’s disease suggesting that UA may operate as a protective factor for midbrain dopaminergic neurons. Consistent with this view, UA exerted partial but long-term protection in a culture model in which these neurons die spontaneously. The rescued neurons were functional as they accumulated dopamine, efficiently. The use of the fluorescent probe dihydrorhodamine-123 revealed that UA operated by an antioxidant mechanism. The iron chelating agent desferrioxamine, the H2O2 scavenger enzyme catalase and the inhibitor of lipid peroxidation Trolox mimicked the effects of UA, suggesting that UA neutralized reactive oxygen species produced via a Fenton-type chemical reaction. UA was, however, not significantly accumulated into neurons, which indicates that the antioxidant effect occurred probably extracellularly. Structure – activity relationships among purine derivatives revealed that the antioxidant properties of UA resulted from the presence of a 8-one substituent in its chemical structure. Of interest, the stimulation of L-type Ca2+ channels by high K+-induced depolarization and the ensuing activation of extracellular signal-regulated kinases 1/2 strongly improved the neuroprotective effect of UA whereas the depolarizing signal alone had no effect. In summary, our data indicate that UA may interfere directly with the disease’s pathomechanism.
days in vitro
extracellular signal-regulated kinases
glial fibrillary acidic protein
reactive oxygen species
Parkinson’s disease (PD) is a debilitating neurodegenerative disorder of ageing characterized by invalidating motor symptoms resulting from the loss of dopaminergic neurons in the substantia nigra (Agid 1991; Dauer and Przedborski 2003). Different epidemiological studies indicate that uric acid (UA), the end-product of purine nucleoside catabolism may influence the progression of the disease; (i) Individuals with high plasma urate levels have a markedly reduced risk of developing PD (de Lau et al. 2005; Weisskopf et al. 2007). (ii) Further, among patients with early PD, higher plasma or cerebrospinal fluid urate levels predict a slower clinical progression (Schiess and Oh 2008; Schwarzschild et al. 2008). (iii) A prospective study also demonstrated that individuals with gout, a rheumatic disease resulting from hyperuricemia, had a significantly reduced occurrence of PD (Alonso et al. 2007; Gao et al. 2008). Conversely, UA levels have been reported to be reduced in the substantia nigra of PD patients (Church and Ward 1993; Fitzmaurice et al. 2003) and probenecid an agent that stimulates the excretion of the purine metabolite in urine was found to potentiate the effects of the dopaminergic toxin MPTP in mice (Petroske et al. 2001; Alvarez-Fischer et al. 2008) which further suggests that UA may interfere with the disease’s pathomechanism.
A number of studies have shown that UA has a strong antioxidant capacity. More specifically, UA was reported to neutralize reactive species such as peroxynitrites (Keller et al. 1998) and hydroxyl radicals (Davies et al. 1986) and to prevent free radical-mediated chain reactions that results in lipid peroxidation (Muraoka and Miura 2003; Allameh et al. 2008), leading to the speculation that UA could protect dopaminergic neurons through an antioxidant mechanism (de Lau et al. 2005). This is particularly pertinent if we consider that oxidative stress is probably pivotal in the disease’s process (Hirsch 1992; Jenner and Olanow 1998; Bharath et al. 2002). Yet, the mechanisms by which UA could protect dopaminergic neurons from oxidative stress-mediated insults remain rather elusive.
In this study, we wished therefore (i) to demonstrate that UA was able to afford long-term protection to dopaminergic neurons in a paradigm in which these neurons die as the result of low-level oxidative stress; (ii) to explore the mechanism of this neuroprotective effect; (iii) to prove that the rescued neurons were functional; (iv) to define the structural requirements conferring neuroprotection to UA; and (v) to determine whether its protective action could be possibly improved by other pharmacological treatments.
Material and methods
Pharmacological reagents including UA and its structural analogues were obtained from Sigma-Aldrich (St Quentin Fallavier, France). Dihydrorhodamine-123 (DHR-123) and calcium-green acetomethylester, the cell permeant probes used for the detection of reactive oxygen species (ROS) and cytosolic calcium (Ca2+cyt), respectively, were purchased from Molecular Probes (Invitrogen, Cergy Pontoise, France). Tritiated compounds including [3H]-dopamine (DA) and [methyl-3H]-thymidine were obtained from GE Healthcare (Orsay, France).
