for identifying mechanisms to enhance rhodopsin folding, and animal models will provide a means for testing these mechanisms in-vivo.

Many other disorders are associated with protein misfolding and ER retention (Schroder and Kaufman 2005). In many cases, ER exit can be promoted by ligands or small molecules (pharmacological chaperones). For example, p-glycoprotein mutants can be rescued by the substrate cyclosporine A, CFTR can be rescued by corr-2b, and a hERG potassium channel mutant can be rescued by channel blockers. P23H rhodopsin provides yet another example. In fact, many retinal dystrophies are caused by defective biosynthesis of proteins; for example, misfolding mutations of peripherin/rds, ABCA4, and ELOVL4 (Grayson and Molday 2005) are associated with retinal dystrophies, and could be rescued by similar mechanisms. Indeed, higher-order assembly of the digenic RP-associated peripherin/rds mutant L185P requires co-expression of its interaction partner rom-1 (Goldberg and Molday 1996). Even where no ligand is known, it may be possible to identify ligands by screening, or based on structural data.

Our understanding of the mechanisms underlying rescue of RD by dark rearing is incomplete. We lack an explanation for the rescuing effects of disruption of signal transduction (observed in mice and suggested in X. laevis). Additionally, the specific role of N-terminal cleavage needs to be addressed. One can envision mechanisms by which cleavage could promote ER exit, as the mutant residue and both N-linked glycosylation sites are removed. ER quality control mechanisms involving retention and surveillance of glycosylated proteins by lectin chaperones are well documented (Schroder and Kaufman 2005). Is eliminating glycosylation or removal of the P23H residue a key event in rescue? Does cleavage represent a cause or consequence of ER exit? What is the protease? Is promoting truncation another approach to RP therapy?

Most importantly, we must identify the mechanisms relevant to human disease. In 1980, a small clinical trial of light deprivation of two RP patients of unreported genotype showed no significant benefits (Berson 1980). A great deal of information has accumulated since these studies were conducted, and it may soon be appropriate to revisit this approach in trials directed at genetic subsets of RP.

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Chapter 59

A Hypoplastic Retinal Lamination in the

Purpurin Knock Down Embryo in Zebrafish

Mikiko Nagashima, Junichi Saito, Kazuhiro Mawatari, Yusuke Mori, Toru Matsukawa, Yoshiki Koriyama, and Satoru Kato

Abstract Recently, we cloned a photoreceptor-specific purpurin cDNA from axotomized goldfish retina. In the present study, we investigate the structure of zebrafish purpurin genomic DNA and its function during retinal development. First, we cloned a 3.7-kbp genomic DNA fragment including 1.4-kbp 5 -flanking region and 2.3-kbp full-length coding region. In the 1.4-kbp 5 -upstream region, there were some cone-rod homeobox (crx) protein binding motifs. The vector of the 1.4-kbp 5 -flanking region combined with the reporter GFP gene showed specific expression of this gene only in the photoreceptors. Although the first appearance time of purpurin mRNA expression was a little bit later (40 hpf) than that of crx (17–24 hpf), the appearance site was identical to the ventral part of the retina. Next, we made purpurin or crx knock down embryos with morpholino antisense oligonucleotides. The both morphants (purpurin and crx) showed similar abnormal phenotypes in the eye development; small size of eyeball and lacking of retinal lamination. Furthermore, co-injection of crx morpholino and purpurin mRNA significantly rescued these abnormalities. These data strongly indicate that purpurin is a key molecule for the cell differentiation during early retinal development in zebrafish under transcriptional crx regulation.

59.1 Introduction

Purpurin is originally discovered as a retina-specific secretory protein in developing chick retina (Schubert and LaCorbiere 1985). In retinal cell culture, purpurin has cell adhesive and survival effects (Schubert et al. 1986). Recently we found that purpurin was a trigger molecule for optic nerve regeneration in adult goldfish retina (Matsukawa et al. 2004). Following optic nerve transection, the level of purpurin mRNA rapidly increased in the photoreceptor cells 2–5 days and then

S. Kato (B)

Department of Molecular Neurobiology, University of Kanazawa, Kanazawa 920-8640, Japan e-mail: satoru@med.kanazawa-u.ac.jp

Medicine and Biology 664, DOI 10.1007/978-1-4419-1399-9_59,C Springer Science+Business Media, LLC 2010