Clues,from,a,missense,mutation,of,the,adenosine,A1receptor,gene,associated,with,early-onset,Parkinson’s,disease

Sergi Ferré, Leonardo Pardo, Francisco Ciruela

Parkinson’s disease (PD) is a complex neurodegenerative disorder for which rare and common genetic variants contribute to disease risk, onset, and progression.The genetic contribution to PD can be classified mainly in, first,rare DNA variants that are highly penetrant and therefore causal, which are typically associated with monogenic PD; and second, more common risk polymorphic variants, which individually exert a small increase in the risk of the disease, which are usually identified in the most prevalent and apparently sporadic PD (Blauwendraat et al.,2020).

The terms monogenic, familiar, and early-onset PD (EOPD) are often used indistinctly.More specifically, PD is defined as familial or sporadic,according to the presence or absence of a clear family history.Approximately 5–10% can then be classified as familial, but monogenetic PD is rare and only accounts for about 30% of familial cases and 3% to 5% of sporadic cases, while in most cases PD is due to a complex interplay between genetics and the environment (Blauwendraat et al., 2020; Guadagnolo et al., 2021).EOPD is commonly defined as an age of onset below 45 years.Monogenic forms of PD are more frequent in EOPD patients, being more than 10% of cases with onset before 45 years and more than 40% in those with onset before 30 years (Blauwendraat et al., 2020; Guadagnolo et al., 2021).Furthermore, the term monogenic,meaning complete dependence on a mutated gene, is an oversimplification, since even for some highly penetrant rare variants, the presentation of PD is dependent on other genetic and non-genetic factors.Thus, the disease might not manifest itself in some carriers of highly penetrant variants.Furthermore, when manifested, the age of onset or the degree or progression of the disease may differ between carriers in the same family(Blauwendraat et al., 2020).

There are several well-established genes in which mutations cause monogenic PD or constitute riskconferring variants, with autosomal dominant inheritance (such as SNCA and LRRK2) and autosomal recessive inheritance (such as PRKN,PINK1, and DJ1).Collectively, rare variants in more than 20 genes have been identified so far,but the relevance of most of them is still a matter of debate, and more replication and functional validation studies are needed (Blauwendraat et al., 2020; Guadagnolo et al., 2021).Dominant mutations in SNCA, the α-synuclein gene, were identified 20 years ago as the first monogenic cause of PD, which is consistent with the critical pathogenetic role of α-synuclein.Therefore,neuronal loss in the substantia nigra pars compacta, which causes striatal dopamine deficiency, and intracellular inclusions that contain aggregates of α synuclein, which constitute the classical Lewy bodies, are the neuropathological hallmarks of PD.Pathogenic variants in the LRRK2 gene, which encodes leucine-rich repeat kinase 2, are the most common causes of autosomal dominant PD, accounting for 5% of familial and 1% of sporadic cases.Among autosomal recessive monogenic PD, pathogenic variants in the PRKN,PINK1, and DJ-1 genes account for more than 10%of cases of EOPD.Importantly, Lewy bodies are not detected in most PRKN mutations, indicating a difference in the pathogenic processes that lead to this EOPD and sporadic PD (Blauwendraat et al.,2020; Guadagnolo et al., 2021).

Jaberi et al.(2016) reported a genetic study in a family with autosomal recessive EOPD with cognitive decline, identifying two affected siblings with homozygous mutations in the adenosine A1receptor (A1R) gene (ADORA1; G2797.44S) and in the peptidyl-tRNA hydrolase domain containing 1 gene (PTRHD1) that segregated with the disease.Based on circumstantial evidence of a significant role for adenosine and A1R in neuroprotection and neurodegeneration (Cunha, 2016), they suggested the ADORA1 mutation is the strongest candidate causative mutation.However, several months after the publication by Jaberi et al.(2016), another mutation of PTRHD1 (p.His53Tyr) was found as a possible cause of autosomal recessive intellectual disability and EOPD.This was followed by two more recent separate studies from Oman and South African families reporting the association of a 28-nucleotide frameshift deletion in the PTRHD1 coding region with EOPD and intellectual disability(reviewed in Al-Kasbi et al., 2021).Therefore,the PTRHD1 mutation might have been the main cause of EOPD simultaneously associated with the ADORA1 mutation described by Jaberi et al.(2016).However, although the bacterial homolog (pth1)is well characterized, human PTRHD1 does not seem to function as peptidyl-tRNA hydrolase and, currently, its function remains unclear (Al-Kasbi et al., 2021).On the other hand, A1R is known to play a very significant role in mediating the central effects of adenosine, both during physiological and pathological conditions (Cunha,2016).Therefore, the putative role of mutated A1RG279Sin the development of EOPD with cognitive decline described by Jaberi et al.(2016) could not be ruled out.Therefore, it became important to study whether the G2797.44Smutation has functional consequences.G2797.44is located in the middle of the transmembrane domain (TM)7 of A1R, facing the lipid bilayer, thus not being part of the orthosteric binding site.But it is also located near the conserved NP7.50xxY motif, which is essential to form the active conformation of the receptor (Nasrollahi-Shirazi et al., 2020; Sarasola et al., 2022).The G2797.44Smutation would then be foreseeable to lead to differences in agonistinduced activation without changes in agonist binding properties.Furthermore, since mutations in the TMs can affect the ability of G proteincoupled receptors to undergo folding in the endoplasmic reticulum, functional differences could be related to different densities at the plasma membrane.Finally, another possibility could be the differential ability of A1RG279Sto form functional heteromers with other G proteincoupled receptors, more importantly with the dopamine D1receptor (D1R) or with the adenosine A2Areceptor (A2AR).Thus, functional A1R-A2AR and A1R-D1R heteromers exert a very significant role in adenosine-mediated presynaptic and postsynaptic modulation of striatal glutamatergic transmission,respectively (Ferré et al., 2022).

