Evidence for the existence of A2AR-TrkB heteroreceptor complexes in the dorsal hippocampus of the rat brain: Potential implications of A2AR and TrkB interplay upon ageing

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Highlights

  • Several GPCRs can directly interact with receptor tyrosine kinases (RTKs).

  • Adenosine A2A receptors are crucial in modulating BDNF action in the hippocampus.

  • A direct interaction A2AR-Tropomyosin related kinase B receptor (TrkB) is suggested.

  • In situ PLA revealed the existence of A2AR-TrkB complexes in rat hippocampus.

  • This A2AR-TrkB physical interaction may have an impact upon ageing.

Abstract

Adenosine A2A receptors (A2AR) are crucial in facilitating the BDNF action on synaptic transmission in the rat hippocampus primarily upon ageing. Furthermore, it has been suggested that A2AR-Tropomyosin related kinase B receptor (TrkB) crosstalk has a pivotal role in adenosine A2AR-mediated modulation of the BDNF action on hippocampal plasticity. Considering the impact of the above receptors interplay on what concerns BDNF-induced enhancement of synaptic transmission, gaining a better insight into the mechanisms behind this powerful crosstalk becomes of primary interest. Using in situ proximity ligation assay (PLA), the existence of a direct physical interaction between adenosine A2AR and TrkB is demonstrated. The A2AR-TrkB heteroreceptor complexes show a heterogeneous distribution within the rat dorsal hippocampus. High densities of the heteroreceptor complexes were observed in the pyramidal cell layers of CA1-CA3 regions and in the polymorphic layer of the dentate gyrus (DG). The stratum radiatum of the CA1-3 regions showed positive PLA signal in contrast to the oriens region. The molecular and granular layers of the DG also lacked significant densities of PLA positive heteroreceptor complexes, but subgranular zone showed some PLA positive cells. Their allosteric receptor-receptor interactions may significantly modulate BDNF signaling impacting on hippocampal plasticity which is impaired upon ageing.

Introduction

The idea of the existence of direct interactions between two different G protein-coupled receptors (GPCRs) at the level of the plasma membrane was introduced in Sweden at the beginning of the 1980s (Agnati et al., 1980; Fuxe et al., 1981). Currently, a large body of evidence has highlighted the relevance of GPCRs homo-heteroreceptor complexes for molecular integration in the central nervous system (CNS) (Borroto-Escuela et al., 2015a; Fuxe et al., 2014a), which mainly takes place via allosteric receptor-receptor interactions at the plasma membrane level. This integration process makes possible a marked increase of the repertoire of GPCR signalling (Borroto-Escuela et al., 2014; Fuxe et al., 2014b), resulting in biochemical and/or pharmacological activities of the receptor protomers different from those of the corresponding monomers and homomers (Borroto-Escuela et al., 2015b, a). Over the past decade, an increasing amount of evidence has pointed out the ability of different GPCRs to interact not only with themselves but also with receptor tyrosine kinases (RTKs), ion channel receptors and receptor activity-modifying proteins, forming large homo-heteroreceptor complexes (Borroto-Escuela et al., 2017, 2015c; Borroto-Escuela et al., 2015d, 2012; Flajolet et al., 2008; Pei et al., 2004; Wang et al., 2012). In this scenario, the growth-promoting activity of many GPCR ligands implicates the activation of RTKs and their downstream signalling pathways (Luttrell et al., 1999) by the GPCR-induced release of neurotrophic factors and/or crosstalk in the GPCR-RTK downstream signalling pathways (Fuxe et al., 2007), thus paving the way to the emergence of the ‘transactivation’ concept.

