The tissue inhibitors of metalloproteinases (TIMPs): An ancient family with structural and functional diversity

Animal models of age related macular degeneration.

The central retina mediates high acuity vision, and its progressive dysfunction due to macular degeneration is the leading cause of visual disability among adults in industrialized societies. Here, we summarize recent progress in understanding the pathophysiology of macular degeneration and the implications of this new knowledge for treatment and prevention. The past decade has witnessed remarkable advances in this field, including the development of new, non-invasive retinal imaging technologies, the development of animal models for macular disease, and the isolation of many of the genes responsible for both early- and late-onset macular diseases. These advances have set the stage for the development of effective mechanism-based therapies.

Macular degeneration: recent advances and therapeutic opportunities

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in Western countries.1 Considerable efforts have been made to establish AMD animal models.2-9 Even though mice have no macula, existing mouse AMD models have been shown to develop cardinal pathologic features of AMD.3,8-10 Strong evidence indicates the involvement of immunologic processes in AMD.11 Additionally, several studies have successfully demonstrated associations between AMD and various single-nucleotide polymorphisms (SNPs). Many of these SNPs are within genes encoding immunologic molecules.12-23

CX3CR1, the specific receptor for CX3CL1/fractalkine chemokine, is expressed on leukocytes (e.g., lymphocytes, macrophages, NK cells, mass cells), dendritic cells,24,25 brain microglia, and astrocytes.26-28 CX3CR1 expression is also reported in the eye,29 iris, ciliary body,30 retinal microglia, RPE, and Muller cell.31,32 In a functional study, two CX3CR1 SNPs resulted in a decreased number of CX3CL1 binding sites and reduced ligand-binding affinity on peripheral blood mononuclear cells.33 We have reported that these SNPs are associated with AMD.22 Furthermore, we have demonstrated a decreased number of CX3CR1 transcripts and protein in AMD maculae compared with the maculae of normal eyes.22,32 However, no ocular abnormalities have been reported in young adult Cx3cr1-deficient mice.

In addition to CX3CR1, CCL2 (MCP-1, a CC chemokine) is thought to play a homeostatic, immunoregulatory role in AMD pathogenesis.34 Aged mice with deficient Ccl2 or Ccr2, the corresponding receptor, develop many cardinal features of AMD including drusen formation, RPE accumulation of lipofuscin and complement factors, and choroidal neovascularization.8 In addition, CCL2 may function as an antiapoptotic factor, as reported in in vitro systems.35,36

In the present study, we examined the hypothesis that deficiencies in both Cx3cr1 and Ccl2 may induce typical pathologic features of AMD in mice more consistently and at an earlier age of onset than in existing animal models. We found that AMD features in the mice were highly characteristic and reproducible. In addition to inflammatory molecules, we also showed evidence for a potential role of endoplasmic reticulum proteins (ERp) in AMD.

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Murine Ccl2/Cx3cr1 Deficiency Results in Retinal Lesions Mimicking Human Age-Related Macular Degeneration

The inherited macular dystrophies comprise a heterogeneous group of disorders characterised by central visual loss and atrophy of the macula and underlying retinal pigment epithelium (RPE) The different forms of macular degeneration encompass a wide range of clinical, psychophysical and histological findings The complexity of the molecular basis of monogenic macular disease is now beginning to be elucidated with the identification of many of the disease-causing genes Age related macular degeneration (ARMD), the leading cause of blind registration in the developed world, may also have a significant genetic component to its aetiology Genes implicated in monogenic macular dystrophies are good candidate susceptibility genes for ARMD, although to date, with the possible exception of ABCA4, none of these genes have been shown to confer increased risk of ARMD The aim of this paper is to review current knowledge relating to the monogenic macular dystrophies, with discussion of currently mapped genes, chromosomal loci and genotype-phenotype relationships Inherited systemic disorders with a macular dystrophy component will not be discussed

https://jmg.bmj.com/content/jmedgenet/40/9/641.full.pdf

The genetics of inherited macular dystrophies

purpose. Sorsby fundus dystrophy (SFD) is a rare, late-onset macular dystrophy caused by mutations in the tissue inhibitor of metalloproteinases-3 (TIMP3) gene. The known mutations introduce potentially unpaired cysteine residues in the C terminus of the protein and result in the formation of higher-molecular-weight protein complexes of as yet unknown composition and functional consequences in the pathologic course of SFD. To facilitate in vivo investigation of mutant TIMP3, the authors generated a knock-in mouse carrying a disease-related Ser156Cys mutation in the orthologous murine Timp3 gene.

methods. Site-directed mutagenesis and homologous recombination in embryonic stem (ES) cells was used to generate mutant ES cells carrying the Timp3 S156C allele. Chimeric animals were obtained, of which two displayed germline transmission of the mutated allele. Molecular genetic, biochemical, electron microscopic, and electrodiagnostic techniques were used for characterization.

results. At 8 months of age, knock-in mice showed abnormalities in the inner aspect of Bruch’s membrane and in the organization of the adjacent basal microvilli of the retinal pigment epithelium (RPE). Changes resembling those in the mutant animals were also present to some extent in normal littermates, but only at an advanced age of 30 months. Long-term electrodiagnostic recordings indicated normal retinal function throughout life. The biochemical characteristics of the mutant protein appear similar in humans and knock-in mice, suggesting common molecular pathways in the two species. The localization of the mutant protein in the eye is normal, although there is evidence of increased Timp3 levels in Bruch’s membrane of mutant animals.

conclusions. The knock-in mice display early features of age-related changes in Bruch’s membrane and the RPE that may represent the primary clinical manifestations of SFD. In addition, our immunolabeling studies and biochemical data support a model proposing that site-specific excess rather than absence or deficiency of functional Timp3 may be the primary consequence of the known Timp3 mutations.

A mouse model for Sorsby fundus dystrophy.

The tissue inhibitor of metalloproteinases-3 (TIMP3) is a multifunctional protein tightly associated with the extracellular matrix (ECM). A specific type of mutation in TIMP3 which results in potentially unpaired cysteine residues at the C-terminus of the protein has been shown to cause Sorsby fundus dystrophy (SFD), an autosomal dominant retinopathy of late onset. An early finding in SFD is a striking accumulation of protein and lipid material in Bruch's membrane, a multilayered ECM structure located between the choroid and the RPE. To study the molecular mechanisms underlying SFD pathology, we recently generated two mouse lines, one deficient in Timp3 (Timp3(-/-)) and one carrying an SFD-related mutation in the orthologous murine Timp3 gene (Timp3(S156C/S156C)). We now established immortalized fibroblast cells from the mutant mouse strains and provide evidence that the various cell lines display distinct morphological and physiological features that are dependent on the mutational status of the Timp3 protein in the secreted ECM. We show that matrix metalloproteinase (MMP) activity and inhibitory properties of Timp3 are not affected by the SFD-associated mutation. We further demonstrate that Timp3(S156C) protein accumulates in the ECM of the mutant fibroblast cells and that this accumulation is not due to a prolonged turnover rate of mutant vs. normal Timp3. We also show that the relative abundance of mutant and normal Timp3 in the ECM has no measurable effects on cellular phenotypes. Together, these findings suggest (i) a functional role of normal Timp3 in pathways determining cellular morphology and (ii) a loss of this particular function as a consequence of the Ser156Cys mutation. We therefore hypothesize that SFD pathogenesis is due to a loss-of-function mutation in TIMP3.

Galina Soboleva

Papers

A mouse model for Sorsby fundus dystrophy.

Sorsby fundus dystrophy mutation Timp3S156C affects the morphological and biochemical phenotype but not metalloproteinase homeostasis