Zinc for GNAO1 encephalopathy: preclinical profiling and a clinical case

Zinc for GNAO1 encephalopathy: preclinical profiling and a clinical case | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Zinc for GNAO1 encephalopathy: preclinical profiling and a clinical case Yonika A. Larasati, Moritz Thiel, Alexey Koval, Denis N. Silachev, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3771723/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract De novo mutations in GNAO1 – the gene encoding the major neuronal G-protein Gαo – cause pediatric encephalopathies largely refractory to available therapies. Zn 2+ emerged to restore GTP hydrolysis and cellular interactions of pathogenic Gαo; dietary Zn 2+ supplementation improves lifespan and motoric function in a Drosophila disease model. Here we show that 16 different pathogenic missense mutations cluster in three distinct groups in their responsiveness to Zn 2+ , and provide the safety study in a mouse disease model. We further describe treatment of a 3 years-old patient with a common GNAO1 mutation c607G > A, p.Gly203Arg with oral 50mg Zn 2+ daily, as applied in Wilson’s disease. During 11 months of treatment, the patient shows cessation of daily hyperkinetic crises, improved Burke-Fahn Marsden Dystonia Rating Scale movement score and general well-being, and an excellent safety profile. Our findings warrant a large-scale clinical trial and might set the new standard of care for GNAO1 encephalopathy. Health sciences/Neurology/Neurological disorders/Encephalopathy Biological sciences/Cell biology/Cell signalling Health sciences/Molecular medicine Biological sciences/Drug discovery GNAO1 encephalopathy Gαo zinc dietary supplementation rare disease animal model clinical case Figures Figure 1 Figure 2 Full Text First identified in 2013, GNAO1 -related neurodevelopmental disorders are caused by mutations in the GNAO1 gene encoding the major neuronal G protein, Gαo 1 . Gαo is one of 16 human Gα-subunits that, together with Gβγ, form heterotrimeric G-protein complexes that are the primary transducers of G protein-coupled receptors (GPCRs). Ligand-activated GPCRs facilitate the exchange of GDP for GTP in Gα, thereby promoting the dissociation of Gα-GTP from Gβγ and the receptor 2 . Gα-GTP and Gβγ can then engage with distinct downstream effectors to transmit the GPCR signal inside the cell. With time, Gα hydrolyzes its GTP back to GDP, and this GTPase activity can be accelerated by the Regulator of G-protein Signaling (RGS) proteins 3 . As of today, about 200 patients worldwide are known to be affected by GNAO1 encephalopathy 4 , identifying it as an ultrarare disease. However, this number is expected to grow with the wider availability of whole exome / genome sequencing in undiagnosed patients. Patients demonstrate a broad range of symptoms, including epilepsy, movement disorders, hypotonia, developmental delay, and brain atrophy 4 – 7 . Current treatments attempt to alleviate the epileptic and movement disorder symptoms, but patients are largely refractory to the available treatments 4 , 8 . With more than 80 pathogenic (mostly missense) variants of Gαo identified to date (ncbi.nlm.nih.gov/clinvar/?term = gnao1%5Bgene%5D&redir = gene), in-depth molecular characterization is crucial to understand the etiology of the disease caused by the individual variants 9 . Earlier studies have revealed that different pathogenic Gαo exhibit a variety of molecular defects, such as accelerated GTP uptake / defective GTP hydrolysis that lead to constitutive GTP loading of Gαo, structural defects that lead to abnormal interaction with RGS proteins and Gβγ, decreased plasma membrane localization, defective GPCR coupling, and neomorphic binding to Ric8A/B proteins 10 – 17 . Powered by the understanding of the molecular defects of pathogenic Gαo, we have identified zinc salts as a potential therapy for patients with the most common variants of GNAO1 encephalopathies: G203R, R209C, and E246K 12 . Replacing Mg 2+ in the active center and inducing structural rearrangements, Zn 2+ restores the GTPase activities of the mutants and their cellular interactions, without influencing wild-type Gαo 12 . Zinc supplementation improves motor function and longevity in a Drosophila model of GNAO1 encephalopathy carrying the G203R variant; our studies using this model further indicate the need for continuous dietary zinc supplementation to achieve the therapeutic effect 12 . We reveal that Zn 2+ counteracts the constitutive GTP binding of pathogenic Gαo variants by different mechanisms. Testing the biochemical properties of 16 variants (Supplementary Table S1 ), we find that most mutants have increased GTP binding rates ( k bind ) and defective GTP hydrolysis (reduced k hydr ) in comparison to wild-type Gαo (Fig. 1A,B and Supplementary Fig. S1 ), agreeing with our previous studies 12 – 15 , 18 . Evaluation of the effects of increasing concentrations of ZnCl 2 on the GTP binding/hydrolysis permits to group the variants into three classes. The first includes the variants that are, like Gαo wild-type, unaffected by Zn 2+ in GTP binding / hydrolysis: L23P, C215Y, and I344del, adding up to the T241_N242insPQ variant we identified previously 13 (Supplementary Fig. S2 and Fig. 1C). The second class represents the variants whose GTP binding remains unaffected, but whose GTP hydrolysis is restored by Zn 2+ , adding K46R to the G203R, R209C, and E246K mutants we studied earlier 12 (Supplementary Fig.S3 and Fig. 1D). And the third category emerges to be the most populated and includes K46N, H57P, T182I, R209H, Y231C, E237K, and Y291N, in addition to P170R studied earlier 15 . Zn 2+ reduces GTP uptake by these variants in both GTP binding and hydrolysis assays, the effect mediated by the > 3-fold reduction in the affinity of Gαo to GTP (Supplementary Fig.S4 and Fig. 1E,F) 15 . Interestingly, this stratification of pathogenic GNAO1 mutations correlates with the clinical severity of the disease manifestations. Analysis of 26 class I, 63 class II, and 40 class III patients (Fig. 1F and Supplementary Table S1 ) reveals that the onset of disease differs strongly and significantly among the classes, class II being the most severe (average onset of 6.8 months) and class I – the least severe (average onset of 4.3 years), class III being the intermediate (6.8 years, Fig. 1G). Noteworthy, the biochemical perturbations of the class I mutants, which are biochemically unresponsive to zinc, are also milder (Fig. 1A,B). In contrast, for the pathogenic variants leading to more severe clinical phenotypes underlined by the biochemical severity, Zn 2+ alleviates their constitutive GTP binding through one of the two mechanisms, both expected to bring clinical benefit: reduction in the GTP uptake or restoration of GTP hydrolysis. These findings lay the basis for the patient stratification in the clinical applications of zinc. Zinc has been approved for treatment of diverse disorders including Wilson’s disease and various neurological conditions 19 , 20 , with the daily dose of elemental zinc in