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. 2016 Dec 15;197(12):4626-4638.
doi: 10.4049/jimmunol.1601488. Epub 2016 Nov 14.

Overexpression of Soluble Fas Ligand Following Adeno-Associated Virus Gene Therapy Prevents Retinal Ganglion Cell Death in Chronic and Acute Murine Models of Glaucoma

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Overexpression of Soluble Fas Ligand Following Adeno-Associated Virus Gene Therapy Prevents Retinal Ganglion Cell Death in Chronic and Acute Murine Models of Glaucoma

Anitha Krishnan et al. J Immunol. .
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Abstract

Glaucoma is a multifactorial disease resulting in the death of retinal ganglion cells (RGCs) and irreversible blindness. Glaucoma-associated RGC death depends on the proapoptotic and proinflammatory activity of membrane-bound Fas ligand (mFasL). In contrast to mFasL, the natural cleavage product, soluble Fas ligand (sFasL) inhibits mFasL-mediated apoptosis and inflammation and, therefore, is an mFasL antagonist. DBA/2J mice spontaneously develop glaucoma and, predictably, RGC destruction is exacerbated by expression of a mutated membrane-only FasL gene that lacks the extracellular cleavage site. Remarkably, one-time intraocular adeno-associated virus-mediated gene delivery of sFasL provides complete and sustained neuroprotection in the chronic DBA/2J and acute microbead-induced models of glaucoma, even in the presence of elevated intraocular pressure. This protection correlated with inhibition of glial activation, reduced production of TNF-α, and decreased apoptosis of RGCs and loss of axons. These data indicate that cleavage of FasL under homeostatic conditions, and the ensuing release of sFasL, normally limits the neurodestructive activity of FasL. The data further support the notion that sFasL, and not mFasL, contributes to the immune-privileged status of the eye.

