A small peptide antagonist of the Fas receptor inhibits neuroinflammation and prevents axon degeneration and retinal ganglion cell death in an inducible mouse model of glaucoma

All animal experiments were approved by the Institutional Animal Care and Use Committee at Schepens Eye Research Institute and were performed under the guidelines of the Association of Research in Vision and Ophthalmology (Rockville, MD). The 8-week-old C57BL/6J WT mice (Stock No: 000664) and B6.MRL-Fas^lpr/J Fas receptor-deficient mice (Stock No: 000482) were purchased from Jackson Laboratories (Bar Harbor, ME) and housed and maintained under cyclic light (12 L-30 lux: 12D) conditions in an AAALAC-approved animal facility at the Schepens Eye Research Institute. To avoid sex bias, equal numbers of male and female mice were included in each experimental group.

Mice were anesthetized by intraperitoneal injection of a mixture of ketamine (100 mg/kg; Ketaset; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (9 mg/kg; TranquiVed; Vedco, Inc., St. Joseph, MO) supplemented by topical application of proparacaine (0.5%; Bausch & Lomb, Tampa, FL). Elevation of IOP was induced unilaterally by injection of polystyrene microbeads (FluoSpheres; Invitrogen, Carlsbad, CA; 15-μm diameter) into the anterior chamber of the right eye of each animal under a surgical microscope, as previously reported [31]. Briefly, microbeads were prepared at a concentration of 5.0 × 10⁶ beads/ml in sterile physiologic saline. The right cornea was gently punctured near the center using a sharp glass micropipette (World Precision Instruments Inc., Sarasota, FL). A small volume (2 μL) of microbeads was injected through this preformed hole into the anterior chamber followed by injection of an air bubble via the micropipette connected with a Hamilton syringe. Any mice that developed signs of inflammation (clouding of the cornea, edematous cornea, etc.) were excluded from the study.

IOP was measured with a rebound TonoLab tonometer (Colonial Medical Supply, Espoo, Finland), as previously described [31, 33]. Mice were anesthetized by 3% isoflurane in 100% oxygen (induction) followed by 1.5% isoflurane in 100% oxygen (maintenance) delivered with a precision vaporizer. IOP measurement was initiated within 2 to 3 min after animals lost toe pinch reflex or tail pinch response. Anesthetized mice were placed on a platform, and the tip of the pressure sensor was placed approximately 1/8 in. from the central cornea. Average IOP was displayed automatically after six measurements after elimination of the highest and lowest values. This machine-generated mean was considered as one reading, and six readings were obtained for each eye. All IOPs were taken at the same time of day (between 10:00 and 12:00 h) due to the variation of IOP throughout the day.

The intravitreal injections, just posterior to the limbus and parallel to the conjunctival vessels, were performed as previously described [31, 36]. Mice received a 1-μl intravitreal injection containing ONL1204 (2 mg/ml) or vehicle control on day 0 (just prior to injection of microbeads) or day 7 after microbeads injection.

For quantification of axons, optic nerves were dissected and fixed in Karnovsky’s reagent (50% in phosphate buffer) overnight. Semi-thin cross-sections of the nerve were taken at 1.0 mm posterior to the globe and stained with 1% p-phenylenediamine (PPD) for evaluation by light microscopy. Ten non-overlapping photomicrographs were taken at × 100 magnification covering the entire area of the optic nerve cross-section. Using ImageJ software, a 50 μM × 50 μM square was placed on each × 100 image and all axons within the square (0.0025 mm²) were counted using the threshold and analyze particles function in image J as previously described [31]. The average axon counts in the 10 images were used to calculate the axon density per square millimeter of the optic nerve (ON). Individuals blinded to the experimental groups performed all axon counts.

