An Update on the Phenotype, Genotype and Neurobiology of ADCY5‐Related Disease

Abstract

Adenylyl cyclase 5 (ADCY5)‐related phenotypes comprise an expanding disease continuum, but much remains to be understood about the underlying pathogenic mechanisms of the disease. ADCY5‐related disease comprises a spectrum of hyperkinetic disorders involving chorea, myoclonus, and/or dystonia, often with paroxysmal exacerbations. Hypotonia, developmental delay, and intellectual disability may be present. The causative gene encodes adenylyl cyclase, the enzyme responsible for the conversion of adenosine triphosphate (ATP) to cyclic adenosine‐3′,5′‐monophosphate (cAMP). cAMP is a second messenger that exerts a wide variety of effects via several intracellular signaling pathways. ADCY5 is the most commonly expressed isoform of adenylyl cyclase in medium spiny neurons (MSNs) of the striatum, and it integrates and controls dopaminergic signaling. Through cAMP pathway, ADCY5 is a key regulator of the cortical and thalamic signaling that control initiation of voluntary movements and prevention of involuntary movements. Gain‐of‐function mutations in ADCY5 have been recently linked to a rare genetic disorder called ADCY5‐related dyskinesia, where dysregulation of the cAMP pathway leads to reduced inhibitory activity and involuntary hyperkinetic movements. Here, we present an update on the neurobiology of ADCY5, together with a detailed overview of the reported clinical phenotypes and genotypes. Although a range of therapeutic approaches has been trialed, there are currently no disease‐modifying treatments. Improved in vitro and in vivo laboratory models will no doubt increase our understanding of the pathogenesis of this rare genetic movement disorder, which will improve diagnosis, and also facilitate the development of precision medicine approaches for this, and other forms of hyperkinesia. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

Full text

REVIEW
An Update on the Phenotype, Genotype and Neurobiology
of ADCY5-Related Disease
Arianna Ferrini, PhD,
1
Dora Steel, MRCPCH,
1,2
Katy Barwick, PhD,
1
and Manju A. Kurian, PhD
1
*
1
Developmental Neurosciences, UCL Great Ormond Street Institute of Child Health, Zayed Centre for Research
into Rare Disease in Children, London, United Kingdom
2
Department of Neurology, Great Ormond Street Hospital, London, United Kingdom
ABSTRACT: Adenylyl cyclase 5 (ADCY5)-related phe-
notypes comprise an expanding disease continuum, but
much remains to be understood about the underlying
pathogenic mechanisms of the disease. ADCY5-related
disease comprises a spectrum of hyperkinetic disorders
involving chorea, myoclonus, and/or dystonia, often with
paroxysmal exacerbations. Hypotonia, developmental
delay, and intellectual disability may be present. The
causative gene encodes adenylyl cyclase, the enzyme
responsible for the conversion of adenosine triphosphate
(ATP) to cyclic adenosine-30,50-monophosphate (cAMP).
cAMP is a second messenger that exerts a wide variety of
effects via several intracellular signaling pathways.
ADCY5 is the most commonly expressed isoform of
adenylyl cyclase in medium spiny neurons (MSNs) of the
striatum, and it integrates and controls dopaminergic sig-
naling. Through cAMP pathway, ADCY5 is a key regulator
of the cortical and thalamic signaling that control initiation
of voluntary movements and prevention of involuntary
movements. Gain-of-function mutations in ADCY5 have
been recently linked to a rare genetic disorder called
ADCY5-related dyskinesia, where dysregulation of the
cAMP pathway leads to reduced inhibitory activity and
involuntary hyperkinetic movements. Here, we present an
update on the neurobiology of ADCY5, together with a
detailed overview of the reported clinical phenotypes and
genotypes. Although a range of therapeutic approaches
has been trialed, there are currently no disease-modifying
treatments. Improved in vitro and in vivo laboratory
models will no doubt increase our understanding of the
pathogenesis of this rare genetic movement disorder,
which will improve diagnosis, and also facilitate the devel-
opment of precision medicine approaches for this, and
other forms of hyperkinesia. © 2021 The Authors. Move-
ment Disorders published by Wiley Periodicals LLC on
behalf of International Parkinson and Movement Disorder
Society
Key Words: ADCY5; dyskinesia; hyperkinesia; move-
ment disorder; precision medicine
Adenylyl cyclases (ACs) comprise a family of molecules
involved in the conversion of adenosine triphosphate
(ATP) to cyclic adenosine-30,50-monophosphate (cAMP),
a secondary messenger that exerts a wide variety of effects
via several intracellular signaling pathways. Isoform 5 of
adenylyl cyclase (ADCY5) is highly expressed in the brain
and myocardium.
1
Brain-specific expression of ADCY5 is
extremely selective, with high levels of expression in the
striatum, nucleus accumbens, and olfactory tubercle.
