International Journal of

Molecular Sciences
Review

Lipid-Based Molecules on Signaling Pathways in Autism
Spectrum Disorder
Kunio Yui * , George Imataka

and Shigemi Yoshihara

Department of Pediatrics, Dokkyo Medical University, Mibu 321-0293, Tochigi, Japan* Correspondence: yui16@bell.ocn.ne.jp

Abstract: The signaling pathways associated with lipid metabolism contribute to the pathophysiologyof autism spectrum disorder (ASD) and provide insights for devising new therapeutic strategies.
Prostaglandin E2 is a membrane-derived lipid molecule that contributes to developing ASD associatedwith canonical Wnt signaling. Cyclooxygenase-2 plays a key role in neuroinflammation and is
implicated in the pathogenesis of neurodevelopmental diseases, such as ASD. The endocannabinoid
system maintains a balance between inflammatory and redox status and synaptic plasticity and is apotential target for ASD pathophysiology. Redox signaling refers to specific and usually reversible
oxidation–reduction reactions, some of which are also involved in pathways accounting for theabnormal behavior observed in ASD. Redox signaling and redox status-sensitive transcription factors
contribute to the pathophysiology of ASD. Cannabinoids regulate the redox balance by altering
the levels and activity of antioxidant molecules via ROS-producing NADPH oxidase (NOX) andROS-scavenging superoxide dismutase enzymes. These signaling cascades integrate a broad range ofneurodevelopmental processes that may be involved in the pathophysiology of ASD. Based on thesepathways, we highlight putative targets that may be used for devising novel therapeutic interventions
for ASD.

Citation: Yui, K.; Imataka, G.;

Keywords: autism spectrum disorder; lipid modified signaling pathway; endocannabinoid system;
prostaglandins; cyclooxygenase-2

Yoshihara, S. Lipid-Based Moleculeson Signaling Pathways in Autism
Spectrum Disorder. Int. J. Mol. Sci.
2022, 23, 9803. https://doi.org/
10.3390/ijms23179803
Academic Editor: Theoharis
C. Theoharides
Received: 28 July 2022
Accepted: 23 August 2022
Published: 29 August 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims inpublished maps and institutional affiliations.

Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Autism spectrum disorder (ASD) is a set of heterogeneous neurodevelopmental conditions [1] associated with a strong genetic component and involving many risk factors [2].
Genes affect synaptic development, leading to neurobiological theories focusing on theconnectivity and neural effects of gene expression [2]. There are few useful non-curative
treatments. Improved strategies for early identification with phenotypic characteristics andbiological markers might hopefully improve the effectiveness of treatment [3].
The pathophysiology of ASD includes alterations in neuronal circuits induced by
defects in various signaling pathways. Most studies on ASD pathogenesis have indicated a
combination of genetic mutations and various altered signaling pathways, such as those
involving mTOR [4], Wnt, Prostaglandin E2 (PGE2) signaling [5], and endocannabinoid signaling [6]. Although significant progress has been made in understanding the pathogenesis
and etiology of ASD, few effective therapies exist. Hence, further research is imperative to
identify potential therapeutic targets.
The combination of various core mutations and consequent dysfunction of cell signaling pathways has already been reported in the pathogenesis of ASD in preclinical animalmodels and patient samples. In this review, we highlight putative therapeutic targets basedon these pathways. These putative targets may be used for novel therapeutic interventions for ASD in the future. Timely, specific, and accurate modulation of these pathways
may contribute to our understanding of ASD pathogenesis. This review provides new
perspectives on the various cellular signaling pathways involved in the regulation of lipid
metabolism in ASD pathogenesis.

Int. J. Mol. Sci. 2022, 23, 9803. https://doi.org/10.3390/ijms23179803

https://www.mdpi.com/journal/ijms

physiological processes implicated in ASD [6,7]. Esterified omega-6 polyunsat
fatty acids (PUFAs), such as arachidonic acid (AA), present on the inner surface of t
membrane, are converted by phospholipase A2 (PLA2) into the free form, which
Int. J. Mol. Sci. 2022, 23, 9803 ther metabolized by cyclooxygenases (COXs) and lipoxygenases (LOXs)
2 of 20into bio
mediators, such as prostanoids and leukotrienes (LTs) [8]. These mediators hav
proposed as novel preventive and therapeutic targets for inflammatory diseas
2. Signaling
Pathways
Lipid Metabolites
cludingsome types
of Associated
ASD [8]. with
Disrupted
COX2/PGE2 signaling has been repor
Lipids act as regulatory molecules that contribute to the growth and maintenance of
ASD pathology [9]. PGE2 treatment induces the expression of Wnt target genes
brain functions. Altered fatty acid metabolic pathways may be involved in ASD pathoneuroectodermal(NE-4C)stem
cells, prostaglandin
PGE2 interacts
withis involved
canonical
Wnt sig
genesis. A natural
lipid-derived
molecule,
E2 (PGE2),in thethrough
PKA and
PI-3K,
which playroles
their migrationand proliferation
physiologicalprocesses
implicatedin ASD[6,7].in
Esterified
omega-6 polyunsaturated
fatty in th
acids(PUFAs),such
asarachidonic
acid
(AA),
present
on
the
inner
surfaceofthecell
via the expression levels of Wnt-target genes being upregulated in responsetomembrane, are converted by phospholipase A2 (PLA2) into the free form, which is further
inducing
crosstalk between PGE2 and Wnt signaling in neuronal cells [7]. Mor
metabolized by cyclooxygenases (COXs) and lipoxygenases (LOXs) into bioactive mediaPGE2
acts
these kinases
to converge
the mediators
Wnt pathway
duringprenatal
tors,
suchvia
as prostanoids
and leukotrienes(LTs)with[8]. These
have been
proposed
as noveland therapeutictargets
inflammatory
diseases, including
opment[7].preventive
These mechanisms
could
beforpossible
pathogenetic
factorssome
underlyin
types
of
ASD
[8].
Disrupted
COX2/PGE2
signalinghas
beenreportedinASD
pathology[9].
rodevelopmental disorders such as ASD [7].
PGE2 treatment induces the expression of Wnt target genes [7]. In neuroectodermal (NE-4C)
Additionally,
PGE2 receptor
EP4Wntsignalingbe PKA
part andof theneuroprotecti
stem
cells, PGE2 interacts
with canonicalsignalingmay
through
PI-3K,
which
anti-inflammatory
inproliferation
models ofinneurodegenerative
disease[10].
play roles in their pathways
migration andthe brain via the expression
levelsof Vario
Wnt-target
genes
being
upregulatedin
response
to
PGE2,
inducing
crosstalkbetween
PGE2
netic and environmental factors influence PGE2 levels and increase the risk of deve
Wnt signaling in neuronal cells [7]. Moreover, PGE2 acts via these kinases to converge
ASDand[11].
with the Wnt pathway during prenatal development [7]. These mechanisms could be
Apart pathogenetic
from these,
the underlying
endocannabinoid
systemdisorders(ECS) may
involvedinpossible
factors
neurodevelopmental
such asbe
ASD[7].
Additionally,[12].
PGE2Thereceptor
EP4issignaling
may benetworkpart of the ofneuroprotective
and antipathophysiology
ECS
a complex
lipid signaling
pathway
inflammatory
pathwaysinmodelsofneurodegenerative
disease[10].
Variousgenetic
andsisting of AA, 2-arachidonoylglycerol-derived compounds, and their G protein-co
environmental factors influence PGE2 levels and increase the risk of developing ASD [11].
receptorsApart(cannabinoid
receptors CB1 and CB2) [13] Moreover, ECS is a lipid cellfrom these, the endocannabinoid system (ECS) may be involved in ASD pathoing system
involved
andofhomeostasisof the brain.
Synapticpla
physiology[12]. The in
ECStheis aphysiology
complex network
lipid signaling pathways
consistingofAA, 2-arachidonoylglycerol-derived
compounds,
their
G protein-coupledneuroendocrine
functions, reproduction,
andandtheimmuneresponsereceptors
also require(cannabinoid
receptors
CB1
and
CB2)
[13]
Moreover,
ECS
is
a
lipid
cell
signalingsystemfunctioning of the ECS [14]. Arachidonic acid-related signaling pathwaysin-are prevolved in the physiology and homeostasis of the brain. Synaptic plasticity, neuroendocrinein Figure1. reproduction, and the immune response also require proper functioning of thefunctions,
ECS [14]. Arachidonic acid-related signaling pathways are presented in Figure 1.

