Pharmacological blockade of A2A receptors prevents MPTP-induced dopaminergic neuron degeneration and glial activation
Systemic administration of the A2A receptor antagonist SCH58261 prevented the degeneration of nigrostriatal TH-positive neurons induced by repeated MPTP exposure in mice. Changes in number of TH-positive neurons correlated with changes in Nissl-stained (cresyl violet-positive) cells, indicating that MPTP treatment resulted in actual loss of dopaminergic neurons, which were rescued by SCH58261.
A neuroprotective effect of A2A receptor antagonists was previously observed upon acute administration of high MPTP doses in mice (Chen et al. 2001; Ikeda et al. 2002; Pierri et al. 2005; Yu et al. 2008). Here, we report that neuroprotection with A2A antagonism can also be achieved upon multiple low-doses MPTP exposure. A number of studies have provided evidence that a repeated daily MPTP administration protocol similar to the one used here, presents histopathological features that more closely reproduce the human PD neuropathology, including apoptotic death of dopaminergic neurons (Jackson-Lewis et al. 1995; Tatton and Kish 1997). Therefore, this study further substantiates the neuroprotective potential of A2A antagonism in PD. To this regard, it is noteworthy that A2AR antagonists were shown to inhibit apoptotic neuronal death in hippocampal neurons (Silva et al. 2007).
Based on their differential location in the Str or other brain regions, A2A receptors may hold different levels of expression and intracellular signaling, reflecting A2A receptor multiple functions (Kull et al. 2000; Pedata et al. 2003; Rosin et al. 2003; Rebola et al. 2005; Shen et al. 2008). According to such varied roles of the A2A receptor, diverse effects have been attributed to A2A antagonists, ranging from symptomatic antiparkinsonian actions to neuroprotection in various neurodegenerative conditions (Alfinito et al. 2003; Blum et al. 2003; Chen et al. 1999; Melani et al. 2003; Popoli et al. 2002). Motor effects of A2A receptor antagonists are likely mediated by A2A receptors located on striatal neurons projecting to globus pallidus, whereas several mechanisms have been hypothesized for their neuroprotective effects, involving either neuronal or glial A2A receptors, though no single mechanism has yet been proven to prevail (Popoli et al. 1995; Chen et al. 2001; Carta et al. 2003; Melani et al. 2003; Pedata et al. 2003; Huang et al. 2006; Schwarzschild et al. 2006; Yu et al. 2008). Noticeably, in this study neuroprotection by SCH58261 was achieved at doses similar to those effective in other neurodegenerative conditions, but several times lower than doses displaying a symptomatic efficacy in PD (Chen et al. 2001; Dall’Igna et al. 2003; Melani et al. 2003; Pinna et al. 2007), supporting the concept that different mechanisms might account for A2A-mediated neuroprotection or symptomatic effects.
SCH58261 fully prevented astroglia and microglia activation in the SNc, while only partially inhibiting astroglia and microglia reactivity in the Str, in line with a partial protection of dopaminergic terminals. Noteworthy, A2A receptor antagonism prevented both astroglia and microglia activation in the SNc and Str at all time points evaluated, in accordance with blockade of neurodegeneration.
Although the mechanism through which A2A receptor blockade produces neuroprotective effects in PD models is unclear, the modulation of neuroinflammation has been proposed as a likely target for neuroprotection (Hunot and Hirsch 2003). Several findings have suggested that neuroinflammation may play an active role in the pathogenesis of neurodegeneration in PD, as focal inflammation has been described in the SNc of PD patients and MPTP-treated primates (McGeer et al. 1988; Barcia et al. 2004). Intriguingly, blockade of microglia reactivity in mice rescued dopamine neurons from acute MPTP toxicity (Wu et al. 2002). Moreover, in mice acutely treated with MPTP, dopamine neuron neuroprotection by pre-treatment with an A2A antagonist was associated with an attenuation of astroglia and microglia activation in SNc and Str (Ikeda et al. 2002; Pierri et al. 2005), consistent with a causal relation between the two events.
Selective deletion of neuron-specific forebrain A2A receptors prevents MPTP-induced dopamine neuron degeneration and glial activation
Previous studies evaluating A2A receptor-mediated neuroprotection have hypothesized several mechanisms that might underlie this process. To determine the role of neuronal versus glial A2A receptors in neuroprotection of dopamine neurons, we exploited genetically modified mice with selective depletion of A2A receptors from fb neurons (Bastia et al. 2005; Shen et al. 2008). Importantly, in the fbnA2AKO mice, deletion of A2A receptor is not only spatially restricted (to fbn A2A receptor) but is also temporally limited to postnatal A2A receptors (Bastia et al. 2005; Yu et al. 2008), thus avoiding potential confounds of compensatory responses to A2A receptor gene disruption during development as might occur in constitutive A2A KO mice.
