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1.  Focal damage to macaque photoreceptors produces persistent visual loss 
Experimental eye research  2013;119:88-96.
Insertion of light-gated channels into inner retina neurons restores neural light responses, light evoked potentials, visual optomotor responses and visually-guided maze behavior in mice blinded by retinal degeneration. This method of vision restoration bypasses damaged outer retina, providing stimulation directly to retinal ganglion cells in inner retina. The approach is similar to that of electronic visual protheses, but may offer some advantages, such as avoidance of complex surgery and direct targeting of many thousands of neurons. However, the promise of this technique for restoring human vision remains uncertain because rodent animal models, in which it has been largely developed, are not ideal for evaluating visual perception. On the other hand, psychophysical vision studies in macaque can be used to evaluate different approaches to vision restoration in humans. Furthermore, it has not been possible to test vision restoration in macaques, the optimal model for human-like vision, because there has been no macaque model of outer retina degeneration. In this study, we describe development of a macaque model of photoreceptor degeneration that can in future studies be used to test restoration of perception by visual prostheses. Our results show that perceptual deficits caused by focal light damage are restricted to locations at which photoreceptors are damaged, that optical coherence tomography (OCT) can be used to track such lesions, and that adaptive optics retinal imaging, which we recently used for in vivo recording of ganglion cell function, can be used in future studies to examine these lesions.
doi:10.1016/j.exer.2013.11.001
PMCID: PMC4329982  PMID: 24316158
retina; light damage; ganglion cells; macaque; adaptive optics
2.  Tumor necrosis factor alpha has an early protective effect on retinal ganglion cells after optic nerve crush 
Background
Glaucoma is an optic neuropathy that is characterized by the loss of retinal ganglion cells (RGCs) initiated by damage to axons in the optic nerve. The degeneration and death of RGCs has been thought to occur in two waves. The first is axogenic, caused by direct insult to the axon. The second is somatic, and is thought to be caused by the production of inflammatory cytokines from the activated retinal innate immune cells. One of the cytokines consistently linked to glaucoma and RGC damage has been TNFα. Despite strong evidence implicating this protein in neurodegeneration, a direct injection of TNFα does not mimic the rapid loss of RGCs observed after acute optic nerve trauma or exposure to excitotoxins. This suggests that our understanding of TNFα signaling is incomplete.
Methods
RGC death was induced by optic nerve crush in mice. The role of TNFα in this process was examined by quantitative PCR of Tnfα gene expression, and quantification of cell loss in Tnfα−/− mice or in wild-type animals receiving an intraocular injection of exongenous TNFα either before or after crush. Signaling pathways downstream of TNFα were examined by immunolabeling for JUN protein accumulation or activation of EGFP expression in NFκB reporter mice.
Results
Optic nerve crush caused a modest increase in Tnfα gene expression, with kinetics similar to the activation of both macroglia and microglia. A pre-injection of TNFα attenuated ganglion cell loss after crush, while ganglion cell loss was more severe in Tnfα−/− mice. Conversely, over the long term, a single exposure to TNFα induced extrinsic apoptosis in RGCs. Müller cells responded to exogenous TNFα by accumulating JUN and activating NFκB.
Conclusion
Early after optic nerve crush, TNFα appears to have a protective role for RGCs, which may be mediated through Müller cells.
doi:10.1186/s12974-014-0194-3
PMCID: PMC4245774  PMID: 25407441
Secondary degeneration; Neuroinflammation; TNFα; Retinal ganglion cell; Macroglia
3.  DBA/2J Mice Are Susceptible to Diabetic Nephropathy and Diabetic Exacerbation of IOP Elevation 
PLoS ONE  2014;9(9):e107291.
Some pathological manifestations of diabetes in the eye include retinopathy, cataracts and elevated intraocular pressure (IOP). Loss of retinal ganglion cells (RGCs) in non-proliferative stages of diabetic retinopathy and small increases in IOP in diabetic patients has raised the possibility that diabetes affects the development and progression of ocular hypertension and glaucoma. The Ins2Akita mutation is known to cause diabetes and retinopathy on a C57BL/6J (B6) background by as early as 3 months of age. Here, the impact of the Akita mutation on glaucoma was assessed using DBA/2J (D2) mice, a widely used mouse model of ocular hypertension induced glaucoma. In D2.Ins2Akita/+ mice, the contribution of diabetes to vascular permeability, IOP elevation, RGC loss, and glaucoma development was assessed. D2.Ins2Akita/+ mice developed a severe diabetic nephropathy and early mortality between 6–8 months of age. This agrees with previous reports showing that the D2 background is more susceptible to diabetes than the B6 background. In addition, D2.Ins2Akita/+ mice had vascular leakage, astrocyte reactivity and a significant increase in IOP. However no RGC loss and no anterograde axonal transport dysfunction were found at 8.5 months of age. Therefore, our data show that despite severe diabetes and an increased IOP compared to controls, RGCs do not lose axon transport or degenerate. This may be due to a DBA/2J-specific genetic modifier(s) that could provide novel and important avenues for developing new therapies for diabetic retinopathy and possibly glaucoma.
doi:10.1371/journal.pone.0107291
PMCID: PMC4160242  PMID: 25207540
4.  Intrinsic axonal degeneration pathways are critical for glaucomatous damage 
Experimental neurology  2012;246:10.1016/j.expneurol.2012.01.014.
