CAPN5mutation 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.

Metabolite therapy guided by liquid biopsy proteomics delays retinal neurodegeneration

“…Histology -Mice were sacrificed, and the eyes enucleated as previously described [34][35][36]. Eyes were embedded in paraffin, sectioned, and stained with hematoxylin and eosin by Exaclibur Pathology, Inc.…”

Small-angle X-ray scattering of calpain-5 reveals a highly open conformation among calpains

“…Interestingly, the previously described human p.R243L mutant of calpain-5 appears to be more active than the wild type and require less calcium for activation (Wert et al, 2015). Since the p.R243L mutation does not fall within loop C1, the mutation more likely affects the second part of calpain activation—calcium binding to the catalytic domain and subsequent sub-domain rearrangements that form the active site.…”

“…Calpains are implicated in a number of disease processes (Goll et al, 2003; Sorimachi et al, 1997; Zatz and Starling, 2005); and mutations in calpain-5 cause ADNIV, an autoimmune inflammatory retinal disease (Bassuk et al, 2015; Mahajan et al, 2012; Wert et al, 2015). Specific inhibitors of calpains (e.g., SNJ-1945) were designed and optimized for calpain-1 (Cuerrier et al, 2006); however, little structural information is available to facilitate optimization of calpain-5 inhibitors.…”

Capn5 expression in the healthy and regenerating zebrafish retina

“…[12][13][14] Mutations in CAPN5 are associated with the devastating retinal degenerative disease autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV). 15,16,17 ADNIV is a hereditary autoimmune disease of the eye that is characterized by abnormal retinal pigmentation, retinal neovascularization, photoreceptor degeneration, vitreous hemorrhage, intraocular fibrosis, and tractional retinal detachment. As the disease progresses it phenocopies more commonly known ocular diseases such as non-infectious uveitis, glaucoma, diabetic retinopathy and retinitis pigmentosa.…”

“…As the disease progresses it phenocopies more commonly known ocular diseases such as non-infectious uveitis, glaucoma, diabetic retinopathy and retinitis pigmentosa. 15,18 The three point mutations that have been identified in ADNIV patients (p.Arg243Leu, p.Leu244Pro, and p.Lys250Asn) are located in the calcium-sensitive domain 2 near the active site and are thought to cause the mislocalization of CAPN5 from the cell membrane to the cytosol. 16,17 Thus, ADNIV is thought to result from gain-of-function mutations in CAPN5 that lower its threshold for activation by calcium.…”