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NMDA receptor-mediated excitotoxicity is thought to play a pivotal role in the pathogenesis of Huntington's disease (HD). The neurotrophin brain-derived neurotrophic factor (BDNF), which is also highly involved in HD and whose effects are modulated by adenosine A2ARs, influences the activity and expression of striatal NMDA receptors. In electrophysiology experiments, we investigated the role of BDNF toward NMDA-induced effects in HD models, and the possible involvement of A2ARs. In corticostriatal slices from wild-type mice and age-matched symptomatic R6/2 mice (a model of HD), NMDA application (75 μM) induced a transient or a permanent (i.e., toxic) reduction of field potential amplitude, respectively. BDNF (10 ng/mL) potentiated NMDA effects in wild-type, while it protected from NMDA toxicity in R6/2 mice. Both effects of BDNF were prevented by A2AR blockade. The protective effect of BDNF against NMDA-induced toxicity was reproduced in a cellular model of HD. These findings may have very important implications for the neuroprotective potential of BDNF and A2AR ligands in HD.
Huntington's disease (HD) is an inherited neurodegenerative disorder caused by a mutation in the exon 1 of the huntingtin (htt) gene (The Huntington's Disease Collaborative Research Group's 1993), namely an abnormal expansion of a CAG codon, resulting in devastating cognitive disturbance, motor impairment, and premature death of affected individuals (Vonsattel and DiFiglia 1998).
The expanded CAG trinucleotide encodes a long polyglutamine (poliQ) tract, which forms aggregates in nucleus and cytoplasm of neurons both in patients and in experimental models of the disease (Reddy et al. 1999). As the neurodegeneration mainly involves the striatum and the cerebral cortex (Vonsattel et al. 1985), the brain areas of the paradigmatic corticostriatal glutamatergic pathway, N-methyl-D-aspartic acid receptor (NMDAR)-mediated excitotoxicity is thought to play a pivotal role in the selective neuronal death that occurs in GABA-ergic medium-sized spiny neurons (MSNs) of the striatum. In agreement, an increased sensitivity to NMDA-induced toxicity has been consistently reported in the striatum of HD mice (Levine et al. 1999; Cepeda et al. 2001; Zeron et al. 2002; Martire et al. 2007). Functional NMDARs are tetrameric structures composed of two NR1 and at least two NR2 (A-D) subunits (Dingledine et al. 1999). The NR2A and NR2B subunits of NMDAR mediate cell survival and cell death, respectively (Liu et al. 2007); indeed, the over-expression of NR2B subunits potentiates striatal neurodegeneration in HD mice (Heng et al. 2009). In the striatum of R6/2 mice, a transgenic model of HD, we found a reduction in the NR2A/NR2B ratio (an index of vulnerability to excitotoxic cell death, Ali and Levine 2006), which correlated with an increased susceptibility to NMDA-induced toxicity (Martire et al. 2010), and which was influenced by adenosine A2A receptor (A2AR) stimulation (Ferrante et al. 2010) and blockade (Martire et al. 2010).
Brain-derived neurotrophic factor (BDNF), a glycoprotein belonging to neurotrophins, is very important for the survival of striatal neurons and for the activity of corticostriatal synapses (Cattaneo et al. 2005), where it controls glutamate release and allows striatal neurons to survive to excitotoxin-induced neurodegeneration (Bemelmans et al. 1999). An impairment in the synthesis and striatal transport of BDNF is considered a major determinant in the pathogenesis of HD (Zuccato et al. 2001; Zuccato and Cattaneo 2007). Interestingly, BDNF has been reported to influence NMDAR signaling in models of HD: a huge remodeling of post-synaptic density and a specific reduction of synaptic alpha CaMKII have been observed in double transgenic R6/1:BDNF(±) mice (Torres-Peraza et al. 2008). Moreover, BDNF functions and NMDA-mediated toxicity in the striatum are both strongly modulated by A2ARs (Martire et al. 2007; Potenza et al. 2007).
