IL1A enhances TNF-induced retinal ganglion cell death.

Glaucoma is a neurodegenerative disease that leads to the death of retinal ganglion cells (RGCs). A growing body of literature suggests a role for neuroinflammation in RGC death after glaucoma-relevant insults. For instance, it was shown that deficiency of three proinflammatory cytokines, complement component 1, subcomponent q ( C1q ), interleukin 1 alpha ( Il1a ), and tumor necrosis factor ( Tnf ), resulted in near complete protection of RGCs after two glaucoma-relevant insults, optic nerve injury and ocular hypertension. While TNF and C1Q have been extensively investigated in glaucoma-relevant model systems, the role of IL1A in RGC is not as well defined. Thus, we investigated the direct neurotoxicity of IL1A on RGCs in vivo. Intravitreal injection of IL1A did not result in RGC death at either 14 days or 12 weeks after insult. Consistent with previous studies, TNF injection did not result in significant RGC loss at 14 days but did after 12 weeks. Interestingly, IL1A+TNF resulted in a relatively rapid RGC death, driving significant RGC loss two weeks after injection. JUN activation and SARM1 have been implicated in RGC death in glaucoma and after cytokine insult. Using mice deficient in JUN or SARM1, we show RGC loss after IL1A+TNF insult is JUN-independent and SARM1-dependent. Furthermore, RNA-seq analysis showed that RGC death by SARM1 deficiency does not stop the neuroinflammatory response to IL1A+TNF. These findings indicate that IL1A can potentiate TNF-induced RGC death after combined insult is likely driven by a SARM1-dependent RGC intrinsic signaling pathway.

BAX activation in mouse retinal ganglion cells occurs in two temporally and mechanistically distinct steps.

BACKGROUND: Pro-apoptotic BAX is a central mediator of retinal ganglion cell (RGC) death after optic nerve damage. BAX activation occurs in two stages including translocation of latent BAX to the mitochondrial outer membrane (MOM) and then permeabilization of the MOM to facilitate the release of apoptotic signaling molecules. As a critical component of RGC death, BAX is an attractive target for neuroprotective therapies and an understanding of the kinetics of BAX activation and the mechanisms controlling the two stages of this process in RGCs is potentially valuable in informing the development of a neuroprotective strategy. METHODS: The kinetics of BAX translocation were assessed by both static and live-cell imaging of a GFP-BAX fusion protein introduced into RGCs using AAV2-mediated gene transfer in mice. Activation of BAX was achieved using an acute optic nerve crush (ONC) protocol. Live-cell imaging of GFP-BAX was achieved using explants of mouse retina harvested 7 days after ONC. Kinetics of translocation in RGCs were compared to GFP-BAX translocation in 661W tissue culture cells. Permeabilization of GFP-BAX was assessed by staining with the 6A7 monoclonal antibody, which recognizes a conformational change in this protein after MOM insertion. Assessment of individual kinases associated with both stages of activation was made using small molecule inhibitors injected into the vitreous either independently or in concert with ONC surgery. The contribution of the Dual Leucine Zipper-JUN-N-Terminal Kinase cascade was evaluated using mice with a double conditional knock-out of both Mkk4 and Mkk7. RESULTS: ONC induces the translocation of GFP-BAX in RGCs at a slower rate and with less intracellular synchronicity than 661W cells, but exhibits less variability among mitochondrial foci within a single cell. GFP-BAX was also found to translocate in all compartments of an RGC including the dendritic arbor and axon. Approximately 6% of translocating RGCs exhibited retrotranslocation of BAX immediately following translocation. Unlike tissue culture cells, which exhibit simultaneous translocation and permeabilization, RGCs exhibited a significant delay between these two stages, similar to detached cells undergoing anoikis. Translocation, with minimal permeabilization could be induced in a subset of RGCs using an inhibitor of Focal Adhesion Kinase (PF573228). Permeabilization after ONC, in a majority of RGCs, could be inhibited with a broad spectrum kinase inhibitor (sunitinib) or a selective inhibitor for p38/MAPK14 (SB203580). Intervention of DLK-JNK axis signaling abrogated GFP-BAX translocation after ONC. CONCLUSIONS: A comparison between BAX activation kinetics in tissue culture cells and in cells of a complex tissue environment shows distinct differences indicating that caution should be used when translating findings from one condition to the other. RGCs exhibit both a delay between translocation and permeabilization and the ability for translocated BAX to be retrotranslocated, suggesting several stages at which intervention of the activation process could be exploited in the design of a therapeutic strategy.

