Localization of complement 1 inhibitor (C1INH/SERPING1) in human eyes with age-related macular degeneration

Age-related macular degeneration (AMD) is a common degenerative disease resulting in injury to the retina, retinal pigment epithelium and choriocapillaris. Recent data from histopathology, animal models and genetic studies have implicated altered regulation of the complement system as a major factor in the incidence and progression of this disease. A variant in the gene SERPING1, which encodes C1INH, an inhibitor of the classical and lectin pathways of complement activation, was recently shown to be associated with AMD. In this study we sought to determine the localization of C1INH in human donor eyes. Immunofluorescence studies using a monoclonal antibody directed against C1INH revealed localization to photoreceptor cells, inner nuclear layer neurons, choriocapillaris, and choroidal extracellular matrix. Drusen did not exhibit labeling. Genotype at rs2511989 did not appear to affect C1INH abundance or localization, nor was it associated with significant molecular weight differences when evaluated by Western blot. In a small number of eyes (n = 7 AMD and n = 7 control) AMD affection status was correlated with increased abundance of choroidal C1INH. These results indicate that C1INH protein is present in the retina and choroid, where it may regulate complement activation

Calpain-5 Mutations Cause Autoimmune Uveitis, Retinal Neovascularization, and Photoreceptor Degeneration

<div><p>Autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) is an autoimmune condition of the eye that sequentially mimics uveitis, retinitis pigmentosa, and proliferative diabetic retinopathy as it progresses to complete blindness. We identified two different missense mutations in the <em>CAPN5</em> gene in three ADNIV kindreds. <em>CAPN5</em> encodes calpain-5, a calcium-activated cysteine protease that is expressed in retinal photoreceptor cells. Both mutations cause mislocalization from the cell membrane to the cytosol, and structural modeling reveals that both mutations lie within a calcium-sensitive domain near the active site. <em>CAPN5</em> is only the second member of the large calpain gene family to cause a human Mendelian disorder, and this is the first report of a specific molecular cause for autoimmune eye disease. Further investigation of these mutations is likely to provide insight into the pathophysiologic mechanisms of common diseases ranging from autoimmune disorders to diabetic retinopathy.</p> </div

ADNIV pedigrees.

<p>A–C. Three families with clinical features of ADNIV exhibit a dominant pattern of inheritance. Black symbols represent clinically affected subjects. Open symbols represent unaffected subjects. Deceased individuals are marked by a slash.</p

ADNIV phenotype.

<p>A–B. Clusters of autoimmune reactive leukocytes are visible in the vitreous cavity (inset, arrows). C. Electroretinography shows loss of the b-wave. D. Fundus image of the normal retina. E. Fundus image of the ADNIV retina shows pigmentary degeneration (arrow) similar to retinitis pigmentosa. F. Fluorescein angiography reveals cystoid macular edema at the fovea (arrow), a consequence of autoimmune intraocular inflammation. G. Preretinal fibrosis leads to tractional retinal detachments. H. Vitreous hemorrhage (arrow) from retinal neovascularization. I. Phthisis bulbi and involution of eye tissues in end-stage ADNIV.</p

Protein structure modeling of calpain-5 and ADNIV mutants.

<p>A. Both ADNIV mutations (red arrows) are located in exon 6, which encodes a portion of the catalytic domain and two of three catalytic residues (blue arrows). Primary protein sequence analysis shows the ADNIV-1/2 and ADNIV-3 mutations to be 8–9 amino acids upstream of the catalytic histidine residue. Secondary structure modeling shows that the two mutations are within a putative alpha helical domain. One mutated codon results in the loss of a basic residue, while the other introduces a proline into the putative alpha helix. B. Alignment of calpain-5 orthologs shows very high evolutionary conservation of the mutated residues (red arrows). Amino acid mismatches are color-highlighted. C. Twelve human calpain paralogs show significant differences in exon 6 (Black, 100% similarity; Dark grey, 80–100% similarity; Light grey, 60–80% similarity; White, less than 60% similarity). D. Three-dimensional modeling of the catalytic domain shows the location of the active site cleft (red outline). E. Magnified view of the active site cleft shows the catalytic triad (dashed line – blue text) and location of the two mutations (red arrows and text). Both mutations are located within a peptide loop that is homologous to a flexible loop of calpain-1 that undergoes a calcium-induced conformational change in association with regulation of the active site cleft (see text).</p

The <i>CAPN5</i> gene harbors mutations in exon 6 of ADNIV subjects.

<p>A. The ADNIV locus was previously mapped to chromosome 11q13 (green bar). STRP and SNP array mapping narrowed the interval (red bar). B. <i>CAPN5</i> gene structure is composed of 13 exons. C. Chromatogram of normal <i>CAPN5</i> DNA sequence in exon 6. D–E. Chromatograms of ADNIV subjects shows mutations in <i>CAPN5</i> exon 6. F. SSCP distinguishes between normal sequence and ADNIV mutations.</p

Calpain-5 expression in human retinal photoreceptor cells and cultured cells.

<p>A. No significant signal was detected in control retinal sections probed with secondary antibody or primary antibody blocked with recombinant calpain-5. DAPI highlights the cell nuclei (blue) B. Calpain-5 was detected in photoreceptor cells (green). A punctate expression pattern was most prominent overlying the photoreceptor nuclei in the outer nuclear layer (ONL) and inner and outer segments (IS, OS). C. Anti-myc antibody western blot detects a single species (black arrow) of the appropriate size in HEK293T cells transfected with a vector bearing normal, myc-tagged <i>CAPN5</i>. D. No significant anti-myc signal was detected in the nuclei (white arrowhead) or cytoplasm (white arrow) of control cells that were treated with transfection reagent alone, vector alone, or secondary antibody alone. E. Anti-myc antibody shows that calpain-5 (green) is expressed in a ruffled pattern (white arrow) that obscures the underlying nuclei (white arrowhead) suggesting a location near the cell surface in these very thin cultured cells. F. Transfection with a mutant <i>CAPN5</i> (Arg243Leu) exhibits a more uniform anti-myc signal that does not obscure the nuclei (white arrowhead) and is thus more compatible with localization to the cytoplasm (white arrow).</p

The role of group I metabotropic glutamate receptor's in neuronal excitotoxicity in Alzheimer's disease

Neurodegenerative diseases such as Huntington's disease, ischemia, and Alzheimer's disease (AD) are major causes of death. Recently, metabotropic glutamate receptors (mGluRs), a group of seven-transmembrane-domain proteins that couple to G-proteins, have become of interest for studies of pathogenesis. Group I mGluRs control the levels of second messengers such as inositol 1,4,5-triphosphate (IP3) Cal(2+) ions and cAMP. They elicit the release of arachidonic acid via intracellular Ca2+ mobilization from intracellular stores such as mitochondria and endoplasmic reticulum. This facilitates the release of glutamate and could trigger the formation of neurofibrillary tangles, a pathological hallmark of AD. mGluRs regulate neuronal injury and survival, possibly through a series of downstream protein kinase and cysteine protease signaling pathways that affect mitochondrially mediated programmed cell death. They may also play a role in glutamate-induced neuronal death by facilitating Cal(2+) mobilization. Hence, mGluRs have become a target for neuroprotective drug development. They represent a pharmacological path to a relatively subtle amelioration of neurotoxicity because they serve a modulatory rather than a direct role in excitatory glutamatergic transmission