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Abstract Sequence variants in LOXL1 coding for the secreted enzyme lysyl oxidase homolog 1 (LOXL1) associate with pseudoexfoliation (PEX) syndrome, a condition that is characterized by the deposition of extracellular fibrillar PEX material in the anterior eye and other parts of the body. Since the specific role of LOXL1 in the pathogenesis of PEX is unclear, and an increase in its expression was reported for early stages of PEX syndrome, we generated and studied transgenic mice with ocular overexpression of its mouse ortholog Loxl1. The chicken βB1-crystallin promoter was used to overexpress Loxl1 in the lenses of βB1-crystallin-Loxl1 transgenic mice. Transgenic lenses contained high levels of the protein LOXL1 and its mRNA, which were both not detectable in lenses of wildtype littermates. In wildtype mice, immunoreactivity for LOXL1 was mainly seen extracellularly in region of the ciliary zonules. βB1-crystallin-Loxl1 littermates showed an additional diffuse immunostaining in lens fibers and capsule, and in the inner limiting membrane and retina indicating secretion of soluble LOXL1 from transgenic lenses. In addition, lens fibers of transgenic animals contained multiple distinct spots of very intense LOXL1 immunoreactivity. By transmission electron microscopy, those spots correlated with electron-dense round or oval bodies of 20–50 nm in diameter which were localized in the rough endoplasmic reticulum and not seen in wildtype lenses. Immunogold electron microscopy confirmed that the electron-dense bodies contained LOXL1 indicating aggregation of insoluble LOXL1. Similar structures were seen in the extracellular lens capsule suggesting their secretion from lens fibers. Otherwise, no changes were seen between the eyes of βB1-crystallin-Loxl1 mice and their wildtype littermates, neither by light microscopy and funduscopy of whole eyes, nor by scanning and quantitative transmission electron microscopy of ciliary epithelium and zonules. At one month of age, intraocular pressure was significantly higher in transgenic mice than in wildtype littermates. No differences in IOP were seen though at 2–5 months of age. We conclude that LOXL1 has a strong tendency to aggregate in the rER when expressed in vivo at high amounts. A similar scenario, involving intracellular aggregation of LOXL1 and secretion of LOXL1 aggregates into the extracellular space, may be involved in the early pathogenetic events in eyes of PEX patients. Highlights • Transgenic overexpression of Loxl1 causes the formation of protein aggregates. • Protein aggregates in the Loxl1 transgenic mouse eye contain LOXL1. • LOXL1 aggregates are formed in the rER. • LOXL1 aggregates are released into the extracellular space. [ABSTRACT FROM AUTHOR]

Although rare, uveal melanoma (UM) is the most common primary intraocular tumor in adults. About half of UM patients develop metastatic disease typically in the liver and die within a short period, due to ineffective systemic therapies. UM has unique and distinct genetic features predictive of metastasis. Animal models are required to improve our understanding of therapeutic options in disseminated UM. Since spontaneous murine UM models are lacking, our aim was to analyze the suitability of the established transgenic melanoma mouse model Tg( Grm1 ) as a new UM model system. We demonstrated that adult Grm1 transgenic mice develop choroidal thickening and uveal melanocytic neoplasia with expression of the melanocytic markers S100B and MelanA. Further, we showed that GRM1 is expressed in human UM, similar to skin melanoma. This study presents a new mouse model for spontaneous UM and suggests that the glutamate signaling pathway is a possible target for UM therapy. [ABSTRACT FROM AUTHOR]

Abstract: Norrin is a retinal signaling molecule which is expressed in Müller glia and binds to Frizzled-4 to activate canonical Wnt/β-catenin signaling. Norrin is part of an essential signaling system that controls the formation of retinal capillaries during development. To evaluate neuroprotective properties of Norrin independently from its function during retinal angiogenesis, we generated transgenic mice (Rpe65–Norrin) that constitutively express Norrin in the retinal pigmented epithelium. Substantial amounts of Norrin were secreted into the outer retina, which triggered retinal Wnt/β-catenin signaling in conjunction with an increase in the expression of endothelin-2 (EDN2), endothelin receptor B (EDNRB), and glial fibrillary acidic protein (GFAP). Photoreceptors of Norrin-overexpressing mice were significantly less vulnerable to light-induced damage compared to their wild-type littermates. Following light damage, we observed less apoptotic death of photoreceptors and a better retinal function than in controls. The protective effects were abolished if either Wnt/β-catenin or EDN2 signaling was blocked by intravitreal injection of Dickkopf-1 or BQ788, respectively. Light-damaged retinae from transgenic mice contained higher amounts of brain-derived neurotrophic factor (BDNF) and pAkt than those of wild-type littermates. We conclude that constitutive overexpression of Norrin protects photoreceptors from light damage, an effect that is mediated by Wnt/β-catenin and EDN2 signaling and involves neurotrophic activities of BDNF. The findings suggest that Norrin and its associated signaling pathways have strong potentials to attenuate photoreceptor death following injury. [Copyright &y& Elsevier]

