Kin-Sang Cho

Re-establishing the regenerative potential of central nervous system axons in postnatal mice.

At a certain point in development, axons in the mammalian central nervous system lose their ability to regenerate after injury. Using the optic nerve model, we show that this growth failure coincides with two developmental events: the loss of Bcl-2 expression by neurons and the maturation of astrocytes. Before postnatal day 4, when astrocytes are immature, overexpression of Bcl-2 alone supported robust and rapid optic nerve regeneration over long distances, leading to innervation of brain targets by day 4 in mice. As astrocytes matured after postnatal day 4, axonal regeneration was inhibited in mice overexpressing Bcl-2. Concurrent induction of Bcl-2 and attenuation of reactive gliosis reversed the failure of CNS axonal re-elongation in postnatal mice and led to rapid axonal regeneration over long distances and reinnervation of the brain targets by a majority of severed optic nerve fibers up to 2 weeks of age. These results suggest that an early postnatal downregulation of Bcl-2 and post-traumatic reactive gliosis are two important elements of axon regenerative failure in the CNS.

Differential Effects of Unfolded Protein Response Pathways on Axon Injury-Induced Death of Retinal Ganglion Cells

Loss of retinal ganglion cells (RGCs) accounts for visual function deficits after optic nerve injury, but how axonal insults lead to neuronal death remains elusive. By using an optic nerve crush model that results in the death of the majority of RGCs, we demonstrate that axotomy induces differential activation of distinct pathways of the unfolded protein response in axotomized RGCs. Optic nerve injury provokes a sustained CCAAT/enhancer binding homologous protein (CHOP) upregulation, and deletion of CHOP promotes RGC survival. In contrast, IRE/XBP-1 is only transiently activated, and forced XBP-1 activation dramatically protects RGCs from axon injury-induced death. Importantly, such differential activations of CHOP and XBP-1 and their distinct effects on neuronal cell death are also observed in RGCs with other types of axonal insults, such as vincristine treatment and intraocular pressure elevation, suggesting a new protective strategy for neurodegeneration associated with axonal damage.

Optic Neuropathy Due to Microbead-Induced Elevated Intraocular Pressure in the Mouse

PURPOSE To characterize a glaucoma model of mice, the authors adopted and modified a method of inducing the chronic elevation of intraocular pressure (IOP) by anterior chamber injection of polystyrene microbeads. METHODS Chronic elevation of IOP was induced unilaterally in adult C57BL/6J mice by injecting polystyrene microbeads to the anterior chamber. Effectiveness of microbeads of different sizes (small, 10 μm; large, 15 μm) on inducing IOP elevation was compared, and IOP was measured every other day using a tonometer. After maintaining elevated IOP for 2, 4, or 8 weeks, the degree of RGC and axon degeneration was assessed quantitatively using electron microscopy, fluorogold, retrograde labeling, and immunohistochemistry. RESULTS Eighty-one of 87 mice that received anterior chamber injection of microbeads exhibited consistent IOP elevation above that of control eyes. Injection of small microbeads induced longer and higher peak value of IOP elevation compared with that of the large microbeads. A single injection of small microbeads resulted in a 4-week elevation of IOP that was maintained to an 8-week period after a second injection of microbeads in week 4. As the duration of IOP elevation increased, RGC bodies and their axons degenerated progressively and reached an approximately 50% loss after an 8-week elevation of IOP. CONCLUSIONS Anterior chamber injection of microbeads effectively induced IOP elevation and glaucomatous optic neuropathy in mice. Development of an inducible mouse model of elevated IOP will allow applications of mouse genetic technology to the investigation of the mechanisms and the evaluation of treatment strategies of glaucoma.

