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\begin{document}

\title{Towards the convergent therapeutic potential of GPCRs in autism spectrum
disorders}



\author[1]{Anil Annamneedi}%
\author[1]{Caroline Gora}%
\author[1]{Ana Dudas}%
\author[1]{Xavier Leray}%
\author[1]{Véronique Bozon}%
\author[1]{Pascale Crepieux}%
\author[1]{Lucie P. Pellissier}%
\affil[1]{Physiologie de la Reproduction et des Comportements}%


\vspace{-1em}



  
  \date{June 17, 2024}


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\selectlanguage{english}
\begin{abstract}
Autism spectrum disorders (ASD) are diagnosed in 1/100 childbirth
worldwide, based on two core symptoms, deficits in social interaction
and communication and stereotyped behaviours. G protein-coupled
receptors (GPCRs) are the largest family of cell-surface receptors that
mediate the transfer of extracellular signals to convergent
intracellular signalling and downstream cellular responses that are
dysregulated in ASD. Despite hundreds of GPCRs are expressed in the
brain, only 23 GPCRs are genetically associated to ASD according to the
Simons Foundation Autism Research Initiative (SFARI) gene database:
oxytocin OTR, vasopressin V1A, V1B, metabotropic glutamate mGlu5, mGlu7,
GABAB, dopamine D1, D2, D3, serotoninergic 5-HT1B, \selectlanguage{greek}β\selectlanguage{english}2-adrenoceptor,
cholinergic M3, adenosine A2A, A3, angiotensin AT2, cannabinoid CB1,
chemokine CX3CR1, orphan GPR37, GPR85 and olfactory OR1C1, OR2M4,
OR2T10, OR52M1. Here, we review the therapeutical potential of these 23
GPCRs, in addition to 5-HT2A, 5-HT6 and 5-HT7 for their relevance to
ASD. We discuss their genetic association with ASD, the effects of their
genetic and pharmacological manipulation in animal models and humans,
their existing pharmacopeia towards core symptoms of ASD and rank them
based on these evidences. Among these 23 GPCRs, we highlight that OTR,
V1A, mGlu5, D2, 5-HT2A, CB1, and GPR37 are the best therapeutic targets.
We conclude that the dysregulation of GPCRs and their signalling is a
convergent pathological mechanism of ASD and their therapeutic potential
has only begun as multiple GPCRs could mitigate ASD.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Autism spectrum disorder (ASD) is a neurodevelopmental disorder
diagnosed around the age of 3 with a worldwide prevalence of around
1/100 child births (Zeidan et al., 2022). Core clinical symptoms are
defined by the Diagnostic and Statistical Manual of Mental Disorders,
Fifth Edition (DSM-V): social interaction and communication deficits and
stereotyped, restrained or compulsive behaviours. ASD often associates
with comorbid symptoms, such as anxiety, epilepsy, sleep disturbances,
motor coordination impairment, gastrointestinal disorders or
intellectual disability. Its aetiology remains partially resolved, as
70\% of cases remain sporadic, highlighting the polygenic and
environmental complexity of the disease. To date, no pharmacological
treatment exists for the core social symptoms of autism. Clinical trials
failed because of an important placebo effect, lack of efficacy and the
large diversity of patients. New advances need to overcome these
challenges. First, subtypes of patients determined on their genetic or
neuropathological mechanism profile should be tested rather than
patients from the whole spectrum. Second, the identification of robust
therapeutic targets and the development of potent drugs should thwart
the placebo effect. More than a thousand of candidate genes are listed
in the Simons Foundation Autism Research Initiative (SFARI) database
(\emph{https://gene.sfari.org}), and rather than a single mutation,
accumulation of deleterious alleles and copy number variants may
underlie the pathological process among affected individuals (Manoli and
State, 2021). Recent genome wide association studies of large ASD
cohorts robustly identified hundreds of candidate genes that fall in two
main convergent neurobiological mechanisms, namely `gene expression
regulation' or `neuronal communication, signalling or plasticity' (De
Rubeis et al., 2014; Satterstrom et al., 2020; Pintacuda et al., 2023).
G protein-coupled receptors (GPCRs) are master regulators of these
convergent mechanisms. In addition, their fine-tuned pharmacology and
their diversity could represent the greatest therapeutic options for ASD
to lead to successful clinical trials. In this review, we demonstrate
why this receptor family meets all criteria of convergent therapeutic
targets for ASD.

\textbf{GPCRs and their signalling are dysregulated in ASD}

Canonical GPCRs display an extracellular domain composed of the
N-terminus and three extracellular loops (EL1-3) that connect seven
transmembrane (TM) helices. Occupancy of the ligand binding pocket leads
to conformational changes of the helices that transmit activation to the
intracellular domain, composed of three intracellular loops (IL1-3) and
a C-terminus region with an 8\textsuperscript{th} helix parallel to the
plasma membrane (\textbf{Figure 1} ). This intracellular domain is
involved in the recruitment and activation of direct transducers that
activate downstream intracellular signalling. GPCRs are translated
inside the membrane of the endoplasmic reticulum (ER), thus actively
exported to the plasma membrane. Due to their molecular complexity, they
are often prone to misfolding or lack of ER export, which may result in
cell toxicity (Beerepoot et al., 2017). Most GPCRs and their transducers
are expressed in the Central Nervous System (CNS) (Regard et al., 2008;
Marti-Solano et al., 2020), with over two hundred variants in GPCR genes
associated with ASD (SFARI). Furthermore, transcriptomic data and
meta-analysis from prefrontal cortex tissue showed that GPCRs are the
most frequently dysregulated genes in ASD and revealed around 200 GPCRs
potentially linked to ASD (Hormozdiari et al., 2015; Monfared et al.,
2021). Among these, serotonin \emph{HTR2A} , adenosine \emph{ADORA1} and
adrenergic \emph{ADRA1D} are the most dysregulated.

GPCRs and their downstream signalling pathways, are also affected in
ASD. Upon activation, GPCRs couple to several heterotrimeric G protein
(\selectlanguage{greek}α, β \selectlanguage{english}and \selectlanguage{greek}γ \selectlanguage{english}subunits) and recruit \selectlanguage{greek}β-\selectlanguage{english}arrestins that impact on the
kinetics of intracellular signalling pathways such as extracellular
signal-regulated kinases (ERK) or promote receptor endocytosis
(\textbf{Figure 1A} ). G\selectlanguage{greek}α\selectlanguage{english}\textsubscript{s/olf} protein activates
adenylyl cyclases that hydrolyse ATP into cAMP, which is degraded by
phosphodiesterases into AMP. cAMP activates exchange protein activated
by cAMP (EPAC), calcium ion channels and protein kinase A (PKA).
Conversely, G\selectlanguage{greek}α\selectlanguage{english}\textsubscript{i/o} inhibits adenylyl cyclases and cAMP
production. G\selectlanguage{greek}α\selectlanguage{english}\textsubscript{q/11} proteins activate phospholipase C\selectlanguage{greek}β,
\selectlanguage{english}which hydrolyses phosphatidylinositol-4,5-bisphosphate
(PIP\textsubscript{2}) into diacylglycerol (DAG) and
inositol-1,4,5-triphosphate (IP\textsubscript{3}). Released
IP\textsubscript{3} binds to ryanodine receptors on the endoplasmic
reticulum, which leads to calcium release from this subcellular
location. Both calcium and DAG activate protein kinase C (PKC) and its
effectors, such as Akt (or protein kinase B) or ERK.
G\selectlanguage{greek}α\selectlanguage{english}\textsubscript{12/13} activates Rho guanine nucleotide exchange Factor
(GEF) and RhoA, which acts on the cytoskeleton to promote neurite
formation. G\selectlanguage{greek}βγ \selectlanguage{english}proteins also participate in downstream signalling
through activation of G protein-coupled inwardly-rectifying potassium
channels (GIRK) or other channels. The most affected transducers in ASD
are G\selectlanguage{greek}α\selectlanguage{english}\textsubscript{i} and G\selectlanguage{greek}α\selectlanguage{english}\textsubscript{12/13} proteins (Monfared
et al., 2021). Following activation, GPCRs are internalized in
intracellular compartments (e.g., endosomes) together with their
transducers and participate in the signalling cascades (Vilardaga et
al., 2022). Downstream intracellular signalling network leads to
integrated cellular processes, translation of specific mRNAs (Musnier et
al., 2012; Le\selectlanguage{ngerman}ón et al., 2014; Tréfier et al., 2018) and gene
transcription via the transcription factor cAMP-responsive element
binding protein (CREB), among many others. Globally, GPCRs are key
master upstream regulators of Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin, ERK, PKC, Pi3K/Akt, CREB,
PTEN and mTOR intracellular pathways that are convergently dysregulated
in ASD (O'Roak et al., 2012; Gazestani et al., 2019; Pintacuda et al.,
2023). In an attempt to estimate their potential as master regulators of
genes involved in ASD, we confronted `Kyoto Encyclopedia of Genes and
Genomes' (KEGG) pathways, 'Reactome' pathways, `Gene Ontology' (GO)
terms and our knowledge (\textbf{supporting information} ) to the 1045
ASD candidate genes from the SFARI list. We identified 23 GPCRs and 129
genes linked to GPCRs (\textbf{Figure 2} , \textbf{Table S1} ) in this
list, accounting for at least 15\% of the candidate genes.
Interestingly, 2 orphan GPR37 and GPR85 and 4 olfactory OR1C1, OR2M4,
OR2T10, OR52M1 receptors with relatively unknown functions in the brain
belong to this list. This proportion might increase with future gain of
knowledge on signalling and cellular processes under the control of
GPCRs, especially their effect on translation and transcription.