1,2
This anatomic specificity likely underlies the impact of
ADCY5 mutations on the control of movement. Muta-
tions in ADCY5 have been linked to a range of complex
movement disorders often associated with neu-
rodevelopmental phenotypes. There are currently no clear
disease-modifying treatments for ADCY5-related disease,
although the role of caffeine is currently being explored.
Therefore, understanding the molecular mechanisms with
appropriate laboratory models is of the utmost impor-
tance. Here, we review the neurobiology of ADCY5 as
well as the clinical presentation and molecular genetic
---------------------------------------------------------
This is an open access article under the terms of the Creative
Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly
cited.
*Correspondence to: Professor Manju A. Kurian, UCL Professor of
Neurogenetics and NIHR Research Professor Honorary Consultant in
Paediatric Neurology, Developmental Neurosciences, UCL Great
Ormond Street Institute of Child Health, Zayed Centre for Research into
Rare Disease in Children, 20 Guilford Street, London WC1N 1EH, UK;
E-mail: manju.kurian@ucl.ac.uk
Relevant conflicts of interests/financial disclosures: Nothing to report.
Received: 18 September 2020; Revised: 23 November 2020;
Accepted: 21 December 2020
Published online in Wiley Online Library
(wileyonlinelibrary.com). DOI: 10.1002/mds.28495
Movement Disorders, 2021 1

features of ADCY5-related movement disorders. We also
review the current management of disease as well as possi-
ble future therapeutic strategies that could be developed
using new in vitro models and genome editing tools.
Biology of Adenylyl Cyclases
The family of human ACs encompasses 10 different
isoforms. Of these, nine are membrane-bound, and one
is a soluble isoform. The structure of membrane-bound
ACs (AC1-AC9) includes an intracellular N-terminus,
two repeats of six transmembrane helices domains (TM1
and TM2), two intracellular catalytic domains of 40 kDa
each (C1 and C2) and an intracellular C-terminus
(Fig. 1). AC activity is regulated by heteromeric G
protein-coupled receptors. All isoforms of membrane-
bound ACs are stimulated by the GTP-bound αsubunit
of G
sα
and inhibited by the αsubunit of G
iα
. Once acti-
vated, ACs catalyze the conversion of ATP to cAMP and
pyrophosphate. The generated cAMP then propagates
downstream signaling via specific cAMP-binding pro-
teins (eg, cAMP-dependent kinases, transcription factors,
or ion transporters).
Knock-out and overexpression cellular models have pro-
vided insights into the tissue distribution and differential
expression of the various AC isoforms.
3-6
It is not surpris-
ing that given the crucial importance of signaling integra-
tion in the brain, all nine membrane-bound ACs are
expressed in the central nervous system. Although some
isoforms are widespread (eg, AC6 and AC7), some are
only expressed in specific regions (eg, AC3 in the olfactory
cilia and AC5 in the striatum) (Supplementary Table S1).
Dopaminergic Signaling
in the Striatum
The striatum within the subcortical basal ganglia is a crit-
ical component of motor and reward systems. GABAergic
medium spiny neurons (MSNs) constitute 95% of the cellu-
lar population of the striatum, and they are the central
receiving station of the basal ganglia.
7
They are innervated
by excitatory glutamatergic fibers from the cortex and thal-
amus and by modulatory dopaminergic fibers from the
midbrain. MSNs are defined by their expression of the
dopamine- and cAMP-regulated phosphoprotein DARPP-
32.
8
There are two distinct populations of MSNs, based on
their neurochemical characterization and projection targets;
DRD1-expressing MSNs of the striatonigral direct path-
way and DRD2-expressing MSNs of the striatopallidal
indirect pathway.
Direct pathway MSNs innervate the output nuclei of
the basal ganglia, which are the internal segment of the
globus pallidus (GPi) and the substantia nigra pars reti-
culata (SNr). Indirect pathway MSNs project to the exter-
nal segment of the globus pallidus (GPe) and the
subthalamic nucleus (STN). Direct and indirect MSNs in
FIG. 1. General structure of adenylyl cyclase proteins. Adenylyl cyclases are transmembrane proteins that consist of two bundles of six transmembrane
domains. They are regulated by heterotrimeric G proteins coupled to membrane receptors. G protein complexes consist of α,β, and γsubunits. When
the receptor is activated by an hormonal stimulus, it undergoes a conformational change that causes the αsubunit to dissociate from the complex and
become bound to GTP. The Gα-GTP complex binds to and activates adenylyl cyclase. Activated adenylyl cyclase catalyzes the conversion of ATP to
cAMP. cAMP is a second messenger which activates downstream signaling regulating several intracellular pathways. GPCR = G-protein coupled
receptor; TM = transmembrane domain; C = catalytic domain; GTP = guanosine triphosphate; ATP = adenosine triphosphate; cAMP = cyclic adeno-
sine-30,50-monophosphate. [Color figure can be viewed at wileyonlinelibrary.com]
2Movement Disorders, 2021
FERRINI ET AL

the striatum exert opposite effects on the control of move-
ment. Activation of DRD1 stimulates striatopallidal path-
way MSNs resulting in disinhibition of thalamocortical
neurons and excitation of the motor cortex, which leads
to movement. On the other hand, on activation of the
indirect pathway, DRD2 inhibits striatonigral pathway
MSNs leading to inhibition of thalamocortical neurons
and the motor cortex, which leads to suppression of move-
ment and prevention of unwanted movements (Fig. 2A).
