Generation and Characterization of the Drosophila melanogaster paralytic Gene Knock-Out as a Model for Dravet Syndrome.

Andrea Tapia,Máximo Ibo Galindo,Carlo N Giachello,Martina Palomino-Schätzlein,Richard A Baines

doi:10.3390/life11111261

Abstract

Dravet syndrome is a severe rare epileptic disease caused by mutations in the SCN1A gene coding for the Nav1.1 protein, a voltage-gated sodium channel alpha subunit. We have made a knock-out of the paralytic gene, the single Drosophila melanogaster gene encoding this type of protein, by homologous recombination. These flies showed a heat-induced seizing phenotype, and sudden death in long term seizures. In addition to seizures, neuromuscular alterations were observed in climbing, flight, and walking tests. Moreover, they also manifested some cognitive alterations, such as anxiety and problems in learning. Electrophysiological analyses from larval motor neurons showed a decrease in cell capacitance and membrane excitability, while persistent sodium current increased. To detect alterations in metabolism, we performed an NMR metabolomic profiling of heads, which revealed higher levels in some amino acids, succinate, and lactate; and also an increase in the abundance of GABA, which is the main neurotransmitter implicated in Dravet syndrome. All these changes in the paralytic knock-out flies indicate that this is a good model for epilepsy and specifically for Dravet syndrome. This model could be a new tool to understand the pathophysiology of the disease and to find biomarkers, genetic modifiers and new treatments.

Highlights

DS has an early onset in the first few months of life, usually triggered by fever, with a hemiclonic or clonic seizure, affecting one or both sides of the body, respectively, which is followed by further episodes that can be prolonged and even result in status epilepticus (SE, duration over 5 min or repeated short episodes)
We designed two arms of homology, an A arm of 951 bp ending in the base pair just upstream of the start codon, and a B arm of 782 bp, starting 10 bp downstream of the start codon (Figure 1B), and both arms were cloned into the pTV[Cherry] vector to achieve the gene editing
Due to the design of the strategy, the homologous recombination would generate a deletion of 12 bp, including the start codon, followed by the insertion of the construct, which would prevent the translation of the full length protein

Summary

Introduction

Dravet syndrome (DS, ORPHA:33069), or epileptic encephalopathy with early onset. 6 (EIEE6), or severe childhood myoclonic epilepsy (SMEI), is a childhood epilepsy [1,2,3]. DS has an early onset in the first few months of life, usually triggered by fever, with a hemiclonic or clonic seizure, affecting one or both sides of the body, respectively, which is followed by further episodes that can be prolonged and even result in status epilepticus (SE, duration over 5 min or repeated short episodes). Patients usually suffer more febrile or afebrile seizures that can be of different types from the initial ones: tonic-clonic, myoclonic, absence. They can suffer cognitive delay, movement disorders and, more seriously, sudden death in a relatively high proportion (>10%). DS is a disease with a very low prevalence, it is estimated that one infant suffers it for every

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Results

Conclusion