\textbf{GPCRs are the most druggable targets for ASD}

GPCRs respond to diverse natural signals ranging from photons, amino
acids, peptides up to large glycosylated proteins. This receptor family
comprises more than 800 GPCRs, subdivided into five classes according to
the International Union of Basic \& Clinical Pharmacology (IUPHAR)
nomenclature and classification (Alexander et al., 2021): the largest
rhodopsin-like class A, the secretin/class B, the glutamate/class C, the
Frizzled class and the adhesion class. Sensory GPCRs (olfactory, vision,
taste and pheromone receptors), which account for most GPCR genes, are
included mostly in class A and a few in class C. Although hundreds of
GPCRs remain without any identified ligand, 24\% of these so-called
``orphan receptors'' (including olfactory receptors) are dysregulated in
ASD. In addition to natural ligands, drugs can modulate GPCR activity,
inducing diverse pharmacological profiles (\textbf{Figure 1B} ). They
can either be chemical compounds, peptides, large autoantibodies and
more recently, antibody fragments (Muji\selectlanguage{polish}ć-\selectlanguage{english}Deli\selectlanguage{polish}ć \selectlanguage{english}et al., 2014).
Orthosteric agonists, inverse agonists and antagonists occupy the
natural ligand binding pocket and activate, inactivate the receptor
and/or prevent the binding of the endogenous ligand respectively. In
contrast, by binding to allosteric sites, positive (PAM) or negative
(NAM) allosteric modulators enhance or decrease GPCR activity only in
the presence of agonists. While their intrinsic instability has been a
major issue for resolving their 3 dimensional-structure, many GPCR
structures in inactive, intermediate and active conformations became
available with the development of cryo-electron microscopy. These recent
advances facilitate \emph{in silico} design of more selective drug.
Finally, GPCRs are modulated by interacting partners. They can form
cell-specific homo- or hetero-oligomers depending on the GPCR
composition and subcellular localisation in a particular cell type. Each
GPCR in the oligomer or expressed in the same cell may influence the
signalling network of other GPCRs, opening a new area of GPCR
pharmacology. Their activity is also modulated through reciprocal
functional interactions with scaffolding protein partners (\emph{e.g.,}
Shank1-3), ion channels and tyrosine kinase receptors. Different ligands
may favour one coupling or protein partner interaction, leading to what
is called `signalling bias' (\textbf{Figure 1A} ). This pharmacological
property of GPCRs is of great interest for therapeutic applications, as
one biased drug can induce one signalling cascade over the others, hence
possibly avoiding side effects. Considering their unique
characteristics, GPCRs offer many levels of leveraging as therapeutic
targets for neurological disorders.

More than 30\% of the drugs approved on the global market target a GPCR
in various disorders, including neurological conditions for 20\% of them
(Hauser et al., 2017; Alexander et al., 2021). This proportion will
increase as nearly half of GPCRs in the CNS remain orphan. So far, no
pharmaceutical agent has reached the market to improve primary symptoms
of ASD. The few GPCRs tested in clinical trials are
mGlu\textsubscript{5}, GABA\textsubscript{B}, V\textsubscript{1A} and
CB\textsubscript{1} and are all listed in the SFARI database
(\textbf{Table S1} ). Therefore, the therapeutic potential of GPCRs has
only begun. In this review, we explored the therapeutical values of
these 23 GPCRs identified in the SFARI list, in addition to
5-HT\textsubscript{2A}, 5-HT\textsubscript{6} and
5-HT\textsubscript{7}receptors for their relevance to ASD
(\textbf{supporting information} ). We studied the potential deleterious
effect of the different variants associated with ASD, their
dysregulation in ASD post-mortem tissues and their pharmacogenomic to
conclude on their potential involvement in the aetiology of ASD
(\textbf{Table S2} ). We addressed the behavioural consequences of their
genetic and pharmacological manipulation in animal models (\textbf{Table
S3} ) and whether these models recapitulate the validity criteria
defined for psychiatric diseases applied to ASD (Chadman et al., 2019).
We analysed their pharmacological landscape (\emph{e.g.,} specific drugs
on the market or in clinical trials), the availability of GPCR
structures, and when available, the results of clinical trials
(\textbf{Table S4} ). We also reported their known downstream signalling
and their interacting partners (\textbf{Table S5} ). Finally, we
reviewed their cellular localisation and levels of expression in the CNS
(\textbf{Figures 2-3, Table S6} ). Based on these evidences, we conclude
on the therapeutic potential of these 26 GPCRs (\textbf{Figure 4} ).

Oxytocin and vasopressin receptors

Oxytocin (OT) was first described in the 1960s for its effects on
reproduction and maternal behaviours (Froemke and Young, 2021).
Extremely conserved in mammals, OT and its paralog arginine vasopressin
(AVP) in the CNS modulate social recognition and memory, defensive
behaviours, trust, empathy and maternal attachment (Macdonald and
Macdonald, 2010; Rae et al., 2022). Mice lacking OT peptide
(\emph{Oxt}KO mice) may display impairment in social memory and
aggressive and anxious-like behaviours (\textbf{Table S3} ), but these
phenotypes are inconsistent across different laboratories and parental
genotypes. Administration of OT in the lateral ventricles or in the
medial amygdala of \emph{Oxt} KO mice normalizes anxious-like behaviour
and restores social recognition (Ferguson et al., 2001; Mantella et al.,
2003). Lastly, Brattleboro rats, which carry a frameshift deletion in
the\emph{Avp} gene leading to the lack of AVP, display defective social
preference (Surget and Belzung, 2008) whereas \emph{Avp} KO mice are
lethal, in the absence of peripheral AVP administration (Zelena, 2017).

Several studies have associated the OT-AVP family with autism spectrum
disorders, because they regulate social behaviours and OT plasma levels
are lower in ASD children (Cataldo et al., 2018; John and Jaeggi, 2021;
Rae et al., 2022). Logically, many studies have investigated the
therapeutic potential of OT or AVP for ASD. Intranasal administration of
OT at low dose improves emotion recognition in young men with autistic
condition (Guastella et al., 2010a). Furthermore, OT inhalation
increased trust and interactions in adults with ASD (Andari et al.,
2010) and reduced severe repetitive behaviours (Hollander et al., 2003).
Unexpectedly, administration of OT failed to improve social abilities
over placebo in phase 2 clinical trials and induced frequent side
effects in several studies (Leppanen et al., 2018; Sikich et al., 2021;
Witte et al., 2022). In fact, OT administration may not be effective in
all patients, but rather only in subtypes of patients with OT
deficiency. Interestingly, administration of AVP in humans improves the
recognition of happy and angry social faces compared to neutral faces
(Guastella et al., 2010b). A phase 2 clinical trial showed that four
weeks of intranasal administration of AVP in 30 ASD children improved
their social skills and reduced anxiety and repetitive behaviours, with
minimal side effects (Parker et al., 2019). Whereas AVP remains to be
tested in a larger cohort of patients, the first results indicate that
it might be more efficient than OT to provide pro-social effects. Thus,
despite mitigated results, the OT-AVP family remains of interest for
ASD. Actually, OT and AVP might not be idealistic treatments as both
bind and activate with nanomolar affinity the four highly conserved
oxytocin receptor (OTR), vasopressin V\textsubscript{1A} and
V\textsubscript{1B} receptors in the CNS and V\textsubscript{2} receptor
in the periphery. Therefore, in the following section, we review the
therapeutic potential of OTR, V\textsubscript{1A} and
V\textsubscript{1B} for treatment in ASD.

