A small peptide antagonist of the Fas receptor inhibits neuroinflammation and prevents axon degeneration and retinal ganglion cell death in an inducible mouse model of glaucoma.

BACKGROUND: Glaucoma is a complex, multifactorial disease where apoptosis, microglia activation, and inflammation have been linked to the death of retinal ganglion cells (RGCs) and axon degeneration. We demonstrated previously that FasL-Fas signaling was required for axon degeneration and death of RGCs in chronic and inducible mouse models of glaucoma and that Fas activation triggered RGC apoptosis, glial activation, and inflammation. Here, we investigated whether targeting the Fas receptor with a small peptide antagonist, ONL1204, has anti-inflammatory and neuroprotective effects in a microbead-induced mouse model of glaucoma. METHODS: Intracameral injection of microbeads was used to elevate intraocular pressure (IOP) in Fas-deficient (Faslpr) mice and WT C57BL/6J mice that received an intravitreal injection of the Fas inhibitor, ONL1204 (2 µg/1 µl) (or vehicle only), on day 0 or day 7 after microbead injection. The IOP was monitored by rebound tonometry, and at 28 days post-microbead injection, Brn3a-stained RGCs and paraphenylenediamine (PPD)-stained axons were analyzed. The effects of ONL1204 on retinal microglia activation and the expression of inflammatory genes were analyzed by immunostaining of retinal flatmounts and quantitative PCR (qPCR). RESULTS: Rebound tonometry showed equivalent elevation of IOP in all groups of microbead-injected mice. At 28 days post-microbead injection, the RGC and axon counts from microbead-injected Faslpr mice were equivalent to saline-injected (no IOP elevation) controls. Treatment with ONL1204 also significantly reduced RGC death and loss of axons in microbead-injected WT mice when compared to vehicle-treated controls, even when administered after IOP elevation. Confocal analysis of Iba1-stained retinal flatmounts and qPCR demonstrated that ONL1204 also abrogated microglia activation and inhibited the induction of multiple genes implicated in glaucoma, including cytokines and chemokines (GFAP, Caspase-8, TNFα, IL-1ß, IL-6, IL-18, MIP-1α, MIP-1ß, MIP-2, MCPI, and IP10), components of the complement cascade (C3, C1Q), Toll-like receptor pathway (TLR4), and inflammasome pathway (NLRP3). CONCLUSIONS: These results serve as proof-of-principal that the small peptide inhibitor of the Fas receptor, ONL1204, can provide robust neuroprotection in an inducible mouse model of glaucoma, even when administered after IOP elevation. Moreover, Fas signaling contributes to the pathogenesis of glaucoma through activation of both apoptotic and inflammatory pathways.

An Evaluation of the Repeatability of Visual Function Following Surgical Repair of Macula-Off Rhegmatogenous Retinal Detachment.

Purpose: To evaluate the reliability and reproducibility of visual function assessments for patients with macula-off rhegmatogenous retinal detachment (RRD). Methods: This prospective study included patients with unilateral macula-off RRD of <10-day duration successfully treated with a single, uncomplicated surgery at least 1 year following repair. Visual function assessments were performed at time of enrollment and 1 month later. Testing included Early Treatment Diabetic Retinopathy Study (ETDRS) best-corrected visual acuity (BCVA), low-luminance visual acuity (LLVA), low-contrast visual acuity (VA) 2.5% and 5%, contrast sensitivity assessment with Mars and Gabor patches, reading speed (acuity, speed, and critical print size), color vision testing (protan, deutan, and tritan), and microperimetry. Spectral-domain ocular coherence tomography (SD-OCT) was performed. Paired t-statistics were used to compare values between visits and between the study and fellow eyes. Results: Fourteen patients (9 male, 5 female) with a mean age of 69 years at time of surgery were evaluated. Correlation coefficients across the two visits were highest for ETDRS BCVA (0.97), tritan color vision testing (0.96), and low-contrast VA 5% (0.96), while the average t-statistic was largest for low-luminance deficit (4.2), ETDRS BCVA (4.1), and reading speed critical print size (3.7). ETDRS BCVA did not correlate with SD-OCT findings. Conclusions: ETDRS BCVA can be considered a highly reliable and reproducible outcome measure. LLVA, protan color discrimination, contrast sensitivity, and reading speed may be useful secondary outcome measures. Translational Relevance: This study provides guidance on the selection of visual function outcome measures for clinical trials of patients with macula-off RRD.

Cell Death in AMD: The Rationale for Targeting Fas.

Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the developed world. While great advances have been made in the treatment of the neovascular ("wet") form of the disease, there is still a significant need for therapies that prevent the vision loss associated with the advanced forms of dry, atrophic AMD. In this atrophic form, retinal pigment epithelial (RPE) and photoreceptor cell death is the ultimate cause of vision loss. In this review, we summarize the cell death pathways and their relation to RPE and retinal cell death in AMD. We review the data that support targeting programmed cell death through inhibition of the Fas receptor as a novel approach to preserve these structures and that this effect results from inhibiting both canonical death pathway activation and reducing the associated inflammatory response. These data lay the groundwork for current clinical strategies targeting the Fas pathway in this devastating disease.

Inhibitor of apoptosis proteins as intracellular signaling intermediates.

Inhibitor of apoptosis (IAP) proteins have often been considered inhibitors of cell death due to early reports that described their ability to directly bind and inhibit caspases, the primary factors that implement apoptosis. However, a greater understanding is evolving regarding the vital roles played by IAPs as transduction intermediates in a diverse set of signaling cascades associated with functions ranging from the innate immune response to cell migration to cell-cycle regulation. In this review, we discuss the functions of IAPs in signaling, focusing primarily on the cellular IAP (c-IAP) proteins. The c-IAPs are important components in tumor necrosis factor receptor superfamily signaling cascades, which include activation of the NF-κB transcription factor family. As these receptors modulate cell proliferation and cell death, the involvement of the c-IAPs in these pathways provides an additional means of controlling cellular fate beyond simply inhibiting caspase activity. Additionally, IAP-binding proteins, such as Smac and caspases, which have been described as having cell death-independent roles, may affect c-IAP activity in intracellular signaling. Collectively, the multi-faceted functions and complex regulation of the c-IAPs illustrate their importance as intracellular signaling intermediates.

Copper is a potent inhibitor of both the canonical and non-canonical NFκB pathways.

Copper is an essential trace element that plays key roles in many metabolic processes. Homeostatic regulation of intracellular copper is normally tightly controlled, but deregulated copper levels are found in numerous metabolic and neurodegenerative diseases, as well as in a range of neoplasms. There are conflicting reports regarding the exact role of copper in the regulation of NFκB-responsive genes, specifically whether copper leads to increased activation of the NFκB pathways, or downregulation. Here we show that increased intracellular levels of copper, using the ionophore clioquinol, leads to a potent inhibition of NFκB pathways, induced by multiple distinct stimuli. Addition of copper to cells inhibits ubiquitin-mediated degradation of IκBα by preventing its phoshorylation by the upstream IKK complex. Intriguingly, copper-dependent inhibition of NFκB can be reversed by the addition of the reducing agent, N-acetylcysteine (NAC). These results suggest that the oxidative properties of excess copper prevent NFκB activation by blocking IκBα destruction, and that NFκB activity should be assessed in diseases associated with copper excess.