calpain and Vitreoretinopathy--Proliferative

Neovascular inflammatory vitreoretinopathy (NIV) is a rare eye disease that ultimately leads to complete blindness and is caused by mutations in the gene encoding calpain-5 (CAPN5), with six pathogenic mutations identified. In transfected SH-SY5Y cells, five of the mutations resulted in decreased membrane association, diminished S-acylation, and reduced calcium-induced autoproteolysis of CAPN5. CAPN5 proteolysis of the autoimmune regulator AIRE was impacted by several NIV mutations. R243, L244, K250 and the adjacent V249 are on β-strands in the protease core 2 domain. Conformational changes induced by Ca

Topics: Calpain; Humans; Mutation; Neuroblastoma; Vitreoretinopathy, Proliferative

Topics: Adult; Calpain; Female; High-Throughput Nucleotide Sequencing; Humans; Male; Mutation; Vitreoretinopathy, Proliferative; Young Adult

Autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) is a rare autoimmune condition that typically presents as progressive uveitis and vitreoretinal degeneration between the second and third decades of life. Though traditionally attributed to inherited mutations of the CAPN5 gene, few reports of de novo variants exist. This report of vision and hearing loss in a 3 year-old girl describes the youngest documented case of ADNIV due to a de novo pathogenic c.865C>T (p.Arg289Trp) CAPN5 variant, illustrating the early stages of this enigmatic disease process.

Topics: Calpain; Child, Preschool; Female; Genetic Predisposition to Disease; Humans; Mutation; Vitreoretinopathy, Proliferative

CAPN5 Neovascular Inflammatory Vitreoretinopathy (CAPN5-NIV; OMIM 193235) is a poorly-understood rare, progressive inflammatory intraocular disease with limited therapeutic options. To profile disease effector proteins in CAPN5-NIV patient vitreous, liquid vitreous biopsies were collected from two groups: eyes from control subjects (n = 4) with idiopathic macular holes (IMH) and eyes from test subjects (n = 12) with different stages of CAPN5-NIV. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein expression changes were evaluated by principal component analysis, 1-way ANOVA (significant p-value < 0.05), hierarchical clustering, gene ontology, and pathway representation. There were 216 differentially-expressed proteins (between CAPN5-NIV and control vitreous), including those unique to and abundant in each clinical stage. Gene ontology analysis revealed decreased synaptic signaling proteins in CAPN5-NIV vitreous compared to controls. Pathway analysis revealed that inflammatory mediators of the acute phase response and the complement cascade were highly-represented. The CAPN5-NIV vitreous proteome displayed characteristic enrichment of proteins and pathways previously-associated with non-infectious posterior uveitis, rhegmatogenous retinal detachment (RRD), age-related macular degeneration (AMD), proliferative diabetic retinopathy (PDR), and proliferative vitreoretinopathy (PVR). This study expands our knowledge of affected molecular pathways in CAPN5-NIV using unbiased, shotgun proteomic analysis rather than targeted detection platforms. The high-levels and representation of acute phase response proteins suggests a functional role for the innate immune system in CAPN5-NIV pathogenesis.

Topics: Adult; Aged; Calpain; Chromatography, Liquid; Diabetic Retinopathy; Female; Humans; Male; Middle Aged; Proteome; Proteomics; Retinal Degeneration; Retinal Detachment; Retinal Perforations; Tandem Mass Spectrometry; Vitreoretinopathy, Proliferative; Vitreous Body

Topics: Aged; Calpain; Eye Diseases; Female; Humans; Retinal Diseases; Tissue Adhesions; Tomography, Optical Coherence; Vitreoretinopathy, Proliferative; Vitreous Body

Mutations that activate the protease calpain-5 (

Topics: Alleles; Amino Acid Sequence; Calpain; Child, Preschool; Developmental Disabilities; Electroencephalography; Exome Sequencing; Genetic Association Studies; Genotype; Hearing Loss; Humans; Male; Models, Molecular; Mutation; Pedigree; Phenotype; Protein Conformation; Vitreoretinopathy, Proliferative

To identify the causative gene and investigate the corresponding mechanisms for an autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) family.. Clinical examination and genetic analysis were performed in a Chinese ADNIV family. To dissect the molecular consequence, we used gene targeting to knock-in a patient's specific mutation in the mouse genome. Immunostaining and immunoprecipitation were harnessed to analyze the colocalization and interaction of CAPN5 with SLIT2 in photoreceptors. The purified SLIT2-N, SLIT2-C fragments, and the conditioned medium from 661W cells with the overexpression of CAPN5 were treated on ARPE-19 cells. The viability of ARPE-19 cells was determined by MTT assays. The activation of protein kinase A (PKA) was analyzed by immunofluorescence and Western blotting in 661W and ARPE-19 cells as well as in frozen retina tissue from wildtype (WT) and knock-in mice.. We identified a novel CAPN5 mutation (p.R289W) in a Chinese family and generated the knock-in CAPN5R289W mouse. This mutation caused abnormal proliferative RPE in both humans and mice. CAPN5 directly cleaved WT SLIT2 in vitro, but not the mutant SLIT2 (p.R1113I). CAPN5 interacted with the SLIT2 in mouse retinal photoreceptors (661W cells) and increased cleavage and secretion of the SLIT2 fragments (SLIT2-N and SLIT2-C). Conditioned medium induced higher levels of secreted SLIT2 fragments, which promoted PKA activation and promoted proliferation of ARPE-19 cells.. The novel CAPN5 mutation (p.R289W) is responsible for the present ADNIV family. The mutant CAPN5 stimulated secretion and cleavage of SLIT2 fragments that may act as a bystander to regulate abnormal RPE cell proliferation for ADNIV.

