Evaluation of Rpe65 mutant mice as models for Leber congenital amaurosis, type 2, an early onset blindness Public
Wright, Charles Baker (2013)
Published
Abstract
The initial steps in vision start with light detection by visual pigment located in the neural retina and recycling of the visual pigment in the adjacent retinal pigment epithelium (RPE). RPE65 is a critical enzyme in the recycling of 11-cis-retinal, a cofactor that is required for light-sensitivity in the retina. When RPE65 is mutated, it causes a severe childhood-onset blindness called Leber congenital amaurosis, type 2 (LCA2). The Rpe65 gene knockout (KO) mouse was developed for the study and treatment of LCA2 and resembles the human disease in many important ways, but the KO mouse has a large Rpe65 gene deletion and such mutations are rarely found in humans. New mouse models, tvrm148 and rd12, contain point mutations in the Rpe65 gene that are similar to, or identical to, mutations found in humans. I hypothesized that the tvrm148 or rd12 point mutations would result in visual phenotypes different from the KO. I found that the tvrm148 mutation in Rpe65 caused a loss in visual function that was virtually identical to the KO because of low levels of RPE65 protein that had no enzymatic activity. In contrast, the rd12 mutation caused a much faster loss of visual function compared to tvrm148 or KO mice and inherited in a semidominant fashion, unlike the KO and many typical LCA2 patients. These mice produced no detectable RPE65 protein and no RPE65 enzymatic activity. The rd12 mice accumulated mutant Rpe65 mRNA that inefficiently associated with ribosomes for translation, and I conclude this contributes to the unusually aggressive form of blindness. These findings supported my hypothesis that point mutations could produce visual defects that differed from the KO mutation. These findings are important because treatments being developed for the disease are based on work in the KO mouse, and my work suggests that these treatments may not be as effective for all human mutations. Furthermore, because the rd12 mutation is found in humans and because my findings showed that it produced a more aggressive blindness, I recommend these patients need to be monitored for an unusually aggressive form of LCA2 that might be more difficult to treat.
Table of Contents
Table of Contents
Chapter 1--Introduction........... 1
1.1--Purpose and central hypothesis........... 3
1.2--The visual system: anatomy, biochemistry, and visual assessment........... 8
1.2.1--Anatomy of the eye........... 8
1.2.2--Biochemistry........... 121.2.3--Visual assessment techniques........... 17
1.3--History of the visual cycle........... 21
1.4--RPE65: structure, function, and mechanism........... 26
1.5--RPE65 and its association with Leber congenital amaurosis (LCA)........... 34
1.6--Mouse lines with Rpe65 gene mutations........... 41
1.6.1--Rpe65KO (KO) mouse........... 41 1.6.2--Rpe65rd12 (rd12) mouse (R44X)........... 44 1.6.3--Rpe65R91W (R91W) mouse........... 471.6.4--Rpe65tvrm148 (tvrm148) mouse (F229S)........... 52
1.6.5--RPE65 polymorphism in C57BL/6 lines........... 53
1.6.6--Comparison of the KO mouse with the diet-induced vitamin A-deficient (VAD) mouse........... 54
1.7--Phenotypic rescue of RPE65-deficient mice........... 56
1.7.1--Viral vector gene delivery........... 58
1.7.2--9-cis-retinoid supplementation........... 621.7.3--Nanoparticle vector and electroporation gene delivery........... 64
1.8--Introduction summary........... 65
Chapter 2--Cellular pathologies in RPE65-deficient mice........... 69
2.1--Cells mediating residual visual function in RPE65-deficient mice........... 71
2.2--Rod photoreceptor cell death........... 75
2.3--Cone photoreceptor cell death........... 79
2.4--RPE dysfunction and stress........... 85
Chapter 3--Complementation test of Rpe65 knockout and tvrm148........... 94
3.1--Abstract........... 96 3.2--Introduction........... 97 3.3--Methods........... 1023.3.1--Experimental animals........... 102
3.3.2--Computer predictions........... 102
3.3.3--Electroretinography (ERG)........... 103
3.3.4--Retinoid analysis........... 104
3.3.5--Optokinetic tracking (OKT)........... 105
3.3.6--Histology and morphometrics........... 106
3.3.7--Quantitative real time polymerase chain reaction (qRT-PCR)........... 107
3.3.8--DNA sequencing of Rpe65........... 108
3.3.9--Immunoblotting........... 1113.3.10--Statistical analysis........... 114
3.4--Results........... 1143.4.1--Predictions of tvrm148 mutation pathogenicity........... 114
3.4.2--Complementation test........... 115
3.4.3--Retinoid levels in tvrm148, knockout, and normal mouse eyes........... 118
