Orphan cytochrome P450 20a1 CRISPR/Cas9 mutants and neurobehavioral phenotypes in zebrafish

Nadja R Brun,John J Stegeman,Matthew C Salanga,Jared V Goldstone,David C Lamb,Francisco X Mora-Zamorano

doi:10.1038/s41598-021-03068-3

Nadja R Brun, John J Stegeman + Show 4 more

Open Access

https://doi.org/10.1038/s41598-021-03068-3

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Abstract

Orphan cytochrome P450 (CYP) enzymes are those for which biological substrates and function(s) are unknown. Cytochrome P450 20A1 (CYP20A1) is the last human orphan P450 enzyme, and orthologs occur as single genes in every vertebrate genome sequenced to date. The occurrence of high levels of CYP20A1 transcripts in human substantia nigra and hippocampus and abundant maternal transcripts in zebrafish eggs strongly suggest roles both in the brain and during early embryonic development. Patients with chromosome 2 microdeletions including CYP20A1 show hyperactivity and bouts of anxiety, among other conditions. Here, we created zebrafish cyp20a1 mutants using CRISPR/Cas9, providing vertebrate models with which to study the role of CYP20A1 in behavior and other neurodevelopmental functions. The homozygous cyp20a1 null mutants exhibited significant behavioral differences from wild-type zebrafish, both in larval and adult animals. Larval cyp20a1-/- mutants exhibited a strong increase in light-simulated movement (i.e., light–dark assay), which was interpreted as hyperactivity. Further, the larvae exhibited mild hypoactivity during the adaptation period of the optomotor assays. Adult cyp20a1 null fish showed a pronounced delay in adapting to new environments, which is consistent with an anxiety paradigm. Taken together with our earlier morpholino cyp20a1 knockdown results, the results described herein suggest that the orphan CYP20A1 has a neurophysiological role.

Highlights

Cytochromes P450 (CYP; P450), a superfamily of enzymes found in every branch of life, catalyze a vast array of oxidation reactions, as well as the reduction and rearrangement of endogenous and exogenous c ompounds[1]
Due to the unavailability of specific antibodies, we were unable to confirm that the Cyp20a1 protein was completely absent from these lines, without the heme-binding domain any P450 protein would be inactive
Our specific focus on behavior was prompted by the possible neurological implications of Cytochrome P450 20A1 (CYP20A1) RNA expression levels in the hippocampus and substantia nigra in the human brain[5], early larval zebrafish[11], and in the developing mouse embryo[13]

Summary

Introduction

Cytochromes P450 (CYP; P450), a superfamily of enzymes found in every branch of life, catalyze a vast array of oxidation reactions, as well as the reduction and rearrangement of endogenous and exogenous c ompounds[1]. Expression is especially abundant in the hippocampus and substantia nigra regions of the brain[5], regions that are prominently associated with learning and memory, and which are involved in neurodegenerative diseases including hyperactivity disorders (e.g., ADHD), panic disorders, social anxiety, and bipolar disorders. Such disorders affect > 10% of the Scientific Reports | (2021) 11:23892. These findings suggest the participation of CYP20A1 in vertebrate development, as well as its potential involvement in endocrine and neuronal processes

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