Fig 1.
Localization of vector-expressed CLRN1-Venus following subretinal delivery.
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.
Fig 2.
Localization of vector-expressed CLRN1 following intravitreal delivery.
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.
Fig 3.
Evaluation of retinal function and CLRN1-HA expression following subretinal delivery.
A. Bar graphs show the average maximum ERG b-wave amplitudes in scotopic, dark-adapted conditions of untreated wild-type control eyes, compared to AAV-treated eyes that received decreasing doses of AAV-CLRN1-HA vector (2 months post-injection). Maximum b-wave amplitudes in AAV-treated eyes were dose dependent, and were significantly lower than untreated controls at 1010, 109, and 108 vg (*, p<0.05, **, p<0.001). B. Localization of CLRN1-HA protein following subretinal delivery of the AAV2quad smCBA vector. CLRN1-HA fluorescence (green) was detected by immunohistochemistry in photoreceptor IS region, ONL and OPL, and was absent from the outer segments (labelled with a rhodopsin antibody, red). The eyes received diluted vector (108 vg). Scale bar: 20 μm.
Fig 4.
Immunofluorescent and quantification of arrestin-1 (red) in light-adapted Clrn1 KO and WT retinal sections.
A. Bottom images: Immunofluorescence distribution of arrestin-1 after light exposure translocation. Top plots: corresponding profiles of fluorescent signal intensity scanned across the photoreceptor layer. Note the increased arrestin-1 signal in the OPL of Clrn1 KO mice (left panel) compared to WT OPL (right panel). Nuclei are stained blue with DAPI. B. Bar graph showing the relative signal intensities of arrestin-1 and α-transducin in the OPL of Clrn1 KO and WT mice, expressed as a percentage of the total signal intensity in the photoreceptor layer (*p<0.05, n = 22 for arrestin, and n = 11 for transducin).
Fig 5.
Representative electron microscopic images showing photoreceptor ribbon synapse ultrastructure in WT (left panel) and Clrn1 KO (right panel) mice.
Arrows point to synaptic ribbons and asterisks indicate horizontal cells; M, mitochondria. Scale bar, 500 nm.