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
(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.
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 8th helix parallel to the
plasma membrane (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 HTR2A , adenosine ADORA1 and
adrenergic 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
(α, β and γ subunits) and recruit β-arrestins that impact on the
kinetics of intracellular signalling pathways such as extracellular
signal-regulated kinases (ERK) or promote receptor endocytosis
(Figure 1A ). Gαs/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, Gαi/o inhibits adenylyl cyclases and cAMP
production. Gαq/11 proteins activate phospholipase Cβ,
which hydrolyses phosphatidylinositol-4,5-bisphosphate
(PIP2) into diacylglycerol (DAG) and
inositol-1,4,5-triphosphate (IP3). Released
IP3 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.
Gα12/13 activates Rho guanine nucleotide exchange Factor
(GEF) and RhoA, which acts on the cytoskeleton to promote neurite
formation. Gβγ 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 Gαi and Gα12/13 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; León 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/β-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 (supporting information ) to the 1045
ASD candidate genes from the SFARI list. We identified 23 GPCRs and 129
genes linked to GPCRs (Figure 2 , 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.
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 (Figure 1B ). They can
either be chemical compounds, peptides, large autoantibodies and more
recently, antibody fragments (Mujić-Delić 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 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 (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’ (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
mGlu5, GABAB, V1A and
CB1 and are all listed in the SFARI database
(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-HT2A, 5-HT6 and 5-HT7receptors for their relevance to ASD (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 (Table S2 ). We addressed the behavioural
consequences of their genetic and pharmacological manipulation in animal
models (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
(e.g., specific drugs on the market or in clinical trials), the
availability of GPCR structures, and when available, the results of
clinical trials (Table S4 ). We also reported their known
downstream signalling and their interacting partners (Table
S5 ). Finally, we reviewed their cellular localisation and levels of
expression in the CNS (Figures 2-3, Table S6 ). Based on these
evidences, we conclude on the therapeutic potential of these 26 GPCRs
(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 (OxtKO mice) may display impairment in social memory and aggressive and
anxious-like behaviours (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 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 theAvp gene leading to the lack of AVP, display defective social
preference (Surget and Belzung, 2008) whereas 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 V1A and
V1B receptors in the CNS and V2 receptor
in the periphery. Therefore, in the following section, we review the
therapeutic potential of OTR, V1A and
V1B for treatment in ASD.
Oxytocin receptor
The oxytocin receptor gene (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 (Table S4 ), such as the
potent peptide agonist Thr4Gly7-OT
(TGOT) (Elands et al., 1988), the Gαq-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 (Figure 3 ) and might
be sexually dimorphic depending on the brain region and species (Dumais
et al., 2013). In humans, 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). Oxtr KO
mice have an autism-like phenotype, with both social deficits and
stereotyped behaviours whereas heterozygous mice express only social
deficits. Oxtr KO also display deficits in social memory and pup
vocalisations following maternal separation and aggressive behaviour
(Table S3 ). Whereas increased self-grooming, anxious-like
behaviours and cognitive inflexibility have been observed, results are
inconsistent through laboratories or mouse lines. However, OxtrKO 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 Oxtr KO mice via
V1A receptors (Sala et al., 2011). This finding
highlights the crosstalk within this GPCR family. More than twenty
variants in the OXTR gene have been associated with ASD
(Table S2 ). Interestingly, variants are mostly located outside
the receptor coding region, leading to potential receptor expression
dysregulation.
Vasopressin V1A and V1Breceptors
AVP, well known as the antidiuretic hormone via the activation of
V2 receptors, binds V1A and
V1B receptors in the CNS. AVPR1A andAVPR1B genes encode each, only one transcript variant and the
V1A and V1B receptors respectively.