\textbf{Oxytocin receptor}

The oxytocin receptor gene (\emph{OXTR} ) spans over 4 exons and encodes
5 splicing transcript variants that differ in their 5' untranslated
region (UTR) leading to only one receptor, OTR. Decades of research
identified several agonists of OTR (\textbf{Table S4} ), such as the
potent peptide agonist Thr\textsuperscript{4}Gly\textsuperscript{7}-OT
(TGOT) (Elands et al., 1988), the G\selectlanguage{greek}α\selectlanguage{english}\textsubscript{q}-biased agonist
carbetocin (Passoni et al., 2016) and the first chemical agonist LIT001
(Frantz et al., 2018). However, all these ligands also bind vasopressin
receptors. OTR expression is found in CNS regions critical for the
regulation of social behaviour and emotion (\textbf{Figure 3} ) and
might be sexually dimorphic depending on the brain region and species
(Dumais et al., 2013). In humans, \emph{OXTR} transcript levels peak
after birth, during all infancy and reduce in adolescents and adults
(Kang et al., 2011). This corresponds to oxytocinergic neuron
development in the same critical period as observed in mice (Soumier et
al., 2022). OTR is involved in complex social behaviours, like maternal
care, social recognition, aggression, mating but also in pair bonding,
empathy and could exert anxiolytic effects (Jurek and Neumann, 2018).
\emph{Oxtr} KO mice have an autism-like phenotype, with both social
deficits and stereotyped behaviours whereas heterozygous mice express
only social deficits. \emph{Oxtr} KO also display deficits in social
memory and pup vocalisations following maternal separation and
aggressive behaviour (\textbf{Table S3} ). Whereas increased
self-grooming, anxious-like behaviours and cognitive inflexibility have
been observed, results are inconsistent through laboratories or mouse
lines. However, \emph{Oxtr}KO in monogamous prairie voles leads to
deficits in social novelty and increased repetitive behaviours, but no
impairment in social interactions, vocalisations or maternal behaviour
(Horie et al., 2019; Berendzen et al., 2022). Interestingly,
intraventricular administration of OT or AVP restores the social
deficits in \emph{Oxtr} KO mice via V\textsubscript{1A} receptors (Sala
et al., 2011). This finding highlights the crosstalk within this GPCR
family. More than twenty variants in the \emph{OXTR} gene have been
associated with ASD (\textbf{Table S2} ). Interestingly, variants are
mostly located outside the receptor coding region, leading to potential
receptor expression dysregulation.

\textbf{Vasopressin V\textsubscript{1A} and
V\textsubscript{1B}receptors}

AVP, well known as the antidiuretic hormone via the activation of
V\textsubscript{2} receptors, binds V\textsubscript{1A} and
V\textsubscript{1B} receptors in the CNS. \emph{AVPR1A} and\emph{AVPR1B}
genes encode each, only one transcript variant and the
V\textsubscript{1A} and V1\textsubscript{B} receptors respectively.

\textbf{V\textsubscript{1A} receptor} is involved in maternal care,
social recognition, affiliative behaviour and pair bonding (Koshimizu et
al., 2012). Administration of the V\textsubscript{1A} antagonist
d(CH2)5Tyr(Me)AVP into the medial amygdala of rats affects maternal
memory (Nephew and Bridges, 2008). Furthermore, \emph{Avpr1a} KO mice
and hamsters display defective social memory, interaction and
communication, reduced anxious-like behaviours and inconsistent levels
of aggressive behaviour across species (\textbf{Table S3} ). Several
studies have associated the length of the promoter and the 5'UTR of
the\emph{AVPR1A} gene, which regulate V\textsubscript{1A} expression
levels, with important social deficits. Indeed, most variants associated
with ASD risk are identified in these regions (\textbf{Table S2} ),
which either influence human relationships and altruism (Walum et al.,
2008; Meyer-Lindenberg et al., 2009), personality in primates (Hopkins
et al., 2012) or social behaviour in rodents (Hammock et al., 2005).
Recently, administration of the selective V\textsubscript{1A}
antagonists RG7713 or balovaptan improved socialisation and
communication in men with ASD (Umbricht et al., 2017; Bolognani et al.,
2019; Schnider et al., 2020). Despite these promising results in phase 2
clinical trials, balovaptan failed to improve social abilities over
placebo in phase 3 (Jacob et al., 2022). Further investigations are
still required to understand the therapeutic potential of
V\textsubscript{1A.}as it is not yet clear whether it should be
activated or inhibited to improve social skills.

\textbf{V\textsubscript{1B} receptor} deletion in mice (\emph{Avpr1b}KO)
leads to increased dominance, decreased aggressive behaviour and
vocalisations and impaired motivation and social memory (\textbf{Table
S3} ). Three independent studies have identified variants in
the\emph{AVPR1B} gene linked to ASD (\textbf{Table S2} ), mood disorders
and aggressive behaviour. Accordingly, administration of the antagonist
nelivaptan (\textbf{Table S4} ) normalizes aggressive, chasing and
anxious-like behaviours in rodents (Blanchard et al., 2005; Salom\selectlanguage{ngerman}é et
al., 2006). Oral administration of nelivaptan is currently in clinical
trials for anxiety and depression.

In conclusion, data in animals and humans support that OTR and
V\textsubscript{1A} receptors may be involved in the aetiology of autism
and are major therapeutic targets for ASD, whileV\textsubscript{1B}might
be of interest for aggressive and anxious-like behaviours. Nevertheless,
so far, clinical trials failed to bypass the placebo effect observed in
patients. Regarding their conservation, their crosstalk and the
existence of homo- and hetero-oligomers of these three receptors
(Terrillon et al., 2003; Dekan et al., 2021), further investigations are
needed to identify the most suitable targets (\emph{e.g.} , which
receptor or oligomer, which signalling pathway) and respective ligands
of this family.

Metabotropic glutamate and GABA receptors

Glutamate and \selectlanguage{greek}γ-\selectlanguage{english}aminobutyric acid (GABA) are the two major
neurotransmitters in the CNS. They bind their cognate class C GPCRs,
metabotropic glutamate mGluRs and GABA\textsubscript{B} receptors
respectively. In contrast to class A GPCRs, glutamate and GABA bind to
the large extracellular N-terminal domain called the Venus Fly Trap,
which closes upon activation. In addition, they form constitutive
oligomers, which lead to specific rearrangements of subunits during
activation. They are mainly expressed in pre- and postsynaptic
compartments in the brain (\textbf{Figures 2-3} ) and participates in
the excitatory and inhibitory balance in the CNS (Nelson and Valakh,
2015), which is hypothesised to be dysregulated in ASD.

\textbf{mGlu\textsubscript{5}}

The \emph{GRM5} gene encodes two splice variants of
mGlu\textsubscript{5} (mGlu\textsubscript{5a} and
mGlu\textsubscript{5b}), with the mGlu\textsubscript{5b} receptor
expressed predominantly during the adult stage (\textbf{Table S6} ).
Activation of mGlu\textsubscript{5} induces synaptic plasticity, which
requires \emph{de novo} mRNA translation through phosphorylation of
eIF2\selectlanguage{greek}α \selectlanguage{english}(Di Prisco et al., 2014). \emph{Grm5} KO mice display ASD-related
core symptoms (\textbf{Table S3} ), deficits in social interaction,
increased stereotyped and compulsive behaviours. Furthermore, they show
hyperactivity, reduced anxious-like behaviours and sensorimotor gating
deficits (Brody et al., 2004; Xu et al., 2021). Five independent studies
have identified over twenty rare variants in the \emph{GRM5} gene of ASD
patients (\textbf{Table S2} ), highlighting \emph{GRM5} as one of the
most susceptible genes in ASD (Nisar et al., 2022). Alterations in
mGlu\textsubscript{5} receptor signalling or expression affect synaptic
and neuronal development, trademarks of ASD and intellectual disability
(D'Antoni et al., 2014). Higher mGlu\textsubscript{5} protein expression
was reported in different brain regions including cerebellar vermis
region and superior frontal cortex in children with ASD (Fatemi et al.,
2011) and in prefrontal cortex of patients with monogenic Fragile X
syndrome (FXS) (Lohith et al., 2013). In contrast, lower
mGlu\textsubscript{5} mRNA and protein expression was reported in the
dorsolateral prefrontal cortex of ASD patients (Chana et al., 2015).
Thus, administration of the selective antagonist mavoglurant and the NAM
basimglurant in the FXS mouse model (\emph{Fmr1} KO) (\textbf{Table S4}
) improved its broad range of phenotypes (Scharf et al., 2015). However,
administration of these compounds failed to provide similar therapeutic
benefits in FXS patients in phase 2b/3 clinical trials (Jacquemont et
al., 2011, 2014; Lozano et al., 2015). These compounds are still in
clinical trials for dyskinesia, obsessive--compulsive disorders and
depression. Altogether, data favour the role of mGlu\textsubscript{5} in
ASD pathogenesis and explain why it is one of the first GPCRs targeted
for ASD. However, targeting mGlu\textsubscript{5} even with a selective
negative allosteric modulator did not pass the placebo effect in
patients. This might be attributed to differences in receptor
expression.

