👤 Masaru Inatani

Cataracts are opacifications of the lens that cause loss of visual acuity and ultimately of eyesight. Age-related cataract develops in most elderly people, but the mechanisms of cataract onset are incompletely understood. The Ihara Cataract Rat (ICR) is an animal model of hereditary cataracts showing cortical opacity that commonly develops prematurely. We identified putative mechanisms of cataract onset in the ICR rat model by measuring gene expression changes before and after cortical cataract development and conducting point mutation analysis. Genes differentially expressed between 4-week-old animals without cortical cataracts and 8-10-week-old animals with cortical cataracts were selected from microarray analysis. Three connections were identified by STRING analysis: (i) Epithelial-Mesenchymal Transition (EMT), including Col1a2, and Pik3r1. (ii) Lens homeostasis, including Aqp5, and Cpm. (iii) Lipid metabolism, including Scd1, Srebf1, and Pnpla3. Subsequently, mutation points were selected by comparing ICR rats with 12 different rats that do not develop cataracts. The apolipoprotein Apoc3 was mutated in ICR rats. Analyses of gene expression changes and point and mutations suggested that abnormalities in EMT or lipid metabolism could contribute to cataract development in ICR rats. Show less

Cataract causes vision loss and blindness due to formation of opacities of the lens. The regulatory mechanisms of cataract formation and progression remain unclear, and no effective drug treatments are clinically available. In the present study, we tested the effect of ataxia telangiectasia mutated (Atm) inhibitors using an ex vivo model in which rat lenses were cultured in galactose-containing medium to induce opacity formation. After lens opacities were induced by galactose, the lenses were further incubated with the Atm inhibitors AZD0156 or KU55933, which decreased lens opacity. Subsequently, we used microarray analysis to investigate the underlying molecular mechanisms of action, and extracted genes that were upregulated by galactose-induced opacity, but not by inhibitor treatment. Quantitative measurement of mRNA levels and subsequent STRING analysis revealed that a functional network consisting primarily of actin family and actin-binding proteins was upregulated by galactose treatment and downregulated by both Atm inhibitors. In particular, Acta2 is a known marker of epithelial-mesenchymal transition (EMT) in epithelial cells, and other genes connected in this functional network (Actn1, Tagln, Thbs1, and Angptl4) also suggested involvement of EMT. Abnormal differentiation of lens epithelial cells via EMT could contribute to formation of opacities; therefore, suppression of these genes by Atm inhibition is a potential therapeutic target for reducing opacities and alleviating cataract-related visual impairment. Show less

PURPOSE. Heparan sulfate (HS) is abundantly expressed in the developing neural retina; however, its role in the intraretinal axon guidance of retinal ganglion cells (RGCs) remains unclear. In this study, the authors examined whether HS was essential for the axon guidance of RGCs toward the optic nerve head. METHODS. The authors conditionally ablated the gene encoding the exostosin-1 (Ext1) enzyme, using the dickkopf homolog 3 (Dkk3)-Cre transgene, which disrupted HS expression in the mouse retina during directed pathfinding by RGC axons toward the optic nerve head. In situ hybridization, immunohistochemistry, DiI tracing, binding assay, and retinal explant assays were performed to evaluate the phenotypes of the mutants and the roles of HS in intraretinal axon guidance. RESULTS. Despite no gross abnormality in RGC distribution, the mutant RGC axons exhibited severe intraretinal guidance errors, including optic nerve hypoplasia, ectopic axon penetration through the full thickness of the neural retina and into the subretinal space, and disturbance of the centrifugal projection of RGC axons toward the optic nerve head. These abnormal phenotypes shared similarities with the RGC axon misguidance caused by mutations of genes encoding Netrin-1 and Slit-1/2. Explant assays revealed that the mutant RGCs exhibited disturbed Netrin-1-dependent axon outgrowth and Slit-2-dependent repulsion. CONCLUSIONS. The present study demonstrated that RGC axon projection toward the optic nerve head requires the expression of HS in the neural retina, suggesting that HS in the retina functions as an essential modulator of Netrin-1 and Slit-mediated intraretinal RGC axon guidance. Show less

Development of the mammalian central nervous system proceeds roughly in four major steps, namely the patterning of the neural tube, generation of neurons from neural stem cells and their migration to genetically predetermined destinations, extension of axons and dendrites toward target neurons to form neural circuits, and formation of synaptic contacts. Earlier studies on spatiotemporal expression patterns and in vitro function of heparan sulfate (HS) suggested that HS is functionally involved in various aspects of neural development. Recent studies using knockout of genes involved in HS biosynthesis have provided more physiologically relevant information as to the role of HS in mammalian neural development. This chapter reviews the current understanding of the in vivo function of HS deduced from the phenotypes of conditional Ext1 knockout mice. Show less