Wilson’s disease treatment of 50mg daily for children under 6 years of age 21 . We argued that a similar dose should be applied for the treatment of GNAO1 patients. Prior to the off-label clinical applications, we aimed at assessing the safety profile of zinc in a mouse model of the disease. Zinc toxicity has not been evaluated in neonates and young pups, whereas zinc supplementation has been well-studied in adult mice. For example, maintenance of adult mice for up to 14 months on water supplemented with 0.5g/L elemental zinc in the form of ZnSO 4 resulted in no adverse effects in the animals 22 ; the maximum tolerated dose (MTD) for adult mice is reported as 75mM (12.11g/L) ZnSO 4 in drinking water 23 . We thus first tested the near-MTD doses of ZnSO 4 supplied in drinking water to C57BL/6 mice all the way from birth to adulthood: 4-8g/L (ca. 1000-2000mg/kg/day of ZnSO 4 ; the human equivalent dose can be estimated (following fda.gov/media/72309/download) as 81-162mg/kg/day. The presence of ZnSO 4 in the drinking water of lactating dams resulted in a transient delay in the body weight gain of the pups with onset at post-natal day (PND)7 and complete resolution by ca. PND17 (Supplementary Fig.S5A). This delay is fully compensated and is not reflected in the body weights of adult males and females by the end of the 3-month measurement period (Fig. 2A,B). No differences were observed in the appearance of animals treated with the highest dose of ZnSO 4 (8g/L, Supplementary Fig.S6). Finally, after 3 months of this treatment, the animals were sacrificed and the weight and appearance of major organs were evaluated at necropsy (Supplementary Figs S5B and S7). No difference in organ weight or appearance was observed. Taken together, these results indicate only a low and transient toxicity of the near-MTD doses of Zn 2+ and their suitability for long-term treatment. Only three mouse strains with pathogenic GNAO1 mutations have been described: R209H, C215Y, and G203R 18 , 24 . Of note, G203R/+ mice die neonatally 24 . We thus used the C215Y mouse line available to us, in which behavioral disturbances in the form of hyperactive behavior have been described 24 . As the C215Y mutant was not responsive to Zn 2+ in our in vitro biochemical assays (Fig. 1C,F), we did not expect to observe an improvement in the behavior of the mutant mice upon chronic ZnSO 4 administration. However, we argued that this mouse disease model could be a precious model to assess the safety profile of zinc supplementation in a GNAO1 disease condition. We find that continuous supplementation of ZnSO 4 (2g/L in the drinking water) leads to no behavioral disturbances. Intriguingly, we see a noticeable improvement in the mouse performance in the rotarod test that can be observed in the three genotypes: C215Y/+ , C215Y/C215Y , and the control wild-type littermates (Fig. 2C). These results indicate that ZnSO 4 supplementation can improve motor skills in mice regardless of their genetic background. Given the known association between mutations in the GNAO1 gene and cognitive impairment in patients, the mice's exploratory behavior was also assessed using the novel object recognition test. In this test, all groups of mice, regardless of genotype, showed increased exploratory activity on the second day of the test. This was manifested by a significant increase in the time spent exploring objects (Fig. 2D and Supplementary Fig.S8A). In the open field test, where C215Y/+ and C215Y/C215Y mice reveal the hyperkinetic activity 24 , zinc supplementation did not change the behavioral readouts for either mutant genotype nor wild-type littermates (Supplementary Fig.S8B-D). Altogether, we conclude that zinc supplementation did not lead to any deterioration in the mouse model of GNAO1 encephalopathy; a general, genotype-independent improvement in motor and cognitive skills was achieved by the treatment. With this background, we applied zinc supplementation therapy to a 3.4 years-old patient with the c607G > A, p.Gly203Arg mutation (see online Methods for additional description of the patient). During the first year of life, the boy presented a severe epileptic encephalopathy without any head control nor achievement of any motoric milestones. Severe dystonia, choreoathetosis and repetitive hyperkinetic crises were prominent during the first months. Orofacial dystonia with tongue involvement caused severe feeding difficulties requiring percutaneous endoscopic gastrostomy (PEG) at the age of 12 months. A severe sleep disorder was present as well. The epilepsy was resistant to multiple antiseizure drugs. The disease progressed and daily hyperkinetic crises were the major cause of morbidity. Pharmacotherapy of the movement disorder comprised benzodiazepines, gabapentin, baclofen, clonidine and cannabinoids, all with minor or only short-lasting effects. In the course of a severe bacterial infection with respiratory impairment and ascites, the boy showed acute deterioration of his global condition with severe disease progression. He was administered to a hospice at the age of 3.4 years. With the family’s agreement we started an off-label zinc therapy. The initial dose was 40mg Zn 2+ daily (2.7mg/kg/day) administered as zinc gluconate which was increased to 50mg Zn 2+ daily (3.5mg/kg/day) administered as zinc acetate dihydrate. To date, the boy has been on zinc for 11 months. After grinding, the medication is provided through PEG as a suspension in water. The medication is well tolerated and no adverse events have been reported to date. Hemoglobin and copper serum levels were stable while the zinc serum level increased but did not exceed the upper limit (Fig. 2E). The patient’s condition has stabilized and an improvement in quality of life is reported by the parents: the daily hyperkinetic crises stopped and the patient started to smile again. The application of emergency medication against hyperkinetic exacerbations could be reduced. The patient shows longer periods of awareness during daytime and the night sleep could be improved. The parents report that their son now enjoys their interaction without any distraction by seizures or involuntary movements after many months of the severe impairment. The Burke-Fahn Marsden Dystonia Rating Scale movement score (BFMDRS-M) improved by 46,5 eight months after the start of the medication (Fig. 2E). The Gross Motor Function measure-66 (GMFM-66) did not show any changes. Morphine was the only new concomitant medication given to reduce agitation due to shortness of breath in the palliative setting. This is the first GNAO1 patient who receives zinc in high dosages equal to the dosages recommended in Wilson’s disease under controlled settings of a natural history study. This treatment trial has been supported by an extensive preclinical assessment, in vitro and in animal models, of the efficacy, mechanism of action, and safety of zinc administration in order to cure the molecular defects caused by pathogenic GNAO1 mutations. The therapy has been feasible and no adverse events have been reported so far. It