Beyond the classical ‘transactivation process’, where the GPCR is not in direct interaction with the RTK, nowadays, there is a new awareness that RTKs and GPCRs possess the capacity for transactivation using RTKs themselves as signalling platforms via direct receptor–receptor interactions (Borroto-Escuela et al., 2015c, d; Borroto-Escuela et al., 2012; Di Palma et al., 2019; Flajolet et al., 2008; Fuxe et al., 2007). In this line of reasoning, among all GPCRs able to trigger RTKs activation, the adenosine receptor A2AR is particularly intriguing. This GPCR can transactivate, in the absence of neurotrophins, the high-affinity receptor of brain-derived neurotrophic factor (BDNF), namely, the tropomyosin related kinase B receptor (TrkB) (Lee and Chao, 2001). Interestingly, these two receptors are coexpressed in different brain areas, including the hippocampus, which is a region noteworthy for its relevance on cognition (Rosin et al., 1998; Spencer-Segal et al., 2011) since it is involved in learning, memory processes (Ambrogini et al., 2013; Gimenez-Llort et al., 2007; Lee et al., 2012; Zeng et al., 2012), and adult neurogenesis (Ambrogini et al., 2004). In addition, much work has been focused on the hippocampus as a proxy to study the ageing process, considering that age-related decline in memory and learning performance dependent on this brain area is consistently found across species and tasks (Barnes, 1979; Colombo et al., 1997; Erickson and Barnes, 2003; Mabry et al., 1996; Oler and Markus, 1998; Tanila et al., 1997; Temido-Ferreira et al., 2019). In this regard, the transactivation of TrkB receptors by A2AR was found to have a pivotal role in gating the BDNF action at hippocampal synapses inducing long-term potentiation (LTP) development (Diogenes et al., 2004; Fontinha et al., 2008) from young to aged animals (Costenla et al., 2011; Diogenes et al., 2007; Temido-Ferreira et al., 2019). Notably, the effect of this neurotrophin on rat hippocampal plasticity is affected by ageing, and this seems to be related to the antithetical differential density of TrkB and A2A receptors induced by age (Costenla et al., 2011; Diogenes et al., 2007; Lopes et al., 1999; Rebola et al., 2003; Silhol et al., 2005; Temido-Ferreira et al., 2019; Webster et al., 2006). Indeed, both receptors expression and signalling are profoundly altered in the hippocampus during the ageing with a concomitant age-related down-regulation of TrkB receptors (Diogenes et al., 2007; Silhol et al., 2005) and an up-regulation of A2A ones (Costenla et al., 2011; Diogenes et al., 2007; Lopes et al., 1999; Rebola et al., 2003). This relationship between age-related reorganisation in the density of these receptors with a consequent increase of A2A-receptor-mediated adenosinergic tonus was found to be crucial in triggering the BDNF action upon hippocampal synaptic transmission in aged rats despite the marked loss of TrkB receptors (Costenla et al., 2011; Diogenes et al., 2007; Temido-Ferreira et al., 2019). Thus, considering the impact of the above receptors interplay on what concerns BDNF-induced enhancement of synaptic transmission in the hippocampus primarily upon ageing, gaining a better insight into the mechanisms behind this powerful adenosine/A2AR and BDNF/TrkB crosstalk becomes of primary interest (Diogenes et al., 2007). In this context, in the light of the capacity of RTKs and GPCRs to physically interact (Fuxe et al., 2007), and that TrkB and A2A receptors were found to associate into complexes in lipid raft and nonlipid raft membranes on rat motor neurons (Mojsilovic-Petrovic et al., 2006), it is plausible to propose the existence of A2AR-TrkB heteroreceptor complexes in the hippocampus as a further interplay tool beyond the classical ‘transactivation process’ (Lee and Chao, 2001; Sebastiao et al., 2011). Therefore, the current work aims to take advantage of a cutting-edge technique as the in situ proximity ligation assay (in situ PLA) (Borroto-Escuela et al., 2016, 2018; Borroto-Escuela et al., 2013, 2012) to determine the existence of A2AR-TrkB heteroreceptor complexes and their distribution in the rat dorsal hippocampus.

Section snippets

Animals

All studies involving animals were performed in accordance with the European Communities Council Directive (Cons 123/2006/3) guidelines. Male Sprague-Dawley rats, ten weeks old, weighing 310–350 g were obtained from Charles River Laboratories (Germany). The animals were housed one week before experiments under a 12-h light/dark cycle, with an ambient temperature of 21 ± 2 °C and a relative humidity of 50 ± 5 %. Food and water available ad libitum.

Rats (n = 4) were anaesthetised with sodium

Results

PLA positive clusters/puncta representing A2AR-TrkB heteroreceptor complexes were observed in the dorsal hippocampus, including both the CA regions and the dentate gyrus. However, a heterogeneous distribution of these PLA positive heteroreceptor complexes was detected (Fig. 1, Fig. 2, Fig. 3).

Discussion

In the current paper, using a cutting-edge technique to study receptor-receptor physical interactions as the in situ PLA, we provided evidence for the existence of A2AR-TrkB heteroreceptor complexes in the rat dorsal hippocampus, showing a specific distribution pattern of the density of these complexes within this brain region. In particular, our data are in line with previous immunoprecipitation findings by Mojsilovic-Petrovic et al. (2006) gathered from lipid raft and nonlipid raft membranes

Declaration of Competing Interest

The authors have no affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.

Acknowledgements

The authors are supported by grants from the Swedish Medical Research Council (62X-00715-50-3) to KF, and by AFA Försäkring (130328) to KF and DOBE. DOBE belong to Academia de Biólogos Cubanos.

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