NEURAL REGENERATION RESEARCH www.nrronline.org Perspective Clues from a missense mutation coupled receptors, more importantly with the dopamine D1 receptor (D1R) or with the adenosine A2A receptor (A2AR). Thus, functional A1R-A2AR and of the adenosine A1 receptor A1R-D1R heteromers exert a very significant role in adenosine-mediated presynaptic and postsynaptic gene associated with early-onset modulation of striatal glutamatergic transmission, respectively (Ferré et al., 2022). Parkinson’s disease Three different studies have been conducted in mammalian transfected cells (HEK- 293 and HEK-293T cells) to address these questions. Using immunohistochemical and co- immunoprecipitation techniques in co-transfected Sergi Ferré, Leonardo Pardo, Francisco Ciruela* cells, Jaberi et al. (2016) found no differences in the density of A1RG279S in the plasma membrane P a r k i n s o n ’s d i s e a s e ( P D ) i s a c o m p l e x dominant PD, accounting for 5% of familial and and its molecular interactions with the dopamine neurodegenerative disorder for which rare 1% of sporadic cases. Among autosomal recessive D1R, compared to wild-type A1R (i.e., A1RWT). This and common genetic variants contribute to monogenic PD, pathogenic variants in the PRKN, was confirmed by Nasrollahi-Shirazi et al. (2020) disease risk, onset, and progression. The genetic PINK1, and DJ-1 genes account for more than 10% using radioligand binding, flow cytometric analysis, contribution to PD can be classified mainly in, first, of cases of EOPD. Importantly, Lewy bodies are and bioluminescent resonance energy transfer rare DNA variants that are highly penetrant and not detected in most PRKN mutations, indicating (BRET). With radioligand binding experiments, therefore causal, which are typically associated a difference in the pathogenic processes that lead the same authors found no differences in the with monogenic PD; and second, more common to this EOPD and sporadic PD (Blauwendraat et al., binding properties of A1R ligands, while signaling risk polymorphic variants, which individually exert 2020; Guadagnolo et al., 2021). experiments implied that the G2797.44S mutation a small increase in the risk of the disease, which increases the constitutive activity and agonist- Jaberi et al. (2016) reported a genetic study in are usually identified in the most prevalent and induced efficacy of A 1R. This was attributed to a family with autosomal recessive EOPD with apparently sporadic PD (Blauwendraat et al., an enhanced conformational flexibility due to a cognitive decline, identifying two affected siblings 2020). reduced kinetic stability of A1RG279S versus A1RWT, with homozygous mutations in the adenosine A1 The terms monogenic, familiar, and early-onset receptor (A1R) gene (ADORA1; G2797.44S) and in as shown by analyzing the time-dependent PD (EOPD) are often used indistinctly. More the peptidyl-tRNA hydrolase domain containing 1 loss of radiolabeled antagonist binding at specifically, PD is defined as familial or sporadic, gene (PTRHD1) that segregated with the disease. different temperatures, and as supported by according to the presence or absence of a clear Based on circumstantial evidence of a significant molecular dynamic simulations (Nasrollahi- family history. Approximately 5–10% can then be role for adenosine and A1R in neuroprotection and Shirazi et al., 2020). In our recent study, using classified as familial, but monogenetic PD is rare neurodegeneration (Cunha, 2016), they suggested immunofluorescence, biotinylation, and NanoBRET and only accounts for about 30% of familial cases the ADORA1 mutation is the strongest candidate techniques, we also found that A 1R G279S stably and 3% to 5% of sporadic cases, while in most causative mutation. However, several months after expressed in HEK-293T cells shows an equivalent cases PD is due to a complex interplay between the publication by Jaberi et al. (2016), another subcellular distribution and cell surface density genetics and the environment (Blauwendraat mutation of PTRHD1 (p.His53Tyr) was found as a as A 1R WT (Sarasola et al., 2022). Furthermore, et al., 2020; Guadagnolo et al., 2021). EOPD is possible cause of autosomal recessive intellectual NanoBRET experiments also showed that a commonly defined as an age of onset below disability and EOPD. This was followed by two fluorescent selective A 1R ligand had the same 45 years. Monogenic forms of PD are more more recent separate studies from Oman and affinity for A 1 R G279S as for A 1 R WT. However, in frequent in EOPD patients, being more than South African families reporting the association of contradiction to the results obtained by Nasrollahi- 10% of