is possible that the beneficial effects cannot be attributed exclusively to the zinc treatment, but apart from morphine, which was started in the hospice setting, the patient did not receive any new therapies other than zinc during the treatment period of eight months. We conclude that further studies on the feasibility and safety of zinc in larger cohorts of patients with GNAO1 -associated disorders are now required. Declarations Acknowledgments We thank Dr. Gonzalo Solis for the fruitful discussion on this project. Mouse experiments were supported with the grant number 21-15-00138 from the Russian Science Foundation to VLK and DNS, and biochemical experiments – with a grant from GNAO1 España to VLK. References Nakamura, K., Kodera, H., Akita, T., Shiina, M., Kato, M., Hoshino, H., Terashima, H., Osaka, H., Nakamura, S., Tohyama, J., Kumada, T., Furukawa, T., Iwata, S., Shiihara, T., Kubota, M., Miyatake, S., Koshimizu, E., Nishiyama, K., Nakashima, M., Tsurusaki, Y., Miyake, N., Hayasaka, K., Ogata, K., Fukuda, A., Matsumoto, N. & Saitsu, H. De Novo mutations in GNAO1, encoding a Galphao subunit of heterotrimeric G proteins, cause epileptic encephalopathy. Am J Hum Genet 93 , 496-505 (2013). Gilman, A. G. G proteins: transducers of receptor-generated signals. Annu Rev Biochem 56 , 615-649 (1987). Dohlman, H. G. & Thorner, J. RGS proteins and signaling by heterotrimeric G proteins. J Biol Chem 272 , 3871-3874 (1997). González, M., Kloosterhuis, K., Pol, L., Baas, F. & Mikkers, H. Phenotypic Diversity in GNAO1 Patients: A Comprehensive Overview of Variants and Phenotypes. Human Mutation 2023 , 1-16 (2023). Briere, L., Thiel, M., Sweetser, D. A., Koy, A. & Axeen, E. GNAO1-Related Disorder. GeneReviews (2023). Schirinzi, T., Garone, G., Travaglini, L., Vasco, G., Galosi, S., Rios, L., Castiglioni, C., Barassi, C., Battaglia, D., Gambardella, M. L., Cantonetti, L., Graziola, F., Marras, C. E., Castelli, E., Bertini, E., Capuano, A. & Leuzzi, V. Phenomenology and clinical course of movement disorder in GNAO1 variants: Results from an analytical review. Parkinsonism Relat Disord 61 , 19-25 (2019). Wirth, T., Garone, G., Kurian, M. A., Piton, A., Millan, F., Telegrafi, A., Drouot, N., Rudolf, G., Chelly, J., Marks, W., Burglen, L., Demailly, D., Coubes, P., Castro-Jimenez, M., Joriot, S., Ghoumid, J., Belin, J., Faucheux, J. M., Blumkin, L., Hull, M., Parnes, M., Ravelli, C., Poulen, G., Calmels, N., Nemeth, A. H., Smith, M., Barnicoat, A., Ewenczyk, C., Méneret, A., Roze, E., Keren, B., Mignot, C., Beroud, C., Acosta, F., Jr., Nowak, C., Wilson, W. G., Steel, D., Capuano, A., Vidailhet, M., Lin, J. P., Tranchant, C., Cif, L., Doummar, D. & Anheim, M. Highlighting the Dystonic Phenotype Related to GNAO1. Mov Disord 37 , 1547-1554 (2022). Axeen, E., Bell, E., Robichaux Viehoever, A., Schreiber, J. M., Sidiropoulos, C. & Goodkin, H. P. Results of the First GNAO1-Related Neurodevelopmental Disorders Caregiver Survey. Pediatr Neurol 121 , 28-32 (2021). Katanaev, V. L., Valnohova, J., Silachev, D. N., Larasati, Y. A. & Koval, A. Pediatric GNAO1 encephalopathies: from molecular etiology of the disease to drug discovery. Neural Regeneration Research 18 , 2188-2189 (2023). Solis, G. P., Kozhanova, T. V., Koval, A., Zhilina, S. S., Mescheryakova, T. I., Abramov, A. A., Ishmuratov, E. V., Bolshakova, E. S., Osipova, K. V., Ayvazyan, S. O., Lebon, S., Kanivets, I. V., Pyankov, D. V., Troccaz, S., Silachev, D. N., Zavadenko, N. N., Prityko, A. G. & Katanaev, V. L. Pediatric Encephalopathy: Clinical, Biochemical and Cellular Insights into the Role of Gln52 of GNAO1 and GNAI1 for the Dominant Disease. Cells 10 , 2749 (2021). Muntean, B. S., Masuho, I., Dao, M., Sutton, L. P., Zucca, S., Iwamoto, H., Patil, D. N., Wang, D., Birnbaumer, L., Blakely, R. D., Grill, B. & Martemyanov, K. A. Gαo is a major determinant of cAMP signaling in the pathophysiology of movement disorders. Cell Rep 34 , 108718 (2021). Larasati, Y. A., Savitsky, M., Koval, A., Solis, G. P., Valnohova, J. & Katanaev, V. L. Restoration of the GTPase activity and cellular interactions of Gα(o) mutants by Zn(2+) in GNAO1 encephalopathy models. Sci Adv 8 , eabn9350 (2022). Koval, A., Larasati, Y. A., Savitsky, M., Solis, G. P., Good, J. M., Quinodoz, M., Rivolta, C., Superti-Furga, A. & Katanaev, V. L. In-depth molecular profiling of an intronic GNAO1 mutant as the basis for personalized high-throughput drug screening. Med 4 , 311-325.e317 (2023). Solis, G. P., Koval, A., Valnohova, J., Savitsky, M. & Katanaev, V. L. Ric8 proteins as the neomorphic partners of Gαo in GNAO1 encephalopathies. bioRxiv , 2023.2003.2027.534359 (2023). Larasati, Y. A., Solis, G. P., Koval, A., Griffiths, S. T., Berentsen, R., Aukrust, I., Lesca, G., Chatron, N., Ville, D., Korff, C. M. & Katanaev, V. L. Clinical Cases and the Molecular Profiling of a Novel Childhood Encephalopathy-Causing GNAO1 Mutation P170R. Cells 12 (2023). Knight, K. M., Obarow, E. G., Wei, W., Mani, S., Esteller, M. I., Cui, M., Ma, N., Martin, S. A., Brinson, E., Hewitt, N., Soden, G. M., Logothetis, D. E., Vaidehi, N. & Dohlman, H. G. Molecular annotation of G protein variants in a neurological disorder. Cell Rep 42 , 113578 (2023). Domínguez-Carral, J., Ludlam, W. G., Junyent Segarra, M., Fornaguera Marti, M., Balsells, S., Muchart, J., Čokolić Petrović, D., Espinoza, I., Ortigoza-Escobar, J. D. & Martemyanov, K. A. Severity of GNAO1-Related Disorder Correlates with Changes in G-Protein Function. Ann Neurol 94 , 987-1004 (2023). Larrivee, C. L., Feng, H., Quinn, J. A., Shaw, V. S., Leipprandt, J. R., Demireva, E. Y., Xie, H. & Neubig, R. R. Mice with GNAO1 R209H Movement Disorder Variant Display Hyperlocomotion Alleviated by Risperidone. J Pharmacol Exp Ther 373 , 24-33 (2020). Członkowska, A., Litwin, T., Dusek, P., Ferenci, P., Lutsenko, S., Medici, V., Rybakowski, J. K., Weiss, K. H. & Schilsky, M. L. Wilson disease. Nat Rev Dis Primers 4 , 21 (2018). Grabrucker, A. M., Rowan, M. & Garner, C. C. Brain-Delivery of Zinc-Ions as Potential Treatment for Neurological Diseases: Mini Review. Drug Deliv Lett 1 , 13-23 (2011). Ranucci, G., Di Dato, F., Spagnuolo, M. I., Vajro, P. & Iorio, R. Zinc monotherapy is effective in Wilson's disease patients with mild liver disease diagnosed in childhood: a retrospective study. Orphanet J Rare Dis 9 , 41 (2014). Aughey, E., Grant, L., Furman, B. L. & Dryden, W. F. The effects of oral zinc supplementation in the mouse. Journal of Comparative Pathology 87 , 1-14 (1977). Souffriau, J., Timmermans, S., Vanderhaeghen, T., Wallaeys, C., Van Looveren, K., Aelbrecht, L., Dewaele, S., Vandewalle, J., Goossens, E., Verbanck, S., Boyen, F., Eggermont, M., De Commer, L., De Rycke, R., De Bruyne, M., Tito, R., Ballegeer, M., Vandevyver, S., Velho, T., Moita, L. F., Hochepied, T., De Bosscher, K., Raes, J., Van Immerseel, F., Beyaert, R. & Libert, C. Zinc inhibits lethal inflammatory shock by preventing microbe-induced interferon signature in intestinal epithelium. EMBO Mol Med 12 , e11917 (2020). Silachev, D., Koval, A., Savitsky, M., Padmasola, G., Quairiaux, C., Thorel, F. & Katanaev, V. L. Mouse models characterize GNAO1 encephalopathy as a neurodevelopmental disorder leading to motor anomalies: from a severe G203R to a milder C215Y mutation. Acta Neuropathol Commun 10 , 9 (2022). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryFiguresS1S8.pdf Supplementary Figures LarasatietalSupplementaryXXext.docx Supplementary text Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3771723","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":268993394,"identity":"7bdd68e2-bba5-4ad9-8718-40b634dda2a8","order_by":0,"name":"Yonika A. 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Katanaev","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFUlEQVRIiWNgGAWjYDACdgYGiQQGCQYGHrhIAwNDAoghgUMLM0ILYwNYhOcAEVogKmFaJBKgUji0GBxmfnjjYY5FPgPP4ecPfu6wkzO4+cbswYNfNgz8sxtwaGEztkjcJmHZwNtm2Nh7JtnY4HaOuUFiXxqDxJ0DWLVINjOYSQC1GDDwMxgCtTEnzpydAxTpOcxgAHchuhb2b1At7B8b/7bV18+ceQak5T9OLfzMPFBbeHsMm3nbDifwSwBFEn4cwKelGOQXAzaeM4WzZduOG/bzpJVJJDYk80jcwK6Fjb19482f2+oM+HnSN3x821Ytz8Z+eJvkjz92cvwzsGtB6EXhMbYhUgOx4A+pGkbBKBgFo2AYAwCVRVWXIG9iswAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-7909-5617","institution":"University of Geneva, Faculty of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Vladimir","middleName":"L.","lastName":"Katanaev","suffix":""}],"badges":[],"createdAt":"2023-12-18 11:51:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3771723/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3771723/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50173820,"identity":"3ebd133f-2463-42dd-b17c-a20747569ba3","added_by":"auto","created_at":"2024-01-25 16:03:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3399820,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThree classes of pathogenic Gαo mutations by their sensitivity to Zn\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e) GTP binding (\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ebind\u003c/em\u003e\u003c/sub\u003e, A) and GTP hydrolysis (\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ehydr\u003c/em\u003e\u003c/sub\u003e, B) rates of Gαo: wild-type and 14 pathogenic mutants. (\u003cstrong\u003eC\u003c/strong\u003e) Class I pathogenic Gαo mutants, like wild-type Gαo, do not display any changes in GTP hydrolysis upon adding increasing concentrations of ZnCl\u003csub\u003e2\u003c/sub\u003e. (\u003cstrong\u003eD\u003c/strong\u003e) Class II pathogenic Gαo mutants restore their GTP hydrolysis capacity upon addition of ZnCl\u003csub\u003e2\u003c/sub\u003e, in a dose-dependent manner. (\u003cstrong\u003eE\u003c/strong\u003e) As an example of class III pathogenic Gαo mutants, T182I displays reduced affinity to GTP upon addition of ZnCl\u003csub\u003e2\u003c/sub\u003e. 1μM BODIPY-GTPγS was titrated with recombinant Gαo[T182I] in the absence or presence of 50μM ZnCl\u003csub\u003e2\u003c/sub\u003e. The maximum values of BODIPY-GTPγS fluorescence (60sec after addition of T182I) were plotted against the concentration of recombinant Gαo[T182I] to calculate the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ed \u003c/em\u003e\u003c/sub\u003eof BODIPY-GTPγS to Gαo[T182I]. Data in (A-E) are mean ± SEM (n≥3); statistical analysis was performed by one-way ANOVA followed by Holm-Sidak test, significance is shown as **** p\u0026lt;0.0001; n.s.: not significant. (\u003cstrong\u003eF\u003c/strong\u003e) List of mutants analyzed in this study (14 mutations) and in two previous publications (2 mutations) categorized into 3 classes by the responsiveness to Zn\u003csup\u003e2+\u003c/sup\u003e. (\u003cstrong\u003eG\u003c/strong\u003e) The 3 classes of pathogenic Gαo mutants differ by the severity of disease they cause, measured as the individual patients’ disease onset (in days). Data are mean ± SEM (n=26 to 63, see Supplementary TableS1); statistical significance by t-test is shown.\u003c/p\u003e","description":"","filename":"Figure1Dec15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3771723/v1/283a98c98034950f7b1d54f3.jpg"},{"id":50173824,"identity":"b5851156-9995-487f-bc8e-6a88c386a9e7","added_by":"auto","created_at":"2024-01-25 16:03:01","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3230491,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePreclinical and clinical assessment of zinc supplementation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e) Body weight monitoring for continued treatment of C57BL/6 mice with the indicated concentrations of ZnSO\u003csub\u003e4\u003c/sub\u003e added to the drinking water shown separately for females (A) and males (B) reveals no significant changes between the control and ZnSO\u003csub\u003e4\u003c/sub\u003e-treated groups. (\u003cstrong\u003eC\u003c/strong\u003e) In the rotarod test, ZnSO\u003csub\u003e4\u003c/sub\u003e-treated mice showed an improvement in motor skills, irrespective of the C215Y/+, C215Y/C215Y, or +/+ genotype. A quantitative assessment of the falling time parameter is shown as an average for 4 test days. The number of animals in each experimental group is shown on the bars. (\u003cstrong\u003eD\u003c/strong\u003e) Treatment with ZnSO\u003csub\u003e4\u003c/sub\u003e increased the exploratory activity of mice, regardless of the C215Y/+, C215Y/C215Y, or +/+ genotype, in the recognition task test. Day 2 performance is shown; data for day 1 are provided in Supplementary Fig.S8A. Data in (A-D) are shown as mean ± SD. #p \u0026lt; 0.05, ##p \u0026lt; 0.01 and *p \u0026lt; 0.05 as determined by two-way ANOVA with Sidak's multiple comparisons test. (\u003cstrong\u003eE\u003c/strong\u003e) Clinical characteristics of the \u003cem\u003eGNAO1\u003c/em\u003e patient before and after continuous treatment with zinc.\u003c/p\u003e","description":"","filename":"Figure2Dec15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3771723/v1/c3692595a7045849543e81cd.jpg"},{"id":50186905,"identity":"87675ca7-a13b-4043-a59a-29db2db0fbf1","added_by":"auto","created_at":"2024-01-25 20:48:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":520813,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3771723/v1/805ec7ef-69b6-4cc2-9979-79b55d8d92db.pdf"},{"id":50173822,"identity":"f010060d-9626-40e6-9440-d2f6353fe929","added_by":"auto","created_at":"2024-01-25 16:03:01","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12376017,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figures\u003c/p\u003e","description":"","filename":"SupplementaryFiguresS1S8.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3771723/v1/0a9b921bf1aa9a35f8ed392e.pdf"},{"id":50174426,"identity":"f3c117f6-24a3-4f77-a408-df0836ae1427","added_by":"auto","created_at":"2024-01-25 16:11:01","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":180983,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary text\u003c/p\u003e","description":"","filename":"LarasatietalSupplementaryXXext.docx","url":"https://assets-eu.researchsquare.com/files/rs-3771723/v1/c6c19473e369a83bf0d5fd6b.