CLRN1 Is Nonessential in the Mouse Retina but Is Required for Cochlear Hair Cell Development

Abstract

Mutations in the CLRN1 gene cause Usher syndrome type 3 (USH3), a human disease characterized by progressive blindness and deafness. Clarin 1, the protein product of CLRN1, is a four-transmembrane protein predicted to be associated with ribbon synapses of photoreceptors and cochlear hair cells, and recently demonstrated to be associated with the cytoskeleton. To study Clrn1, we created a Clrn1 knockout (KO) mouse and characterized the histological and functional consequences of Clrn1 deletion in the retina and cochlea. Clrn1 KO mice do not develop a retinal degeneration phenotype, but exhibit progressive loss of sensory hair cells in the cochlea and deterioration of the organ of Corti by 4 months. Hair cell stereocilia in KO animals were longer and disorganized by 4 months, and some Clrn1 KO mice exhibited circling behavior by 5–6 months of age. Clrn1 mRNA expression was localized in the retina using in situ hybridization (ISH), laser capture microdissection (LCM), and RT–PCR. Retinal Clrn1 transcripts were found throughout development and adulthood by RT–PCR, although expression peaked at P7 and declined to undetectable levels in adult retina by ISH. LCM localized Clrn1 transcripts to the retinas inner nuclear layer, and WT levels of retinal Clrn1 expression were observed in photoreceptor-less retinas. Examination of Clrn1 KO mice suggests that CLRN1 is unnecessary in the murine retina but essential for normal cochlear development and function. This may reflect a redundancy in the mouse retina not present in human retina. In contrast to mouse KO models of USH1 and USH2, our data indicate that Clrn1 expression in the retina is restricted to the Müller glia. This is a novel finding, as most retinal degeneration associated proteins are expressed in photoreceptors, not in glia. If CLRN1 expression in humans is comparable to the expression pattern observed in mice, this is the first report of an inner retinal protein that, when mutated, causes retinal degeneration.

Clarin-1 gene transfer rescues auditory synaptopathy in model of Usher syndrome

“…Indeed, almost all USH3A patients develop normal speech, and, although an elevation of hearing thresholds is diagnosed in most patients before the age of 10 years, some patients display only mild to moderate hearing threshold elevation at the time of detection, at an age of 30-40 years (42,43). The discrepancy in hair cell phenotype between humans (42, 43) and Clrn1 total knockout mice (7,11) suggests that compensatory mechanisms, possibly involving genes encoding other members of the clarin family (CLRN2 and CLRN3), operate much more efficiently, particularly during embryogenesis and early postnatal development, in humans than in mice. USH1 and USH2 patients present congenital hearing loss.…”

“…Studies of mutant mice lacking exon 1 of Clrn1 (7,8) and knockdown approaches in zebrafish (9,10) have revealed structural and functional defects of F-actin-enriched stereocilia in hair cells. Additional roles for clarin-1 have also been sought, as mouse Clrn1 transcripts have been detected in both hair cells and the auditory primary neurons, from embryonic day 16 (E16) to adult stages (1,11). Clarin-1 has also been detected at the apical and basal synaptic poles of hair cells and photoreceptor cells in the zebrafish (9,12).…”

Alternative splice variants of the USH3A gene Clarin 1 (CLRN1)

“…6,8 Computer analyses predict a four-transmembrane domain tetraspanin-like secondary/tertiary structure for the CLRN1 protein. 7,8 When transfected into and expressed in cultured baby hamster kidney (BHK) cells under CMV promoter, the wild-type full-length CLRN1 protein is trafficked to the plasma membrane, [9][10][11] and endogenous CLRN1 protein in UB/OC-1 immortal auditory hair cell line is trafficked to the post-trans Golgi vesicles, 12 whereas the mutated forms are retained in the endoplasmic reticulum (ER). 9,11 CLRN1 has also been reported to be involved with F-actin organization 11 and synaptic maturation.…”

“…12 The localization has been characterized in the mouse cochlea, where Clrn1 is expressed in hair cells and spiral ganglion cells, 7,12 and the absence of Clrn1 in null mice leads to disorganization of the hair cell stereocilia. 10,13 In the murine retina, Clrn1 expression was found in Müller glia, 10 whereas immunohistochemical analyses suggest protein localization in the photoreceptor connecting cilia, inner segments, and ribbon synapses. 12 The pathophysiology of USH3 remains unexplained.…”

Usher Syndrome: Genes, Proteins, Models, Molecular Mechanisms, and Therapies

“…Clarin-1 shows a sequence homologous to stargazin, an auxiliary subunit of ion channels in the synapse (Osten and Stern-Bach, 2006;Tomita et al, 2007). Presently, several research groups are intensively focusing on understanding this gene (Aarnisalo et al, 2007;Geller et al, 2009;Geng et al, 2009;Tian et al, 2009;Zallocchi et al, 2009). However, the biological function and cellular expression of clarin-1 still remain elusive.…”