Gαo is a major determinant of cAMP signaling in the
Gαo is a major determinant of cAMP signaling in the
Gαo is a major determinant of cAMP signaling in the
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Cell Rep. Author manuscript; available in PMC 2021 February 24.
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2Department of Biomedical Science and Brain Institute, Charles E. Schmidt College of Medicine,
Florida Atlantic University, Jupiter, FL 33458, USA
3Center for Integrative Brain Research, Seattle Children’s Research Institute, Seattle, WA 98101,
USA
4Neurobiology Laboratory, National Institute of Environmental Health Sciences, Durham, NC
27709, USA
5Institute
of Biomedical Research (BIOMED), Catholic University of Argentina, Buenos Aires
C1107AAZ, Argentina
6Department of Pediatrics, University of Washington, Seattle, WA 98101, USA
7Lead contact
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SUMMARY
The G protein alpha subunit o (Gαo) is one of the most abundant proteins in the nervous system,
and pathogenic mutations in its gene (GNAO1) cause movement disorder. However, the function
of Gαo is ill defined mechanistically. Here, we show that Gαo dictates neuromodulatory
responsiveness of striatal neurons and is required for movement control. Using in vivo optical
sensors and enzymatic assays, we determine that Gαo provides a separate transduction channel
that modulates coupling of both inhibitory and stimulatory dopamine receptors to the cyclic AMP
(cAMP)-generating enzyme adenylyl cyclase. Through a combination of cell-based assays and
rodent models, we demonstrate that GNAO1-associated mutations alter Gαo function in a neuron-
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Overall, our findings suggest that Gαo and its pathological variants function in specific circuits to
regulate neuromodulatory signals essential for executing motor programs.
Graphical Abstract
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In Brief
Muntean et al. describe biochemical, cellular, and physiological mechanisms by which the
heterotrimeric G protein subunit Gαo controls neuromodulatory signaling in the striatum and
elucidate mechanisms by which Gαo mutations compromise movements in GNAO1 disorder.
INTRODUCTION
G protein-coupled receptors (GPCRs) mediate communication between neurons to regulate
complex behaviors (Betke et al., 2012; Bjarnadóttir et al., 2006; Pierce et al., 2002). All
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One of the most mysterious and intensely studied Gα proteins is G protein alpha subunit o
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(Gαo), for which a number of mechanisms (Purvanov et al., 2010; Solis et al., 2017) and
effectors (Campbell et al., 1993; Ewald et al., 1989; VanDongen et al., 1988) have been
proposed. Gαo is one of the most abundant proteins in the brain, highly conserved in
evolution and critical for nervous system function (Solis et al., 2017; Sternweis and
Robishaw, 1984; Strittmatter et al., 1990; Wolfgang et al., 1990). A particularly controversial
subject is the role of Gαo in regulation of cyclic AMP (cAMP), a ubiquitous second
messenger regulated by a number of GPCRs. cAMP is critical for many fundamental
neuronal processes, including neuromodulation, synaptic plasticity, and excitability
(Håkansson et al., 2004; Kandel, 2012). Early in vitro studies showed that Gαo does not
directly modulate the activity of the cAMP-producing enzyme adenylyl cyclase (AC) (Wong
et al., 1992), with the exception of modest effects on the AC1 isoform (Taussig et al., 1994).
Nonetheless, in cellular systems, Gαo impacts cAMP production (Feng et al., 2017;
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Ghahremani et al., 1999). How this occurs remains unclear, primarily due to a lack of tools
to disentangle influences of multiple G protein species concurrently activated by GPCRs in
an endogenous setting (Sadana and Dessauer, 2009; Sunahara et al., 1996).
Interest in Gαo has been spurred by the discovery that mutations in its gene, GNAO1, cause
neurological disorders (early in-fantile epileptic encephalopathy [Online Mendelian
Inheritance in Man (OMIM): 615473] and neurodevelopmental disorder with involuntary
movements [OMIM: 617493]), collectively referred as GNAO1 encephalopathy (Ananth et
al., 2016; Nakamura et al., 2013). Clinical features of this disease include delayed
psychomotor development, intractable seizures, and hyperkinetic involuntary movements. To
date, 26 unique pathogenic variants in Gαo have been reported (Kelly et al., 2019; Mihalek
et al., 2017). Knockin mouse models carrying orthologous mutations to clinical variants in
Gαo recapitulate movement dysregulation phenotypes (Feng et al., 2019; Larrivee et al.,
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In this study, we establish the role of Gαo in neuromodulatory control of striatal circuitry
and movement in mice. By recording cAMP dynamics in individual neuronal populations
using real-time optical sensors, we defined the contribution of Gαo in processing dopamine
and adenosine signals. We further delineated underlying signaling mechanisms in cell-based
and enzymatic biochemical assays. On the basis of these findings, we probed how
pathogenic mutations causing the GNAO1 disorder affect G protein signaling, neuron
function, and behavior leading to functional classification of disease mechanisms. Together,
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our findings resolve the mechanistic role Gαo plays in controlling GPCR signaling to cAMP
and demonstrate how its disruption manifests in disease.
RESULTS
Gαo acts in striatal neurons to enable coordination of movements
The striatum is a key brain structure for motor control implicated in the pathophysiology of
many movement disorders (Giordano et al., 2018). To begin probing the role of Gαo in
S1A). The resulting Gnao1flox/flox:RGS9Cre striatal knockout (Str KO) animals were viable
and fertile and did not show gross hyperactivity (Figures S1B and S1C) reported in the
global knockout (Jiang et al., 1998).
We assessed motor functions of Str KO mice and their wild-type (WT) littermates
(Gnao1flox/flox) in a panel of neurological tests. When evaluating dystonia features, we found
that in contrast to normal outward hindlimb extension by WT mice, Str KO clasped their
hindlimbs inward to the abdomen (Figure 1B). The Str KO animals also showed profound
deficits when challenged to coordinate their movement sequences when walking backward
on a rotating beam (Figure 1C). Similarly, the Str KO displayed ineffective hindlimb use in
the ledge test when compared to the WT littermates (Figure 1D). The performance of Str KO
mice was also significantly reduced in both vertical and horizontal pole tests (Figures S1D
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Finally, we probed the role of Gαo in the acquisition and retention of motor skills using
accelerated rotarod. This test revealed that Str KO mice had significantly reduced
improvements in performance compared with WT when assayed across multiple consecutive
trials (Figures 1E and S1F). The motor learning impairment in Str KO animals persisted over
prolonged periods of time (Figures S1G–S1I). Importantly, the observed motor deficits were
not due to physical inability, as both genotypes had equal grip strength (Figure S1J).