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Zinc for GNAO1 encephalopathy: preclinical profiling and a clinical case","fulltext":[{"header":"Full Text","content":"\u003cp\u003eFirst identified in 2013, \u003cem\u003eGNAO1\u003c/em\u003e-related neurodevelopmental disorders are caused by mutations in the \u003cem\u003eGNAO1\u003c/em\u003e gene encoding the major neuronal G protein, G\u0026alpha;o\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. G\u0026alpha;o is one of 16 human G\u0026alpha;-subunits that, together with G\u0026beta;\u0026gamma;, form heterotrimeric G-protein complexes that are the primary transducers of G protein-coupled receptors (GPCRs). Ligand-activated GPCRs facilitate the exchange of GDP for GTP in G\u0026alpha;, thereby promoting the dissociation of G\u0026alpha;-GTP from G\u0026beta;\u0026gamma; and the receptor\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. G\u0026alpha;-GTP and G\u0026beta;\u0026gamma; can then engage with distinct downstream effectors to transmit the GPCR signal inside the cell. With time, G\u0026alpha; hydrolyzes its GTP back to GDP, and this GTPase activity can be accelerated by the Regulator of G-protein Signaling (RGS) proteins\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAs of today, about 200 patients worldwide are known to be affected by \u003cem\u003eGNAO1\u003c/em\u003e encephalopathy\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, identifying it as an ultrarare disease. However, this number is expected to grow with the wider availability of whole exome / genome sequencing in undiagnosed patients. Patients demonstrate a broad range of symptoms, including epilepsy, movement disorders, hypotonia, developmental delay, and brain atrophy\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Current treatments attempt to alleviate the epileptic and movement disorder symptoms, but patients are largely refractory to the available treatments\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. With more than 80 pathogenic (mostly missense) variants of G\u0026alpha;o identified to date (ncbi.nlm.nih.gov/clinvar/?term\u0026thinsp;=\u0026thinsp;gnao1%5Bgene%5D\u0026amp;redir\u0026thinsp;=\u0026thinsp;gene), in-depth molecular characterization is crucial to understand the etiology of the disease caused by the individual variants\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Earlier studies have revealed that different pathogenic G\u0026alpha;o exhibit a variety of molecular defects, such as accelerated GTP uptake / defective GTP hydrolysis that lead to constitutive GTP loading of G\u0026alpha;o, structural defects that lead to abnormal interaction with RGS proteins and G\u0026beta;\u0026gamma;, decreased plasma membrane localization, defective GPCR coupling, and neomorphic binding to Ric8A/B proteins\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003ePowered by the understanding of the molecular defects of pathogenic G\u0026alpha;o, we have identified zinc salts as a potential therapy for patients with the most common variants of \u003cem\u003eGNAO1\u003c/em\u003e encephalopathies: G203R, R209C, and E246K\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Replacing Mg\u003csup\u003e2+\u003c/sup\u003e in the active center and inducing structural rearrangements, Zn\u003csup\u003e2+\u003c/sup\u003e restores the GTPase activities of the mutants and their cellular interactions, without influencing wild-type G\u0026alpha;o\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Zinc supplementation improves motor function and longevity in a \u003cem\u003eDrosophila\u003c/em\u003e model of \u003cem\u003eGNAO1\u003c/em\u003e encephalopathy carrying the G203R variant; our studies using this model further indicate the need for continuous dietary zinc supplementation to achieve the therapeutic effect\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWe reveal that Zn\u003csup\u003e2+\u003c/sup\u003e counteracts the constitutive GTP binding of pathogenic G\u0026alpha;o variants by different mechanisms. Testing the biochemical properties of 16 variants (Supplementary Table\u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e), we find that most mutants have increased GTP binding rates (\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ebind\u003c/em\u003e\u003c/sub\u003e) and defective GTP hydrolysis (reduced \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ehydr\u003c/em\u003e\u003c/sub\u003e) in comparison to wild-type G\u0026alpha;o (Fig.\u0026nbsp;1A,B and Supplementary Fig.\u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e), agreeing with our previous studies\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Evaluation of the effects of increasing concentrations of ZnCl\u003csub\u003e2\u003c/sub\u003e on the GTP binding/hydrolysis permits to group the variants into three classes. The first includes the variants that are, like G\u0026alpha;o wild-type, unaffected by Zn\u003csup\u003e2+\u003c/sup\u003e in GTP binding / hydrolysis: L23P, C215Y, and I344del, adding up to the T241_N242insPQ variant we identified previously\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e (Supplementary Fig.\u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e and Fig.\u0026nbsp;1C). The second class represents the variants whose GTP binding remains unaffected, but whose GTP hydrolysis is restored by Zn\u003csup\u003e2+\u003c/sup\u003e, adding K46R to the G203R, R209C, and E246K mutants we studied earlier\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e (Supplementary Fig.S3 and Fig.\u0026nbsp;1D). And the third category emerges to be the most populated and includes K46N, H57P, T182I, R209H, Y231C, E237K, and Y291N, in addition to P170R studied earlier\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Zn\u003csup\u003e2+\u003c/sup\u003e reduces GTP uptake by these variants in both GTP binding and hydrolysis assays, the effect mediated by the \u0026gt;\u0026thinsp;3-fold reduction in the affinity of G\u0026alpha;o to GTP (Supplementary Fig.S4 and Fig.\u0026nbsp;1E,F)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eInterestingly, this stratification of pathogenic \u003cem\u003eGNAO1\u003c/em\u003e mutations correlates with the clinical severity of the disease manifestations. Analysis of 26 class I, 63 class II, and 40 class III patients (Fig.\u0026nbsp;1F and Supplementary Table\u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e) reveals that the onset of disease differs strongly and significantly among the classes, class II being the most severe (average onset of 6.8 months) and class I \u0026ndash; the least severe (average onset of 4.3 years), class III being the intermediate (6.8 years, Fig.\u0026nbsp;1G). Noteworthy, the biochemical perturbations of the class I mutants, which are biochemically unresponsive to zinc, are also milder (Fig.\u0026nbsp;1A,B). In contrast, for the pathogenic variants leading to more severe clinical phenotypes underlined by the biochemical severity, Zn\u003csup\u003e2+\u003c/sup\u003e alleviates their constitutive GTP binding through one of the two mechanisms, both expected to bring clinical benefit: reduction in the GTP uptake or restoration of GTP hydrolysis.