V1A receptor is involved in maternal care,
social recognition, affiliative behaviour and pair bonding (Koshimizu et
al., 2012). Administration of the V1A antagonist
d(CH2)5Tyr(Me)AVP into the medial amygdala of rats affects maternal
memory (Nephew and Bridges, 2008). Furthermore, Avpr1a KO mice
and hamsters display defective social memory, interaction and
communication, reduced anxious-like behaviours and inconsistent levels
of aggressive behaviour across species (Table S3 ). Several
studies have associated the length of the promoter and the 5’UTR of theAVPR1A gene, which regulate V1A expression
levels, with important social deficits. Indeed, most variants associated
with ASD risk are identified in these regions (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 V1A 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 V1A.as it is not yet clear
whether it should be activated or inhibited to improve social skills.
V1B receptor deletion in mice (Avpr1bKO) leads to increased dominance, decreased aggressive behaviour and
vocalisations and impaired motivation and social memory (Table
S3 ). Three independent studies have identified variants in theAVPR1B gene linked to ASD (Table S2 ), mood disorders and
aggressive behaviour. Accordingly, administration of the antagonist
nelivaptan (Table S4 ) normalizes aggressive, chasing and
anxious-like behaviours in rodents (Blanchard et al., 2005; Salomé 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
V1A receptors may be involved in the aetiology of autism
and are major therapeutic targets for ASD, whileV1Bmight 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
(e.g. , which receptor or oligomer, which signalling pathway) and
respective ligands of this family.
Metabotropic
glutamate and GABA receptors
Glutamate and γ-aminobutyric acid (GABA) are the two major
neurotransmitters in the CNS. They bind their cognate class C GPCRs,
metabotropic glutamate mGluRs and GABAB 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 (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.
mGlu5
The GRM5 gene encodes two splice variants of
mGlu5 (mGlu5a and
mGlu5b), with the mGlu5b receptor
expressed predominantly during the adult stage (Table S6 ).
Activation of mGlu5 induces synaptic plasticity, which
requires de novo mRNA translation through phosphorylation of
eIF2α (Di Prisco et al., 2014). Grm5 KO mice display ASD-related
core symptoms (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 GRM5 gene of ASD
patients (Table S2 ), highlighting GRM5 as one of the
most susceptible genes in ASD (Nisar et al., 2022). Alterations in
mGlu5 receptor signalling or expression affect synaptic
and neuronal development, trademarks of ASD and intellectual disability
(D’Antoni et al., 2014). Higher mGlu5 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
mGlu5 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 (Fmr1 KO) (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 mGlu5 in
ASD pathogenesis and explain why it is one of the first GPCRs targeted
for ASD. However, targeting mGlu5 even with a selective
negative allosteric modulator did not pass the placebo effect in
patients. This might be attributed to differences in receptor
expression.
mGlu7
The GRM7 gene encodes two isoforms (mGluR7a,
mGluR7b) that differ in their C-terminus, potentially
leading to different protein-protein interactions and receptor coupling.
mGlu7 is expressed during the critical
neurodevelopmental period, when it augments synapse formation and
stabilisation (Song et al., 2021). Compared to other mGluRs,
mGlu7 has less affinity for glutamate, hence is
considered as an “emergency brake”. mGlu7 are
predominantly localised at presynaptic sites that regulate
neurotransmitters release of glutamate or GABA. Interestingly, the
non-selective mGlu4,6,7 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). Grm7 KO mice display intact social interaction,
but social memory deficits (Table S3 ), which might be explained
by their global learning defects. Overall, Grm7 KO mice and mice
carrying the Ile154Thr mGlu7 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 GRM7 gene have been
associated with ASD (Table S2 ). In particular, the Ile154Thr,
Arg658Trp and Thr675Lys mutants lead to reduced mGlu7surface expression and/or degradation. Dysregulated levels of
mGlu7 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 mGlu7in post-mortem motor cortex samples from patients with Rett syndrome
(RTT) and in a mouse model of RTT (Mecp2 KO) (Bedogni et al.,
2016; Gogliotti et al., 2017). In conclusion, mGlu7 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.