cases with onset before 45 years and a 28-nucleotide frameshift deletion in the PTRHD1 Shirazi et al. (2020), using NanoBiT technology in more than 40% in those with onset before 30 coding region with EOPD and intellectual disability transiently transfected HEK-293T cells, we did not years (Blauwendraat et al., 2020; Guadagnolo et (reviewed in Al-Kasbi et al., 2021). Therefore, observe a significant difference in the functional al., 2021). Furthermore, the term monogenic, the PTRHD1 mutation might have been the main response of A1R agonists, in their ability to couple meaning complete dependence on a mutated cause of EOPD simultaneously associated with the with transducer proteins (Gαi, Gαq, Gα12/13, Gαs, gene, is an oversimplification, since even for some ADORA1 mutation described by Jaberi et al. (2016). β-arrestin2, or GRK2) (Sarasola et al., 2022). highly penetrant rare variants, the presentation of After the report by Jaberi et al. (2016), we However, although the bacterial homolog (pth1) PD is dependent on other genetic and non-genetic postulated that a possible mechanistic explanation is well characterized, human PTRHD1 does not factors. Thus, the disease might not manifest of the pathogenetic link of the G2797.44S mutation seem to function as peptidyl-tRNA hydrolase itself in some carriers of highly penetrant variants. with EOPD could be a loss of function of the A1R and, currently, its function remains unclear (Al- Furthermore, when manifested, the age of onset in its interactions with the A 2AR in the cortico- Kasbi et al., 2021). On the other hand, A 1R is or the degree or progression of the disease striatal glutamatergic terminals (Fernández- known to play a very significant role in mediating may differ between carriers in the same family Dueñas et al., 2017). As we recently reviewed, the central effects of adenosine, both during (Blauwendraat et al., 2020). adenosine plays a very significant role in local physiological and pathological conditions (Cunha, There are several well-established genes in which 2016). Therefore, the putative role of mutated striatal modulation of cortico-striatal glutamate mutations cause monogenic PD or constitute risk- A1RG279S in the development of EOPD with cognitive release and, secondarily, of acetylcholine and conferring variants, with autosomal dominant decline described by Jaberi et al. (2016) could dopamine release (Ferré et al., 2022). This inheritance (such as SNCA and LRRK2) and not be ruled out. Therefore, it became important modulation is largely mediated by A1R-A2AR and autosomal recessive inheritance (such as PRKN, to study whether the G279 7.44S mutation has A2AR-cannabinoid CB1 receptor (CB1R) heteromers PINK1, and DJ1). Collectively, rare variants in functional consequences. G279 7.44 is located in localized in cortico-striatal glutamatergic terminals more than 20 genes have been identified so far, the middle of the transmembrane domain (TM) (Figure 1; Ferré et al., 2022). Predominant but the relevance of most of them is still a matter 7 of A1R, facing the lipid bilayer, thus not being activation of A1Rs or A2ARs in the cortico-striatal of debate, and more replication and functional part of the orthosteric binding site. But it is also terminal results in inhibition or facilitation of validation studies are needed (Blauwendraat et located near the conserved NP7.50xxY motif, which glutamate release, respectively, depending on al., 2020; Guadagnolo et al., 2021). Dominant is essential to form the active conformation of the the degree of constitutive activity of A 2AR, on mutations in SNCA, the α-synuclein gene, were receptor (Nasrollahi-Shirazi et al., 2020; Sarasola the extracellular level of adenosine, and on the identified 20 years ago as the first monogenic et al., 2022). The G2797.44S mutation would then level of endocannabinoids. We have previously cause of PD, which is consistent with the critical be foreseeable to lead to differences in agonist- demonstrated that the constitutive activity of A2AR pathogenetic role of α-synuclein. Therefore, induced activation without changes in agonist disappears in the A1R-A2AR heteromer, but not in neuronal loss in the substantia nigra pars binding properties. Furthermore, since mutations the A 2AR-CB 1R heteromer (Köfalvi et al., 2020). compacta, which causes striatal dopamine in the TMs can affect the ability of G protein- Then, the ability of endocannabinoids and other CB1R agonists to inhibit glutamate release depends deficiency, and intracellular inclusions that contain coupled receptors to undergo folding in the on their ability to counteract the constitutive A2AR- aggregates of α synuclein, which constitute the