Topics: Animals; Blotting, Western; Calpain; Cell Line; Cell Proliferation; Child; Cyclic AMP-Dependent Protein Kinases; Electroretinography; Female; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation; Gene Knock-In Techniques; Humans; Intercellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Mutation; Nerve Tissue Proteins; Pedigree; Photoreceptor Cells, Vertebrate; Plasmids; Real-Time Polymerase Chain Reaction; Retinal Pigment Epithelium; Tomography, Optical Coherence; Vitreoretinopathy, Proliferative

Calpain-5 is a calcium-activated protease expressed in the retina. Mutations in calpain-5 cause autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM#193235). The structure of calpain-5 has not been determined, thus hindering the investigation of its proteolytic targets and pathological role in ADNIV. Herein, we report models of the proteolytic core of calpain-5 (mini-calpain-5) containing two globular domains (termed DIIa-IIb) connected by a short, flexible linker, consistent with small-angle X-ray scattering (SAXS) data. Structural modeling in the absence of calcium suggests that mini-calpain-5 adopts a more open conformation when compared to previously determined structures of other calpain cores. This open conformation, achieved by a rotation of DIIa and DIIb with respect to each other, prevents formation of the active site and constrains the enzyme in an inactivated form. The relative domain rotation of 60-100° we found for mini-calpain-5 (a non-classical calpain) is significantly greater than the largest rotation previously observed for a classical calpain (i.e., 55.0° for mini-calpain-9). Together with our prediction that, in the full-length form, a long loop in DIIb (loop C1), a few residues downstream of the inter-domain linker, likely interacts with the shorter, acidic, inactivating loop on domain-III (DIII), these structural insights illuminate the complexity of calpain regulation. Moreover, our studies argue that pursuing higher resolution structural studies are necessary to understand the complex activity regulation prevalent in the calpain family and for the design of specific calpain inhibitors.

Topics: Amino Acid Sequence; Binding Sites; Calcium; Calpain; Catalytic Domain; Humans; Models, Molecular; Mutation; Protein Conformation; Protein Structure, Tertiary; Retina; Scattering, Small Angle; Vitreoretinopathy, Proliferative; X-Rays

CAPN5 mutations have been linked to autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV), a blinding autoimmune eye disease. Here, we link a new CAPN5 mutation to ADNIV and model the three-dimensional structure of the resulting mutant protein. In our study, a kindred with inflammatory vitreoretinopathy was evaluated by clinical eye examinations, DNA sequencing, and protein structural modeling to investigate the disease-causing mutation. Two daughters of an affected mother demonstrated symptoms of stage III ADNIV, with posterior uveitis, cystoid macular edema, intraocular fibrosis, retinal neovascularization, retinal degeneration, and cataract. The women also harbored a novel guanine to thymine (c.750G>T, p.Lys250Asn) missense mutation in exon 6 of CAPN5, a gene that encodes a calcium-activated cysteine protease, calpain-5. Modeling based on the structures of all known calpains revealed the mutation falls within a calcium-sensitive flexible gating loop that controls access to the catalytic groove. Three-dimensional modeling placed the new mutation in a region adjacent to two previously identified disease-causing mutations, all three of which likely disrupt hydrogen bonding within the gating loop, yielding a CAPN5 with altered enzymatic activity. This is the third case of a CAPN5 mutation leading to inherited uveitis and neovascular vitreoretinopathy, suggesting patients with ADNIV features should be tested for CAPN5 mutations. Structural modeling of novel variants can be used to support mechanistic consequences of the disease-causing variants.

Topics: Base Sequence; Calpain; Computational Biology; DNA Primers; Female; Fluorescein Angiography; Humans; Models, Molecular; Molecular Sequence Data; Mutation, Missense; Pedigree; Phenotype; Protein Conformation; Retinal Detachment; Sequence Analysis, DNA; Tomography, Optical Coherence; Uveitis; Vitreoretinopathy, Proliferative

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.

Topics: Adult; Amino Acid Sequence; Animals; Calpain; Disease Models, Animal; Exome; Humans; Inflammation Mediators; Lentivirus; Mice, Inbred C57BL; Mice, Transgenic; Molecular Sequence Data; Mutation, Missense; Photoreceptor Cells, Vertebrate; Retina; Transduction, Genetic; Vitreoretinopathy, Proliferative