PUBLICATION

New insights into the early mechanisms of epileptogenesis in a zebrafish model of Dravet syndrome

Authors

Tiraboschi, E., Martina, S., van der Ent, W., Grzyb, K., Gawel, K., Cordero-Maldonado, M.L., Poovathingal, S.K., Heintz, S., Satheesh, S.V., Brattespe, J., Xu, J., Suster, M., Skupin, A., Esguerra, C.V.

ID

ZDB-PUB-200226-7

Date

2020

Source

Epilepsia 61(3): 549-560 (Journal)

Registered Authors

Cordero-Maldonado, Maria Lorena, Esguerra, Camila V., Gawel, Kinga Aurelia, Suster, Maximiliano, van der Ent, Wietske

Keywords

Dravet syndrome, epileptogenesis, fenfluramine, sodium channel, zebrafish

Datasets

GEO:GSE145801

MeSH Terms

Animals
Epilepsies, Myoclonic/genetics*
Epilepsies, Myoclonic/metabolism
Epilepsies, Myoclonic/pathology
Epilepsies, Myoclonic/physiopathology
RNA-Seq
Anticonvulsants/pharmacology
Brain/drug effects
Brain/metabolism
Brain/pathology
Brain/physiopathology*
Single-Cell Analysis
GABAergic Neurons/drug effects
GABAergic Neurons/metabolism
GABAergic Neurons/pathology
Mutation, Missense
Diazepam/pharmacology
Real-Time Polymerase Chain Reaction
Zebrafish
Electroencephalography
Fenfluramine/pharmacology
Locomotion/drug effects
Zebrafish Proteins/genetics*
Zebrafish Proteins/metabolism
Cell Proliferation/drug effects
Disease Models, Animal
Neuronal Plasticity/drug effects
Neuronal Plasticity/genetics*
NAV1.1 Voltage-Gated Sodium Channel/genetics*
NAV1.1 Voltage-Gated Sodium Channel/metabolism
Gliosis/genetics
Gliosis/pathology
Serotonin 5-HT2 Receptor Agonists/pharmacology
Gene Expression Profiling
CRISPR-Cas Systems

(all 35)

PubMed

32096222 Full text @ Epilepsia

Abstract

Objective To pinpoint the earliest cellular defects underlying seizure onset (epileptogenic period) during perinatal brain development in a new zebrafish model of Dravet syndrome (DS) and to investigate potential disease-modifying activity of the 5HT₂ receptor agonist fenfluramine.

Methods We used CRISPR/Cas9 mutagenesis to introduce a missense mutation, designed to perturb ion transport function in all channel isoforms, into scn1lab, the zebrafish orthologue of SCN1A (encoding voltage-gated sodium channel alpha subunit 1). We performed behavioral analysis and electroencephalographic recordings to measure convulsions and epileptiform discharges, followed by single-cell RNA-Seq, morphometric analysis of transgenic reporter-labeled γ-aminobutyric acidergic (GABAergic) neurons, and pharmacological profiling of mutant larvae.

Results Homozygous mutant (scn1lab^mut/mut ) larvae displayed spontaneous seizures with interictal, preictal, and ictal discharges (mean = 7.5 per 20-minute recording; P < .0001; one-way analysis of variance). Drop-Seq analysis revealed a 2:1 shift in the ratio of glutamatergic to GABAergic neurons in scn1lab^mut/mut larval brains versus wild type (WT), with dynamic changes in neuronal, glial, and progenitor cell populations. To explore disease pathophysiology further, we quantified dendritic arborization in GABAergic neurons and observed a 40% reduction in arbor number compared to WT (P < .001; n = 15 mutant, n = 16 WT). We postulate that the significant reduction in inhibitory arbors causes an inhibitory to excitatory neurotransmitter imbalance that contributes to seizures and enhanced electrical brain activity in scn1lab^mut/mut larvae (high-frequency range), with subsequent GABAergic neuronal loss and astrogliosis. Chronic fenfluramine administration completely restored dendritic arbor numbers to normal in scn1lab^mut/mut larvae, whereas similar treatment with the benzodiazepine diazepam attenuated seizures, but was ineffective in restoring neuronal cytoarchitecture. BrdU labeling revealed cell overproliferation in scn1lab^mut/mut larval brains that were rescued by fenfluramine but not diazepam.

Significance Our findings provide novel insights into early mechanisms of DS pathogenesis, describe dynamic cell population changes in the scn1lab^mut/mut brain, and present first-time evidence for potential disease modification by fenfluramine.

Genes / Markers

Figures

Show all Figures

Expression

Phenotype

Fish	Conditions	Stage	Phenotype	Figure
AB	chemical treatment: fenfluramine	Day 6	optic tectum mitotic S phase increased occurrence, abnormal	Fig. 5
scn1lab^{uio202/uio202} (AB)	standard conditions	Protruding-mouth	optic tectum GABAergic neuron decreased branchiness, abnormal	Fig. 4
scn1lab^{uio202/uio202} (AB)	standard conditions	Day 4	swimming increased occurrence, abnormal	Fig. S2
scn1lab^{uio202/uio202} (AB)	standard conditions	Day 5	optic tectum transmission of nerve impulse increased process quality, abnormal	Fig. 1
scn1lab^{uio202/uio202} (AB)	standard conditions	Day 5	pigmentation increased spatial extent of a process, abnormal	Fig. 1
scn1lab^{uio202/uio202} (AB)	standard conditions	Day 5	swim bladder inflation decreased occurrence, abnormal	Fig. 1
scn1lab^{uio202/uio202} (AB)	standard conditions	Day 5	swim bladder uninflated, abnormal	Fig. 1
scn1lab^{uio202/uio202} (AB)	standard conditions	Day 5	trunk bent, abnormal	Fig. 1
scn1lab^{uio202/uio202} (AB)	standard conditions	Day 6	brain elavl3 expression increased amount, abnormal	Fig. 3
scn1lab^{uio202/uio202} (AB)	standard conditions	Day 6	brain gad1b expression increased amount, abnormal	Fig. 3

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
uio201Tg	Tg(nccr.i56i-i56ii-dlx6a:GAL4-2A-GFP)	Transgenic Insertion
uio202		Indel	scn1lab

Human Disease / Model

Human Disease	Fish	Conditions	Evidence
Dravet syndrome	scn1lab^{uio202/uio202} (AB)	standard conditions	TAS

Sequence Targeting Reagents

Target	Reagent	Reagent Type
scn1lab	CRISPR2-scn1lab	CRISPR

Fish

Fish
AB
scn1lab^{uio202/uio202} (AB)

Antibodies

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
GAL4	EFG	GAL4
GFP	EFG	GFP

Mapping

Tiraboschi et al., 2020 ZDB-PUB-200226-7 PMID:32096222

New insights into the early mechanisms of epileptogenesis in a zebrafish model of Dravet syndrome

Tiraboschi et al., 2020

ZDB-PUB-200226-7
PMID:32096222