BAX activation in mouse retinal ganglion cells occurs in two temporally and mechanistically distinct steps.

Background Pro-apoptotic BAX is a central mediator of retinal ganglion cell (RGC) death after optic nerve damage. BAX activation occurs in two stages including translocation of latent BAX to the mitochondrial outer membrane (MOM) and then permeabilization of the MOM to facilitate the release of apoptotic signaling molecules. As a critical component of RGC death, BAX is an attractive target for neuroprotective therapies and an understanding of the kinetics of BAX activation and the mechanisms controlling the two stages of this process in RGCs is potentially valuable in informing the development of a neuroprotective strategy. Methods The kinetics of BAX translocation were assessed by both static and live-cell imaging of a GFP-BAX fusion protein introduced into RGCs using AAV2-mediated gene transfer in mice. Activation of BAX was achieved using an acute optic nerve crush (ONC) protocol. Live-cell imaging of GFP-BAX was achieved using explants of mouse retina harvested 7 days after ONC. Kinetics of translocation in RGCs were compared to GFP-BAX translocation in 661W tissue culture cells. Permeabilization of GFP-BAX was assessed by staining with the 6A7 monoclonal antibody, which recognizes a conformational change in this protein after MOM insertion. Assessment of individual kinases associated with both stages of activation was made using small molecule inhibitors injected into the vitreous either independently or in concert with ONC surgery. The contribution of the Dual Leucine Zipper-JUN-N-Terminal Kinase cascade was evaluated using mice with a double conditional knock-out of both Mkk4 and Mkk7 . Results ONC induces the translocation of GFP-BAX in RGCs at a slower rate and with less intracellular synchronicity than 661W cells, but exhibits less variability among mitochondrial foci within a single cell. GFP-BAX was also found to translocate in all compartments of an RGC including the dendritic arbor and axon. Approximately 6% of translocating RGCs exhibited retrotranslocation of BAX immediately following translocation. Unlike tissue culture cells, which exhibit simultaneous translocation and permeabilization, RGCs exhibited a significant delay between these two stages, similar to detached cells undergoing anoikis. Translocation, with minimal permeabilization could be induced in a subset of RGCs using an inhibitor of Focal Adhesion Kinase (PF573228). Permeabilization after ONC, in a majority of RGCs, could be inhibited with a broad spectrum kinase inhibitor (sunitinib) or a selective inhibitor for p38/MAPK14 (SB203580). Intervention of DLK-JNK axis signaling abrogated GFP-BAX translocation after ONC. Conclusions A comparison between BAX activation kinetics in tissue culture cells and in cells of a complex tissue environment shows distinct differences indicating that caution should be used when translating findings from one condition to the other. RGCs exhibit both a delay between translocation and permeabilization and the ability for translocated BAX to be retrotranslocated, suggesting several stages at which intervention of the activation process could be exploited in the design of a therapeutic strategy.

TNF-α and NF-κB signaling play a critical role in cigarette smoke-induced epithelial-mesenchymal transition of retinal pigment epithelial cells in proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is characterized by the growth and contraction of cellular membranes within the vitreous cavity and on both surfaces of the retina, resulting in recurrent retinal detachments and poor visual outcomes. Proinflammatory cytokines like tumor necrosis factor alpha (TNFα) have been associated with PVR and the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. Cigarette smoke is the only known modifiable risk factor for PVR, but the mechanisms are unclear. The purpose of this study was to examine the impact of cigarette smoke on the proinflammatory TNFα/NF-κB/Snail pathway in RPE cells to better understand the mechanisms through which cigarette smoke increases the risk of PVR. Human ARPE-19 cells were exposed to cigarette smoke extract (CSE), for 4 to 24-hours and TNFα, Snail, IL-6, IL-8, and α-SMA levels were analyzed by qPCR and/or Western blot. The severity of PVR formation was assessed in a murine model of PVR after intravitreal injection of ARPE-19 cells pre-treated with CSE or not. Fundus imaging, OCT imaging, and histologic analysis 4 weeks after injection were used to examine PVR severity. ARPE-19 cells exposed to CSE expressed higher levels of TNFα, SNAIL, IL6 and IL8 mRNA as well as SNAIL, Vimentin and α-SMA protein. Inhibition of TNFα and NF-κB pathways blocked the effect of CSE. In vivo, intravitreal injection of ARPE-19 cells treated with CSE resulted in more severe PVR compared to mice injected with untreated RPE cells. These studies suggest that the TNFα pathway is involved in the mechanism whereby cigarette smoke increases PVR. Further investigation into the role of TNFα/NF-κB/Snail in driving PVR and pharmacological targeting of these pathways in disease are warranted.

