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.

Neuronal protein phosphatase 1ß regulates glutamate release, cortical myelination, node of Ranvier formation, and action potential propagation in the optic nerve.

Precise regulation of protein phosphorylation is critical for many cellular processes, and dysfunction in this process has been linked to various neurological disorders and diseases. Protein phosphatase 1 (PP1) is a ubiquitously expressed serine/threonine phosphatase with three major isoforms, (α, ß, γ) and hundreds of known substrates. Previously, we reported that PP1α and PP1γ are essential for the known role of PP1 in synaptic physiology and learning/memory, while PP1ß displayed a surprising opposing function. De novo mutations in PP1ß cause neurodevelopmental disorders in humans, but the mechanisms involved are currently unknown. A Cre-Lox system was used to delete PP1ß specifically in neurons in order to study its effects on developing mice. These animals fail to survive to 3 postnatal weeks, and exhibit deficits in cortical myelination and glutamate release. There was defective compound action potential (CAP) propagation in the optic nerve of the null mice, which was traced to a deficit in the formation of nodes of Ranvier. Finally, it was found that phosphorylation of the PP1ß-specific substrate, myosin light chain 2 (MLC2), is significantly enhanced in PP1ß null optic nerves. Several novel important in vivo roles of PP1ß in neurons were discovered, and these data will aid future investigations in delineating the mechanisms by which de novo mutations in PP1ß lead to intellectual and developmental delays in patients.

Microglia Depletion leads to Increased Susceptibility to Ocular Hypertension-Dependent Glaucoma.

In recent years, microglia have been highlighted for playing integral roles in neurodegenerative diseases, like glaucoma. To better understand the role of microglia during chronic ocular hypertension, we depleted microglia from aged (9-12 months old) DBA/2J (D2) mice, which exhibit age-related increases in intraocular pressure, using a dietary CSF1R antagonist, PLX5622. Retinal ganglion cell (RGC) somas were counted, and optic nerve cross-sections stained and assessed for glaucomatous damage. Sustained administration of dietary PLX5622 significantly reduced the numbers of retinal microglia. Dietary PLX5622 did not lead to changes in intraocular pressure in D2 or normotensive DBA/2J-Gpnmb+ (D2-Gpnmb+) control mice. While PLX5622-treated D2-Gpnmb+ did not develop optic nerve damage, PLX5622-treated D2 mice showed a significant increase in moderate-to-severe optic nerve damage compared to D2 mice fed a control diet. In conclusion, global reduction of microglia exacerbated glaucomatous neurodegeneration in D2 mice suggesting microglia play an overall beneficial role in protecting from ocular hypertension associated RGC loss.

Age- and glaucoma-induced changes to the ocular glymphatic system.

The ocular glymphatic system supports bidirectional fluid transport along the optic nerve, thereby removes metabolic wastes including amyloid-ß. To better understand this biological process, we examined the distributions of intravitreally and intracisternally infused tracers in full-length optic nerves from different age groups of mice. Aging was linked to globally impaired ocular glymphatic fluid transport, similar to what has seen previously in the brain. Aging also reduced the pupillary responsiveness to light stimulation and abolished light-induced facilitation in anterograde ocular glymphatic flow. In contrast to normal aging, in the DBA/2 J model of glaucoma, we found a pathological increase of glymphatic fluid transport to the anterior optic nerve that was associated with dilation of the perivascular spaces. Thus, aging and glaucoma have fundamentally different effects on ocular glymphatic fluid transport. Manipulation of glymphatic fluid transport might therefore present a new target for the treatment of glaucoma.

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.

Vascular derived endothelin receptor A controls endothelin-induced retinal ganglion cell death.