\textbf{mGlu\textsubscript{7}}

The \emph{GRM7} gene encodes two isoforms (mGluR\textsubscript{7a},
mGluR\textsubscript{7b}) that differ in their C-terminus, potentially
leading to different protein-protein interactions and receptor coupling.
mGlu\textsubscript{7} is expressed during the critical
neurodevelopmental period, when it augments synapse formation and
stabilisation (Song et al., 2021). Compared to other mGluRs,
mGlu\textsubscript{7} has less affinity for glutamate, hence is
considered as an ``emergency brake''. mGlu\textsubscript{7} are
predominantly localised at presynaptic sites that regulate
neurotransmitters release of glutamate or GABA. Interestingly, the
non-selective mGlu\textsubscript{4,6,7} agonist L-AP4 negatively
regulates glutamate or GABA release whereas the selective PAM AMN082
positively affects the extracellular glutamate levels and negatively the
GABA levels (Manahan-Vaughan and Reymann, 1995; Mitsukawa et al., 2005;
Li et al., 2008). \emph{Grm7} KO mice display intact social interaction,
but social memory deficits (\textbf{Table S3} ), which might be
explained by their global learning defects. Overall, \emph{Grm7} KO mice
and mice carrying the Ile154Thr mGlu\textsubscript{7} mutation
identified in ASD patients recapitulate comorbid symptoms, such as
anxious-like behaviours, motor coordination impairment and seizures
(Fisher et al., 2020, 2021). 21 SNPs and CNVs in the \emph{GRM7} gene
have been associated with ASD (\textbf{Table S2} ). In particular, the
Ile154Thr, Arg658Trp and Thr675Lys mutants lead to reduced
mGlu\textsubscript{7}surface expression and/or degradation. Dysregulated
levels of mGlu\textsubscript{7} results in lack of axonal growth due to
altered cAMP-PKA-ERK signalling and reduced number of synapses in
primary neuronal cultures, which is rescued by the PAM AMN082 (Song et
al., 2021). This is in line with reduced expression of
mGlu\textsubscript{7}in post-mortem motor cortex samples from patients
with Rett syndrome (RTT) and in a mouse model of RTT (\emph{Mecp2} KO)
(Bedogni et al., 2016; Gogliotti et al., 2017). In conclusion,
mGlu\textsubscript{7} is a promising target as it could contribute to
ASD pathogenesis. Furthermore, selective agonists or PAM (e.g., AMN082)
already exist and normalise comorbid symptoms in mouse models of ASD via
the regulation of glutamate and GABA release, and possibly in patients
as well.

\textbf{GABA\textsubscript{B} receptor}

Metabotropic GABA\textsubscript{B} receptors are obligatory
hetero-oligomers of GABA\textsubscript{B1} and
GABA\textsubscript{B2}through their C-terminus coiled-coiled domain.
Presynaptic GABA\textsubscript{B} receptors suppress neurotransmitter
release whereas postsynaptic receptors induce slow inhibitory
postsynaptic currents, which shunt the excitatory currents (L\selectlanguage{ngerman}üscher et
al., 1997). GABA\textsubscript{B} receptor deletion in mice
(\emph{Gabbr1-Gabbr2}double KO) leads to stress-induced social
withdrawal, emotional behavioural disturbances and increased anxious-
and anti-depressive-like behaviours (\textbf{Table S3} ). The effect of
\emph{Gabbr2} deletion alone has not been reported yet. However,
\emph{Xenopus tropicalis}tadpole larvae carrying the Ala567Thr,
Ser695Ile and Ile705Asn GABA\textsubscript{B2} mutants identified in ASD
and epileptic patients, display increased seizure-like behaviour and
altered swimming patterns that are partially rescued by the selective
GABA\textsubscript{B}agonist baclofen (Yoo et al., 2017). Of note, when
expressed in heterologous HEK293 cells, these three mutants disrupt
GABA\textsubscript{B} activation. Four genomic studies revealed the
association of the \emph{GABBR2} gene with ASD and RTT (\textbf{Table
S2} ). Administration of baclofen normalises the behaviours observed in
a mouse model of FXS, in an idiopathic BTBR mouse model of ASD and in
the C58 inbred mouse strain (Henderson et al., 2012; Silverman et al.,
2015). Despite its first beneficial effect and its good tolerance in FXS
and ASD patients, baclofen clinical trials were discontinued after phase
2 for its lack of efficacy (Berry-Kravis et al., 2012;
Veenstra-VanderWeele et al., 2017). Nevertheless, baclofen is currently
tested as an adjuvant therapy to risperidone for irritability
(Mahdavinasab et al., 2019). Finally, in agreement with the unbalanced
GABA and glutamate transmission hypothesis in ASD, reduced expression
levels of the GABA\textsubscript{B} receptor were observed in the
cerebellum, in the cingulate cortex and in the fusiform gyrus of ASD
patients (Fatemi et al., 2009; Oblak et al., 2010).

In conclusion, GABA\textsubscript{B} receptor remains a promising
therapeutic target for ASD according to the genomic and genetic data in
patients and in animal models. However, its efficacy might be greater in
combination with the administration of other ligands, such as
risperidone for irritability or drugs targeting mGluRs to restore the
excitatory and inhibitory balance.

Biogenic amine receptors

Biogenic amine receptors are class A GPCRs that interact with endogenous
aminergic ligands, such as adrenaline, noradrenaline, dopamine and
serotonin (5-hydroxytryptamine, 5-HT).

\textbf{Dopamine receptors}

Dopaminergic D\textsubscript{1}-like (D\textsubscript{1} and
D\textsubscript{5}) and D\textsubscript{2}-like (D\textsubscript{2},
D\textsubscript{3} and D\textsubscript{4}) receptors regulate broad
functions: locomotion including voluntary movement, reward processing,
learning, motivated behaviour, action selection, sleep, attention, and
decision making (Mishra et al., 2018), some of which, when dysregulated,
are comorbid symptoms of ASD (DiCarlo et al., 2022).

\textbf{D\textsubscript{1} receptors} are particularly enriched in
D\textsubscript{1} striato-nigral GABAergic medium spiny neurons of the
striatum. They have the lowest dopamine affinity among all the
dopaminergic receptors, suggesting that they are activated by high
phasic dopamine release, while D\textsubscript{2}-like receptors might
detect low tonic dopamine levels (Beaulieu and Gainetdinov, 2011).
D\textsubscript{1} receptor may have a role, although controversial, in
social behaviour (Scerbina et al., 2012; Campi et al., 2014). Rat
carrying the Ile116Ser mutation in the D\textsubscript{1} receptor
exhibited ASD-like social symptoms with reduced social interaction
(sociability and social novelty) and ultrasonic vocalisations in pups
while calling their mothers (\textbf{Table S3} ). However, no
stereotyped behaviours were observed for this rat model. This mutant has
reduced expression at the cell surface and impaired G protein coupling.
Administration of the D\textsubscript{1} receptor antagonist SCH23390
ameliorated stereotyped behaviours in mice lacking the tyrosine
hydroxylase that catalyses dopamine synthesis (Chartoff et al., 2001).
Furthermore, administration of the approved antipsychotic antagonist
flupentixol at low doses reduced the rate of deliberate self-harm
injuries in schizophrenic patients (Ruhrmann et al., 2007; Witt et al.,
2021). The antagonist ecopipam is currently in phase 2 clinical trials
for the treatment of Tourette's syndrome, characterised by repetitive
tics (Gilbert et al., 2018). Conversely, excessive activation of the
D\textsubscript{1} receptor induces an autistic-like phenotype in WT
mice (Lee et al., 2018). Lastly, one study reported that three common
SNPs located in the 5'UTR of the \emph{DRD1} gene (\textbf{Table S2} )
are associated with severe impairments in social interaction, non-verbal
communication and increased motor stereotypies.

\textbf{D\textsubscript{2} receptors} encoded by the \emph{DRD2} gene
comprise two splicing isoforms, short D\textsubscript{2S} and long
D\textsubscript{2L} differing in their IL3. D\textsubscript{2S} serves
as an auto-receptor regulating dopamine release and dopamine synthesis
while D\textsubscript{2L} is a postsynaptic receptor (Negyessy and
Goldman-Rakic, 2005). These receptors are mainly expressed in neurons,
with the highest levels in GABAergic indirect
D\textsubscript{2}striato-pallidal medium spiny neurons of the striatum,
but also in astrocytes and oligodendrocytes (\textbf{Figure 2} ).
D\emph{rd2} KO mice show great impairments in social behaviour
(sociability and social novelty), impaired social olfaction and
stereotyped behaviours (\textbf{Table S3} ). Moreover, \emph{Drd2}
heterozygous mice exposed to early maternal separation stress also
display social interaction deficits and stereotyped behaviours. This
phenotype seems exclusively mediated by the dorsal striatum as specific
knock-down of the D\textsubscript{2} receptor in this structure is
sufficient to recapitulate all the behavioural impairments reported in
\emph{Drd2} KO mice (Lee et al., 2018). Conversely, D\textsubscript{2}
receptor overexpression in the striatum and olfactory tubercle revealed
impairment in sociability only in female mice and vocalisation. Among
the dopamine receptors, the \emph{DRD2} gene displays the highest number
of SNPs associated with ASD (\textbf{Table S2} ). Currently, the only
available approved treatments for ASD patients are antipsychotics
(\textbf{Table S4} ), such as aripiprazole or risperidone, which
antagonise D\textsubscript{2} receptor in addition to other GPCRs, to
treat irritability, aggressive and repetitive behaviours (McDougle et
al., 2005; Varni et al., 2012). Additionally, two other
D\textsubscript{2} antagonists, pimozide and olanzapine, are
antipsychotics used in clinics for schizophrenia and Tourette's
syndrome, leading to potential amelioration of speech impairment
(Maguire et al., 2004; Pringsheim and Marras, 2009).