The striatum contains two populations of medium spiny neurons (MSNs) characterized by
divergent projection sites and differential expression of dopamine receptor subtypes: Drd1-
expressing direct-pathway neurons (dMSNs) and Drd2-expressing indirect-pathway neurons
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(iMSNs) (Gerfen et al., 1990). Synchronized activity between these two neuronal
populations is thought to coordinate motor programs (Jin et al., 2014; Tecuapetla et al.,
2014, 2016). Therefore, we next sought to determine the identity of striatal output neurons
requiring Gαo for motor control. First, we eliminated Gαo selectively in dMSNs (dMSN
KO) by crossing Gnao1flox/flox with a Drd1aCre driver line (Figure 1F and S1A). Assessment
of dystonic features in dMSN KO revealed no differences in hindlimb clasping score
compared to WT littermates (Figure 1G). The dMSN KO mice also performed similarly as
WT littermates during the backward walking challenge (Figure 1H), ledge test (Figure 1I),
vertical pole climb (Figure S1K), and horizontal pole assay (Figure S1L). Interestingly,
dMSN KO phenocopied only the motor learning deficits observed in Str KO mice, showing
a significantly decreased performance on rotarod relative to WT littermates (Figures S1M
and 1J). Grip strength was again similar between genotypes (Figure S1N), and motor
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learning deficits persisted over extended period of time similar to what was observed in Str
KO (Figures S1O–S1Q).
We next probed the role of Gαo in iMSNs by crossing Gnao1flox/flox mice with a Drd2Cre
driver line (iMSN KO) (Figures 1K and S1A). In contrast to dMSN KO mice, iMSN KO
mice phenocopied Str KO across our neurological panel of coordination and balance tests.
First, iMSN KO displayed dystonic features, as evidenced by a greater hindlimb clasping
score compared to WT littermates (Figure 1L). iMSN KO also showed deficits in the
backward walking challenge, ledge test, vertical pole climb, and horizontal pole tests
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(Figures 1M, 1N, S1R, and S1S) but no motor learning deficits in the rotarod task (Figures
S1T and 1O). Together, these findings indicate that striatal Gαo is required for proper motor
function differentially programming movements through actions in dMSN and iMSN
populations.
Loss of Gαo causes population-specific changes in the activity of striatal neurons and
their responses to neuromodulatory inputs
To identify the cellular correlate underlying changes in the motor behavior, we analyzed the
activity of striatal neurons by patch-clamp electrophysiology in brain slices. dMSNs and
iMSNs were identified by stereotaxic injection of a Cre-dependent adeno-associated virus
(AAV) encoding EYFP into dorsomedial striatum of adult mice (Figure S2A). Measurement
of intrinsic membrane properties of iMSNs showed no significant alterations in firing
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frequency, in both iMSN KO and dMSN KO (Figures S2F–S2H) consistent with the
postsynaptic nature of a change. We next examined the contribution of AMPA receptors
(AMPARs) and NMDA receptors (NMDAR) to changes in EPSCs (Figures 2G and 2H). We
found that the AMPAR component was significantly increased in both iMSN KO and dMSN
KO, whereas the NDMA currents were not significantly changed (Figures S2I and S2J). As
a result, deletion of Gαo significantly increased the AMPAR/NMDAR ratio in iMSN KO
(Figure 2I) and dMSN KO (Figure 2J). We further ascertained the postsynaptic nature of
observed changes by examining the dynamics of dopamine input onto MSNs using cyclic
voltammetry and found no changes in evoked dopamine dynamics in either dMSN KO or
iMSN KO slices (Figures S2K and S2L). Together, our electrophysiological data indicate
that Gαo acts postsynaptically in MSNs to significantly impact synaptic properties of both
MSN populations and selectively influence intrinsic excitability of dMSNs.
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We next assessed the impact of Gαo on regulation of MSNs by two key neuromodulators in
the region, dopamine and adenosine. Since neuromodulators do not elicit readily recordable
changes in the electrophysiological properties of MSNs (Gerfen and Surmeier, 2011), we
used an optical strategy (Muntean et al., 2018). We introduced a fluorescence resonance
energy transfer (FRET)-based cAMP biosensor into MSNs cultured from neonatal Str KO or
WT mice and studied real-time dynamics of cAMP modulation. Segregated expression of
dopamine and adenosine receptors enabled the classification of MSNs based on the direction
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of the cAMP response, which was stimulated by dopamine (via DR1) and decreased by
adenosine (via A1R) in dMSNs but inhibited by dopamine (via DR2) and increased by
adenosine (via A2AR) in iMSNs (Figure 2K). Initial analysis in Str KO revealed that both
dMSNs and iMSNs exhibited significant elevation in cAMP (Figure 2L). In dMSNs,
dopamine increased the cAMP signal in a dose-dependent manner (Figure S2M). This
relationship had a prominent leftward shift and augmented maximal-response-amplitude
neurons (Figure 2M). dMSNs also exhibited a dose-dependent inhibition of cAMP in
response to adenosine (Figure S2N). There was no shift in the dose-response of this
inhibitory effect; however, these neurons also exhibited reduced maximal response
amplitude (Figure 2N). Thus, in dMSN neurons, Gαo affects both the efficacy and potency
of responses to dopamine while only modulating adenosine efficacy.
dopamine, and only its efficacy was reduced (Figures 2O and S2O). In contrast, iMSN KO
displayed a leftward-shifted dose response following stimulation by adenosine as well as
reduction in its maximal amplitude (Figures 2P and S2P). Thus, in iMSN neurons, Gαo
affects both efficacy and potency of responses to adenosine while only modulating dopamine
efficacy. Taken together, these data indicate that Gαo plays pivotal role in controlling the
potency and efficacy of stimulatory neuromodulation while only affecting the efficacy of
inhibitory inputs in both populations of striatal neurons.
Gαo tunes efficacy and potency of GPCR signaling to cAMP via Gβγ
To obtain insight into how Gαo drives changes in cAMP induced by the Gαo loss, we
analyzed players and reactions involved in this process. First, we confirmed changes in the
baseline cAMP levels by biochemical ELISA-based approach. Striatal tissue samples from
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Str KO animals showed a significant increase in total cAMP levels compared to WT mice
(Figure 3A). Western blotting confirmed a significant loss of Gαo in Str KO tissue samples,
but no effects on expression of key proteins involved in cAMP generation, such as Gαolf,
Gβ2, or AC5 (Figure S3A). Furthermore, Str KO tissues did not show significant effects on
levels or activation of kinases affected by dopamine, including Akt, ERK1/2, and GSK3β
(Figure S3B). Thus, Str KOs show selective cAMP elevation that is likely driven by
alterations in signaling rather than changes in expression of key molecular players.
et al., 1996). We found that purified recombinant Gαo charged with a non-hydrolyzable
GTP analog (Gαo-GTPγS) did not significantly affect cAMP production, consistent with
previous work showing Gαo does not affect the activity of AC5 (Taussig et al., 1994). In
contrast, addition of Gαi-GTPγS readily inhibited cAMP production (Figure 3B). Control
experiments confirmed equivalent activity of our recombinant Gαo-GTPγS and Gαi-
GTPγS preparations based on their interaction with the Gi/o effector PDE6γ (Figure S3C).
