While all forms of glaucoma are characterized by a specific pattern of retinal ganglion cell death, they are clinically divided into several distinct subclasses, including normal tension glaucoma, primary open angle glaucoma, congenital glaucoma, and secondary glaucoma. For each type of glaucoma there are likely numerous molecular pathways that control susceptibility to the disease. Given this complexity, a single animal model will never precisely model all aspects of all the different types of human glaucoma. Therefore, multiple animal models have been utilized to study glaucoma but more are needed. Because of the powerful genetic tools available to use in the laboratory mouse, it has proven to be a highly useful mammalian system for studying the pathophysiology of human disease. The similarity between human and mouse eyes coupled with the ability to use a combination of advanced cell biological and genetic tools in mice have led to a large increase in the number of studies using mice to model specific glaucoma phenotypes. Over the last decade, numerous new mouse models and genetic tools have emerged, providing important insight into the cell biology and genetics of glaucoma. In this review, we describe available mouse genetic models that can be used to study glaucoma-relevant disease/pathobiology. Furthermore, we discuss how these models have been used to gain insights into ocular hypertension (a major risk factor for glaucoma) and glaucomatous retinal ganglion cell death. Finally, the potential for developing new mouse models and using advanced genetic tools and resources for studying glaucoma are discussed.
neuroinflammation; IOP; axonal degeneration; trabecular meshwork; mouse genetics; genomics; neurodegeneration; DBA/2J
Mutations in the gene encoding collagen type IV alpha 1 (COL4A1) cause multisystem disorders including anterior segment dysgenesis (ASD) and optic nerve hypoplasia. The penetrance and severity of individual phenotypes depends on genetic context. Here, we tested the effects of a Col4a1 mutation in two different genetic backgrounds to compare how genetic context influences ocular dysgenesis, IOP, and progression to glaucoma.
Col4a1 mutant mice maintained on a C57BL/6J background were crossed to either 129S6/SvEvTac or CAST/EiJ and the F1 progeny were analyzed by slit-lamp biomicroscopy and optical coherence tomography. We also measured IOPs and compared tissue sections of eyes and optic nerves.
We found that the CAST/EiJ inbred strain has a relatively uniform and profound suppression on the effects of Col4a1 mutation and that mutant CASTB6F1 mice were generally only very mildly affected. In contrast, mutant 129B6F1 mice had more variable and severe ASD and IOP dysregulation that were associated with glaucomatous signs including lost or damaged retinal ganglion cell axons and excavation of the optic nerve head.
Ocular defects in Col4a1 mutant mice model ASD and glaucoma that are observed in a subset of patients with COL4A1 mutations. We demonstrate that different inbred strains of mice give graded severities of ASD and we detected elevated IOP and glaucomatous damage in 129B6F1, but not CASTB6F1 mice that carried a Col4a1 mutation. These data demonstrate that genetic context differences are one factor that may contribute to the variable penetrance and severity of ASD and glaucoma in patients with COL4A1 mutations.
type IV collagen; glaucoma anterior segment; basement membrane; genetic context; phenotypic variation
Mutations in collagen, type IV, alpha 1 (COL4A1), a major component of basement membranes, cause multisystem disorders in humans and mice. In the eye, these include anterior segment dysgenesis, optic nerve hypoplasia and retinal vascular tortuosity. Here we investigate the retinal pathology in mice carrying dominant-negative Col4a1 mutations. To this end, we examined retinas longitudinally in vivo using fluorescein angiography, funduscopy and optical coherence tomography. We assessed retinal function by electroretinography and studied the retinal ultrastructural pathology. Retinal examinations revealed serous chorioretinopathy, retinal hemorrhages, fibrosis or signs of pathogenic angiogenesis with chorioretinal anastomosis in up to approximately 90% of Col4a1 mutant eyes depending on age and the specific mutation. To identify the cell-type responsible for pathogenesis we generated a conditional Col4a1 mutation and determined that primary vascular defects underlie Col4a1-associated retinopathy. We also found focal activation of Müller cells and increased expression of pro-angiogenic factors in retinas from Col4a1+/Δex41mice. Together, our findings suggest that patients with COL4A1 and COL4A2 mutations may be at elevated risk of retinal hemorrhages and that retinal examinations may be useful for identifying patients with COL4A1 and COL4A2 mutations who are also at elevated risk of hemorrhagic strokes.
Injury to retinal ganglion cell (RGC) axons triggers rapid activation of Jun N-terminal kinase (JNK) signaling, a major prodeath pathway in injured RGCs. Of the multiple kinases that can activate JNK, dual leucine kinase (Dlk) is known to regulate both apoptosis and Wallerian degeneration triggered by axonal insult. Here we tested the importance of Dlk in regulating somal and axonal degeneration of RGCs following axonal injury. Removal of DLK from the developing optic cup did not grossly affect developmental RGC death or inner plexiform layer organization. In the adult, Dlk deficiency significantly delayed axonal-injury induced RGC death. The activation of JUN was also attenuated in Dlk deficient retinas. Dlk deficiency attenuated the activation of the somal pool of JNK but did not prevent activation of the axonal pool of JNK after axonal injury, indicating that JNK activation in different cellular compartments of an RGC following axonal injury is regulated by distinct upstream kinases. In contrast to its robust influence on somal degeneration, Dlk deficiency did not alter RGC axonal degeneration after axonal injury as assessed using physiological readouts of optic nerve function.
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.
retina; light damage; ganglion cells; macaque; adaptive optics
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.
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.
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.
Early after optic nerve crush, TNFα appears to have a protective role for RGCs, which may be mediated through Müller cells.
Secondary degeneration; Neuroinflammation; TNFα; Retinal ganglion cell; Macroglia
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.
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.
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.
glaucoma; cell death; regeneration; ER stress; trauma; axonal injury; AP1
To investigate the role of Pou4f1 and Pou4f2 in the survival of adult retinal ganglion cells (RGCs).
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.
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.
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.
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.
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.
JNK; axonal injury; apoptosis; retinal ganglion cell; cJUN; mouse; neurodegeneration; neuroprotection; glaucoma
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.
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.
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.
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.
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.
(170.4460) Ophthalmic optics and devices; (110.1080) Active or adaptive optics; (330.7324) Visual optics, comparative animal models
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
(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
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.