Glaucoma is a neurodegenerative disease affecting 70 million people worldwide. For some time, analysis of human glaucoma and animal models suggested that RGC axonal injury in the optic nerve head (where RGC axons exit the eye) is an important early event in glaucomatous neurodegeneration. During the last decade advances in molecular biology and genome manipulation have allowed this hypothesis to be tested more critically, at least in animal models. Data indicate that RGC axon degeneration precedes soma death. Preventing soma death using mouse models that are mutant for BAX, a proapoptotic gene, is not sufficient to prevent the degeneration of RGC axons. This indicates that different degeneration processes occur in different compartments of the RGC during glaucoma. Furthermore, the Wallerian degeneration slow allele (Wlds) slows or prevents RGC axon degeneration in rodent models of glaucoma. These experiments and many others, now strongly support the hypothesis that axon degeneration is a critical pathological event in glaucomatous neurodegeneration. However, the events that lead from a glaucomatous insult (e.g. elevated intraocular pressure) to axon damage in glaucoma are not well defined. For developing new therapies, it will be necessary to clearly define and order the molecular events that lead from glaucomatous insults to axon degeneration.
doi:10.1016/j.expneurol.2012.01.014
PMCID: PMC3831512  PMID: 22285251
5.  JUN regulates early transcriptional responses to axonal injury in retinal ganglion cells 
Experimental eye research  2013;112:106-117.
The AP1 family transcription factor JUN is an important molecule in the neuronal response to injury. In retinal ganglion cells (RGCs), JUN is upregulated soon after axonal injury and disrupting JUN activity delays RGC death. JUN is known to participate in the control of many different injury response pathways in neurons, including pathways controlling cell death and axonal regeneration. The role of JUN in regulating genes involved in cell death, ER stress, and regeneration was tested to determine the overall importance of JUN in regulating RGC response to axonal injury. Genes from each of these pathways were transcriptionally controlled following axonal injury and Jun deficiency altered the expression of many of these genes. The differentially expressed genes included, Atf3, Ddit3, Ecel1, Gadd45α, Gal, Hrk, Pten, Socs3, and Sprr1a. Two of these genes, Hrk and Atf3, were tested for importance in RGC death using null alleles of each gene. Disruption of the prodeath Bcl2 family member Hrk did not affect the rate or amount of RGC death after axonal trauma. Deficiency in the ATF/CREB family transcription factor Atf3 did lessen the amount of RGC death after injury, though it did not provide long term protection to RGCs. Since JUN’s dimerization partner determines its transcriptional targets, the expression of several candidate AP1 family members were examined. Multiple AP1 family members were induced by axonal injury and had a different expression profile in Jun deficient retinas compared to wildtype retinas (Fosl1, Fosl2 and Jund). Overall, JUN appears to play a multifaceted role in regulating RGC response to axonal injury.
doi:10.1016/j.exer.2013.04.021
PMCID: PMC3700614  PMID: 23648575
glaucoma; cell death; regeneration; ER stress; trauma; axonal injury; AP1
6.  Pou4f1 and Pou4f2 Are Dispensable for the Long-Term Survival of Adult Retinal Ganglion Cells in Mice 
PLoS ONE  2014;9(4):e94173.
Purpose
To investigate the role of Pou4f1 and Pou4f2 in the survival of adult retinal ganglion cells (RGCs).
Methods
Conditional alleles of Pou4f1 and Pou4f2 were generated (Pou4f1loxP and Pou4f2loxP respectively) for the removal of Pou4f1 and Pou4f2 in adult retinas. A tamoxifen-inducible Cre was used to delete Pou4f1 and Pou4f2 in adult mice and retinal sections and flat mounts were subjected to immunohistochemistry to confirm the deletion of both alleles and to quantify the changes in the number of RGCs and other retinal neurons. To determine the effect of loss of Pou4f1 and Pou4f2 on RGC survival after axonal injury, controlled optic nerve crush (CONC) was performed and RGC death was assessed.
Results
Pou4f1 and Pou4f2 were ablated two weeks after tamoxifen treatment. Retinal interneurons and Müller glial cells are not affected by the ablation of Pou4f1 or Pou4f2 or both. Although the deletion of both Pou4f1 and Pou4f2 slightly delays the death of RGCs at 3 days post-CONC in adult mice, it does not affect the cell death progress afterwards. Moreoever, deletion of Pou4f1 or Pou4f2 or both has no impact on the long-term viability of RGCs at up to 6 months post-tamoxifen treatment.
