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The uricosuric agent probenecid is co-administered with the dopaminergic neurotoxin MPTP to produce a chronic mouse model of Parkinson's disease. It has been proposed that probenecid serves to elevate concentrations of MPTP in the brain by reducing renal elimination of the toxin. However, this mechanism has never been formally demonstrated to date and is questioned by our previous data showing that intracerebral concentrations of MPP+, the active metabolite of MPTP, are not modified by co-injection of probenecid. In this study, we investigated the potentiating effects of probenecid in vivo and in vitro arguing against the possibility of altered metabolism or impaired renal elimination of MPTP. We find that probenecid (i) is toxic in itself to several neuronal populations apart from dopaminergic neurons, and (ii) that it also potentiates the effects of other mitochondrial complex I inhibitors such as rotenone. On a mechanistic level, we show that probenecid is able to lower intracellular ATP concentrations and that its toxic action on neuronal cells can be reversed by extracellular ATP. Probenecid can potentiate the effect of mitochondrial toxins due to its impact on ATP metabolism and could therefore be useful to model atypical parkinsonian syndromes.
The uricosuric agent probenecid is used in the treatment of gout. It operates by inhibiting the reabsorption of uric acid by the urate transporter 1 (URAT1), a member of the renal organic anion transporter family (Hediger et al. 2005). As an inhibitor of organic anion transporter 1, probenecid also reduces renal elimination and thus enhances plasma concentrations of other pharmacological agents such as penicillin and other antibiotics (Beyer et al. 1951; Masereeuw et al. 2000). Regarding the CNS, probenecid crosses the blood–brain barrier by a low-affinity system, probably by diffusion (Yuwiler 1982).
In the field of Parkinson's disease (PD) research, probenecid is co-administered with MPTP to produce the so-called ‘chronic’ mouse model of the disease. In this experimental paradigm, about 40–45% of the dopaminergic (DA) neurons from the substantia nigra pars compacta are lost by 3 weeks post-treatment compared to 25% in the subchronic model without probenecid (Petroske et al. 2001; Meredith et al. 2008a,b). The loss of dopaminergic neurons remains substantial 6 months after intoxication with only a minimal recovery (Petroske et al. 2001; Meredith et al. 2008a,b) in contrast to the subchronic and acute MPTP models where substantial recovery does occur (Petroske et al. 2001; Meredith et al. 2008a,b). However, the mechanism by which probenecid enhances the effects of MPTP is not fully understood.
After systemic administration, MPTP crosses the blood–brain barrier and reaches the CNS. There it is metabolized into its active metabolite, MPP+. MPP+ is taken up by dopaminergic neurons via the dopamine transporter (DAT) and inhibits complex I of the mitochondrial respiratory chain. This leads to subsequent energy failure with ATP depletion (Schmidt and Ferger 2001). It has been demonstrated that probenecid strongly reduces the clearance of MPTP and its metabolites by the kidney (Lau et al. 1988, 1990). This has led to the speculation that probenecid might operate by enhancing the concentration of MPTP/MPP+ available to brain cells (Lau et al. 1990; Petroske et al. 2001). However, this has never been formally demonstrated to date.
Previously, we failed to detect enhanced MPP+ levels in the brain after a co-administration of MPTP and probenecid (Alvarez-Fischer et al. 2008) and thus questioned the ‘renal’ hypothesis of the probenecid action. Indeed, some studies reported unspecifically altered oxygen tissue consumption after probenecid exposition (Choi and Kim 1992) and an interference with cellular oxidative metabolism (Masereeuw et al. 2000). Moreover, probenecid has been reported to alter extracellular concentrations of lactate (MacMillan 1987; Kuhr et al. 1988; Korf 1996), a crucial aerobic energy substrate that enables neurons to endure activation (Schurr et al. 1999). Finally, there is a broad literature on the inhibiting properties of probenecid on the multidrug-resistance transporter (Darby et al. 2003) and pannexin 1 hemichannels (Silverman et al. 2008, 2009; Ma et al. 2009), two proteins that have been associated with ATP release in the CNS (Darby et al. 2003; Silverman et al. 2008, 2009; Ma et al. 2009).
The aim of this study was, therefore, to explore how probenecid contributes to the neural toxicity of MPTP. To this end, we measured striatal MPP+ levels with and without co-administration of probenecid. Furthermore, we used two culture systems where dopaminergic neuronal death is caused by a chronic exposure to MPP+, the active metabolite of MPTP, or to rotenone, a plant toxin that operates like MPP+ by blocking mitochondrial complex I activity. Our data suggest that probenecid can potentiate the effect of the two mitochondrial toxins directly because of its impact on cellular energy metabolism.
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- Materials and methods
- Supporting Information
In this study, we demonstrate that striatal MPP+ levels are not altered by co-administration of probenecid as was originally proposed (Lau et al. 1988, 1990) although it yielded the predicted effectiveness on nigro-striatal damage. We show that the toxic effects of probenecid are also detectable in vitro which argues against an effect of probenecid on renal MPTP/MPP+ elimination and alterations in the conversion of MPTP to MPP+. We show that probenecid is toxic in itself and that its toxicity, with or without MPP+, is dose-dependent. This is true for primary neuronal cultures containing glia although highly enriched in neurons [they contain 5% glial cells (Michel et al. 1999)] and for SH-SY5Y human neuroblastoma cells, suggesting that toxic effects of probenecid occur independently of glial cells.
