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Keratoconus (KC) is the most common corneal ectatic disorder affecting >300,000 people in the US. KC normally has its onset in adolescence, progressively worsening through the third to fourth decades of life. KC patients report significant impaired vision-related quality of life. Genetic factors play an important role in KC pathogenesis. To identify novel genes in familial KC patients, we performed whole exome and genome sequencing in a four-generation family. We identified potential variants in the PPIP5K2 and PCSK1 genes. Using in vitro cellular model and in vivo gene-trap mouse model, we found critical evidence to support the role of PPIP5K2 in normal corneal function and KC pathogenesis. The gene-trap mouse showed irregular corneal surfaces and pathological corneal thinning resembling KC. For the first time, we have integrated corneal tomography and pachymetry mapping into characterization of mouse corneal phenotypes which could be widely implemented in basic and translational research for KC diagnosis and therapy in the future.

Introduction:Neuroprotective therapeutics are needed to treat glaucoma, an optic neuropathy that results in death of retinal ganglion cells (RGCs). Areas covered:The BDNF/TrkB pathway is important for RGC survival. Temporal and spatial alterations in the BDNF/TrkB pathway occur in development and in response to acute optic nerve injury and to glaucoma. In animal models, BDNF supplementation is successful at slowing RGC death after acute optic nerve injury and in glaucoma, however, the BDNF/TrkB signaling is not the only pathway supporting long term RGC survival. Expert commentary:Much remains to be discovered about the interaction between retrograde, anterograde, and retinal BDNF/TrkB signaling pathways in both neurons and glia. An ideal therapeutic agent for glaucoma likely has several modes of action that target multiple mechanisms of neurodegeneration including the BDNF/TrkB pathway. [ABSTRACT FROM PUBLISHER]

Purpose: Sigma receptors 1 (σR1) and 2 (σR2) are thought to be two distinct proteins which share the ability to bind multiple ligands, several of which are common to both receptors. Whether σR1 and σR2 share overlapping biological functions is unknown. Recently, progesterone receptor membrane component 1 (PGRMC1) was shown to contain the putative σR2 binding site. PGRMC1 has not been studied in retina. We hypothesize that biological interactions between σR1 and PGRMC1 will be evidenced by compensatory upregulation of PGRMC1 inσR1−/−mice. Methods: Immunofluorescence, RT-PCR, and immunoblotting methods were used to analyze expression of PGRMC1 in wild-type mouse retina. Tissues fromσR1−/−mice were used to investigate whether a biological interaction exists between σR1 and PGRMC1. Results: In the eye, PGRMC1 is expressed in corneal epithelium, lens, ciliary body epithelium, and retina. In retina, PGRMC1 is present in Müller cells and retinal pigment epithelium. This expression pattern is similar, but not identical to σR1. PGRMC1 protein levels in neural retina and eye cup fromσR1−/−mice did not differ from wild-type mice. Nonocular tissues, lung, heart, and kidney showed similarPgrmc1gene expression in wild-type andσR1−/−mice. In contrast, liver, brain, and intestine showed increasedPgrmc1gene expression inσR1−/−mice. Conclusion: Despite potential biological overlap, deletion of σR1 did not result in a compensatory change in PGRMC1 protein levels inσR1−/−mouse retina. IncreasedPgrmc1gene expression in organs with high lipid content such as liver, brain, and intestine indicates a possible tissue-specific interaction between σR1 and PGRMC1. The current studies establish the presence of PGRMC1 in retina and lay the foundation for analysis of its biological function. [ABSTRACT FROM AUTHOR]

Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults in United States. Research indicates an association between oxidative stress and the development of diabetes complications. However, clinical trials with general antioxidants have failed to prove effective in diabetic patients. Mounting evidence from experimental studies that continue to elucidate the damaging effects of oxidative stress and inflammation in both vascular and neural retina suggest its critical role in the pathogenesis of DR. This review will outline the current management of DR as well as present potential experimental therapeutic interventions, focusing on molecules that link oxidative stress to inflammation to provide potential therapeutic targets for treatment or prevention of DR. Understanding the biochemical changes and the molecular events under diabetic conditions could provide new effective therapeutic tools to combat the disease. [ABSTRACT FROM AUTHOR]