Conclusion
Pou4f1 and Pou4f2 are involved in the acute response to damage to RGCs but are dispensable for the long-term survival of adult RGC in mice.
doi:10.1371/journal.pone.0094173
PMCID: PMC3988073  PMID: 24736625
7.  BCL2L1 (BCL-x) promotes survival of adult and developing retinal ganglion cells 
The Bcl-2 family is responsible for regulating cell death pathways in neurons during development, after injury and in disease. The activation of the pro-death family member BAX is often the final step before cell death in neurons. Pro-survival family members such as BCL-X (BCL2L1) act to inhibit BAX activation. Overexpression studies have suggested that BCL-X could play an important physiological role in mediating neuronal viability. Loss-of-function studies performed in vivo have implicated BCL-X as a mediator of neuronal survival during the early stages of neurodevelopment. To assess whether BCL-X is needed to promote the survival of neurons in the central nervous system throughout life, Bcl-x was conditionally removed from the optic cup or throughout the adult mouse. During development BCL-X was required for the survival of differentiating retinal ganglion cells (RGCs) leading up to their normal window of developmental death. Despite its expression in adult RGCs, BCL-X was not required for maintaining RGC viability in adult retinas. However, the loss of BCL-X in adult RGCs did significantly increase the rate of death of RGCs after axonal injury. Thus, in developing and injured RGCs there appears to be an active cell survival program preventing neuronal death.
doi:10.1016/j.mcn.2012.07.006
PMCID: PMC3436941  PMID: 22836101
8.  JNK2 and JNK3 are major regulators of axonal injury-induced retinal ganglion cell death 
Neurobiology of Disease  2012;46(2):393-401.
Glaucoma is a neurodegenerative disease characterized by the apoptotic death of retinal ganglion cells (RGCs). The primary insult to RGCs in glaucoma is thought to occur to their axons as they exit the eye in the optic nerve head. However, pathological signaling pathways that exert central roles in triggering RGC death following axonal injury remain unidentified. It is likely that the first changes to occur following axonal injury are signal relay events that transduce the injury signal from the axon to the cell body. Here we focus on the c-Jun N-terminal kinase (JNK1-3) family, a signaling pathway implicated in axonal injury signaling and neurodegenerative apoptosis, and likely to function as a central node in axonal injury-induced RGC death. We show that JNK signaling is activated immediately after axonal injury in RGC axons at the site of injury. Following its early activation, sustained JNK signaling is observed in axonally-injured RGCs in the form of JUN phosphorylation and upregulation. Using mice lacking specific Jnk isoforms, we show that Jnk2 and Jnk3 are the isoforms activated in injured axons. Combined deficiency of Jnk2 and Jnk3 provides robust long-term protection against axonal injury-induced RGC death and prevents downregulation of the RGC marker, BRN3B, and phosphorylation of JUN. Finally, using Jun deficient mice, we show that JUN-dependent pathways are important for axonal injury-induced RGC death. Together these data demonstrate that JNK signaling is the major early pathway triggering RGC death after axonal injury and may directly link axon injury to transcriptional activity that controls RGC death.
doi:10.1016/j.nbd.2012.02.003
PMCID: PMC3323666  PMID: 22353563
JNK; axonal injury; apoptosis; retinal ganglion cell; cJUN; mouse; neurodegeneration; neuroprotection; glaucoma
9.  Mutations in a P-Type ATPase Gene Cause Axonal Degeneration 
PLoS Genetics  2012;8(8):e1002853.
Neuronal loss and axonal degeneration are important pathological features of many neurodegenerative diseases. The molecular mechanisms underlying the majority of axonal degeneration conditions remain unknown. To better understand axonal degeneration, we studied a mouse mutant wabbler-lethal (wl). Wabbler-lethal (wl) mutant mice develop progressive ataxia with pronounced neurodegeneration in the central and peripheral nervous system. Previous studies have led to a debate as to whether myelinopathy or axonopathy is the primary cause of neurodegeneration observed in wl mice. Here we provide clear evidence that wabbler-lethal mutants develop an axonopathy, and that this axonopathy is modulated by Wlds and Bax mutations. In addition, we have identified the gene harboring the disease-causing mutations as Atp8a2. We studied three wl alleles and found that all result from mutations in the Atp8a2 gene. Our analysis shows that ATP8A2 possesses phosphatidylserine translocase activity and is involved in localization of phosphatidylserine to the inner leaflet of the plasma membrane. Atp8a2 is widely expressed in the brain, spinal cord, and retina. We assessed two of the mutant alleles of Atp8a2 and found they are both nonfunctional for the phosphatidylserine translocase activity. Thus, our data demonstrate for the first time that mutation of a mammalian phosphatidylserine translocase causes axon degeneration and neurodegenerative disease.
Author Summary
Axonal degeneration is an important pathological feature of many neurodegenerative diseases, such as Alzheimer disease, Parkinson's disease, and amyotrophic lateral sclerosis. In most of these disease conditions, molecular mechanisms of axonal degeneration remain largely unknown. Spontaneous mouse mutants are important in human disease studies. Identification of a disease-causing gene in mice can lead to the identification of the human ortholog as the disease gene in humans. This approach has the power to identify unexpected genes and pathways involved in disease. Our study centered on wabbler lethal (wl) mutant mice, which display axonal degeneration in both the central and peripheral nervous systems. We identified the disease-causing gene in mice with different wl mutations. The mutations are in Atp8a2, a gene encoding a phosphatidylserine translocase. This protein functions to keep phosphatidylserine enriched to the inner leaflet of the plasma membrane. Our study demonstrates a new role for phospholipid asymmetry in maintaining axon health, and it also reveals a novel function for phosphatidyleserine translocase in neurodegenerative diseases.
doi:10.1371/journal.pgen.1002853
PMCID: PMC3415440  PMID: 22912588
10.  The Bcl-2 family member BIM has multiple glaucoma-relevant functions in DBA/2J mice 
Scientific Reports  2012;2:530.