The aim of this study was to further investigate the role of BDNF toward NMDA-induced effects in HD models, and the possible involvement of A2ARs.
Here we report that 1) BDNF oppositely modulates NMDA-mediated toxicity in the striatum of transgenic HD (R6/2) versus wild-type (WT) mice, respectively, showing protective and worsening effects on synaptic transmission in the two genotypes; 2) BDNF effects are always counteracted by TrkB or A2AR blockade, confirming the permissive role of A2AR in BDNF signaling; and 3) a similar protective effect against excitotoxicity is reproduced in a cellular model of HD (ST14A/Q120 cell line).
These findings may have very important implications for the neuroprotective potential of both BDNF and A2AR ligands in HD.
- Top of page
- Materials and methods
- Supporting Information
BDNF, which exerts an important role on the preservation of medium-sized spiny neurons (Gavalda et al. 2004) and is highly involved in the pathogenesis of HD (Zuccato et al. 2001; Gauthier et al. 2004; del Toro et al. 2006), influences phosphorylation, activity, expression, and trafficking of NMDA receptors in different brain areas, including the striatum of HD mice (Caldeira et al. 2007; Crozier et al. 2008; Torres-Peraza et al. 2008). Moreover, the turnover of BDNF in the striatum of symptomatic R6/2 mice is reduced, as demonstrated by the decrease in pro-BDNF levels associated with no changes in the mature form of BDNF (Traficante et al. 2007). Furthermore, the activity of BDNF is strictly linked to the functionality of A2ARs, which exert both a facilitatory (Diógenes et al. 2004; Pousinha et al. 2006) and a permissive (Tebano et al. 2008) role on the neurotrophin effects. It is reported in literature that A2AR density is significantly decreased in HD since the early stage of the disease (Glass et al. 2000).
In agreement with previous reports showing a decrease in A2AR mRNA and A2AR density in the striatum of symptomatic R6/2 mice (Cha et al. 1999; Tarditi et al. 2006), in this study, we further demonstrate the down-regulation of the A2AR protein (Figure S3). In addition, an aberrant A2A receptor signaling has been found both in an in vitro model of the disease and in peripheral circulating cells from HD patients (Varani et al. 2001, 2003).
Thus, the role of BDNF toward NMDA-induced effects in HD mice, and the possible involvement of A2ARs, was worth considering.
In WT mice, BDNF clearly potentiated NMDA effects, worsening the synaptic recovery. This finding is in line with the view that BDNF generally facilitates NMDA-dependent effects and that physiological BDNF release may be necessary to allow a normal NMDA receptor transmission and plasticity (Pattwell et al. 2012). On the contrary, BNDF displayed a frankly protective effect in R6/2 mice, as revealed by the significant reduction of NMDA-mediated toxicity.
The different effect elicited by BDNF in the two genotypes was not related to changes in AMPAR-dependent basal synaptic transmission, as suggested by the similar I/O plot found in WT and R6/2 mice. Furthermore, the different outcome could not be ascribed to a different synaptic effect of BDNF on its own, as the neurotrophin failed to affect the FP amplitude in corticostriatal slices from both WT and R6/2 mice. This lack of efficacy was further confirmed in whole-cell patch-clamp recordings, which showed that BDNF did not influence excitatory currents in the striatum and has only a minimal effect on inhibitory current.
Alternatively, the attainment of a neuroprotective effect in the HD striatum might depend on the dysregulation of TrkB receptor isoforms that occurs under pathological conditions; in particular, excitotoxicity has been reported to down-regulate TrkB.FL protein levels and to inhibit their signaling activity in parallel with the up-regulation of TrkB.T isoforms, a shift that may favor the neuroprotective effects of BDNF (Gomes et al. 2012).
As already published by our group (Martire et al. 2010), the full-length form of TrkB (TrkB.FL) is not changed in the striatum of R6/2 mice with respect to WT littermates, and is also comparable in ST14A Q15 and Q120 cells (Figure S4). Even if it cannot be excluded that the truncated form of TrkB (TrkB.T) could influence in some way the signaling of TrkB.FL, in our experiments, the effects exerted by BDNF in both R6/2 and WT mice, as in Q120 cells, were blocked by K252a, which selectively inhibits the tyrosine kinase activity present only in TrkB.FL (Tapley et al. 1992).
