Challenges and  new  perspectives

Different sources of adenosine, as well as different sources of ATP, which in turn can be metabolized into adenosine (see section 2; Figure 1), can participate in the activation of different adenosine receptors, as well as in different pathways and functions in the brain. Therefore, determining the adenosine source and receptors subtype, which are singularly associated with specific physiological process or disease status, in different definable extracellular domains within the brain parenchyma (that is, neuronal and/or synaptic, astrocytic, microglial or vascular domains), is crucial in order to develop new therapeutic strategies for brain disorders (Chen et al., 2013).

The difficulty in distinguishing the several sources of extracellular adenosine under physiological and pathological conditions is a major challenge and caveat in the adenosinergic field. The difficulties start with the different handling of preparations, which generally produce a massive extracellular accumulation of adenosine that occurs after different types of insults (Latini and Pedata, 2001). The challenges continue with the lack

1

of tools to provide a reliable quantification of adenosine in a nanomolar range, like the ones observed in physiological conditions, and the possibility to perform the quantifications in specific cellular and subcellular domains, like the synaptic cleft.

A further major issue contributing to the inability to pinpoint the contribution of the different sources of endogenous extracellular adenosine is the inability to determine the location and role of the different ectonucleotidases. This is probably related to the general lack of pharmacological tools to specifically manipulate particular enzymes in this large family of enzymes (Zimmermann, 2000), as well as to the lack of tools to accurate recognize them. In fact, we know considerably more about the molecular biology of ectonucleotidases than of their localization and kinetic properties in native tissues that ultimately define their physiological role (Cunha, 2005).

Despite the fact that adenosine receptor ligands are metabolically stable, they are able to reach all receptors (and they are found on many cells in the body) contributing to a substantial risk of inducing side effects. In addition, because of their generally high affinity, would provide prolonged stimulation. Alternatively, instead of directly targeting adenosine receptors, an optimal therapeutic approach would be to manipulate a specific source of endogenous adenosine, which could provide some degree of specificity (Chen et al., 2013). An elegant use of network pharmacology is the development of a novel type of prodrug that needs to be metabolized in order to become available as an adenosine receptor agonist (El-Tayeb et al., 2009). Accordingly, a  5ʹ-phosphate prodrug of adenosine receptor agonist was generated in order to be hydrolized at sites where CD73 is highly expressed, in order to release the active adenosine receptor agonist (Flögel et al., 2012). This prodrug approach not only allows the site-specific action within the tissues where CD73 is enriched but may also avoid some of the side effects.

The adenosinergic field has greatly improved and gained prominence due to the awareness of the gaps and the technical limitations that were still hampering the field.

1 This led to the enhancement and optimization of experimental tools and techniques to

perform a multiplicity of different studies and thus on resultant expansion of the available data. Hopefully, in a near future, this will be reflected by an increased consequential knowledge and on new therapeutic strategies.

GOALS

Adenosine is a prototypic neuromodulator that fine-tunes on-going synaptic transmission, controlling the flow of information through different neuronal circuits in the brain (Dunwiddie and Masino, 2001). Some of adenosine receptors’   roles  in the brain are well known, however, the source of adenosine is a caveat in the adenosinergic field.

Importantly, under physiological conditions adenosine tonus is not very prominent, and the adopted strategy to study the source of extracellular adenosine was trough CD73 knockout (KO) mice. CD73 is the key enzyme that catabolizes the last step of the catabolism of ATP to adenosine and we proposed to refine the characterization and role of CD73 in the central nervous system (CNS).

On chapter 2 we started to investigate the presence of CD73 in the different brain areas, as well as, its cellular and subcellular localization in physiological settings.

On chapter 3 we aim to explore the role of CD73 in locomotion, memory and learning paradigms, taking advantage of CD73 KO mice.

Due to the results obtained on chapters 2 and 3, with a segregation of CD73 on basal ganglia, on chapter 4 we decided to explore the role of CD73 in different pathological conditions that are patent in the striatum, namely drug addiction, psychomotor activity and  Parkinson’s  disease,  by taking advantage of CD73 KO mice.

Thanks to the results obtained on the previous chapters that pointed CD73 with a particular role on the activation of A_2AR, on chapter 5 we proposed to explore if CD73 provides the particular pool of extracellular adenosine selectively responsible for activating striatal A2AR.

Adenosine has a huge impact in the hippocampus and causes different and most often opposite actions by activating different receptors, namely A₁R and A_2AR that, in addition can be co-localized, at least in nerve terminals (Cunha, 2008). Thus, it becomes of upmost importance to understand how the differential activation of the different adenosine receptors can be effectively controlled to meet the needs of the system.

In deleterious conditions like in epilepsy, adenosine has a huge impact, being known to act as a powerful endogenous anticonvulsive, mainly through A₁R activation (Boison, 2012). In order to define and characterize the involvement of the adenosine catabolic mediator CD73 and A2AR in epilepsy, on chapter 6 we compared mice models with selective cellular deletions of A_2AR (neuronal or astrocytic) and mice deficient in CD73 on a mice model of mesial temporal lobe epilepsy.

Since hippocampal A_2AR are known to be involved in important functions like long-term potentiation (Rebola et al., 2008; Fontinha et al., 2009) and different types of memories (Wei et al., 2010), on chapter 7 we proposed to explore the role of hippocampal A2AR in different behavior paradigms and to investigate a possible local (synaptic) synthesis of A2AR in the hippocampus.

CHAPTER 2

DISTRIBUTION OF CD73 IN THE BRAIN