Axonal insult induces retinal ganglion cell (RGC) death through a BAX-dependent process. The pro-apoptotic Bcl-2 family member BIM is known to induce BAX activation. BIM expression increased in RGCs after axonal injury and its induction was dependent on JUN. Partial and complete Bim deficiency delayed RGC death after mechanical optic nerve injury. However, in a mouse model of glaucoma, DBA/2J mice, Bim deficiency did not prevent RGC death in eyes with severe optic nerve degeneration. In a subset of DBA/2J mice, Bim deficiency altered disease progression resulting in less severe nerve damage. Bim deficient mice exhibited altered optic nerve head morphology and significantly lessened intraocular pressure elevation. Thus, a decrease in axonal degeneration in Bim deficient DBA/2J mice may not be caused by a direct role of Bim in RGCs. These data suggest that BIM has multiple roles in glaucoma pathophysiology, potentially affecting susceptibility to glaucoma through several mechanisms.
doi:10.1038/srep00530
PMCID: PMC3404412  PMID: 22833783
11.  Radiation treatment inhibits monocyte entry into the optic nerve head and prevents neuronal damage in a mouse model of glaucoma 
The Journal of Clinical Investigation  2012;122(4):1246-1261.
Glaucoma is a common ocular disorder that is a leading cause of blindness worldwide. It is characterized by the dysfunction and loss of retinal ganglion cells (RGCs). Although many studies have implicated various molecules in glaucoma, no mechanism has been shown to be responsible for the earliest detectable damage to RGCs and their axons in the optic nerve. Here, we show that the leukocyte transendothelial migration pathway is activated in the optic nerve head at the earliest stages of disease in an inherited mouse model of glaucoma. This resulted in proinflammatory monocytes entering the optic nerve prior to detectable neuronal damage. A 1-time x-ray treatment prevented monocyte entry and subsequent glaucomatous damage. A single x-ray treatment of an individual eye in young mice provided that eye with long-term protection from glaucoma but had no effect on the contralateral eye. Localized radiation treatment prevented detectable neuronal damage and dysfunction in treated eyes, despite the continued presence of other glaucomatous stresses and signaling pathways. Injection of endothelin-2, a damaging mediator produced by the monocytes, into irradiated eyes, combined with the other glaucomatous stresses, restored neural damage with a topography characteristic of glaucoma. Together, these data support a model of glaucomatous damage involving monocyte entry into the optic nerve.
doi:10.1172/JCI61135
PMCID: PMC3314470  PMID: 22426214
12.  Adaptive optics retinal imaging in the living mouse eye 
Biomedical Optics Express  2012;3(4):715-734.
Correction of the eye’s monochromatic aberrations using adaptive optics (AO) can improve the resolution of in vivo mouse retinal images [Biss et al., Opt. Lett. 32(6), 659 (2007) and Alt et al., Proc. SPIE 7550, 755019 (2010)], but previous attempts have been limited by poor spot quality in the Shack-Hartmann wavefront sensor (SHWS). Recent advances in mouse eye wavefront sensing using an adjustable focus beacon with an annular beam profile have improved the wavefront sensor spot quality [Geng et al., Biomed. Opt. Express 2(4), 717 (2011)], and we have incorporated them into a fluorescence adaptive optics scanning laser ophthalmoscope (AOSLO). The performance of the instrument was tested on the living mouse eye, and images of multiple retinal structures, including the photoreceptor mosaic, nerve fiber bundles, fine capillaries and fluorescently labeled ganglion cells were obtained. The in vivo transverse and axial resolutions of the fluorescence channel of the AOSLO were estimated from the full width half maximum (FWHM) of the line and point spread functions (LSF and PSF), and were found to be better than 0.79 μm ± 0.03 μm (STD)(45% wider than the diffraction limit) and 10.8 μm ± 0.7 μm (STD)(two times the diffraction limit), respectively. The axial positional accuracy was estimated to be 0.36 μm. This resolution and positional accuracy has allowed us to classify many ganglion cell types, such as bistratified ganglion cells, in vivo.
doi:10.1364/BOE.3.000715
PMCID: PMC3345801  PMID: 22574260
(170.4460) Ophthalmic optics and devices; (110.1080) Active or adaptive optics; (330.7324) Visual optics, comparative animal models
13.  Ocular Fibroblast Diversity: Implications for Inflammation and Ocular Wound Healing 
Fibroblasts mediate immune function and may account for differences in susceptibility of the different ocular tissues to become inflamed. Recognizing these differences will promote the development of novel therapeutic strategies for diseases of the eye.
Purpose.
Various ocular and orbital tissues differ in their manifestations of inflammation, although the reasons for this are unclear. Such differences may be due to behaviors exhibited by resident cell types, including fibroblasts. Fibroblasts mediate immune function and produce inflammatory mediators. Chronic stimulation of ocular fibroblasts can lead to prolonged inflammation and, in turn, to impaired vision and blindness. Interleukin (IL)-1β, which is produced by various cells during inflammation, is a potent activator of fibroblasts and inducer of the expression of inflammatory mediators. The hypothesis for this study was that that human fibroblasts derived from distinct ocular tissues differ in their responses to IL-1β and that variations in the IL-1 signaling pathway account for these differences.