Cultures of mesencephalic dopaminergic neurons
Animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council 1996), the European Directive N886/609, and the guidelines of the local institutional committee for animal care and use. Cultures of dopaminergic neurons were prepared from the ventral mesencephalon of Wistar rat embryos dissected at embryonic day 15.5 as described previously (Troadec et al. 2002). Mesencephalic cells in suspension were plated onto polyethylenimine (1 mg/mL; Sigma-Aldrich) pre-coated culture plates (24 wells) and maintained in 500 μL of chemically defined serum-free medium consisting of equal volumes of Dulbecco’s minimal essential medium and Ham’s F12 nutrient mixture (Invitrogen). This medium was supplemented with 10 μg/mL insulin, 30 mM glucose and 100 U/mL penicillin and streptomycin. To favour cell attachment, foetal calf serum (10%) was also added to the medium but only the first hour after plating. Cultures were fed every 2 days by replacing twice 350 μL of the culture medium. Control culture media and culture media supplemented with purine derivatives and/or high K+ were equilibrated for 1 h in the incubator at 37°C before being added to the cultures. Other pharmacological treatments were applied directly to the cultures.
Immunofluorescence detection protocols
The survival of dopaminergic and more generally of mesencephalic neurons was assessed by tyrosine hydroxylase (TH) and microtubule-associated protein-2 (MAP-2) immunofluorescent detection respectively. The mouse anti-TH (Diasorin, Stillwater, MN, USA) and anti-MAP-2 monoclonal antibodies (clone AP-20; Sigma-Aldrich) were diluted 1 : 5000 and 1 : 50, respectively, in phosphate-buffered saline (PBS) containing 0.2% Triton X-100 (Guerreiro et al. 2008). Subsequent incubations were performed with a secondary anti-mouse IgG cyanin 3 conjugate (1 : 500; Sigma-Aldrich). Glial cells and more specifically astrocytes were detected using vimentin (Clone V9, Sigma-Aldrich) and glial fibrillary acidic protein (GFAP) (Dako France, Trappes, France) antibodies, respectively, as described before (Troadec et al. 2002). A rabbit polyclonal antibody raised against UA conjugated to a protein carrier (#ab53000; Abcam, Cambridge, UK) was used to visualize UA within cultured neurons. Briefly, the cultures were exposed overnight to the anti-UA antibody diluted 1 : 1000 in PBS containing 0.2% Triton X-100 followed by a detection step with an Alexa Fluor® 488 goat anti-rabbit IgG.
Dopamine uptake quantification
The functional integrity of dopaminergic neurons was evaluated by their ability to accumulate tritiated DA (50 nM; 40 Ci/mmol) by active transport as described (Guerreiro et al. 2008). Blank values were obtained in the presence of 3 μM GBR-12909 (Sigma-Aldrich).
Uptake of [methyl-3H]-thymidine
[Methyl-3H]-thymidine, a marker of DNA synthesis was used to label proliferating cells (Traver et al. 2006). Mesencephalic cultures exposed for 48 h in vitro to [3H]-thymidine (40 Ci/mmol; 0.5 μCi/well) in the presence of the test compounds were then allowed to recover until day in vitro (DIV) 5 in the same conditions of treatment but in the absence of the tritiated deoxynucleoside. The detection of TH and GFAP positive cells was performed as described before. Thymidine positive nuclei were visualized using the Hypercoat LM-1 emulsion (GE Healthcare, Orsay, France) after an incubation of 4 days at 4°C.
Quantification of reactive oxygen species and cytosolic-free calcium levels
Cytoplasmic-free calcium and ROS levels were measured using calcium-green acetomethylester, (Guerreiro et al. 2008) and DHR-123 (Traver et al. 2005) as fluorescent probes respectively. Calcium measurements were performed in the early phase of the degenerative process (4 DIV) whereas ROS were quantified at a later stage when oxidative stress is at its peak (5–6 DIV).