3.4.4--Inheritance of tvrm148........... 120
3.4.5--Visual acuity affected by null mutations........... 124
3.4.6--Comparison of slow loss of visual acuity in tvrm148/tvrm148 and KO/KO mice........... 126
3.4.7--Histology and morphometrics--slight losses in mutants........... 128
3.4.8--RNA quantitation of tvrm148 mutation........... 135
3.4.9--RPE65 protein expression in tvrm148/tvrm148 mice........... 135
3.5--Discussion........... 138Chapter 4--The rd12allele exerts a semidominant negative effect on visual function in mice........... 146
4.1--Abstract........... 148 4.2--Introduction........... 149 4.3--Methods........... 1514.3.1--Experimental animals........... 151
4.3.2--Optokinetic tracking (OKT)........... 155
4.3.3--Retinoid analysis........... 155
4.3.4--Electroretinography (ERG)........... 155
4.3.5--Histology and morphometrics........... 156
4.3.6--RNA extraction and qRT-PCR........... 156
4.3.7--qRT-PCR standard curve........... 157
4.3.8--DNA sequencing of Rpe65 mRNA........... 161
4.3.9--Immunoblotting........... 1614.3.10--Fluorescence in situ hybridization........... 162
4.3.11--RNA structure prediction........... 163
4.3.12--Linear sucrose gradient fractionation........... 163
4.3.13--Statistical analysis........... 164
4.4--Results........... 1664.4.1--Complementation test........... 166
4.4.2--Visual acuity........... 169
4.4.3--ERG measurements of homozygous mutant mice........... 173
4.4.4--ERG measurements of heterozygous mutant mice........... 177
4.4.5--Histology and morphometrics........... 182
4.4.6--RPE65 protein levels in rd12 mice........... 189
4.4.7--Rpe65 mRNA levels in rd12 mice........... 1934.4.9--Predicted Rpe65 mRNA secondary structures........... 197
4.4.10--FISH of Rpe65 mRNA........... 200
4.4.11--Impaired association of mutant mRNA with ribosomes........... 204
4.5--Discussion........... 213Chapter 5--Discussion and future directions........... 225
5.1--Summary of novel findings........... 227
5.1.1--Novel tvrm148 findings........... 227
5.1.2--Novel rd12 findings........... 228
5.1.3--Comparisons of the three mouse strains........... 229
5.2--Limitations of this study........... 230
5.3--Future directions........... 232
5.3.1--Preliminary data for future studies........... 232
5.3.2--Future planned experiments........... 241
5.4--Final Conclusions........... 244
References........... 246 Appendix--Protocols........... 262A.1--Electroretinography (ERG)........... 263
A.2--Optokinetic tracking (OKT)........... 267
A.3--Fluorescence in situ hybridization (FISH)........... 271
A.4--Whole cell RNA extraction........... 281
A.5--Cell fractionation and RNA extraction of nuclear/cytoplasmic fractions........... 284
A.6--Extraction of soluble and insoluble protein for SDS-PAGE........... 288
A.7--Linear sucrose gradient fractionation and RNA extraction........... 291
A.8--qRT-PCR........... 295 A.9--Immunoblotting........... 300
Fig. 1.1--Anatomy of the eye........... 10
Fig. 1.2--Anatomy of the mammalian retina........... 11
Fig. 1.3--Biochemistry of the visual cycle........... 14
Fig. 1.4--The isomerization of 11-cis-RAL to all-trans-RAL by light initiates the phototransduction cascade........... 15
Fig. 1.5--OKTs and ERGs are visual assessment methods for mice........... 19
Fig. 1.6--The retina only recovers sensitivity to light if the RPE is left attached........... 22
Fig. 1.7--Alignment of human RPE65 and BCMO1 protein sequences........... 29
Fig. 1.8--Tertiary structure of RPE65 with iron (II) cofactor and mouse mutations labeled........... 30
Fig. 1.9--Known human and mouse mutations in RPE65........... 39
Fig. 1.10--The R91W and F229S amino acid substitutions are predicted to distort RPE65 protein tertiary structure........... 50
Fig. 2.1--Rod photoreceptors are subject to two phases of cell death........... 78
Fig. 2.2--Cone cell death is associated with altered cone opsin trafficking in the absence of 11-cis-RAL........... 83
Fig. 2.3--Fundus images of wild type and Rpe65 mutant mice........... 87
Fig. 2.4--RPE cells experience only slight morphological aberrations during RPE65 deficiency........... 92
Fig. 3.1--Rpe65 resides outside the markers D3Mit147 and D3Mit19 on the sequence map of chromosome 3........... 100
Fig. 3.2--The complementation test........... 101
Fig. 3.3. Qiagen Rpe65 primers were validated by DNA sequencing of PCR product........... 109
Fig. 3.4--There was only one mutation in Rpe65 in tvrm148 mice........... 113
Fig. 3.5--The tvrm148 and Rpe65KO alleles did not complement........... 117
Fig. 3.6--tvrm148/tvrm148 mice, but not tvrm148/+ mice, had a loss of ERG response........... 122