\textbf{D\textsubscript{3} receptor} expression is conserved between
humans and rodents and controls habituation to novelty (Mishra et al.,
2018). \emph{Drd3} KO mice display hyperactive and addictive behaviours,
with particular vulnerability to alcohol and drug abuse (\textbf{Table
S3} ), but their social skills or stereotyped behaviours have not been
reported yet. Three independent studies identified three SNPs in
the\emph{DRD3} gene associated with ASD (\textbf{Table S2} ). The
antipsychotic cariprazine, a partial agonist for D\textsubscript{3} that
also binds D\textsubscript{2} receptors with lower affinity, is approved
for the treatment of schizophrenia and bipolar disorder. Interestingly,
administration of cariprazine improved social behaviours in a
dose-dependent way in male rat models of ASD exposed to valproic
acid\emph{in utero} (Román et al., 2021), which makes this ligand a
potential treatment for ASD.

In conclusion, alteration in any of these three dopaminergic receptors
result in autistic-like symptoms in animal models and in genetic
association with ASD. However, approved ligands targeting the
D\textsubscript{2} receptor are already on the market to ameliorate
autistic symptoms, especially repetitive behaviours, and could be tested
for social symptoms, highlighting this receptor as a promising target
for ASD treatment. Nonetheless, other dopamine receptors might be of
interest; for example, the less known \emph{DRD5} gene that display the
highest number of distinct missense and loss of function variants in the
general population (Hauser et al., 2018).

\textbf{Serotonin 5-HT\textsubscript{1B}}

Dysregulation in 5-HT levels in different CNS structure has been
observed in ASD (Pourhamzeh et al., 2022) while enhanced 5-HT release
restores social deficit in several ASD mouse models (Walsh et al.,
2021). The dup15q11-q13 mouse model of ASD displays reduced
serotoninergic activity of the dorsal raphe nucleus, associated with low
5-HT levels in all CNS regions and impaired social interaction (Farook
et al., 2012; Nakai et al., 2017). As more than 25\% of the ASD patients
show increased 5-HT blood levels, 5-HT is considered as a biomarker for
a subgroup of patients (Gabriele et al., 2014; Muller et al., 2016). All
14 serotonin receptors encode a GPCR, except the channel receptor
5-HT\textsubscript{3}. They modulate cognition, memory, sleep, appetite,
respiration, thermo-regulation and mood (Berger et al., 2009).
5-HT\textsubscript{1B} belongs to the 5-HT\textsubscript{1} receptor
family that are encoded by 7 genes (\emph{HTR1A-F} ). It exerts a
consistent effect on anxious-like behaviours, as administration of the
selective full agonist CP94253 or antagonists SB 216641 and GR 127935
(\textbf{Table S4} ) leads to anxiogenic or anxiolytic effect in
rodents. In addition, administration of CP94253 reduced aggressive
behaviour in resident male mice, whereas anpirtoline, that also targets
5-HT\textsubscript{3} channels, restored isolation-induced impairments,
increased pain threshold and exerted anti-depressive effects in mice
(Schlicker et al., 1992; Fish et al., 1999). In agreement with
pharmacological studies, \emph{Htr1B} KO mice display decreased
anxious-like behaviours, exacerbated aggressive behaviour, deficits in
maternal behaviour, improved cognitive flexibility and vulnerability to
drug abuse (\textbf{Table S3} ). So far, only two variants associated
with ASD have been reported from two independent studies (\textbf{Table
S2} ).

Beside the \emph{HTR1B} gene, evidence has highlighted that
the\emph{HTR2A} gene fulfils the SFARI criteria as a strong candidate
and targeting 5-HT\textsubscript{6} or 5-HT\textsubscript{7} improve
core symptoms in mouse models of ASD (\textbf{supporting information} ).
Thus, potentially the 14 serotonin receptors are of interest for ASD.
Highly promising drugs targeting multiple 5-HT receptors, such as
arylpiperazine derivative drugs (Lacivita et al., 2021) and the first
5-HT\textsubscript{7} biased agonist (El Khamlichi et al., 2022) could
be tested to improve core ASD symptoms. Currently, antipsychotic
(aripiprazole and risperidone) administration to treat irritability,
aggressive and repetitive behaviours in ASD patients partially activates
5-HT\textsubscript{1A} and inhibits 5-HT\textsubscript{2A}.

\selectlanguage{greek}\textbf{β2-adrenoceptor}\selectlanguage{english}

Noradrenaline and adrenaline activate the \selectlanguage{greek}α\selectlanguage{english}\textsubscript{1},
\selectlanguage{greek}α\selectlanguage{english}\textsubscript{2}, \selectlanguage{greek}β\selectlanguage{english}\textsubscript{1}, \selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} and
\selectlanguage{greek}β\selectlanguage{english}\textsubscript{3}-adrenoceptors with different potencies. In the CNS,
they control cognition, memory, emotions and stress-induced behaviours.
Only the \emph{ADRB2} gene is present in the SFARI list and encodes the
\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2}-adrenoceptor. Despite its vital cardiac
function,\emph{Adrb2} KO mice are fertile and viable and display
increased anxious-like behaviours and decreased depressive-like
behaviours (\textbf{Table S3} ). Increased adrenergic neuron activity
from the locus coeruleus or increased noradrenaline plasma concentration
has been associated with aberrant attention and decreased interest in
ASD individuals (Bast et al., 2018; Beversdorf, 2020). Accordingly, two
common SNPs associated with ASD (\textbf{Table S2} ) show enhanced
isoproterenol agonist-induced response. Furthermore, studies have
suggested an association between prenatal exposure to
\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2}-adrenoceptor agonists and ASD (Gidaya et al., 2016).
Interestingly, administration of the approved
\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2}/\selectlanguage{greek}β\selectlanguage{english}\textsubscript{3}-adrenergic antagonist propranolol
improves verbal responses and social interactions and decreases anxiety
in ASD patients (Hegarty et al., 2017). Therefore, the
\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2}-adrenoceptor is an interesting target for ASD and the
approved drug propranolol (\textbf{Table S4} ) may help to normalise
core social symptoms. Further investigations should also address the
potential interest of the other members of this family, such
as\emph{ADRA1D,} one of the most downregulated genes in the prefrontal
cortex of ASD patients (Monfared et al., 2021) whose targeting with
clonidine improves hyperarousal, hyperactivity and social relationships
in individuals with ASD (Ming et al., 2008).

Other class A receptors

\par\null

\textbf{Adenosine receptors}

Adenosine receptors are divided into four different subtypes, namely
A\textsubscript{1}, A\textsubscript{2A}, A\textsubscript{2B} and
A\textsubscript{3}, among which A\textsubscript{1} and
A\textsubscript{2A} show the highest affinity for adenosine (Alexander
et al., 2021). Brain adenosine receptors have important roles in
different processes, such as neuroplasticity, sleep-wake cycle,
locomotion, and cognition (Wei et al., 2011).

\textbf{A\textsubscript{2A} receptor} encoded by the \emph{ADORA2A}gene,
stimulates glutamate release at presynaptic terminals and myelination by
oligodendrocytes (De Nuccio et al., 2019). Post-synaptic
A\textsubscript{2A} receptors are highly enriched in
D\textsubscript{2}GABAergic striato-pallidal medium spiny neurons of the
striatum to modulate locomotion and anxious-like behaviours (Coelho et
al., 2014). Accordingly, \emph{Adora2a} KO mice display motor impairment
and anxious-like behaviours (\textbf{Table S3} ). A\textsubscript{2A}
might act as a regulator of other GPCRs (\textbf{Table S5} ), as it
forms many different hetero-oligomers with D\textsubscript{2},
mGlu\textsubscript{5,} \selectlanguage{greek}δ \selectlanguage{english}opioid receptors and orphan GPR88 (Ciruela et
al., 2011; Pellissier et al., 2018; Laboute et al., 2020). During brain
development, GABAergic synapses, which release adenosine and ATP in
addition to GABA, are the first synapses to be formed and are crucial
for the construction of the neural network. Activation of
A\textsubscript{2A} receptors is necessary and sufficient to prune
GABAergic synapses during this period (Gomez-Castro et al., 2021). In
contrast, any impairment in A\textsubscript{2A} signalling or expression
may result in GABAergic synapse alteration and cognitive deficits in
adults, as observed in animals administered with
A\textsubscript{2A}antagonists during development. Spontaneous
stereotypies often result from unbalanced cortical glutamatergic and
GABAergic afferences (glutamate hyperactivity) on the striatum and
decreased activation of the efferent subthalamic nucleus, as observed in
ASD patients and animal models (Li and Pozzo-Miller, 2020).
Consistently, administration of the selective A\textsubscript{2A}
agonist CGS21680 normalises aberrant vertical repetitive behaviours in
BTBR and C58 inbred mice (Amodeo et al., 2018; Lewis et al., 2019) via
its action on D\textsubscript{2}medium spiny neurons, which in turn
restore the neurotransmission on efferent subthalamic nucleus. So far,
only one study has associated four SNPs in the \emph{ADORA2A} gene with
autism and severe anxiety (\textbf{Table S2} ).

