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

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

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

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

Abstract 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

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