Establishment of zebrafish model of attention-deficit/hyperactivity disorder

Epilepsy is a common neurological disorder, which affects about 50 million people worldwide. It is marked by abnormal electrical activity in the brain, i.e. seizures, and is typically characterized by either a sudden brief period of altered or lost consciousness, involuntary movements or convulsions. Approximately 30% of patients are currently resistant to available medication, leaving only neurosurgery for a minority of these patients as a last treatment option.

Distinct de novo mutations in the sodium channel, voltage gated, type I alpha subunit, SCN1A are known to be the origin of Dravet syndrome (DS), a rare (ca. 1/20.000 to < 1/40.000 live births) and severe myoclonic epilepsy of infancy that persists throughout adulthood. According to the International League Against Epilepsy (ILAE) classification, DS is defined by “febrile and afebrile generalized and unilateral, clonic or tonic–clonic, seizures that occur in the first year of life in an otherwise normal infant and are later associated with myoclonus, atypical absences, and partial seizures. Developmental delay becomes apparent within the second year of life and is followed by definite cognitive impairment and personality disorders”. As DS is one of the most pharmacoresistant epilepsy syndromes, therefore new, effective antiepileptic drugs with novel modes of action would be of significant value for the expansion of current treatment possibilities. The search for new drugs with improved activity and fewer adverse effects therefore remains highly relevant.

Zebrafish offer several advantages over other epilepsy model organisms, such as rapid ex utero development, transparent embryonic and larval stages, and high fecundity. Moreover, relatively high genetic homology between zebrafish and human (ca. 70%) and numberless easy and robust methods for genetic manipulation make zebrafish an ideal model of functional genomics; furthermore, its small size, cost-effective maintenance and minimal ethical administrative requirements make zebrafish as a powerful tool for early drug discovery. Therefore, we used zebrafish to model and characterize DS and borderline DS by knocking down zebrafish scn1Lab (encoding sodium channel, voltage-gated, type I like, alpha b) and chd2 (encoding chromodomain helicase DNA binding protein 2), respectively.

We knocked down scn1Lab using morpholino (MO) oligos, which resulted in spontaneous seizure-like behavior (hyperactivity) and abnormal brain activity. Furthermore, we showed that these larvae are remarkably sensitive to hyperthermia, similar to what has been described for mouse models of DS, as well as for human DS patients. Pharmacological evaluation revealed that sodium valproate and fenfluramine significantly reduce epileptiform discharges in scn1Lab morphants (MO injected zebrafish larvae). Our findings for this zebrafish model of DS are in accordance with clinical data for human DS patients. Moreover, this is the first study demonstrating effective seizure inhibition of fenfluramine in an animal model of Dravet syndrome. Importantly, this scn1Lab larval zebrafish model can be potentially used to identify compounds that decrease seizure frequency for the treatment of DS.

We also investigated the role of CHD2/chd2. The group of P. De Jonghe, identified three de novo loss-of-function mutation in CHD2 in three Dravet-syndrome-affected individuals without an SCN1A mutation. All three individuals with a CHD2 mutation had intellectual disability and fever-sensitive generalized seizures, as well as prominent myoclonic seizures starting in the second year of life or later. To validate the functional relevance of CHD2 haploinsufficiency, we knocked down chd2 in zebrafish by using targeted morpholino antisense oligomers. chd2 knockdown larvae exhibited altered locomotor activity, and the epileptic nature of this seizure-like behavior was confirmed by field-potential recordings that revealed epileptiform discharges similar to seizures in affected persons. Both altered locomotor activity and epileptiform discharges were absent in appropriate control larvae. Our study provides evidence that de novo loss-of-function mutations in CHD2 are a cause of epileptic encephalopathy with generalized seizures. Importantly, the chd2 morphants represent that zebrafish is a relevant model in functional genomics, i.e. by knocking down zebrafish chd2 we could prove that this gene is implicated in patients with DS without an SCN1A mutation.