These results suggest it is unlikely that the increase in the cAMP levels we see in Gαo KO
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Interestingly, we found that Gβγ subunits effectively reduced the efficacy, but not potency,
of the Gαi-mediated inhibitory effect on cAMP production (Figures 3C–3E). This effect is
reminiscent of the reduced efficacy of inhibitory cAMP modulation by dopamine in iMSN
(via D2R) and adenosine in dMSNs (via A1R). Because Gαo is an effective liberator of
functional Gβγ subunits (Digby et al., 2008), it is possible loss of Gαo could result in
excess Gβγ signaling activity (Yoda et al., 2015). To test this hypothesis, we compared the
AC activities in striatal membrane preparations from WT and Str KO mice. These
experiments revealed prominent leftward shift in forskolin dose dependence in Str KO
relative to WT (Figures 3F–3H), a hallmark effect produced by Gβγ binding to AC5,
sensitizing it to the stimulatory influence of Gαs/olf and forskolin (Gao et al., 2007; Xie et
al., 2012). To test the sensitizing effect more directly, we employed biochemical assays
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stimulating membrane preparations with selective agonists for either D1R (SKF81297;
dMSN) or A2AR (CGS21680; iMSN) and measuring the effect on the cAMP production.
Indeed, we found significantly greater cAMP production in membranes from Str KO upon
activation of D1R or A2R (Figure S3D). Addition of a Gβγ blocker reduced AC5 activity,
producing a rightward shift in the dose-response curve to stimulation by recombinant Gαs-
GTPγS while completely reversing response augmentation in Str KO membranes back to
WT levels (Figures S3E and S3F). To probe the biochemical mechanism further, we
immunoprecipitated AC5 from striatal tissue and determined its association with Gβγ. We
observed significantly increased interaction of Gβ1 with AC5 from Str KO tissue as
compared to WT controls (Figure 3I). Collectively, biochemical data support the mechanism
whereby Gαo modulates AC5 responsiveness to stimulatory Gαs/olf inputs by controlling
the release of Gβγ.
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Collectively, these results indicate that Gαo controls cAMP production by modulating the
levels of Gβγ bound to AC5, which enhances efficacy and potency of the stimulatory cAMP
influence while reducing efficacy of the inhibitory influence of GPCRs on cAMP
generation.
GNAO1 encephalopathy (Kelly et al., 2019; Mihalek et al., 2017) using our recently
developed molecular deconvolution platform (Masuho et al., 2018). Structural modeling
showed that these mutations mapped to highly conserved motifs among the Gα protein
family residing predominantly around the phosphate-binding loop (P loop), switch II, and
switch III regions (Figure 4A), suggesting potential influence on Gαo activation (Bosch et
al., 2012) and its interaction with guanine nucleotides (Nakamura et al., 2013).
Western blotting of transfected HEK293 cell lysates showed that all Gαo mutants tested
were expressed at levels similar to WT Gαo, indicating that mutations did not significantly
compromise protein folding and/or stability (Figure 4B). We next assessed the functional
activity of Gαo mutants based on their ability to propagate signals using D2R as a model
GPCR. Using the bioluminescence resonance transfer (BRET) strategy that monitors G
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We further examined the effects of representative mutations in both the P loop (G42R) and
switch regions (G203R and R209C) on D2R-catalyzed G protein activation using a
bimolecular strategy that monitors heterotrimer dissociation directly via changes in BRET
between NanoLuc-tagged Gαo and the Venus-tagged Gβγ (Figure S4A). We found that all
three mutations resulted in a significantly decreased level of trimer dissociation (Figures
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S4B and S4C) confirming the data with the indirect Gβγ-release measurements. These
observations indicate that all Gαo mutants have impaired activation by GPCRs.
To understand what deficits in G protein cycle underlie the observed loss of signaling we
examined the heterotrimeric complex formation of each mutant Gαo with Gβγ. We used the
same Gβγ-release BRET assay but measured a suppression of the baseline signal upon
introduction of exogenous Gαo (Figure 4F). We found that mutations proximal to the P loop
(G42R, S47G, and I56T) resulted in prominent deficits in Gβγ binding. G203R led to small
but significant reduction in signal (Figure 4G). When trimer formation was corrected for
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fluctuations in expression levels, we found that only G42R, S47G and I56T mutants
significantly affected Gβγ binding compared to WT Gαo (Figure 4H). Thus, association
with Gβγ is only compromised by mutations in the P loop of Gαo.
The dominant nature of GNAO1 disorder implies that mutant proteins are expressed
alongside a WT Gαo copy. Therefore, we next tested Gαo variants for the interference with
the dopamine responses of WT Gαo (Figure 4I). We found that five switch region mutants
(G203R, G204R, R209C, Q233P, E246K) significantly suppressed BRET responses
indicating that they interfere with D2R-mediated activation of the WT Gαo (Figures 4J and
4K). To assess how these dominant-negative effects occur, we developed an assay to
measure association of G protein heterotrimers with the D2R. This approach relies on
complementation between SmBit-tagged D2R and LgBit-tagged Gβγ, producing
luminescent NLuc upon interaction (Figure 4L). We found that all Gαo mutants showed
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two of the most prevalent Gαo variants that also feature prominent effects on signaling
observed in reconstituted studies, G203R (dominant negative with strong GPCR trapping)
and R209C (dominant negative without increased GPCR association). The mutants were
introduced into neurons cultured from Gnao1flox/flox mice containing WT Gαo to better
model the heterozygosity situation occurring in the brains of GNAO1 patients. Dose-
response studies were performed to evaluate the effects on both potency and efficacy of
signaling (Figures 5A, 5E, S5A, and S5C). Strikingly, these studies revealed that Gαo
mutants produced unique effects differentially impacting dopamine signaling in a MSN-
subtype-selective fashion (Figures 5B and 5F). The R209C mutant affected iMSN responses
exclusively, lowering the efficacy of dopamine signaling. In contrast, the G203R mutant
affected both populations increasing potency of the response in dMSN and lowering efficacy
in iMSNs.
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These changes were mirrored by alterations in adenosine responses (Figures 5C, 5G, S5B,
and S5D). Here, the R209C mutation affected dMSN exclusively, diminishing the efficacy of
adenosine signaling (Figures 5D and 5H). In contrast, G203R mutation affected both
populations, reducing efficacy in dMSNs while increasing potency of adenosine responses in
iMSNs (Figures 5D and 5H). In summary, these experiments indicate that dominant-negative
that the dominant-negative influence of G203R and R209C variants in either MSN
subpopulation is sufficient to disrupt signaling that leads to profound movement control
deficits.