Measurement of intracellular uric acid
Intracellular UA was assessed with the Kit PAP150 (BioMérieux, Lyon, France) using the protocol described by Duarte et al. (2005) and based on the manufacturer’s instructions. This procedure consisted in measuring H2O2 formed through the enzymatic conversion of UA into allantoin by uricase. Briefly, cultured cells were washed twice with PBS and scrapped off 16 mm cultures wells using 70 μL of distilled water. Cell material from two culture wells was pooled and 100 μL of the total cell suspension was mixed with 900 μL of 50 mM Tris buffer, pH 8.0, containing 80 IU/L uricase, 200 IU/L peroxidase, 1000 IU/L ascorbate oxidase, 250 μM 4-aminoantipyrine, 30 μM potassium ferrocyanate and 2 mM 3,5-dichloro-2-hydroxybenzene sulfonic acid. The absorbance was measured at 520 nm using a Beckman Coulter DU 640B spectrophotometer (Fullerton, CA, USA).
Western immunoblotting of extracellular signal-regulated kinases 1/2
After acute exposure (15 min) of the cultures to the test treatments, the cells were recovered in a lysis buffer, pH 8, containing 10 mM Tris/HCl, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40 (Sigma-Aldrich), 0.8% sodium deoxycholate, 250 IU/mL benzonase and 1% of the complete miniprotease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Samples were electrophoresed through a 4–12% acrylamide gel (NuPAGE Novex Tris–Acetate Mini Gels; Invitrogen) and blotted onto a nitrocellulose membrane. The membranes were incubated with a phospho-extracellular signal-regulated kinases 1/2 (ERK1/2) antibody (1 : 300; New England Biolabs, Ipswich, MA, USA) and developed with the enhanced chemiluminescence detection kit (Pierce, Rockford, IL, USA). Membranes were exposed 5 min to the Re-blot Plus Mild stripping solution (Millipore, Temicula, CA, USA), washed three times with 0.1% Tween in PBS, incubated with an anti-ERK1/2 antibody (1 : 1000; New England Biolabs) and then developed as described above.
All data points presented are the mean ± SEM of five to eight independent experiments. The statistical significance of differences between groups was analysed with the SigmaStat 3.5 Software from Systat (San Jose, CA, USA) using one-way anova followed by Dunnett’s (for multiple comparisons against a single reference group) or Bonferroni (for all pairwise comparisons) post hoc tests. The Student’s t-test was applied when comparing a single treatment group to its corresponding control.
Uric acid affords substantial protection to degenerating dopaminergic neurons
Counts of mesencephalic dopaminergic neurons identified by TH immunofluorescence confirmed that these neurons degenerate progressively in our culture system; 40% of the initial population was lost after 4–5 DIV and the degenerative process was generally complete by 8 DIV (Fig. 1a). When UA was applied to the cultures, the loss of TH+ cells was reduced in a concentration-dependent manner (Fig. 1b). The TH+ neurons rescued by UA were morphologically normal (Fig. 1c) and functional as they accumulated [3H]-DA efficiently via active transport (Fig. 1b). The EC50, estimated graphically for both parameters, was ∼50 μM. Concentrations < 30 μM were ineffective. Protection against DA cell loss was not complete as in the presence of an optimal concentration of 200 μM UA only ∼50% of the TH+ neurons initially present in the cultures survived at 8 DIV (Fig. 1a). Interestingly, when the treatment was delayed after plating, UA still saved a significant proportion of dopaminergic neurons, but only if added no later than day 4 (Fig. 1d). Conversely, when the cultures were treated only transiently with UA between days 0 and 4 and then left in standard culture medium, no effect of UA was observed at DIV 8.
Uric acid operates by reducing intracellular oxidative stress
We explored the possibility that UA could operate as an antioxidant using the ROS-sensitive probe DHR-123 as described (Traver et al. 2005). At a time when the death process is extensive, i.e. after 5–6 DIV, ROS levels increased dramatically in untreated cultures. In the presence of 200 μM UA, a concentration which is optimally effective for dopaminergic cell survival, ROS production was decreased by more than 50% whereas a concentration of 10 μM which has no protective effect failed to reduce the intensity of fluorescent signal (Fig. 2a and b). An illustration that depicts the antioxidant effect of UA is given in Fig. 2c.
