CAPN5 mutation in hereditary uveitis: the R243L mutation increases calpain catalytic activity and triggers intraocular inflammation in a mouse model

Abstract

A single amino acid mutation near the active site of the CAPN5 protease was linked to the inherited blinding disorder, autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM #193235). In homology modeling with other calpains, this R243L CAPN5 mutation was situated in a mobile loop that gates substrate access to the calcium-regulated active site. In in vitro activity assays, the mutation increased calpain protease activity and made it far more active at low concentrations of calcium. To test whether the disease allele could yield an animal model of ADNIV, we created transgenic mice expressing human (h) CAPN5(R243L) only in the retina. The resulting hCAPN5(R243L) transgenic mice developed a phenotype consistent with human uveitis and ADNIV, at the clinical, histological and molecular levels. The fundus of hCAPN5(R243L) mice showed enhanced autofluorescence (AF) and pigment changes indicative of reactive retinal pigment epithelial cells and photoreceptor degeneration. Electroretinography showed mutant mouse eyes had a selective loss of the b-wave indicating an inner-retina signaling defect. Histological analysis of mutant mouse eyes showed protein extravasation from dilated vessels into the anterior chamber and vitreous, vitreous inflammation, vitreous and retinal fibrosis and retinal degeneration. Analysis of gene expression changes in the hCAPN5(R243L) mouse retina showed upregulation of several markers, including members of the Toll-like receptor pathway, chemokines and cytokines, indicative of both an innate and adaptive immune response. Since many forms of uveitis share phenotypic characteristics of ADNIV, this mouse offers a model with therapeutic testing utility for ADNIV and uveitis patients.

Figure 1.

Generation of a recombinant mini-CAPN1/5 hybrid with loop 2 of proteolytic domain IIb (DIIb) swapped from CAPN5 into the homologous region in mini-CAPN1. The mini-calpain system allows the catalytic activity of domain II to be isolated and assayed, since full-length calpains are generally not stable. (A) Cartoon representation of the rat mini-CAPN5 structure with calcium bound (green spheres), highlighting the active site residues (yellow sticks), the four flexible loops (pink) and the ADNIV mutations (red dotted circle). (B) Sequence alignment of domain II of rat mini-CAPN1 and human mini-CAPN5 highlighting the flexible loops (pink box). (C) Schematic representation showing proteins used in our modified mini-calpain system. Mini-CAPN1, a hybrid mini-CAPN1/5 with loop 2 of mini-CAPN1 swapped out with that of mini-CAPN5 and a hybrid mini-CAPN1/5 harboring the p.R243L ADNIV mutation.

Figure 2.

A mini-CAPN1/5 hybrid is active in in vitro assays. A mini-CAPN1/5 is sensitive to a calpain inhibitor as well as mutation of the catalytic cysteine residue (C81S) that renders all calpains enzymatically inactive. (A) Time course of substrate peptide (AC-LLY-AFC) shows catalytic activity of CAPN1/5 hybrid with (green) and without (blue) a calpain peptide inhibitor (Z-LLY-AFC). (B) Catalytic activity of CAPN1/5 hybrid bearing a mutation of the catalytic cysteine residue (C81S; green) renders it inactive. RFU, relative fluorescent units.

Figure 3.

R243L ADNIV mutation increases catalytic activity of a recombinant mini-CAPN1/5 hybrid. The R243L mutation renders the mini-CAPN1/5 hybrid over 300% more sensitive to calcium (activity is a ratio normalized to mini-CAPN1; control is the reaction without calpain). (A) A time-course comparison of the CAPN1/5 hybrid and a hybrid bearing the R243L mutation, showing catalysis of a standard peptide substrate at 0.01 mm calcium, (B) 0.10 mm calcium, (C) 1.0 mm calcium and (D) 100 mm calcium. RFU, relative fluorescent units.

Figure 4.

Transgenic ADNIV mutant construct. (A) A p4.2 construct designed for opsin promoter-driven expression of a Myc-tagged human CAPN5^R243L transgene (pA, polyadenylation signal; AmpR, ampicillin-resistant selection gene; pUC ori, origin of replication). (B) The hCAPN5^R243L transgene expression was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in two transgenic mice as compared with a WT control mouse and a non-transgenic mouse from the same litter. (C) Anti-Myc immunoblot confirms transgene expression in the retinal extract.

Figure 5.