ADCY5 is the most expressed AC isoform in MSNs,
and it has been estimated that it accounts for more than
80% of cAMP production.
1,9
ADCY5 is located mostly
in DRD1-expressing MSNs.
10
Stimulation of DRD1
activates G
sα
-mediated ADCY5 activity increasing
cAMP levels whereas stimulation of DRD2 activates
G
iα
-mediated ADCY5 activity decreasing cAMP levels.
9
Increased intracellular cAMP levels are linked to pro-
tein kinase A (PKA)-mediated downstream signaling
(Fig. 2B). In MSNs, increased levels of cAMP and PKA
activity lead to increased phosphorylation of DARPP-
32 and transcription factor cAMP-responsive element-
binding protein (CREB) with a broad range of
downstream effects on neuronal function. Disruption of
cAMP signaling therefore contributes to post-synaptic
MSN dysfunction, which may underpin movement dis-
orders such as dystonia, chorea, and parkinsonism.
11
Together with ADCY5, other genes with key roles in
MSN-related cAMP signaling (such as PDE10A,
GNA01,GNAL1, and GPR88) have also been associ-
ated with overlapping motor phenotypes.
11
Clinical Features of ADCY5-Related
Disease
ADCY5 mutations were first implicated in neurologi-
cal disorders in 2012,
12
and they are associated with
heterogeneous syndromes. Movement disorders are
often a prominent feature of the clinical phenotype.
Classically, the condition presents in early childhood
with an initially fluctuating or paroxysmal hyperkinetic
movement disorder characterized by dystonia, chorea,
and/or myoclonus. There may be a progression with
age from paroxysmal to continuous abnormal move-
ments.
13
Symptoms vary greatly in severity between
patients. Exacerbations can vary in length from minutes
to hours or days, and the most widely reported trigger
is fatigue. Other triggers include anxiety, excitement,
and intercurrent illness. Axial hypotonia is often also
present and eye movements may be abnormal.
14
Although most other movement disorders remit during
sleep, patients with ADCY5-related disorders often
experience episodes of abnormal movement throughout
the night, resulting in significant disturbance.
15,16
A
recent study confirmed that ADCY5-related nocturnal
paroxysmal dyskinesia is not directly elicited by sleep
or because of a primary sleep disorder.
15
Rather,
patients showed nocturnal paroxysmal dyskinesia that
emerged during night-time awakenings with subsequent
delayed sleep (as opposed to movements associated
with drowsiness or delayed sleep onset). Patients were
also found to have long and often violent paroxysmal
dyskinesia on morning awakening. Except for sleep effi-
ciency and specific sleep measures related to prolonged
nocturnal awakenings, sleep architecture (proportion of
each sleep stage, respiratory events, periodic leg move-
ments, and muscle activity) is otherwise normal in
patients with ADCY5 mutations.
The primary disease phenotype has been labelled
“familial benign chorea”
17
or “familial dyskinesia with
facial myokymia”.
12
The term “myokymia”is technically
a misnomer; it describes twitching arising from a pathol-
ogy of the muscle or neuromuscular junction, whereas
the movements seen in ADCY5-related conditions are
believed to originate from the central nervous system
18
.
The perioral muscle twitching observed in patients with
ADCY5 gene mutations were initially described as
myokymia. However, a subsequent electromyography
study showed a complex electrophysiological pattern
with no evidence of myokymia.
18
Based on the clinical
phenomenology and electrophysiological findings, the
facial twitching and truncal jerks in these patients are
now considered to be dyskinesia or myoclonus-chorea.
Variant presentations reported in a small number of
patients include generalized myoclonus-dystonia,
19
spas-
tic paraparesis,
20
and, in one report, alternating hemiple-
gia of childhood.
13
The course of the condition is
generally stable after onset, and life expectancy is
believed to be normal.
21
Although the movement disor-
der can be significantly disabling, and poorly responsive
to many drugs, there are also some reports of spontane-
ous improvement in adolescence or adulthood.
14,22
The
majority of affected individuals have normal intelligence,
but intellectual disability does occur in a minority,
14
and
acquisition of early milestones is often delayed by the
movement disorder.
21
There is an impression that the
incidence of mood disorder and psychotic illness may be
increased, but this has not been reliably quantified.