A sulfated carbohydrate epitope inhibits axon regeneration after injury

Chondroitin sulfate proteoglycans (CSPGs) represent a major barrier to regenerating axons in the central nervous system (CNS), but the structural diversity of their polysaccharides has hampered efforts to dissect the structure-activity relationships underlying their physiological activity. By taking advantage of our ability to chemically synthesize specific oligosaccharides, we demonstrate that a sugar epitope on CSPGs, chondroitin sulfate-E (CS-E), potently inhibits axon growth. Removal of the CS-E motif significantly attenuates the inhibitory activity of CSPGs on axon growth. Furthermore, CS-E functions as a protein recognition element to engage receptors including the transmembrane protein tyrosine phosphatase PTPσ, thereby triggering downstream pathways that inhibit axon growth. Finally, masking the CS-E motif using a CS-E-specific antibody reversed the inhibitory activity of CSPGs and stimulated axon regeneration in vivo. These results demonstrate that a specific sugar epitope within chondroitin sulfate polysaccharides can direct important physiological processes and provide new therapeutic strategies to regenerate axons after CNS injury.

Synergistic effect of Nogo-neutralizing antibody IN-1 and ciliary neurotrophic factor on axonal regeneration in adult rodent visual systems.

The presence of Nogo axon regeneration inhibitory molecules in the central nervous system (CNS) and the counteracting effect of IN-1 antibodies have been widely reported. In this study, we examined the effect of IN-1-producing hybridoma cells on axon regeneration in adult rodent retinal ganglion cells (RGCs) after various types of optic nerve (ON) injury, evaluating therein whether ciliary neurotrophic factor (CNTF) potentiated the effect of IN-1. We found that application of IN-1 alone failed to enhance regeneration of intracranially or intraorbitally transected RGC axons in a peripheral nerve (PN) graft. IN-1 hybridoma cells also failed to significantly promote intraorbitally crushed ON axons to reenter the distal part of the ON. However, a combined application of IN-1 and CNTF had a synergistic effect in both intracranial PN and intraorbital ON crush paradigms. This study suggests that the action of IN-1 antibodies in promoting axon regeneration in the CNS could be more effective when coupled with other appropriate factors.

Microbead-Induced Ocular Hypertensive Mouse Model for Screening and Testing of Aqueous Production Suppressants for Glaucoma

PURPOSE To characterize the microbead-induced ocular hypertension (OHT) mouse model and investigate its potential use for preclinical screening and evaluation of ocular hypotensive agents, we tested the models responses to major antiglaucoma drugs. METHODS Adult C57BL/6J mice were induced to develop OHT unilaterally by intracameral injection of microbeads. The effects of the most commonly used ocular hypotensive drugs, including timolol, brimonidine, brinzolamide, pilocarpine, and latanoprost, on IOP and glaucomatous neural damage were evaluated. Degeneration of retinal ganglion cells (RGCs) and optic nerve axons were quantitatively assessed using immunofluorescence labeling and histochemistry. Thickness of the ganglion cell complex (GCC) was also assessed with spectral-domain optical coherence tomography (SD-OCT). RESULTS A microbead-induced OHT model promptly responded to drugs, such as timolol, brimonidine, and brinzolamide, that lower IOP through suppressing aqueous humor production and showed improved RGC and axon survival as compared to vehicle controls. Accordingly, SD-OCT detected significantly less reduction of GCC thickness in mice treated with all three aqueous production suppressants as compared to the vehicle contol-treated group. In contrast, drugs that increase aqueous outflow, such as pilocarpine and latanoprost, failed to decrease IOP in the microbead-induced OHT mice. CONCLUSIONS Microbead-induced OHT mice carry dysfunctional aqueous outflow facility and therefore offer a unique model that allows selective screening of aqueous production suppressant antiglaucoma drugs or for studying the mechanisms regulating aqueous humor production. Our data set the stage for using GCC thickness assessed by SD-OCT as an imaging biomarker for noninvasive tracking of neuronal benefits of glaucoma therapy in this model.

Neuroglobin is an endogenous neuroprotectant for retinal ganglion cells against glaucomatous damage.

