Compositional Analysis of Extracellular Aggregates in the Eyes of Patients With Exfoliation Syndrome and Exfoliation Glaucoma.

Purpose: Exfoliation syndrome (XFS) is a condition characterized by the production of insoluble fibrillar aggregates (exfoliation material; XFM) in the eye and elsewhere. Many patients with XFS progress to exfoliation glaucoma (XFG), a significant cause of global blindness. We used quantitative mass spectrometry to analyze the composition of XFM in lens capsule specimens and in aqueous humor (AH) samples from patients with XFS, patients with XFG and unaffected individuals. Methods: Pieces of lens capsule and samples of AH were obtained with consent from patients undergoing cataract surgery. Tryptic digests of capsule or AH were analyzed by high-performance liquid chromatography-mass spectrometry and relative differences between samples were quantified using the tandem mass tag technique. The distribution of XFM on the capsular surface was visualized by SEM and super-resolution light microscopy. Results: A small set of proteins was consistently upregulated in capsule samples from patients with XFS and patients with XFG, including microfibril components fibrillin-1, latent transforming growth factor-ß-binding protein-2 and latent transforming growth factor-ß-binding protein-3. Lysyl oxidase-like 1, a cross-linking enzyme associated with XFS in genetic studies, was an abundant XFM constituent. Ligands of the transforming growth factor-ß superfamily were prominent, including LEFTY2, a protein best known for its role in establishing the embryonic body axis. Elevated levels of LEFTY2 were also detected in AH from patients with XFG, a finding confirmed subsequently by ELISA. Conclusions: This analysis verified the presence of suspected XFM proteins and identified novel components. Quantitative comparisons between patient samples revealed a consistent XFM proteome characterized by strong expression of fibrillin-1, lysyl oxidase-like-1, and LEFTY2. Elevated levels of LEFTY2 in the AH of patients with XFG may serve as a biomarker for the disease.

Enzyme Replacement Therapy Ameliorates Multiple Symptoms of Murine Homocystinuria.

Classical homocystinuria (HCU) is the most common inherited disorder of sulfur amino acid metabolism caused by deficiency in cystathionine beta-synthase (CBS) activity and characterized by severe elevation of homocysteine in blood and tissues. Treatment with dietary methionine restriction is not optimal, and poor compliance leads to serious complications. We developed an enzyme replacement therapy (ERT) and studied its efficacy in a severe form of HCU in mouse (the I278T model). Treatment was initiated before or after the onset of clinical symptoms in an effort to prevent or reverse the phenotype. ERT substantially reduced and sustained plasma homocysteine concentration at around 100 µM and normalized plasma cysteine for up to 9 months of treatment. Biochemical balance was also restored in the liver, kidney, and brain. Furthermore, ERT corrected liver glucose and lipid metabolism. The treatment prevented or reversed facial alopecia, fragile and lean phenotype, and low bone mass. In addition, structurally defective ciliary zonules in the eyes of I278T mice contained low density and/or broken fibers, while administration of ERT from birth partially rescued the ocular phenotype. In conclusion, ERT maintained an improved metabolic pattern and ameliorated many of the clinical complications in the I278T mouse model of HCU.

Somatic Variants in the Human Lens Epithelium: A Preliminary Assessment.

PURPOSE: We hypothesize that somatic mutations accumulate in cells of the human lens and may contribute to the development of cortical or posterior sub-capsular cataracts. Here, we used a Next-generation sequencing (NGS) strategy to screen for low-allelic frequency variants in DNA extracted from human lens epithelial samples. METHODS: Next-Generation sequencing of 151 cancer-related genes (WUCaMP2 panel) was performed on DNA extracted from post-mortem or surgical specimens obtained from 24 individuals. Usually, pairwise comparisons were made between two or more ocular samples from the same individual, allowing putative somatic variants detected in lens samples to be differentiated from germline variants. RESULTS: Use of a targeted hybridization approach enabled high sequence coverage (>1000-fold) of the WUCaMP2 genes. In addition to high-frequency variants (corresponding to homozygous or heterozygous SNPs and Indels), somatic variants with allelic frequencies of 1-4% were detected in the lens epithelial samples. The presence of one such variant, a T > C point substitution at position 32907082 in BRCA2, was verified subsequently using droplet digital PCR. CONCLUSIONS: Low-allelic fraction variants are present in the human lens epithelium, at frequencies consistent with the presence of millimeter-sized clones.

Birc7: A Late Fiber Gene of the Crystalline Lens.

