MKS and NPHP modules cooperate to establish basal body/transition zone membrane associations and ciliary gate function during ciliogenesis

Eight proteins, defects in which are associated with Meckel-Gruber syndrome and nephronophthisis ciliopathies, work together as two functional modules at the transition zone to establish basal body/transition zone connections with the membrane and barricade entry of non-ciliary components into this organelle

AAV-Mediated Clarin-1 Expression in the Mouse Retina: Implications for USH3A Gene Therapy

<div><p>Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by mutations in clarin-1 <i>(CLRN1)</i> gene, leading to progressive retinal degeneration and sensorineural deafness. Efforts to develop therapies for preventing photoreceptor cell loss are hampered by the lack of a retinal phenotype in the existing USH3 mouse models and by conflicting reports regarding the endogenous retinal localization of clarin-1, a transmembrane protein of unknown function. In this study, we used an AAV-based approach to express CLRN1 in the mouse retina in order to determine the pattern of its subcellular localization in different cell types. We found that all major classes of retinal cells express AAV-delivered CLRN1 driven by the ubiquitous, constitutive small chicken β-actin promoter, which has important implications for the design of future USH3 gene therapy studies. Within photoreceptor cells, AAV-expressed <i>CLRN1</i> is mainly localized at the inner segment region and outer plexiform layer, similar to the endogenous expression of other usher proteins. Subretinal delivery using a full strength viral titer led to significant loss of retinal function as evidenced by ERG analysis, suggesting that there is a critical limit for CLRN1 expression in photoreceptor cells. Taken together, these results suggest that CLRN1 expression is potentially supported by a variety of retinal cells, and the right combination of AAV vector dose, promoter, and delivery method needs to be selected to develop safe therapies for USH3 disorder.</p></div

Localization of vector-expressed CLRN1 following intravitreal delivery.

<p>A. Representative image of retinal cross-section showing CLRN1-Venus fusion fluorescence. Strong expression is seen at the OPL, the stratified dendrites within the IPL, and inner retinal neurons. Scale bar: 30 μm. B. Higher magnification view (40X) of CLRN1-Venus expression showing intense fluorescence at the Müller cells and their apical processes at the outer limiting membrane (arrow). C. Low magnification (10X) image showing the localization of CLRN1-Venus expression along the nerve fiber layer and optic nerve extension of ganglion cell axons. D. Localization of vector-expressed CLRN1-HA protein by immunostaining with an anti-HA antibody (40X magnification). Note the expression in numerous Müller cells with fine processes extending between photoreceptors inner segments. An isolated patch of RPE cells with strong labeling of the apical processes is also present (arrow). Abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; IPL, inner plexiform layer; NFL, nerve fiber layer.</p

Localization of vector-expressed CLRN1-Venus following subretinal delivery.

<p>A. CLRN1-Venus fusion fluorescence was detected on the apical side of RPE cell membranes (arrows), and in photoreceptor IS, ONL and OPL. Scale bar: 20 μm. Panel B shows a continuous area of intense apical RPE CLRN1-Venus (arrow). C. Higher magnification image showing strong CLRN1-Venus fluorescence in specific regions within photoreceptor cells: IS, cell body membrane and OPL. Nuclei are stained blue with DAPI. Scale bar: 30 μm. Abbreviations: RPE, Retinal Pigment Epithelium; IS, inner segment; OS, outer segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer.</p