Verhoeven-Duif, N. M. | bioRxiv Science

bioRxiv2019

NaV1.1 and NaV1.6 selective compounds reduce the behavior phenotype in a novel zebrafish model for Dravet Syndrome

Dravet syndrome is caused by dominant loss-of-function mutations in SCN1A which cause reduced activity of Nav1.1 leading to lack of neuronal inhibition. On the other hand, gain-of-function mutations in SCN8A can lead to a severe epileptic encephalopathy subtype by over activating NaV1.6 channels. These observations suggest that Nav1.1 and Nav1.6 represent two opposing sides of the neuronal balance between inhibition and activation. Here, we hypothesize that Dravet syndrome may be treated by either enhancing Nav1.1 or reducing Nav1.6 activity. To test this hypothesis we generated and characterized a novel DS zebrafish model and tested new compounds that selectively activate or inhibit the human NaV1.1 or NaV1.6 channel respectively. We used CRISPR/Cas9 to generate two separate Scn1Lab knockout lines as an alternative to previous knock-down models. Using an optimized locomotor assay, spontaneous burst movements were detected that were unique to Scn1Lab knockouts and disappear when introducing human SCN1A mRNA. Besides the behavioral phenotype, Scn1Lab knockouts show sudden, electrical discharges in the brain that indicate epileptic seizures in zebrafish. Scn1Lab knockouts showed increased sensitivity to the convulsant pentylenetetrazole and a reduction in whole organism GABA levels. Drug screenings further validated a Dravet syndrome phenotype. We tested the NaV1.1 activator AA43279 and our newly synthesized NaV1.6 inhibitors MV1369 and MV1312 in the Scn1Lab knockouts. Both type of compounds significantly reduced the number of burst movements. Our results show that selective inhibition of NaV1.6 could be just as efficient as selective activation of NaV1.1 and these approaches could prove to be novel potential treatment strategies for Dravet syndrome and other (genetic) epilepsies. Compounds tested in zebrafish however, should always be further validated in other model systems, preferably human derived.

genetics↗

bioRxiv2019

Pyridox(am)ine 5’-phosphate oxidase deficiency induces seizures in Drosophila melanogaster

Pyridox(am)ine 5-phosphate oxidase (PNPO) is a rate-limiting enzyme in converting dietary vitamin B6 (VB6) to pyridoxal 5-phosphate (PLP), the biologically active form of VB6, and involved in the synthesis of neuro-transmitters including GABA, dopamine, and serotonin. In humans, PNPO mutations have been increasingly identified in neonatal epileptic encephalopathy and more recently also in early-onset epilepsy. Till now, little is known about the neurobiological mechanisms underlying PNPO-deficiency-induced seizures due to the lack of animal models. Previously we identified a c.95 C > A missense mutation in sgll - the Drosophila homolog of human PNPO (hPNPO) and found mutant (sgll95) flies exhibiting a lethal phenotype on a diet devoid of VB6. Here we report the establishment of both sgll95 and ubiquitous sgll knockdown (KD) flies as valid animal models of PNPO-deficiency-induced epilepsy. Both sgll95 and sgll KD flies exhibit spontaneous seizures before they die. Electrophysiological recordings reveal that seizures caused by PNPO deficiency have characteristics similar to that in flies treated with GABA antagonist picrotoxin. Both seizures and lethality are associated with low PLP levels and can be rescued by ubiquitous expression of wild-type sgll or hPNPO, suggesting the functional conservation of the PNPO enzyme between humans and flies. Results from cell type-specific sgll KD further demonstrate that PNPO in the brain is necessary for seizure prevention and survival. Our establishment of the first animal model of PNPO deficiency will lead to better understanding of VB6 biology, the PNPO gene and its mutations discovered in patients, and can be a cost-effective system to test therapeutic strategies.

neuroscience↗