PURPOSE: A distinct subset of genes, so-called "late fiber genes," is expressed in cells bordering the central, organelle-free zone (OFZ) of the lens. The purpose of this study was to identify additional members of this group. METHODS: Fiber cells were harvested from various layers of the lens by laser micro-dissection and subjected to microarray, in situ hybridization, and Western blot analysis. RESULTS: Expression of Livin, a member of the inhibitor of apoptosis protein (IAP) family encoded by Birc7, was strongly upregulated in deep cortical fiber cells. The depth-dependent distribution of Livin mRNA was confirmed by quantitative PCR and in situ hybridization. The onset of Livin expression coincided with loss of organelles from primary fiber cells. Livin expression peaked at 1 month but was sustained even in aged lenses. Antibodies raised against mouse Livin labeled multiple bands on immunoblots, reflecting progressive proteolysis of the parent molecule during differentiation. Mice harboring a floxed Birc7 allele were generated and used to conditionally delete Birc7 in lens. Lenses from knockout mice grew normally and retained their transparency, suggesting that Livin does not have an indispensable role in fiber cell differentiation. CONCLUSIONS: Birc7 is a late fiber gene of the mouse lens. In tumor cells, Livin acts as an antiapoptotic protein, but its function in the lens is enigmatic. Livin is a RING domain protein with putative E3 ubiquitin ligase activity. Its expression in cells bordering the OFZ is consistent with a role in organelle degradation, a process in which the ubiquitin proteasome pathway has been implicated previously.

The penny pusher: a cellular model of lens growth.

PURPOSE: The mechanisms that regulate the number of cells in the lens and, therefore, its size and shape are unknown. We examined the dynamic relationship between proliferative behavior in the epithelial layer and macroscopic lens growth. METHODS: The distribution of S-phase cells across the epithelium was visualized by confocal microscopy and cell populations were determined from orthographic projections of the lens surface. RESULTS: The number of S-phase cells in the mouse lens epithelium fell exponentially, to an asymptotic value of approximately 200 cells by 6 months. Mitosis became increasingly restricted to a 300-µm-wide swath of equatorial epithelium, the germinative zone (GZ), within which two peaks in labeling index were detected. Postnatally, the cell population increased to approximately 50,000 cells at 4 weeks of age. Thereafter, the number of cells declined, despite continued growth in lens dimensions. This apparently paradoxical observation was explained by a time-dependent increase in the surface area of cells at all locations. The cell biological measurements were incorporated into a physical model, the Penny Pusher. In this simple model, cells were considered to be of a single type, the proliferative behavior of which depended solely on latitude. Simulations using the Penny Pusher predicted the emergence of cell clones and were in good agreement with data obtained from earlier lineage-tracing studies. CONCLUSIONS: The Penny Pusher, a simple stochastic model, offers a useful conceptual framework for the investigation of lens growth mechanisms and provides a plausible alternative to growth models that postulate the existence of lens stem cells.

Cadm1 expression and function in the mouse lens.

PURPOSE: The immunoglobulin superfamily member Cadm1 is a single-pass, type 1 membrane protein that mediates calcium-independent, cell-cell adhesion. Cadm1 has been implicated in tumor formation and synaptogenesis. A recent analysis of mouse lens cell membranes identified Cadm1 as a major constituent of the fiber cell membrane proteome. Here the authors examined the expression and function of Cadm1 in the mouse lens. METHODS: Cadm1 expression was analyzed by Western blotting and immunofluorescence. The morphology of individual wild-type and Cadm1-null lens cells was visualized by confocal microscopy. RESULTS: Cadm1 was present in epithelial and superficial fiber cells as a heavily glycosylated protein with an apparent molecular mass of ≈80 kDa. Analysis of proteins extracted from various strata of the lens indicated that Cadm1 was degraded during fiber cell differentiation, at approximately the same time as the lens organelles, an observation confirmed by confocal microscopy. In epithelial cells, Cadm1 was enriched in basolateral membranes, whereas, in fiber cells, expression was restricted to the lateral membranes. Lenses from Cadm1-null mice were of normal size and transparency. The three-dimensional morphology of the cells in the epithelial layer was unaltered in the absence of Cadm1. However, in contrast to wild-type lens fiber cells, Cadm1-null fiber cells had an irregular, highly undulating morphology. CONCLUSIONS: Cadm1 is an abundant component of the lens fiber cell membrane. Although not essential for lens transparency, Cadm1 has an indispensable role in establishing and maintaining the characteristic three-dimensional architecture of the lens fiber cell mass.

A method for determining cell number in the undisturbed epithelium of the mouse lens.