Neuroglobin (NGB), a newly discovered member of the globin superfamily, may regulate neuronal survival under hypoxia or oxidative stress. Although NGB is greatly expressed in retinal neurons, the biological functions of NGB in retinal diseases remain largely unknown. We investigated the role of NGB in an experimental model of glaucoma, a neurodegenerative disorder that usually involves elevation of intraocular pressure (IOP). Elevated IOP is thought to induce oxidative stress in retinal ganglion cells (RGCs), thereby causing RGC death and, eventually, blindness. We found that NGB plays a critical role in increasing RGC resistance to ocular hypertension and glaucomatous damage. Elevation of IOP stimulated a transient up-regulation of endogenous NGB in RGCs. Constitutive overexpression of NGB in transgenic mice prevented RGC damage induced by glutamate cytotoxicity in vitro and/or by chronic IOP elevation in vivo. Moreover, overexpression of NGB attenuated ocular hypertension-induced superoxide production and the associated decrease in ATP levels in mice, suggesting that NGB acts as an endogenous neuroprotectant to reduce oxidative stress and improve mitochondrial function, thereby promoting RGC survival. Thus, NGB may modulate RGC susceptibility to glaucomatous neural damage. Manipulating the expression and bioactivity of NGB may represent a novel therapeutic strategy for glaucoma.

Ephrin-A3 Suppresses Wnt Signaling to Control Retinal Stem Cell Potency†‡§

The ciliary epithelium (CE) of adult mammals has been reported to provide a source of retinal stem cells (RSCs) that can give rise to all retinal cell types in vitro. A recent study, however, suggests that CE‐derived cells possess properties of pigmented ciliary epithelial cells and display little neurogenic potential. Here we show that the neurogenic potential of CE‐derived cells is negatively regulated by ephrin‐A3, which is upregulated in the CE of postnatal mice and presents a strong prohibitory niche for adult RSCs. Addition of ephrin‐A3 inhibits proliferation of CE‐derived RSCs and increases pigment 349 cell 359. In contrast, absence of ephrin‐A3 promotes proliferation and increases expression of neural progenitor cell markers and photoreceptor progeny. The negative effects of ephrin‐A3 on CE‐derived RSCs are mediated through activation of an EphA4 receptor and suppression of Wnt3a/β‐catenin signaling. Together, our data suggest that CE‐derived RSCs contain the intrinsic machinery to generate photoreceptors and other retinal neurons, while the CE of adult mice expresses negative regulators that prohibit the proliferation and neural differentiation of RSCs. Manipulating ephrin and Wnt/β‐catenin signaling may, thus, represent a viable approach in activating the endogenous neurogenic potential of CE‐derived RSCs for treating photoreceptor damage and retinal degenerative disorders. STEM CELLS2013;31:349–359

Postnatal onset of retinal degeneration by loss of embryonic Ezh2 repression of Six1.

Some adult-onset disorders may be linked to dysregulated embryonic development, yet the mechanisms underlying this association remain poorly understood. Congenital retinal degenerative diseases are blinding disorders characterized by postnatal degeneration of photoreceptors, and affect nearly 2 million individuals worldwide, but ∼50% do not have a known mutation, implicating contributions of epigenetic factors. We found that embryonic deletion of the histone methyltransferase (HMT) Ezh2 from all retinal progenitors resulted in progressive photoreceptor degeneration throughout postnatal life, via derepression of fetal expression of Six1 and its targets. Forced expression of Six1 in the postnatal retina was sufficient to induce photoreceptor degeneration. Ezh2, although enriched in the embryonic retina, was not present in the mature retina; these data reveal an Ezh2-mediated feed-forward pathway that is required for maintaining photoreceptor homeostasis in the adult and suggest novel targets for retinal degeneration therapy.

Electrical Stimulation as a Means for Improving Vision.

Evolving research has provided evidence that noninvasive electrical stimulation (ES) of the eye may be a promising therapy for either preserving or restoring vision in several retinal and optic nerve diseases. In this review, we focus on minimally invasive strategies for the delivery of ES and accordingly summarize the current literature on transcorneal, transorbital, and transpalpebral ES in both animal experiments and clinical studies. Various mechanisms are believed to underlie the effects of ES, including increased production of neurotrophic agents, improved chorioretinal blood circulation, and inhibition of proinflammatory cytokines. Different animal models have demonstrated favorable effects of ES on both the retina and the optic nerve. Promising effects of ES have also been demonstrated in clinical studies; however, all current studies have a lack of randomization and/or a control group (sham). There is thus a pressing need for a deeper understanding of the underlying mechanisms that govern clinical success and optimization of stimulation parameters in animal studies. In addition, such research should be followed by large, prospective, clinical studies to explore the full potential of ES. Through this review, we aim to provide insight to guide future research on ES as a potential therapy for improving vision.