Introduction
While the rd12 mutation results in a slow retinal degeneration, the morphological changes have an early onset. At 3 weeks, lipid-like droplets are found in the RPE (Pang et al., 2005) and the PNA-lectin positive cone count is reduced to 55 % (Li et al., 2011), with cone opsin lost prior to cone cell death. By 3 months, cone counts are further reduced by 16 % and S-opsin expression is lost (Li et al., 2011).
By 7 months the POS are distinctively shorter and the ONL is reduced to 6-7 cells thick (Pang et al., 2005, Wright et al., 2014). By 2 years the POS are almost absent and the ONL is reduced to 3-4 layers (Pang et al., 2005).
Methods
Animals
Two breeding pairs of rd12 mice (B6(A)-rpe65rd12/J) were obtained from the Jackson Laboratory, US. The progenies of these mice were used for all subsequent experiment. C57 mice were used as control mice (WT).
Fixation of Tissue
3-month-old adult Rd12 and WT animals were fixated by perfusion with 4%
paraformaldehyde as described in paragraph 2.3.1.
Sectioning and immunostaining
As described in paragraph 2.3.2, eyes were cryopreserving, sectioned and immunolabelled. Anti-RPE65, raised in rabbit (diluted 1:200), provided by Dr T.
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Michael Redmond, National Eye Institute, Bethesda, Maryland, US (Ma et al., 2001, Pang et al., 2005), anti-RGR raised in rabbit (1:800 dilution, kind gift from Henry Fong, UCLA), rhodopsin raised in rabbit (1:1000, Abcam) and anti-M/L opsin raised in rabbit (1:15000, Milipore) were used as a primary antibody with a secondary antibody raised in donkey against rabbit IgG tagged with Alexa Fluor 568 (ab10042; Abcam) diluted at 1:2000. Anti-M/L opsin was double labelled with anti-S-opsin raised in goat (SC-14363, Santa Cruz), diluted to 1:200 with secondary antibody anti-goat raised in donkey tagged with FITC diluted to 1:200 (Jackson ImmunoResearch, West Grove, PA, USA).
Imaging sections
Labelled sections were imaged in a z-stack using the Zeiss 700 microscope.
These z-stacks were then flattened to extract a maximum intensity projection using zen software.
Results
Validation of the model
To validate the absence of RPE65 in our rd12 mice, retinal sections from rd12 and WT mice were labelled for RPE65. In the WT retina, RPE65 labelling is specific to the RPE layer. No RPE65 labelling is seen in the rd12 retina (Figure 4.1). RGR was also labelled, to investigate if expression of other isomerase enzymes in the visual cycle were disrupted due to the rd12 mutation. RGR is an opsin expressed in the RPE and Müller glia (Jiang et al., 1993). In the dark, RGR contains all-trans-retinal (Hao and Fong, 1999) and controls the visual cycle
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production of 11-cis retinol until illumination (Radu et al., 2008). RGR labelling is seen in the RPE of both WT and rd12 mice (Figure 4.2).
Figure 4.1 Absence of RPE65 labelling in the rd12 retina.
This figure displays RPE65 labelling (red) and transmitted light in WT and rd12 retina. RPE65 (red) is seen in the RPE in WT retina. No RPE65 labelling is seen in the rd12 retina. Scale bar indicates 50 µm.
171 Figure 4.2 RGR labelling in rd12 retina.
RGR labelling (red) is seen in the RPE in both WT and rd12 retina. Scale bar indicates 50 µm.
172 Loss of rods and cones in the rd12 retina
At 3 months, rhodopsin levels are still comparable to WT levels (Figure 4.3).
Cones are more notably affected by the rd12 mutation. No M/L-opsin was detected (Figure 4.4). S-opsin can still be seen, although, the number of cones expressing S-opsin was notably reduced and S-opsin is seen mislocated to the PIS, ONL and outer plexiform layer (OPL). S-opsin expression is stronger in the ventral retina, and, it is known that S-cones are more abundant in the ventral retina (Ortin-Martinez et al., 2014).
Figure 4.3 Rhodopsin labelling in rd12 retina.
Rhodopsin labelling (red) is seen in the POS in both 3 month WT and rd12 retina.
Scale bar represents 20 µm.
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Figure 4.4 Cone opsin labelling in 3-month rd12 retina.
Top images display nuclei staining (blue) with M/ L- (red) and S-opsin (green) labelling in 3-month WT and rd12 retinas. M/L-opsin labelling is not detected in 3-month rd12 retina. The number of cones expressing opsin is reduced and S-opsin is mislocated to the PIS and the outer plexiform layer. Scale bar indicates 50 µm. Sagittal section of rd12 retina shows loss of M/L-opsin and S-opsin mislocation.
Discussion
The rd12 mouse was validated by immunohistochemistry. No RPE65 was identified in the rd12 RPE. This antibody was kindly provided by Dr T. Michael Redmond, National Eye Institute, Bethesda, Maryland, US (Ma et al., 2001) and was also used by Pang and colleagues to validate the rd12 mouse (Pang et al., 2005).
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RGR was also labelled to investigate other visual cycle protein levels and was seen in the RPE layer in rd12 mice. Since this experiment, Zheng and colleagues have found 7 visual cycle-associated proteins which have altered proteomics, including: CRALBP, retinol-binding protein 1 (RBP-1), IRBP, RDH-2, RDH-5, LRAT, and ezrin-radixin-moesin-binding phosphoprotein 50 (EBP-50) (Zheng et al., 2015).
The loss of the visual cycle has a destructive effect on outer-retinal photoreceptors. At 3 months, M/L-opsin is lost. Li and colleagues showed that the number of cones is significantly reduced by P21 in the rd12 retina (Li et al., 2011).
In WT rat development rpe65 reaches its peak and steady state expression around P10-P12, which coincides with peak opsin expression in rat and mouse (Bowes et al., 1988, Manes et al., 1998, Hamel et al., 1993). Li and colleagues showed that P14 PNA-positive cone counts are similar to WT, but, after this, cone loss is rapid. A slower degeneration of S-cones in the rd12 retina was observed.
However, the opsin was mislocated to the PIS, ONL and OPL. This mislocation of cone opsins is also seen in the rpe65-/- mouse and can be appropriately localised with the treatment of chromophore (Rohrer et al., 2005). Chromophore has been shown to be required for cone opsin trafficking to outer-segments (Bandyopadhyay et al., 2013). This slower loss of S-opsin, seen in these results, is contradicting results reported by Li and colleagues, who found no S-opsin at 2 months (Li et al., 2011), however, their study looked in flatmounts, which may not detect S-opsin that has been mislocated to the ONL and OPL. Also, a different antibody was used, which may not detect a mis-folded protein. Pang and colleagues described how rpe65 gene therapy at P35 protected M/L-cone loss but S-cones could only be saved with earlier treatment at P14 (Pang et al., 2010).
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However, treatment with chromophore was more successful in correcting the localisation of M/L-opsin than S-opsin (Rohrer et al., 2005).
Rods on the other hand, are lost at a slower rate. Rhodopsin staining intensity in the POS appeared similar in rd12 compared to WT. This is supported by results showing no significant alterations in rhodopsin mRNA levels in the rpe65-/- up to 8 weeks (Znoiko et al., 2005). Unlike cones, chromophore is not required for rhodopsin trafficking. While rhodopsin protein levels are strong, rhodopsin spectral absorbance is not detectable (Pang et al., 2005), presumable due to the absence of chromophore.