The anterior face of the mouse lens is covered by a layer of epithelial cells. The epithelial cells serve a barrier function at the lens surface and as a progenitor population from which lens fiber cells, the predominant cell type of the lens, are derived. Decreased epithelial cell density is commonly observed during aging and cataract formation in humans and animal models and may contribute directly to tissue opacification. However, the loss of cells from the epithelium is often not easy to quantify, in part because the cells are arrayed across a near-spherical surface and, as a consequence, are difficult to image and count. Here, we describe a technique for determining epithelial cell number in the undisturbed lens of the mouse, a popular cataract model. The method utilizes orthographic projections of confocal images collected from the anterior and equatorial regions of the lens. The overlapping projections are brought into register using the unique distribution of proliferating cells as fiduciary points. Cell counts are performed using a computer-assisted method. This approach offers several advantages over flat-mount methods employed previously.

The stratified syncytium of the vertebrate lens.

The fusion of cells to generate syncytial tissues is a crucial event in the development of many organisms. In the lens of the vertebrate eye, proteins and other macromolecules diffuse from cell to cell via the large molecule diffusion pathway (LMDP). We used the tamoxifen-induced expression of GFP to investigate the nature and role of the LMDP in living, intact lenses. Our data indicate that the LMPD preferentially connects cells lying within a stratum of the lens cortex and that formation of the LMPD depends on the expression of Lim2, a claudin-like molecule. The conduits for intercellular protein exchange are most likely regions of partial cellular fusion, which are commonly observed in wild-type lenses but rare or absent in Lim2-deficient lenses. The observation that lens tissue constitutes a stratified syncytium has implications for the transparency, refractive function and pathophysiology of the tissue.

Inducible gene expression in the lens using tamoxifen and a GFP reporter.

In recent years, P1 phage Cre-recombinase has become an indispensable tool for conditional activation and/or inactivation of genes in the mouse. By fusing Cre to a tamoxifen-sensitive form of the estrogen receptor (Cre-ER) temporal regulation of gene expression can be achieved. Here, we report the initial characterization of the Cre-ER system for controlling gene expression in the lens of the mouse. Cre-ER mice were crossed with a reporter strain (Z/EG; lacZ/EGFP). Following i.p. injection of tamoxifen into Cre-ER;Z/EG mice, green fluorescent protein (GFP) expression was observed in 1-5% of lens epithelial and fiber cells. GFP expression was maintained for at least 6 months although, in the fibers, GFP appeared to diffuse from the cells as they were internalized into the lens. The rate of elongation of the fiber cells was determined by measuring the length of GFP-expressing cells at intervals after tamoxifen injection. The results of this calculation suggested that tamoxifen treatment induced GFP expression predominantly in lens epithelial cells. The GFP-positive fibers were apparently derived from this cell population. The strong induced expression of GFP in a small proportion of lens cells allowed individual lens cells to be identified in the intact tissue and should serve as a useful lineage marker to track the fate of lens cells and their descendents.

Proteolytic mechanisms underlying mitochondrial degradation in the ocular lens.

PURPOSE: To remove light-scattering structures from the visual axis, all intracellular organelles are eliminated from cells in the center of the developing ocular lens. Organelle degradation is accompanied by an increase in VEIDase (caspase-6-like) activity, but data from caspase-null mice suggest that the lens VEIDase is not caspase-6. The goal of the present work was to identify the lens VEIDase and determine whether it plays a role in organelle breakdown. METHODS: The approximate molecular mass of the lens VEIDase was determined by size-exclusion chromatography. Three proteasome inhibitors (NLVS, MG132, and clasto-lactacystin beta-lactone) were tested for their ability to inhibit lens VEIDase activity. Lens lysates were immunodepleted of proteasomes using an antibody against the 20S proteasome. To inhibit the ubiquitin-proteasome pathway (UPP) in vivo, lactacystin was injected into the vitreous humor of the developing chicken eye. The effect of lactacystin on mitochondrial degradation was assessed by examining the disappearance of succinate-ubiquinone oxidoreductase, an integral protein of the inner mitochondrial membrane. RESULTS: The lens VEIDase eluted at approximately 700 kDa from a size-exclusion column and was inhibited by the proteasome inhibitors NLVS, MG132, and clasto-lactacystin beta-lactone. In vivo, the trypsin-like activity of the proteasome was reduced by 60% to 70% after lactacystin injection. Proteasome inhibition was associated with the accumulation of ubiquitinated proteins and reversible opacification of the lens cortex. In lactacystin-injected eyes, the programmed degradation of succinate-ubiquinone oxidoreductase was inhibited in the central lens fiber cells. CONCLUSIONS: These data suggest that lens VEIDase activity is attributable to the proteasome and that the UPP may function in the removal of organelle components during lens fiber cell differentiation.