Salinomycin inhibits proliferative vitreoretinopathy formation in a mouse model.

Proliferative vitreoretinopathy (PVR) is a progressive disease that develops in a subset of patients who undergo surgery for retinal detachment repair, and results in significant vision loss. PVR is characterized by the migration of retinal pigment epithelial (RPE) cells into the vitreous cavity, where they undergo epithelial-to-mesenchymal transition and form contractile membranes within the vitreous and along the retina, resulting in recurrent retinal detachments. Currently, surgical intervention is the only treatment for PVR and there are no pharmacological agents that effectively inhibit or prevent PVR formation. Here, we show that a single intravitreal injection of the polyether ionophore salinomycin (SNC) effectively inhibits the formation of PVR in a mouse model with no evidence of retinal toxicity. After 4 weeks, fundus photography and optical coherence tomography (OCT) demonstrated development of mean PVR grade of 3.5 (SD: 1.3) in mouse eyes injected with RPE cells/DMSO (vehicle), compared to mean PVR grade of 1.6 (SD: 1.3) in eyes injected with RPE cells/SNC (p = 0.001). Additionally, immunohistochemistry analysis showed RPE cells/SNC treatment reduced both fibrotic (αSMA, FN1, Vim) and inflammatory (GFAP, CD3, CD20) markers compared to control RPE cells/DMSO treatment. Finally, qPCR analysis confirmed that Tgfß, Tnfα, Mcp1 (inflammatory/cytokine markers), and Fn1, Col1a1 and Acta2 (fibrotic markers) were significantly attenuated in the RPE cells/SNC group compared to RPE/DMSO control. These results suggest that SNC is a potential pharmacologic agent for the prevention of PVR in humans and warrants further investigation.

Endothelin 1-induced retinal ganglion cell death is largely mediated by JUN activation.

Glaucoma is a neurodegenerative disease characterized by loss of retinal ganglion cells (RGCs), the output neurons of the retina. Multiple lines of evidence show the endothelin (EDN, also known as ET) system is important in glaucomatous neurodegeneration. To date, the molecular mechanisms within RGCs driving EDN-induced RGC death have not been clarified. The pro-apoptotic transcription factor JUN (the canonical target of JNK signaling) and the endoplasmic reticulum stress effector and transcription factor DNA damage inducible transcript 3 (DDIT3, also known as CHOP) have been shown to act downstream of EDN receptors. Previous studies demonstrated that JUN and DDIT3 were important regulators of RGC death after glaucoma-relevant injures. Here, we characterized EDN insult in vivo and investigated the role of JUN and DDIT3 in EDN-induced RGC death. To accomplish this, EDN1 ligand was intravitreally injected into the eyes of wildtype, Six3-cre+Junfl/fl (Jun-/-), Ddit3 null (Ddit3-/-), and Ddit3-/-Jun-/- mice. Intravitreal EDN1 was sufficient to drive RGC death in vivo. EDN1 insult caused JUN activation in RGCs, and deletion of Jun from the neural retina attenuated RGC death after EDN insult. However, deletion of Ddit3 did not confer significant protection to RGCs after EDN1 insult. These results indicate that EDN caused RGC death via a JUN-dependent mechanism. In addition, EDN signaling is known to elicit potent vasoconstriction. JUN signaling was shown to drive neuronal death after ischemic insult. Therefore, the effects of intravitreal EDN1 on retinal vessel diameter and hypoxia were explored. Intravitreal EDN1 caused transient retinal vasoconstriction and regions of RGC and Müller glia hypoxia. Thus, it remains a possibility that EDN elicits a hypoxic insult to RGCs, causing apoptosis via JNK-JUN signaling. The importance of EDN-induced vasoconstriction and hypoxia in causing RGC death after EDN insult and in models of glaucoma requires further investigation.

