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Opposing roles for membrane bound and soluble Fas ligand in glaucoma-associated retinal ganglion cell death.

Gregory MS, Hackett CG, Abernathy EF, Lee KS, Saff RR, Hohlbaum AM, Moody KS, Hobson MW, Jones A, Kolovou P, Karray S, Giani A, John SW, Chen DF, Marshak-Rothstein A, Ksander BR - PLoS ONE (2011)

Bottom Line: Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis.We now show that FasL also plays a major role in retinal neurotoxicity.By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, The Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNFα triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma.

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Loss of RGCs in ΔCS mice is dependent upon the Fas/FasL pathway.All Western blots displayed are representative of three independent experiments: (A) CD3 activated T cell lysates and supernatants, (B) whole eye lysates from individual eyes of: WT, ΔCS, and FasL KO mice, (C) densitometry of eye lysate Western blots (* and ** P<0.05), (D–G) WT or ΔCS mice received an intravitreal injection of saline, or TNFα. Retinal sections were obtained 7 days later and RGCs were stained with βIII-tubulin. Arrow heads highlight the loss of RGCs. (H) All βIII-tubulin positive RGCs were counted in each retinal section (5 sections per eye; 10 eyes per group) and the average number RGCs per retinal section was calculated. (I) WT or ΔCS×lpr mice received intravitreal injections of saline, or TNFα. The number of RGCs was determined 7 days later in retinal sections. Green = Beta tubulin III and blue = nuclear stain. N = 10 per treatment group. (* p>0.05).
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pone-0017659-g002: Loss of RGCs in ΔCS mice is dependent upon the Fas/FasL pathway.All Western blots displayed are representative of three independent experiments: (A) CD3 activated T cell lysates and supernatants, (B) whole eye lysates from individual eyes of: WT, ΔCS, and FasL KO mice, (C) densitometry of eye lysate Western blots (* and ** P<0.05), (D–G) WT or ΔCS mice received an intravitreal injection of saline, or TNFα. Retinal sections were obtained 7 days later and RGCs were stained with βIII-tubulin. Arrow heads highlight the loss of RGCs. (H) All βIII-tubulin positive RGCs were counted in each retinal section (5 sections per eye; 10 eyes per group) and the average number RGCs per retinal section was calculated. (I) WT or ΔCS×lpr mice received intravitreal injections of saline, or TNFα. The number of RGCs was determined 7 days later in retinal sections. Green = Beta tubulin III and blue = nuclear stain. N = 10 per treatment group. (* p>0.05).

Mentions: To examine the importance of sFasL in ocular homeostasis and RGC degeneration, we constructed a membrane-only FasL gene-targeted mouse in which the FasL metalloproteinase cleavage sites in exon 2 were mutated (Figure S1). This mouse line was designated as ΔCS. WT and ΔCS mice were tested for expression of mFasL and sFasL. Cell lysates and culture supernatants prepared from activated T cells were analyzed by Western blot. A 38 kDa mFasL band was detected in the WT lysate and a 27 kDa sFasL band was detected in the WT supernatant. By contrast, cell lysates from the ΔCS T cells contained more of the 38 kDa mFasL protein and the ΔCS supernatant contained no detectable sFasL (Figure 2A). Whole eye lysates were also prepared from WT and ΔCS mutant mice to test for ocular expression of mFasL and sFasL by Western blot (Figure 2B). Two mFasL bands (38 kD and 34 kD) were detected at low levels in whole eye lysates from WT mice. As an important specificity control, both mFasL bands were missing from eye lysates that were prepared from FasL knockout mice [27]. Therefore, the two bands most likely represent differential glycosylation, previously reported for mFasL [24], [28], [29]. In comparison to WT mice the expression of both the 38 kD and 34 kD mFasL bands were significantly increased in ΔCS mice (Figure 2B and 2C). It is important to note, that sFasL was not detected by Western blot in the whole lysates from WT mice, possibly reflecting clearance from the eye or instability of the soluble form.


