Knock-Down of Cathepsin D Affects the Retinal Pigment Epithelium, Impairs Swim-Bladder Ontogenesis and Causes Premature Death in Zebrafish

Abstract

The lysosomal aspartic protease Cathepsin D (CD) is ubiquitously expressed in eukaryotic organisms. CD activity is essential to accomplish the acid-dependent extensive or partial proteolysis of protein substrates within endosomal and lysosomal compartments therein delivered via endocytosis, phagocytosis or autophagocytosis. CD may also act at physiological pH on small-size substrates in the cytosol and in the extracellular milieu. Mouse and fruit fly CD knock-out models have highlighted the multi-pathophysiological roles of CD in tissue homeostasis and organ development. Here we report the first phenotypic description of the lack of CD expression during zebrafish (Danio rerio) development obtained by morpholino-mediated knock-down of CD mRNA. Since the un-fertilized eggs were shown to be supplied with maternal CD mRNA, only a morpholino targeting a sequence containing the starting ATG codon was effective. The main phenotypic alterations produced by CD knock-down in zebrafish were: 1. abnormal development of the eye and of retinal pigment epithelium; 2. absence of the swim-bladder; 3. skin hyper-pigmentation; 4. reduced growth and premature death. Rescue experiments confirmed the involvement of CD in the developmental processes leading to these phenotypic alterations. Our findings add to the list of CD functions in organ development and patho-physiology in vertebrates.

Developing Therapeutic Antibodies for Neurodegenerative Disease

“…Nevertheless, recent advances have been made in the clinic that show promise for small molecule inhibition of BACE1 [47], yet most of these approaches still lack the specificity that an antibody approach will offer. In particular, there are clear risks associated with cathepsin-D inhibition [48][49][50], and the risks associated with BACE2 inhibition are not yet evident. Recent genetic studies have also raised the concern that complete inhibition of BACE1 itself may be deleterious [51][52][53].…”

Tumor Angiogenesis: Fishing for Screening Models

“…Zebrafish have also proven to be a helpful system in studying miRNA functions during developmental processes [44]. Identification and characterization of miRNAs in zebrafish have provided important information regarding the conservation of miRNA genes in vertebrates.…”

“…The role of a miRNA in larvae and adult animals can be investigated by using Cre-loxP or TALEN technologies that allow stable and conditional miRNA down or overexpression in a tissue-specific manner [48]. Several vascular specific miRNAs have been identified and studied in zebrafish inter alia miR-24, miR-92, miR-126, and miR-221 [44,48]. Some of them act as anti-angiogenic factors.…”

Knockdown of cathepsin D in zebrafish fertilized eggs determines congenital myopathy

“…The pathophysiological role of CD in organ development and homoeostasis can be inferred from the phenotype of knockdown and knockout animal models, as described in mouse [1], fruitfly [2] and zebrafish [3]. One main function of CD is to degrade long-lived proteins transferred into the lysosomes via endocytosis, phagocytosis and autophagy [4,5].…”

“…In our previous work, we found that zebrafish zygotes lacking CD develop to larvae that present with several phenotypic alterations, including failure of yolk absorption, reduced body length, microphthalmia and defective development of the digestive tract, the swim bladder and the retina-pigmented epithelium [3]. In this work, we focused our attention on the development of the somitic musculature in CD-knockdown zebrafish.…”