Gαo is a major determinant of cAMP signaling in the pathophysiology of movement disorders

Abstract

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-type-specific fashion via a combination of a dominant-negative and loss-of-function mechanisms. Overall, our findings suggest that Gαo and its pathological variants function in specific circuits to regulate neuromodulatory signals essential for executing motor programs.

Keywords: GPCR; Gαo; cAMP; disease mechanisms; heterotrimeric G proteins; movement disorder; mutations; neuromodulation; striatum; synaptic plasticity.

Figure 1.. Gnao1 expression in dMSNs required for motor learning in mice

(A) Schematic of targeting Gnao1 deletion in striatal neurons. (B) Hindlimb clasping pathology score for Gnao1^flox/flox (WT; n = 7) and Gnao1^flox/flox:RGS9^Cre (Str KO; n = 4) mice (nonparametric t test p = 0.0030, Kolmogorov-Smirnov D = 1.000). (C) Latency to fall off a rotating beam while walking backward for Gnao1^flox/flox (WT; n = 7) and Gnao1^flox/flox:RGS9^Cre (Str KO; n = 4) mice (nonparametric t test p = 0.0030, Kolmogorov-Smirnov D = 1.000). (D) Ledge test pathology score for Gnao1^flox/flox (WT; n = 7) and Gnao1^flox/flox:RGS9^Cre (Str KO; n = 4) mice (nonparametric t test p = 0.0030, Kolmogorov-Smirnov D = 1.000). (E) Accelerating rotarod learning rate for Gnao1^flox/flox (WT; n = 12) and Gnao1^flox/flox:RGS9^Cre (Str KO; n = 10) mice (nonparametric t test p = 0.0157, Kolmogorov-Smirnov D = 0.6667). (F) Schematic of targeting Gnao1 deletion in dMSNs. (G) Hindlimb clasping pathology score for Gnao1^flox/flox (WT; n = 8) and Gnao1^flox/flox:Drd1a^Cre (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 Gnao1^flox/flox (WT; n = 8) and Gnao1^flox/flox:Drd1a^Cre (dMSN KO; n = 7) mice (nonparametric t test p = 0.6476, Kolmogorov-Smirnov D = 0.3214). (I) Ledge test pathology score for Gnao1^flox/flox (WT; n = 7) and Gnao1^flox/flox:Drd1a^Cre (dMSN KO; n = 8) mice (nonparametric t test p = 0.2821, Kolmogorov-Smirnov D = 0.3036). (J) Accelerating rotarod learning rate for Gnao1^flox/flox (WT; n = 11) and Gnao1^flox/flox:Drd1a^Cre (dMSN KO; n = 8) mice (nonparametric t test p = 0.0041, Kolmogorov-Smirnov D = 0.8182). (K) Schematic of targeting Gnao1 deletion in iMSNs. (L) Hindlimb clasping pathology score for Gnao1^flox/flox (WT; n = 8) and Gnao1^flox/flox:Drd2^Cre (iMSN KO; n = 10) mice (p < 0.0001, Kolmogorov-Smirnov D = 1.000). (M) Latency to fall off a rotating beam while walking backward for Gnao1^flox/flox (WT; n = 8) and Gnao1^flox/flox:Drd2^Cre (iMSN KO; n = 10) mice (nonparametric t test p = 0.0012, Kolmogorov-Smirnov D = 0.8000). (N) Ledge test pathology score for Gnao1^flox/flox (WT; n = 8) and Gnao1^flox/flox:Drd2^Cre (iMSN KO; n = 10) mice (nonparametric t test p < 0.0001, Kolmogorov-Smirnov D = 1.000). (O) Accelerating rotarod learning rate for Gnao1^flox/flox (WT; n = 8) and Gnao1^flox/flox:Drd2^Cre (iMSN KO; n = 7) mice (nonparametric t test p = 0.4218, Kolmogorov-Smirnov D = 0.4286). All data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 2.. Gnao1 deletion differentially affects signaling of dMSNs and iMSNs