\u003c/p\u003e\n\u003cp\u003eThese findings lay the basis for the patient stratification in the clinical applications of zinc. Zinc has been approved for treatment of diverse disorders including Wilson\u0026rsquo;s disease and various neurological conditions\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, with the daily dose of elemental zinc in Wilson\u0026rsquo;s disease treatment of 50mg daily for children under 6 years of age\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. We argued that a similar dose should be applied for the treatment of \u003cem\u003eGNAO1\u003c/em\u003e patients. Prior to the off-label clinical applications, we aimed at assessing the safety profile of zinc in a mouse model of the disease. Zinc toxicity has not been evaluated in neonates and young pups, whereas zinc supplementation has been well-studied in adult mice. For example, maintenance of adult mice for up to 14 months on water supplemented with 0.5g/L elemental zinc in the form of ZnSO\u003csub\u003e4\u003c/sub\u003e resulted in no adverse effects in the animals\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e; the maximum tolerated dose (MTD) for adult mice is reported as 75mM (12.11g/L) ZnSO\u003csub\u003e4\u003c/sub\u003e in drinking water\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. We thus first tested the near-MTD doses of ZnSO\u003csub\u003e4\u003c/sub\u003e supplied in drinking water to C57BL/6 mice all the way from birth to adulthood: 4-8g/L (ca. 1000-2000mg/kg/day of ZnSO\u003csub\u003e4\u003c/sub\u003e; the human equivalent dose can be estimated (following fda.gov/media/72309/download) as 81-162mg/kg/day. The presence of ZnSO\u003csub\u003e4\u003c/sub\u003e in the drinking water of lactating dams resulted in a transient delay in the body weight gain of the pups with onset at post-natal day (PND)7 and complete resolution by ca. PND17 (Supplementary Fig.S5A). This delay is fully compensated and is not reflected in the body weights of adult males and females by the end of the 3-month measurement period (Fig.\u0026nbsp;2A,B). No differences were observed in the appearance of animals treated with the highest dose of ZnSO\u003csub\u003e4\u003c/sub\u003e (8g/L, Supplementary Fig.S6). Finally, after 3 months of this treatment, the animals were sacrificed and the weight and appearance of major organs were evaluated at necropsy (Supplementary Figs S5B and S7). No difference in organ weight or appearance was observed. Taken together, these results indicate only a low and transient toxicity of the near-MTD doses of Zn\u003csup\u003e2+\u003c/sup\u003e and their suitability for long-term treatment.\u003c/p\u003e\n\u003cp\u003eOnly three mouse strains with pathogenic \u003cem\u003eGNAO1\u003c/em\u003e mutations have been described: R209H, C215Y, and G203R\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Of note, \u003cem\u003eG203R/+\u003c/em\u003e mice die neonatally\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. We thus used the C215Y mouse line available to us, in which behavioral disturbances in the form of hyperactive behavior have been described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. As the C215Y mutant was not responsive to Zn\u003csup\u003e2+\u003c/sup\u003e in our \u003cem\u003ein vitro\u003c/em\u003e biochemical assays (Fig.\u0026nbsp;1C,F), we did not expect to observe an improvement in the behavior of the mutant mice upon chronic ZnSO\u003csub\u003e4\u003c/sub\u003e administration. However, we argued that this mouse disease model could be a precious model to assess the safety profile of zinc supplementation in a \u003cem\u003eGNAO1\u003c/em\u003e disease condition. We find that continuous supplementation of ZnSO\u003csub\u003e4\u003c/sub\u003e (2g/L in the drinking water) leads to no behavioral disturbances. Intriguingly, we see a noticeable improvement in the mouse performance in the rotarod test that can be observed in the three genotypes: \u003cem\u003eC215Y/+\u003c/em\u003e, \u003cem\u003eC215Y/C215Y\u003c/em\u003e, and the control wild-type littermates (Fig.\u0026nbsp;2C). These results indicate that ZnSO\u003csub\u003e4\u003c/sub\u003e supplementation can improve motor skills in mice regardless of their genetic background. Given the known association between mutations in the \u003cem\u003eGNAO1\u003c/em\u003e gene and cognitive impairment in patients, the mice's exploratory behavior was also assessed using the novel object recognition test. In this test, all groups of mice, regardless of genotype, showed increased exploratory activity on the second day of the test. This was manifested by a significant increase in the time spent exploring objects (Fig.\u0026nbsp;2D and Supplementary Fig.S8A). In the open field test, where \u003cem\u003eC215Y/+\u003c/em\u003e and \u003cem\u003eC215Y/C215Y\u003c/em\u003e mice reveal the hyperkinetic activity\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, zinc supplementation did not change the behavioral readouts for either mutant genotype nor wild-type littermates (Supplementary Fig.S8B-D). Altogether, we conclude that zinc supplementation did not lead to any deterioration in the mouse model of \u003cem\u003eGNAO1\u003c/em\u003e encephalopathy; a general, genotype-independent improvement in motor and cognitive skills was achieved by the treatment.\u003c/p\u003e\n\u003cp\u003eWith this background, we applied zinc supplementation therapy to a 3.4 years-old patient with the c607G\u0026thinsp;\u0026gt;\u0026thinsp;A, p.Gly203Arg mutation (see online Methods for additional description of the patient). During the first year of life, the boy presented a severe epileptic encephalopathy without any head control nor achievement of any motoric milestones. Severe dystonia, choreoathetosis and repetitive hyperkinetic crises were prominent during the first months. Orofacial dystonia with tongue involvement caused severe feeding difficulties requiring percutaneous endoscopic gastrostomy (PEG) at the age of 12 months. A severe sleep disorder was present as well. The epilepsy was resistant to multiple antiseizure drugs. The disease progressed and daily hyperkinetic crises were the major cause of morbidity. Pharmacotherapy of the movement disorder comprised benzodiazepines, gabapentin, baclofen, clonidine and cannabinoids, all with minor or only short-lasting effects.\u003c/p\u003e\n\u003cp\u003eIn the course of a severe bacterial infection with respiratory impairment and ascites, the boy showed acute deterioration of his global condition with severe disease progression. He was administered to a hospice at the age of 3.4 years. With the family\u0026rsquo;s agreement we started an off-label zinc therapy. The initial dose was 40mg Zn\u003csup\u003e2+\u003c/sup\u003e daily (2.7mg/kg/day) administered as zinc gluconate which was increased to 50mg Zn\u003csup\u003e2+\u003c/sup\u003e daily (3.5mg/kg/day) administered as zinc acetate dihydrate. To date, the boy has been on zinc for 11 months. After grinding, the medication is provided through PEG as a suspension in water. The medication is well tolerated and no adverse events have been reported to date. Hemoglobin and copper serum levels were stable while the zinc serum level increased but did not exceed the upper limit (Fig.