23,24
ADCY5 encodes a specific adenylyl cyclase that is also
highly expressed in the myocardium,
2
and there have
been reports of cardiac complications such as congestive
heart failure in patients.
12
Brain imaging is normal,
21
and diagnosis is usually confirmed by genetic testing.
The first brain autopsy findings in a molecularly
proven case of ADCY5-dyskinesia (age of death,
46 years) have been recently reported.
23
In this study,
gross pathology was unremarkable with the exception
of mild pallor of the substantia nigra. Compared to
control subjects, there was no loss or decrease in size of
neurons in the patient. Increased immunoreactivity for
ADCY5 was found in neurons in multiple brain
regions. Interestingly, tau deposits were found in the
Movement Disorders, 2021 3
UPDATE ON ADCY5-RELATED DYSKINESIA

FIG. 2. Basal ganglia motor circuits. (A) Direct and indirect pathways of the basal ganglia. Direct and indirect MSNs in the striatum have opposite
effects on the control of movement. MSNs of the direct pathway innervate the internal segment of the globus pallidus (GPi) and the substantia nigra
pars reticulata (SNr). This results in the disinhibition of thalamocortical neurons and excitation of the motor cortex, which leads to movement. MSNsof
the indirect pathway project to the external segment of the globus pallidus (GPe) and the subthalamic nucleus (STN). Activation of indirect pathway
leads to the inhibition of thalamocortical neurons and the motor cortex, which leads to suppression of movement and prevention of unwanted move-
ments. (B) Role of ADCY5 in the integration of direct and indirect pathway signaling in medium spiny neurons. ADCY5 is mainly expressed on
DRD1-MSNs. Activation of DRD1 has a stimulatory effect and activates G
sα
-mediated ADCY5 activity, increasing intracellular cAMP levels. Increased
cAMP levels are linked to PKA-mediated downstream signaling. Activation of DRD2 has an inhibitory effect and activates G
iα
-mediated ADCY5 activity
decreasing cAMP levels. SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; GPe, globus pallidus external segment;
GPi, globus pallidus internal segment; STN, subthalamic nucleus. Green arrows, activation; red arrows, inhibition. [Color figure can be viewed at
wileyonlinelibrary.com]
4Movement Disorders, 2021
FERRINI ET AL

deep cortical sulci, midbrain, and hippocampus with
minimal amyloid pathology and no Lewy bodies. This
was somewhat surprising and further studies on molec-
ularly proven cases of ADCY5-related dyskinesia will
be needed to assess whether the disease has a tauopathy
component.
Molecular Genetic Features
of ADCY5-Related Disease
ADCY5 consists of 1261 amino acids encoded by a
21-exon gene located on chromosome 3p21.1. Muta-
tions in the ADCY5 gene were initially identified in
2001 in a single five-generation German kindred and
formerly described as familial dyskinesia and facial
myokymia.
25
Inheritance of ADCY5 mutations is usu-
ally autosomal dominant, and no reports of incomplete
penetrance have been published to date. There are sev-
eral reports of somatic mosaicism, where mosaic car-
riers may present with symptoms, although these are
often less severe.
23
Mosaic parents may also be asymp-
tomatic.
20
Autosomal recessive inheritance has been
reported in two families; in both, heterozygous carriers
were asymptomatic and homozygous individuals expe-
rienced a phenotype closely resembling the autosomal
dominant disease form.
26,27
Table 1 presents an over-
view of all the reported mutations and associated phe-
notypes. The biological basis for the observed clinical
heterogeneity needs further investigation because there
is no strikingly clear genotype–phenotype correlation. It
is possible that the p.R418W mutation is linked to a
more severe phenotype, whereas the p.A726T is associ-
ated with a milder phenotype. The p.R418W variant,
together with other mutations at this residue (p.R418Q,
p.R418G) are recurrent mutations in the majority of
reported cases, suggesting a mutational hotspot at Argi-
nine 418. In the last few years, exome sequencing has
emerged as a very powerful tool to identify causative
genes for rare Mendelian diseases.
12,30
Diagnostic
exome sequencing has also provided insights into the
molecular diversity of ADCY5-related dyskinesia and
identified several de novo mutations or previously
undiagnosed cases.
20,30,32
Interestingly, one of these de
novo variants was found again at the 418 site, con-
firming a markedly increased degree of intrinsic muta-
bility of this genomic site.
32
ADCY5 has two transmembrane domains (TM1 and
TM2), comprising six helices of hydrophobic amino
acids, and two cytoplasmic domains (C1 and C2). C1
and C2 are brought together to form an ATP-binding
site with a catalytic pocket for the hydrolysis of ATP.