Immediately following euthanasia, eyes were enucleated and fixed in 4% paraformaldehyde for 2 h at room temperature. The retina was detached from the eyecup, and four radial incisions reaching approximately 2/3 of the radius of the retina were made to create a butterfly shape. Retinal flatmounts were washed with PBS/T (0.1% Triton X-100) and permeabilized with 0.1% Triton X-100 in 20% superblock blocking buffer (2 ml superblock (Thermo Fisher cat no- 37580) + 8 ml PBS/T + 10μl Triton X) for 30 min at room temperature. Following permeabilization, the retinas were blocked in blocking solution (20% superblock + 10% goat serum) for 1 h at room temperature. Retinas were then incubated at 4 °C overnight with a primary Ab against Brn3a, a RGC-specific marker (Millipore Cat no- 1585, Billerica, MA), or against Iba1, a microglia/macrophage marker (Wako, Chemicals USA, Inc. Cat# 019-19741). An Alexa Fluor 555—conjugated for Brn3a— and Alexa Fluor 488—conjugated for IBA1 (Invitrogen)—was used as secondary Ab. Nuclei were counterstained with DAPI (vector stain).

To quantitate retinal ganglion cells, × 60 oil immersion was used and 16 non-overlapping images were taken (4–5 images per quadrant) using the × 60 oil immersion objective of Leica TCS SP5 confocal microscope system. All Brn3a-stained RGCs were quantitated using an automated counting platform we previously developed using CellProfiler software [37]. ImageJ software was used to calculate the area of each image, and the average number of RGCs in the 16 images was used to calculate the RGC density per square millimeter of retina. Individuals blinded to the experimental groups performed all RGC counts.

To quantitate Iba1+ microglia/macrophages, image stacks of retinal flatmounts were acquired using the × 20 oil immersion objective (zoom 1.7, 35 μm depth (includes GCL and IPL)) of the Leica TCS SP5 confocal microscope system. The retina was divided into four quadrants, and one mid-peripheral region was imaged per quadrant for a total of four images per retina (480 μm by 480 μm per region). Microglial cells were counted manually by an individual blinded to the treatment groups using ImageJ software as previously described [38]. The longest cell process length, which is a marker of cell quiescence, was used as a morphometric descriptor to analyze microglia activation using NeruonJ Fiji Plugin as previously described [39]. Individuals blinded to the experimental groups performed all microglia quantification.

RNA was isolated from the neural retina using a QIAGEN RNeasy Mini Kit (catalog number 74104), according to the manufacturer’s protocol. RNA was treated with DNase (catalog number AM222; Invitrogen) to ensure no contamination of genomic DNA. A total of 500 ng of RNA was reverse-transcribed (Thermo fisher Cat no 11756050 Superscript IV VILO master mix) according to the manufacturer’s instructions. cDNA was diluted 1:4 and then used for each amplification reaction. cDNA was treated with RNase H (18021-014; Invitrogen) to ensure the absence of ssRNA. Quantitative PCR (qPCR) reactions were performed in a 10 μl total volume using the FastStart Universal SYBR Green Master (Rox) (4913914001; Sigma) according to the manufacturer’s protocol. PCR cycles consisted of a denaturation step at 95 °C for 10 min, followed by 50 cycles of 95 °C for 15 s and 60 °C for 60 s. Each sample was subjected to melting curve analysis to confirm amplification specificity. Samples were run in duplicate, and each experiment included nontemplate control wells. Samples were normalized to house-keeping genes and expressed as the relative expression using the δ-delta Ct method. Relative expression to two house-keeping genes β2 microglobulin and PPIA was quantified using the formula: relative expression δ-delta CT = 2^(avg. gene cT−avg. saline-treated cT). Fold changes were calculated with respect to saline-injected control eyes. All of the primers used are listed in Table 1.

The ability of ONL1204 to inhibit FasL-mediated apoptosis of murine A20 B lymphoma cells was evaluated in vitro. Microvesicle preparations were isolated from transfected Neuro2a cells that expressed either murine mFasL (mFasL VP) or the vector control (Neo VP) as described previously [40]. A20 lymphoma cells were incubated for 4 h with titrations of ONL 1204 or the vehicle control together with a 1:100 dilution of either mFasL VP or Neo VP and then cultured overnight in the presence of ³H-thymidine. Survival was assessed by ³H-thymidine incorporation using the formula (cpm of mFasL VP + ONL1204 or vehicle)/cpm Neo VP + ONL1204 or vehicle).