The \textbf{A\textsubscript{3} receptor} promotes expression of the
serotonin transporter (SERT) to the cell surface (Campbell et al.,
2013). Thus, lack of A\textsubscript{3} signalling decreases SERT cell
surface expression and leads to extracellular accumulation of serotonin,
as observed in ASD patients (see section 4 on serotonin). Actually, two
variants in the \emph{ADORA3} gene are associated with ASD,
(\textbf{Table S2} ) with impaired adenosine binding (Campbell et al.,
2013). Consequently, \emph{Adora3} KO mice show anxious-like and despair
behaviours (\textbf{Table S3} ).

In conclusion, A\textsubscript{2A} shows the greatest promise to
mitigate repetitive behaviours and anxiety in ASD. However, most of
A\textsubscript{2A} agonists have failed in clinical trials (Guerrero,
2018) due to severe side effects, including CNS excitotoxicity
(\textbf{Table S4} ). Only few of them have been approved, such as the
agonist regadenoson. An alternative strategy would be to consider other
members of this family, such as the \emph{ADORA1} gene, which is one of
the most downregulated GPCR genes in patients (Monfared et al., 2021)
and whose targeting in combination to A\textsubscript{2A} agonists
improves stereotyped behaviours (Lewis et al., 2019). A better
specificity could also be achieved by targeting
A\textsubscript{2A}hetero-oligomers such as
D\textsubscript{2}-A\textsubscript{2A}-mGluR\textsubscript{5} to avoid
side effects.

\textbf{Angiotensin AT\textsubscript{2} receptor}

Angiotensin receptors are divided into AT\textsubscript{1} and
AT\textsubscript{2} subtypes. They are activated by different maturation
products of angiotensinogen peptides, namely angiotensin II and III.
Only the AT\textsubscript{2} receptor is implicated in different
neurological disorders such as ASD, schizophrenia, Parkinson's disease
(PD) and Alzheimer's disease (Firouzabadi et al., 2016;
Szczepanska-Sadowska et al., 2022). Its functions in the brain remain
elusive. To date, no data from genetic or pharmacological manipulation
support a role of AT\textsubscript{2} in social or stereotyped
behaviours (\textbf{Table S3} ). However, \emph{Agtr2} KO mice show
impaired reward processing and locomotion. Interestingly, administration
of the AT\textsubscript{2} receptor selective agonist C21/M024 improves
cognition in a mouse model of Alzheimer's disease (Jing et al., 2012).
Finally, four independent studies have associated the \emph{AGTR2} gene
on the chromosome X with ASD and X-linked intellectual disability
(\textbf{Table S2} ). Therefore, together with its unknown expression
and function in the CNS, further studies are required to conclude on the
therapeutic potential of AT\textsubscript{2}.

\textbf{Cannabinoid CB\textsubscript{1} receptor}

Endogenous cannabinoids regulate dopamine circuits that are crucial for
reward processes linked to addiction and for synaptic transmission
through neurotransmitter release modulation (Zhang et al., 2004).
Cannabinoid receptors are composed of CB\textsubscript{1} and
CB\textsubscript{2}. CB\textsubscript{1} mediates the central effects of
cannabis and its derivatives. \emph{Cnr1} KO mice show deficits in
social interaction and communication, two core symptoms of ASD
(\textbf{Table S3} ). They also exhibit anxiogenic, context-dependent
social aggressive and depressive-like behaviours and improved social
memory. Interestingly, administration of endocannabinoids improves
social interactions via the potentiation of reward processes and
inhibition of social anxiety in BTBR and \emph{Fmr1} KO mice (Wei et
al., 2017). Three independent studies have reported more than thirty
variants in the \emph{CNR1} gene associated with ASD (\textbf{Table S2}
). Some states in the USA have already authorised cannabis to treat
self-injurious or aggressive behaviours in ASD patients. While the first
results of clinical trials with a combination of cannabidiol and
delta-9-THC showed no side effects, but mitigated results (Aran et al.,
2021), few case studies have shown improvement of core and comorbid
symptoms in children (Carreira et al., 2022). Thus, further testing is
required and will be obtained with the administration of
CB\textsubscript{1} NAM cannabidiol or endocannabinoid mix that are
currently in clinical trials for ASD (Aran et al., 2021). In conclusion,
multiple evidences highlight CB\textsubscript{1} receptor as one of the
most promising GPCR target to treat core and associated symptoms in ASD.

\textbf{Chemokine CX\textsubscript{3}CR1 receptor}

Chemokine receptors are a vast family of GPCRs involved in the immune
system. Both secreted and membrane-bound chemokine CX3CL1 activate the
C-X3-C motif chemokine receptor 1 (CX\textsubscript{3}CR1 or GPR13). In
humans, the \emph{CX3CR1} gene encodes 4 transcript variants and two
protein isoforms that differ in their N-terminus domain (Marti-Solano et
al., 2020). CX\textsubscript{3}CR1 is expressed on microglia where it is
activated by CX3CL1 release from neurons upon inflammatory response and
during synaptic maturation and pruning (Jung et al., 2000; Soriano et
al., 2002; Zhan et al., 2014). \emph{Cx3cr1} KO mice display social
interaction deficits and increased motor stereotypies (\textbf{Table S3}
), associated with decreased functional brain connectivity from the
prefrontal cortex, similarly to observations in ASD patients. Moreover,
in animals exposed to social isolation, levels of
\emph{Cx3cr1}transcripts were increased in the prefrontal cortex,
nucleus accumbens and hippocampus (Zhou et al., 2020). Three rare
missense deleterious mutations in the \emph{CX3CR1} gene have been
associated with schizophrenia and ASD (\textbf{Table S2} ).

In conclusion, CX\textsubscript{3}CR1 plays a major role in
neuron-microglia mutual interaction, highlighting the growing evidence
of microglia in neurodevelopmental disorders, including ASD (Lukens and
Eyo, 2022). CX\textsubscript{3}CR1 is a promising target to treat ASD.
However, development of specific compounds will be necessary to
demonstrate its beneficial effect.

\textbf{Muscarinic acetylcholine M\textsubscript{3} receptor}

In addition to ionotropic receptors, acetylcholine activates five
muscarinic M\textsubscript{1}-M\textsubscript{5} GPCRs. Whereas many
receptor ligands, including allosteric modulators, have been reported,
only few of them are selective for a receptor subtype (\textbf{Table S4}
). The \emph{CHRM3} gene is complex, spans over 550 kb and includes 7
exons, with only exon 7 encoding the M\textsubscript{3} receptor. It has
10 described and 21 predicted transcript variants. Like other muscarinic
receptors, M\textsubscript{3} modulates excitatory transmission,
neuronal development including cellular proliferation and survival,
neuronal differentiation and controls food intake, learning and memory
(Yamada et al., 2001; Poulin et al., 2010). \emph{Chrm3} KO mice or
knock-in of a mutant receptor whose IL3 cannot be phosphorylated,
significantly altered hippocampus-dependent contextual fear memory
formation and decreased paradoxical sleep (\textbf{Table S3} ). However,
no study has investigated the ASD-like symptoms in these animals nor the
therapeutic potential of muscarinic ligands. Seven variants have been
associated with ASD in six independent studies (\textbf{Table S2} ),
suggesting the potential involvement of M\textsubscript{3} in ASD
aetiology. Interstitial deletion in the 1q43 region, which mostly
affects the \emph{CHRM3} gene, is associated with ASD, intellectual
disability, seizures, microcephaly and congenital malformation (van
Bever et al., 2005; Hiraki et al., 2008). Whereas reduced cholinergic
enzyme activity has been observed in cortical areas of ASD patients
(Perry et al., 2001), further evidence is needed to conclude on the
potential interest of muscarinic receptors as therapeutic targets for
ASD.

Orphan and olfactory receptors

Hundreds of orphan and olfactory GPCRs are expressed in the CNS and
represent new potential therapeutic targets for neurological disorders
(Khan and He, 2017) including ASD. Interestingly, orphan GPR37 and GPR85
are the top dysregulated GPCR genes in ASD tissues (Monfared et al.,
2021). Except their classification by sequence homology to the class A
of GPCRs, the study of orphan or olfactory receptors remains challenging
due to the lack of any identified ligand or poorly known function.

\textbf{GPR37}

GPR37 or parkin-associated endothelin-like receptor (Pael-R) is closely
related to endothelin GPCRs. Several potential natural peptides have
been reported activating GPR37 (\textbf{Table S4} ), but remains to be
confirmed. GPR37 is characterized by a poor export from ER to plasma
membrane in heterologous cell lines, which is either rescued by deletion
of its long N-terminus domain, oligomerization with
A\textsubscript{2A}or D\textsubscript{2} receptors, or interaction with
syntenin 1 through their PDZ domain (Dunham et al., 2009; Hertz et al.,
2019). GPR37 is up-regulated during oligodendrocyte differentiation
where it inhibits late-stage differentiation and myelination (Yang et
al., 2016). GPR37 is also located in dopaminergic axon terminals of the
substantia nigra where it controls dopamine release through a direct
interaction with the dopamine transporter (Marazziti et al., 2007).
\emph{Gpr37} KO mice display obsessive compulsive behaviours, decreased
locomotion, reduced colon motility and abnormal sensorimotor gating
(\textbf{Table S3} ). They may have increased anxious-like behaviours,
but this phenotype varies depending on the tests, sex and housing
conditions. Conversely, transgenic mice overexpressing \emph{Gpr37} show
increased methamphetamine-induced stereotyped behaviours, motor
coordination and locomotion (Imai et al., 2007). Despite social
interactions remain to be investigated, \emph{Gpr37} mice rather display
a large variety of comorbid symptoms of ASD associated with altered
striatal dopamine signalling, a feature of ASD (Li and Pozzo-Miller,
2020). Interestingly, variants of the dopamine transporter gene, its
direct interactor, are also associated with ASD (DiCarlo et al., 2019)
and lead to similar alterations of dopamine transmission in the
striatum. The \emph{GPR37}gene has been identified in the first autism
locus (AUTS1) on chromosome 7q31--33. Since then, nine variants in this
gene have been associated with ASD (\textbf{Table S2} ). Therefore,
several pieces of evidence confirm that GPR37 might be an interesting
target for ASD. However, selective ligands should be developed and
tested in preclinical models to further strengthen its therapeutic
potential for ASD.