We further demonstrate that probenecid does not alter DAT function and conclude that the effects of probenecid are not because of enhanced intracellular levels of MPP+ within dopaminergic neurons. The findings obtained with rotenone also argue against an effect restricted to MPTP or to its metabolite MPP+. Consequently, the toxicity-enhancing effects of probenecid are neither restricted to a specific toxin nor to a specific neuronal cell type.
Our data also show that probenecid lowers intracellular concentrations of ATP that was additive to that of MPTP/MPP+. Indeed, Masereeuw and co-workers (Masereeuw et al. 2000) demonstrated that probenecid impairs mitochondrial oxidative phosphorylation through an uncoupling effect in renal tubular cells (Masereeuw et al. 2000). We also demonstrated in vitro a decrease in the respiration of primary neuronal cultures, both in the basal resting state and under stimulation by the protonophore cccp. This reduction in the respiration rate is expected to induce a parallel decrease of ATP production. However, in our study, it was not accompanied with uncoupling as proposed by Masereeuw and co-workers (Masereeuw et al. 2000). The presence of a complex I defect shown with the dipstick assay fitted with the reduced respiration observed in intact cells respiring on glucose and pyruvate and, thus, on electrons essentially entering the respiratory chain at the complex I level. By treating isolated mitochondria with probenecid we showed that probenecid does not operate like MPP+ or rotenone by direct blockade of mitochondrial complex I. In fact, probenecid may rather operate up-stream of complex I inhibition. However, with our set of experiments we could not detect inhibition of the glycolytic pathway as probenecid neither reduced lactate production nor impaired neuronal glucose uptake.
Supporting the view that two distinct mechanisms were involved in the effects of probenecid and MPP+, we observed that reductions in complex I activity of similar intensities had very different impacts on neuronal survival whether they resulted from probenecid or MPP+ treatments. However, we could not address the presence of other cellular targets for probenecid, including alteration of substrate oxidation downstream of pyruvate, such as pyruvate transport, pyruvate dehydrogenase or the citric acid cycle, nor could we investigate increased ATP lysis, which would add to the decreased cellular ATP levels. Such mechanisms would be compatible with the observation that high concentrations of glucose were sufficient to restore neuronal survival in the presence of toxic amounts of probenecid. Indeed, glucose might simply operate by restoring the glycolytic flux upstream of complex I and the action of probenecid.
In addition, we show that the deleterious effects of probenecid on cell survival are reversed by extracellular ATP. Extracellular ATP can operate as a potent signalling molecule (Apolloni et al. 2009; Tu and Wang 2009) and important physiological functions have been ascribed to this nucleotide in the CNS (Abbracchio et al. 2009). In particular, ATP can operate as a neurotransmitter or a trophic molecule through the activation of its cognate receptors, namely purinergic type 2 receptors (Michel et al. 2007; Tu and Wang 2009). Of interest, extracellular ATP has been previously reported to prevent degeneration in a model system of spontaneous dopaminergic cell death in midbrain cultures (Michel et al. 2007; Tu and Wang 2009). Furthermore, Sato and colleagues (Sato et al. 2006) have shown that Parkin, a protein that is mutated in familial forms of PD, is required to generate currents through purinergic type 2X receptors, a subclass of ionotropic ATP receptors (Sato et al. 2006).
Probenecid is not known to interfere with calcium-dependent exocytosis which is the principal mechanism that controls ATP release in neuronal cells (Abbracchio et al. 2009). Yet, pannexin 1 hemichannels and the multidrug resistance protein that are also susceptible to mediate ATP release (Darby et al. 2003; Silverman et al. 2008, 2009; Ma et al. 2009; Ransford et al. 2009) are inhibited by probenecid (Darby et al. 2003; Dallas et al. 2006; Silverman et al. 2008, 2009; Ma et al. 2009; Ransford et al. 2009). Thus, we speculate that the deleterious mechanism of probenecid resulted not only from a reduced production of intracellular ATP because of impairment of the mitochondrial function but also from impaired neuronal release of ATP by a mechanism that remains to be established.
In conclusion, our results suggest that probenecid does not potentiate MPTP toxicity as originally proposed by increasing MPTP/MPP+ accumulation and retention within the brain but most likely by affecting ATP production and release. As the effects of probenecid are neither toxin-specific nor limited to a specific neuronal phenotype, this compound may be useful in other toxin-based animal models of PD or related disorders, such as rotenone/annonacin to mimic atypical parkinsonian syndromes such as progressive supranuclear palsy or multiple system atrophy in rodents (Höglinger et al. 2003, 2005; Lannuzel et al. 2003; Champy et al. 2004; Escobar-Khondiker et al. 2007).