The heparan sulfate (HS) is a component of proteoglycans in the extracellular matrix and on cell surfaces, modulating developmental processes. The aim of this study is to investigate whether the defect of HS in the periocular mesenchyme impairs ocular morphogenesis. First, using Protein 0-Cre transgenic mice, we ablated Ext1, which encodes an indispensable enzyme for HS synthesis, in the developing periocular mesenchyme. The expression of Ext1 messenger RNA (mRNA) and HS were observed by RT-PCR and immunohistochemistry, respectively. The phenotypes in the mutant were evaluated by light microscopy and immunohistochemistry for cellular makers. Second, the distribution of the mutant periocular mesenchymal cells was tracked using a Rosa26 Cre-reporter gene. No mutant embryos (Protein 0-Cre;Ext1(flox/flox)) were identified after embryonic day 14.5 (E14.5). RT-PCR showed that an intense band amplified from Ext1 was observed in cDNAs from the control periocular mesenchymal cells at E13.5; however, the band for Ext1 was hardly detectable in cDNA from the mutant embryo, indicating that the mRNA was missing in the mutant periocular mesenchyme at E13.5. The HS expression was disrupted in the periocular mesenchyme of the mutant ocular tissues. The HS deficiency resulted in microphthalmia with reduced axial lengths, lens diameters, and vitreous sizes compared with the littermate eyes. The mutant embryos showed agenesis of the anterior chamber, where cells expressing Cre recombinase were distributed. Moreover, the mutants showed phenotypic alterations in the neural ectoderm including dysgenesis of the presumptive ciliary body and agenesis of the optic nerve head. These findings demonstrate that HS in the periocular mesenchyme plays a critical role in normal ocular morphogenesis, indicating reciprocal interactions between the periocular mesenchyme and the neural ectoderm. Show less

During human embryogenesis, neural crest cells migrate to the anterior chamber of the eye and then differentiate into the inner layers of the cornea, the iridocorneal angle, and the anterior portion of the iris. When proper development does not occur, this causes iridocorneal angle dysgenesis and intraocular pressure (IOP) elevation, which ultimately results in developmental glaucoma. Here, we show that heparan sulfate (HS) deficiency in mouse neural crest cells causes anterior chamber dysgenesis, including corneal endothelium defects, corneal stroma hypoplasia, and iridocorneal angle dysgenesis. These dysfunctions are phenotypes of the human developmental glaucoma, Peters anomaly. In the neural crest cells of mice embryos, disruption of the gene encoding exostosin 1 (Ext1), which is an indispensable enzyme for HS synthesis, resulted in disturbed TGF-beta2 signaling. This led to reduced phosphorylation of Smad2 and downregulated expression of forkhead box C1 (Foxc1) and paired-like homeodomain transcription factor 2 (Pitx2), transcription factors that have been identified as the causative genes for developmental glaucoma. Furthermore, impaired interactions between HS and TGF-beta2 induced developmental glaucoma, which was manifested as an IOP elevation caused by iridocorneal angle dysgenesis. These findings suggest that HS is necessary for neural crest cells to form the anterior chamber via TGF-beta2 signaling. Disturbances of HS synthesis might therefore contribute to the pathology of developmental glaucoma. Show less

There is increasing evidence that heparan sulfate (HS) plays an essential role in various axon guidance processes. These observations, however, have not addressed whether HS is required cell autonomously as an axonal coreceptor or as an environmental factor that modulates the localization of guidance molecules in the terrain in which growing axons navigate. Here we demonstrate that netrin-1-mediated commissural axon guidance requires cell-autonomous expression of HS in commissural neurons in vivo. We used the Wnt1-Cre transgene to drive region-specific ablation of Ext1, which encodes an enzyme essential for HS synthesis, in the dorsal part of the spinal cord. Remarkably, Wnt1-Cre-mediated ablation of Ext1 causes commissural axon pathfinding defects that share similarities with those of Netrin-1-deficient and DCC (deleted in colorectal cancer)-deficient mice. Neither Ext1-deficient dorsal spinal cord explants nor wild-type explants in which HS expression was ablated could extend axons in response to netrin-1. Intracellular signaling downstream of netrin-1 and DCC was defective in Ext1-deficient commissural neurons and in DCC-transfected HEK293T cells from which HS was removed. These results demonstrate that the expression of HS by commissural neurons is essential for these neurons to transduce netrin-1 signals, thus providing evidence for a cell-autonomous role of HS in netrin-1/DCC-mediated axon guidance. Show less

Heparan sulfate (HS) is required for morphogen signaling during Drosophila pattern formation, but little is known about its physiological importance in mammalian development. To define the developmental role of HS in mammalian species, we conditionally disrupted the HS-polymerizing enzyme EXT1 in the embryonic mouse brain. The EXT1-null brain exhibited patterning defects that are composites of those caused by mutations of multiple HS-binding morphogens. Furthermore, the EXT1-null brain displayed severe guidance errors in major commissural tracts, revealing a pivotal role of HS in midline axon guidance. These findings demonstrate that HS is essential for mammalian brain development. Show less

Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPGs) play significant roles in various biological processes. There is a wealth of circumstantial and experimental evidence suggesting the roles of HS in mammalian neural development. HS synthesis is governed by a series of enzymes. Among them, two enzymes, EXT1 and EXT2, catalyze polymerization of glucuronic acid and N-acetylglucosamine, the crucial step of HS synthesis. To obtain insight into the roles of HS in neural development, we examined the spatiotemporal expression patterns of EXT1 and EXT2 during mice brain development. RT-PCR analyses showed that expression of EXT1 and EXT2 peaks during early postnatal period in the cerebrum and around birth in the cerebellum. In situ hybridization revealed that in the embryonic brain, EXT1 and EXT2 were localized primarily in the neuroepithelial cells surrounding the lateral ventricles, the mesencephalic vesicle, and the fourth ventricle. In the early postnatal stage, intense expression of EXT1 and EXT2 was observed in the cerebral cortex and the hippocampus formation. In the postnatal cerebellum, expression of EXT1 and EXT2 was mainly observed in external and internal granular layers. Our results demonstrate that EXT1 and EXT2 are highly expressed in the developing brain, and that their expression is developmentally regulated, suggesting that HS is involved in various neurodevelopmental processes. Show less