However, further investigations about the role of TrkB.T and p75, the lower affinity BDNF receptor, are worth performing in future studies.
The main defect of K252a is that it is not selective for TrkB receptors, but is a membrane-permeable inhibitor of all Trk receptors (high-affinity neurotrophin receptors) tyrosine kinases; undesirable effects on other tyrosine kinases, at least at the concentration used in our experiments (200 nM), can be ruled out and thus K252a is still an effective blocking agent against the biological actions of BDNF and other neurotrophins (Knüsel and Hefti 1992; Tapley et al. 1992).
Moreover, in almost all our experiments, K252a has been used to block the effects induced by exogenous BDNF applied to slices or cells. Those effects were always fully blocked by K252a and then they can be reasonably considered as mediated by TrkB signaling. Unfortunately, a more selective chemical agent against TrkB receptor is not yet available.
In ST14A/Q120 cells, an in vitro model of HD, BDNF had a significant protective effect on NMDA toxicity, while it was ineffective on control (Q15) cells. Such an effect was blocked by both K252a and ZM 241385, showing the involvement of both TrkB and A2ARs. The levels of this latter receptor were found reduced in Q120 cells (Figure S4 and Varani et al. 2001).
Despite a comparable level of NR1 subunit in Q15 and Q120 cells (Figure S4), the profound changes that NMDA receptors undergo in models of HD might contribute to the shift in the effects of BDNF. For instance, we recently reported a reduction in the NR2A/NR2B ratio in the striatum of R6/2 mice (Martire et al. 2010), and it is well accepted that changes in subunit composition result in NMDA receptors with very different functional characteristics (Cull-Candy et al. 2001). Interestingly, in the present electrophysiological experiments, the opposite influence of BDNF toward NMDA-induced synaptic effects strongly resembles the similar pro-toxic and neuroprotective actions induced by the A2AR agonist CGS21680, respectively, in WT and R6/2 genotypes (Martire et al. 2007). This leads to suppose that A2ARs are involved in the dual modulatory role of the neurotrophin. Indeed, consistently with the well-known role of A2ARs in regulating BDNF levels and effects (Diógenes et al. 2004; Minghetti et al. 2007; Tebano et al. 2008), the opposite effect of exogenous BDNF on striatal NMDA toxicity was blocked by the A2AR antagonist ZM241385.
Moreover, considering that the stimulation of A2ARs can lead to TrkB receptor transactivation (Lee and Chao 2001), we wondered if the previously discovered genotype-dependent shift from the pro-toxic to the protective effects of CGS21680 (Martire et al. 2007) could be mediated by Trk receptors. This was apparently not the case, given that the Trk tyrosine kinase inhibitor K252a failed to block the beneficial effect of CGS21680 on NMDA toxicity in R6/2 mice. Thus, while BNDF effects require the activation of A2ARs, the effects of A2ARs on NMDA do not depend on Trk receptors functioning.
As the protective effects of BDNF were observed in conditions in which NMDA exerted toxic effects, we wanted to verify whether BDNF could become protective also in WT striata in presence of a toxic concentration of NMDA. This was clearly not the case, as in control striata, BDNF did not exert any protection toward a degree of NMDA toxicity comparable with that achieved in R6/2 mice. At the same time, however, BDNF lost its potentiating effects in a condition of stronger NMDAR stimulation. This seems in line with the above-reported observations that BDNF facilitates a “normal” NMDA receptor transmission.
In conclusion, our data show that BDNF exerts neuroprotective effects toward NMDA-dependent toxicity in experimental models of HD, that such effects of BDNF seem specifically related to the pathological genotype, and that they require endogenous A2AR activation.
Considering the pivotal role of NMDA receptors, BDNF, and A2ARs in the pathogenesis of HD, these data have obvious (potential) implications for therapeutics.