We previously demonstrated that a small peptide antagonist of the Fas receptor (Met12) inhibits Fas-induced Caspase 8 activation and cell death of photoreceptors and retinal pigment epithelial cells in models of retinal detachment and NaIO₃ model of oxidative stress respectively [34, 35]. In this study, we used a new derivative of Met12, ONL1204, with improved pharmaceutical properties. To confirm that ONL1204 blocks Fas death receptor signaling, we treated Fas+ murine A20 B lymphoma cells with membrane-FasL-expressing microvesicles in the presence of increasing concentrations of ONL1204 (Fig. 1). We previously demonstrated that microvesicles isolated from transfected Neuro2a cells expressing murine membrane-bound FasL (mFasL-VP) can serve as a cell-free source of mFasL that is highly efficient in killing Fas+ murine A20 B lymphoma cells [40]. Microvesicles isolated from Neuro2a cells transfected with the vector control (Neo-VP) do not express mFasL and serve as a negative control. Herein, A20 cells were treated with mFasL-VP at a 1:100 dilution for 4 h, and ³H-Thymidine incorporation indicated significant cell death with only 8.0% survival when compared to A20 cells incubated with media alone (Fig. 1). By contrast, no significant cell death was observed in A20 cells treated with Neo-VP, resulting in 100% survival when compared to A20 cells incubated with media alone. To determine whether ONL1204 could block apoptosis triggered by mFasL-VP, A20 cells were treated with mFasL-VP at a 1:100 dilution for 4 h in the presence of increasing concentrations of ONL1204 or vehicle control. Our results showed that FasL-induced apoptosis was inhibited by ONL1204 in a dose-dependent manner, while vehicle only had no effect (Fig. 1). These results demonstrate that ONL1204 can block activation of the Fas death receptor signaling pathway and prevent mFasL-induced apoptosis.

Using genetically modified mice, we demonstrated previously that the membrane-bound form of FasL (mFasL) is neurotoxic and accelerates RGC death and axon degeneration in inducible and chronic mouse models of glaucoma [31, 36]. By contrast, overexpression of the soluble form of FasL (sFasL) via AAV-mediated gene delivery prevented RGC death and axon degeneration [31]. While these previous studies revealed opposing roles for mFasL and sFasL in the pathogenesis of glaucoma, the requirement of the Fas signaling pathway for the development of glaucoma was never demonstrated. Therefore, to determine whether Fas signaling was required for the development of glaucoma, we utilized a well-defined microbead-induced mouse model of elevated IOP to induce elevated IOP in C57BL/6J WT mice and Fas-deficient LPR mice (Fas^lpr)) [41]. As previously described [31], a single anterior chamber injection of 15 μm polystyrene microbeads resulted in elevated IOP for up to 21 days in C57BL/6J WT as compared to saline controls (Fig. 2a). The IOP was monitored by rebound tonometry, and there was no significant difference in the time course or magnitude of the microbead-induced elevated IOP between Fas^lpr mice or C57BL/6J WT mice, indicating Fas signaling was not involved in the elevation of IOP. At 4 weeks post-microbead injection, RGC density was measured in retinal whole mounts stained with a RGC-specific anti-Brn3a antibody [37] (Fig. 2b, c) and axon density was measured in optic nerve sections stained with PPD [31] (Fig. 2d, e). Quantification of RGCs revealed a significant decrease in RGC density in microbead-injected WT mice as compared to saline-injected controls (Fig. 2c). However, in the absence of Fas signaling, the RGC density in microbead-injected Fas^lpr mice was equal to that of saline-injected controls (Fig. 2c). Similar results were observed in the optic nerve where Fas deficiency afforded complete protection of axons in microbead-injected Fas^lpr mice as compared to the microbead-injected C57BL/6 WT mice (Fig. 2d, e). Taken together, these results demonstrate that Fas signaling is required for the death of RGCs and loss of axons in the microbead-induced mouse model of glaucoma.