Quantifying the number of axons in the optic nerve is of interest in many research questions. Here, we show that a stereological method allows simple, efficient, precise and unbiased determination of the total axon number in the murine optic nerve. Axons in semi-thin optic nerve cross sections from untreated eyes (n = 21) and eyes subjected to retinal damage by intravitreous NMDA injections (n = 32) or PBS controls (n = 5) were manually identified, counted and digitally labeled by hand. A stereological procedure was empirically tested with systematic combinations of different sampling methods (simple random sampling without replacement, systematic uniform random sampling, stratified random sampling) and sampling parameters. Extensive numerical Monte Carlo experiments were performed to evaluate their large-sample properties. Our results demonstrate reliable determination of total axon number and superior performance compared to other methods at a small fraction of the time required for a full manual count. We specify suitable sampling parameters for the adoption of an efficient stereological sampling scheme, give empirical estimates of the additionally introduced sampling variance to facilitate experimental planning, and offer AxonCounter, an easy-to-use plugin implementing these stereological methods for the multi-platform image processing application NIH ImageJ. [ABSTRACT FROM AUTHOR]

There is an ongoing controversy regarding the role of WDR36 sequence variants in the pathogenesis of primary open-angle glaucoma (POAG). WDR36 is a nucleolar protein involved in the maturation of 18S rRNA. The function of WDR36 is essential as homozygous Wdr36 -deficient mouse embryos die before reaching the blastocyst stage. Here we provide a detailed analysis of the phenotype of heterozygous Wdr36 -deficient mice. Loss of one Wdr36 allele causes a substantial reduction in the expression of Wdr36 mRNA. In the eyes of Wdr36 +/− animals, the structure of the tissues involved in aqueous humor circulation and of the optic nerve head are not different from that of control littermates. In addition, one-year-old Wdr36 +/− animals do not differ from wild-type animals with regards to intraocular pressure and number of optic nerve axons. The susceptibility of retinal ganglion cells to excitotoxic damage induced by NMDA is similar in Wdr36 +/− and wild-type animals. Moreover, the amount of optic nerve axonal damage induced by high IOP is not different between Wdr36 +/− and wild-type mice. Transgenic overexpression of mutated Del605-607 Wdr36 in Wdr36 +/− animals does not cause changes in the number of optic nerve axons or susceptibility to excitotoxic damage. In addition, analysis of 18S rRNA maturation in Del605-607 Wdr36 +/− or Wdr36 +/− mice does not show obvious differences in rRNA processing or in the amounts of precursor forms when compared to wild-type animals. Our data obtained in Wdr36 +/− mice do not support the assumption of a causative role for WDR36 in the pathogenesis of POAG. [ABSTRACT FROM AUTHOR]

Mutations in the myocilin gene (MYOC) are causative for 10% of cases with juvenile open-angle glaucoma and 3-4% of those with primary open-angle glaucoma. Myocilin is a secreted protein with relatively ill-defined matricellular properties. Despite its high expression in the eye, myocilin-deficient mice have originally been reported to have no obvious ocular phenotype. Here we revisited the ocular phenotype of myocilin-deficient mice and detected a higher number of neurons in their inner (INL) and outer (ONL) nuclear layers, as well as a higher number of retinal ganglion cells (RGC) and their axons. The increase in retinal neurons appears to be caused by a decrease in programmed developmental cell death, as apoptosis of retinal neurons between postnatal days 4 and 10 was found to be attenuated when compared to that of wildtype littermates. In contrast, when Myoc-/- mice were crossed with βB1-crystallin-MYOC mice with ectopic overexpression of myocilin in the eye, no differences in developmental apoptosis, RGC number and INL thickness were observed when compared to wildtype littermates. The amounts of the anti-apoptotic Bcl-2-like protein 1 (BCL2L1, Bcl-xL) and its mRNA were increased in retinae of Myoc-/- mice, while lower amounts of BCL2L1 and its mRNA were detected in mixed Myoc-/-/βB1-crystallin-MYOC mice. The structural differences between Myoc-/- mice and wildtype littermates did not result in functional differences as measured by electroretinography. Noteworthy though mixed Myoc-/-/βB1-crystallin-MYOC mice with ocular overexpression of myocilin had significant cone function deficits. Myocilin appears to modulate apoptotic death of retinal neurons likely by interacting with the intrinsic apoptotic pathway. [ABSTRACT FROM AUTHOR]