Abstract
Purpose :
Recent in vitro and in vivo studies using pharmacologic or transgenic inhibition of caspase-8 supported cell type-specific processes regulating different fates of RGCs and astroglia during glaucomatous neurodegeneration. We hypothesized that cFLIP, an anti-apoptotic gene target of NF-κB, functions as a molecular switch between caspase-8-mediated cell death/survival/inflammation pathways and regulates cell type-specific caspase-8 functions, such as inflammatory responses of astroglia (highly express cFLIP) vs apoptosis of RGCs (poorly express cFLIP). This study analyzed the outcomes of astroglia-targeting transgenic models on neurodegenerative inflammation in experimental glaucoma for further testing.
Methods :
Ocular hypertension was induced by anterior chamber microbead injections in mice with conditional deletion of cFLIP (or cFLIPL to determine isoform-specificity) in astroglia (crossbreds of cFLIPf/f or cFLIPLf/f with GFAP-cre/ERT2) and background controls (cFLIPf/f or cFLIPLf/f). Morphologic and molecular analyses of the retina and optic nerve assessed the distribution of different glial states (by specific marker labeling) and cytokine/chemokine profiles (by multiplex immunoassays). We also analyzed neuron survival (by RGC axon/soma counting) and function (by PERG).
Results :
Analyses of retinal whole mounts and optic nerve sections exhibited >four-fold decrease in the number of C3+/TNFα+ astroglia in GFAP/cFLIP (and GFAP/FLIPL) mice compared to their controls at 12-week of ocular hypertension (P<0.001). Similarly, there was >three-fold reduction of pro-inflammatory cytokines (including IL-2, TNF-α, IFN-γ) in samples from ocular hypertensive GFAP/cFLIP (and GFAP/FLIPL) relative to ocular hypertensive controls (n:4; P=0.02). We also detected a greater number of remaining RGCs and axons in ocular hypertensive GFAP/cFLIP and GFAP/cFLIPL mice than controls (23% vs 36% neuron loss; P<0.001).
Conclusions :
Our observations support that caspase-8 functions (related to its dead effector domain, scaffold function, or enzymatic activity) are controlled by caspase-8/cFLIP interaction. Deletion of cFLIP (or cFLIPL) in astroglia can prevent/reverse neurodegenerative inflammation by selectively targeting proinflammatory/neurotoxic glia (that are primed for inflammatory caspase-8 functions through TNF-α/TNFR, Fas/FasL, TLRs, and inflammasome) and protect RGCs against inflammatory injury in experimental glaucoma.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.