Uric acid prevents neurodegenerative changes caused by a Fenton-type reaction
Neuroprotection by UA was reproduced by desferrioxamine (10 μM) which predominantly chelates ferric iron and by catalase (500 IU/mL) (Fig. 3a) which indicates that hydroxyl radicals produced via a Fenton-type reaction kill dopaminergic neurons in this culture system (Halliwell and Gutteridge 1984). Consistent with this view, neuroprotection was also achieved by Trolox (10 μM), a cell-permeable water-soluble derivative of vitamin E known to prevent hydroxyl radical-mediated lipid peroxidation. Desferrioxamine, catalase and Trolox were equally potent to UA in reducing ROS levels in degenerating neurons (Fig. 3b). Note that the survival effect of UA (200 μM) was not improved by any of these antioxidants (unshown results). Curiously, ascorbic acid (100 μM) which has been reported to be as potent as UA to prevent oxidative stress in some in vitro assays (Whiteman and Halliwell 1996) failed to reduce ROS production and to afford protection in our culture system (Fig. 3).
Uric acid does not neutralize directly intracellular oxidative stress
The use of a biochemical assay revealed that intracellular levels of UA were not significantly increased in cultures exposed chronically to the purine derivative (Fig. 4a). This suggests that UA did not require to be accumulated into dopaminergic neurons to exert its neuroprotective effect. A visual confirmation of this finding was obtained with an antibody raised against UA conjugated to a protein carrier (Fig. 4b). Indeed, the fluorescent signal was identical in control and UA-treated cultures. Note that virtually no fluorescent signal was detected when the first antibody was omitted.
Neuroprotection by structural analogues of uric acid
We also tested the neuroprotective effects of some of the close structural analogues of UA, xanthine, hypoxanthine and guanine, which are also its immediate precursors in the catabolic pathway of purines (Fig. 5). None of these compounds was able to reproduce the protective action of UA. Not surprisingly, they were also ineffective in reducing ROS production. At variance, the synthetic 1,7-dimethyl derivative of UA reported previously to be protective in models of brain focal ischemia in mice (Haberman et al. 2007) retained the protective effects of its parent compound. The antioxidant potential of this compound was also comparable to that of UA in our model system.
The neuroprotective effect of UA is potentiated by high K+-induced depolarization
The rescuing effects of UA being only partial in our model system, we wished to determine whether dopaminergic cell survival could be improved by another treatment applied concurrently. Because dopaminergic neurons survive better when they are slightly depolarized (Michel et al. 2007), we submitted UA-treated cultures to high (30 mM) K+-induced depolarization in the presence of 1 μM MK-801 to avoid secondary excitotoxic stress (Salthun-Lassalle et al. 2004). In this particular set of experiments, MK-801 was therefore used with all other culture conditions. Our results show that the effect of UA at 8 DIV was strongly enhanced by the depolarizing treatment combining high K+ and MK-801 (Fig. 6a). Importantly, the survival rate of TH+ neurons was not modified by the presence of MK-801 either in control cultures (Fig. 6a) or in cultures exposed to UA alone. In a representative experiment, the number of TH+ neurons/well in UA-treated cultures was estimated to 1670 ± 122 and 1759 ± 144 in the absence or presence of MK-801 respectively. There is a possibility that the increase in TH+ cell numbers could result, at least in part, from a mitogenic effect of elevated K+ on TH+ neuroblasts or their precursor cells maintained in the presence of UA. This possibility was however, excluded as we failed to demonstrate the presence of dividing TH+ cells in cultures challenged with a marker of DNA synthesis [methyl-3H]-thymidine (Fig. 6b). In fact, the tritiated label was present only in cells with a glial phenotype detectable by vimentin or GFAP immunostaining (Fig 6b). In agreement with our previous observation (Troadec et al. 2002), the number of dividing cells expressing at least one of these two markers represented < 5% of all mesencephalic cells. The comparison of the number of surviving TH+ neurons at 0 and at 8 DIV suggested that the combined application of UA and 30 mM K+ rescued virtually all dopaminergic neurons plated in these cultures. Interestingly, the depolarizing signal alone afforded no protection, at all (Fig. 6a).
