Endoplasmic reticulum quality control in health and disease: roles of kdelr3 and ADTKD-REN mutations in zebrafish

The endoplasmic reticulum quality control (ERQC) system is a process that involves chaperone-mediated folding, vesicular trafficking, ER-associated degradation (ERAD), ER-phagy, and activation of the unfolded protein response (UPR), to maintain the proteostasis within the cell. Alterations due to unfolded protein accumulation, aberrant protein trafficking or chronic activation of the UPR are increasingly recognized as the origin and driver of several pathologies, including neurodegenerative diseases, type 2 diabetes, Wolcott-Rallison Syndrome and cancer. In this thesis, we investigated two complementary processes of ER proteostasis in zebrafish (Danio rerio): the physiological role of the KDEL receptor family member KDELR3, involved in chaperone anterograde retrieval, and the pathological condition cause by ADTKD-REN due to renin mutations that lead to ER accumulation and organelle mistargeting in human patients. Comparisons of protein sequence, structure and phylogeny showed a remarkable evolutionary conservation of zebrafish kdelr3 compared to human KDELR3. Spatial and temporal expression analysis showed that zebrafish kdelr3 is expressed in early embryogenesis and organogenesis, particularly mesenchymal and epithelial tissues. To better characterize the possible in vivo roles of kdelr3 in the ERQC, we generated a zebrafish knock-out line for this gene through CRISPR/Cas9-editing technique. The new kdelr3 depleted line displayed transient posterior trunk and tail-bud defects during embryogenesis, craniofacial cartilage alterations, downregulation of collagen genes, abnormal vasculature patterning and enhanced caudal fin asymmetrical regrowth. At the molecular level, the absence of kdelr3 led to selective alterations of UPR pathways, with a higher susceptibility to tunicamycin and brefeldin A, molecules able to induce ER-stress through accumulation of unfolded protein or protein trafficking disruption. Concomitantly, kdelr3 deficiency was characterized by upregulation of inflammatory markers, aberrant redistribution of neutrophils and increased levels of non-apoptotic cell death and cellular proliferation. To model ER proteostasis failure in a disease context, we generated stable Tol2-based transgenic zebrafish lines expressing wild-type or ADTKD-REN–associated human REN variants (p.L16R, p.L16del, p.E48K, p.L381P) under control of a specific zebrafish ren promoter that selectively marks juxtaglomerular (JG) cells. Indeed, protein structural and sequence analyses confirmed conservation of the catalytic core between zebrafish and human renin, supporting the use of zebrafish humanized lines to closely mimic human patient pathology. Mutant transgenic lines displayed mutation- and dosage-dependent alterations of pronephric proximal tubule morphology, characterized by reduced convolution and abnormal looping. Moreover, the p.L381P variant induced ultrastructural dilation of ER cisternae in renin-expressing cells, consistent with altered protein processing. Despite these morphological abnormalities, early glomerular filtration assessed by dextran clearance and JG cells number remained preserved. Pharmacological RAAS blockade with captopril enhanced morphological sensitivity in transgenic backgrounds, indicating increased susceptibility to systemic stress. Together, these complementary models show that perturbations at different levels of the ERQC cascade– disruption of chaperone retrieval by kdelr3 loss or expression of an unfolded secretory cargo in ADTKDREN– are translated into specific developmental, structural and stress responsive phenotypes in vivo.