Figure 1. Arachidonic acid is the ordinary substrate for eicosanoid synthesis. The cyclooxygenase(COX) pathways form prostaglandins (PGs) and the COX pathways form leukotrienes (LTs) and thecytP450 pathways form various epoxy, hydroxy and dihydroxy derivatives.

Int. J. Mol. Sci. 2022, 23, 9803

3 of 20

3. Prostaglandin E2 (PGE2)
3.1. Characteristics
Prostaglandin E2 (PGE2) is a well-known membrane-derived lipid signaling moleculethat plays an important role in neuronal development. Abnormal PGE2 levels are due
to environmental insults during prenatal development [5,15]. Various genetic and environmental factors can influence PGE2 levels, thereby increasing the risk of developing
ASD [11]. Altered PGE2 signaling is associated with the pathological changes observedin the nervous system [7]. Furthermore, PGE2 is associated with canonical Wnt signaling
through the protein kinase A (PKA), phosphatidylinositide 3-kinase (PI-3K), and protein
kinase C (PKC) pathways [7]. Enhanced levels of PGE2 increase Wnt-dependent motility
and the proliferation of neuroectodermal stem cells [8]. PGE2 can influence the expressionof genes from the canonical Wnt signaling pathway via β-catenin, which is the major downstream regulator of Wnt-dependent gene transcription [11]. These cellular mechanisms
may contribute to ASD development [11].
3.2. PGE2 in ASD
Cyclooxygenase-2 (COX-2) is the main regulator of PGE2 synthesis [9]. Abnormal
COX-2/PGE2 signaling is associated with behavioral symptoms of ASD [6]. Male micelacking cyclooxygenase-1 and cyclooxygenase-2 (COX−1-/- and COX−2-/- ) exhibit altered
COX/PGE2 signaling and, hence, are a potential model of ASD [9].
Collectively, PGE2 upregulates the canonical Wnt signaling pathway, which is associated with the development of ASD. The association between PGE2 and Wnt signaling
during prenatal development of the nervous system, where PKA and PI-3K might act
as mediators, suggests that PGE2 alters Wnt-dependent migration and proliferation ofneuroectodermal stem cells, which has implications for ASD [7].
3.3. Perspectives on ASD Therapy
Neurodevelopmental disorders, such as ASD, are associated with increased activity ofthe brain catalase of phospholipase A2 (PLA2) isoforms. As PGE2 biosynthesis has been
associated with PLA2 expression [16], new synthetic inhibitors of PLA2 have been used fortreating neuroinflammation-associated neurodevelopmental disorders in cells [17].
4. Cyclooxygenase-2 (COX2)
Cyclooxygenase-2 (COX-2) plays a key role in neuroinflammation and the pathogenesis
of neurodegenerative diseases such as ASD [18]. Endogenous cannabinoids are substrates for
COX-2 and can be oxygenated by COX-2 to form new classes of prostaglandins [18]. COX-2 insynaptic signaling may provide a mechanistic basis for designing new drugs aimed at preventing, treating, or alleviating neuroinflammation-associated neurological disorders [18].
For reference, accumulating evidence supports an association between the neuropsychiatricdisorders involving ASD and inflammation, and thus anti-inflammatory agents, such as
the COX-2 inhibitors (celecoxib), may contribute to a novel avenue for the prevention andtreatment of neuropsychiatric illness [19]. The brain morphologic aberrations in ASD may
be a result of neural pruning processes and neuroinflammation. Treatment with celecoxibcombined with risperidone in a 10-week trial in 40 patients diagnosed with ASD induced
efficacy according to the Aberrant Behavior Checklist—Community Edition (ABC-C) inthe domains of irritability, social withdrawal, and stereotypy in children with ASD [19].
Autistic children display immune system dysregulation and show an altered immune
response of peripheral blood mononuclear cells (PBMCs). The mRNA level for cannabinoid
receptor type 2 (CB2) was significantly increased in peripheral blood mononuclear cells in
ASD children as compared to healthy subjects. Protein levels of CB-2 were also significantly
increased in autistic children. These findings indicate CB2 receptor as potential therapeutictarget for the pharmacological management of ASD [20].
COX-2 knockout mice exhibited dysregulated expression of several ASD-related
genes [9]. COX-2 deficient mice, lacking the activity of the PGE2-producing enzyme,

Int. J. Mol. Sci. 2022, 23, 9803

logical management of ASD [20].
COX-2 knockout mice exhibited dysregulated expression of several ASD-r
genes [9]. COX-2 deficient mice, lacking the activity of the PGE2-producing en
display ASD-linked behaviors such as hyperactivity, repetitive behaviors, altered
4 of 20
strength, and awkward social interaction [6].
5. The Association between PGE2 and COX-2 in ASD
display ASD-linked behaviors such as hyperactivity, repetitive behaviors, altered motor
Regulation of PGE2 synthesis via the modulation of COX-2 activity has beenstrength, and awkward social interaction [6].

cated in the pathogenesis of neurodevelopmental diseases such as ASD [9]. Cyc

genase-2(COX-2)is themainof PGE2 synthesis [9]. Abnormal COX-2
5. TheAssociation
betweenPGE2
and regulator
COX-2 in ASD

signaling
isPGE2
associatedof hasASD
Alteration i
Regulation ofsynthesiswith
via thebehavioral
modulation ofsymptoms
COX-2 activity
been[6].
implicatedCOX-2/PGE2
induces differential
ASD-related
gene [9].
in thepathogenesis pathwayof neurodevelopmental
diseases expression
such as ASDof[9].
Cyclooxygenase-2(COX-2) isIrregular
the main regulator
of PGE2
synthesisleads[9]. Abnormal
COX-2/PGE2
signalingis social a
COX-2/PGE2
signaling
to ASD-related
behaviorsandassociatedwithbehavioral
symptoms
of
ASD
[6].
Alteration
intheCOX-2/PGE2
pathway
malities. Thus, COX-2 may be related to ASD-like behavioral symptoms in assoc
induces differential expression of ASD-related gene [9].
with PGE2. The associations between COX-2 and PGE2 are presented in Figure
Irregular COX-2/PGE2 signaling leads to ASD-related behaviors and social abnornote, prostaglandin families (prostaglandin J2) are endogenous toxic products
malities. Thus, COX-2 may be related to ASD-like behavioral symptoms in association
and areactively
neuronal
dysfunction
with clooxygenases
PGE2. The associations
between
COX-2involved
and PGE2 in
are thepresented
in Figure
2. Of note, induce
pro-inflammatory
stimuli. The
production
oftoxic
PGE2
and upregulationin the cyc
prostaglandinfamilies (prostaglandin
J2) areendogenous
productsof cyclooxygenases
and areactively
involvedin the neuronal
dysfunction
inducedTheby pro-inflammatory
stimuli.
genase(COX)
pathway
were inducedby PLA2.
arachidonic acid(AA) prod
The productionof PGE2ofandupregulationin the(COX)
pathway
were[21].
and participation
intracellular
PLA2
incyclooxygenase
these events were
also
evaluatedinduced by PLA2. The arachidonic acid (AA) production and participation of intracellular
PLA2 in these events were also evaluated [21].

