Fig 2: 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 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).(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, 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 Gnao1flox/flox:Drd1aCre (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 Gnao1flox/flox (WT; n = 8) and Gnao1flox/flox:Drd2Cre (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 Gnao1flox/flox (WT; n = 8) and Gnao1flox/flox:Drd2Cre (iMSN KO; n = 10) mice (nonparametric t test p = 0.0012, Kolmogorov-Smirnov D = 0.8000).(N) Ledge test pathology score for Gnao1flox/flox (WT; n = 8) and Gnao1flox/flox:Drd2Cre (iMSN KO; n = 10) mice (nonparametric t test p < 0.0001, Kolmogorov-Smirnov D = 1.000).(O) Accelerating rotarod learning rate for Gnao1flox/flox (WT; n = 8) and Gnao1flox/flox:Drd2Cre (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.

Fig 3: Dissection of pathological mechanisms of GNAO1 clinical variants(A) Mapping the genetic variation on the structural model of Gao. The homology model of Gao was constructed on the basis of the crystal structure of the Gai1 (1GP2).(B) Expression levels of Gao mutants analyzed by western blotting with anti-Gao antibody.(C) The assay design for GPCR-G protein coupling. HEK293T/17 cells were transfected with plasmids encoding FLAG-D2R, Gao, Venus-Gß1?2, and masGRK3ct-Nluc-hemagglutinin (HA). Dopamine application to the transfected cells induces the dissociation of Gao from Venus-Gß1?2, which increases the BRET ratio through the interaction of Venus-Gß1?2 with masGRK3ct-Nluc-HA.(D) Effect of mutations on GPCR-mediated G protein activation.(E) Correlation analysis of GPCR-mediated G protein activation and Gao expression levels.(F) The assay design for trimer formation. In the absence of exogenous Ga subunit, transfected masGRK3ct-Nluc-HA and Venus-Gß1?2 produces masGRK3ct-Nluc-HA-bound Venus-Gß1?2 and results in high basal BRET signal (left). Exogenous expression of Gao sequesters Venus-Gß1?2 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 Gao or with WT Gao is designated as 0% or 100% trimer formation.(H) Correlation analysis of trimer formation versus Gao expression level quantified from western blotting experiments.(I) The assay design for the dominant-negative activity of Gao mutants. WT Gao and mutant Gao were transfected with FLAG-D2R and BRET sensors. Dominant-negative mutants can suppress the coupling of D2R and WT Gao.(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 Ga mutants.(K) Effect of mutations on agonist-mediated G protein activation. The activity of the Ga 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, Gao, LgBiT-Gß1, and G?2. Dopamine application to the transfected cells induces the interaction between dopamine-activated D2R-myc-SmBiT and Go trimer consisted of exogenous Gao, LgBiT-Gß1, and G?2, resulting in reconstitution of functional Nluc.(M) Time course of agonist-induced D2R and Gao interaction.(N) Effect of mutations on agonist-induced D2R and Gao 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.

Fig 4: Model of Gao mechanism in processing of GPCR signals to cAMP and its alteration by pathogenic GNAO1 mutations(A) Canonical role of Gao acting as signaling modifier through regulation of Gß?. By interacting with an allosteric site on AC5, Gß? increases the potency of Gas/olf stimulation and diminishes efficacy of Gai-mediated inhibition. This results in reducing inhibitory tone of Gai inputs while sensitizing Gaolf 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.

Fig 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 Gao variant.(B) Gnao1flox/flox dMSNs transfected with indicated Gao dose-response curve to dopamine (top), quantification of maximum cAMP amplitude to 10 µM dopamine (middle), and EC50 to dopamine (bottom). n (# neurons) = no Ga (13), WT Gao (10), G203R (8), R209C (10).(C) Mean cAMP response to 10 µM adenosine in Gnao1flox/flox dMSNs transfected with indicated Gao variant.(D) Gnao1flox/flox dMSNs transfected with indicated Gao dose-response curve to adenosine (top), quantification of maximum cAMP amplitude to 10 µM adenosine (middle), and EC50 to adenosine (bottom). n (# neurons) = no Ga (13), WT Gao (12), G203R (13), R209C (14).(E) Mean cAMP response to 10 µM dopamine in Gnao1flox/flox iMSNs transfected with indicated Gao.(F) Gnao1flox/flox iMSNs transfected with indicated Gao dose-response curve to dopamine (top), quantification of maximum cAMP amplitude to 10 µM dopamine (middle), and EC50 to dopamine (bottom). n (# neurons) = no Ga (12), WT Gao (6), G203R (8), R209C (7).(G) Mean cAMP response to 10 µM adenosine in Gnao1flox/flox iMSNs transfected with indicated Gao.(H) Gnao1flox/flox iMSNs transfected with indicated Gao dose-response curve to adenosine (top), quantification of maximum cAMP amplitude to 10 µM adenosine (middle), and EC50 to adenosine (bottom). n (# neurons) = no Ga (12), WT Gao (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.