It is interesting to note that the protective effect of UA was not restricted to dopaminergic neurons as the purine derivative also promoted the survival of other populations of mesencephalic neurons characterized by their immunoreactivity for MAP-2 (Fig. 6c). The effect of the depolarizing signal in the presence of UA appeared, however, restricted to dopaminergic neurons (Fig. 6c).
Mechanisms underlying high K+-mediated survival promotion of dopaminergic neurons in the presence of UA
The protective effect of the depolarizing signal in the presence of UA was not accompanied by a further reduction of ROS levels (Fig. 6a and d) suggesting that it was attributable to another mechanism. In fact, the survival promoting effect of high K+ was associated to a moderate increase in intracellular calcium levels (Fig. 6e). Preventing this rise with the specific blocker of L-type voltage-gated calcium channels nifedipine (20 μM) abolished the survival promoting effect of the elevation of K+ but had no effect on that provided by UA indicating that the calcium elevation was accountable for the effect of the depolarizing signal. Note that the same rise was also detected in cultures where the depolarizing signal was applied in the absence of UA. Yet, this elevation was not sufficient per se to protect dopaminergic neurons.
High K+-induced depolarization also caused an increase in ERK1/2 activation (Fig. 6f). Blockade of this increase with PD98059 (20 μM) or nifedipine abolished the survival effect of the depolarizing signal without affecting that of UA (Fig.6a). Unlike nifedipine, PD98059 had no influence on the Ca2+ elevation caused by K+-induced depolarization (Fig. 6e). This suggests that the rise in calcium and the activation of ERK1/2 occurred sequentially to promote dopaminergic cell survival. A schematic representation of the mechanisms by which UA may protect midbrain dopaminergic neurons is given in Fig. 7.
We showed here that UA the final product of purine nucleoside metabolism in humans and higher primates exerts partial but long-term protective effects in a culture model of spontaneous dopaminergic cell death. UA prevented the activation of a death pathway involving ROS by a mechanism that did not require its accumulation into neurons. Structure–activity relationship revealed that the 8-one moiety in its chemical structure was crucial for neuroprotection. Interestingly, UA-mediated neuroprotection was improved substantially by high K+-induced depolarization through a mechanism involving Ca2+ elevation and subsequently ERK1/2 activation.
Uric acid affords robust but partial protection to dopamine neurons
Mesencephalic dopaminergic neurons die spontaneously when maintained in a serum-free environment that contains trace amounts of iron (Troadec et al. 2002). This study showed that a significant proportion of these neurons could be rescued in a functional state when UA was applied chronically to the cultures. Of importance, UA was also protective if added when the loss of dopaminergic neurons was already ongoing. To be effective, however, the treatment with UA had to be initiated before 4 DIV which indicates that beyond a certain time-point, dopaminergic neurons become engaged in a cell death pathway that cannot be blocked any longer by UA. Du et al. (2007) reported previously that astrocytes intervene crucially in the neuroprotective effect of UA in a model system of spinal cord neurons dying by excitotoxic stress. Obviously, the effect of UA on dopaminergic neurons did not depend on astrocytes as the density of glial cells was very low in our culture paradigm. Accordingly, the protective effect of UA persisted when glial cells were totally removed from the cultures by a treatment with the antimitotic cytarabine (data not shown). We may therefore surmise that the protective mechanism of UA varied with the nature of the lethal insults applied to the cultures and with the phenotypes of neurons as well.