GABAB receptor
Metabotropic GABAB receptors are obligatory
hetero-oligomers of GABAB1 and GABAB2through their C-terminus coiled-coiled domain. Presynaptic
GABAB receptors suppress neurotransmitter release
whereas postsynaptic receptors induce slow inhibitory postsynaptic
currents, which shunt the excitatory currents (Lüscher et al., 1997).
GABAB receptor deletion in mice (Gabbr1-Gabbr2double KO) leads to stress-induced social withdrawal, emotional
behavioural disturbances and increased anxious- and anti-depressive-like
behaviours (Table S3 ). The effect of Gabbr2 deletion
alone has not been reported yet. However, Xenopus tropicalistadpole larvae carrying the Ala567Thr, Ser695Ile and Ile705Asn
GABAB2 mutants identified in ASD and epileptic patients,
display increased seizure-like behaviour and altered swimming patterns
that are partially rescued by the selective GABABagonist baclofen (Yoo et al., 2017). Of note, when expressed in
heterologous HEK293 cells, these three mutants disrupt
GABAB activation. Four genomic studies revealed the
association of the GABBR2 gene with ASD and RTT (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 GABAB 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, GABAB 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).
Dopamine receptors
Dopaminergic D1-like (D1 and
D5) and D2-like (D2,
D3 and D4) 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).
D1 receptors are particularly enriched in
D1 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 D2-like receptors might
detect low tonic dopamine levels (Beaulieu and Gainetdinov, 2011).
D1 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 D1 receptor
exhibited ASD-like social symptoms with reduced social interaction
(sociability and social novelty) and ultrasonic vocalisations in pups
while calling their mothers (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 D1 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
D1 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 DRD1 gene (Table S2 )
are associated with severe impairments in social interaction, non-verbal
communication and increased motor stereotypies.
D2 receptors encoded by the DRD2 gene
comprise two splicing isoforms, short D2S and long
D2L differing in their IL3. D2S serves
as an auto-receptor regulating dopamine release and dopamine synthesis
while D2L is a postsynaptic receptor (Negyessy and
Goldman-Rakic, 2005). These receptors are mainly expressed in neurons,
with the highest levels in GABAergic indirect D2striato-pallidal medium spiny neurons of the striatum, but also in
astrocytes and oligodendrocytes (Figure 2 ). Drd2 KO mice
show great impairments in social behaviour (sociability and social
novelty), impaired social olfaction and stereotyped behaviours
(Table S3 ). Moreover, 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
D2 receptor in this structure is sufficient to
recapitulate all the behavioural impairments reported in Drd2 KO
mice (Lee et al., 2018). Conversely, D2 receptor
overexpression in the striatum and olfactory tubercle revealed
impairment in sociability only in female mice and vocalisation. Among
the dopamine receptors, the DRD2 gene displays the highest number
of SNPs associated with ASD (Table S2 ). Currently, the only
available approved treatments for ASD patients are antipsychotics
(Table S4 ), such as aripiprazole or risperidone, which
antagonise D2 receptor in addition to other GPCRs, to
treat irritability, aggressive and repetitive behaviours (McDougle et
al., 2005; Varni et al., 2012). Additionally, two other
D2 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).
D3 receptor expression is conserved between
humans and rodents and controls habituation to novelty (Mishra et al.,
2018). Drd3 KO mice display hyperactive and addictive behaviours,
with particular vulnerability to alcohol and drug abuse (Table
S3 ), but their social skills or stereotyped behaviours have not been
reported yet. Three independent studies identified three SNPs in theDRD3 gene associated with ASD (Table S2 ). The
antipsychotic cariprazine, a partial agonist for D3 that
also binds D2 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 acidin 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
D2 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 DRD5 gene that display the
highest number of distinct missense and loss of function variants in the
general population (Hauser et al., 2018).
Serotonin 5-HT1B
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-HT3. They modulate cognition, memory, sleep, appetite,
respiration, thermo-regulation and mood (Berger et al., 2009).