Three different studies have been conducted in mammalian transfected cells (HEK-293 and HEK-293T cells) to address these questions.Using immunohistochemical and coimmunoprecipitation techniques in co-transfected cells, Jaberi et al.(2016) found no differences in the density of A1RG279Sin the plasma membrane and its molecular interactions with the dopamine D1R, compared to wild-type A1R (i.e., A1RWT).This was confirmed by Nasrollahi-Shirazi et al.(2020)using radioligand binding, flow cytometric analysis,and bioluminescent resonance energy transfer(BRET).With radioligand binding experiments,the same authors found no differences in the binding properties of A1R ligands, while signaling experiments implied that the G2797.44Smutation increases the constitutive activity and agonistinduced efficacy of A1R.This was attributed to an enhanced conformational flexibility due to a reduced kinetic stability of A1RG279SversusA1RWT,as shown by analyzing the time-dependent loss of radiolabeled antagonist binding at different temperatures, and as supported by molecular dynamic simulations (Nasrollahi-Shirazi et al., 2020).In our recent study, using immunofluorescence, biotinylation, and NanoBRET techniques, we also found that A1RG279Sstably expressed in HEK-293T cells shows an equivalent subcellular distribution and cell surface density as A1RWT(Sarasola et al., 2022).Furthermore,NanoBRET experiments also showed that a fluorescent selective A1R ligand had the same affinity for A1RG279Sas for A1RWT.However, in contradiction to the results obtained by Nasrollahi-Shirazi et al.(2020), using NanoBiT technology in transiently transfected HEK-293T cells, we did not observe a significant difference in the functional response of A1R agonists, in their ability to couple with transducer proteins (Gαi, Gαq, Gα12/13, Gαs,β-arrestin2, or GRK2) (Sarasola et al., 2022).

After the report by Jaberi et al.(2016), we postulated that a possible mechanistic explanation of the pathogenetic link of the G2797.44Smutation with EOPD could be a loss of function of the A1R in its interactions with the A2AR in the corticostriatal glutamatergic terminals (Fernández-Dueñas et al., 2017).As we recently reviewed,adenosine plays a very significant role in local striatal modulation of cortico-striatal glutamate release and, secondarily, of acetylcholine and dopamine release (Ferré et al., 2022).This modulation is largely mediated by A1R-A2AR and A2AR-cannabinoid CB1receptor (CB1R) heteromers localized in cortico-striatal glutamatergic terminals(Figure 1; Ferré et al., 2022).Predominant activation of A1Rs or A2ARs in the cortico-striatal terminal results in inhibition or facilitation of glutamate release, respectively, depending on the degree of constitutive activity of A2AR, on the extracellular level of adenosine, and on the level of endocannabinoids.We have previously demonstrated that the constitutive activity of A2AR disappears in the A1R-A2AR heteromer, but not in the A2AR-CB1R heteromer (Kӧfalvi et al., 2020).Then, the ability of endocannabinoids and other CB1R agonists to inhibit glutamate release depends on their ability to counteract the constitutive A2ARmediated adenylyl cyclase activation in the A2ARCB1R heteromer (Figure 1; Kӧfalvi et al., 2020).