Endothelin (EDN, also known as ET) signaling has been suggested to be an important mediator of retinal ganglion cell (RGC) death in glaucoma. Antagonism of EDN receptors (EDNRA and EDNRB, also known as ET-A and ET-B) prevented RGC death in mouse models of chronic ocular hypertension, and intravitreal injection of EDN ligand was sufficient to drive RGC death. However, it remains unclear which cell types EDN ligands directly affect to elicit RGC death. Multiple cell types in the retina and optic nerve express EDNRA and EDNRB and thus could respond to EDN ligands in the context of glaucoma. Here, we systematically deleted Edn receptors from specific cell types to identify the critical EDN receptor mediating RGC death in vivo. Deletion of both Ednra and Ednrb from retinal neurons (including RGCs) and macroglia did not prevent RGC loss after exposure to EDN1 ligands, suggesting EDN1 ligands cause RGC death via an indirect mechanism involving a secondary cell type. Deletion of Ednra from the full body, and then specifically from vascular mural cells, prevented EDN1-induced vasoconstriction and RGC death. Together, these data suggest EDN ligands cause RGC death via a mechanism initiated by vascular mural cells. It is possible RGC death is a consequence of vascular mural cell-induced vasoconstriction and its pathological sequelae. These results highlight the potential importance of neurovascular dysfunction in glaucoma.

Transcriptional control of retinal ganglion cell death after axonal injury.

Injury to the axons of retinal ganglion cells (RGCs) is a key pathological event in glaucomatous neurodegeneration. The transcription factors JUN (the target of the c-Jun N-terminal kinases, JNKs) and DDIT3/CHOP (a mediator of the endoplasmic reticulum stress response) have been shown to control the majority of proapoptotic signaling after mechanical axonal injury in RGCs and in other models of neurodegeneration. The downstream transcriptional networks controlled by JUN and DDIT3, which are critical for RGC death, however, are not well defined. To determine these networks, RNA was isolated from the retinas of wild-type mice and mice deficient in Jun, Ddit3, and both Jun and Ddit3 three days after mechanical optic nerve crush injury (CONC). RNA-sequencing data analysis was performed and immunohistochemistry was used to validate potential transcriptional signaling changes after axonal injury. This study identified downstream transcriptional changes after injury including both neuronal survival and proinflammatory signaling that were attenuated to differing degrees by loss of Ddit3, Jun, and Ddit3/Jun. These data suggest proinflammatory signaling in the retina might be secondary to activation of pro-death pathways in RGCs after acute axonal injury. These results determine the downstream transcriptional networks important for apoptotic signaling which may be important for ordering and staging the pro-degenerative signals after mechanical axonal injury.

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.

An ocular glymphatic clearance system removes ß-amyloid from the rodent eye.

Despite high metabolic activity, the retina and optic nerve head lack traditional lymphatic drainage. We here identified an ocular glymphatic clearance route for fluid and wastes via the proximal optic nerve in rodents. ß-amyloid (Aß) was cleared from the retina and vitreous via a pathway dependent on glial water channel aquaporin-4 (AQP4) and driven by the ocular-cranial pressure difference. After traversing the lamina barrier, intra-axonal Aß was cleared via the perivenous space and subsequently drained to lymphatic vessels. Light-induced pupil constriction enhanced efflux, whereas atropine or raising intracranial pressure blocked efflux. In two distinct murine models of glaucoma, Aß leaked from the eye via defects in the lamina barrier instead of directional axonal efflux. The results suggest that, in rodents, the removal of fluid and metabolites from the intraocular space occurs through a glymphatic pathway that might be impaired in glaucoma.

Gabor domain optical coherence microscopy combined with laser scanning confocal fluorescence microscopy.

We report on the development of fluorescence Gabor domain optical coherence microscopy (Fluo GD-OCM), a combination of GD-OCM with laser scanning confocal fluorescence microscopy (LSCFM) for synchronous micro-structural and fluorescence imaging. The dynamic focusing capability of GD-OCM provided the adaptive illumination environment for both modalities without any mechanical movement. Using Fluo GD-OCM, we imaged ex vivo DsRed-expressing cells in the brain of a transgenic mouse, as well as Cy3-labeled ganglion cells and Cy3-labeled astrocytes from a mouse retina. The self-registration of images taken by the two different imaging modalities showed the potential for a correlative study of subjects and double identification of the target.

DDIT3 (CHOP) contributes to retinal ganglion cell somal loss but not axonal degeneration in DBA/2J mice.

