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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.

A growing body of literature suggests a role for neuroinflammation in retinal ganglion cell (RGC) death in glaucoma. For instance, deficiency of three proinflammatory cytokines, complement component 1, subcomponent q ( ), interleukin 1 alpha ( ), and tumor necrosis factor ( ), resulted in significant protection of RGCs after glaucoma-relevant insults. While TNF and C1Q have been extensively investigated in glaucoma-relevant model systems, the role of IL1A in RGCs is not well defined. Eyes of 2-4 month-old C57BL/6J mice or mice deficient in either or were intravitreally injected with IL1A alone, TNF alone, or IL1A and TNF together. Retinal flat mounts were assessed for RGC survival using immunostaining of RBPMS. Bulk RNA-sequencing and differential expression analyses of retinal tissue was performed to determine molecular changes in response to IL1A, TNF, and IL1A combined with TNF within C57BL/6J and deficient mice. Intravitreal injection of IL1A did not result in RGC death at either 14 days or 12 weeks. Consistent with previous studies, TNF injection did not cause significant RGC loss at 14 days but did after 12 weeks. Together, IL1A+TNF resulted in a relatively rapid RGC death, driving significant loss 2 weeks after injection. We identified molecular changes which occur in response to IL1A and to combined IL1A+TNF treatment with limited changes identified in TNF alone treated eyes. Using mice deficient in or , we showed RGC loss after IL1A+TNF insult is JUN-independent and SARM1-dependent. Furthermore, RNA-seq analysis showed deficiency does not stop the neuroinflammatory response to IL1A+TNF. We identified a novel role of IL1A, we found that IL1A acted as a sensitizer to TNF-induced death. Co-injection of IL1A and TNF resulted in rapid RGC death, with significant RGC loss 14 days after injection. TNF+IL1A-induced RGC death did not depend on JUN activation and was rather SARM1 dependent. Also, RNA-seq analyses indicated that while deficiency protected from IL1A+TNF induced RGC loss it did not significantly alter microglia and astrocyte responses. Altogether, these findings indicate that IL1A potentiates SARM1-dependent TNF-induced RGC death .

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.

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/2 J (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/2 J- 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.

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.

Excitotoxicity leads to the activation of a cytotoxic cascade that causes neuronal death. In the retina, retinal ganglion cells (RGCs) die after an excitotoxic insult. Multiple pathways have been proposed to contribute to RGC death after an excitotoxic insult, including TNF signaling, JNK activation, and ER stress. To test the importance of these pathways in RGC death after excitotoxic injury, the excitotoxin N-methyl-D-aspartate (NMDA) was intravitreally injected into mice deficient in components of these pathways. Absence of Tnf or its canonical downstream mediator, Bid , did not confer short- or long-term protection to RGCs. Despite known activation in RGCs and a prominent role in mediating RGC death after other insults, attenuating JNK signaling did not prevent RGC death after excitotoxic insult. Additionally, deficiency of the ER stress protein DDIT3 (CHOP), which has been shown to be involved in RGC death, did not lessen NMDA induced RGC death. Furthermore, absence of both Jun (JNK’s canonical target) and Ddit3 , which together provide robust, long-term protection to RGC somas after axonal insult, did not lessen RGC death. Collectively, these results indicate that the drivers of excitotoxic injury remain to be identified and/or multiple cell death pathways are activated in response to injury.

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.

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 Tgfβ , 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.

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.

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.