Three-dimensional reconstruction of cells in the living lens: the relationship between cell length and volume.

During terminal differentiation, lens fiber cells increase in length by 100-1000 fold. The mechanism of cellular elongation is not well understood but previous measurements on differentiating embryonic fiber cells indicated that cell volume increases in direct proportion to cell length. Fiber cells are tightly packed within the lens. Under these circumstances, a cellular volume increase might be transduced into an increase in length. Such considerations have led to the suggestion that volume increase may be the driving force for cell elongation. In the present study, we tested this model using a strain of mice in which a GFP transgene is expressed sporadically in the lens. We employed a combination of confocal microscopy, image deconvolution, and volume-rendering techniques to visualize the structure of individual GFP-expressing cells within the living lens. We examined their morphology at various stages of differentiation; from the central epithelium to young fiber cells. At each stage, cell length and volume were determined. In contrast to earlier studies, our data indicated that increases in cell length were not matched by a proportional increase in volume. Rather, the initial phase of elongation (in which cells increased in length from <10 to >150 microm) appeared to result largely from a change in cell shape. The present observations suggest that the driving force for elongation of primary and secondary fiber cells may differ fundamentally.

Regulation of tissue oxygen levels in the mammalian lens.

Opacification of the lens nucleus is a major cause of blindness and is thought to result from oxidation of key cellular components. Thus, long-term preservation of lens clarity may depend on the maintenance of hypoxia in the lens nucleus. We mapped the distribution of dissolved oxygen within isolated bovine lenses and also measured the rate of oxygen consumption (QO2) by lenses, or parts thereof. To assess the contribution of mitochondrial metabolism to the lens oxygen budget, we tested the effect of mitochondrial inhibitors on (QO2) and partial pressure of oxygen (PO2). The distribution of mitochondria was mapped in living lenses by 2-photon microscopy. We found that a steep gradient of PO2 was maintained within the tissue, leading to PO2 < 2 mmHg in the core. Mitochondrial respiration accounted for approximately 90% of the oxygen consumed by the lens; however, PO2 gradients extended beyond the boundaries of the mitochondria-containing cell layer, indicating the presence of non-mitochondrial oxygen consumers. Time constants for oxygen consumption in various regions of the lens and an effective oxygen diffusion coefficient were calculated from a diffusion-consumption model. Typical values were 3 x 10(-5) cm(2) s(-1) for the effective diffusion coefficient and a 5 min time constant for oxygen consumption. Surprisingly, the calculated time constants did not differ between differentiating fibres (DF) that contained mitochondria and mature fibres (MF) that did not. Based on these parameters, DF cells were responsible for approximately 88% of lens oxygen consumption. A modest reduction in tissue temperature resulted in a marked decrease in (QO2) and the subsequent flooding of the lens core with oxygen. This phenomenon may be of clinical relevance because cold, oxygen-rich solutions are often infused into the eye during intraocular surgery. Such procedures are associated with a strikingly high incidence of postsurgical nuclear cataract.

Intravitreal gene therapy reduces lysosomal storage in specific areas of the CNS in mucopolysaccharidosis VII mice.

The mucopolysaccharidoses (MPSs) are lysosomal storage diseases resulting from impaired catabolism of sulfated glycosaminoglycans. MPS VII mice lack lysosomal beta-glucuronidase (GUSB) activity, leading to the accumulation of partially degraded chondroitin, dermatan, and heparan sulfates in most tissues. Consequently, these mice develop most of the symptoms exhibited by human MPS VII patients, including progressive visual and cognitive deficits. To investigate the effects of reducing lysosomal storage in nervous tissues, we injected recombinant adeno-associated virus encoding GUSB directly into the vitreous humor of young adult mice. Interestingly, GUSB activity was subsequently detected in the brains of the recipients. At 8-12 weeks after treatment, increased GUSB activity and reduced lysosomal distension were found in regions of the thalamus and tectum that received inputs from the injected eye. Lysosomal storage was also reduced in adjacent nonvisual regions, including the hippocampus, as well as in the visual cortex. The findings suggest that both diffusion and trans-synaptic transfer contribute to the dissemination of enzyme activity within the CNS. Intravitreal injection may thus provide a means of delivering certain therapeutic gene products to specific areas within the CNS.