endoplasmic reticulum, functional differences mediated adenylyl cyclase activation in the A2AR- classical Lewy bodies, are the neuropathological could be related to different densities at the CB1R heteromer (Figure 1; Köfalvi et al., 2020). hallmarks of PD. Pathogenic variants in the LRRK2 plasma membrane. Finally, another possibility gene, which encodes leucine-rich repeat kinase could be the differential ability of A1RG279S to form Additionally, significant allosteric interactions take 2, are the most common causes of autosomal functional heteromers with other G protein- place between ligands that bind to orthosteric 2204 ｜NEURAL REGENERATION RESEARCH｜Vol 18｜No. 10｜October 2023 Perspective NEURAL REGENERATION RESEARCH www.nrronline.org A B C under 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. Figure 1 ｜ Schematic representation of cortico-striatal glutamatergic terminals and their modulatory A1 receptor References (A1R)-A2A receptor (A2AR) and A2AR-CB1 receptor (CB1R) heteromers. Al-Kasbi G, Al-Saegh A, Al-Qassabi A, Al-Jabry T, Zadjali Arrows represent receptor activation or facilitation of glutamate (GLU) release. Red arrows represent constitutive F, Al-Yahyaee S, Al-Maawali A (2021) Biallelic PTRHD1 activation of the A2AR. Lines with perpendicular ending segments represent inhibitory allosteric modulation or inhibition frameshift variants associated with intellectual of glutamate release. The lower and higher degrees of activation, facilitation, or inhibition are represented by broken and thicker arrows and lines, respectively. Predominant activation of A1R or A2AR promotes inhibition or facilitation disability, spasticity, and parkinsonism. Mov Disord Clin of GLU release, respectively. (A) Low degree of GLU release under physiological conditions, with low extracellular Pract 8:1253-1257. concentrations of adenosine (ADO), which promotes a predominant activation of A1R in the A1R-A2AR heteromer; the Blandini F, Porter RH, Greenamyre JT (1996) Glutamate constitutive activation of A2AR in the A2AR-CB1R heteromer depends on the degree of inhibitory control by CB1R. (B) A2AR loses its constitutive activity in the A1R-A2AR heteromer and plays a role with pathologically high concentrations of ADO, and Parkinson’s disease. Mol Neurobiol 12:73-94. which facilitates the release of GLU. (C) The absence of heteromerization of A1RG279S with A2AR reveals a non-inhibited Blauwendraat C, Nalls MA, Singleton AB (2020) The constitutive and agonist-induced activation of A2AR and a facilitatory effect on the release of GLU. Although adenosine receptors and receptor heteromers are proposed to be predominantly dimeric and tetrameric, respectively, they are genetic architecture of Parkinson’s disease. Lancet represented as monomers and dimers for the sake of simplicity. Created using Illustrator 27.1.1. Neurol 19:170-178. Campanelli F, Natale G, Marino G, Ghiglieri V, Calabresi P (2022) Striatal glutamatergic hyperactivity in sites of the A 1 R-A 2A R heteromer. On the one similar mutations of A1R in other clinical conditions hand, the binding of an A1R agonist decreases the where alterations in the function of A 1R-A 2AR Parkinson’s disease. Neurobiol Dis 168:105697. potency and efficacy of an A2AR agonist (Sarasola et heteromers in cortico-striatal terminals have been Cunha RA (2016) How does adenosine control neuronal al., 2022). Since adenosine has higher affinity for proposed, such as restless legs syndrome (Ferré dysfunction and neurodegeneration? J Neurochem A1R than for A2AR, this allosteric interaction ensures et al., 2022) or other neuropsychiatric disorders 139:1019-1055. that, under physiological variations of extracellular associated with alterations in cortico-striatal Ferré S, Sarasola LI, Quiroz C, Ciruela F (2022) Presynaptic adenosine, the functional effect of A1R activation transmission. in the cortico-striatal terminal predominates over adenosine receptor heteromers as key modulators of the effect of A2AR activation (Figure 1A). On the The present work was supported by the intramural glutamatergic and dopaminergic neurotransmission in other hand, higher pathological levels of adenosine funds of the National Institute in drug abuse (ZIA the striatum. Neuropharmacology 223:109329. can overcome the allosteric interaction imposed DA000493) (to SF); projects PID2019-109240RB-I00 Fernández-Dueñas V, Pérez-Arévalo A, Altafaj X, Ferré S, by activated A1R and promote an opposite effect, (to LP) and PID2020-118511RB-I00 (to FC) were through a reciprocal antagonistic interaction, by founded by MCIN/AEI/10.13039/501100011033, Ciruela F (2017) Adenosine A1-A2A receptor heteromer which binding of an A2AR agonist decreases