Zebrafish Models to Understand CDKL5 Deficiency and the Role of Mosaicism
Abstract number :
3.152
Submission category :
3. Neurophysiology / 3F. Animal Studies
Year :
2023
Submission ID :
1131
Source :
www.aesnet.org
Presentation date :
12/4/2023 12:00:00 AM
Published date :
Authors :
First Author: Cristina Baker, B.Sc. – Boston Childrens Hospital
Presenting Author: Christopher McGraw, MD, PhD – Mass General Hospital, Boston Childrens, HMS
Guoqi Zhang, MD – Neurology – Boston Childrens Hospital; Annapurna Poduri, MD, MPH – Neurology – Boston Childrens Hospital; Christopher McGraw, MD, PhD – Neurology – Massachusetts General Hospital
Rationale: CDKL5 Deficiency Disorder (CDD) is a rare X-linked developmental epileptic encephalopathy (DEE) due to loss-of-function (LOF) mutations in the gene CDKL5. The hallmarks of CDD include early onset severe seizures, visual dysfunction, among other features. Zebrafish is an established model of genetic epilepsy and visual dysfunction. Although initial reports suggest that cdkl5 LOF in zebrafish does not cause prominent seizure phenotypes, questions remain regarding the optimal strategy for modeling cdkl5 deficiency in zebrafish (which lack an X chromosome), and visual function has not yet been evaluated in these lines. Here we propose to evaluate three different zebrafish models of cdkl5 deficiency, including a novel mosaic conditional by inversion (COIN) knock-in, to determine whether phenotypic severity is affected by mosaicism and choice of allele. Animals are phenotyped across two CDD-relevant domains, including seizure-related activity and visual dysfunction.
Methods: Fish. The cdkl5 sa21938 fish line (harboring a nonsense variant in exon 11) was obtained from ZIRC. A cdkl5 knock-in allele which causes early truncation in exon 4 without triggering nonsense mediated decay was generated by the GeneWeld technique. To achieve somatic recombination resembling X-chromosome inactivation, we designed a novel knock-in mosaic COIN cassette targeting exon 4 (“mCOIN KI”), which integrates without altering cdkl5 expression. Upon cre activity, recombination results either in inversion (leading to truncated mRNA and protein) or deletion (causing no effect on cdkl5 mRNA or protein). Fish are crossed by hsp:cre fish for recombination. Phenotyping. Fish are evaluated for seizure-like activity by tectal LFP recordings, and by calcium fluorescence imaging using a fluorescent plate reader (FDSS7000EX) and time-lapse spinning disc confocal microscopy (IXM). Visual function is assessed using the visual motor response (VMR) assay, visual evoked potentials (VEP), and optokinetic response (OKR) assay.
Results: The cdkl5 sa21938 line shows no differences in spontaneous epileptiform abnormalities by tectal LFP. VMR shows subtly reduced activity during last 30 sec of dark phase and first 30 sec of light phase in HOM. Preliminary data also suggest reductions in VEP, and increased rate of OKR in HET/HOM fish. For cdkl5 mCOIN KI fish, we established one founder (N=1 out of 3 F0 fish with germline transmission). Following heat-shock, PCR and Sanger sequencing confirms in vivo evidence of deletion and inversion of the inserted construct. Establishment of the cdkl5 KI line and additional characterizations using calcium fluorescence are still pending.
Conclusions: The cdkl5 sa21938 zebrafish line lacks prominent epileptiform abnormalities but may have visual abnormalities that deserve further investigation. Pending investigations in the cdkl5 KI and mosaic COIN KI fish will address whether genetic compensation or mosaicism may modulate CDD-related phenotypes in zebrafish.
Funding: This work was supported by NIH NINDS K08NS118107, and Loulou Foundation Program of Excellence Pilot Award.
Neurophysiology