\u0026nbsp;2E). The patient\u0026rsquo;s condition has stabilized and an improvement in quality of life is reported by the parents: the daily hyperkinetic crises stopped and the patient started to smile again. The application of emergency medication against hyperkinetic exacerbations could be reduced. The patient shows longer periods of awareness during daytime and the night sleep could be improved. The parents report that their son now enjoys their interaction without any distraction by seizures or involuntary movements after many months of the severe impairment. The Burke-Fahn Marsden Dystonia Rating Scale movement score (BFMDRS-M) improved by 46,5 eight months after the start of the medication (Fig.\u0026nbsp;2E). The Gross Motor Function measure-66 (GMFM-66) did not show any changes. Morphine was the only new concomitant medication given to reduce agitation due to shortness of breath in the palliative setting.\u003c/p\u003e\n\u003cp\u003eThis is the first \u003cem\u003eGNAO1\u003c/em\u003e patient who receives zinc in high dosages equal to the dosages recommended in Wilson\u0026rsquo;s disease under controlled settings of a natural history study. This treatment trial has been supported by an extensive preclinical assessment, \u003cem\u003ein vitro\u003c/em\u003e and in animal models, of the efficacy, mechanism of action, and safety of zinc administration in order to cure the molecular defects caused by pathogenic \u003cem\u003eGNAO1\u003c/em\u003e mutations. The therapy has been feasible and no adverse events have been reported so far. It is possible that the beneficial effects cannot be attributed exclusively to the zinc treatment, but apart from morphine, which was started in the hospice setting, the patient did not receive any new therapies other than zinc during the treatment period of eight months. We conclude that further studies on the feasibility and safety of zinc in larger cohorts of patients with \u003cem\u003eGNAO1\u003c/em\u003e-associated disorders are now required.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe thank Dr. Gonzalo Solis for the fruitful discussion on this project. Mouse experiments were supported with the \u003cem\u003egrant number 21-15-00138 from the Russian Science Foundation to VLK and DNS, and biochemical experiments \u0026ndash; with a grant from\u003c/em\u003e GNAO1 Espa\u0026ntilde;a to VLK.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNakamura, K., Kodera, H., Akita, T., Shiina, M., Kato, M., Hoshino, H., Terashima, H., Osaka, H., Nakamura, S., Tohyama, J., Kumada, T., Furukawa, T., Iwata, S., Shiihara, T., Kubota, M., Miyatake, S., Koshimizu, E., Nishiyama, K., Nakashima, M., Tsurusaki, Y., Miyake, N., Hayasaka, K., Ogata, K., Fukuda, A., Matsumoto, N. \u0026amp; Saitsu, H. De Novo mutations in GNAO1, encoding a Galphao subunit of heterotrimeric G proteins, cause epileptic encephalopathy. \u003cem\u003eAm J Hum Genet\u003c/em\u003e \u003cstrong\u003e93\u003c/strong\u003e, 496-505 (2013).\u003c/li\u003e\n\u003cli\u003eGilman, A. G. G proteins: transducers of receptor-generated signals. \u003cem\u003eAnnu Rev Biochem\u003c/em\u003e \u003cstrong\u003e56\u003c/strong\u003e, 615-649 (1987).\u003c/li\u003e\n\u003cli\u003eDohlman, H. G. \u0026amp; Thorner, J. RGS proteins and signaling by heterotrimeric G proteins. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e272\u003c/strong\u003e, 3871-3874 (1997).\u003c/li\u003e\n\u003cli\u003eGonz\u0026aacute;lez, M., Kloosterhuis, K., Pol, L., Baas, F. \u0026amp; Mikkers, H. Phenotypic Diversity in GNAO1 Patients: A Comprehensive Overview of Variants and Phenotypes. \u003cem\u003eHuman Mutation\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, 1-16 (2023).\u003c/li\u003e\n\u003cli\u003eBriere, L., Thiel, M., Sweetser, D. A., Koy, A. \u0026amp; Axeen, E. GNAO1-Related Disorder. \u003cem\u003eGeneReviews\u003c/em\u003e (2023).\u003c/li\u003e\n\u003cli\u003eSchirinzi, T., Garone, G., Travaglini, L., Vasco, G., Galosi, S., Rios, L., Castiglioni, C., Barassi, C., Battaglia, D., Gambardella, M. L., Cantonetti, L., Graziola, F., Marras, C. E., Castelli, E., Bertini, E., Capuano, A. \u0026amp; Leuzzi, V. Phenomenology and clinical course of movement disorder in GNAO1 variants: Results from an analytical review. \u003cem\u003eParkinsonism Relat Disord\u003c/em\u003e \u003cstrong\u003e61\u003c/strong\u003e, 19-25 (2019).\u003c/li\u003e\n\u003cli\u003eWirth, T., Garone, G., Kurian, M. A., Piton, A., Millan, F., Telegrafi, A., Drouot, N., Rudolf, G., Chelly, J., Marks, W., Burglen, L., Demailly, D., Coubes, P., Castro-Jimenez, M., Joriot, S., Ghoumid, J., Belin, J., Faucheux, J. M., Blumkin, L., Hull, M., Parnes, M., Ravelli, C., Poulen, G., Calmels, N., Nemeth, A. H., Smith, M., Barnicoat, A., Ewenczyk, C., M\u0026eacute;neret, A., Roze, E., Keren, B., Mignot, C., Beroud, C., Acosta, F., Jr., Nowak, C., Wilson, W. G., Steel, D., Capuano, A., Vidailhet, M., Lin, J. P., Tranchant, C., Cif, L., Doummar, D. \u0026amp; Anheim, M. Highlighting the Dystonic Phenotype Related to GNAO1. \u003cem\u003eMov Disord\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 1547-1554 (2022).\u003c/li\u003e\n\u003cli\u003eAxeen, E., Bell, E., Robichaux Viehoever, A., Schreiber, J. M., Sidiropoulos, C. \u0026amp; Goodkin, H. P. Results of the First GNAO1-Related Neurodevelopmental Disorders Caregiver Survey. \u003cem\u003ePediatr Neurol\u003c/em\u003e \u003cstrong\u003e121\u003c/strong\u003e, 28-32 (2021).\u003c/li\u003e\n\u003cli\u003eKatanaev, V. L., Valnohova, J., Silachev, D. N., Larasati, Y. A. \u0026amp; Koval, A. Pediatric GNAO1 encephalopathies: from molecular etiology of the disease to drug discovery. \u003cem\u003eNeural Regeneration Research\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 2188-2189 (2023).\u003c/li\u003e\n\u003cli\u003eSolis, G. P., Kozhanova, T. V., Koval, A., Zhilina, S. S., Mescheryakova, T. I., Abramov, A. A., Ishmuratov, E. V., Bolshakova, E. S., Osipova, K. V., Ayvazyan, S. O., Lebon, S., Kanivets, I. V., Pyankov, D. V., Troccaz, S., Silachev, D. N., Zavadenko, N. N., Prityko, A. G. \u0026amp; Katanaev, V. L. Pediatric Encephalopathy: Clinical, Biochemical and Cellular Insights into the Role of Gln52 of GNAO1 and GNAI1 for the Dominant Disease. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 2749 (2021).\u003c/li\u003e\n\u003cli\u003eMuntean, B. S., Masuho, I., Dao, M., Sutton, L. P., Zucca, S., Iwamoto, H., Patil, D. N., Wang, D., Birnbaumer, L., Blakely, R. D., Grill, B. \u0026amp; Martemyanov, K. A. G\u0026alpha;o is a major determinant of cAMP signaling in the pathophysiology of movement disorders. \u003cem\u003eCell Rep\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 108718 (2021).\u003c/li\u003e\n\u003cli\u003eLarasati, Y. A., Savitsky, M., Koval, A., Solis, G. P., Valnohova, J. \u0026amp; Katanaev, V. L. Restoration of the GTPase activity and cellular interactions of G\u0026alpha;(o) mutants by Zn(2+) in GNAO1 encephalopathy models. \u003cem\u003eSci Adv\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, eabn9350 (2022).