A1R “ERDF A way of making Europe”. as a possible target for early-onset Parkinson’s disease. function (Figure 1B; Ferré et al., 2022). The same Front Neurosci 11:652. type of allosteric interaction, but in the A2AR-CB1R Sergi Ferré, Leonardo Pardo, Guadagnolo D, Piane M, Torrisi MR, Pizzuti A, Petrucci S heteromer, can promote the counteraction of Francisco Ciruela* (2021) Genotype-phenotype correlations in monogenic the antagonistic effect of CB1R agonists on A2AR- Integrative Neurobiology Section, National mediated signaling in the A2AR-CB1R heteromer Parkinson disease: a review on clinical and molecular Institute on Drug Abuse, Intramural Research (Ferré et al., 2022), altogether maximizing A2AR- Program, National Institutes of Health, Baltimore, findings. Front Neurol 12:648588. mediated glutamate release (Figure 1B). Jaberi E, Rohani M, Shahidi GA, Nafissi S, Arefian E, MD, USA (Ferré S) In fact, we were able to demonstrate, using Laboratory of Computational Medicine, Soleimani M, Moghadam A, Arzenani MK, Keramatian NanoBiT technology, that A 1 R G279S does not Biostatistics Unit, Faculty of Medicine, Universitat F, Klotzle B, Fan JB, Turk C, Steemers F, Elahi E (2016) form heteromers with A2AR. Molecular dynamic Autònoma Barcelona, Bellaterra, Barcelona, Spain Mutation in ADORA1 identified as likely cause of early- simulations allowed us to propose an indirect (Pardo L) onset parkinsonism and cognitive dysfunction. Mov mechanism by which the G279 7.44S mutation in TM 7 of A 1R weakens the TM 5/6 interface of Pharmacology Unit, Department of Pathology and Disord 31:1004-1011. the A1R-A2AR heteromer. As expected, the lack of Experimental Therapeutics, School of Medicine Köfalvi A, Moreno E, Cordomí A, Cai NS, Fernández- A1R-A2AR heteromerization was associated with and Health Sciences, Institute of Neurosciences, Dueñas V, Ferreira SG, Guixà-González R, Sánchez-Soto the disappearance of the A 1R agonist-induced University of Barcelona. Neuropharmacology M, Yano H, Casadó-Anguera V, Cunha RA, Sebastião allosteric modulation of A 2AR signaling and the & Pain Group, Neuroscience Program, Bellvitge restoration of the constitutive activity of the AM, Ciruela F, Pardo L, Casadó V, Ferré S (2020) Control Institute for Biomedical Research, L’Hospitalet de A2AR (Sarasola et al., 2022). Therefore, this could of glutamate release by complexes of adenosine and Llobregat, Spain (Ciruela F) confer an increased sensitivity of cortico-striatal cannabinoid receptors. BMC Biol 18:9. *Correspondence to: Francisco Ciruela, PhD, glutamatergic terminals (Figure 1C), which could Nasrollahi-Shirazi S, Szöllösi D, Yang Q, Muratspahic E, El- enhance the well-established striatal glutamatergic fciruela@ub.edu. https://orcid.org/0000-0003-0832-3739 Kasaby A, Sucic S, Stockner T, Nanoff C, Freissmuth M hyperactivity of PD (Blandini et al., 1996; Campanelli et al., 2022). This hyperglutamatergic (Francisco Ciruela) (2020) Functional impact of the G279S substitution state involves pre- and postsynaptic mechanisms Date of submission: December 5, 2022 in the adenosine A1-receptor (A1R-G279S7.44), a and has been suggested to be a critical mechanism Date of decision: December 30, 2022 mutation associated with Parkinson’s disease. Mol underlying different striatal alterations associated Date of acceptance: January 10, 2023 Pharmacol 98:250-266. with PD in the early and advanced symptomatic Date of web publication: March 3, 2023 Sarasola LI, Del Torrent CL, Pérez-Arévalo A, Argerich J, stages of the disease (Campanelli et al., 2022). Casajuana-Martín N, Chevigné A, Fernández-Dueñas Although we do not yet know the pathogenetic https://doi.org/10.4103/1673-5374.369113 V, Ferré S, Pardo L, Ciruela F (2022) The ADORA1 contribution of the G2797.44S mutation of A1R in How to cite this article: Ferré S, Pardo L, Ciruela F EOPD described by Jaberi et al. (2016), to our mutation linked to early-onset Parkinson’s disease (2023) Clues from a missense mutation of the knowledge, this is the first example of a single alters adenosine A1-A2A receptor heteromer formation adenosine A1 receptor gene associated with early- missense mutation that specifically results in and function. Biomed Pharmacother 156:113896. the impairment of G protein-coupled receptor onset Parkinson’s disease. Neural Regen Res heteromerization, which probably results in 18(10):2204-2205. pathological implications. It would then be Open access statement: This is an open P-Reviewer: Cunha R; C-Editors: Zhao M, Liu WJ, Qiu Y; important to look for G2797.44S or other functionally access journal, and articles are distributed T-Editor: Jia Y NEURAL REGENERATION RESEARCH｜Vol 18｜No. 10｜October 2023｜2205