Figure 2. Cyclooxygenases 1 and 2 (COX-1 and COX-2), which are key enzymes in the converFigure
2. Cyclooxygenases
1 and
2 (COX-1
and
COX-2),
are key enzymes
in the conv
sion ofarachidonic
acid into bioactive
prostanoidsplay
a centralrolewhichin the inflammatorycascade.
of arachidonic
into bioactive
playproductsa centralin the inflammatoryca
Prostaglandinfamiliesacid(postaglandin
J2) are prostanoids
endogenous toxic
of rolecyclooxygenases,
and
Prostaglandinfamilies(postaglandin
J2) areendogenous
toxic
productsof cyclooxygenase
becausetheir levels are
significantly
increased uponbrain
injury, they are
actively
involvedin neutheirlevelsbyaresignificantly stimuli.
increasedinjury,theyanare
actively invol
ronalbecause
dysfunction
inducedpro-inflammatory
Theuponline of brain
evidence
indicates
importantneuronal
dysfunction
inducedby pro-inflammatory
stimuli. Thereceptor
line oftype
evidence
indicatesinterplay
betweenthe cannabinoid
receptor
type 1 (CB1) and cannabinoid
2 (CB2)
portant interplay(ECB)
betweentheinflammatory
cannabinoidand
receptor
type 1 (CB1)and cannabinoidand endocannabinoids
are key
redox-dependent
processes.
The endocan-receptor(CB2)and (ECS)
endocannabinoids(ECB)
are stress
key inflammatory
and redox-dependent
nobinoidsystemmay also control
oxidativeby directly or indirectly
modulating CB1 processe
and CB2 signaling or CB receptor-independent processes via COX-2-dependent eicosanoid pathway.
Modulation of cellular redox homeostasis is induced by the ECS. The reactive oxygen species (ROS)
act as second messengers and exert oxidative activity to influence immune, inflammatory, and othersignaling processes.

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As PGE2 plays an important role in brain development, abnormalities in the COX2/PGE2 signaling pathway due to genetic or environmental causes are linked to ASD [22]and may result in autism-related behaviors, such as increased hyperactivity, anxiety, repetitive behavior, motor deficits, and social abnormalities, as observed in several cases of ASD
in the developing brain [9].
Cyclooxygenase-2 (COX-2) is the main regulator of PGE2 synthesis [9]. Abnormal
COX-2/PGE2 signaling is associated with behavioral symptoms of ASD [9]. Male micelacking cyclooxygenase-1 and cyclooxygenase-2 (COX−1-/- and COX−2-/- ) exhibit altered
COX/PGE2 signaling and, hence, are a potential model of ASD [9].
With respect to pathophysiology of ASD, abnormal COX-2/PGE2 signaling is associated with behavioral symptoms of ASD [9]. Male mice lacking cyclooxygenase-1 andcyclooxygenase-2 (COX−1-/- and COX−2-/- ) exhibit altered COX-2/PGE2 signaling and,hence, are a potential model of ASD [9].
Significantly higher levels of both COX-2 and PGE2 have been reported in the plasma
samples of autistic patients. Increased plasma PGE2 levels were concomitant with significantly lower levels of α-synuclein [23], which plays a critical role in synaptic functions,
including synaptic pool preservation, vesicular stabilization, and synaptic plasticity [23].
The combination of α-Syn, COX-2, and PGE2/its EP2 receptor may be a predictive diagnostic biomarker of ASD in relation to mitochondrial dysfunction and glutamate excitotoxicity
as etiological mechanisms of ASD, indicating that the above-described diagnostic markers
might contribute to early intervention [23].
In summary, combining α-synuclein, COX-2, and the PGE2/PEA receptor could play
an important role in brain development, with abnormalities in the COX-2/PGE2 signaling
pathway due to genetic or environmental causes linked to ASD [22].
6. Endocannabinoid Signaling
6.1. The Role of Endocannabinoids in Brain Function
The endocannabinoid system (ECS) consists of endogenous cannabinoids and metabolic
enzymes that play a critical homeostatic role in modulating polyunsaturated omega fatty
acid (PUFA) signaling to maintain a balanced inflammatory and redox state [24].
PUFAs may be linked to the ECS and involved in the maintenance of brain plasticity,
memory and learning, blood flow, and the genesis of neural cells [25]. The ECS is apervasive neuromodulatory system that plays an important role in the development of thecentral nervous system, synaptic plasticity, the response to endogenous and environmental
insults [24,26], neurotransmission, and microglial activation [27]. Cannabigerol, one of the
major phytocannabinoids present in Cannabis sativa L., may regulate signaling and exert
therapeutic effects via cannabinoid receptors [27]. ECS’s action is mediated by CB1 and CB2
receptors, which form heteromeric complexes (CB1–CB2 heteroreceptor complexes) [27].
ECS-induced metabolic outcomes may support key mediators and inflammatory resolution
pathways critical for maintaining homeostasis [25].
ECS signaling regulates proliferation, differentiation, and the death of brain cells andhas important consequences for neural development and brain repair via cannabinoid(CB) receptors [28]. Furthermore, crosstalk of the brain ECS with the immune system
maintains the neuroprotective and anti-inflammatory actions [28]. Thus, ECS-mediatedmaintenance of synaptic plasticity and neuronal cell function may contribute to endogenous
stem cell-based neuroprotective strategies [28].
The two cannabinoid receptors, CB1 and CB2, are derivatives of PUFAω-6 AA andω-3 fatty acids Wu [23]. The ECS plays a role in modulating behavioral traits [29]. The
modulation of anandamide (AEA), one of the main endocannabinoids in the brain, has beenshown to regulate social behaviors in animal models of ASD [29]. Social deficits, repetitive
behaviors, and abnormal emotional behaviors in VPA-treated offspring improved after
treatment with an inhibitor of AEA-degrading enzyme [29].

