Restoration of the GTPase activity of Gαo mutants by Zn2+ in GNAO1 encephalopathy models

Abstract

GNAO1 encephalopathy is a rare pediatric disease characterized by motor dysfunction, developmental delay, and epileptic seizures1-3. De novo point mutations in the gene encoding Gαo, the major neuronal G protein, lie at the core of this dominant genetic malady4. Half of the clinical case mutations fall on codons Gly203, Arg209, or Glu246 near the GTP binding/hydrolysis pocket of Gαo1-3. We here show that these pathologic mutations strongly speed up GTP uptake and inactivate GTP hydrolysis by Gαo, resulting in constitutive GTP binding by the G protein. Molecular dynamics simulations indicate that the mutations cause displacement of Gln205, the key to GTP hydrolysis. Decreased interactions with cellular partners including RGS19 suggest that despite the enhanced GTP residence, the mutants fail to fully adopt the activated conformation and thus transmit the signal. Through a high-throughput screening of approved drugs aiming at correction of this core biochemical dysfunction, we identify zinc pyrithione and Zn2+ ions as agents restoring the active conformation, GTPase activity, and cellular interactions of the encephalopathy mutants, with a negligible effect on wild type Gαo. We describe a Drosophila model of GNAO1 encephalopathy and show that dietary zinc supplementation restores the motor function and longevity of the mutant flies. With zinc supplements frequently recommended for diverse human neurological conditions, our work spanning from identification of the core biochemical defect in Gαo mutants and cellular interactions analysis to high-throughput screening and animal validation of the deficiency-correcting drug defines the potential therapy for GNAO1 encephalopathy patients.

Mouse models characterize GNAO1 encephalopathy as a neurodevelopmental disorder leading to motor anomalies: from a severe G203R to a milder C215Y mutation

“…Such an approach has so far been proven successful in two studies in mice: an GNAO1[R209H]/ + mouse model [ 33 ] and the two mouse models described in the current manuscript: GNAO1[G203R]/ + and GNAO1[C215Y]/ + . Further, similar modeling has proven successful in Drosophila ( Gαo[G203R]/ + model with reduced locomotion and life span, responding to a pharmacological rescue [ 35 ]). The modeling of GNAO1 encephalopathy has also been performed in C.elegans , with the nematodes heterozygous for S47G or A221D mutations revealing the dominant "unlaid eggs" phenotype indicative of effects in motor neurons, while the heterozygous G42R or R209C mutations produced the dominant aldicarb hypersensitivity effects [ 36 , 37 ].…”

Pediatric Encephalopathy: Clinical, Biochemical and Cellular Insights into the Role of Gln52 of GNAO1 and GNAI1 for the Dominant Disease

“…We wish to underline that the incompetence to interact with guanine nucleotides we ascribe to the Gln52 mutations in GNAO1 and GNAI1 is unlikely to be the general molecular feature for all the >30 point mutations identified in either gene in the pediatric encephalopathy patients. Indeed, we have explicitly shown that many encephalopathy mutants of Gαo are competent to uptake GTP in the BODIPY-GTPγS experiments ( [45] and our unpublished observations), just as it has been shown by others for the Gαo[Arg209His] mutant [46] or for Gαi1[Glu245Lys] (corresponding to Glu246 in Gαo, see Supplementary Figure S1) [47]. While the feature of differential effects of different encephalopathy mutations on the GTP binding and hydrolysis needs a dedicated and separate investigation, we conclude that some of the encephalopathy mutants, represented by the Gln52 mutations in Gαo and Gαi1 we described here, are deficient in these basic features of a G protein, and we further suggest that this basic biochemical deficiency and the devastating cellular outcomes of it are at the core of the dominant nature of these particular heterozygous mutations causing pediatric encephalopathy.…”

Phenotypic Assessment of Pathogenic Variants in GNAO1 and Response to Caffeine in C. elegans Models of the Disease

“…In this scenario, simple model organisms represent a powerful tool of investigation. In a humanized Drosophila model of GNAO1 encephalopathy, heterozygous flies carrying the G203R variant recapitulated some of the clinical features of the disease, manifesting motor dysfunction, reduced life span, and brain abnormalities [19,40]. Remarkably, these phenotypes were improved by dietary zinc supplementation [19].…”

“…Recent data from our study and others suggest that GNAO1 mutations act through a combination of loss-of-function (LOF) and dominant-negative (DN) mechanisms, depending on the particular residue involved and the specific amino acid substitution [7,[17][18][19]. However, a comprehensive understanding of the genotype/phenotype correlations and the underlying pathogenic mechanisms is still lacking, which hinders the discovery of effective therapies.…”

“…Recently, we have shown that caffeine dramatically improves the aberrant motor function of engineered nematodes that carry mutations in goa-1, the C. elegans orthologues of GNAO1, by blocking a putative adenosine receptor (AR) in the worm [18]. In the same year, Larasati and colleagues showed that dietary zinc supplementation restores both the motor function and the longevity of humanized flies harboring disease-causing GNAO1 variants [19].…”