A Mouse Model of Proliferative Vitreoretinopathy Induced by Intravitreal Injection of Gas and RPE Cells.

Purpose: Develop a reproducible proliferative vitreoretinopathy (PVR) mouse model that mimics human PVR pathology. Methods: Mice received intravitreal injections of SF6 gas, followed by retinal pigment epithelial cells 1 week later. PVR progression was monitored using fundus photography and optical coherence tomography imaging, and histologic analysis of the retina as an endpoint. We developed a PVR grading scheme tailored for this model. Results: We report that mice that received gas before retinal pigment epithelial injection developed more severe PVR. In the 1 × 104 retinal pigment epithelial cell group, after 1 week, 0 of 11 mice in the no gas group developed grade 4 or greater PVR compared with 5 of 13 mice in the SF6 gas group (P = 0.02); after 4 weeks, 3 of 11 mice in the no gas group developed grade 5 or greater PVR compared with 11 of 14 mice in the SF6 gas group (P = 0.01). We were able to visualize contractile membranes both on the retinal surface as well as within the vitreous of PVR eyes, and demonstrated through immunohistochemical staining that these membranes expressed fibrotic markers alpha smooth muscle actin, vimentin, and fibronectin, as well as other markers known to be found in human PVR membranes. Conclusions: This mouse PVR model is reproducible and mimics aspects of PVR pathology reported in the rabbit PVR model and human PVR. The major advantage is the ability to study PVR development in different genetic backgrounds to further elucidate aspects of PVR pathogenesis. Additionally, large-scale experiments for testing pharmacologic agents to treat and prevent PVR progression is more feasible compared with other animal models. Translational Relevance: This model will provide a platform for screening potential drug therapies to treat and prevent PVR, as well as elucidate different molecular pathways involved in PVR pathogenesis.

Mkk4 and Mkk7 are important for retinal development and axonal injury-induced retinal ganglion cell death.

The mitogen-activated protein kinase (MAPK) pathway has been shown to be involved in both neurodevelopment and neurodegeneration. c-Jun N-terminal kinase (JNK), a MAPK important in retinal development and after optic nerve crush injury, is regulated by two upstream kinases: MKK4 and MKK7. The specific requirements of MKK4 and MKK7 in retinal development and retinal ganglion cell (RGC) death after axonal injury, however, are currently undefined. Optic nerve injury is an important insult in many neurologic conditions including traumatic, ischemic, inflammatory, and glaucomatous optic neuropathies. Mice deficient in Mkk4, Mkk7, and both Mkk4 and Mkk7 were generated. Immunohistochemistry was used to study the distribution and structure of retinal cell types and to assess RGC survival after optic nerve injury (mechanical controlled optic nerve crush (CONC)). Adult Mkk4- and Mkk7-deficient retinas had all retinal cell types, and with the exception of small areas of disrupted photoreceptor lamination in Mkk4-deficient mice, the retinas of both mutants were grossly normal. Deficiency of Mkk4 or Mkk7 reduced JNK signaling in RGCs after axonal injury and resulted in a significantly greater percentage of surviving RGCs 35 days after CONC as compared to wild-type controls (Mkk4: 51.5%, Mkk7: 29.1%, WT: 15.2%; p < 0.001). Combined deficiency of Mkk4 and Mkk7 caused failure of optic nerve formation, irregular retinal axonal trajectories, disruption of retinal lamination, clumping of RGC bodies, and dendritic fasciculation of dopaminergic amacrine cells. These results suggest that MKK4 and MKK7 may serve redundant and unique roles in molecular signaling important for retinal development and injury response following axonal insult.

Role of SARM1 and DR6 in retinal ganglion cell axonal and somal degeneration following axonal injury.