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6.2. The Role of eCBs in the Development of BrainAs described above, the main endocannabinoid in the brain, AEA, has been reported
to alter social behaviors in genetic models of ASD [29]. Such effects were mediated byenhancing the mechanism of removal of postsynaptic α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid receptor (AMPAR) subunits GluA1/2 underlying AEA signaling in
the PFC in a VPA-induced model of ASD [29]. eCBs exert epigenetic effects on neurotransmitter signaling and regulate their expression. Moreover, eCBs play a role in epigenetic
modifications, such as DNA methylation and histone modifications [30]. As eCBs play animportant role during neurodevelopment, the interaction between the ECS and epigenetic
modifications may play a regulatory role in neurodevelopmental disorders [30]. Thus, eCB
can epigenetically modulate neurodevelopment [30]. A recent study based on a murine
model of ASD (Shank3B–/– mice showing substantial social interaction impairment) showedthat BLA-NAc glutamatergic circuit activation reduces sociability and social reward seeking
without increased anxiety or rewarding effects [31].
2-arachidonoylglycerol (2-AG)-mediated cannabinoid signaling regulates the BLA–
NAc glutamatergic circuit [31], which was thus shown to be important for social functionin this animal model. 2-AG may be a promising target for the treatment of social domain
symptoms in ASD [31].
Studies based on animal models have revealed important roles for ECS combined with
G protein-coupled cannabinoid receptors. Furthermore, ECS’s endogenous lipid-derived
agonists may contribute to social anxiety and social reward as two key aspects of social
behavior [32]. Functional MRI studies in humans support such a role of anandamide [32].
A previous clinical study reported that 18 autistic patients aged between 7 and 18 years
were treated with cannabidiol (CBD) and CBD-enriched cannabis sativa extract, including
0.04 to 0.12 mg/kg/day CDB, for 6–9 months. CBD-enriched CE ameliorated multiple ASD
symptoms in 30% of patients [33].
Collectively, the role of eCB signaling may be related to social impairment in neuropsychiatric diseases such as ASD. With respect to the therapeutic potential of eCBs, the
ECS plays a key role in neurodevelopment via inflammatory responses and, thus, therenowned anti-inflammatory properties of cannabinoids might be useful in treating ASD
pathology [32].
6.3. The Role of Cannabinoid Compounds of Cannabigerol, CB1 and CB2 Receptors, andCannabidivarin in ASD Symptoms
Cannabigerol (CBG) is the precursor molecule for the most abundant phytocannabinoids [34]. CBG is one of the major phytocannabinoids in cannabis and acts as a competitive
partial agonist ligand [34]. CBG, related to cannabinoid receptors, such as the CB1–CB2 heteroreceptor complex in activated microglia, and CGB are potential targets for the treatmentof neurodegenerative diseases [34]. CBG has a potent inhibitory action on oxidative stress,
which in turn inhibits IκB-α phosphorylation and the translocation of nuclear factor-κB(NF-κB) [35]. In other words, CBG has a potent inhibitory effect on oxidative stress, acting via downregulation of the expression of the main oxidative markers and preventingIκB-α phosphorylation and translocation of NF-κB via modulation of the MAP kinase pathway [35]. Thus, the antioxidant properties of CBG may help treat oxidative stress-relateddisorders, such as ASD.
The microglia harbor a completely functional ECS signaling system [12]. In addition,
the ECS is closely involved in regulating microglia polarization [12]. Hence, the ECS may
affect inflammation due to its role in regulating microglia. The activation of CB2 receptors
not only affects the migration, proliferation, and release of proinflammatory cytokines frommicroglia but also promotes the transformation of microglial cells to the anti-inflammatory
phenotype [12].

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With respect to the role of CB1 and CB2, CBG binds CB1 and CB2 but functions as
a competitive antagonist for CB1. CB1 is important for neuronal differentiation, normal
axon migration, and the establishment of neuronal connectivity [12]. Early exposure oflipopolysaccharide (LPS) in rats impairs communication and cognition due to decreased
ozendocannabinoid (eCB)–CB1 binding, elevated major endocannabinoid ananadamide(AEA) levels, and increased fatty acid amide hydrolase (FAAH) in the amygdala [12]. Oraladministration of the FAAH inhibitor PF-04457845 (1 mg/kg) may normalize LPS-inducedsocial behavior changes [12]. A nonpsychoactive cannabinoid such as cannabidivarin(CBDV) might be beneficial therapeutic effects on social novelty, short-term memory, andhyperactivity in the VPA-induced autism rat model [12]. These neuroprotective effects are
mediated by the upregulated expression of CB2 protein in the hippocampus and enhanced
activity of microglia. ECS intervention may regulate neuroimmune inflammation via CB2
to alleviate ASD symptoms [12,29]. Exogenous insults, such as exposure to valproic acid,can also dysregulate resting CBD levels and CBD components [29]. Collectively, alteredendocannabinoid signaling and immune dysfunction may contribute to ASD pathogenesis
and the modulation of social behavior [29].
6.4. ASD and Endocannabinoids: The Role of Cannabidivarin and 2-AG
Anandamide-mediated signaling may act selectively in reducing social anxiety and enhancing social rewards in relation to ASD [27]. Importantly, treatment with the cannabinoid
agonist WIN 55,212-2 (1.2 mg/kg) persistently hampers social interactions and behavior [29]. According to a recent review article, CBD is considered as a most promisingtherapeutic target due to its wide range of pharmacological activities involving antioxidant, anti-inflammatory, and neuroprotective properties in neurological and neuroplastic
diseases [36]. The endocannabinoid retrogrades signaling pathway is widely expressedin the central nervous system in regulating synaptic plasticity (excitatory and inhibitory).
Cannabidiol (CBD)-enriched cannabis extracts have been widely used to treat children withrefractory epilepsy [37]. Additionally, CBD was useful as an anxiolytic- and antidepressantlike compound, possibly due to the effects on DNA methylation and microRNAs [38].
Although limited effects of CBD on ASD symptoms, a previous study reported clinicaleffects of CBD on ASD symptoms [39]. Moreover, according to a former review article, proinflammatory cytokines, such as IL-6, IL12, IL-1β, and TNF-α, released by immune cells in
the CNS, contribute to the development of neuroinflammation and neurodegeneration [39].
During inflammation, endogenous eCBs, such as arachidonyl ethanolamide (AEA) and2-arachidonyl glycerol (2-AG), are released from immune cells and neurons in the CNS
and play a neuroprotective role. Therefore, eCB is associated with therapeutic effects in
the regulation of immune responses. mRNA and CB2 receptors were upregulated in the
blood in children with autistic disorder, suggesting the involvement of the ECS in thedevelopment of ASD [39].
The phytocannabinoid cannabidivarin (CBDV) has the ability to ameliorate behavioral abnormalities resembling core and associated symptoms of ASD [40]. Restoration ofhippocampal eCB signaling and neuroinflammation may contribute to these effects [40].
This ability to restore ECS abnormalities might contribute to its beneficial effects on
ASD-like behaviors via the expression of CB2 receptors [38]. As a cellular mechanism,
2-arachidonoylglycerol (2-AG) eCB signaling reduced basolateral amygdala-nucleus accumbens (BLA-NAc), glutamatergic activity, and pharmacological 2-AG augmentation,
blocking social interaction deficits [31]. These data reveal the BLA-NAc circuit as a criticalregulator of social function and suggest that pharmacological 2-AG augmentation could be
a promising approach to the treatment of social domain symptoms in ASDs [31].

