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. 2014 May 15;23(10):2665-77.
doi: 10.1093/hmg/ddt661. Epub 2013 Dec 30.

Functional validation of a human CAPN5 exome variant by lentiviral transduction into mouse retina

Katherine J Wert  1 , Jessica M Skeie, Alexander G Bassuk, Alicia K Olivier, Stephen H Tsang, Vinit B Mahajan
Affiliations

Functional validation of a human CAPN5 exome variant by lentiviral transduction into mouse retina

Katherine J Wert et al. Hum Mol Genet. .

Abstract

Exome sequencing indicated that the gene encoding the calpain-5 protease, CAPN5, is the likely cause of retinal degeneration and autoimmune uveitis in human patients with autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM #193235). To explore the mechanism of ADNIV, a human CAPN5 disease allele was expressed in mouse retinas with a lentiviral vector created to express either the wild-type human (h) CAPN5 or the ADNIV mutant hCAPN5-R243L allele under a rhodopsin promoter with tandem green fluorescent protein (GFP) expression. Vectors were injected into the subretinal space of perinatal mice. Mouse phenotypes were analyzed using electroretinography, histology and inflammatory gene expression profiling. Mouse calpain-5 showed high homology to its human ortholog with >98% sequence identity that includes the ADNIV mutant residue. Calpain-5 protein was expressed in the inner and outer segments of the photoreceptors and in the outer plexiform layer. Expression of the hCAPN5-R243L allele caused loss of the electroretinogram b-wave, photoreceptor degeneration and induction of immune cell infiltration and inflammatory genes in the retina, recapitulating major features of the ADNIV phenotype. Intraocular neovascularization and fibrosis were not observed during the study period. Our study shows that expression of the hCAPN5-R243L disease allele elicits an ADNIV-like disease in mice. It further suggests that ADNIV is due to CAPN5 gain-of-function rather than haploinsufficiency, and retinal expression may be sufficient to generate an autoimmune response. Genetic models of ADNIV in the mouse can be used to explore protease mechanisms in retinal degeneration and inflammation as well as preclinical therapeutic testing.