5-HT1B belongs to the 5-HT1 receptor
family that are encoded by 7 genes (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
(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-HT3 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, Htr1B KO mice display decreased
anxious-like behaviours, exacerbated aggressive behaviour, deficits in
maternal behaviour, improved cognitive flexibility and vulnerability to
drug abuse (Table S3 ). So far, only two variants associated
with ASD have been reported from two independent studies (Table
S2 ).
Beside the HTR1B gene, evidence has highlighted that theHTR2A gene fulfils the SFARI criteria as a strong candidate and
targeting 5-HT6 or 5-HT7 improve core
symptoms in mouse models of ASD (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-HT7 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-HT1A and inhibits 5-HT2A.
β2-adrenoceptor
Noradrenaline and adrenaline activate the α1,
α2, β1, β2 and
β3-adrenoceptors with different potencies. In the CNS,
they control cognition, memory, emotions and stress-induced behaviours.
Only the ADRB2 gene is present in the SFARI list and encodes the
β2-adrenoceptor. Despite its vital cardiac function,Adrb2 KO mice are fertile and viable and display increased
anxious-like behaviours and decreased depressive-like behaviours
(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 (Table S2 ) show enhanced
isoproterenol agonist-induced response. Furthermore, studies have
suggested an association between prenatal exposure to
β2-adrenoceptor agonists and ASD (Gidaya et al., 2016).
Interestingly, administration of the approved
β2/β3-adrenergic antagonist propranolol
improves verbal responses and social interactions and decreases anxiety
in ASD patients (Hegarty et al., 2017). Therefore, the
β2-adrenoceptor is an interesting target for ASD and the
approved drug propranolol (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 asADRA1D, 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
Adenosine receptors
Adenosine receptors are divided into four different subtypes, namely
A1, A2A, A2B and
A3, among which A1 and
A2A 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).
A2A receptor encoded by the ADORA2Agene, stimulates glutamate release at presynaptic terminals and
myelination by oligodendrocytes (De Nuccio et al., 2019). Post-synaptic
A2A receptors are highly enriched in D2GABAergic striato-pallidal medium spiny neurons of the striatum to
modulate locomotion and anxious-like behaviours (Coelho et al., 2014).
Accordingly, Adora2a KO mice display motor impairment and
anxious-like behaviours (Table S3 ). A2A might
act as a regulator of other GPCRs (Table S5 ), as it forms many
different hetero-oligomers with D2,
mGlu5, δ 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
A2A receptors is necessary and sufficient to prune
GABAergic synapses during this period (Gomez-Castro et al., 2021). In
contrast, any impairment in A2A signalling or expression
may result in GABAergic synapse alteration and cognitive deficits in
adults, as observed in animals administered with A2Aantagonists 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 A2A 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 D2medium spiny neurons, which in turn restore the neurotransmission on
efferent subthalamic nucleus. So far, only one study has associated four
SNPs in the ADORA2A gene with autism and severe anxiety
(Table S2 ).
The A3 receptor promotes expression of the
serotonin transporter (SERT) to the cell surface (Campbell et al.,
2013). Thus, lack of A3 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 ADORA3 gene are associated with ASD,
(Table S2 ) with impaired adenosine binding (Campbell et al.,
2013). Consequently, Adora3 KO mice show anxious-like and despair
behaviours (Table S3 ).
In conclusion, A2A shows the greatest promise to
mitigate repetitive behaviours and anxiety in ASD. However, most of
A2A agonists have failed in clinical trials (Guerrero,
2018) due to severe side effects, including CNS excitotoxicity
(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 ADORA1 gene, which is one of
the most downregulated GPCR genes in patients (Monfared et al., 2021)
and whose targeting in combination to A2A agonists
improves stereotyped behaviours (Lewis et al., 2019). A better
specificity could also be achieved by targeting A2Ahetero-oligomers such as
D2-A2A-mGluR5 to avoid
side effects.
Angiotensin AT2 receptor
Angiotensin receptors are divided into AT1 and
AT2 subtypes. They are activated by different maturation
products of angiotensinogen peptides, namely angiotensin II and III.
