The Use of Dominant Negative Strategy in Zebrafish to Study CDKL5 Deficiency Disorder (2020)

Varela, T., Martins, G., Varela, D., Vitorino, M., Conceição, N., & Cancela, M.L. (2020, November 20). The use of dominant negative strategy in zebrafish to study CDKL5 deficiency disorder [Poster presentation].

Annual Meeting of the Portuguese Society of Human Genetics, Lisbon, Portugal.


CDKL5 deficiency disorder (CDD) is an X-linked neurodevelopmental condition caused by mutations in cyclin-dependent kinase-like 5 (CDKL5) gene resulting in the loss-of-function of its encoded protein - a serine/threonine kinase- essential for normal brain development and function. Patients suffering from this rare disorder present early-onset seizures, which begin in the first months of life and a regression in neurological and motor development. Although a genetic cause for this condition was identified, the association between the type/location of mutations and patient’s phenotype and the mechanisms responsible for its onset remain unclear. Mouse models developed to mimic CDD are unable to develop seizures, a central feature of this condition in humans. Therefore, the use of other models such as zebrafish represent an alternative tool to study this syndrome. In this work we investigated the effect of mutant CDKL5 using a dominant negative approach in zebrafish thus expecting to contribute to unveil the molecular pathways involved in the onset of CDD. For that, we produced CDKL5 RNA harboring the mutation c.1708G>T found in a CDD patient presenting recurrent seizures, gross motor hypotonia and poor eye contact, and injected it into zebrafish embryos to look for possible phenotypes analogous to those observed in the human clinical condition. The resulting mutant protein (p.E570X) is truncated and lacks the Cterminus motifs responsible for its nuclear localization and exportation. Bioinformatic analysis confirmed the high degree of conservation between human and zebrafish CDKL5 gene structure and protein sequence. Motor behavior was evaluated using the Zantiks equipment and a seizure behavior was observed in wildtype 5dpf larvae exposed to PTZ, a seizure-inducing drug. We are currently performing this behavioral analysis in larvae expressing the CDKL5 dominant negative form. Altogether, our results support the use of zebrafish as a valid alternative model to study CDD.