Microglia Are Neurotoxic or Neuroprotective During the Development of Glaucoma Depending Upon the Expression of Membrane and Soluble FasL

Abstract

Purpose: : The death of retinal ganglion cells (RGCs) is the common endpoint in all forms of glaucoma. While current data indicates RGC apoptosis occurs during glaucoma, the exact molecular mechanisms of cell death are unclear. The pro-apoptotic molecule Fas Ligand (FasL) is expressed on microglia within the retina. However, FasL is a membrane-bound protein (mFasL) that can also be cleaved to release a soluble molecule with antagonistic activity (sFasL). We hypothesize the form of FasL expressed on microglia determines their function during glaucoma. Neurotoxic microglia express mFasL that triggers apoptosis of RGCs; neuroprotective microglia express sFasL that prevents RGC apoptosis.

Methods: : mFasL-only knock-in mice (ΔCS.1) were constructed by mutating the cleavage site in the FasL gene. ΔCS.1 mice only express mFasL and no sFasL. Two mouse models of glaucoma were used: (i) inducible IOP using an anterior chamber injection of microbeads, and (ii) intravitreal injection of TNF-α (1ng), a known mediator of RGC death in glaucoma. Intravitreal injection of recombinant sFasL was used to block RGC death. Eyes were enucleated and RGCs were stained with anti-β-tubulin and counted. IHC analysis included staining for: Fas, FasL, microglia (Iba), astrocytes (anti-GFAP), and nerve fibers (SMI32).

Results: : Intravitreal injection of TNFα triggered a loss of RGCs beginning at 4 wks in WT, but not FasL KO mice, indicating FasL is required for RGC death. By contrast, ΔCS.1 mice displayed an accelerated loss of RGCs and nerve fibers at 1 wk; this loss was prevented in ΔCS.1 x lpr mice that lacked the Fas receptor. Furthermore, TUNEL staining confirmed death of Fas+ RGCs was due to apoptosis and IHC revealed increased mFasL expression on microglia. Most importantly, the accelerated death of RGCs was prevented with one intravitreal injection of recombinant soluble FasL, indicating that the form of FasL expressed in the retina is critical in the pathogenesis of glaucoma. Similar to the TNFα model, accelerated RGC loss was also observed in ΔCS.1 mice following three weeks of elevated IOP as compared to WT.

Conclusions: : These data demonstrate the ratio of membrane / soluble FasL expressed on microglia determines whether these cells are neurotoxic or neuroprotective during glaucoma. In addition, these data identifies sFasL as a possible neuroprotective agent for RGCs.

Keywords: microglia • ganglion cells • apoptosis/cell death