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Figures

Figure 1.
Figure 1.
ADNIV Clinical Case. (A) The fundus image shows a normal vasculature, optic nerve and very little pigmentary change in the left retina in an early stage I–II case of ADNIV. (B) The visual field, however, reveals constriction of all isopters, which indicates reduced retinal light sensitivity to various intensities of light. (C) This was confirmed by electroretinography. Loss of maximal b-wave amplitudes (b) for the ADNIV human patient (black) compared with a normal control (blue) indicates a signaling defect in both rod and cone visual function. No significant change in the a-wave amplitudes (a) were noted between the ADNIV patient (black) and the control (blue) (D) Over two decades, stage III–IV disease develops. The retina shows signs of degeneration with extensive pigmentary changes (arrows) and atrophic changes throughout the fundus. The optic nerve becomes pale and the retinal vessels are attenuated. (E) Later, optical coherence tomography reveals fibrotic membranes, cystoid macular edema and distortion of the retinal layers.
Figure 2.
Figure 2.
The mouse calpain-5 protein is highly homologous to its human ortholog. (A) Diagram of mouse Capn5 exonic structure. (B) Representative RNA-Seq from mouse retina. Vertical bars above the corresponding exon represent reads from massive parallel sequencing of mouse retina. Note that exon 6 is the ortholog to the human exon for which CAPN5 mutations have been identified in ADNIV patients. (C) Domain map shows the catalytic domain is encoded by exon 6. ADNIV mutations occur near the catalytic residues. (D) Protein alignment between calpain-5 in mouse and human species shows high conservation, including the site of R243L mutation.
Figure 3.
Figure 3.
Calpain-5 is located in the mouse and human retina. (A) Retinal section of a wild-type C57BL/6J (B6) control mouse. (B) Retinal section of a wild-type B6 control mouse with CAPN5 protein located in the outer and inner segments of the photoreceptor cells. (C) Magnified view of the white-dashed box in B, showing calpain-5 localization in the outer plexiform layer (arrow). (D) Calpain-5 localization in the human retina outer plexiform layer (arrows). Red or blue, DAPI; green, mCAPN5 or hCAPN5. RGC, retinal ganglion cells; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, photoreceptor inner segments; OS, photoreceptor outer segments. Scale bar = 20 µm.
Figure 4.
Figure 4.
Expression of lentiviral construct after subretinal injection in mice. (A) Lentiviral construct map of wild-type human (h) CAPN5 and mutant ADNIV hCAPN5-R243L with green fluorescent protein (GFP) tag. (B) Autofluorescence fundus image at 5 months of age of a B6 non-injected eye, and (C) the fellow B6 eye that was injected at post-natal day 5 with the wild-type hCAPN5 lentivirus showing punctate GFP expression (arrow). LTR, long terminal repeat; Rho, mouse rhodopsin promoter; EIFa, elongation factor promoter.
Figure 5.
Figure 5.
Electroretinography shows reduced visual function in retinas injected with mutant CAPN5 lentiviral vector. (A) Representative traces of scotopic rod-cone maximum ERG responses at 3 months of age in control (blue), hCAPN5 (green) and hCAPN5-R243L (red) mice. (B) Representative traces of scotopic dim-light rod-only ERG responses at 3 months of age in control (blue), hCAPN5 (green) and hCAPN5-R243L (red) mice. (C) Representative traces of photopic cone-specific ERG responses at 3 months of age in control (blue), hCAPN5 (green) and hCAPN5-R243L (red) mice. (D) Cartoon depicting the injected eye of the mouse and uninjected fellow eye, with colors corresponding to injected solution (blue, control saline or GFP lentivirus without transgene; green, wild-type hCAPN5 lentivirus; red, ADNIV mutant hCAPN5-R243L lentivirus). (E) Differences between the injected (dark blue, saline; light blue, GFP lentiviral vector without transgene; green, wild-type hCAPN5 lentiviral vector; red, mutant hCAPN5-R243L lentiviral vector) and uninjected eyes for scotopic maximum rod-cone b-wave amplitudes at 3 months of age. (F) Differences between the injected (dark blue, saline; light blue, GFP lentiviral vector without transgene; green, wild-type hCAPN5 lentiviral vector; red, mutant hCAPN5-R243L lentiviral vector) and uninjected eyes for scotopic maximum rod-cone a-wave amplitudes at 3 months of age. (G) Differences between the injected (dark blue, saline; light blue, GFP lentiviral vector without transgene; green, wild-type hCAPN5 lentiviral vector; red, mutant hCAPN5-R243L lentiviral vector) and uninjected eyes for photopic, cone-specific b-wave amplitudes at 3 months of age. Statistical significance analyzed for difference between groups using a paired t-test analysis (*P < 0.05; **P < 0.01; ***P < 0.001). n = 3, 3, 10 and 19 for the saline, GFP lentivirus, hCAPN5 lentivirus and hCAPN5-R243L lentivirus-injected eyes, respectively.
Figure 6.
Figure 6.
Photoreceptor degeneration after mutant CAPN5 overexpression. (A) Retina of a hematoxylin and eosin (H&E)-stained wild-type B6 control mouse eye at 6 months of age, (B) a wild-type hCAPN5 lentiviral-injected eye and (C) a mutant hCAPN5-R243L lentiviral-injected eye. Scale bar = 600 µm. RGC, retinal ganglion cells; INL, inner nuclear layer; ONL, outer nuclear layer. (D) Quantification of outer nuclear layer (ONL) and inner nuclear layer (INL) thickness in control, hCAPN5 lentiviral-injected and mutant hCAPN5-R243L lentiviral-injected eyes at 6 months of age. *P < 0.05; ***P < 0.001. n ≥ 4 mice. (E) Fluorescein angiography of a B6 eye injected with the mutant hCAPN5-R243L lentivirus compared with (F) the uninjected fellow eye.
Figure 7.
Figure 7.
T-cell-related markers in the retina after subretinal injection of mutant CAPN5 lentiviral vector. (A) Immunohistochemistry of CD3-positive T cells in spleen. (B) Wild-type hCAPN5 lentiviral-injected eye had no CD3-positive cells present. (C and D) hCAPN5-R243L lentiviral-injected eyes had several CD3-positive cells in immunohistochemistry labeled sections (arrows). (E) T-cell markers, CD34 and CD4, are up-regulated in the hCAPN5-R243L retinas compared with the wild-type hCAPN5 retinas (* P < 0.05).
Figure 8.
Figure 8.
Inflammatory gene expression after subretinal injection of mutant CAPN5 lentiviral vector. Retinas from hCAPN5-R243L-injected mice were compared with the wild-type hCAPN5-injected mouse retinas using real-time quantitative PCR arrays. Four mice were analyzed for each condition. Up-regulation of cytokines such as Il-12b suggests a Th17 differentiation pathway, where Il-10 would have suggested regulatory T-cell (Treg) differentiation but was found unchanged. The transcription factor, Nfatc, was increased in the mutant injected retina. Some genes were down-regulated including Ccl2, Cx3cl1, Csf3 and Socs3. Normalized samples were analyzed in triplicate, and the SEM was on average only 1.1% of the total average Ct values per condition (*P < 0.05, **P < 0.01).
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