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Collectively, BLA-NAc circuit activation decreases sociability and is regulated by eCB
signaling. NAc-specific 2-AG augmentation and BLA-NAc circuit inhibition normalizedsocial deficits in the Shank3B–/– model of ASDs [31]. Therefore, 2-AG augmentation
reduced social deficits in Shank3B–/– mice via normalization of hyperactive GABAergic
and glutamatergic signaling in the NAc [31]. These findings reveal the BLA-NAc circuit
to be a critical regulator of social function. Importantly, 2-AG augmentation suggests a
promising approach to the treatment of social domain symptoms in ASDs [31].
6.5. Therapeutic Potential of the Endocannabinoid System for ASD
Cannabis is derived from Cannabis sativa, one of the world’s oldest propagated plants,
and its extracts are available for medical use [41]. According to a previous review article,
cannabis eased hyperactivity, sleep disorders, self-injury, anxiety, behavioral problems, andcommunication in 188 patients with ASD [41]. A previous clinical trial indicated that awhole-plant cannabis extract containing cannabidiol and ∆9-tetrahydrocannabinol significantly improved total SRS scores in 150 subjects with ASD compared to 36 control subjects
who received a placebo (n = 36) [42]. According to another study, cannabinoid cannabidivarin (CBDV) alters the balance of striatal ”excitatory–inhibitory” metabolites [41]. ASD
symptoms were linked to abnormalities in the striatum and its functional circuitry and, thus,
the function of the striatum displayed both hyper- and hypoconnectivity, with numerous
cortical regions involved. CBDV shifted atypical connections towards neurotypical findings
at the baseline [43]. Therefore, CBDV may play a role in the reduction of hyperconnectivity
in ASD.
Interestingly, cannabinoids (CBs) reduced the frequency of seizures in autistic patients
via the excitatory/inhibitory imbalance in the excitatory/inhibitory system, such as a
CB-related increase in glutamate (the excitatory system) in subcortical regions (i.e., basal
ganglia) and a decrease in cortical regions (i.e., dorso-medial prefrontal cortex). CBD
increased GABA transmission (the inhibitory system) in critical and subcortical regions ofnormal development subjects but decreased it in the same areas of the ASD group [44].
Collectively, CB may have important pharmacological potential for the behavioral
symptoms associated with ASD and ASD-related seizures. Although they have been shown
to reduce seizure frequency via the excitatory/inhibitory system [44], further investigations
are warranted to identify the conclusive clinical effects of CB and additional underlying
cellular mechanisms.
6.6. Endocannabinoid and Valproic Acid-Induced Model of ASD
A previous study reported reduced endocannabinoid (eCB) signaling in children
with autism and in an animal model of VAP-induced ASD. 2-AG deficiency induced behavioral defects caused by the absence of its primary synthetic enzyme, diacylglycerol
lipase α (DGLα), in dendritic mesoporous silica nanoparticles (dMSNs) [45]. These cellularand circuit-level changes were associated with reduced sociability, excessive repetitive
grooming, and social impairment [45]. Furthermore, elevation of 2-AG improved ASD-likefeatures in animal models [46]. Thus, disruption of 2-AG signaling in dMSN circuits could
contribute to the social and repetitive behavioral phenotypes associated with ASD [46]. In
autistic children and rat models of VPA-induced ASD, increased degradation of enzymes
and upregulated expression of cannabinoid receptors may lead to repetitive and stereotypical behaviors, hyperactivity, altered sociability and social preferences, and impaired
cognitive functioning [46]. These defects are reportedly improved following acute, as
well as chronic, treatment with JZL184 [46], which is the primary enzyme responsible fordegrading the eCB2-AG [47]. JZL184 treatment enhanced intrinsic 2-AG levels and ameliorated autistic behavior, such as repetitive and stereotypical behaviors, hyperactivity in the
open field test, sociability and social preference in the three-chamber test, and cognitive
functioning in VPA-exposed offspring [46]. Thus, JZL184 modulates neurotransmission
and neuronal plasticity and may contribute to the pathophysiology of ASD [46]. Thesefindings are important for potential therapeutic targets for ASD. JZL184-induced elevation

Int. J. Mol. Sci. 2022, 23, 9803

9 of 20in 2-AG levels confers protection against neuroinflammation, which ameliorates ASD-likebehavior [46]. Thus, reduced CBs were observed in autistic children and a murine model of
ASD, boosting 2-AG-ameliorated ASD-like phenotypes, suggesting a novel approach for
ASD treatment [46].
7. Redox Signaling
7.1. The Role of Redox Signaling
Redox signaling refers to the specific and usually reversible oxidation/reduction reactions involved in cellular signaling pathways [48]. Redox signaling regulates several
physiological processes (e.g., excitation–contraction coupling) and is involved in a wide variety of pathophysiological, homoeostatic, and stress response pathways [48]. Individuals
with ASD exhibit enhanced ROS signaling following phosphatidylinositol 3-kinase/protein
kinase 3 (PI3K/AKT) signaling pathway stimulation [49]. Environmentally, cellular levelsof reactive oxygen species (ROS) may be affected by prenatal neuroinflammatory dysregulation in neural stem redox signaling [49], suggesting an effect of neuroinflammatory
dysregulation on neural stem ROS levels. The genetic mutations were recognized in antioxidant and immune responses and ROS generation [49]. Rescue of maternal inflammatory
response revealed that brain overgrowth and pathway activation can occur following
inhibition of prenatal nicotinamide adenine dinucleotide phosphate (NADPH) oxidases(NOXs), which are enzymes that generate superoxide or hydrogen peroxide from molecularoxygen [50]. Importantly, environmental factors, such as mild maternal inflammation, andsubsequent alterations in the neural stem cell redox balance can significantly change brain
development in infants, leading to brain overgrowth and abnormal behaviors associatedwith ASD [49].
7.2. Reactive Nitrogen and Oxygen Species
Reactive oxygen species (ROS) act as secondary messengers regulating redox-sensitive
signaling pathways, which elicit specific cellular responses [51]. As redox signaling is
an intrinsic component of metabolism in humans, its disruption may contribute to thedevelopment of various neuronal disorders [51]. Redox signaling is a tightly regulated
process that controls cell growth, differentiation, and death [48]. An imbalance between
ROS and oxidative stress may contribute to many pathological conditions, including
ASD [49,50]. As S-nitrosylation plays an important role in biological processes in neuronaldifferentiation and maturation [52], S-nitrosylation-mediated redox signaling may act asa molecular switch, whereby redox-mediated post-translational modifications modulate
neurogenesis and neurodegeneration via a common transcriptional signaling cascade [53].
Additionally, ROS act as secondary messengers and affect oxidative activity to influenceimmune, inflammatory, and other signaling processes and redox-sensitive transcription
factors [54]. These mechanisms contribute to reduced antioxidative properties in ASD [54].
The role of ROS in COX-2 and cannabinoids is presented in Figure 3.

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FigureFigure 3.
3. Oxidative
Oxidative stressstress related
related oxygen
oxygen species
species activate
activate redox signaling
signaling which
which regulates
regulates several
severalphysiologicalphysiological processes
processes inin aa wide
wide variety
variety ofof endocannabinoid
endocannabinoid syatem
syatem inin pathophysiologicalpathophysiological andandhomoeostatic
homoeostatic pathways
pathways such
such asas COX-2,
COX-2, inducing
inducing neuriinflammation
neuriinflammation inin relation
relation toto ASD.
ASD.

7.3. The
The Association
Association between
between Redox
Redox Signaling
Signaling andand Iron
Iron
7.3.
The redox
redox state
state isis predominantly
predominantly reliant
reliant onon an
an ironiron redox
redox couple
couple andand isis maintained
maintainedThe
withinstrictphysiologicallimits.
Iron
acts
as
a
redox-active
cofactorinmanybiologicalwithin strict physiological limits. Iron acts as a redox-active cofactor in many biologicalprocesses andand plays
plays anan important
important rolerole in cell growth [55]. Iron
Iron homeostasis
homeostasis is
is vital
vital totoprocesses
human health,health, andand ironiron imbalance
imbalance can lead to various disorders [56].
human
A recent study
αA
study reported
reported thatthatsynucleinopathy
synucleinopathy(the(thepathologic
pathologicaccumulation
accumulationof ofsynuclein)
inducesthecellularironsequestration
response,
leadingtoneurobehavioral
α-synuclein) induces the cellular iron sequestration response, leading to neurobehavioral
deficits [55].
role
in in
biologicalprocesses
in neuronal
deficits[55]. AsAs S-nitrosylation
S-nitrosylationplays
playsananimportant
importantrolebiologicalprocesses
in neudifferentiation
andmaturation[52],
S-nitrosylation-mediatedredox
signaling
may actasronal differentiation and maturation [52], S-nitrosylation-mediated redox signalingmayamolecular
switch,wherebyredox-mediatedpost-translational
modifications
modulate
act as a molecular switch, whereby redox-mediated post-translational modifications
neurogenesis
and neurodegeneration
via a common
signaling cascade[53].
modulateneurogenesis
and neurodegeneration
via transcriptional
a common transcriptionalsignalingcascade [53].
8. Copper Signaling
8.1.
The Roleof Copper Signaling
8.
CopperSignaling
Human copper transporters are subject of a complex multilayer regulation by various
8.1. The Role of Copper Signaling
metabolic signals [57]. Ceruloplasmin (Cp) signaling has a broad and growing influence
Humancoppertransporters
subjectof a complex
multilayer
regulation
by varion humanhealth,such
as a role inareiron
homeostasis
by protecting
tissuesfrom oxidativeous
metabolicsignals[57].
Ceruloplasmin(Cp)signaling
has
a
broad
and
growing
indamage [58]. In mammalian systems, intracellular Cp trafficking is influenced by the redox
fluenceonhumanhealth,
such
as
a
role
in
iron
homeostasis
byprotecting
tissuesfromstate that triggers Cp-induced signals [59]. Importantly, copper signaling may prove to beoxidative
damageIn mammalian systems,
intracellularCp trafficking
is influenced
an effective
therapy[58].
for neurodegeneration[57]. Notably,
the Cp-dependentcommunication
by
the
redox
state
that
triggers
Cp-induced
signals[59].
Importantly,
copperbetween neurons and astrocytes is associated with various brain diseases [60]. signaling
may prove to be an effective therapy for neurodegeneration [57]. Notably, theCp-dependent communication between neurons and astrocytes is associated with various brain diseases [60].