33
As illustrated in Figure 3, the majority of the reported
mutations are located in C1 and C2 domains, suggesting
how they might affect the strength of enzyme-substrate
binding or the C1-C2 interaction to form the catalytic
pocket. For example, the most common mutation on
residue arginine 418 lies in the cytoplasmic domain C1
and replaces a branched positively charged amino acid
with the negatively charged amino acid tryptophan,
likely affecting the normal formation of the catalytic
pocket. It can be hypothesized that a gain-of-function
mutation facilitates the interaction between C1 and C2,
leading to enhanced cAMP production. Mutations out-
side C1 and C2 such as M1029L in the TM2 domain
are likely linked to rearrangement of the protein struc-
ture, which may eventually lead to C1 and C2 being in
closer proximity. Although most of the reported
ADCY5 mutations are missense, some frameshift muta-
tions are reported, such as the deletion p.K694_M696
in the intracellular catalytic portion. It is not clear
whether such mutations lead to loss- or gain-of-func-
tion; within in the transmembrane domain, it is possible
that these mutations could enhance the affinity between
C1 and C2, leading to aberrant dimerization and ligand-
independent interaction.
It is important to acknowledge that ADCY5-related
dyskinesia is not only clinically but also genetically het-
erogeneous. As discussed, although most of the reported
mutations are postulated to lead to a gain-of-function,
there are several studies suggesting that, at least for cer-
tain mutations, loss-of function may instead be the path-
ophysiological mechanism, especially in families where
autosomal recessive inheritance is observed.
26,27
For
example, the missense mutation described by Bohlega
et al on the C1 domain has been predicted to be damag-
ing by different in silico tools.
26
Based on the recessive
inheritance pattern, it is conceivable that this biallelic
change leads to loss of normal protein function. Further-
more, Carapito et al
34
reported a single de novo muta-
tion (c.2088 + 1G > A in a 50donor splice-site of
ADCY5) segregating with disease. This mutation is
predicted to induce mRNA degradation, suggesting that
ADCY5 haploinsufficiency may also be a possible
mechanism of disease. Therefore, it appears that
ADCY5-related dyskinesia can result from either a gain-
or loss-of-function mechanism, although the underlying
pathogenic processes accounting for these differences
are not yet fully understood. Further investigation is
needed to better delineate the link between ADCY5
mutations, effect on protein function and different dis-
ease phenotypes.
Proposed Molecular Mechanisms
Given the relatively recent identification of patho-
genic mutations in ADCY5, there are only very few
reports of in vitro functional studies assessing the
impact of mutant protein function. Chen and colleagues
performed some pivotal in vitro functional studies using
HEK293 cells overexpressing ADCY5. They showed
Movement Disorders, 2021 5
UPDATE ON ADCY5-RELATED DYSKINESIA

TABLE 1. Overview of reported ADCY5 mutations with associated clinical phenotype
Variant Features
ReferencesTranscript cDNA Protein Inheritance Mutation type Clinical phenotype
NM_183357.2 c.409_428del p.G137Cfs*184 Autosomal
recessive
Compound
heterozygous
Frameshift Generalized dystonia with
superimposed myoclonus
27
NM_183357.2 c.3037C > T p.R1013C Autosomal
recessive
Compound
heterozygous
Missense Generalized dystonia with
superimposed myoclonus
27
NM_183357.2 c.1252C > T p.R418W Autosomal
dominant/de
novo
Missense
Gain of function
Infantile- or early childhood-
onset axial hypotonia with
limb hypertonia, intermittent
tremors, paroxysmal
dyskinesia, myoclonus both
at rest and with activity,
involuntary choreic and
dystonic movements,
occasional facial
movements but not obvious
myokymia
Normal brain MRI
Delayed motor and speech
milestones
Mild cognitive delay
Abnormal saccades
Nonparoxysmal generalized
chorea (Benign hereditary
chorea, BHC)
Sleep disturbances
14,16,17,18,20,21,
28,29,30,31,47,48,49
Chen DH et al. 2015
Meijer et al. 2016
Friedman et al. 2016
NM_183357.2 c.1253G > A p.R418Q Autosomal
dominant
Missense Dystonia
Generalized chorea, mild
myoclonic jerks
Delayed motor milestones
21
Chen et al. 2015
Chang et al. 2016
Friedman et al. 2016
NM_183357.2 c.1252C > G p.R418G Autosomal
dominant
Missense Delayed motor and speech
milestones
Axial hypotonia, mild
generalized chorea and
dystonic posturing of the
limbs (tiptoe walking)
Anxiety and obsessive
compulsive disorders
18,20,21
NM_183357.2 c.1313G > C p.R438P Autosomal
dominant
Missense Paroxysmal dyskinesia, axial
hypotonia, dystonia, tremor
Normal motor and speech
development
Friedman et al. 2016
NM_183357.2 c.1378A > T p.I460F De novo Missense Lower face dyskinesias,
tongue thrusting, dysarthric
speech, phasic retro- and
laterocollis, and axial
dystonia.