\u003c/li\u003e\n\u003cli\u003eKoval, A., Larasati, Y. A., Savitsky, M., Solis, G. P., Good, J. M., Quinodoz, M., Rivolta, C., Superti-Furga, A. \u0026amp; Katanaev, V. L. In-depth molecular profiling of an intronic GNAO1 mutant as the basis for personalized high-throughput drug screening. \u003cem\u003eMed\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 311-325.e317 (2023).\u003c/li\u003e\n\u003cli\u003eSolis, G. P., Koval, A., Valnohova, J., Savitsky, M. \u0026amp; Katanaev, V. L. Ric8 proteins as the neomorphic partners of G\u0026alpha;o in GNAO1 encephalopathies. \u003cem\u003ebioRxiv\u003c/em\u003e, 2023.2003.2027.534359 (2023).\u003c/li\u003e\n\u003cli\u003eLarasati, Y. A., Solis, G. P., Koval, A., Griffiths, S. T., Berentsen, R., Aukrust, I., Lesca, G., Chatron, N., Ville, D., Korff, C. M. \u0026amp; Katanaev, V. L. Clinical Cases and the Molecular Profiling of a Novel Childhood Encephalopathy-Causing GNAO1 Mutation P170R. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e (2023).\u003c/li\u003e\n\u003cli\u003eKnight, K. M., Obarow, E. G., Wei, W., Mani, S., Esteller, M. I., Cui, M., Ma, N., Martin, S. A., Brinson, E., Hewitt, N., Soden, G. M., Logothetis, D. E., Vaidehi, N. \u0026amp; Dohlman, H. G. Molecular annotation of G protein variants in a neurological disorder. \u003cem\u003eCell Rep\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 113578 (2023).\u003c/li\u003e\n\u003cli\u003eDom\u0026iacute;nguez-Carral, J., Ludlam, W. G., Junyent Segarra, M., Fornaguera Marti, M., Balsells, S., Muchart, J., Čokolić Petrović, D., Espinoza, I., Ortigoza-Escobar, J. D. \u0026amp; Martemyanov, K. A. Severity of GNAO1-Related Disorder Correlates with Changes in G-Protein Function. \u003cem\u003eAnn Neurol\u003c/em\u003e \u003cstrong\u003e94\u003c/strong\u003e, 987-1004 (2023).\u003c/li\u003e\n\u003cli\u003eLarrivee, C. L., Feng, H., Quinn, J. A., Shaw, V. S., Leipprandt, J. R., Demireva, E. Y., Xie, H. \u0026amp; Neubig, R. R. Mice with GNAO1 R209H Movement Disorder Variant Display Hyperlocomotion Alleviated by Risperidone. \u003cem\u003eJ Pharmacol Exp Ther\u003c/em\u003e \u003cstrong\u003e373\u003c/strong\u003e, 24-33 (2020).\u003c/li\u003e\n\u003cli\u003eCzłonkowska, A., Litwin, T., Dusek, P., Ferenci, P., Lutsenko, S., Medici, V., Rybakowski, J. K., Weiss, K. H. \u0026amp; Schilsky, M. L. Wilson disease. \u003cem\u003eNat Rev Dis Primers\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 21 (2018).\u003c/li\u003e\n\u003cli\u003eGrabrucker, A. M., Rowan, M. \u0026amp; Garner, C. C. Brain-Delivery of Zinc-Ions as Potential Treatment for Neurological Diseases: Mini Review. \u003cem\u003eDrug Deliv Lett\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 13-23 (2011).\u003c/li\u003e\n\u003cli\u003eRanucci, G., Di Dato, F., Spagnuolo, M. I., Vajro, P. \u0026amp; Iorio, R. Zinc monotherapy is effective in Wilson\u0026apos;s disease patients with mild liver disease diagnosed in childhood: a retrospective study. \u003cem\u003eOrphanet J Rare Dis\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 41 (2014).\u003c/li\u003e\n\u003cli\u003eAughey, E., Grant, L., Furman, B. L. \u0026amp; Dryden, W. F. The effects of oral zinc supplementation in the mouse. \u003cem\u003eJournal of Comparative Pathology\u003c/em\u003e \u003cstrong\u003e87\u003c/strong\u003e, 1-14 (1977).\u003c/li\u003e\n\u003cli\u003eSouffriau, J., Timmermans, S., Vanderhaeghen, T., Wallaeys, C., Van Looveren, K., Aelbrecht, L., Dewaele, S., Vandewalle, J., Goossens, E., Verbanck, S., Boyen, F., Eggermont, M., De Commer, L., De Rycke, R., De Bruyne, M., Tito, R., Ballegeer, M., Vandevyver, S., Velho, T., Moita, L. F., Hochepied, T., De Bosscher, K., Raes, J., Van Immerseel, F., Beyaert, R. \u0026amp; Libert, C. Zinc inhibits lethal inflammatory shock by preventing microbe-induced interferon signature in intestinal epithelium. \u003cem\u003eEMBO Mol Med\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, e11917 (2020).\u003c/li\u003e\n\u003cli\u003eSilachev, D., Koval, A., Savitsky, M., Padmasola, G., Quairiaux, C., Thorel, F. \u0026amp; Katanaev, V. L. Mouse models characterize GNAO1 encephalopathy as a neurodevelopmental disorder leading to motor anomalies: from a severe G203R to a milder C215Y mutation. \u003cem\u003eActa Neuropathol Commun\u003c/em\u003e\u003cstrong\u003e10\u003c/strong\u003e, 9 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"GNAO1 encephalopathy, Gαo, zinc, dietary supplementation, rare disease, animal model, clinical case","lastPublishedDoi":"10.21203/rs.3.rs-3771723/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3771723/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eDe novo\u003c/em\u003e mutations in \u003cem\u003eGNAO1\u003c/em\u003e \u0026ndash; the gene encoding the major neuronal G-protein Gαo \u0026ndash; cause pediatric encephalopathies largely refractory to available therapies. Zn\u003csup\u003e2+\u003c/sup\u003e emerged to restore GTP hydrolysis and cellular interactions of pathogenic Gαo; dietary Zn\u003csup\u003e2+\u003c/sup\u003e supplementation improves lifespan and motoric function in a \u003cem\u003eDrosophila\u003c/em\u003e disease model. Here we show that 16 different pathogenic missense mutations cluster in three distinct groups in their responsiveness to Zn\u003csup\u003e2+\u003c/sup\u003e, and provide the safety study in a mouse disease model. We further describe treatment of a 3 years-old patient with a common \u003cem\u003eGNAO1\u003c/em\u003e mutation c607G\u0026thinsp;\u0026gt;\u0026thinsp;A, p.Gly203Arg with oral 50mg Zn\u003csup\u003e2+\u003c/sup\u003e daily, as applied in Wilson\u0026rsquo;s disease. During 11 months of treatment, the patient shows cessation of daily hyperkinetic crises, improved Burke-Fahn Marsden Dystonia Rating Scale movement score and general well-being, and an excellent safety profile. Our findings warrant a large-scale clinical trial and might set the new standard of care for \u003cem\u003eGNAO1\u003c/em\u003e encephalopathy.\u003c/p\u003e","manuscriptTitle":"Zinc for GNAO1 encephalopathy: preclinical profiling and a clinical case","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-25 16:02:56","doi":"10.21203/rs.3.rs-3771723/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2f8d9858-756b-4716-a5d6-9a25888f02d6","owner":[],"postedDate":"January 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28340776,"name":"Health sciences/Neurology/Neurological disorders/Encephalopathy"},{"id":28340777,"name":"Biological sciences/Cell biology/Cell signalling"},{"id":28340778,"name":"Health sciences/Molecular medicine"},{"id":28340779,"name":"Biological sciences/Drug discovery"}],"tags":[],"updatedAt":"2024-01-25T16:02:56+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-25 16:02:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3771723","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3771723","identity":"rs-3771723","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}