Opposing roles for membrane bound and soluble Fas ligand in glaucoma-associated retinal ganglion cell death.

Gregory MS, Hackett CG, Abernathy EF, Lee KS, Saff RR, Hohlbaum AM, Moody KS, Hobson MW, Jones A, Kolovou P, Karray S, Giani A, John SW, Chen DF, Marshak-Rothstein A, Ksander BR - PLoS ONE (2011)

Loss of RGCs in ΔCS mice is dependent upon the Fas/FasL pathway.All Western blots displayed are representative of three independent experiments: (A) CD3 activated T cell lysates and supernatants, (B) whole eye lysates from individual eyes of: WT, ΔCS, and FasL KO mice, (C) densitometry of eye lysate Western blots (* and ** P<0.05), (D–G) WT or ΔCS mice received an intravitreal injection of saline, or TNFα. Retinal sections were obtained 7 days later and RGCs were stained with βIII-tubulin. Arrow heads highlight the loss of RGCs. (H) All βIII-tubulin positive RGCs were counted in each retinal section (5 sections per eye; 10 eyes per group) and the average number RGCs per retinal section was calculated. (I) WT or ΔCS×lpr mice received intravitreal injections of saline, or TNFα. The number of RGCs was determined 7 days later in retinal sections. Green = Beta tubulin III and blue = nuclear stain. N = 10 per treatment group. (* p>0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3066205&req=5

pone-0017659-g002: Loss of RGCs in ΔCS mice is dependent upon the Fas/FasL pathway.All Western blots displayed are representative of three independent experiments: (A) CD3 activated T cell lysates and supernatants, (B) whole eye lysates from individual eyes of: WT, ΔCS, and FasL KO mice, (C) densitometry of eye lysate Western blots (* and ** P<0.05), (D–G) WT or ΔCS mice received an intravitreal injection of saline, or TNFα. Retinal sections were obtained 7 days later and RGCs were stained with βIII-tubulin. Arrow heads highlight the loss of RGCs. (H) All βIII-tubulin positive RGCs were counted in each retinal section (5 sections per eye; 10 eyes per group) and the average number RGCs per retinal section was calculated. (I) WT or ΔCS×lpr mice received intravitreal injections of saline, or TNFα. The number of RGCs was determined 7 days later in retinal sections. Green = Beta tubulin III and blue = nuclear stain. N = 10 per treatment group. (* p>0.05).
Mentions: To examine the importance of sFasL in ocular homeostasis and RGC degeneration, we constructed a membrane-only FasL gene-targeted mouse in which the FasL metalloproteinase cleavage sites in exon 2 were mutated (Figure S1). This mouse line was designated as ΔCS. WT and ΔCS mice were tested for expression of mFasL and sFasL. Cell lysates and culture supernatants prepared from activated T cells were analyzed by Western blot. A 38 kDa mFasL band was detected in the WT lysate and a 27 kDa sFasL band was detected in the WT supernatant. By contrast, cell lysates from the ΔCS T cells contained more of the 38 kDa mFasL protein and the ΔCS supernatant contained no detectable sFasL (Figure 2A). Whole eye lysates were also prepared from WT and ΔCS mutant mice to test for ocular expression of mFasL and sFasL by Western blot (Figure 2B). Two mFasL bands (38 kD and 34 kD) were detected at low levels in whole eye lysates from WT mice. As an important specificity control, both mFasL bands were missing from eye lysates that were prepared from FasL knockout mice [27]. Therefore, the two bands most likely represent differential glycosylation, previously reported for mFasL [24], [28], [29]. In comparison to WT mice the expression of both the 38 kD and 34 kD mFasL bands were significantly increased in ΔCS mice (Figure 2B and 2C). It is important to note, that sFasL was not detected by Western blot in the whole lysates from WT mice, possibly reflecting clearance from the eye or instability of the soluble form.

Bottom Line: Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis.We now show that FasL also plays a major role in retinal neurotoxicity.By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, The Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNFα triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma.

Show MeSH
Related in: MedlinePlus