DISCUSSION
Role of Gαo in canonical GPCR signaling to cAMP
A number of studies demonstrated key roles of neuromodulators acting on distinct GPCRs at
discrete points in striatal circuitry in shaping behavioral outcomes related to action selection
and reward programming (Castro and Bruchas, 2019; Kravitz and Kreitzer, 2012; Lovinger,
2010). However, the role of individual G protein signals initiated by these inputs remains
poorly defined. The key contribution of this study is defining the role of Gαo in controlling
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responses causing the leftward shift on dopamine dose dependence, consistent with a
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Finally, we found that loss of Gαo in iMSNs was responsible for inducing dystonia-like
features often observed GNAO1 patients (Mihalek et al., 2017), which could be considered a
part of hyperkinetic motor program constrained by this neuronal population (Durieux et al.,
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2009). The selective involvement of Gαo supports a dominant role of iMSN circuitry in
dystonia (Berardelli et al., 1998), including observed alterations in electrophysiological
properties of iMSNs in mouse models of dystonia (Sciamanna et al., 2020). In this
connection, it is also interesting to note that deep brain stimulation of the external global
pallidus, a major projection site of iMSNs (Gerfen et al., 1990), improves motor epochs in
primary dystonia patients (Houeto et al., 2007).
Our observations introduce Gαo in the genetic network of players in striatal neurons that are
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essential for motor function in both human disease and mouse models. Pathogenic mutations
in the stimulatory striatal G protein Gαolf (encoded by GNAL) cause dystonia (Fuchs et al.,
2013), while Gnal haploin-sufficiency in mice impairs motor learning (Pelosi et al., 2017).
The Gβγ subunits also play critical roles in movement as observed in patients with Gβ1
mutation (Lohmann et al., 2017) and Gγ7 KO mice (Schwindinger et al., 2003). Movement
disorder is also associated with ADCY5 mutations (OMIM: 606703), and again, motor
learning deficits are observed in AC5 KO mice (Iwamoto et al., 2003; Kheirbek et al., 2009).
Finally, striatal PDE10A is associated with motor deficits in patients (OMIM: 616921) and
mice (Siuciak et al., 2006). In addition to these players, which are all important elements in
the GPCR-cAMP axis, our findings that Gαo affects cAMP homeostasis bolsters the case
that striatal cAMP signaling is critical for motor control (Qian et al., 2015).
Our evaluation of Gαo variants that cause GNAO1 disorder provides insights into
mechanisms underlying pathological signal processing. All GNAO1 variants that we tested
displayed a loss-of-function (LOF) behavior in transmission of GPCR signals. We found that
these deficits arose from distinct mechanisms affecting G protein cycle, including
impairment in binding to Gβγ and inability to promote downstream signaling. In addition,
several mutants displayed clear dominant-negative effects interfering with the function of the
normal Gαo. Many of these effects are not mutually exclusive and show a spectrum of the
severities. Thus, on the basis of these observations, we propose a model that GNAO1
mutations disrupt GPCR signaling through a combination of two principal mechanisms, loss
of signaling ability and dominant-negative interference (Figure 7B).
Our conclusions regarding molecular mechanisms underlying disruption in Gαo function are
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Our observations contradict the findings from a recent survey of GNAO1 mutations that
classified variants as LOF, gain of function (GOF), or normal function (NF) based on their
ability to suppress cAMP production in reconstituted cellular systems (Feng et al., 2017).
Several considerations may explain this discrepancy. First, the effects of GNAO1-related
mutations were previously studied in combination with the C351G point mutation
engineered to impart pertussis toxin (PTX) insensitivity. This mutation alters the C-terminal
region critical for GPCR engagement (Flock et al., 2017). As a result, this manipulation
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might have altered signaling outcomes, which could be particularly relevant in the case of
dominant-negative variants that we show in many cases are explained by enhanced GPCR
interactions. Given our results that Gαo affects cAMP production indirectly, mutation of the
C-terminal region and concomitant inactivation of endogenous proteins by PTX may have
also led to artificial outcomes due to cross-talk in complex signaling network that regulates
AC activity. Finally, the proposed classification makes it unclear how Gαo variants with no
detectable deficits (NF) contribute to an established disease pathology. Our results indicate
that one such mutation, R209C, previously considered NF, in fact results in LOF
accompanied by a dominant-negative activity at the levels of signaling, neuronal function,
and mouse behavior.
depending on the type of mutation. For example, the R209C mutation disrupts inhibitory
signals and thus selectively skews iMSN responses to dopamine and dMSN responses to
adenosine. The G203R mutation has strong dominant-negative activity perturbing
integration of dopamine and adenosine in both dMSNs and iMSNs. This implies that each
GNAO1 variant likely adjusts signaling pressure in a unique manner to misalign striatal
coordination and imbalance motor control (Figure 7). Thus, disease variants likely produce
circuit-selective effects depending on the particular mechanisms of their signaling
disruptions. This would inherently generate diversity in the adverse outcome stemming from
different variations in Gαo. In accordance with the range of severity in movement disorders
observed in GNAO1 patients (Kelly et al., 2019), we suggest that the pathology is a
continuous spectrum rather than isolated manifestations. As such, we believe phenotype-
genotype correlations in GNAO1 disorder are likely quite nuanced and need to take into
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STAR★METHODS
RESOURCE AVAILABILITY
Lead contact—Further information and requests for reagents and resources may be
directed to, and will be fulfilled by, the Lead Contact, Kirill A. Martemyanov
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(kirill@scripps.edu).
Materials availability—Constructs generated in this study are available upon request from
the Lead Contact.
Data and code availability—Data generated in this study are available upon request
from the Lead Contact.
Animal subjects—All experimental procedures and work utilizing mice were approved by
The Scripps Research Institute’s IACUC committee in compliance with guidelines set by the
NIH. The mice were maintained under standard housing conditions in a pathogen-free
facility under a 12/12 light/dark cycle where all mice had continuous access to food and
water. The generation of Gnao1flox/flox and RGS9Cre mouse lines have been previously
described (Chamero et al., 2011; Dang et al., 2006). Drd1aCre (Drd1-Cre; EY262; stock#
017264-UCD) and Drd2Cre (Drd2-Cre; ER43; Stock #: 017268-UCD) mouse lines were
obtained from the Mutant Mouse Resource & Research Centers (MMRRC). Conditional
knockout mice were generated by two rounds of crossing Gnao1flox/flox with RGS9Cre,
Drd1aCre, or Drd2Cre to obtain homozygous Gnao1flox/flox:RGS9Cre,
Gnao1flox/flox:Drd1aCre, or Gnao1flox/flox:Drd2Cre and their wild-type control littermates
(Gnao1flox/flox). Mice were identified through standard PCR genotyping methods as
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previously described for each line. Behavioral studies exclusively utilized male mice.
Biochemical studies utilized both male and female mice. All experiments were performed on
mice between the age of 2–4 months.
standard P1000 pipette in the presence of DNase I (0.05 U/ul), cells were filtered through a
40 um cell strainer, and the plated on Poly-D-Lysine coated glass coverslips. Neuronal
cultures were maintained at 37°C/5% CO2 in a humidified incubator whereupon half of the
growth media was replenished every three days. Transfection of TEpacVV biosensor along
with mutated GNAO1 constructs was performed utilizing Lipofectamine 2000, as previously
described (Masuho et al., 2018).