Methods.
Human fibroblasts were isolated from the lacrimal gland, cornea, and Tenon's capsule and treated with IL-1β in vitro. Cytokine and prostaglandin (PG)E2 production were measured by ELISA and EIA. Cyclooxygenase (Cox)-2 expression was detected by Western blot. Components of the IL-1 signaling pathway were detected by flow cytometry, ELISA, Western blot, and immunofluorescence.
Results.
Cytokine and PGE2 production and Cox-2 expression were greatest in corneal fibroblasts. VEGF production was greatest in Tenon's capsule fibroblasts. Variations in IL-1 receptor and receptor antagonist expression, IκBα degradation and p65 nuclear translocation, however, did not account for these differences, but overexpression of the NF-κB member RelB dampened Cox-2 expression in all three fibroblast types.
Conclusions.
The results highlight the inherent differences between ocular fibroblast strains and provide crucial insight into novel, tissue-specific treatments for ocular inflammation and disease, such as RelB overexpression.
doi:10.1167/iovs.10-7066
PMCID: PMC3175934  PMID: 21571679
14.  Intravitreal Injection of AAV2 Transduces Macaque Inner Retina 
Intravitreally injected AAV2 transduced inner retinal cells in a restricted region at the macaque fovea. Because macaque and human eyes are similar, the results suggest a need to improve transduction methods in gene therapy for the human inner retina.
Purpose.
Adeno-associated virus serotype 2 (AAV2) has been shown to be effective in transducing inner retinal neurons after intravitreal injection in several species. However, results in nonprimates may not be predictive of transduction in the human inner retina, because of differences in eye size and the specialized morphology of the high-acuity human fovea. This was a study of inner retina transduction in the macaque, a primate with ocular characteristics most similar to that of humans.
Methods.
In vivo imaging and histology were used to examine GFP expression in the macaque inner retina after intravitreal injection of AAV vectors containing five distinct promoters.
Results.
AAV2 produced pronounced GFP expression in inner retinal cells of the fovea, no expression in the central retina beyond the fovea, and variable expression in the peripheral retina. AAV2 vector incorporating the neuronal promoter human connexin 36 (hCx36) transduced ganglion cells within a dense annulus around the fovea center, whereas AAV2 containing the ubiquitous promoter hybrid cytomegalovirus (CMV) enhancer/chicken-β-actin (CBA) transduced both Müller and ganglion cells in a dense circular disc centered on the fovea. With three shorter promoters—human synapsin (hSYN) and the shortened CBA and hCx36 promoters (smCBA and hCx36sh)—AAV2 produced visible transduction, as seen in fundus images, only when the retina was altered by ganglion cell loss or enzymatic vitreolysis.
Conclusions.
The results in the macaque suggest that intravitreal injection of AAV2 would produce high levels of gene expression at the human fovea, important in retinal gene therapy, but not in the central retina beyond the fovea.
doi:10.1167/iovs.10-6250
PMCID: PMC3088562  PMID: 21310920
15.  Datgan, a reusable software system for facile interrogation and visualization of complex transcription profiling data 
BMC Genomics  2011;12:429.
Background
We introduce Glaucoma Discovery Platform (GDP), an online environment for facile visualization and interrogation of complex transcription profiling datasets for glaucoma. We also report the availability of Datgan, the suite of scripts that was developed to construct GDP. This reusable software system complements existing repositories such as NCBI GEO or EBI ArrayExpress as it allows the construction of searchable databases to maximize understanding of user-selected transcription profiling datasets.
Description
Datgan scripts were used to construct both the underlying data tables and the web interface that form GDP. GDP is populated using data from a mouse model of glaucoma. The data was generated using the DBA/2J strain, a widely used mouse model of glaucoma. The DBA/2J-Gpnmb+ strain provided a genetically matched control strain that does not develop glaucoma. We separately assessed both the retina and the optic nerve head, important tissues in glaucoma. We used hierarchical clustering to identify early molecular stages of glaucoma that could not be identified using morphological assessment of disease. GDP has two components. First, an interactive search and retrieve component provides the ability to assess gene(s) of interest in all identified stages of disease in both the retina and optic nerve head. The output is returned in graphical and tabular format with statistically significant differences highlighted for easy visual analysis. Second, a bulk download component allows lists of differentially expressed genes to be retrieved as a series of files compatible with Excel. To facilitate access to additional information available for genes of interest, GDP is linked to selected external resources including Mouse Genome Informatics and Online Medelian Inheritance in Man (OMIM).
Conclusion
Datgan-constructed databases allow user-friendly access to datasets that involve temporally ordered stages of disease or developmental stages. Datgan and GDP are available from http://glaucomadb.jax.org/glaucoma.
doi:10.1186/1471-2164-12-429
PMCID: PMC3171729  PMID: 21864367
16.  BBC3 (PUMA) regulates developmental apoptosis but not axonal injury induced death in the retina 
Background
Naturally occurring apoptosis is a developmental process that shapes the retina by eliminating overproduced neurons. In the absence of the proapoptotic Bcl-2 family member BAX, developmental apoptosis in the retina is disrupted and extra neurons survive. It is unknown how BAX is activated or if this regulation varies between neuronal types and subtypes. Since the Bcl-2 family members BIM, BID, and BBC3 (PUMA) are powerful direct activators of BAX, we used mice deficient for each of these genes to investigate their importance in developmental apoptosis.