Glaucoma is an age-related neurodegenerative disease characterized by the progressive loss of retinal ganglion cells (RGCs). Chronic ocular hypertension, an important risk factor for glaucoma, leads to RGC axonal injury at the optic nerve head. This insult triggers molecularly distinct cascades governing RGC somal apoptosis and axonal degeneration. The molecular mechanisms activated by ocular hypertensive insult that drive both RGC somal apoptosis and axonal degeneration are incompletely understood. The cellular response to endoplasmic reticulum stress and induction of pro-apoptotic DNA damage inducible transcript 3 (DDIT3, also known as CHOP) have been implicated as drivers of neurodegeneration in many disease models, including glaucoma. RGCs express DDIT3 after glaucoma-relevant insults, and importantly, DDIT3 has been shown to contribute to both RGC somal apoptosis and axonal degeneration after acute induction of ocular hypertension. However, the role of DDIT3 in RGC somal and axonal degeneration has not been critically tested in a model of age-related chronic ocular hypertension. Here, we investigated the role of DDIT3 in glaucomatous RGC death using an age-related, naturally occurring ocular hypertensive mouse model of glaucoma, DBA/2J mice (D2). To accomplish this, a null allele of Ddit3 was backcrossed onto the D2 background. Homozygous Ddit3 deletion did not alter gross retinal or optic nerve head morphology, nor did it change the ocular hypertensive profile of D2 mice. In D2 mice, Ddit3 deletion conferred mild protection to RGC somas, but did not significantly prevent RGC axonal degeneration. Together, these data suggest that DDIT3 plays a minor role in perpetuating RGC somal apoptosis caused by chronic ocular hypertension-induced axonal injury, but does not significantly contribute to distal axonal degeneration.

The polyether ionophore salinomycin targets multiple cellular pathways to block proliferative vitreoretinopathy pathology.

Proliferative vitreoretinopathy (PVR) is characterized by membranes that form in the vitreous cavity and on both surfaces of the retina, which results in the formation of tractional membranes that can cause retinal detachment and intrinsic fibrosis of the retina, leading to retina foreshortening. Currently, there are no pharmacologic therapies that are effective in inhibiting or preventing PVR formation. One of the key aspects of PVR pathogenesis is retinal pigment epithelial (RPE) cell epithelial mesenchymal transition (EMT). Here we show that the polyether ionophore compound salinomycin (SNC) effectively inhibits TGFß-induced EMT of RPE cells. SNC blocks the activation of TGFß-induced downstream targets alpha smooth muscle actin (αSMA) and collagen 1 (Col1A1). Additionally, SNC inhibits TGFß-induced RPE cell migration and contraction. We show that SNC functions to inhibit RPE EMT by targeting both the pTAK1/p38 and Smad2 signaling pathways upon TGFß stimulation. Additionally, SNC is able to inhibit αSMA and Col1A1 expression in RPE cells that have already undergone TGFß-induced EMT. Together, these results suggest that SNC could be an effective therapeutic compound in both the prevention and treatment of PVR.

Axon injury signaling and compartmentalized injury response in glaucoma.

Axonal degeneration is an active, highly controlled process that contributes to beneficial processes, such as developmental pruning, but also to neurodegeneration. In glaucoma, ocular hypertension leads to vision loss by killing the output neurons of the retina, the retinal ganglion cells (RGCs). Multiple processes have been proposed to contribute to and/or mediate axonal injury in glaucoma, including: neuroinflammation, loss of neurotrophic factors, dysregulation of the neurovascular unit, and disruption of the axonal cytoskeleton. While the inciting injury to RGCs in glaucoma is complex and potentially heterogeneous, axonal injury is ultimately thought to be the key insult that drives glaucomatous neurodegeneration. Glaucomatous neurodegeneration is a complex process, with multiple molecular signals contributing to RGC somal loss and axonal degeneration. Furthermore, the propagation of the axonal injury signal is complex, with injury triggering programs of degeneration in both the somal and axonal compartment. Further complicating this process is the involvement of multiple cell types that are known to participate in the process of axonal and neuronal degeneration after glaucomatous injury. Here, we review the axonal signaling that occurs after injury and the molecular signaling programs currently known to be important for somal and axonal degeneration after glaucoma-relevant axonal injuries.

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

There was an error introduced into Figures 4, 5, and 7 during the proofing stage which has since been corrected.

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.