METHOD DETAILS
Behavioral studies
Rotarod: Rotarod performance was tested using a five-station rotarod treadmill (IITC Life
Sciences, USA) with an acceleration from 8 to 20rpm. Rotarod testing consisted of six trials
per day with 5 min between intertrial intervals, while daily testing consisted of four trials per
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day up to 6 consecutive days. Each trial ended when a mouse fell off the rod or completed
one full revolution on the rod or reached 120 s and the time was scored as the latency to fall.
Grip strength tests—Grip strength was measured as the peak force using a grip strength
meter (Ugo Basile Italy). Both forearms of mouse grasped the grid and the tail was pulled
horizontally until the mouse released its hold entirely. Three separate readings were recorded
for each mouse, with a corresponding 20 s between each trial.
Wood Dale, IL) and distance traveled was recorded using Anymaze video-tracking software.
Mice were placed in the center of the chambers and distance traveled was measure for 60
mins and analyzed in 10 min bins.
Hindlimb clasping—As previously described (Guyenet et al., 2010), mice (males and
females, approximately 3 months old) were held by base of tail, lifted in the air, and
observed for 30 s. Animals were scored as followed: no clasping (0), clasping of 1 hindlimb
part of the time (1), clasping of 1 hindlimb of the entire time (2), clasping of both hindlimbs
part of the time (3), and clasping of both hindlimbs of the entire time (4). Animals were
tested and scored once a day for 3 days.
Backward walking—Mice (males and females, approximately 3 months old) were placed
into RotaRod apparatus (IITC Life Science Inc., Woodland Hills, CA USA) and made to
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walk backward. RotaRod was fitted to ensure that mice were not able to turn around and
walk forward. Animals had walked backward from 1 s at 8.1 RPM and cut off time was 10 s
at 9.15 RPM. Each mouse was tested once a day for 3 days. Latency to fall was recorded.
Ledge test—As previously described (Guyenet et al., 2010), mice (males and females,
approximately 3 months old) were individually placed onto lip of house cage (Allentown
Inc., Allentown, NJ USA) and observed for balancing and movement. Animals were scored
as followed: balancing and walking well (0), good balance but teetering walk (1), teetering
in balance and walk (2), teetering in balance but unable to walk (3), and falling off (4).
Animals were tested and scored once a day for 3 days.
Vertical pole—As previously described (Matsuura et al., 1997), mice (males and females,
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approximately 3 months old) were placed nose facing up on a wooden pole (1 cm diameter)
at 50 cm in height from bottom of mouse cage (Allentown Inc., Allentown NJ USA). In
order to successfully complete this task with a score of 0, subjects had to turn around (nose
facing down) and procced down the pole. Subjects that had turned around and climbed down
the pole with some difficulty had received a score of 1. Subjects that had climbed down the
pole without turning around had a score of 2. Animals which slid down the pole had a score
of 3 and animals that fell off the pole had scored 4. Due to the nature of this study, there was
no cut off time. Animals were tested and scored three times on the same day.
Horizontal pole—As previously described (Farr et al., 2006), mice (males and females,
approximately 3 months old) were placed at 50 cm away from home cage (facing toward
home cage) on a 1 cm diameter wooden pole. Mice were scored as followed: normal gait
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and balance to home cage (0), normal gait but unbalanced to home cage (1), both poor gait
and balance to home cage (2), unable to complete task due to lack of movement (3), and
falling off pole (4). Cut off time for sessions was 120 s. Animals were tested and scored
three times on the same day.
were obtained from Integrated DNA Technologies (Coralville, IA, USA) for in vitro
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and 525–600 nm (Venus FRET acceptor). Image stacks containing XYZ planes were
acquired at 10 s intervals through a 25x objective lens. Quantification of fluorescence
intensity was performed on neuronal cell bodies using ImageJ to calculate FRET from the
inverse ratio of donor:acceptor. Absolute cAMP values were determined from interpolation
of a cAMP standard curve in permeabilized CAMPER neurons (Muntean et al., 2018).
Dopamine and adenosine were added in phasic puffs in the pH 7.2 recording buffer which
consisted of (in mM): CaCl2 (1.3), MgCl2 (0.5), MgSO4 (0.4), KH2PO4 (0.4), NaHCO3
(4.2), NaCl (138), Na2HPO4 (0.3), D-Glucose (5.6), and HEPES (20). In dMSNs D1R
stimulates and A1R inhibits cAMP whereas in iMSNs A2AR stimulates and D2R inhibits
cAMP. Therefore, sign of cAMP response classified neurons as dMSN or iMSN without the
need for additional reagents to isolate the cAMP signaling pathway.
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to the pooled fractions to remove histidine tag and incubated overnight at 4°C. Histidine tag
free Gαo was collected as flow through (FT) upon loading sample on HisTALON Superflow
Cartridge. FT was further loaded onto a Mono Q 4.6/100 PE column (GE Healthcare) and
eluted over a 500 mM NaCl gradient. The eluted protein was further purified using Hiload
26/60 Superdex 75 column (GE Healthcare) which was pre-equilibrated with buffer B (20
mM HEPES (pH 8), 200 mM NaCl, 2 mM DTT, 5 mM MgCl2, 10 μM GDP). The purity of
the protein was analyzed by SDS-PAGE, pooled, concentrated to 20 mg/ml and stored at
Adenylyl cyclase assay—As previously described (Orlandi et al., 2019; Xie et al.,
2012), striatal membranes were isolated from flash frozen (liquid nitrogen) 2 mm tissue
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cAMP measurements—For assessment of total brain cAMP level, dorsal striatal tissue
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punches (2 mm) were flash frozen in liquid nitrogen followed by homogenization in 0.1M
HCl. Levels of cAMP were then determined (tissue punches and adenylyl cyclase assays) by
diluting samples (between 1:20 and 1:50) in 0.1 M HCl followed by quantification with a
competitive cAMP enzyme immunoassay following the acetylated protocol described in the
manufacturer’s guidelines (Direct cAMP ELISA kit, ENZO Life Sciences, Farmingdale,
NY).
mM NaCl, Roche cOmplete protease inhibitor, Sigma Phosphatase Inhibitor Cocktails 2 and
3, and 0.5% non-ionic detergent C12E9), sonicated for 15 s, and slowly rotated for 15 min at
4°C followed by centrifugation at 16,000 × g for 15 min at 4°C. The supernatant of each
sample was diluted to the same concentration and 400 μg was incubated with 3 μg anti-AC5
antibody (Xie et al., 2015) and 50 μL Dynabeads Protein G (ThermoFisher) for 1 hr while
rotating at 4°C. The beads were then washed three times with 1 mL of lysis buffer followed
by elution with urea sample buffer, incubation for 15 min at 42°C, and Western analysis.
