Humanization of Drosophila Gαo to Model GNAO1 Paediatric Encephalopathies.

Mikhail Savitsky,Vladimir L Katanaev,Gonzalo P Solis,Mikhail Kryuchkov

doi:10.3390/biomedicines8100395

Mikhail Savitsky, Vladimir L Katanaev + Show 2 more

Open Access

https://doi.org/10.3390/biomedicines8100395

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Abstract

Several hundred genes have been identified to contribute to epilepsy—the disease affecting 65 million people worldwide. One of these genes is GNAO1 encoding Gαo, the major neuronal α-subunit of heterotrimeric G proteins. An avalanche of dominant de novo mutations in GNAO1 have been recently described in paediatric epileptic patients, suffering, in addition to epilepsy, from motor dysfunction and developmental delay. Although occurring in amino acids conserved from humans to Drosophila, these mutations and their functional consequences have only been poorly analysed at the biochemical or neuronal levels. Adequate animal models to study the molecular aetiology of GNAO1 encephalopathies have also so far been lacking. As the first step towards modeling the disease in Drosophila, we here describe the humanization of the Gαo locus in the fruit fly. A two-step CRISPR/Cas9-mediated replacement was conducted, first substituting the coding exons 2–3 of Gαo with respective human GNAO1 sequences. At the next step, the remaining exons 4–7 were similarly replaced, keeping intact the gene Cyp49a1 embedded in between, as well as the non-coding exons, exon 1 and the surrounding regulatory sequences. The resulting flies, homozygous for the humanized GNAO1 loci, are viable and fertile without any visible phenotypes; their body weight, locomotion, and longevity are also normal. Human Gαo-specific antibodies confirm the endogenous-level expression of the humanized Gαo, which fully replaces the Drosophila functions. The genetic model we established will make it easy to incorporate encephalopathic GNAO1 mutations and will permit intensive investigations into the molecular aetiology of the human disease through the powerful toolkit of Drosophila genetics.

Highlights

Epilepsy is a chronic disease of multigenic origin, characterized by appearance, mostly in an unpredicted manner, of seizures, and sometimes complicated by other neurological or neurodevelopmental deficits [1]
With 65 millions of people worldwide currently suffering from epilepsy and the inadequacy of the current pharmacological approaches to certain subtypes of the disease, the need to advance our understanding of the aetiology of this disease is clear, as is the urgency to develop novel medical treatments [2]
One of the proteins that have recently emerged as an important player in epilepsy is Gαo—the major neuronal α-subunit of heterotrimeric G proteins, encoded by the gene GNAO1

Summary

Introduction

Epilepsy is a chronic disease of multigenic origin, characterized by appearance, mostly in an unpredicted manner, of seizures, and sometimes complicated by other neurological or neurodevelopmental deficits [1]. Dorsal root ganglion cells derived from Gαo KO mice presented a reduced inhibition of Ca2+ channel curr gonist-induced activation of opioid receptors [20]. Gαo transcript and protein were present at all stages of embryonic development with a marked increase during the period of active axonogenesis [35,36] As improper synaptic plasticity and process outgrowth are both implicated in the aetiology of epilepsy [1], the possible dominant effects of the epileptic Gαo mutations on these novel Golgi-emanating mechanisms discovered by us need to be investigated in detail, as are the molecular events happening at PM. As the first step towards modeling GNAO1 paediatric encephalopathies in the fruit fly, we here describe the humanization of the Drosophila Gαo locus, finding the human protein fully replaces the insect one’s functions without any aberrations

Donor Plasmid pLdhGao23R for the First Round of Humanization dGαo

Donor Plasmid for the Second Round of Humanization dGαo

Molecular Analysis

Immunochemistry and Microscopy

Behavioural Assays

Results and Discussion