Elevated glutamate levels have been reported in humans with diabetic retinopathy. Retinal Müller glial cells regulate glutamate levels via the GLAST transporter and system xc − (cystine-glutamate exchanger). We have investigated whether transporter function and gene and/or protein expression are altered in mouse Müller cells cultured under conditions of hyperglycemia or oxidative stress (two factors implicated in diabetic retinopathy). Cells were subjected to hyperglycemic conditions (35 mM glucose) over an 8-day period or to oxidative stress conditions (induced by exposure to various concentrations of xanthine:xanthine oxidase) for 6 h. The Na+-dependent and –independent uptake of [3H] glutamate was assessed as a measure of GLAST and system xc − function, respectively. Hyperglycemia did not alter the uptake of [3H] glutamate by GLAST or system xc −; neither gene nor protein expression decreased. Oxidative stress (70:14 or 100:20 μM xanthine:mU/ml xanthine oxidase) decreased GLAST activity by ~10% but increased system xc − activity by 43% and 89%, respectively. Kinetic analysis showed an oxidative-stress-induced change in Vmax, but not Km. Oxidative stress caused a 2.4-fold increase in mRNA encoding xCT, the unique component of system xc −. Of the two isoforms of xCT (40 and 50 kDa), oxidative stress induced a 3.6-fold increase in the 40-kDa form localized to the plasma membrane. This is the first report of the differential expression and localization of xCT isoforms as caused by cellular stress. Increased system xc − activity in Müller cells subjected to conditions associated with diabetic retinopathy may be beneficial, as this exchanger is important for the synthesis of the antioxidant glutathione. [ABSTRACT FROM AUTHOR]

Accumulation of the nerve growth factor precursor (proNGF) and its receptor p75(NTR) have been associated with several neurodegenerative diseases in both brain and retina. However, whether proNGF contributes to microvascular degeneration remain unexplored. This study seeks to investigate the mechanism by which proNGF/p75(NTR) induce endothelial cell (EC) death and development of acellular capillaries, a surrogate marker of retinal ischemia. Stable overexpression of the cleavage-resistant proNGF and molecular silencing of p75(NTR) were utilized in human retinal EC and rat retinas in vivo. Stable overexpression of proNGF decreased NGF levels and induced retinal vascular cell death evident by 1.9-fold increase in acellular capillaries and activation of JNK and cleaved-PARP that were mitigated by p75(NTR)shRNA. In vitro, overexpression of proNGF did not alter TNF-α level, reduced NGF, however induced EC apoptosis evident by activation of JNK and p38 MAPK, cleaved-PARP. Silencing p75(NTR) using siRNA restored expression of NGF and TrkA activation and prevented EC apoptosis. Treatment of EC with human-mutant proNGF induced apoptosis that coincided with marked protein interaction and nuclear translocation of p75(NTR) and the neurotrophin receptor interacting factor. These effects were abolished by a selective p75(NTR) antagonist. Therefore, targeting p75(NTR) represents a potential therapeutic strategy for diseases associated with aberrant expression of proNGF.

Purpose: To determine the effects of endogenous elevation of homocysteine on the retina using the cystathionine beta-synthase (cbs) mutant mouse., Methods: Retinal homocysteine in cbs mutant mice was measured by high-performance liquid chromatography (HPLC). Retinal cryosections from cbs(-/-) mice and cbs(+/-) mice were examined for histologic changes by light and electron microscopy. Morphometric analysis was performed on retinas of cbs(+/-) mice maintained on a high-methionine diet (cbs(+/-) HM). Changes in retinal gene expression were screened by microarray., Results: HPLC analysis revealed an approximate twofold elevation in retinal homocysteine in cbs(+/-) mice and an approximate sevenfold elevation in cbs(-/-) mice. Distinct alterations in the ganglion, inner plexiform, inner nuclear, and epithelial layers were observed in retinas of cbs(-/-) and 1-year-old cbs(+/-) mice. Retinas of cbs(+/-) HM mice demonstrated an approximate 20% decrease in cells of the ganglion cell layer (GCL), which occurred as early as 5-weeks after onset of the HM diet. Microarray analysis revealed alterations in expression of several genes, including increased expression of Aven, Egr1, and Bat3 in retinas of cbs(+/-) HM mice., Conclusions: This study provides the first analysis of morphologic and molecular effects of endogenous elevations of retinal homocysteine in an in vivo model. Increased retinal homocysteine alters inner and outer retinal layers in cbs homozygous mice and older cbs heterozygous mice, and it primarily affects the cells of the GCL in younger heterozygous mice. Elevated retinal homocysteine alters expression of genes involved in endoplasmic reticular stress, N-methyl-d-aspartate (NMDA) receptor activation, cell cycle, and apoptosis.