Western blotting—For brain samples, tissue punches of the striatum were homogenized
in ice-cold buffer (137 mM NaCl, 20 mM Tris (pH 8.0), 1% NP-40, 10% glycerol and 0.1%
sodium dodecyl sulfate) with the addition of protease and phosphatase inhibitors (Roche,
Rock-ford, IL) and then sonicated. Protein concentration of tissue lysates was determined by
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Pierce 660 nm Protein Assay Reagent (Thermo Fisher, Waltham, MA) and samples were
diluted to the same concentration and denatured in SDS buffer. For cultured cells, each
sample of about 5 × 106 cells were lysed in 500 ul of sample buffer (125 mM Tris-HCl, pH
6.8, 4 M urea, 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, bromophenol blue (0.16 mg/
ml)). Western blotting analysis of proteins was performed after samples were resolved by
SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. Blots were
blocked with 5% skim milk in PBS containing 0.1% Tween 20 (PBST) for 30–90 min at
room temperature (22–26°C). To detect the proteins of interest membranes were incubated
with the following primary antibodies: AC5 (1:3000) (Xie et al., 2015), Gαo (Cell signaling,
cs-3975, 1:1000), Gαolf (Corvol et al., 2001), Gβ1 (Lee et al., 2004), Gβ2 (C-16) (Santa
Cruz, sc-380), GAPDH (Millipore AB2302, 1:25,000), pAkt473 (Cell signaling, cs-4060,
1:1000), Akt (Cell signaling, cs-2920, 1:1000), pERK (Cell signaling, cs-9101, 1:1000),
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ERK Cell signaling, cs-4695, 1:1000), GSK-3 (Cell signaling, cs-5676, 1:1000) and pGSK-3
(Cell signaling, cs-9331, 1:1000). Blots were washed in PBST and incubated with secondary
antibodies conjugated with horseradish peroxidase in PBST containing 1% skimmed milk.
All protein signals were visualized using Kwik Quant Imager (Kindle Biosciences) and band
intensities were determined using NIH ImageJ software.
without the first methionine (NM_002074)) and Venus 1–155-Gγ2 (amino acids 1–155 of
Venus fused to a GGSGGG linker at the N terminus of Gγ2 (NM_053064)) were gifts from
Dr. Nevin A. Lambert (Hollins et al., 2009). The masGRK3ct-Nluc-HA constructs were
constructed by introducing HA tag at the C terminus of masGRK3ct-Nluc-HA reported
previously (Masuho et al., 2015b). Nluc was inserted between residues 91 and 92 of GαoA
(NM_020988) with SGGGGSGGGGS linker at the N terminus and C terminus of the Nluc
to make GαoA-Nluc. D2R-myc-SmBiT was generated by introducing myc epitope tag,
VSQGSSGGGGSGGGGSSG linger, and SmBiT tag at the C-terminal end of D2R. GαoA-
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Nluc and D2R-myc-SmBiT were inserted into the mammalian expression vector
pcDNA3.1(+). pcDNA3.1(+) was purchased from Thermo Fisher Scientific. GenBank
accession number for each sequence is given in brackets.
dissociation assay, Flag-D2R (1), GαoA-Nluc (0.1), Venus 156–239-Gβ1 (1), and Venus 1–
155-Gγ2 (1) were transfected. The number in brackets indicates the ratio of transfected
DNA (ratio 1 = 0.42 μg). An empty vector (pcDNA3.1(+)) was used to normalize the
amount of transfected DNA.
BRET buffer. Approximately 50,000 to 100,000 cells per well were distributed in 96-well
flatbottomed white microplates (Greiner Bio-One). The substrate for Nano luciferase (Nluc),
furimazine, were purchased from Promega and used according to the manufacturer’s
instruction. BRET measurements were made using a microplate reader (POLARstar Omega;
BMG Labtech) equipped with two emission photomultiplier tubes, allowing detection of two
emissions simultaneously with a highest possible resolution of 20 ms per data point. All
measurements were performed at room temperature. The BRET signal is determined by
calculating the ratio of the light emitted by the Venus-Gβ1γ2 (535 nm with a 30 nm band
path width) over the light emitted by the masGRK3ct-Nluc-HA (475 nm with a 30 nm band
path width). The average baseline value (basal BRET ratio) recorded prior to agonist
stimulation was subtracted from the experimental BRET signal values.
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anesthetized with isoflurane and the head was fixed on a Kopf stereotaxic apparatus. The
animals were kept warm (~37°C) for the whole duration of the surgery via a heating pad
connected to a DC temperature controller provided with a feedback system (FHC Inc.). An
eye lubricant was applied to prevent corneal drying during the surgery. Adeno-associated
virus (AAV) encoding the fluorescent protein EYFP (AAV5-EF1a-DIO-EYFP) was obtained
from the Vector Core at the University of North Carolina at Chapel Hill (UNC Vector Core,
USA). AAV encoding GNAO1 variants (AAV9-Syn-DIO-Gαo-IRES-mCherry) were
obtained from VectorBuilder (Chicago, IL). Viral injections were targeted to the dorsal
striatum (AP +0.7, ML ± 1.5 relative to bregma, DV −1.7 relative to dura). The injection
volume (300 nl) and flow rate (50 nl/min) were controlled with an injection pump (Cemyx
Nanojet, USA). The needle was left in place for 5 min after the injection and then slowly
withdrawn. Mice were allowed to recover for at least 15 days before electrophysiological
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experiments.
Slice electrophysiology—Mice aged 8–12 weeks old were anesthetized with isoflurane
and decapitated. The brain was quickly removed and rested for 30 s in ice-cold oxygenated
NMDG cutting solution containing (in mM): 93 NMDG, 2.5 KCl, 1.2 NaH2PO4, 30
NaHCO3, 20 HEPES, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, 0.5 CaCl2$, 10
MgCl2, (adjusted to 7.2–7.4 pH with HCl). Coronal slices (300 μm thick) containing the
striatum were cut on a vibratome (VT1200S, Leica) and incubated for 30 min at 34°C in
oxygenated ACSF containing the following (in mM): 126 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2,
18 NaHCO3, 1.2 NaH2PO4, 10 glucose, then allowed to recover for at least 1 hr at room
temperature before recording. Whole cell recordings were obtained from MSNs in the dorsal
striatum using a Scientifica SliceScope system. Pipets (4–6 MΩ) were pulled by P-1000
(Sutter Instruments, CA) and filled with an intracellular solution containing the following (in
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mM): 119 K-MeSO4, 12 KCl, 1 MgCl2, 0.1 CaCl2, 10 HEPES, 1 EGTA, 0.4 Na-GTP, 2
Mg-ATP, (280–300 mOsm, pH 7.3 adjusted with KOH). Recordings were performed in a
chamber perfused with ACSF at a rate of 2 ml/min and maintained at 32°C. Somatic EYFP
expression was verified in cell-attach mode to confirm cell identity before breaking into
whole-cell mode. Current-clamp recordings were performed to quantify intrinsic membrane
properties from fluorescent MSNs from Drd1aCre and Drd2Cre which were used as controls
and compared with MSNs from Gnao1flox/flox:Drd1aCre and Gnao1flox/flox:Drd2Cre. Spikes
were evoked using current step injections (500-ms duration at 0.2 Hz, −200 to +500 pA
range with increasing 20 pA steps). Rheobase current was defined as the first current step
capable of eliciting one action potential. Input resistance was measured with a 120 pA
hyperpolarizing step from the resting membrane potential. For voltage-clamp experiments,
pipettes (3–5 MΩ) were filled with Cs+ internal solution containing the following (in mM):
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120 CsMeSO3, 15 CsCl, 8 NaCl, 10 TEA-Cl, 10 HEPES, 2–5 QX-314, 0.2 EGTA, 2 Mg-
ATP, 0.3 Na-GTP, pH 7.3 adjusted with CsOH. To record miniature excitatory post-synaptic
currents (mEPSCs) MSNs were clamped at −70 mV and TTX (1 μM) and picrotoxin (100
μM) were added to the recording solution. Evoked EPSCs were recorded using a bipolar
stimulating electrode located ~200 μm away from the recorded soma. AMPA/NMDA ratios
of evoked EPSC were obtained by measuring AMPA-EPSC at −70 mV/NMDA-EPSCs at
+40 mV. NMDAR-mediated EPSCs were measured 60 ms after the stimulus onset. Mean
EPSCs were calculated from an average of 15 sweeps obtained at 0.05 Hz. Acquisition was
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done using Clampex 10.5, MultiClamp 700B amplifier and Digidata 1440A (Molecular
Devices, CA). Data were analyzed with Clampfit 10.5.