To determine the neuroprotective potential of Fas-receptor inhibition in glaucoma, we pretreated C57BL/6J WT mice with ONL1204, prior to induction of elevated IOP. In this study, C57BL/6J WT mice received an intravitreal injection of ONL1204 (2 μg/μl) or vehicle only, immediately preceding injection of microbeads or saline. IOPs were monitored every 3–4 days by rebound tonometry and revealed no significant difference in the time course or magnitude of the microbead-induced elevated IOP between mice treated with ONL1204 or vehicle only, indicating ONL1204 did not affect IOP (Fig. 3a). Quantification of RGCs at 4 weeks post-microbead injection revealed a significant decrease in RGC density in microbead-injected vehicle-treated mice as compared to the saline-injected controls (Fig. 3b, c). However, pretreatment with ONL1204 was neuroprotective, and the RGC density in microbead-injected ONL1204-treated mice was equal to the RGC density in the saline-injected controls (Fig. 3b, c). Similar results were observed in the optic nerve with a significant decrease in axon density detected in microbead-injected, vehicle-treated mice when compared to saline-injected controls, while pretreatment with ONL1204 afforded complete protection of axons with axon density in microbead-injected ONL1204-treated mice equal to that of the saline-injected controls (Fig. 3d, e). Taken together, these results demonstrate that pretreatment with the Fas inhibitor, ONL1204, prior to elevated IOP provides significant neuroprotection to both RGCs and their axons in the microbead-induced mouse model of glaucoma.

While pretreatment with ONL1204 provided significant neuroprotection in the microbead-induced mouse model of glaucoma, the more clinically relevant question is whether treatment with ON1204 can provide neuroprotection even when administered after detection of elevated IOP, as this would be the time at which glaucoma patients would most likely receive treatment. To answer this question, C57BL/6J WT mice received an anterior chamber injection of microbeads or saline, and at 7 days post-microbead injection, all mice receive an intravitreal injection of ONL1204 or vehicle alone. IOPs were monitored every 3–4 days by rebound tonometry, verifying that IOP was elevated prior to intravitreal injection of the drug or vehicle. The IOP data revealed no significant difference in the time course or magnitude of the IOP between mice treated with ONL1204 or vehicle at 7 days post-microbead injection (Fig. 4a). At 4 weeks post-microbead injection, quantification of RGCs revealed significant preservation of RGCs in the ONL1204-treated mice when compared to mice treated with vehicle alone (Fig. 4b, c). Significant protection of axons was also observed with axon density in ONL1204-treated mice equivalent to the axon density in saline-treated controls (Fig. 4d, e). In conclusion, these data demonstrate that inhibition of Fas activation provides significant protection to both the RGCs and axons, even when administered after elevated IOP.