Figures

Fig 1
Fig 1. Accelerated loss of RGCs and axons in D2-ΔCS mice
(A) IOP measurements were taken by rebound tonometry in D2-Gp, D2, and D2-ΔCS mice at 3, 6, and 9 months of age (N=16 D2-GP; N=22 D2, N=22 D2-ΔCS). Data is presented as mean IOP ± SEM. (B) Representative confocal images of retinal flat-mounts isolated from D2-Gp, D2, and D2-ΔCS mice at 3 and 6 months of age, stained with β-III tubulin (red) a RGC-specific marker and DAPI a nuclear stain (blue) (Scale bar, 75μm). (C) Quantification of β-III tubulin positive RGCs, represented as RGC density/mm2 retina. N=10 eyes per group. (D) Representative photomicrographs of PPD optic nerve cross sections taken from D2-Gp, D2 and D2-ΔCS at 3 and 6 months of age (Scale bar, 10μm). (E) Quantification of healthy axons, represented as axon density (104)/mm2 ON. N=10 optic nerves per group. Ns, non-significant, ****P<0.0001.
Fig 2
Fig 2. Accelerated apoptosis coincides with RGC loss in the absence of sFasL
Representative TUNEL staining in paraffin embedded retinal sections taken from D2-Gp, D2 and D2-ΔCS mice at (A) 3 and (C) 6 months of age (Scale bar, 100μm). TUNEL = red, DAPI, nuclear marker = blue. GCL, ganglion cells layer; INL, inner nuclear layer; ONL, outer nuclear layer. White arrowhead = TUNEL positive cells in GCL, white arrow = TUNEL positive cells in INL. TUNEL positive cells in the GCL were quantitated at (B) 3 months and (D) 6 months of age, shown as the number of TUNEL positive cells in the GCL/retinal section (9 sections/retina), N=8 per group. ****P<0.0001.
Fig 3
Fig 3. Expression of Fas, FasL, and FADD in D2-ΔCS mice
Quantitative RT-PCR was performed on the neural retina isolated from D2-GP, D2, and D2-ΔCS mice at 3 and 6 months of age to quantitate mRNA levels of: (A) Fas, (B) FasL, and (C) FADD. N=6 per group. Representative Western blots from protein lysates (20μg/sample) prepared from posterior eye cups isolated from D2-GP, D2, and D2-ΔCS mice for (D) FasL at 3 and 6 months (actin is green, FasL is red), (E) Fas at 6 months (actin is green, Fas is red), and (F) FADD at 6 months (actin is green, FADD is green). Protein lysates prepared from L5178Y-R tumor cells transfected with sFasL or mFasL vectors (31) were used as positive controls for FasL and the protein lysate from posterior eyecups of FasL-KO mice (27, 35) was used as a negative control for FasL. Densitometry analysis of mFasL (34 and 38 kD bands), sFasL (26 kD band), Fas (43 kD), and FADD (28 kD) is the average of 3 independent experiments (3 independent blots consisting of 1 posterior segment per group, per experiment) Error bars indicate SEM; N.D.- not detected (below the level of detection by densitometry). *P<0.05, **P<0.01, ****P<0.0001.
Fig 4
Fig 4. Expression of GFAP and TNFα in the retina of D2-ΔCS mice
(A) Representative confocal microscopy images of paraffin embedded retinal sections taken from D2-Gp, D2, and D2-ΔCS mice at (A) 3 and 6 months of age and stained for GFAP (red) and DAPI (blue). (white arrow=GFAP in muller cells, Scale bar, 100μm). Quantitative RT- PCR was performed on the neural retina isolated from D2-GP, D2, and D2-ΔCS mice at 3 and 6 months of age to quantitate mRNA levels of (B) GFAP and (C) TNFα. N=6 per group. Error bar indicates SEM. **<0.01 and ***P<0.001.
Fig 5
Fig 5. Over expression of sFasL in D2 mice
D2 mice received one intravitreal injection of AAV2.sFasL or AAV2.eGFP as a control at 2 months of age. (A) Retinal flat-mount showing expression of AAV2.eGFP throughout the retina (Magnification, 5x and 40x). (B) Cross sectional 3-D reconstruction of the retina. GCL-ganglion cell layer, INL-inner nuclear layer. (C) Western blot from neural retinal lysates (5μg/sample, sFasL is red band, actin is green band) showing overexpression of sFasL (26 kD) at both 2wk and at 8 months post AAV2 injection. N=3 per group. Densitometry is the average of 3 independent experiments (3 independent blots consisting of 1 neural retina per group, per experiment). (D) IOP measurements were taken at 3, 5, 7, and 9 months of age by rebound tonometry in D2 (uninjected), D2-AAV2.eGFP, and D2-AAV2.sFasL. Mean IOP for the D2-Gp non-glaucomatous control group is 12 mmHg ±3 SEM, identified as an solid line on the IOP graph. Data is presented as mean IOP ± SEM IOP (N=10 per group). Representative slit lamp images taken at 9 months of age show pigment dispersion and iris stromal atrophy in D2-Gp, D2 (uninjected), D2-AAV.eGFP, and D2-AAV.sFasl mice. Ns, non-significant, *P<0.05.
Fig 6
Fig 6. Intravitreal AAV2.sFasL protects RGCs and axons in D2 mice
D2 mice received one intravitreal injection of AAV2.sFasL or AAV2.eGFP as a control at 2 months of age. At 10 and 15 months of age, AAV2.sFasL and AAV2.eGFP treated mice, in addition to age-matched D2-uninj (uninjected) and D2-Gp mice were euthanized and retinas and optic nerves processed for analysis of RGC and axon density. (A) Representative confocal images of retinal flat-mounts isolated from 10 month old D2-Gp, D2 uninjected, D2-AAV2.eGFP and D2-AAV2.sFasL mice, stained with β-III tubulin (red) an RGC-specific marker and DAPI a nuclear stain (blue). (Scale bar, 50μm). (B) Quantification of β-III tubulin positive RGCs, represented as RGC density/mm2 retina. N=10 per group (C) Representative photomicrographs of PPD stained optic nerve cross sections taken from 10 month old D2-Gp, D2 uninjected, D2-AAV2.eGFP and D2-AAV2.sFasL mice (Scale bar, 10μm). (D) Quantification of healthy axons, represented as axon density (104)/mm2. N=10 per group (E) Representative confocal images of retinal flat-mounts isolated from 15 month old D2-Gp, D2 uninjected, D2-AAV2.eGFP and D2-AAV2.sFasL mice, stained with β-III tubulin (red) an RGC-specific marker and DAPI a nuclear stain (blue) (Scale bar, 75μm). (F) Quantification of β-III tubulin positive RGCs, represented as RGC density/mm2 retina. N=5 per group. (G) Representative photomicrographs of PPD optic nerve cross sections taken from 15 month old D2-Gp, D2 uninjected, D2-AAV2.eGFP and D2-AAV2.sFasL mice (Scale bar, 10μm). (H) Quantification of healthy axons, represented as axon density (104)/mm2. N=5 per group. *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001
Fig 7
Fig 7. Muller glial cell activation and induction of inflammatory and apoptotic mediators in the retina of D2 mice treated with AAV2.sFasL
(A) Representative confocal microscopy images of paraffin embedded retinal sections taken from D2-Gp, D2-uninj. D2-AAV2-eGFP, and D2-AAV2-sFasL mice at 10 months of age and stained for GFAP (red) and DAPI (blue) (Scale bar, 100μm). Quantitative RT- PCR was performed on the neural retina isolated from D2-Gp, D2-uninj., D2-AAV2-eGFP, and D2-AAV2-sFasL mice at 10 months of age to quantitate mRNA levels of (B) GFAP, (C) TNFα, (D) pro-apoptotic mediators Fas, FADD, and BAX, and (E) anti-apoptotic mediators cFLIP, Bcl2, and cIAP2. N=5–6 per group. Error bar indicates SEM. *P<0.05, **<0.01, ***P<0.001, ****<0.0001.
Fig. 8
Fig. 8. Pre treatment with AAV2.sFasL protects RGC cell death in microbead-induced mouse model of elevated IOP in C57/BL6 mice
(A) Western blot from neural retina lysates (5ug/sample) showing overexpression of sFasL at 26 kD (Red band) and actin (Green band) in saline and microbead injected B6.AAV2.eGFP and B6.AAV2.sFasL mice at 4 weeks post microbead or saline injections. (B) IOP measurements were taken by rebound tonometry from B6.AAV2.eGFP and B6.AAV2.sFasL mice treated with saline or microbeads. Data is presented as mean IOP ± SEM, (N=10 per group). At 28 days post microbead injection the neural retina and optic nerve were processed for quantification of RGC and axon density. (C) Representative confocal microscopic images from retinal flat-mounts stained with βIII tubulin (red), an RGC-specific marker and DAPI a nuclear stain (blue) at 4 weeks post microbead or saline injections (Scale bar, 75μm). (D) Quantification of β-III tubulin positive RGCs, represented as RGC density/mm2 retina (E) Representative photomicrographs of optic nerve cross sections stained with PPD at 4 weeks post microbead or saline injections (Scale bar, 10μm). (F) Quantification of healthy axons, represented as axon density (104)/mm2. N=5 per group. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

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