Only the AT2 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 AT2 in social or stereotyped
behaviours (Table S3 ). However, Agtr2 KO mice show
impaired reward processing and locomotion. Interestingly, administration
of the AT2 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 AGTR2 gene
on the chromosome X with ASD and X-linked intellectual disability
(Table S2 ). Therefore, together with its unknown expression and
function in the CNS, further studies are required to conclude on the
therapeutic potential of AT2.
Cannabinoid CB1 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 CB1 and
CB2. CB1 mediates the central effects of
cannabis and its derivatives. Cnr1 KO mice show deficits in
social interaction and communication, two core symptoms of ASD
(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 Fmr1 KO mice (Wei et
al., 2017). Three independent studies have reported more than thirty
variants in the CNR1 gene associated with ASD (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
CB1 NAM cannabidiol or endocannabinoid mix that are
currently in clinical trials for ASD (Aran et al., 2021). In conclusion,
multiple evidences highlight CB1 receptor as one of the
most promising GPCR target to treat core and associated symptoms in ASD.
Chemokine CX3CR1 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 (CX3CR1 or GPR13). In
humans, the CX3CR1 gene encodes 4 transcript variants and two
protein isoforms that differ in their N-terminus domain (Marti-Solano et
al., 2020). CX3CR1 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). Cx3cr1 KO mice display social
interaction deficits and increased motor stereotypies (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 Cx3cr1transcripts were increased in the prefrontal cortex, nucleus accumbens
and hippocampus (Zhou et al., 2020). Three rare missense deleterious
mutations in the CX3CR1 gene have been associated with
schizophrenia and ASD (Table S2 ).
In conclusion, CX3CR1 plays a major role in
neuron-microglia mutual interaction, highlighting the growing evidence
of microglia in neurodevelopmental disorders, including ASD (Lukens and
Eyo, 2022). CX3CR1 is a promising target to treat ASD.
However, development of specific compounds will be necessary to
demonstrate its beneficial effect.
Muscarinic acetylcholine M3 receptor
In addition to ionotropic receptors, acetylcholine activates five
muscarinic M1-M5 GPCRs. Whereas many
receptor ligands, including allosteric modulators, have been reported,
only few of them are selective for a receptor subtype (Table
S4 ). The CHRM3 gene is complex, spans over 550 kb and includes 7
exons, with only exon 7 encoding the M3 receptor. It has
10 described and 21 predicted transcript variants. Like other muscarinic
receptors, M3 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). 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 (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 (Table S2 ),
suggesting the potential involvement of M3 in ASD
aetiology. Interstitial deletion in the 1q43 region, which mostly
affects the 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.
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 (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 A2Aor D2 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). Gpr37 KO mice
display obsessive compulsive behaviours, decreased locomotion, reduced
colon motility and abnormal sensorimotor gating (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 Gpr37 show increased
methamphetamine-induced stereotyped behaviours, motor coordination and
locomotion (Imai et al., 2007). Despite social interactions remain to be
investigated, 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 GPR37gene 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 (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.
GPR85
GPR85/SREB2 belongs to the super-conserved receptor expressed in the
brain (SREB) family. The 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 (Figure
3 ). It is expressed in all types of neurons and microglia
(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). Gpr85 KO mice display
increased neurogenesis associated with enlarged brain size and increased
cognitive abilities in spatial tasks (Table S3 ). Conversely,
mice overexpressing 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 humanGPR85 gene in Japanese ASD patients (Table S2 ),
including one variant in the 3’UTR. Furthermore, two studies have found
downregulated GPR85 transcripts and decreased GPR85splicing events in the cortex of ASD patients (Voineagu et al., 2011;
Monfared et al., 2021). Interestingly, increased 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.
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 OR1C1 , OR2M4 , OR2T10 andOR52M1 genes with ASD, with the strongest evidence forOR1C1 (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 (Figures 2-3 , 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
(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.