\textbf{GPR85}

GPR85/SREB2 belongs to the super-conserved receptor expressed in the
brain (SREB) family. The \emph{GPR85} gene encodes 7 predicted and 3
transcript variants due to alternative splicing of the 3'UTR. They all
encode the extremely conserved GPR85, which shares 100\% homology and
strong expression throughout the CNS in humans and mice (\textbf{Figure
3} ). It is expressed in all types of neurons and microglia
(\textbf{Figure 2} ). At the molecular level, GPR85 directly interacts
with SHANK3 or PSD95 scaffolding partners through its PDZ domain in its
C-terminus, and indirectly with neuroligin through PSD95 (Fujita-Jimbo
et al., 2015; Jin et al., 2018). In the adult hippocampus, GPR85
negatively regulates neurogenesis and dendritic morphology, thereof
controlling brain size (Chen et al., 2012). \emph{Gpr85} KO mice display
increased neurogenesis associated with enlarged brain size and increased
cognitive abilities in spatial tasks (\textbf{Table S3} ). Conversely,
mice overexpressing \emph{Gpr85} in forebrain neurons show core symptoms
of ASD, social interaction deficits and restrictive behaviours, in
addition to cognitive inabilities, abnormal sensorimotor gating and
reduced dendritic arborisation (Matsumoto et al., 2008; Chen et al.,
2012). Two independent studies reported five variants in the
human\emph{GPR85} gene in Japanese ASD patients (\textbf{Table S2} ),
including one variant in the 3'UTR. Furthermore, two studies have found
downregulated \emph{GPR85} transcripts and decreased
\emph{GPR85}splicing events in the cortex of ASD patients (Voineagu et
al., 2011; Monfared et al., 2021). Interestingly, increased \emph{Gpr85}
mRNA levels have been found in the striatum and prefrontal cortex of
mice overexpressing Shank3 (Jin et al., 2018). Although studies on GPR85
remain sparse and no drug are available, data from mice and patients
converge on its therapeutic potential to improve social interaction
deficits.

\textbf{Olfactory receptors}

In humans,~387 genes encode olfactory receptors (OR) in addition to 462
pseudogenes. ORs, encoded by a single exon, are subdivided in aquatic
ancestry class I receptors clustered on human chromosome 1 (OR1-15) and
the largest terrestrial ancestry class II (OR51-56) located on different
chromosomes (Olender et al., 2020). They detect odorant volatile
molecules, although most of them remain orphan. Since their discovery,
growing evidence have shown OR expression outside the olfactory
epithelium, primarily in testis, then in most tissues, including the
CNS. Their roles in development, chemotaxis, tissue injury and
regeneration are starting to be deciphered. Only few studies have
associated the \emph{OR1C1} , \emph{OR2M4} , \emph{OR2T10}
and\emph{OR52M1} genes with ASD, with the strongest evidence
for\emph{OR1C1} (\textbf{Table S2} ). Furthermore, other OR genes were
also identified in association studies (Ruzzo et al., 2019), in
particular with schizophrenia. Often qualified as `ectopic' outside the
olfactory epithelium, their expression is rather conserved among species
(De la Cruz et al., 2009; Olender et al., 2016). OR1C1, OR2M4 and OR2T10
are present in the CNS, in contrast to OR52M1, which is conserved
between humans and rodents (\textbf{Figures 2-3} , \textbf{Table S4} ).
OR1C1 and OR2T10, specific to apes, are both detected in the cortex,
with OR1C1 also found in the pons, cerebellum, hippocampus and amygdala
(\textbf{Table S6} ). OR2M4 is conserved in apes, cows and pigs and is
detected in neurons of most CNS areas. Their function remains to be
elucidated in the CNS as no ligands nor animal models are available. In
conclusion, the function of orphans and ORs only starts to be elucidated
in the CNS and they could be of interest for ASD in the future with the
development of selective drugs.

\textbf{GENERAL CONCLUSIONS and FUTURE DIRECTIONS}

In this review, we highlighted the involvement of the different GPCRs in
the aetiology of ASD and as potential targets. We analysed the effect of
GPCR variants on their level of expression, ligand binding, receptor
folding or activation of downstream signalling pathways (\textbf{Table
S2} ). Variants located in introns, untranslated regions or coding
regions of mGlu\textsubscript{7}, 5-HT\textsubscript{2A},
CB\textsubscript{1}, GPR37 and GPR85 receptor genes are consistent with
the decreased levels of transcript expression observed in patients
(Monfared et al., 2021). Based on the evidences in ASD patients, their
function and localisation (\textbf{Figures 2-3} ), and their behavioural
predictive validity in animal models (\textbf{Table S3} ), we classified
these GPCRs. OTR, V\textsubscript{1A}, mGlu\textsubscript{5},
D\textsubscript{2}, 5-HT\textsubscript{2A}, CB\textsubscript{1} and
GPR37 receptors fulfil most, if not all, criteria.
V\textsubscript{1A}display a clear potential for social interaction,
D\textsubscript{2} and GPR37 for stereotyped behaviours, and
mGlu\textsubscript{5}, OTR, CB\textsubscript{1} and
5-HT\textsubscript{2A} eventually for both core symptoms. Overall, it is
surprising that out of 800 GPCRs, only 23 GPCRs are included in the
SFARI list, and that all these genes are classified in the second
category, namely `strong candidate gene'. We propose to move OTR,
CB\textsubscript{1} and V\textsubscript{1A} receptors to the first
category `high confidence genes', and add 5-HT\textsubscript{2A}to the
list. In this review, we also suggest GABA\textsubscript{B1},
5-HT\textsubscript{6}, 5-HT\textsubscript{7}, D\textsubscript{4} and
D\textsubscript{5} has potential candidates for ASD. Increasing pieces
of evidence showed the functional crosstalk between GPCRs within a cell,
to control a specific function, such as D\textsubscript{2},
A\textsubscript{2A} and mGlu\textsubscript{5} for the control of motor
activity (Ciruela et al., 2011). In fact, independently of their
physical interaction, GPCRs are not individual entities but should
rather be considered as a set of GPCRs and isoforms working together in
a cell to orchestrate the different signals and regulate downstream
signalling network and related cellular processes. Up to hundreds of
GPCRs are expressed in the same brain structures or cell types, with the
highest diversity in the striatum, cortex and hypothalamus (Vassilatis
et al., 2003; Marti-Solano et al., 2020). In 2018, Babu and colleagues
identified hundreds of missense and CNV variants in the genes coding for
V\textsubscript{1B}, D\textsubscript{1}, D\textsubscript{2}, and
D\textsubscript{3}, 5-HT\textsubscript{2A},
\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2}-adrenoceptors and GABA\textsubscript{B} (Hauser et
al., 2018) that might influence their ligand binding or transducer
recruitment (\textbf{Table S4} ). This area of research based on
receptor bias is known as pharmacogenomics. Considering the major impact
of GPCR signalling (\textbf{Table S5} ) that are altered in ASD (De
Rubeis et al., 2014; Hormozdiari et al., 2015; Gazestani et al., 2019),
any slight modification in a GPCR or in a combination of GPCRs would
lead to drastic signalling defects and neuronal pathogenicity. Here, we
found that at least 15\% of the genes listed in the SFARI database are
in the signalling networks and cellular downstream processes of GPCRs.
Therefore, application of pharmacogenomics to hundreds of GPCRs
expressed in the CNS remains an outstanding hypothesis to fully decipher
the global effect of GPCRs on pathological processes underlying ASD.
Therefore, the impact of GPCRs for autism research has only begun to be
highlighted, and rather than a single entity, GPCRs should be considered
as one global functional unit of GPCRs expressed in a cell that control
signalling networks, in order to understand their contribution to ASD
aetiology.