Results
Bax deficient mice have an increase in retinal ganglion cells (RGCs), bipolar cells and dopaminergic amacrine cells, but not photoreceptors, horizontal cells or cholinergic amacrine cells. The retinas of adult Bim and Bid deficient mice appeared to have no increase in any retinal cell type. Bbc3 deficient mice, either homozygous or heterozygous for a null allele of Bbc3, had an increase in the same cell types as Bax deficient mice. An analogous result may occur in the brain where, similar to Bax deficient mice, Bbc3 deficient mice have a larger gross brain weight compared to wild type mice. In contrast to its developmental role, BBC3 did not appear to be a primary factor in BAX-dependent axonal injury induced neurodegeneration in adult RGCs.
Conclusion
The regulation of BAX activation in the retina appears to be complex, dependent on the developmental stage of the animal, the nature of the insult and even the type of neuron.
doi:10.1186/1750-1326-6-50
PMCID: PMC3149592  PMID: 21762490
17.  The Usher 1B protein, MYO7A, is required for normal localization and function of the visual retinoid cycle enzyme, RPE65 
Human Molecular Genetics  2011;20(13):2560-2570.
Mutations in the MYO7A gene cause a deaf-blindness disorder, known as Usher syndrome 1B.  In the retina, the majority of MYO7A is in the retinal pigmented epithelium (RPE), where many of the reactions of the visual retinoid cycle take place.  We have observed that the retinas of Myo7a-mutant mice are resistant to acute light damage. In exploring the basis of this resistance, we found that Myo7a-mutant mice have lower levels of RPE65, the RPE isomerase that has a key role in the retinoid cycle.  We show for the first time that RPE65 normally undergoes a light-dependent translocation to become more concentrated in the central region of the RPE cells.  This translocation requires MYO7A, so that, in Myo7a-mutant mice, RPE65 is partly mislocalized in the light.  RPE65 is degraded more quickly in Myo7a-mutant mice, perhaps due to its mislocalization, providing a plausible explanation for its lower levels.  Following a 50–60% photobleach, Myo7a-mutant retinas exhibited increased all-trans-retinyl ester levels during the initial stages of dark recovery, consistent with a deficiency in RPE65 activity.  Lastly, MYO7A and RPE65 were co-immunoprecipitated from RPE cell lysate by antibodies against either of the proteins, and the two proteins were partly colocalized, suggesting a direct or indirect interaction.  Together, the results support a role for MYO7A in the translocation of RPE65, illustrating the involvement of a molecular motor in the spatiotemporal organization of the retinoid cycle in vision.
doi:10.1093/hmg/ddr155
PMCID: PMC3110002  PMID: 21493626
18.  Endoplasmic Reticulum Stress as a Primary Pathogenic Mechanism Leading to Age-Related Macular Degeneration 
Age-related macular degeneration (AMD) is a multi-factorial disease and a leading cause of blindness. Proteomic and genetic data suggest that activation or de-repression of the alternate complement cascade of innate immunity is involved in end-stage disease. Several lines of evidence suggest that production of reactive oxygen species and chronic oxidative stress lead to protein and lipid modifications that initiate the complement cascade. Understanding the triggers of these pathogenic pathways and the site of the primary insult will be important for development of targeted therapeutics. Endoplasmic reticulum (ER) stress from misfolded mutant proteins and other sources are an important potential tributary mechanism. We propose that misfolded-protein-induced ER stress in the retinal-pigmented epithelium and/or choroid could lead to chronic oxidative stress, complement deregulation and AMD. Small molecules targeted to ER stress and oxidative stress could allow for a shift from disease treatment to disease prevention.
doi:10.1007/978-1-4419-1399-9_46
PMCID: PMC3068206  PMID: 20238041
19.  Molecular clustering identifies complement and endothelin induction as early events in a mouse model of glaucoma 
The Journal of Clinical Investigation  2011;121(4):1429-1444.
Glaucoma is one of the most common neurodegenerative diseases. Despite this, the earliest stages of this complex disease are still unclear. This study was specifically designed to identify early stages of glaucoma in DBA/2J mice. To do this, we used genome-wide expression profiling of optic nerve head and retina and a series of computational methods. Eyes with no detectable glaucoma by conventional assays were grouped into molecularly defined stages of disease using unbiased hierarchical clustering. These stages represent a temporally ordered sequence of glaucoma states. We then determined networks and biological processes that were altered at these early stages. Early-stage expression changes included upregulation of both the complement cascade and the endothelin system, and so we tested the therapeutic value of separately inhibiting them. Mice with a mutation in complement component 1a (C1qa) were protected from glaucoma. Similarly, inhibition of the endothelin system with bosentan, an endothelin receptor antagonist, was strongly protective against glaucomatous damage. Since endothelin 2 is potently vasoconstrictive and was produced by microglia/macrophages, our data provide what we believe to be a novel link between these cell types and vascular dysfunction in glaucoma. Targeting early molecular events, such as complement and endothelin induction, may provide effective new treatments for human glaucoma.
doi:10.1172/JCI44646
PMCID: PMC3069778  PMID: 21383504
20.  Optical properties of the mouse eye 
Biomedical Optics Express  2011;2(4):717-738.