Abnormal gait
32
NM_183357.2 c.1425C > G p.I475M N/A Missense N/A Only reported in ClinVar
(by Ambry Genetics)
NM_183357.2 c.1646 + 1G > A Altered
splicing
N/A Frameshift N/A Only reported in ClinVar
(by Ambry Genetics)
NM_183357.2 c.1762G > A p.D588N Autosomal
recessive
Missense Axial hypotonia with dystonia
Facial and oral twitching,
myoclonus, dysarthria
Delayed motor and speech
milestones
Normal cognitive function
26
(Continues)
6Movement Disorders, 2021
FERRINI ET AL

that two recurrent ADCY5 mutations (p.A726T and p.
R418W) cause a significant gain-of-function, with an
enhancement of cAMP production in response to
β-adrenergic stimulation compared to wild-type AC5,
supporting their causative role in the pathogenesis of
the disease.
28
Recently, Doyle et al
35
expanded on this,
characterizing five recurrent ADCY5 gain-of-function
mutations. Using a newly developed HEK293 line
depleted of other predominant adenylyl cyclase
isoforms, they demonstrated that ADCY5 mutants show
TABLE 1. Continued
Variant Features
ReferencesTranscript cDNA Protein Inheritance Mutation type Clinical phenotype
NM_183357.2 c.2088 + 1G > T Altered
splicing
Autosomal
dominant
Frameshift
ADCY5
haploinsufficiency
Mild choreiform movements
associated with dystonia
No facial myokymia
Normal psychomotor
development
Chen et al. 2012
Carapito et al. 2014
NM_183357.2 c.2176G > A p.A726T Autosomal
dominant
Missense Familial dyskinesia with facial
myokymia (FDFM), dystonic
movements of neck and
arms, perioral and
periorbital twitches
18
Fernandez et al. 2001
Chen et al. 2012
NM_183357.2 c.2722G > A p.E908K Autosomal
dominant
(mosaic
asymptomatic
parent)
Missense Axial hypotonia with dystonia
Delayed motor and speech
milestones
Spastic paraparesis with
hyperreflexia, hypertonia in
the legs, and extensor
plantar reflexes
20
NM_183357.2 c.2080_2088del p.K694_M696 Autosomal
dominant
Frameshift deletion Severe choreoathetosis
involving face, limbs and
trunk
Profound axial and
appendicular hypotonia with
no dystonia or myoclonus
Significantly delayed cognitive
function
Orolingual dyskenesia
29
NM_183357.2 c.3086 T > A p.M1029K Autosomal
dominant
Missense Severe dystonia, hypotonia,
chorea
Mild cognitive impairment
Familial dyskinesia with facial
myokymia (FDFM)
19
Chen DH et al. 2015
NM_183357.2 c.2180G > A p.R727K Autosomal
dominant
Missense N/A Zech et al. 2017
NM_183357.2 c.1196C > T p.P399L Autosomal
dominant
Missense N/A Zech et al. 2017
NM_183357.2 c.1400A > G p.N467S Autosomal
dominant
Missense N/A Zech et al. 2017
NM_183357.2 c.3177_
3182delTGA
p.D1060del Autosomal
dominant
In-frame deletion N/A Zech et al. 2017
NM_183357.2 c.3625A > G p.M1209V Autosomal
dominant
Missense N/A Zech et al. 2017
NM_183357.2 c.3045C > A p.D1015E N/A Missense Paroxysmal paralysis
Paroxysmal chorea
Mild hypotonia
Repeated attacks of
hemiplegia involving either
side of the body
Mild developmental delay
Westenberger et al. 2016
NM_183357.2 c.3074A > T p.E1025V N/A Missense Paroxysmal paralysis
Paroxysmal dystonia
Repeated attacks of
hemiplegia involving either
side of the body
Mild developmental delay
Westenberger et al. 2016
Movement Disorders, 2021 7
UPDATE ON ADCY5-RELATED DYSKINESIA

an enhanced response to G
αs
-stimulation. They further
demonstrated that increased cAMP at the membrane
results in increased downstream target gene transcrip-
tion, providing potential insights into pathogenic molec-
ular mechanisms. The increased cAMP promotes the
dissociation and activation of protein kinase A catalytic
subunits, which translocate into the nucleus and phos-
phorylate several proteins, including the CREB. This
stimulates an altered transcription which leads to a hyp-
eractivation of the direct pathway (Fig. 4).
In contrast to the gain-of-function effects of missense
mutations, an ADCY5 knock-out mouse generated by
homologous recombination exhibited a hypokinetic
phenotype with parkinsonism features.
36
Interestingly,
the same ADCY5 knock-out mouse was also used to
study ageing and longevity, showing that ADCY5 dis-
ruption increases lifespan by 30% through oxidative
stress protection.