(A) Representative traces of voltage responses of iMSNs to a depolarizing current step (300 pA) recorded by whole-cell patch clamp from Drd2^Cre (WT) and Gnao1^flox/flox:Drd2^Cre (iMSN KO) mice. (B) Quantification of action potentials elicited in response to somatic current injection in Drd2^Cre (WT; n = 7 mice/10 neurons) and Gnao1^flox/flox:Drd2^Cre (iMSN KO; n = 5 mice/10 neurons). (C) Rheobase current in Drd2^Cre (WT; n = 7 mice/10 neurons) and Gnao1^flox/flox:Drd2^Cre (iMSN KO; n = 5 mice/10 neurons) (nonparametric t test; Mann-Whitney test, p = 0.1529). (D) Representative voltage responses from dMSNs in acute brain slices obtained from Drd1a^Cre (WT) and Gnao1^flox/flox:Drd1a2^Cre (dMSN KO). (E) Quantification of action potentials elicited in response to somatic current injection in Drd1a^Cre (WT; n = 7 mice/10 neurons) and Gnao1^flox/flox:Drd1a^Cre (dMSN KO; n = 6 mice/10 neurons). (F) Rheobase current in Drd1a^Cre (WT; n = 7 mice/10 neurons) and Gnao1^flox/flox:Drd1a^Cre (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 Drd2^Cre (WT; n = 6 mice/13 neurons) and Gnao1^flox/flox:Drd2^Cre (iMSN KO; n = 6 mice/11 neurons) (nonparametric t test; Mann-Whitney test, p = 0.0048). (J) Quantification of the AMPA/NMDA ratio from Drd1a^Cre (WT; n = 6 mice/11 neurons) and Gnao1^flox/flox:Drd1a^Cre (dMSN KO; n = 5 mice/11 neurons) (nonparametric t test; Mann-Whitney test, p = 0.0473). (K) Schematic of experimental design and representative images of striatal neurons from Gnao1^flox/flox (WT) and Gnao1^flox/flox:RGS9^Cre (Str KO) pups. Scale bar, 20 μm. (L) Basal cAMP compared between dMSNs from Gnao1^flox/flox (WT; 30 neurons) and Gnao1^flox/flox:RGS9^Cre (dMSN KO; 27 neurons), nonparametric t test; Mann-Whitney test, p < 0.0001. Basal cAMP was compared between iMSNs from Gnao1^flox/flox (WT; 31 neurons) and Gnao1^flox/flox:RGS9^Cre (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 Gnao1^flox/flox (WT; n = 13) and Gnao1^flox/flox:RGS9^Cre (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 Gnao1^flox/flox (WT; n = 13) and Gnao1^flox/flox:RGS9^Cre (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 Gnao1^flox/flox (WT; n = 12) and Gnao1^flox/flox:RGS9^Cre (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 Gnao1^flox/flox (WT; n = 12) and Gnao1^flox/flox:RGS9^Cre (iMSN KO; n = 13) primary striatal neurons; EC₅₀ quantification, nonparametric t test; Mann-Whitney test, p < 0.0001; maximum cAMP amplitude to 100 μm adenosine quantification; nonparametric t test; Mann-Whitney test, p = 0.0001. All data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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 Gnao1^flox/flox (WT; n = 9 mice) and Gnao1^flox/flox:RGS9^Cre (Str KO; n = 9 mice) (nonparametric t test; Mann-Whitney test, p = 0.0028). (B) Inhibition of forskolin-stimulated AC in WT striatal membranes by Gαi-GTPγs (n = 8 experiments) or Gαo-GTPγs (n = 3 experiments). (C) Inhibition of forskolin-stimulated AC in WT striatal membranes by Gαi-GTPγs in the presence of Gβ1γ2 (n = 8 experiments). (D) IC₅₀ to Gαi-GTPγs (n = 8 experiments), nonparametric t test; Mann-Whitney test, p = 0.0927. (E) Efficacy of Gαi-GTPγs inhibition (n = 8 experiments) (nonparametric t test; Mann-Whitney test, p = 0.0002). (F) Forskolin dose response on striatal membrane AC activity from Gnao1^flox/flox (WT) and Gnao1^flox/flox:RGS9^Cre (Str KO) (n = 9 experiments). (G) EC₅₀ to forskolin (n = 9 experiments) (nonparametric t test; Mann-Whitney test, p = 0.0008). (H) Efficacy of forskolin (n = 9 experiments) (nonparametric t test; Mann-Whitney test, p < 0.0001). (I) Gβ1 co-immunoprecipitation with anti-AC5 antibody in striatum from Gnao1^flox/flox (WT; n = 5) and Gnao1^flox/flox:RGS9^Cre (Str KO; n = 5) mice (nonparametric t test; Mann-Whitney test, p = 0.0079). (J) Schematic representation of dopamine (D1R) and acetylcholine (M4R) receptor regulation of cAMP in Gαi KO dMSNs. (K) Dopamine-induced cAMP responses in Gαi KO dMSN in buffer or 1 μM acetylcholine. Data represented as mean ± SD. (L) Maximum cAMP amplitude to varying doses of dopamine in Gαi KO dMSN in buffer (n = 10 neurons/dose) or 1 μM acetylcholine (n ≥ 6 neurons/dose). (M) EC₅₀ quantification to Gαi KO dMSNs in buffer (n = 10 neurons/dose) or 1 μM acetylcholine (n ≥ 6 neurons/dose) (nonparametric t test; Mann-Whitney test, p = 0.0001). (N) Maximum cAMP amplitude to 100 μm dopamine in buffer (n = 10 neurons) or 1 μM acetylcholine (n = 8 neurons) (nonparametric t test; Mann-Whitney test, p < 0.0001). (O) Schematic representation of adenosine (A2AR) and dopamine (D2R) receptor regulation of cAMP in Gαi KO iMSNs. (P) Adenosine-induced cAMP responses in Gαi KO iMSN in buffer or 1 μM dopamine. Data represented as mean ± SD. (Q) Maximum cAMP amplitude to varying doses of adenosine in Gαi KO iMSN in buffer (n = 10 neurons/dose) or 1 μM dopamine (n ≥ 5 neurons/dose). (R) EC₅₀ quantification to Gαi KO iMSN in buffer (n = 10 neurons/dose) or 1 μM dopamine (n ≥ 5 neurons/dose) (nonparametric t test; Mann-Whitney test, p = 0.0059). (S) Maximum cAMP amplitude to 100 μm adenosine in buffer (n = 10 neurons) or 1 μM acetylcholine (n = 8 neurons) (nonparametric t test; Mann-Whitney test, p < 0.0001). Unless indicated otherwise, all data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4.. Dissection of pathological mechanisms of GNAO1 clinical variants