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11 of 208.2. Copper Signaling and ASD
The antioxidant capacity of Cp is implicated in various neurodevelopmental disorders,such as ASD. Cp expression in myelinating glial cells is crucial to prevent oxidative stressand neurodegeneration in the central and peripheral nervous systems [61]. Lower plasma
Cp levels may contribute to ASD pathophysiology in young individuals [62–64]. AA is aknown regulator of signal transduction proteins and acts as a precursor of potent signaling
molecules [63–65]. Importantly, these findings indicate that the signaling mediator Cp
levels are downregulated via a competitive association between plasma omega-3 PUFAs
and omega-6 PUFAs, such as AA [63,65]. Specifically, lower plasma Cp levels due to lower
plasma AA levels may impair various signaling activities.
9. Iron Signaling and ASD
A former clinical study indicated that 93 children with ASD had lower ferritin andhigher mean corpuscular volume (MCV value) compared to 74 children with no neurodevelopmental disorders other than ASD [66]. The ASD gruop showed significantly higher
MCV values due to a deficiency of vitamin B12 and folic acid. Folic acid is essential for thecorrect synthesis of red blood cells [66]. Vitamin B12 is an essential component for DNA
synthesis, and vitamin B12 deficiency causes neurologic and psychiatric symptoms, such as
motor dysfunction, sensory and memory deficits, and cognitive impairment. Collectively,
ASD patients were more likely to have hypoferritinemia based on deficient vitamin B12
as well as folic acid, inducing ASD-related neurological symptoms [66]. Additionally, a
deficiency of iron and vitamin D, as well as iron-deficient anemia, were more common in
ASD subjects as compared to control children [67].
10. The Association between the Endocannabinoid Systems and Redox Signaling
The evidence indicates an important interplay between the cannabinoid receptors
CB1 and CB2, their synthetic and metabolizing enzymes, and various key inflammatory
and redox-dependent processes. The ECS may also control oxidative stress by directly or
indirectly modulating CB1 and CB2 signaling or CB receptor-independent processes via
COX-2-dependent eicosanoid pathways [68]. Modulation of cellular redox homeostasis isinduced by the ECS [69].
According to primary findings, cannabinoids (CBs) regulate the redox balance byaltering the level and activity of antioxidant molecules [70]. CBs target the regulation ofredox-sensitive transcription factors, such as Nrf2 in microglia, indicating an important
key role of Nrf2 in initiating the transcription of antioxidant and cytoprotective genes [70].
CB modulates the expression of the antioxidant enzyme heme oxygenase 1, suggesting arole for CBD in regulating cellular ROS levels [70]. CB can also modulate the importantantioxidation protein superoxide dismutase and its metabolized activities, such as Cu-, Zn-,
and Mn-SOD [70], and upregulate GSH levels, as well as, simultaneously, SOD1 and GPx
activity, based on the finding that the administration of CB reduces the oxidized glutathione
ratio (GSH/GSSG) in myocardial tissue of diabetic mice and prevents GSH depletion in
cardiac tissue following cardiotoxicity [70]. As endocannabinoid (eCB) interrupts free
radical chain reactions via the electrophilic aromatic molecular region [70], eCB is closely
related to antioxidant capacities. The regulatory impact of eCB on reactive oxygen species(ROS) produces nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and ROSscavenging superoxide dismutase enzymes [71]. However, few studies have reported thisassociation between cannabidiol (CBD) and SOD in ASD. Future studies should examine
these associations in ASD.
Collectively, as described above, changes in neuromodulatory eCB signaling were
found in patients with ASD and in VPA-induced rodent models of ASD. The presynaptic
release of glutamate induces postsynaptic mGluR-mediated upregulation of eCB and themain endogenous agonists of cannabinoid (CB) receptors, such as anandamide and 2arachidonoylglycerol (2-AG) [72], indicating a close relationship between the antioxidant
glutamine and ECS.

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With respect to genetic factors of eCB signaling, a recent study reported that the fatty
acid amide hydrolase (FAAH) C385A polymorphism was associated with this signaling in
the brain [73]. Epigenetic modifications of the eCB may be related to DNA methylation or
histone acetylation/deacetylation [73]. Epigenetic modifications of the eCB may be related
to DNA methylation and histone acetylation/deacetylation, and FAAH genes encoding
the CB1 receptor and FAAH hydrolyzing enzyme play a relevant role in neurologicaldisease pathogenesis and progression [73]. Moreover, FAAH genes may be involved in
ASD via network-level changes in neural connectivity associated with genetic variations
in endocannabinoid signaling and genotype-associated neural differences [74]. The ECS
and ROS are key cellular signaling systems in the modulation of diverse cellular functions.
A former review article suggested that CB1 activation and upregulation of brain CB2
receptors reduce oxidative stress in the brain, inducing less tissue damage and less neuroinflammation. Upregulation of CB2 in the peripheral and central nervous systems may
reduce neuroinflammation [75]. Furthermore, increased expression of CB1 receptors leads
to increased oxidative stress, lipid modifications, and inflammation, which, in turn, may
promote the progression of psoriasis into the advanced, arthritic form of the disease [76].
11. Modulation of Endocannabinoid Action in Neuroinflammation Is Related to ASD
Neuroinflammation contributes to the pathogenesis of various neurodevelopmental
disorders, such as ASD. Microglia and astrocytes are the main pathological components inthis process [76]. The endocannabinoid 2-arachidonoylglycerol (2-AG) modulates synaptic
function, neurophysiology, and behavior. 2-AG signaling is terminated by monoacylglycerol lipase (MAGL) [77]. MAGL is broadly expressed throughout the nervous system,and different brain cell types contribute to the regulation of 2-AG activity. Astrocytes
regulate 2-AG content and endocannabinoid-dependent forms of synaptic plasticity andbehavior [77]. Astrocytic MAGL is responsible for modulating 2-AG in neuroinflammation
through prostaglandins. The astrocytic–neuronal interplay thus provides distributed oversight of 2-AG metabolism and function and protects the nervous system from excessive
CB1 receptor activation in neuroinflammatory activation related to ASD [77]. Microglia
modulates brain homeostasis by controlling neuronal proliferation/differentiation andsynaptic activity [78]. As chronic inflammatory activation of microglia is correlated withseveral neurodegenerative diseases, the endocannabinoid (eCB) system cannabinoid 2
receptor (CB2R) signaling shifts the balance of neuroprotective genes, and homeostatic
genes may acquire therapeutic functionality in microglia for neuroinflammation involved in
ASD [79]. Glutamine synthetase (GS), an astrocyte-specific enzyme, plays an important rolein neuroprotection and can be modulated by eCB 2-arachidonoylglycerol (2-AG) throughextracellular signal-regulated protein kinase 1/2 (ERK1/2) [79]. Microglia modulate brain
homeostasis by controlling neuronal proliferation/differentiation and synaptic activity [80].
Chronic inflammatory activation of microglia is correlated with several neurodegenerative
diseases, and functional modulation of microglial phenotypes has been considered asa potential therapeutic strategy [78]. The eCB system’s cannabinoid 2 receptor (CB2R)
signaling shifts the balance of expression between neuroinflammatory genes, neuroprotective genes, and homeostatic genes toward the latter two, by which microglia acquire
therapeutic functionality in the neuroinflammation involved in ASD [79]. The modulationof endocannabinoid action in neuroinflammation in astrocytes and microglia is presented
in Figure 4.