AF and IR imaging of human and mouse WT and hCAPN5^R243L retinas reveals early retinal degeneration. (A) The normal human AF fundus image. (B) Mid- to late-stage ADNIV eyes show increased AF that may be associated with photoreceptor or RPE cell dysfunction. (C) IR imaging of an ADNIV patient shows scattered pigment accumulation (arrows). Image quality is limited by cataract and vitreous membranes. (D) AF image of a control WT mouse at 18 weeks of age. (E) AF imaging of hCAPN5^R243L mice at 9 weeks of age shows increased AF compared with the control. (F) Further autofluorescent increase is observed by 18 weeks of age, as seen in human ADNIV eyes. (G) IR imaging of a control WT mouse fundus. (H) IR imaging of a hCAPN5^R243L mouse fundus at 9 weeks of age, and (I) by 18 weeks of age scattered, melanin-like pigment is accumulating in the hCAPN5^R243L mouse fundus (arrow).

Figure 6.

Human ADNIV pathology. (A) Histology reveals a foreshortened, fibrotic iris with dilated vessels and exudate in the anterior chamber (visualized by Masson's trichrome stain). (B) A cataractous lens with lens epithelial cells (arrow) located along the posterior capsule (visualized by Masson's trichrome stain). (C). RPE cells (arrow) infiltrating the degenerating retina (visualized by H&E stain). (D) Vitreous containing fibrous membranes (arrow) and filled with exudate (visualized by H&E stain). (E) Epiretinal membrane (arrow). (F) Fibrovascular membrane at the optic nerve (arrow). Scale bar = 250 μm.

Figure 7.

The hCAPN5^R243L transgenic mouse develops severe uveitis that phenocopies early-stage human ADNIV. (A) Pupil-optic nerve section of a normal mouse eye. (B) Pupil-optic nerve section of a hCAPN5^R243L transgenic mouse eye at 4 months of age. The anterior segment shows signs of inflammation with protein exudates in the anterior chamber and a contracted iris with dilated vessels. There are membranes and cells in the vitreous chamber. (C) Magnified view of foreshortened iris and dilated vessels (arrow). (D) Posterior epithelial cells (green arrow) are a sign of posterior subcasular cataract. Vitreous vessels (green arrow) are a sign of early vitreous neovascularization. (E) Vitreous membranes (arrow) correlate with fibrotic bands found in the human vitreous. (F) Control retina. (G) Retinal vessels are dilated (arrow). (H) Reactive retinal pigmented epithelial cells (arrow) in early retinal degeneration. (I) Fibrovascular tissue at optic nerve (arrow).

Figure 8.

Electroretinography shows hCAPN5^R243L mice have a specific loss of the inner retinal cells and cone cell function. (A) ERG tracing of ADNIV patient shows reduction of b-wave relative to the a-wave (14). The scotopic b-wave was 83% of normal (normal scotopic b-wave was 450 ± 100 μV in this system). (B) The photopic ERG b-wave was ∼32.5% of normal (339 ± 85 μV). The calibration mark before stimulus shows 100 μV. (C) A representative ERG test on an 8 month-old hCAPN5^R243L transgenic mouse (green) compared with a control mouse (blue) displays the frequently observed functional phenotype. hCAPN5^R243L transgenic mice have a reduced scotopic maximal b-wave amplitude compared with control mice, with the a-wave remaining unchanged. (D) Photopic, cone-specific ERGs showed a reduction in the b-wave maximal amplitude in the hCAPN5^R243L transgenic mice compared with controls. Such a pattern correlates to ADNIV patients.

Figure 9.

IL-6 is upregulated in the hCAPN5^R243L transgenic mouse ciliary body. (A) Control IgG antibody. (B) No IL-6 expression in 4-month-old WT vitreous, as assayed by immunohistochemistry. (C) Vitreous IL-6 expression is upregulated in the hCAPN5^R243L mouse anterior vitreous. CB, ciliary body; Vit, vitreous; R, retina. Scale bar = 25 μm.

Figure 10.

The hCAPN5^R243L transgenic retina shows early changes in inflammatory gene expression before any clinical or histological signs of disease. (A) Volcano plot displays genes in the hCAPN5^R243L retinas at post-natal day (P) 45 that had statistically significant expression changes that were greater than 2-fold (P < 0.05), when compared with P45 control retinas. Four genes (Fos, Bcl6, IL1rap and Nr3c1) were downregulated (blue dots, upper left). (B) Schematic representation shows that several members of the TLR pathways were among the upregulated inflammatory genes.

Figure 11.

An integrated ADNIV disease model. (A) Histology of the CAPN5^R243L uveitis model. (B) The CAPN5^R243L disease allele transgene is expressed in retina photoreceptors. Because of the high calcium levels in the retina for phototransduction, CAPN5 mutations in the retina are particularly sensitive. TLR signaling and cyotokine secretion trigger retinal and iris vessel hyperpermeability and a cell-mediated autoinflammatory reaction that includes vitreous inflammation and fibrosis.