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

Abstract

GNAO1 encephalopathy characterized by a wide spectrum of neurological deficiencies in pediatric patients originates from de novo heterozygous mutations in the gene encoding Gαo, the major neuronal G protein. Efficient treatments and even the proper understanding of the underlying etiology are currently lacking for this dominant disease. Adequate animal models of GNAO1 encephalopathy are urgently needed. Here we describe establishment and characterization of mouse models of the disease based on two point mutations in GNAO1 with different clinical manifestations. One of them is G203R leading to the early-onset epileptic seizures, motor dysfunction, developmental delay and intellectual disability. The other is C215Y producing much milder clinical outcomes, mostly-late-onset hyperkinetic movement disorder. The resultant mouse models show distinct phenotypes: severe neonatal lethality in GNAO1[G203R]/ + mice vs. normal vitality in GNAO1[C215Y]/ + . The latter model further revealed strong hyperactivity and hyperlocomotion in a panel of behavioral assays, without signs of epilepsy, recapitulating the patients’ manifestations. Importantly, despite these differences the two models similarly revealed prenatal brain developmental anomalies, such as enlarged lateral ventricles and decreased numbers of neuronal precursor cells in the cortex. Thus, our work unveils GNAO1 encephalopathy as to a large extent neurodevelopmental malady. We expect that this understanding and the tools we established will be instrumental for future therapeutic developments.

Restoration of the GTPase activity and cellular interactions of Gα o mutants by Zn 2+ in GNAO1 encephalopathy models

“…Some features of the Drosophila model of the disease, namely, the delayed lethality of the G α o[G203R]/G α o[G203R] mutant flies as compared to homozygous G α o null mutants ( 42 , 43 ), also speak in favor of the hypomorph nature of the mutation. Last, from human patients to the Drosophila G α o[G203R]/+ model, agreeing also with mouse ( 9 , 23 ) and nematode ( 56 , 57 ) models of the disease, and further agreeing with some in vitro observations ( 23 ), the encephalopathy GNAO1 mutations have been described in many instances as dominant negative or antimorph in the Muller classification ( 64 ).…”

“…However, this degeneration was very modest as compared to that observed, e.g., in Drosophila models of Alzheimer’s disease ( 47 ), the finding that may be aligned with the fact that the G203R mutant flies did not display any signs of spontaneous epilepsy. Given the neonatal lethality of GNAO1[G203R]/+ mice ( 9 ), we thus establish the first viable animal model of G203R encephalopathy with this Drosophila line capable of recapitulating some of the clinical manifestations of the disease.…”

“…The number of found different, mostly missense point, mutations in GNAO1 causing this malady steadily increases every year since the first 2013 report ( 5 ). However, despite some insights ( 5 , 9 , 19 , 23 , 56 , 57 ), the understanding of the molecular etiology underlying the pathological developments has been largely missing. This delay in the understanding blocks development of therapies to treat the patients.…”

“…This discovery was followed by an avalanche of clinical analyses that cumulatively led to the recognition of GNAO1 encephalopathy as a spectrum of neurodevelopmental disorders manifesting as motor dysfunction, epileptic seizures, and developmental delay first appearing mostly in infancy ( 6 – 8 ). Although Gα o , in addition to neurons, is also strongly expressed in glial cells ( www.proteinatlas.org ), disease modeling in mice ( 9 ) and brain organoids ( 10 ) demonstrated strong effects of GNAO1 mutations on neuronal rather than glial differentiation, highlighting neurons as the main target of Gα o malfunctioning in the disease. Gα o couples to many neuronal GPCRs, such as D2 dopamine, μ-opioid, M2 muscarinic, α2-adrenergic, and more.…”

CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics

“…For blastocyst assay, the zygotes were transferred into 35 mm Petri dishes and cultured in 50-60 μL droplets of KSOM medium (MTI-GlobalStem) under mineral oil at 37 °C and 5% CO2 for 3 days till the blastocyst stage. To produce genome-modified mice, injected eggs were incubated overnight, and viable twocell-stage embryos were implanted into pseudopregnant foster dams (Silaeva et al, 2018).…”