In human and experimental models of glaucoma, activated microglia are detected in the optic nerve head and retina [14‐19], and blocking microglia activation with minocycline [14, 20], anti-TNF [22, 23], or irradiation [42] prevents death of RGCs and axon degeneration. While triggering the Fas receptor is best known for inducing apoptosis, we previously demonstrated that accelerated death of RGCs in mice that only express the membrane form of FasL (mFasL) correlated with increased activation of retinal microglia, suggesting that FasL mediates both RGC apoptosis and glial activation [36]. To determine whether ONL1204-mediated neuroprotection correlates with inhibition of microglia activation in the neural retina, C57BL/6J WT mice were pretreated with ONL1204 just prior to the anterior chamber injection of microbeads. At 28 days post-microbead injection, retinal whole mounts were stained with Iba1 (microglia/macrophage marker). Retinal microglia are located in the ganglion cell layer (GCL), inner plexiform layer (IPL), and outer plexiform layer (OPL). However, we did not detect any changes in the microglia in the OPL during the development of glaucoma in our model system, and since glaucoma specifically targets the RGCs and their axons, we focused our analysis on microglia in the GCL and IPL of the retina. Representative confocal images from saline control groups treated with vehicle or ONL1204 reveal Iba1+ cells with a quiescent phenotype and dendritic morphology (Fig. 5a), while confocal images from microbead-injected mice treated with vehicle only reveal Iba1+ cells with an activated phenotype and amoeboid morphology (Fig. 5a). By contrast, the Iba1+ cells in retinal whole mounts prepared from microbead-injected mice pretreated with ONL1204 maintained a quiescent phenotype with dendritic morphology, similar to that observed in the saline-injected control groups (Fig. 5a). Quantification of microglia density in the GCL/IPL revealed no significant difference in absolute number of Iba1+ cells at 28 days post-microbead injection between all groups (Fig. 5b). However, quantification of microglia activation, using the measurement of the longest process length as previously described [39], revealed a significant shortening of the cell process length in Iba1+ cells from microbead-injected mice treated with vehicle as compared to Iba1+ cells from microbead-injected mice treated with ONL1204 (Fig. 5c). These data indicate that Fas activation mediates both RGC apoptosis and microglia activation.

In human and experimental glaucoma, multiple inflammatory pathways have been implicated in the pathogenesis of disease, including the Toll-like receptor signaling pathway [43], the inflammasome pathway [44‐47], the TNFα pathway [22, 23, 48‐50], and the complement cascade [51‐53]. While triggering of the Fas receptor is best known for inducing apoptosis through the activation of caspase-8, activated caspase-8 can also induce the production of pro-inflammatory mediators [54‐57]. Moreover, caspase-8 activation has also been linked to microglia activation [58] and inflammation in experimental models of glaucoma and inhibiting caspase-8 blocked inflammation and prevented death of RGCs [45]. To explore the role of Fas activation in triggering inflammation in glaucoma, we pretreated C57BL/6J WT mice with ONL1204 just prior to injection of microbeads, and at 28 days post-microbead injection, the neural retina was isolated, and qPCR was performed to assess the expression of several proinflammatory genes associated with human and/or experimental models of glaucoma. We first examined the gene expression of caspase-8, which plays an essential role in Fas receptor signaling cascades that induces both apoptosis and cytokine production [55], as well as GFAP as a measure of glial activation. The qPCR analysis revealed a significant induction in both GFAP and Caspase-8 in microbead-injected mice treated with vehicle only, as compared to saline-treated controls (Fig. 6a). We then examined the gene expression of several proinflammatory cytokines (TNFα, IL-1β, IL-6, and IL-18) (Fig. 6b) and chemokines (MIP-1α, MIP-1β, MIP-2, MCPI, and IP10) (Fig. 6c) that have been implicated in human and experimental models of glaucoma [43, 48, 59‐61]. The qPCR analysis revealed significant induction of each of these genes in the retina of microbead-injected mice treated with vehicle only as compared to the saline-treated controls (Fig. 6b, c). By contrast, pretreatment with ONL1204 prevented the induction of each of these genes and gene expression was equivalent to the saline-treated control (Fig. 6b, c). Additionally, key mediators of the Toll-like receptor pathway, inflammasome pathway, and complement cascade that have been identified in human and experimental glaucoma were also examined, specifically TLR4 [43, 44], NLRP3 [44, 45, 48], and complement components C3 and C1Q [53, 62, 63]. Similar to the proinflammatory cytokines and chemokines, gene expressions of C3, C1Q, TLR4, and NLRP3 were all significantly induced at 28 days post-microbead injection in the retina of microbead-injected mice treated with vehicle only as compared to saline-treated controls (Fig. 6d, e). However, the induction of each of these genes was inhibited in microbead-injected mice that were pretreated with ONL1204 (Fig. 6d, e). Together, these results suggest that Fas activation is upstream to multiple inflammatory pathways that have been implicated in glaucoma and blocking Fas activation with ONL1204 prevents RGC apoptosis, as well as microglia activation and the induction of neurodestructive inflammation.