(A) Mapping the genetic variation on the structural model of Gαo. The homology model of Gαo was constructed on the basis of the crystal structure of the Gαi1 (1GP2). (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, transfected masGRK3ct-Nluc-HA and Venus-Gβ1γ2 produces masGRK3ct-Nluc-HA-bound Venus-Gβ1γ2 and results in high basal BRET signal (left). Exogenous expression of Gαo sequesters Venus-Gβ1γ2 from masGRK3ct-Nluc and decreases the BRET signal (right). (G) Effect of mutations on trimer formation measured by basal BRET ratio. The ratio obtained without Gαo or with WT Gαo is designated as 0% or 100% trimer formation. (H) Correlation analysis of trimer formation versus Gαo expression level quantified from western blotting experiments. (I) The assay design for the dominant-negative activity of Gαo mutants. WT Gαo and mutant Gαo were transfected with FLAG-D2R and BRET sensors. Dominant-negative mutants can suppress the coupling of D2R and WT Gαo. (J) Time course of agonist-mediated G protein activation. The condition transfected with 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 experiments, each performed with three replicates. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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 Gnao1^flox/flox dMSNs transfected with indicated Gαo variant. (B) Gnao1^flox/flox dMSNs 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 (13), WT Gαo (10), G203R (8), R209C (10). (C) Mean cAMP response to 10 μM adenosine in Gnao1^flox/flox dMSNs transfected with indicated Gαo variant. (D) Gnao1^flox/flox dMSNs 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 (13), WT Gαo (12), G203R (13), R209C (14). (E) Mean cAMP response to 10 μM dopamine in Gnao1^flox/flox iMSNs transfected with indicated Gαo. (F) Gnao1^flox/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 Gnao1^flox/flox iMSNs transfected with indicated Gαo. (H) Gnao1^flox/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.

Figure 6.. GNAO1 mutations impair locomotor behavior in mice

(A) Schematic of targeting expression of GNAO1 variants (G203R, R209C, or WT control) in defined striatal neurons by stereotaxic delivery of Cre-dependent AAV in either adult Drd1a^Cre (dMSN Gαo expression) or Drd2^Cre (iMSN Gαo expression) mice. (B) Hindlimb clasping pathology score for Drd1a^Cre 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 Drd2^Cre 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 Drd1a^Cre 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 = 0.0019, WT versus R209C p = 0.0003), and latency to fall off a rotating beam while walking backward for Drd2^Cre 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 Drd1a^Cre 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 Drd2^Cre 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 Drd1a^Cre 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 = 0.6411, WT versus R209C p = 0.5076), and accelerating rotarod learning rate for Drd2^Cre 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 = 0.9979, WT versus R209C p = 0.9093). All data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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 of Gαi inputs while sensitizing Gαolf stimulation of AC5 in striatal neurons. (B) GNAO1 clinical variants manifest a spectrum of pathology through individualized strength in scaled loss-of-function (LOF) and dominant-negative (DN) properties.