The Shack-Hartmann wavefront sensor (SHWS) spots upon which ocular aberration measurements depend have poor quality in mice due to light reflected from multiple retinal layers. We have designed and implemented a SHWS that can favor light from a specific retinal layer and measured monochromatic aberrations in 20 eyes from 10 anesthetized C57BL/6J mice. Using this instrument, we show that mice are myopic, not hyperopic as is frequently reported. We have also measured longitudinal chromatic aberration (LCA) of the mouse eye and found that it follows predictions of the water-filled schematic mouse eye. Results indicate that the optical quality of the mouse eye assessed by measurement of its aberrations is remarkably good, better for retinal imaging than the human eye. The dilated mouse eye has a much larger numerical aperture (NA) than that of the dilated human eye (0.5 NA vs. 0.2 NA), but it has a similar amount of root mean square (RMS) higher order aberrations compared to the dilated human eye. These measurements predict that adaptive optics based on this method of wavefront sensing will provide improvements in retinal image quality and potentially two times higher lateral resolution than that in the human eye.
doi:10.1364/BOE.2.000717
PMCID: PMC3072116  PMID: 21483598
(170.4460) Medical optics and biotechnology: Ophthalmic optics and devices; (330.5370) Vision, color, and visual optics: Physiological optics; (330.4300) Vision system - noninvasive assessment; (110.1080) Active or adaptive optics; (330.7324) Vision, color, and visual optics: Visual optics, comparative animal models
21.  MATH5 controls the acquisition of multiple retinal cell fates 
Molecular Brain  2010;3:36.
Math5-null mutation results in the loss of retinal ganglion cells (RGCs) and in a concurrent increase of amacrine and cone cells. However, it remains unclear whether there is a cell fate switch of Math5-lineage cells in the absence of Math5 and whether MATH5 cell-autonomously regulates the differentiation of the above retinal neurons. Here, we performed a lineage analysis of Math5-expressing cells in developing mouse retinas using a conditional GFP reporter (Z/EG) activated by a Math5-Cre knock-in allele. We show that during normal retinogenesis, Math5-lineage cells mostly develop into RGCs, horizontal cells, cone photoreceptors, rod photoreceptors, and amacrine cells. Interestingly, amacrine cells of Math5-lineage cells are predominately of GABAergic, cholinergic, and A2 subtypes, indicating that Math5 plays a role in amacrine subtype specification. In the absence of Math5, more Math5-lineage cells undergo cell fate conversion from RGCs to the above retinal cell subtypes, and occasionally to cone-bipolar cells and Müller cells. This change in cell fate choices is accompanied by an up-regulation of NEUROD1, RXRγ and BHLHB5, the transcription factors essential for the differentiation of retinal cells other than RGCs. Additionally, loss of Math5 causes the failure of early progenitors to exit cell cycle and leads to a significant increase of Math5-lineage cells remaining in cell cycle. Collectively, these data suggest that Math5 regulates the generation of multiple retinal cell types via different mechanisms during retinogenesis.
doi:10.1186/1756-6606-3-36
PMCID: PMC2994854  PMID: 21087508
22.  Role of myosin VIIa and Rab27a in the motility and localization of RPE melanosomes 
Journal of cell science  2004;117(Pt 26):6473-6483.
Summary
Myosin VIIa functions in the outer retina, and loss of this function causes human blindness in Usher syndrome type 1B (USH1B). In mice with mutant Myo7a, melanosomes in the retinal pigmented epithelium (RPE) are distributed abnormally. In this investigation we detected many proteins in RPE cells that could potentially participate in melanosome transport, but of those tested, only myosin VIIa and Rab27a were found to be required for normal distribution. Two other expressed proteins, melanophilin and myosin Va, both of which are required for normal melanosome distribution in melanocytes, were not required in RPE, despite the association of myosin Va with the RPE melanosome fraction. Both myosin VIIa and myosin Va were immunodetected broadly in sections of the RPE, overlapping with a region of apical filamentous actin. Some 70–80% of the myosin VIIa in RPE cells was detected on melanosome membranes by both subcellular fractionation of RPE cells and quantitative immunoelectron microscopy, consistent with a role for myosin VIIa in melanosome motility. Time-lapse microscopy of melanosomes in primary cultures of mouse RPE cells demonstrated that the melanosomes move in a saltatory manner, interrupting slow movements with short bursts of rapid movement (>1 µm/second). In RPE cells from Myo7a-null mice, both the slow and rapid movements still occurred, except that more melanosomes underwent rapid movements, and each movement extended approximately five times longer (and further). Hence, our studies demonstrate the presence of many potential effectors of melanosome motility and localization in the RPE, with a specific requirement for Rab27a and myosin VIIa, which function by transporting and constraining melanosomes within a region of filamentous actin. The presence of two distinct melanosome velocities in both control and Myo7a-null RPE cells suggests the involvement of at least two motors other than myosin VIIa in melanosome motility, most probably, a microtubule motor and myosin Va.