GPCRs meet all the criteria of therapeutic targets for ASD to bypass the
placebo effect observed in clinical trials. They contribute to the
polygenic ASD aetiology, pathogenic variants are recessive, they are
therapeutically rescuable, and they are \emph{per essence} membrane
receptors that display a large pharmacopeia of safe and efficient drugs.
We analysed the therapeutic potential of these 26 GPCRs and categorized
them as `high', `moderate' and `low' candidates, based on 1) drugs that
are already approved or tested for a related disorder, 2) their
beneficial effects in animal models of ASD or in clinical trials, 3)
their pharmacogenomic profile and 4) their safety (\textbf{Table S4}
,\textbf{S6} ). We classified mGlu\textsubscript{5},
GABA\textsubscript{B}, D\textsubscript{2}, 5-HT\textsubscript{2A},
5-HT\textsubscript{7}, CB\textsubscript{1} as `high' candidates and OTR,
V\textsubscript{1A}, mGlu\textsubscript{7}, D\textsubscript{1},
5-HT\textsubscript{1B} and M\textsubscript{3} as `moderate' candidates
due to the lack of selective ligands. However, the high number of
variants of mGlu\textsubscript{5} and 5-HT\textsubscript{2A} might
compromise their responsiveness to drugs (Hauser et al., 2018) and might
explain why mGlu\textsubscript{5}-targeted clinical trials have failed.
We excluded \selectlanguage{greek}β\selectlanguage{english}\textsubscript{2}-adrenoceptors, A\textsubscript{2A}, and
AT\textsubscript{2} as toxicity or severe side effects have been
reported in clinical trials. Finally, despite their strong potential in
the future, orphan and olfactory receptors belong to the `low' category
as there are no natural ligands or drugs clearly identified. We also
ranked CX3CR\textsubscript{1}, V\textsubscript{1B},
D\textsubscript{3}and A\textsubscript{3} as `low' candidates, as no
selective ligands have been developed and the adequate pharmacological
profile remains to be investigated. Finally, considering their
involvement in ASD aetiology, their therapeutic potential and results of
clinical trials (\textbf{Figure 4} ), we conclude that
D\textsubscript{2}, 5-HT\textsubscript{2A}, CB\textsubscript{1}, OTR,
V\textsubscript{1A}and GPR37 are the most promising targets for clinical
development. D\textsubscript{2}, 5-HT\textsubscript{2A} and
CB\textsubscript{1} are already ongoing for irritability, repetitive
behaviours, aggressive and self-injury behaviours, but could be tested
on other core symptoms, especially CB\textsubscript{1} on social scales.
In the near future, when specific ligands will be developed, OTR,
V\textsubscript{1A} and GPR37 should be tested as well. The recent
development of antibody fragments targeting GPCRs (Muji\selectlanguage{polish}ć-\selectlanguage{english}Deli\selectlanguage{polish}ć \selectlanguage{english}et al.,
2014) and the emergence of high throughput screening by DNA-based
bar-coded chemical libraries (Madsen et al., 2020) should boost the
identification of new drugs to target GPCRs, including orphan and
olfactory receptors. Interestingly, antibody fragments display all types
of pharmacological profiles, and can also be used as chaperones, or
target oligomers of GPCRs. Considering that GPCRs function as a global
GPCR module in a cell, a similar approach might also be applied for
future drug development. Finally, the use of several drugs or of a drug
targeting multiple GPCRs might be relevant for ASD. Such drugs already
exist. For example, aripiprazole, risperidone or cariprazine targets
multiple dopamine, serotonin, histamine and/or adrenoceptors
(\textbf{Table S4} ) or arylpiperazine derivatives target multiple 5-HT
receptors (Lacivita et al., 2021). Based on their fine-tune pharmacology
(biased ligands, oligomerization, global GPCR entities) and their
diversity, GPCRs represent the greatest therapeutic options for ASD and
hold the promise to successful clinical trials.



\textbf{Abbreviations}

ASD autism spectrum disorders

cAMP adenosine 3',5 cyclic monophosphate

CNS central nervous system

CNV copy number variants

CREB cAMP-responsive element binding protein

DAG diacylglycerol

ER endoplasmic reticulum

ERK extracellular signal-regulated kinase

FXS fragile X syndrome

GABA \selectlanguage{greek}γ-\selectlanguage{english}aminobutyric acid

GIRK G protein-coupled inwardly-rectifying potassium channels

GPCR G-protein coupled receptor

GO gene ontology

5-HT 5-hydroxytryptamine

IP\textsubscript{3} inositol 1,3,4- triphosphate

KEGG Kyoto encyclopaedia of genes and genomes pathway

KD knock-down

KI knock-in

KO knock-out

NAM negative allosteric modulator

OR olfactory receptor

PAM positive allosteric modulator

PD Parkinson's disease

PKA protein kinase A

PLC phospholipase CRTK receptor tyrosine kinase

RTT Rett syndrome

SNP single nucleotide polymorphism

Tg transgenic animals

UTR untranslated region

WT wild type

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\textbf{FIGURES}

\textbf{Figure 1} \textbf{GPCR signalling and pharmacology}

\textbf{A} ) GPCR are composed of seven transmembrane domains connecting
the extracellular domain {[}N-terminus, extracellular loops (EL) 1-3{]}
where various ligands bind the receptor (\emph{e.g.,} natural ligands
and chemicals/antibodies), to the intracellular domain {[}intracellular
loops (IL) 1-3, helix H8, C-terminus{]} that recruit the direct
transducers, which activate a signalling network (\emph{e.g.,} Akt, ERK)
and integrated cellular processes. A biased ligand has the particular
pharmacological profile to favour signalling pathways within the complex
receptor signalling network. Here, a G protein-biased ligand that
favours G protein coupling (black arrow) over \selectlanguage{greek}β-\selectlanguage{english}arrestin recruitment
(grey arrow) is shown. The radial graph represents the maximum efficacy
(Emax) of this G protein-biased ligand to stimulate the indicated
signalling responses. The scale is indicated as \% of the efficacy of
the natural ligand. \textbf{B} ) GPCRs display a rich pharmacopoeia of
ligands, with agonists, antagonists and inverse agonists that bind to
the orthosteric binding site (e.g., the binding site of the natural
ligand) to respectively activate or prevent the agonist binding and
inactivate the receptor. Other ligands bind to allosteric sites that
increase or decrease the efficacy or efficiency of the natural ligand,
respectively called positive or negative allosteric modulators.

\textbf{Figure 2} \textbf{At least 15\% of SFARI genes participate in
GPCR activity and signalling processes}

GPCR ligands are synthesized by metabolic enzymes, loaded by their
transporters into synaptic vesicles that fuse with the presynaptic
membrane upon increase of intracellular calcium, leading to
neurotransmitter release in the synaptic cleft. These neurotransmitters
or ligands are either recaptured by membrane transporters, degraded, or
bind and activate their cognate GPCR. Even in the absence of ligand,
GPCRs are present in preformed higher complexes with scaffolding
partners, channels, cytoskeleton and signalling transducers. Upon GPCR
activation, transducers activate enzyme and channel effectors to produce
second messengers. These second messengers activate major kinases and
guanine nucleotide exchange factor (GEF) that tune up or down downstream
cellular processes, including translation and transcription. Syndromic
(in red), high confidence (category 1 in dark orange), strong candidate
(category 2 in light orange) and suggestive evidence (category 3 in
green) genes are coloured according to SFARI gene scoring and colour
code (gene.sfari.org/about-gene-scoring, \textbf{Table S1} ) and
additional GPCR genes are in black. GPCRs are localized at pre,
post-synaptic compartment of neurons, in astrocytes or in unknown or
other cell types according to their expression pattern (see text for
further details).

\textbf{Figure 3} \textbf{GPCR localisation and expression in the human
and mouse brain}

Relative expression and localisation of the 25 GPCRs are presented on
the murine and human brain templates from the protein atlas database
(www.proteinatlas.org). After comparison to protein expression for
consistency (only available for the 5-HT\textsubscript{1B},
5-HT\textsubscript{2A}, 5-HT\textsubscript{7}, A\textsubscript{2A},
V\textsubscript{1B}, M\textsubscript{3}, CB\textsubscript{1},
CX\textsubscript{3}CR1, D\textsubscript{2}, GABA\textsubscript{B2} and
GPR37), relative RNA levels are represented as high (brown, over 20
normalised transcript expression values, expressed as nTPM), moderate
(red, 10-20 nTPM), low (pink, 2-10 nTPM) and just detectable (light
pink, 0.1-2 nTPM) expression in the CNS (expression in the other organs
are indicated in \textbf{Table S4 and S6} ). Brain templates are from
Servier Medical Art.

\textbf{Figure 4 Workflow to assess the relevance to ASD of GPCR genes
extracted from the SFARI database and their therapeutic potential}

Twenty-three GPCRs have been considered in this review, and for each of
them, the data obtained \emph{in vitro} , in animal models (mainly
mouse) and the features of GPCR variants and expression levels
encountered in ASD patients have been discussed side-by-side (see text
for further details).

\textbf{Hosted file}

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\verb`Annamneedi et al_figure3.pptx`  available at \url{https://authorea.com/users/589167/articles/626127-towards-the-convergent-therapeutic-potential-of-gpcrs-in-autism-spectrum-disorders}

\textbf{Hosted file}

\verb`Annamneedi et al_figure4.pptx`  available at \url{https://authorea.com/users/589167/articles/626127-towards-the-convergent-therapeutic-potential-of-gpcrs-in-autism-spectrum-disorders}

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