Caspase-8-mediated inflammation but not apoptosis drives death of retinal ganglion cells and loss of visual function in glaucomaa

Abstract

Background-: Glaucoma is a complex multifactorial disease where apoptosis and inflammation represent two key pathogenic mechanisms. However, the relative contribution of apoptosis versus inflammation in axon degeneration and death of retinal ganglion cells (RGCs) is not well understood. In glaucoma, caspase-8 is linked to RGC apoptosis, as well as glial activation and neuroinflammation. To uncouple these two pathways and determine the extent to which caspase-8-mediated inflammation and/or apoptosis contributes to the death of RGCs, we used the caspase-8 D387A mutant mouse (Casp8 ^DA/DA ) in which a point mutation in the auto-cleavage site blocks caspase-8-mediated apoptosis but does not block caspase-8-mediated inflammation.

Methods-: Intracameral injection of magnetic microbeads was used to elevate the intraocular pressure (IOP) in wild-type, Fas deficient Fas^lpr, and Casp8 ^DA/DA mice. IOP was monitored by rebound tonometry. Two weeks post microbead injection, retinas were collected for microglia activation analysis. Five weeks post microbead injection, visual acuity and RGC function were assessed by optometer reflex (OMR) and pattern electroretinogram (pERG), respectively. Retina and optic nerves were processed for RGC and axon quantification. Two- and five-weeks post microbead injection, expression of the necrosis marker, RIPK3, was assessed by qPCR.

Results-: Wild-type, Fas^lpr, and Casp8 ^DA/DA mice showed similar IOP elevation as compared to saline controls. A significant reduction in both visual acuity and pERG that correlated with a significant loss of RGCs and axons was observed in wild-type but not in Fas^lpr mice. The Casp8 ^DA/DA mice displayed a significant reduction in visual acuity and pERG amplitude and loss of RGCs and axons similar to that in wild-type mice. Immunostaining revealed equal numbers of activated microglia, double positive for P2ry12 and IB4, in the retinas from microbead-injected wild-type and Casp8 ^DA/DA mutant mice. qPCR analysis revealed no induction of RIPK3 in wild-type or Casp8 ^DA/DA mice at two- or five-weeks post microbead injection.

Conclusions-: Our results demonstrate that caspase-8-mediated extrinsic apoptosis is not involved in the death of RGCs in the microbead-induced mouse model of glaucoma implicating caspase-8-mediated inflammation, but not apoptosis, as the driving force in glaucoma progression. Taken together, these results identify the caspase-8-mediated inflammatory pathway as a potential target for neuroprotection in glaucoma.

Keywords: Apoptosis; Caspase 8; Fas ligand; Glaucoma; Inflammation; Neuroinflammation; astrocytes; microglia.

Figure 1. Death receptor signaling via caspase-8 pathway

(a) A schematic diagram of the Fas-mediated death receptor signaling pathway in wild-type and caspase-8 mutant (Casp8^DA/DA) mice. When Fas ligand engages the Fas receptor, pro-caspase-8 is directly recruited to the death-induced signaling complex (DISC) by the adapter proteins FADD and TRADD. Pro-caspase-8 undergoes autocleavage at D387 to produce catalytically active caspase-8 that leads to apoptosis via downstream activation of effector caspases, caspase-3 and caspase-7. The recruitment of pro-caspase-8 to the DISC also upregulates immune cell activation inflammation via MAP-kinase and NF- κB pathways, and autocleavage of caspase-8 is not required for this function. A point mutation at the auto-cleavage site, D387A, prevents caspase-8 activation and blocks caspase-8-mediated apoptosis; however, it does not block caspase-8-mediated inflammation. Schematic created with BioRender.com (b) A DNA gel depicting typical genotyping results of a restriction fragment length polymorphism assay showing expected sizes of HinFI undigested (–) and digested (+) PCR amplicon products produced from genomic DNA of mice from the indicated genotypes. Both wild-type and Casp8^DA/DA produce a 300-base pair PCR amplicon and following restriction digestion with HinFI, two fragments of 150-base pair appear in Casp8^DA/DA but not in wild-type mice.