Uric acid protects dopamine neurons by reducing ROS production
Several observations support the idea that UA protected DA neurons from death by reducing oxidative stress; (i) Dopaminergic cell demise in our preparation resulted from the presence of trace amounts of iron, a transition metal that catalyses the formation of hydroxyl radicals via a Fenton-type reaction (Halliwell and Gutteridge 1984). (ii) Neuroprotection by UA was mimicked by conventional antioxidants including the chain breaking antioxidant Trolox and the hydroxyl radical detoxifying enzyme catalase. Accordingly, ROS levels were reduced substantially in UA-treated mesencephalic cultures. These observations are also consistent with studies showing that UA can operate as a potent antioxidant either in acellular preparations (Ames et al. 1981; Davies et al. 1986) or in a number of paradigms where neuronal cell death is caused by excitotoxic stress (Yu et al. 1998; Du et al. 2007) or metabolic impairment (Duan et al. 2002). Importantly, the antioxidant effect provided by UA was not sufficient in itself to rescue all cultured dopaminergic neurons, suggesting that some of these neurons die either by a mechanism unrelated to oxidative stress or alternatively that the antioxidant effect is not sufficient in itself to provide protection to this subpopulation of dopaminergic neurons. It is also worth noting that UA failed to prevent dopaminergic cell death induced by the nitric oxide donor diethylamine NONOate (unshown results) which confirms that the protective action of the purine derivative did not extend to reactive nitrogen species or their derivatives (Rodríguez-Martín et al. 2002).
Mechanism underlying the antioxidant effect of uric acid
Given that UA has the potential to form stable 1 : 1 and 2 : 1 coordination complexes with either Fe3+ or Fe2+ cations (Davies et al. 1986), one may speculate that UA reproduced the effect of desferrioxamine and thus operated by sequestration of iron. As ferric iron is present at a concentration of 1.5 μM in the culture medium, a protective effect by sequestration would be expected to occur at concentrations on the order of 3 μM. This was not the case as UA promoted dopaminergic cell survival when its concentrations reached at least 30–50 μM. This means that the chelating properties of UA were not responsible for its protective action in our model system. Alternatively, UA was also reported to operate as an inhibitor of lipid peroxidation (Davies et al. 1986; Muraoka and Miura 2003), a mechanism which is more likely to explain the effects of UA in the present paradigm as the concentrations of UA protective in mesencephalic cultures (i.e. 30–500 μM) were very similar to those reported effective against lipid peroxidation in liposomal or microsomal preparations (Davies et al. 1986; Muraoka and Miura 2003). Still consistent with this hypothesis, the inhibitor of lipid peroxidation Trolox mimicked the protective effect of UA for DA neurons in our model system.
The fact that the treatment with UA caused a decrease in intracellular ROS production may signify that UA operated after being accumulated into neurons. Specific UA transporters exist but essentially in renal proximal tubule cells (Endou and Anzai 2008) and passive transport is unlikely owing to the hydrophilic character of UA (Muraoka and Miura 2003). Accordingly, UA levels were not increased in neuronal cells chronically exposed to this compound. This means that UA exerted probably its antioxidant effect extracellularly. Consistent with this hypothesis, the initial source of ROS was also extracellular in our model system as catalase which does not diffuse through plasma membranes (Buettner 1993), provided nevertheless robust protection to dopaminergic neurons. Therefore, we may assume that UA operated by preventing the propagation of oxidative stress from the extracellular to the intracellular milieu, possibly by preserving the integrity of the plasma membrane. Supporting this view, UA has been reported to operate at the lipid-aqueous boundary of membranes in microsomal preparations (Muraoka and Miura 2003). Interestingly, the lowest protective concentrations of UA in mesencephalic cultures, i.e. 30–50 μM, were physiologically relevant as they corresponded to levels of the purine metabolite detected extracellularly in the striatum (Osborne and Hashimoto 2006).
What structural features confer antioxidant effects to uric acid?
To determine what structural features conferred antioxidant properties to UA, we tested the neuroprotective ability of some of its close structural analogues xanthine, hypoxanthine and guanine which are also its precursors in the catabolic pathway of purine nucleosides (Rodwell 1993). Xanthine the immediate precursor of UA, which is produced via the degradation pathway of adenosine or that of guanosine, failed to afford neuroprotection. Likewise, hypoxanthine and guanine the two purines bases which give rise to xanthine via catabolism of adenosine and guanosine, respectively, were ineffective too. In line with this observation, xanthine, hypoxanthine and guanine failed to reduce intracellular oxidative stress. Interestingly, Muraoka and Miura (2003) reported very weak antioxidant properties of xanthine and hypoxanthine in microsomal preparations in comparison to UA. Given that UA differs from xanthine only by a 8-one moiety (Rodwell 1993), we may surmise that this chemical group greatly contributed to the antioxidant activity of UA. In line of this observation, the 1,7-dimethyl derivative analogue of UA which retains the 8-one moiety in its structure was as potent as UA itself. This finding also suggested that N-methylation on nitrogens at positions 1 and 7 did not weaken the antioxidant potential of the purine structure.