Optic neuropathies such as glaucoma are characterized by the degeneration of retinal ganglion cells (RGCs) and the irreversible loss of vision. In these diseases, focal axon injury triggers a propagating axon degeneration and, eventually, cell death. Previous work by us and others identified dual leucine zipper kinase (DLK) and JUN N-terminal kinase (JNK) as key mediators of somal cell death signaling in RGCs following axonal injury. Moreover, others have shown that activation of the DLK/JNK pathway contributes to distal axonal degeneration in some neuronal subtypes and that this activation is dependent on the adaptor protein, sterile alpha and TIR motif containing 1 (SARM1). Given that SARM1 acts upstream of DLK/JNK signaling in axon degeneration, we tested whether SARM1 plays a similar role in RGC somal apoptosis in response to optic nerve injury. Using the mouse optic nerve crush (ONC) model, our results show that SARM1 is critical for RGC axonal degeneration and that axons rescued by SARM1 deficiency are electrophysiologically active. Genetic deletion of SARM1 did not, however, prevent DLK/JNK pathway activation in RGC somas nor did it prevent or delay RGC cell death. These results highlight the importance of SARM1 in RGC axon degeneration and suggest that somal activation of the DLK/JNK pathway is activated by an as-yet-unidentified SARM1-independent signal.

Together JUN and DDIT3 (CHOP) control retinal ganglion cell death after axonal injury.

BACKGROUND: Optic nerve injury is an important pathological component in neurodegenerative diseases such as traumatic optic neuropathies and glaucoma. The molecular signaling pathway(s) critical for retinal ganglion cell (RGC) death after axonal insult, however, is/are not fully defined. RGC death after axonal injury is known to occur by BAX-dependent apoptosis. Two transcription factors JUN (the canonical target of JNK) and DDIT3 (CHOP; a key mediator of the endoplasmic reticulum stress response) are known to be important apoptotic signaling molecules after axonal injury, including in RGCs. However, neither Jun nor Ddit3 deficiency provide complete protection to RGCs after injury. Since Jun and Ddit3 are important apoptotic signaling molecules, we sought to determine if their combined deficiency might provide additive protection to RGCs after axonal injury. METHODS: To determine if DDIT3 regulated the expression of JUN after an axonal insult, mice deficient for Ddit3 were examined after optic nerve crush (ONC). In order to critically test the importance of these genes in RGC death after axonal injury, RGC survival was assessed at multiple time-points after ONC (14, 35, 60, and 120 days after injury) in Jun, Ddit3, and combined Jun/Ddit3 deficient mice. Finally, to directly assess the role of JUN and DDIT3 in axonal degeneration, compound actions potentials were recorded from Jun, Ddit3, and Jun/Ddit3 deficient mice after ONC. RESULTS: Single and combined deficiency of Jun and Ddit3 did not appear to alter gross retinal morphology. Ddit3 deficiency did not alter expression of JUN after axonal injury. Deletion of both Jun and Ddit3 provided significantly greater long-term protection to RGCs as compared to Jun or Ddit3 deficiency alone. Finally, despite the profound protection to RGC somas provided by the deficiency of Jun plus Ddit3, their combined loss did not lessen axonal degeneration. CONCLUSIONS: These results suggest JUN and DDIT3 are independently regulated pro-death signaling molecules in RGCs and together account for the vast majority of apoptotic signaling in RGCs after axonal injury. Thus, JUN and DDIT3 may represent key molecular hubs that integrate upstream signaling events triggered by axonal injury with downstream transcriptional events that ultimately culminate in RGC apoptosis.

JUN is important for ocular hypertension-induced retinal ganglion cell degeneration.

Ocular hypertension, a major risk factor for glaucoma, is thought to trigger glaucomatous neurodegeneration through injury to retinal ganglion cell (RGC) axons. The molecular signaling pathway leading from ocular hypertension to RGC degeneration, however, is not well defined. JNK signaling, a component of the mitogen-activated protein kinase (MAPK) family, and its canonical target, the transcription factor JUN, have been shown to regulate neurodegeneration in many different systems. JUN is expressed after glaucoma-relevant injuries and Jun deficiency protects RGCs after mechanical injury to the optic nerve. Here, we tested the importance of JNK-JUN signaling for RGC death after ocular hypertensive axonal injury in an age-related, mouse model of ocular hypertension. Immunohistochemistry was performed to evaluate JUN expression in ocular hypertensive DBA/2J mice. JUN was expressed in a temporal and spatial pattern consistent with a role in glaucomatous injury. To determine the importance of JUN in ocular hypertension-induced RGC death, a floxed allele of Jun and a retinal expressed cre recombinase (Six3-cre) were backcrossed onto the DBA/2J background. Intraocular pressure (IOP) and gross morphology of the retina and optic nerve head were assessed to determine whether removing Jun from the developing retina altered IOP elevation or retinal development. Jun deficiency in the retina did not alter DBA/2J IOP elevation or retinal development. Optic nerves and retinas were assessed at ages known to have glaucomatous damage in DBA/2J mice. Jun deficiency protected RGC somas from ocular hypertensive injury, but did not protect RGC axons from glaucomatous neurodegeneration. Jun is a major regulator of RGC somal degeneration after glaucomatous ocular hypertensive injury. These results suggest in glaucomatous neurodegeneration, JNK-JUN signaling has a major role as a pro-death signaling pathway between axonal injury and somal degeneration.