37,38
Inhibition of ADCY5 activates
SIRT1/FoxO3a and Raf/MERK/ERK pathway that
upregulates the antioxidant mitochondrial enzyme
MnSOD, resulting in resistance to oxidative stress dur-
ing ageing.
37
It is known that increased levels of cAMP
are associated with oxidative stress.
39
Therefore, a
mechanism by which an overactivation of ADCY5
could lead to neuronal dysfunction may be through
increased oxidative stress in MSNs, potentially leading
to reactive oxygen species (ROS)-induced cell death.
The absence of neuronal loss in both available imaging
studies and on post-mortem analysis would however
not corroborate this theory. It is possible that single-
photon emission computed tomography or positron
emission tomographic neuroimaging might offer better
resolution than magnetic resonance imaging (MRI) to
investigate neuronal degeneration in ADCY5 patients.
40
Another potential mechanism for neuronal dysfunction
could be ATP depletion as a result of increased cAMP
production, leaving the cells depleted of energy. Further
in vitro and in vivo models are needed to test these pro-
posed hypotheses and to better delineate the molecular
mechanisms of disease at both the neuronal and sys-
tems level.
The enzyme adenylyl cyclase 5 receives signals from
striatal GPCRs, including dopamine receptors DRD1,
DRD2, and the A2A adenosine receptor.
9
A potential
reason why stress may trigger worsening of the symp-
toms lies in the hypothesized molecular mechanism.
FIG. 3. Localization of ADCY5 mutations. (A) Schematic representation of ADCY5 mutations in the gene. Localization of the reported patients’muta-
tions in the exons of ADCY5 gene. (B) Localization of ADCY5 mutations on the domains of the protein. TM, transmembrane domain; C1 and
C2, cytoplasmic domains. [Color figure can be viewed at wileyonlinelibrary.com]
8Movement Disorders, 2021
FERRINI ET AL

Stress increases striatal dopamine synthesis and release,
enhancing D1R sensitivity and activating ADCY5
through G
αs
. Mutated ADCY5 with gain-of-function
could increase ATP binding to the catalytic pocket,
increasing downstream levels of cAMP and subsequent
cellular activity.
24
Current and Future Therapeutic
Perspectives
To date, there are no disease-modifying therapies for
ADCY5-related disease that show proven long-term
efficacy. A good response to treatment with benzodiaze-
pines (clonazepam or clobazam) has been reported in
some patients with ADCY5-related dyskinesia,
21,31
and
there has also been a case report of positive response to
methylphenidate.
41
Deep brain stimulation has led to
significant, although partial, improvement in a number
of cases.
29
Most recently, some patients have reported a
dramatic improvement following drinking coffee,
suggesting that caffeine may be a useful treatment for
some.
42
The rationale underlying this phenomenon is
that caffeine is an antagonist of the adenosine A2A
receptors (A2AR) (localized preferentially in striatal
neurons expressing dopamine D2 receptors) that acti-
vate ADCY5.
43
Therefore, caffeine likely inhibits
ADCY5 by inhibiting A2A receptors, leading to clinical
improvement in patients with gain-of-function mutation
and ADCY5 overactivity. A pilot study on caffeine effi-
ciency in ADCY5-related dyskinesia (NCT04351360,
17/04/2020 on http://ClinicalTrials.gov) has been
recently started to determine the percentage of
responders to caffeine. The primary outcome measure
is an improvement in overall involuntary movements of
40% or more; the results of this trial are eagerly
awaited.
Of note, an aggravating factor that is consistently
observed across affected individuals is the presence of
anxiety and exposure to typical life stressors. Further
research will be needed to determine whether the num-
ber and frequency of movements might be reduced with
better stress management techniques or limitation of
stress-inducing activities.
Another recent insight into a targeted therapeutic
approach has been provided by the functional in vitro
studies of Doyle and colleagues
35
withtheirworkonP-
site inhibitors. P-site inhibitors are adenosine nucleotide
analogues that bind to the catalytic pocket of adenylyl
cyclase. It has been shown that the inhibitor SQ 22.536 is
able to hinder ADCY5 activity in ADCY5-overexpressing
HEK cells. However, SQ 22.536 has no ADCY5 specific-
ity, and it is anticipated that the lack of specificity would
lead to detrimental side effects because of the inhibition of
other ADCY isoforms. Further research is needed to iden-
tify better ADCY5-specific P-site inhibitors.
Another therapeutic avenue could involve RNA
manipulation techniques. For example, small interfering
RNA or antisense oligonucleotides are powerful tools
to reduce the expression of a single gene; ideally, domi-
nant, gain-of-function disease such as ADCY5-related
dyskinesia could be treated using such approaches that
specifically silence the mutant allele while leaving the
expression of the wild-type allele unperturbed.