K+-induced depolarization strongly potentiates the effect of UA by a mechanism that does not involve a reduction in ROS production
We demonstrated in this study that UA provided robust protection to dopaminergic neurons but only to a fraction of them. Yet, neuroprotection by UA was strongly enhanced when the cultures were exposed to a low-level depolarizing stimulus produced by elevation of extracellular concentrations of K+. The increase in TH+ neurons was not because of a possible mitogenic effect on precursor cells (Daadi and Weiss 1999) as tritiated thymidine a marker of DNA synthesis was absent from the nucleus of these neurons. The induction of the TH enzyme in neurons that did not express originally detectable levels of the enzyme as reported in other model systems (Traver et al. 2006) was also excluded for the reason that the number TH+ cells exposed to UA in the presence of elevated concentrations of K+ remained identical or slightly below the number of TH+ neuroblasts detectable immediately after plating. Therefore, the effect of the depolarizing signal reflected most likely a true neuroprotective effect. It is worth noting that the elevation of K+ did not further reduce ROS production in UA-treated cultures which indicates that the depolarizing signal did not operate itself via an antioxidant mechanism. This may explain why elevated K+ failed to provide protection to dopaminergic neurons in the absence of UA.
The rescuing effect of K+-induced depolarization is mediated by Ca2+-dependent ERK1/2 activation
The potentiation of the effect of UA by elevated K+ resulted most likely from a small increase in Ca2+cyt as preventing this increase with the L-type voltage-gated calcium channel blocker nifedipine prevented the rescuing effect of the depolarizing signal. Note that these observations are somehow paradoxical if we refer to data showing that UA and its analogues were reported previously to limit the effects of ischemic insults and excitotoxic stress (Yu et al. 1998; Du et al. 2007; Haberman et al. 2007), i.e. pathological conditions causing calcium overload. These results are, however, in line with the concept that calcium has to be maintained slightly above control levels in DA neurons to prevent their demise (Salthun-Lassalle et al. 2004; Guerreiro et al. 2008).
Next, we explored the possibility that the effect of elevated Ca2+cyt in UA-treated cultures could result from the activation of the ERK1/2 kinases, which are involved in neuronal survival in a number of experimental paradigms (Troadec et al. 2002) although this remains controversial (Zhuang and Schnellmann 2006). Blocking the activation of mitogen-activated protein kinase kinase the direct upstream kinase of ERK1/2 with PD98059 had no effect on the elevation of Ca2+cyt caused by high K+-induced depolarization but it prevented the rescuing effect of the depolarizing signal in UA-treated cultures. Nifedipine, however, prevented both the influx of calcium and the activation of ERK1/2 elicited by the depolarizing signal which indicates that Ca2+cyt elevation and ERK1/2 activation were involved sequentially in the survival of the population of dopaminergic neurons for which UA alone was ineffective. Interestingly, neither nifedipine nor PD98059 inhibited the effects of UA suggesting that ERK1/2 activation by calcium was solely responsible for the effect of the depolarizing signal. The nature of the downstream targets of ERK1/2, remains, however, to be established. Ultimately, these results demonstrate that UA acted in concert with a ERK1/2-dependent mechanism elicited by high K+-induced depolarization to rescue dopaminergic neurons. Yet, it appears that the two mechanisms were not mutually substitutable.
In summary, we demonstrate here that UA can exert potent survival promoting effects for dopaminergic neurons through a direct antioxidant mechanism that can be possibly amplified (but not mimicked) by low-level depolarization. On this basis, we may speculate that reduced levels of UA in the substantia nigra of PD patients (Church and Ward 1993; Fitzmaurice et al. 2003) may sensitize dopaminergic neurons to oxidative stress-mediated insults whereas high levels of the purine metabolite may exert protective effects against the disease’s process.
AP performed this work in partial fulfilment of a Master degree in Biology of Aging at University Pierre et Marie Curie. This work was supported by INSERM and UPMC. PPM was supported by Laboratoire Pierre Fabre.
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