Fig. 3.7--tvrm148/tvrm148 mice, but not tvrm148/+ mice, progressively lost visual acuity with age........... 125
Fig. 3.8--tvrm148/tvrm148 mice had residual visual function similar to KO/KO mice........... 127
Fig. 3.9--tvrm148/tvrm148 mice had a thinner retina than tvrm148/+ or +/+ counterparts........... 130
Fig. 3.10--tvrm148/tvrm148 mouse retina layers progressively thinned with age........... 131
Fig. 3.11--tvrm148/tvrm148 mice had significant cone nuclei loss at P60 and P210........... 133
Fig. 3.12--TEM showed lipid droplets indicative of retinyl ester accumulation in the RPE of tvrm148/tvrm148 mice........... 134
Fig. 3.13--tvrm148/tvrm148 mice expressed Rpe65 mRNA at the same level as +/+ mice under steady-state conditions........... 136
Fig. 3.14--Immunoblots and densitometry for RPE65 protein in +/+, tvrm148/+, and tvrm148/tvrm148 mice showed mutant mice have reduced protein
levels........... 137
Fig. 4.1--Flowcharts of hypotheses and experiments in Chapter 4........... 152
Fig. 4.2--qRT-PCR products from Rpe65 mRNA with primers specific to each exon boundary........... 158
Fig. 4.3--Standard curve of Rpe65 amplification by qRT-PCR........... 160
Fig. 4.4--Linear sucrose gradient fractionation........... 165
Fig. 4.5--The Rpe65 knockout and rd12 alleles did not complement........... 167
Fig. 4.6--The rd12 allele caused visual acuity loss in a semidominant fashion........... 170
Fig. 4.7--Raw ERG traces of genotypes in this study........... 174
Fig. 4.8--Mutant mice had reduced dark-adapted a- and b-wave amplitudes compared to +/+ mice from P30-P90........... 175
Fig. 4.9--One copy of the rd12 allele caused small reductions in dark-adapted ERG amplitudes........... 178
Fig. 4.10--Lamb and Pugh modeling of a-wave responses showed rd12/+ mice had normal rod phototransduction kinetics........... 180
Fig. 4.11--KO/KO and rd12/rd12 mice had a thinner retina than +/+ mice........... 183
Fig. 4.12--KO/KO and rd12/rd12 mice had similar ONL and OS thinning with age........... 185
Fig. 4.13--Mice with Rpe65 mutations had large reductions in the number of cone nuclei........... 187
Fig. 4.14. The lower limit of detection of the N-terminal specific antibody is 2.5% wild type RPE65 protein level........... 190
Fig. 4.15--rd12/rd12 mice did not accumulate detectable amounts of RPE65 protein........... 192
Fig. 4.16--rd12 mutant Rpe65 mRNA was exported to the cytoplasm........... 194
Fig. 4.17--Chromatograms of DNA sequences from wild type and rd12 Rpe65 mRNA showed a nonsense mutation in rd12/rd12 mice........... 195
Fig. 4.18--rd12 Rpe65 mRNA had a predicted secondary structure that closely resembled wild type mRNA........... 198
Fig. 4.19--RNA in situ hybridization showed similar localization of Rpe65 mRNA in both +/+ and rd12/rd12 RPE cells........... 201
Fig. 4.20--Possible outcomes of polyribosome profiling........... 206
Fig. 4.21--Raw cycle values from polyribosome profiling........... 208
Fig. 4.22--rd12 Rpe65 mRNA was mostly found in ribosome-free mRNPs........... 209
Fig. 4.23--Rpe65 mRNA is mostly bound by mRNPs in rd12/rd12 mice........... 211
Fig. 4.24. Polyribosome profiles of b-actin are similar between wild type and rd12........... 212
Fig. 4.25--Proposed model for the foundation of the negative semidominant effects exerted by the rd12 allele on visual function........... 222
Fig. 5.1--RNA-sensing pathways activate Type I interferons (IFNs) and NLRP3 inflammasomes........... 234
Fig. 5.2--TLR receptors may be activated in the RPE of rd12/rd12 mice........... 238
Fig. 5.3--RNA sensors TLR3 and TLR7 are proposed mediators for the negative semidominant visual effects induced by the rd12 allele........... 239
Table 1.1--Hypotheses tested concerning the tvrm148 mutation........... 6
Table 1.2--Hypotheses tested concerning the rd12 mutation........... 7
Table 1.3--Mutations in different genes can cause LCA........... 36
Table 1.4--Mouse RPE65 variants and their effects on protein function........... 43
Table 1.5--Current clinical trials for patients with RPE65 mutations........... 57
Table 3.1--Primers for amplification of Rpe65 cDNA........... 112
Table 3.2--Predictions of putative tvrm148 mutation on RPE65 function........... 116
Table 3.3--Retinol isomerase activity is abolished in KO/KO and tvrm148/tvrm148........... 119
Table 4.1--Primer sequences for Rpe65 exon boundary amplification........... 159
Table 4.2--Retinoid levels in +/+, KO/KO, and rd12/rd12 mice........... 168
Table 4.3--All Rpe65 exons were successfully amplified in the mutant rd12 mRNA........... 196
Table 5.1--Predicted phenotypes of offspring of rd12 and innate immune deficient mice 242
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