FIG. 4. Disrupted cAMP pathway in medium spiny neurons with mutated
ADCY5. ADCY5 gain-of-function mutations lead to increased intracellular
cAMP levels. This, in turn, leads to increased activation of PKA and
increased levels of phosphorylated DARPP-32. Eventually, there is a dys-
regulation of the ERK pathway and an altered expression of genes under
the transcription factor cAMP-responsive element-binding protein (CREB)
with a broad range of downstream effects on neuronal function and a hyp-
eractivation of the direct pathway. PKA, protein kinase A; DARPP-32,-
dopamine- and cAMP-regulated neuronal phosphoprotein; PP-1, protein
phosphatase 1; STEP, striatal enriched phosphatase; ERK, extracellular
signal-regulated kinase; CREB, cAMP-response element binding protein.
[Color figure can be viewed at wileyonlinelibrary.com]
Movement Disorders, 2021 9
UPDATE ON ADCY5-RELATED DYSKINESIA

Although several therapeutic approaches to manage
the manifestation of disease have been attempted, these
treatments are still not entirely specific in targeting the
core underlying pathogenesis of this disorder. Hence,
better models enabling a deeper understanding of the
molecular mechanisms involved in the pathogenesis of the
disease and its impact on MSNs are of the utmost impor-
tance. In this respect, in vitro models with neurons derived
from human induced pluripotent stem cells (hiPSCs) can
not only shed light on the molecular mechanisms but also
drive the development of new therapeutic strategies in a
patient-specific manner, as already done for other
pediatric neurological disorders.
44
Recently, DARPP32-
expressing MSNs have been successfully differentiated
from human hiPSCs.
8
Genome editing tools such as the
clustered regularly interspaced short palindromic repeats
(CRISPR)-Cas9 system can be used to correct the muta-
tions in patient-derived cells or to generate isogenic control
lines, allowing detection of disease-specific phenotypes.
44
Therefore, patient-derived MSNs represent an unprece-
dented humanized tool to decipher the exact pathogenesis
of ADCY5-related dyskinesia and identify potential drug
targets for pre-clinical and clinical studies. Crosstalk
between neurons and other cell types with synaptic con-
nectivity is extremely important for neuronal networks,
and co-culture in vitro systems are now widely used to
study cell interactions and improve the maturation of
hiPSC-derived cells. hiPSC-derived MSNs with ADCY5
mutations can be potentially co-cultured with hiPSC-
derived cholinergic interneurons
45
to better mimic the
native microenvironment and the impact of cAMP dys-
regulation not only on MSNs but also on other neuronal
subtypes. Besides 2D monolayer cultures, 3D brain tissue-
like systems, either scaffold-free (eg, organoids) or
scaffold-based (eg, using biomaterials) are emerging as
novel model systems to investigate human brain develop-
ment and disease and could be used to elucidate the molec-
ular and cellular dysfunction in ADCY5-related disorders.
Phenotypic data obtained from these advanced in vitro
models could then be integrated with data obtained from
in vivo models and human patients. As previously dis-
cussed, an ADCY5 knock-out mouse model has been
used to study motor dysfunction in parkinsonism disor-
ders.
36
An ADCY5 knock-in mouse with constitutively
active ADCY5 is still lacking. It could be generated with
CRISPR-Cas9, and may potentially recapitulate the
motor features of patients with gain-of-function muta-
tions. In addition to rodents, other easily manipulable
species could be engineered to generate transgenic ani-
mals for disease modelling. For example, Drosophila
melanogaster is a simple, yet powerful, in vivo system
used to model Parkinson’s disease.
46
This simple organ-
ism could be used to recapitulate the pathogenic muta-
tions of ADCY5 and provide insights into the
pathobiology and genotype/phenotype relationships in
ADCY5-related disorders.
Conclusions
ADCY5-related dyskinesia is an evolving new genetic
disorder with a prominent motor phenotype, and one
of the many post-synaptic disorders now associated
with altered cAMP signaling. Functional studies have
shown increased adenylyl cyclase activity as a patho-
physiological factor in ADCY5-related dyskinesia with
gain-of-function mutations. As additional families are
characterized, the full spectrum of ADCY5 mutations
and their relationship to the phenotype of ADCY5-
related dyskinesia will be better elucidated. Better
cellular and animal disease models, such as the ones
discussed in this review will provide the basis for supe-
rior precision medicine approaches, therefore paving
the way for new treatments for ADCY5-related dyski-
nesia and other similar genetic movement disorders.
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Supporting Data
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UPDATE ON ADCY5-RELATED DYSKINESIA

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Authors’Roles
(1) Research Project: A. Conception, B. Organization, C. Execution; (2) Manuscript: A. Writing of the First Draft,
B. Review and Critique;
A.F.: 1A, 1B, 1C, 2A
D.S.: 1C, 2B
K.B.: 1C, 2B
M.K.: 1A, 1B, 2B
Financial Disclosures
The authors declare that there is no financial or any other type of conflict of interest.