Figure 2. Casp8^DA/DA mutation does not affect magnetic microbead-induced elevated intraocular pressure (IOP)

(a) A graph showing a 35-day follow-up of mean IOP ± SD (mmHg), and (b) cumulative IOP ± SD (mmHg) from saline- or magnetic-microbead-injected WT, Fas-deficient (Fas^lpr), and Casp8^DA/DA mutant mice.Two-way ANOVA and Dunnett’s multiple comparisons test, **** p<0.0001; ns not significant, n=9–14; MB, microbeads; WT, wild-type.

Figure 3. Caspase-8-mediated inflammation, not apoptosis, underlies loss of visual function in glaucoma

(a) A schematic representation of optomotor response (OMR) apparatus used to measure visual acuity (VA) in cycles/degree. The vertical lines rotated counterclockwise to measure VA in the right eye, while the mouse was placed on a pedestal at the center of OMR apparatus. The OMR measured visually-induced head tracking behavior of mice that occurred when the mouse pursued a sequential rotating black-and-white stripes stimulus pattern shown on four screens. (b) A plot showing VA (in cycles/degree) at baseline (day 0) and endpoint (day 35) from saline- or microbead-injected mice from WT, Fas-defcient (Fas^lpr), and Casp8^DA/DA mutant mice as indicated. (c) Graphs showing representative pattern electroretinogram (pERG) amplitude, as a function of time (ms), recordings from saline-(left panels) and microbead-(right panels) injected WT (top panels), Fas^lpr (middle panels) and Casp8^DA/DA (bottom panels) mice as labelled. The difference between peak P1 and trough N2 was used to calculate pERG amplitude (μV). (d) A plot showing pERG amplitude (μV) at baseline (day 0) and endpoint (day 35) from saline- or microbead-injected mice from WT, Fas-defcient (Fas^lpr), and Casp8^DA/DA mutant mice as indicated. Data presented as mean ± SD, one-way ANOVA and Dunnett’s multiple comparisons test, **** p <0.0001, ns not significant; n=9–16; MB, microbeads; WT, wild-type.

Figure 4. RGC loss and axon degeneration are not dependent on caspase 8-mediated apoptosis

Representative (a) confocal microscopy images of retinal flat mounts stained for retinal ganglion cells (RGCs) with Brn3a (scale bar 50μm) and (c) brightfield images of optic nerve sections stained for axons with P-phenylenediamine (PPD, scale bar 50μm) from saline-(top panels) and microbead-(bottom panels) injected WT, Fas^lpr, and Casp8^DA/DA mice showing RGC and axon loss in microbead-injected WT and Casp8^DA/DA but not in Fas^lpr. Graphs showing (b) RGC density data presented as RGC number per square mm retina ± SD and (d) axon density data presented as axon number per square mm of ON ± SD. One-way ANOVA and Dunnett’s multiple comparisons test, **** p <0.0001, ns not significant; n=10–20; MB, microbeads; WT, wild-type.

Figure 5. Casp8^DA/DA mutation does not affect microglia activation post-elevated intraocular pressure

(a) Representative confocal microscopy images of retinal flat mounts co-stained with P2Ry12, isolectin-B4 (IB-4), and DAPI for activated microglia at day 14 post-microbead or saline injection from WT and Casp8^DA/DA mice. (b) Graph showing quantification of P2RY12 and IB4 double positive microglia, revealing significant increase in activated microglia in both WT and casp8^DA/DA mutant mice as compared to that in saline controls. Data represented as mean activated microglia cell number per square mm of retina ± SD. n=4–5, One-way ANOVA and Dunnett’s multiple comparisons test, ****p <0.0001; ns not significant; MB, microbeads; WT, wild-type; scale bar 50 μm.

Figure 6. Casp8^DA/DA mutation does not shunt RGC death to RIPK3-mediated necroptosis

Graphs showing fold change in RIPK3 transcript levels as assessed by quantitative reverse transcription PCR (RT-qPCR) in WT and Casp8DA/DA mice at (a) early, day 14, and (b) late, day 35, timepoints following elevation of intraocular pressure. N=4–5, One-way ANOVA and Dunnett’s multiple comparisons test; ns not significant; MB, microbeads; WT, wild-type.