Our results revealed that selective deletion of A2A receptors from fb neurons totally prevented dopaminergic neuron loss in the SNc following multiple MPTP injections, while partially preventing damage to striatal dopaminergic terminals. A2AR deletion provided a greater protection of SNc neurons than the A2AR antagonist, as expected from a permanent when compared with a temporal pharmacological blockade of the receptor, in line with the reported half-life of SCH58261 of 2–3 h.
A2A receptors are located both pre- and post-synaptically in striatal and cortical neurons, and are expressed in microglial as well as astroglial cells (Küst et al. 1999; Cunha 2001; Rosin et al. 2003; Nishizaki 2004;Rebola et al. 2005). Positive modulation of post-synaptic signaling as well as of pre-synaptic release of neurotransmitters as glutamate and acetylcholine by A2A receptors have been described (Popoli et al. 1995; Marchi et al. 2002; Fredholm et al. 2003; Fuxe et al. 2003; Schwarzschild et al. 2006; Schiffmann et al. 2007). In addition, A2A receptors interfere with glia-mediated synthesis and release of neurotoxic factors such as cyclooxygenase-2, prostaglandins, nitric oxide, and glutamate, which have been hypothesized to play central roles in inflammatory processes and neuronal damage (Fiebich et al. 1996; Li et al. 2001; Saura et al. 2005).
This study, by showing that selective deletion of A2A receptors from fb neurons protects dopaminergic neurons from MPTP toxicity, endorses a primary role of neuronal receptors in mediating neuroprotection in this multiple injections MPTP model of PD. As very low levels of A2A receptors are expressed by dopaminergic neurons in the SNc, it is unlikely that a direct action at this level might mediate neuroprotection from MPTP toxicity in the SNc. Rather, an indirect effect at the pre-synaptic level, through an inhibition of A2A-mediated glutamate release, which contribute to neuronal damage, could be envisaged (Aguirre et al. 2005; Battaglia et al. 2004; Monopoli et al., 1998; Popoli et al. 2002). Interestingly, recent studies have reported a tight cross-talk between adenosine and glial cell line-derived neurotrophic factor receptors, resulting in a fine modulation of glutamate and dopamine release (Gomes et al. 2006, 2009). In the Str, A2AR blockade would impair glial cell line-derived neurotrophic factor-stimulated increase of corticostriatal glutamate release, thus providing a beneficial effect on neurodegeneration. In addition, A2A receptor antagonism on striatopallidal or subthalamic neurons might be protective from MPTP toxicity by modulating excessive activation of subthalamic nucleus, thereby reducing excitotoxic glutamate efflux to SNc neurons (Wallace et al. 2007).
A study by Yu et al. (2008) reported that fbnA2AKO mice from the same line as used here, were not protected from striatal dopamine loss in response to acute MPTP exposure (in a single dose or multiple doses over 4 h). These results open to several compelling interpretations. First, the cellular basis of A2A receptor-dependence of MPTP toxicity might vary depending on the duration of toxin exposure, in line with the different type of neurotoxicity produced by acute when compared with subchronic MPTP. Moreover, it should be taken into account the different parameters were used to evaluate nigrostriatal neurons damage. The drop of striatal dopamine levels assessed by Yu et al. may reflect functional injury to dopaminergic terminals, whereas the measure of dopaminergic nigral neuron employed in our study may reflect an underlying neurodegenerative process in this area. All together results support the concept that A2A receptors display complex actions related to the duration of insult, cellular elements and brain areas targeted by neurodegenerative processes.
Lack of a glial reaction in fbnA2AKO mice, when compared with the robust astroglia and microglia activation in MPTP-treated control mice, indicates that deletion of neuronal A2A receptors may indirectly inhibit the inflammatory response. Glutamate is a main contributor to the complex neuron–glia crosstalk engaged by pathological events, which trigger both microglia and astroglia activation. For instance, by an action on NMDA receptors, glutamate release stimulates mitogen-activated protein kinases. Neuronal as well as glial p38 mitogen-activated protein kinase activation has been involved in cell suffering and apoptotic death, being activated and inducing several inflammatory mediators (Kawasaki et al. 1997; Irving et al. 2000; Piao et al. 2003; Gianfriddo et al. 2004). Therefore, A2A antagonists indirectly through a reduction of glutamate release might counteract glial reactivity and neuroinflammation in both SNc and Str (Melani et al. 2003, 2006). Activated glial cells, by the release of several toxic species as cytokines, free radicals, glutamate, is known to contribute to neuronal damage, and has been suggested to sustain a self-amplifying cycle which perpetuates MPTP toxicity (Hunot and Hirsch 2003). Hence, interruption of such a detrimental vicious cycle might indirectly contribute to A2A receptor-dependent neuroprotection. Accordingly results suggest that in our model, operational glial A2A receptors did not contribute to MPTP toxicity when A2A fb neuronal receptors where deleted. All together, our results suggest that A2A antagonists, by blocking neuronal A2A receptors, might act upstream in the cascade of toxic events and lead to an attenuation of dopamine neuron degeneration in PD.