Early immune responses are independent of RGC dysfunction in glaucoma with complement component C3 being protective.

Various immune response pathways are altered during early, predegenerative stages of glaucoma; however, whether the early immune responses occur secondarily to or independently of neuronal dysfunction is unclear. To investigate this relationship, we used the Wlds allele, which protects from axon dysfunction. We demonstrate that DBA/2J.Wlds mice develop high intraocular pressure (IOP) but are protected from retinal ganglion cell (RGC) dysfunction and neuroglial changes that otherwise occur early in DBA/2J glaucoma. Despite this, immune pathways are still altered in DBA/2J.Wlds mice. This suggests that immune changes are not secondary to RGC dysfunction or altered neuroglial interactions, but may be directly induced by the increased strain imposed by high IOP. One early immune response following IOP elevation is up-regulation of complement C3 in astrocytes of DBA/2J and DBA/2J.Wlds mice. Unexpectedly, because the disruption of other complement components, such as C1Q, is protective in glaucoma, C3 deficiency significantly increased the number of DBA/2J eyes with nerve damage and RGC loss at an early time point after IOP elevation. Transcriptional profiling of C3-deficient cultured astrocytes implicated EGFR signaling as a hub in C3-dependent responses. Treatment with AG1478, an EGFR inhibitor, also significantly increased the number of DBA/2J eyes with glaucoma at the same early time point. These findings suggest that C3 protects from early glaucomatous damage, a process that may involve EGFR signaling and other immune responses in the optic nerve head. Therefore, therapies that target specific components of the complement cascade, rather than global inhibition, may be more applicable for treating human glaucoma.

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.

Phospholipid flippase ATP8A2 is required for normal visual and auditory function and photoreceptor and spiral ganglion cell survival.

ATP8A2 is a P4-ATPase that is highly expressed in the retina, brain, spinal cord and testes. In the retina, ATP8A2 is localized in photoreceptors where it uses ATP to transport phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the exoplasmic to the cytoplasmic leaflet of membranes. Although mutations in ATP8A2 have been reported to cause mental retardation in humans and degeneration of spinal motor neurons in mice, the role of ATP8A2 in sensory systems has not been investigated. We have analyzed the retina and cochlea of ATP8A2-deficient mice to determine the role of ATP8A2 in visual and auditory systems. ATP8A2-deficient mice have shortened photoreceptor outer segments, a reduction in photoresponses and decreased photoreceptor viability. The ultrastructure and phagocytosis of the photoreceptor outer segment appeared normal, but the PS and PE compositions were altered and the rhodopsin content was decreased. The auditory brainstem response threshold was significantly higher and degeneration of spiral ganglion cells was apparent. Our studies indicate that ATP8A2 plays a crucial role in photoreceptor and spiral ganglion cell function and survival by maintaining phospholipid composition and contributing to vesicle trafficking.

Focal damage to macaque photoreceptors produces persistent visual loss.

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.

JUN regulates early transcriptional responses to axonal injury in retinal ganglion cells.

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.

Mutations in a P-type ATPase gene cause axonal degeneration.

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 Wld(s) 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.

The Bcl-2 family member BIM has multiple glaucoma-relevant functions in DBA/2J mice.

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.

Radiation treatment inhibits monocyte entry into the optic nerve head and prevents neuronal damage in a mouse model of glaucoma.

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.

Datgan, a reusable software system for facile interrogation and visualization of complex transcription profiling data.

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.