Figure 1｜Schematic representation of cortico-striatal glutamatergic terminals and their modulatory A1receptor(A1R)-A2Areceptor (A2AR) and A2AR-CB1receptor (CB1R) heteromers.

Additionally, significant allosteric interactions take place between ligands that bind to orthosteric sites of the A1R-A2AR heteromer.On the one hand, the binding of an A1R agonist decreases the potency and efficacy of an A2AR agonist (Sarasola et al., 2022).Since adenosine has higher affinity for A1R than for A2AR, this allosteric interaction ensures that, under physiological variations of extracellular adenosine, the functional effect of A1R activation in the cortico-striatal terminal predominates over the effect of A2AR activation (Figure 1A).On the other hand, higher pathological levels of adenosine can overcome the allosteric interaction imposed by activated A1R and promote an opposite effect,through a reciprocal antagonistic interaction, by which binding of an A2AR agonist decreases A1R function (Figure 1B; Ferré et al., 2022).The same type of allosteric interaction, but in the A2AR-CB1R heteromer, can promote the counteraction of the antagonistic effect of CB1R agonists on A2ARmediated signaling in the A2AR-CB1R heteromer(Ferré et al., 2022), altogether maximizing A2ARmediated glutamate release (Figure 1B).

In fact, we were able to demonstrate, using NanoBiT technology, that A1RG279Sdoes not form heteromers with A2AR.Molecular dynamic simulations allowed us to propose an indirect mechanism by which the G2797.44Smutation in TM 7 of A1R weakens the TM 5/6 interface of the A1R-A2AR heteromer.As expected, the lack of A1R-A2AR heteromerization was associated with the disappearance of the A1R agonist-induced allosteric modulation of A2AR signaling and the restoration of the constitutive activity of the A2AR (Sarasola et al., 2022).Therefore, this could confer an increased sensitivity of cortico-striatal glutamatergic terminals (Figure 1C), which could enhance the well-established striatal glutamatergic hyperactivity of PD (Blandini et al., 1996;Campanelli et al., 2022).This hyperglutamatergic state involves pre- and postsynaptic mechanisms and has been suggested to be a critical mechanism underlying different striatal alterations associated with PD in the early and advanced symptomatic stages of the disease (Campanelli et al., 2022).

Although we do not yet know the pathogenetic contribution of the G2797.44Smutation of A1R in EOPD described by Jaberi et al.(2016), to our knowledge, this is the first example of a single missense mutation that specifically results in the impairment of G protein-coupled receptor heteromerization, which probably results in pathological implications.It would then be important to look for G2797.44Sor other functionally similar mutations of A1R in other clinical conditions where alterations in the function of A1R-A2AR heteromers in cortico-striatal terminals have been proposed, such as restless legs syndrome (Ferré et al., 2022) or other neuropsychiatric disorders associated with alterations in cortico-striatal transmission.

The present work was supported by the intramural funds of the National Institute in drug abuse (ZIA DA000493) (to SF); projects PID2019-109240RB-I00(to LP) and PID2020-118511RB-I00 (to FC) were founded by MCIN/AEI/10.13039/501100011033,“ERDF A way of making Europe”.

Sergi Ferré, Leonardo Pardo,Francisco Ciruela*Integrative Neurobiology Section, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore,MD, USA (Ferré S)Laboratory of Computational Medicine,Biostatistics Unit, Faculty of Medicine, Universitat Autònoma Barcelona, Bellaterra, Barcelona, Spain(Pardo L)Pharmacology Unit, Department of Pathology and Experimental Therapeutics, School of Medicine and Health Sciences, Institute of Neurosciences,University of Barcelona.Neuropharmacology& Pain Group, Neuroscience Program, Bellvitge Institute for Biomedical Research, L’Hospitalet de Llobregat, Spain (Ciruela F)

*Correspondence to:Francisco Ciruela, PhD,fciruela@ub.edu.https://orcid.org/0000-0003-0832-3739(Francisco Ciruela)

Date of submission:December 5, 2022

Date of decision:December 30, 2022

Date of acceptance:January 10, 2023

Date of web publication:March 3, 2023

https://doi.org/10.4103/1673-5374.369113

How to cite this article:Ferré S, Pardo L, Ciruela F(2023) Clues from a missense mutation of theadenosine A1receptor gene associated with earlyonset Parkinson’s disease.Neural Regen Res 18(10):2204-2205.

Open access statement:This is an open access journal, and articles are distributedunder the terms of the Creative Commons AttributionNonCommercial-ShareAlike 4.0 License,which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

Open peer reviewer:Rodrigo Cunha, University of Coimbra, Portugal.

Additional file:Open peer review report 1.