In situ hybridization—As similarly described (Sutton et al., 2016), The ViewRNA 2-plex
In Situ Hybridization Assay kit was utilized to evaluate mRNA expression with probes
selective for Drd1 (NM_010076.3; Assay ID VB6-12478), Drd2 (NM_010077.2; Assay ID
VB6-16550), and Gnao1 exon5_6 (NM_010308; Assay ID VPMFWXD). DAPI mounting
media was used to visualize the nucleus. Confocal images of the dorsal striatum were
acquired through a 10x objective lens on a Leica TCS SP8 MP confocal microscope. Images
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Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
ACKNOWLEDGMENTS
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We thank Natalia Martemyanova for husbandry, maintenance, and genotyping of all the mice examined in this
study, as well as Nickolas K. Skamangas and Hideko Masuho for technical support. This work was supported by
funding from the NIH (grants DA041207 to B.S.M., DA048579 to S.Z., NS072129 to B.G., and DA036596 and
DA026405 to K.A.M.) and the Intramural Research Program of the NIH (project Z01-ES-101643 to L.B.). This
work was also supported by a research fellowship from the Bow Foundation (B.S.M.).
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Highlights
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• Striatal neurons require Gαo for synaptic function, excitability, and motor
control
• Gαo acts to modify both inhibitory and stimulatory GPCR signaling to cAMP
Smirnov D = 1.000).
(C) Latency to fall off a rotating beam while walking backward for Gnao1flox/flox (WT; n =
7) and Gnao1flox/flox:RGS9Cre (Str KO; n = 4) mice (nonparametric t test p = 0.0030,
Kolmogorov-Smirnov D = 1.000).
(D) Ledge test pathology score for Gnao1flox/flox (WT; n = 7) and Gnao1flox/flox:RGS9Cre
(Str KO; n = 4) mice (nonparametric t test p = 0.0030, Kolmogorov-Smirnov D = 1.000).
(E) Accelerating rotarod learning rate for Gnao1flox/flox (WT; n = 12) and
Gnao1flox/flox:RGS9Cre (Str KO; n = 10) mice (nonparametric t test p = 0.0157,
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Kolmogorov-Smirnov D = 0.6667).
(F) Schematic of targeting Gnao1 deletion in dMSNs.
(G) Hindlimb clasping pathology score for Gnao1flox/flox (WT; n = 8) and
Gnao1flox/flox:Drd1aCre (dMSN KO; n = 7) mice (p = 0.5692, Kolmogorov-Smirnov D =
0.2321).
(H) Latency to fall off a rotating beam while walking backward for Gnao1flox/flox (WT; n =
8) and Gnao1flox/flox:Drd1aCre (dMSN KO; n = 7) mice (nonparametric t test p = 0.6476,
Kolmogorov-Smirnov D = 0.3214).
(I) Ledge test pathology score for Gnao1flox/flox (WT; n = 7) and Gnao1flox/flox:Drd1aCre
(dMSN KO; n = 8) mice (nonparametric t test p = 0.2821, Kolmogorov-Smirnov D =
0.3036).
(J) Accelerating rotarod learning rate for Gnao1flox/flox (WT; n = 11) and
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mice/10 neurons).
(F) Rheobase current in Drd1aCre (WT; n = 7 mice/10 neurons) and Gnao1flox/flox:Drd1aCre
(dMSN KO; n = 6 mice/10 neurons) (nonparametric t test; Mann-Whitney test, p = 0.0005).
(G and H) Representative AMPA and NMDA traces obtained from WT, iMSN KO, and
dMSN KO.
(I) Quantification of the AMPA/NMDA ratio from Drd2Cre (WT; n = 6 mice/13 neurons)
and Gnao1flox/flox:Drd2Cre (iMSN KO; n = 6 mice/11 neurons) (nonparametric t test; Mann-
Whitney test, p = 0.0048).
(J) Quantification of the AMPA/NMDA ratio from Drd1aCre (WT; n = 6 mice/11 neurons)
and Gnao1flox/flox:Drd1aCre (dMSN KO; n = 5 mice/11 neurons) (nonparametric t test;
Mann-Whitney test, p = 0.0473).
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(K) Schematic of experimental design and representative images of striatal neurons from
Gnao1flox/flox (WT) and Gnao1flox/flox:RGS9Cre (Str KO) pups. Scale bar, 20 μm.
(L) Basal cAMP compared between dMSNs from Gnao1flox/flox (WT; 30 neurons) and
Gnao1flox/flox:RGS9Cre (dMSN KO; 27 neurons), nonparametric t test; Mann-Whitney test,
p < 0.0001. Basal cAMP was compared between iMSNs from Gnao1flox/flox (WT; 31
neurons) and Gnao1flox/flox:RGS9Cre (iMSN KO; 28 neurons) (nonparametric t test; Mann-
Whitney test, p < 0.0001).
(M) Maximum cAMP amplitude to varying doses of dopamine in dMSNs from
Gnao1flox/flox (WT; n = 13) and Gnao1flox/flox:RGS9Cre (dMSN KO; n = 15) primary striatal
neurons; EC50 quantification, nonparametric t test; Mann-Whitney test, p = 0.0195;
maximum cAMP amplitude to 100-μm dopamine quantification; nonparametric t test; Mann-
Whitney test, p = 0.0026).
(N) Maximum cAMP amplitude to varying doses of adenosine in dMSNs from
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Gnao1flox/flox (WT; n = 13) and Gnao1flox/flox:RGS9Cre (dMSN KO; n = 15) primary striatal
neurons; EC50 quantification, nonparametric t test; Mann-Whitney test, p = 0.0648;
maximum cAMP amplitude to 100 μm adenosine quantification; nonparametric t test; Mann-
Whitney test, p = 0.0464.