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Figure 4. The association among endocannobinoid systems, cannobiboid receptor and GABAnergic
Figure 4. The association among endocannobinoid systems, cannobiboid receptor and GABAnergic
intrrneuron. The endocannabinoid system is emerging as a regulator of microglia, resulting in theintrrneuron.
The endocannabinoid
is emerging
as a regulatorof microglia,
resultingtheneuronal-microglia
communicationsystemsystem.
Cannabinoid
1 (CB1) receptor
signaling
on in
Gamneuronal-microglia
communication
system.
Cannabinoid
1(CB1)
receptorsignalingonGammama-Amino Butyric Acid (GABA) ergic interneurons plays a crucial role in regulating microglial
Amino
Acidto(GABA)
ergic interneurons
plays
a crucial role in regulating microglial activity
activityButyricin relation
pro-inflammatory
states andneurodegeneration.
in relation to pro-inflammatory states and neurodegeneration.

12. The Association between Lipid Metabolism and the Endocannabinoid System and12. The Association between Lipid Metabolism and the Endocannabinoid System andIts Related
Related Signaling
Signaling Pathways
PathwaysIts
The endocannabinoid
endocannabinoidsystemsystem(ECS)(ECS)has
hasbeenbeenrepresentatively
representatively
examinedfieldsThe
examinedin inthethefieldsofofneuroscienceandneurodevelopment.
With
respecttothepathophysiologyofASD,
neuroscience and neurodevelopment. With respect to the pathophysiology of ASD, several
several indicate
studies indicatethat eicosanoids
are lipid-based
signaling
molecules
thatplayinastudiesthat eicosanoids
are lipid-based
signaling
molecules
that playa rolerolepathophysiology
in the pathophysiology
oftoASDto theirinteraction
with cyclooxygenase
theof ASD due
their dueinteraction
withcyclooxygenase(COX)-derived(COX)-derived
prostaglandins
(PGs)
and
PGE2
[80].
AlthoughASD
isacomplexneuroprostaglandins (PGs) and PGE2 [80]. Although ASD is a complex neurodevelopmental
developmental
disorderwithamultifactorial
etiologyofsignaling
pathways,thehydisorder with a multifactorial etiology of signaling pathways, the hypothesis of a brain
pothesis
of
a
brain
link
betweenthecore
behavioral
symptoms
of
ASD
and
a
chronic
link between the core behavioral symptoms of ASD and a chronic neuroinflammatory state
neuroinflammatory
state of thethe mainbrain pathogenesis
may representofthemainpathogenesis
ASD.
ofthe brain may represent
ASD.
The
ECS plays aofkey
roleThein
ECS plays a key role
neurodevelopment
and normal
responses,
andneurodevelopment
and innormalinflammatory responses,
andinflammatory
may provide new
biomarkers
maypromisingprovide new
biomarkers
promisingtherapies in the future [80].
andtherapies
in theandfuture[80].
13. Dysregulation of the ECS in Fragile X Syndrome
Fragilemostcommonheritable
formof mental
retardation
andFragile XXsyndrome
syndrome(FXS)(FXS)isisthethemostcommonheritable
formof mental
retardation
monogenic
causecauseof ASD.
FXS ischaracterizedby motor,cognitive,
and social
alterations,and monogenicof ASD.
FXS
is characterizedby motor,cognitive,
and social
altermostly
with ASDwith
behavioral
The severity ofthese
symptoms
andations, overlapping
mostly overlapping
ASD phenotypes.
behavioral phenotypes.
Theseverityof thesetheirtimingmaybeexacerbatedor
advancedbyenvironmental
adversityinteracting
withsymptoms and their timing may be exacerbated or advanced by environmental adversity
thegenetic mutation[81]. Symptoms
ASDSymptoms
are frequently
observedin patientswith FXS;interactingwith the genetic
mutationof[81].
of ASDare frequently
observedinhowever,behavioral
similarities
anddifferences
between
FXS
and
ASD
are
importantthepatients with FXS; however, behavioral similarities and differences between FXSinandcausesandcorrelations
ofcauses
ASD with
Individuals
withFXSFXS.
andIndividuals
comorbid ASD
ASD areimportantin theand FXS.
correlationsof ASDwithwithhave
FXS
more
severe behavioral
problems
than individuals
FXS alone;patients
and comorbid
ASD have
more severe
behavioralwith
problems
thanhowever,individuals
with with
FXS
FXS
and
comorbid
ASD showedlesssevereimpairmentsin the lesssocialand communication
alone;
however,patients
with FXS
andcomorbid
ASD showed
severeimpairments insymptoms
thanpatientswithnonsyndromic
ASD.
Subjects
with
FXS
present
withSubjects
anxiety
the social and communication symptoms than patients with nonsyndromic ASD.
andseizuresin
additiontocomorbidASD
symptoms,
anddifferencesin
these
conditions
with FXS present with anxiety and seizures in addition to comorbid ASD symptoms, andInt. J. Mol. Sci. 2022, 23, 9803

