Phenotypic analysis of catastrophic childhood epilepsy genes

Abstract

Genetic engineering techniques have contributed to the now widespread use of zebrafish to investigate gene function, but zebrafish-based human disease studies, and particularly for neurological disorders, are limited. Here we used CRISPR-Cas9 to generate 40 single-gene mutant zebrafish lines representing catastrophic childhood epilepsies. We evaluated larval phenotypes using electrophysiological, behavioral, neuro-anatomical, survival and pharmacological assays. Local field potential recordings (LFP) were used to screen ∼3300 larvae. Phenotypes with unprovoked electrographic seizure activity (i.e., epilepsy) were identified in zebrafish lines for 8 genes; ARX, EEF1A, GABRB3, GRIN1, PNPO, SCN1A, STRADA and STXBP1. We also created an open-source database containing sequencing information, survival curves, behavioral profiles and representative electrophysiology data. We offer all zebrafish lines as a resource to the neuroscience community and envision them as a starting point for further functional analysis and/or identification of new therapies.

Mosaic and non-mosaic protocadherin 19 mutation leads to neuronal hyperexcitability in zebrafish

“…We conclude that the nature of the pathology, and its resultant hyperexcitability, generated by mosaic and non-mosaic pcdh19 LOF mutations in zebrafish larvae requires a sensitive detection method (tectal LFP recordings) to consistently identify evidence of hyperexcitability in all lines. Indeed, the lack of seizure-like events using behavioral assays in the setting of a hyperexcitable neuronal network is not an uncommon phenomenon, as recently demonstrated in a large zebrafish epilepsy screen (Griffin et al, 2021).…”

“…The lack of spontaneous behavioral seizures in the mosaic or non-mosaic Pcdh19 LOF fish, despite abnormal electrophysiological findings, does not diminish the significance of the positive findings observed and is consistent with several previously reported models of genes responsible for human epilepsy. A recent large-scale epilepsy screen reported a low prevalence of behavioral seizures in a set of 40 zebrafish lines with variants in catastrophic childhood epilepsy genes as well as surprisingly low prevalence of epileptiform abnormalities in electrophysiological recordings (Griffin et al, 2021). Due to technical limitations, we could not test for the presence of segregated and abnormally clustered cell populations in the brain in the mosaic mutants.…”

“…Furthermore, zebrafish larvae are not sexually dimorphic and thus, this disease model does not allow for the evaluation of sex-dependent differences. Given that the most consistent phenotypic abnormality relied on electrophysiological experiments, we advocate for including such experiments in drug screens for the identification of novel therapeutic compounds to target pcdh19 dysfunction, which are readily feasible in zebrafish larvae (Griffin et al, 2021). Evaluation of some of these aspects in mouse models may allow additional understanding of the role of PCDH19 in brain development and epilepsy (Hoshina et al, 2021; Pederick et al, 2018).…”

“…This distinction into small and large amplitude event can be directly obtained from a histogram of all detected spike amplitudes in which we observe two distinct features: a narrow peak with low signal amplitude (referred to as the SAE) and a second broad peak with large signal amplitude (referred to as the LAE). The threshold discriminating between small and large amplitude events is 0.08-0.12mV depending on the noise (after completion of the manuscript, the authors became aware of a related classification (Griffin et al, 2021)). A final stage of the software groups the detected large amplitude events into bursts in case the following event occurs within a 1 second time window.…”

Panx1 channels promote both anti- and pro-seizure-like activities in the zebrafish via p2rx7 receptors and ATP signaling

“…Evolutionary conservation implies that epilepsy can be investigated in zebrafish 21 – 24 . Models of Dravet Syndrome and 40 single-gene mutant zebrafish lines representing childhood epilepsies exemplify the generation of models by mutagenic screens or gene-targeting 25 , 26 . Further, zebrafish-centric drug discovery has brought antiepileptic drugs to clinical studies and the bedside 27 – 29 .…”