doi:10.1242/jcs.01580
PMCID: PMC2942070  PMID: 15572405
Myosin VIIa; Myosin Va; Rab27a; Melanosome; Usher syndrome; Organelle motility
23.  Laminin Expression in Adult and Developing Retinae: Evidence of Two Novel CNS Laminins 
Components of the extracellular matrix exert myriad effects on tissues throughout the body. In particular, the laminins, a family of heterotrimeric extracellular glycoproteins, have been shown to affect tissue development and integrity in such diverse organs as the kidney, lung, skin, and nervous system. Of these, we have focused on the roles that laminins play in the differentiation and maintenance of the nervous system. Here, we examine the expression of all known laminin chains within one component of the CNS, the retina. We find seven laminin chains—α3, α4, α5, β2, β3, γ2, and γ3—outside the retinal basement membranes. Anatomically, these chains are coexpressed in one or both of two locations: the matrix surrounding photoreceptors and the first synaptic layer where photoreceptors synapse with retinal interneurons. Biochemically, four of these chains are coisolated from retinal extracts in two independent complexes, confirming that two novel heterotrimers—α4β2γ3 and α5β2γ3—are present in the retinal matrix. During development, all four of these chains, along with components of laminin 5 (the α3, β3, and γ2 chains) are also expressed at sites at which they could exert important effects on photoreceptor development. Together, these data suggest the existence of two novel laminin heterotrimers in the CNS, which we term here laminin 14 (composed of the α4, β2, and γ3 chains) and laminin 15 (composed of the α5, β2, and γ3 chains), and lead us to hypothesize that these laminins, along with laminin 5, may play roles in photoreceptor production, stability, and synaptic organization.
PMCID: PMC2924637  PMID: 10964957
retina; synapse; matrix; photoreceptor; interphoto-receptor matrix; laminin
24.  BARHL2 differentially regulates the development of retinal amacrine and ganglion neurons 
Summary
Through transcriptional regulations the BarH family of homeodomain proteins play essential roles in cell fate specification, cell differentiation, migration and survival. Barhl2, a member of the Barh gene family, is expressed in retinal ganglion cells (RGCs), amacrine cells (ACs) and horizontal cells. Here, to investigate the role of Barhl2 in retinal development, Barhl2 deficient mice were generated. Analysis of AC subtypes in Barhl2 deficient retinas suggests that Barhl2 plays a critical role in AC subtype determination. A significant reduction of glycinergic and GABAergic ACs with a substantial increase in the number of cholinergic ACs was observed in Barhl2-null retinas. Barhl2 is also critical for the development of a normal complement of RGCs. Barhl2 deficiency resulted in a 35% increase in RGCs undergoing apoptosis during development. Genetic analysis revealed that Barhl2 functions downstream of the Atoh7-Pou4f3 regulatory pathway and regulates the maturation and/or survival of RGCs. Thus, BARHL2 appears to have numerous roles in retinal development, including regulating neuronal subtype specification, differentiation, and survival.
doi:10.1523/JNEUROSCI.5237-08.2009
PMCID: PMC2756297  PMID: 19339595
BarH; Math5; Atoh7; Pou4f2; Isl1; retinal ganglion cells; amacrine cells; retinal development; transcription factor
25.  In-vivo imaging of retinal nerve fiber layer vasculature: imaging - histology comparison 
BMC Ophthalmology  2009;9:9.
Background
Although it has been suggested that alterations of nerve fiber layer vasculature may be involved in the etiology of eye diseases, including glaucoma, it has not been possible to examine this vasculature in-vivo. This report describes a novel imaging method, fluorescence adaptive optics (FAO) scanning laser ophthalmoscopy (SLO), that makes possible for the first time in-vivo imaging of this vasculature in the living macaque, comparing in-vivo and ex-vivo imaging of this vascular bed.
Methods
We injected sodium fluorescein intravenously in two macaque monkeys while imaging the retina with an FAO-SLO. An argon laser provided the 488 nm excitation source for fluorescence imaging. Reflectance images, obtained simultaneously with near infrared light, permitted precise surface registration of individual frames of the fluorescence imaging. In-vivo imaging was then compared to ex-vivo confocal microscopy of the same tissue.
Results
Superficial focus (innermost retina) at all depths within the NFL revealed a vasculature with extremely long capillaries, thin walls, little variation in caliber and parallel-linked structure oriented parallel to the NFL axons, typical of the radial peripapillary capillaries (RPCs). However, at a deeper focus beneath the NFL, (toward outer retina) the polygonal pattern typical of the ganglion cell layer (inner) and outer retinal vasculature was seen. These distinguishing patterns were also seen on histological examination of the same retinas. Furthermore, the thickness of the RPC beds and the caliber of individual RPCs determined by imaging closely matched that measured in histological sections.
Conclusion
This robust method demonstrates in-vivo, high-resolution, confocal imaging of the vasculature through the full thickness of the NFL in the living macaque, in precise agreement with histology. FAO provides a new tool to examine possible primary or secondary role of the nerve fiber layer vasculature in retinal vascular disorders and other eye diseases, such as glaucoma.
doi:10.1186/1471-2415-9-9
PMCID: PMC2744910  PMID: 19698151

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