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are recognized in patients with FXS and ASD, possibly due to the difference in the roleof fragile X mental retardation 1 protein (FMRP) in FXS and phenotypes of FMRO in
ASD [82]. Social anxiety is a common disorder with negative impacts in multiple domainsof function in clinical groups including those with fragile X syndrome and ASD. Initialsocial avoidance characterized all males with FXS, whereas prolonged social avoidance wasassociated only with boys with FXS who had elevated ASD features [83]. FXS is a leading
inherited cause of ASD and intellectual disability, resulting from a mutation in the FMR1
gene and loss of its protein product FMRP. Although it has this simple genetic origin, FXS
is a phenotypically complex disorder including a range of physical and neurocognitive
disruptions. Circuit hyperexcitability may be a common convergence point accountable
for many wide-ranging phenotypes in FXS [84]. The mechanisms for hyperexcitability in
FXS include changes in excitatory synaptic function and connectivity, reducing inhibitory
neuron activity and resulting in ion channel expression and conductance. FMRP is also an
important regulator of activity-dependent plasticity in the brain and can be a cause andconsequence of hyperexcitable networks in FXS [84].
The new drug ZYN002 is a transdermal cannabidiol gel formulation and targets thedysregulation of the endocannabinoid system [85]. Treatment with ZYN002 was found tobe safe and well-tolerated in a phase 2 open-label study and extension conducted in 20
children and adolescents with FXS. Behavioral symptoms were improved by blockade ofpresynaptic CB1 in the mouse model via blockade of the degradation of 2-AG. A blockadeof the degradation of anandamide improved learning and memory in the mouse model [85].
Fragile X syndrome (FXS) is caused by the silencing of the FMR1 gene, leading to the lossof fragile X mental retardation protein (FMRP), a synaptically expressed RNA-binding
protein regulating translation. The ECS is a key modulator of synaptic plasticity, cognitive
performance, and seizure susceptibility [86]. The cannabinoid receptors CB1 and CB2 areactivated by mGluR5 activation-related phospholipid-derived endocannabinoids. Thus,
blockage of ECS is a potential therapeutic approach to normalize specific alterations in
FXS [86]. As fatty acid amide hydrolase (FAAH) catalyzes the metabolism of the eCB
anandamide, treatment with the FAAH inhibitor URB-597 improved performance in fragile
X syndrome gene fmr1 KO mice, possibly via the anxiolytic effects of FAAH inhibition. The
results indicate that the eCB system is involved in FXS and suggest that the eCB system is apromising target for the treatment of FXS [87]. Collectively, dysregulation of ECS may be a
key element in the pathogenesis of FXS.
14. Limitation of This Review
This review has several limitations: (1) the discussion of neuroinflammation in generalis rudimentary; (2) the clinical use of non-steroid anti-inflammatory drugs and cannabinoids
does not support the conclusion.
(1) With respect to the role of neuroinflammation in ASD, neuroinflammation is
emerging as a key underlying mechanism of ASD [88]. The various inflammatory andanti-inflammatory effects of dietary components and changes in inflammatory status canprime offspring brain development in neurodevelopmental disorders. Although the impact
of maternal nutrients contributes to neuroinflammation changes in offspring, microglia
are key immunocompetent cells that produce and respond to inflammatory cues. During
neurodevelopment, these cues lead to changes in the neurovascular development andmaturation [88]. The regulation of non-neuronal cells and the control of neuroinflammation
depend on the local synthesis of the endogenous lipid amide palmitoylethanolamide (PEA)
and related endocannabinoids. When the imbalance between the synthesis and degradationof PEA is disrupted, the behavior of non-neuronal cells may not be appropriately regulated,and neuroinflammation exceeds the physiological boundaries [89]. Once administered informulations with adequate bioavailability, PEA may provide safe and effective control ofneuroinflammation [89]. Although the role of inflammation in the underlying mechanisms
in specific subgroups/subtypes of ASD (e.g., those with concurrent immunological disorders) has been reported, the role of anti-inflammatory medications as adjuvant therapy is

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unclear [90]. The exact mechanisms of action, including the biological association between
the anti-inflammatory mechanisms and behavior changes of individuals with ASD, are notclear [90]. Robust, large-scale clinical trials are needed to support the efficacy and safety ofanti-inflammatory interventions in ASD [90].
(2) With respect to the clinical use of non-steroid anti-inflammatory drugs and cannabinoids, the current evidence is suggested to be insufficient to support the prescription of
cannabinoids for the treatment of psychiatric disorders involving ASD [91]Cannabis has been used to alleviate symptoms associated with ASD (i.e., hyperactivity,
irritability, and aggressiveness) [92]. Moreover, cannabis and cannabinoids improved
cognition, sensory sensitivity, attention, social interaction, and language [92]. The mostcommon adverse effects were sleep disorders and restlessness [92]. However, comparing
the studies’ results is not possible because the authors used different designs to measure
the evolution of symptoms through cannabis use in autism, including a systematic review
using questionnaires or scales developed by the authors themselves [92]. None of thestudies reported the use of neuropsychological assessments to investigate the cognitive
aspect [92]. Moreover, samples were small, and several participants were lost during thestudy period [92]. Additionally, clinical studies should consider recruiting subjects within
narrower ranges of age and functional level, assess the long-term tolerability and safety ofcannabinoid treatments, and identify target populations within the ASD population thatmight benefit from these treatments [42].
Additinaly, a recent clinical study conducted in 53 children (median age: 11 years)
indicated that cannabidiol may improve symptoms of comorbidities of ASD; however, thelong-term effects should be evaluated in a large-scale study [93].
With respect to the risks of endocannabinoid therapy, cannabinoid signaling may
be involved in the social impairment and repetitive behaviors observed in individuals
with ASD due to a possible role of the EC system in excitatory–inhibitory (E-I) imbalance
and immune dysregulation in ASD [94]. Moreover, the consumption of cannabis induces
a higher risk of gingival and periodontal disease, oral infection, and cancer of the oralcavity [95]. Therefore, the medical use of endocannabinoids is limited. In summary, theclinical effects of endocannabinoid therapy do not support the conclusion.
15. Conclusions
The signaling pathways associated with lipid metabolism help to identify abnormalities and new strategies for treating the pathophysiology of ASD. The available experimental
evidence links to several common signaling pathways, such as prostaglandin-E2 (PGE2)
and cyclooxygenase-2 (COX-2), endocannabinoids (eCBs), redox signaling, iron signaling,
and copper signaling.
The alteration of PGE2 function by environmental insults has been linked to brain
pathology, including ASD. PGE2 is a membrane-derived lipid signaling molecule and plays
an important role in neuronal development, contributing to the risk of developing ASD
associated with canonical Wnt signaling. COX-2 is an enzyme for converting arachidonic
acid to prostaglandins and plays a key role in neuroinflammation, being implicated in thepathogenesis of neurodegenerative diseases, including ASD. The endocannabinoid system(ECS) consists of signaling for the maintenance of a balance between inflammatory andredox states, synaptic plasticity, and neuroendocrine functions. The ECS plays a role inepigenetic modifications, such as DNA methylation and neurodevelopment, via inflammatory responses, contributing to the pathophysiology of ASD. Abnormal COX-2/PGE2
signaling is related to the development of ASD. New synthetic inhibitors of lipolytic enzyme phospholipase A2 (PLA2), which is involved in the inflammatory atherosclerotic
process, may be used for the treatment of neuroinflammation-associated neurodevelopmental disorders in cells. As described in the limitation section, the clinical use of non-steroidal
anti-inflammatory drugs and cannabinoids are limited.
Redox imbalance induces cellular changes, leading to abnormal behaviors in ASD.
Redox signaling refers to the specific and usually reversible oxidation/reduction mod-

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ification of molecules involved in cellular signaling pathways. Environmental factors
related to mild maternal inflammation and subsequent alterations in brain development
may induce cellular changes, leading to the abnormal behaviors in ASD. The final common pathway of enhanced reactive oxygen species (ROS) signaling may act via resultant
PI3K/AKT pathway stimulation. The ECS may also control oxidative stress by directly
or indirectly modulating CB1 and CB2 signaling or CB receptor-independent processes
via COX-2-dependent eicosanoid pathways. The role of ROS in signaling pathways andredox-sensitive transcription factors contributes to the pathophysiology of ASD.
Cannabinoids regulate the redox balance by altering the level and activity of antioxidant molecules via ROS-producing NADPH oxidase (Nox) and ROS-scavenging superoxide
dismutase enzymes.
Neuroinflammation contributes to the pathogenesis of various neurodegenerative
diseases, such as ASD. Microglia and astrocytes are main pathological components in thisprocess in relation to the endocannabinoid systems.
We reviewed the current knowledge on the main signaling pathways that are commonly disturbed in ASD. Additionally, we highlighted some putative druggable targets
along these pathways in relation to novel therapeutic interventions for ASD. Fragile X
syndrome (FXS) is the most common heritable form of mental retardation and monogenic
cause of ASD. FXS is a leading inherited cause of ASD and intellectual disability, resulting
from a mutation in the FMR1 gene and loss of its protein product FMRP.
Accurate modulation of these prominent pathways may contribute to the neurodevelopmental regulation of homeostatic patterns and could be used to treat some ASDs.
Author Contributions: Conceptualization, K.Y.; Investigation, K.Y., and G.I.; Resources, KY.; Writing—original draft preparation, K.Y.; Writing—review and editing, S.Y.; All authors have read and agreed
to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors report that they have no related financial interests or potential
conflict of interest.

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