(O) Maximum cAMP amplitude to varying doses of dopamine in iMSNs from Gnao1flox/flox
(WT; n = 12) and Gnao1flox/flox:RGS9Cre (iMSN KO; n = 13) primary striatal neurons; EC50
quantification, nonparametric t test; Mann-Whitney test, p = 0.7689; maximum cAMP
amplitude to 100 μm dopamine quantification; nonparametric t test; Mann-Whitney test, p =
0.0016.
(P) Maximum cAMP amplitude to varying doses of adenosine in iMSNs from Gnao1flox/flox
(WT; n = 12) and Gnao1flox/flox:RGS9Cre (iMSN KO; n = 13) primary striatal neurons; EC50
quantification, nonparametric t test; Mann-Whitney test, p < 0.0001; maximum cAMP
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Figure 3. Biochemical mechanism of adenylyl cyclase (AC) regulation by Gαo in the striatum
(A) ELISA determination of total cAMP in striatal tissue punches from Gnao1flox/flox (WT;
n = 9 mice) and Gnao1flox/flox:RGS9Cre (Str KO; n = 9 mice) (nonparametric t test; Mann-
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(B) Expression levels of Gαo mutants analyzed by western blotting with anti-Gαo antibody.
(C) The assay design for GPCR-G protein coupling. HEK293T/17 cells were transfected
with plasmids encoding FLAG-D2R, Gαo, Venus-Gβ1γ2, and masGRK3ct-Nluc-
hemagglutinin (HA). Dopamine application to the transfected cells induces the dissociation
of Gαo from Venus-Gβ1γ2, which increases the BRET ratio through the interaction of
Venus-Gβ1γ2 with masGRK3ct-Nluc-HA.
(D) Effect of mutations on GPCR-mediated G protein activation.
(E) Correlation analysis of GPCR-mediated G protein activation and Gαo expression levels.
(F) The assay design for trimer formation. In the absence of exogenous Gα subunit,
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empty vector, pcDNA3.1(+), mimics a single null allele (gray). The lower activity than this
condition indicates the dominant-negative activity of Gα mutants.
(K) Effect of mutations on agonist-mediated G protein activation. The activity of the Gα
mutants was compared to the single null allele condition.
(L) The assay design for agonist-induced GPCR-G protein interaction. HEK293T/17 cells
were transfected with plasmids encoding D2R-myc-SmBiT, Gαo, LgBiT-Gβ1, and Gγ2.
Dopamine application to the transfected cells induces the interaction between dopamine-
activated D2R-myc-SmBiT and Go trimer consisted of exogenous Gαo, LgBiT-Gβ1, and
Gγ2, resulting in reconstitution of functional Nluc.
(M) Time course of agonist-induced D2R and Gαo interaction.
(N) Effect of mutations on agonist-induced D2R and Gαo interaction (n = 3 experiments).
Statistical analyses were performed by one-way ANOVA followed by the Dunnett’s post hoc
comparisons with a control. Values represent means ± SEM from three independent
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experiments, each performed with three replicates. *p < 0.05; **p < 0.01; ***p < 0.001;
****p < 0.0001.
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Figure 5. GNAO1 genetic variants impart circuit-specific alterations in striatal dopamine and
adenosine signal integration
(A) Mean cAMP response to 10 μM dopamine in Gnao1flox/flox dMSNs transfected with
indicated Gαo variant.
(B) Gnao1flox/flox dMSNs transfected with indicated Gαo dose-response curve to dopamine
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indicated Gαo.
(F) Gnao1flox/flox iMSNs transfected with indicated Gαo dose-response curve to dopamine
(top), quantification of maximum cAMP amplitude to 10 μM dopamine (middle), and EC50
to dopamine (bottom). n (# neurons) = no Gα (12), WT Gαo (6), G203R (8), R209C (7).
(G) Mean cAMP response to 10 μM adenosine in Gnao1flox/flox iMSNs transfected with
indicated Gαo.
(H) Gnao1flox/flox iMSNs transfected with indicated Gαo dose-response curve to adenosine
(top), quantification of maximum cAMP amplitude to 10 μM adenosine (middle), and EC50
to adenosine (bottom). n (# neurons) = no Gα (12), WT Gαo (13), G203R (12), R209C (13).
All data are presented as mean ± SEM; one-way ANOVA, Holm-Sidak’s multiple
comparisons test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
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(B) Hindlimb clasping pathology score for Drd1aCre mice expressing WT Gαo (n = 7),
G203R Gαo (n = 7), or R209C Gαo (n = 7) (one-way ANOVA, Dunnett’s multiple
comparisons test; WT versus G203R p = 0.0414, WT versus R209C p = 0.0050), and
hindlimb clasping pathology score for Drd2Cre mice expressing WT Gαo (n = 7), G203R
Gαo (n = 6), or R209C Gαo (n = 7) (one-way ANOVA, Dunnett’s multiple comparisons
test; WT versus G203R p = 0.0003, WT versus R209C p = 0.0021).
(C) Latency to fall off a rotating beam while walking backward for Drd1aCre mice
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expressing WT Gαo (n = 7), G203R Gαo (n = 7), or R209C Gαo (n = 7) (one-way ANOVA,
Dunnett’s multiple comparisons test; WT versus G203R p = 0.0019, WT versus R209C p =
0.0003), and latency to fall off a rotating beam while walking backward for Drd2Cre mice
expressing WT Gαo (n = 7), G203R Gαo (n = 6), or R209C Gαo (n = 7) (one-way ANOVA,
Dunnett’s multiple comparisons test; WT versus G203R p < 0.0001, WT versus R209C p <
0.0001).
(D) Ledge test pathology score for Drd1aCre mice expressing WT Gαo (n = 7), G203R Gαo
(n = 7), or R209C Gαo (n = 7) (one-way ANOVA, Dunnett’s multiple comparisons test; WT
versus G203R p = 0.0015, WT versus R209C p = 0.0041), and ledge test pathology score for
Drd2Cre mice expressing WT Gαo (n = 7), G203R Gαo (n = 6), or R209C Gαo (n = 7)
(one-way ANOVA, Dunnett’s multiple comparisons test; WT versus G203R p < 0.0001, WT
versus R209C p < 0.0001).
(E) Accelerating rotarod learning rate for Drd1aCre mice expressing WT Gαo (n = 7),
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Figure 7. Model of Gαo mechanism in processing of GPCR signals to cAMP and its alteration by
pathogenic GNAO1 mutations
(A) Canonical role of Gαo acting as signaling modifier through regulation of Gβγ. By
interacting with an allosteric site on AC5, Gβγ increases the potency of Gαs/olf stimulation
and diminishes efficacy of Gαi-mediated inhibition. This results in reducing inhibitory tone
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Oligonucleotides
Recombinant DNA