Effects of Morpholino-mediated knockdown of the Angiotensin-converting enzyme 2 (ace-2) on dopamine Neurogenesis and the Hypothalamic Pituitary-adrenal (HPA) Axis in Larval Zebrafish (Danio rerio)

Abstract

While the presence of ACE2 and its expression in brain structures along with its recent link with the novel SARS-CoV-2, their impact on the development of the brain and on the activity of the HPA axis in larval zebrafish is not known. Therefore, the primary objective of this study was to determine how silencing the ace2 gene affects DA neurogenesis and the endocrine stress response in larval zebrafish (Danio rerio). The DA neurogenesis was assessed by nurr1 expression, while endocrine stress response by quantification of Corticotrophin-Releasing-Hormone (crh) and Glucocorticoid receptor (gr) gene expression by RT-PCR. In this investigation, an ACE2 AUG morpholino oligonucleotide was introduced by electroporation into the 96hpf zebrafish in order to silence the expression of the ace2 gene using antisense technology.   Electroporation of the embryo in 1X PBS on 1.5% Agarose gel with ACE2 AUG morpholino oligonucleotide was done to transfer it into the embryo. After successful transfer, no visible morphological changes were observed. A significant reduction in ace2 gene expression confirms the successful oligonucleotide transfer.  nurr1 expression was significantly reduced as ace2 expression was silenced but no significant effect on gr and crh expression. Interestingly a significant increase in locomotor activity was seen after the silencing of ace2 expression. Overall, these results facilitate the prediction of possible neurological manifestations of brain ACE2 dysregulation during and after COVID-19 infection.