Meredith Gregory-Ksander

Introduction: Age-related macular degeneration (AMD), results from choroidal neovascularization (CNV), retinal edema and loss of photoreceptors. Previous studies suggest that Fas Ligand (FasL) on retinal pigment epithelial cells inhibits CNV by inducing apoptosis of infiltrating Fas+ vascular endothelial cells. However, membrane-bound FasL (mFasL) can be cleaved by metalloproteinases to release a soluble fragment (sFasL) and the two isoforms have distinct functions. mFasL induces apoptosis and inflammation while sFasL has been shown to promote cell activation, cell survival, cell proliferation, and cell migration. In this current study, we use a genetic approach to prevent cleavage of mFasL in order to determine the extent to which sFasL and/or mFasL contribute to vascular leakage in a mouse model of laser-induced CNV. Methods: To distinguish the role of mFasL from sFasL in regulating CNV and vascular leakage, we used a 532 nm laser to induce CNV in three types of mice: (i) wild-type (WT) FasL ^WT/WT mice that express low levels of mFasL and high levels of sFasL, (ii) homozygous FasL ^ΔCS/ΔCS mice that express greater than normal levels of mFasL and no sFasL, and (iii) heterozygous FasL ^ΔCS/WT mice that express greater than normal levels of mFasL and less than normal levels of sFasL. Results: Unexpectedly, CNV size and the area of vascular leakage were comparable in homozygous FasL ^ΔCS/ΔCS and WT mice. However, heterozygous FasL ^ΔCS/WT mice developed significantly less vascular leakage and showed accelerated neovessel maturation that depended on the expression of the Fas receptor in LysM-positive myeloid cells. Moreover, the protective phenotype observed in heterozygous FasL ^ΔCS/WT mice correlated with increased retinal expression of PDGFRb and TGFβ1, reduced retinal expression of VEGFA, and increased expression of PEDF, which is known to inhibit VEGF-induced vascular permeability and promote vessel normalization. Discussion: Overall, these data indicate that FasL-mediated protection from CNV depends on expression of both sFasL and mFasL. Moreover, the accelerated neovessel maturation observed in heterozygous FasL ^ΔCS/WT mice depends on Fas signaling in LysM-positive myeloid cells and is highly dependent on the extent of FasL cleavage, identifying Fas ligand cleavage as a potential therapeutic strategy to promote neovessel maturation and prevent vascular leakage in wet AMD.

Aging is a complex process, yet therapeutic discovery has largely relied on hypothesis-driven testing of individual targets. Here, we performed an unbiased genome-scale open reading frame (ORF) screen for oxidative-stress resistance in retinal pigment epithelium (RPE) cells, which are particularly vulnerable to oxidative damage with age. The screen identified nine protective ORFs, including antioxidant factors and a regulator of epithelial-to-mesenchymal transition. Notably, we also identified three factors that reduce transcriptomic age, including the cardiac transcription factor NKX2-5. NKX2-5 conferred robust oxidative resilience in vitro and in vivo . Domain dissection showed that its homeodomain was dispensable for resilience, enabling development of NKX2-5ΔHD, which retained protective activity without detectable toxicity over 14 months. In middle-aged mice, subretinal NKX2-5ΔHD restored visual and electrophysiological function, and its systemic delivery improved grip strength and reduced frailty in late-aged mice. These findings establish genome-scale ORF screening as a strategy to discover transferable resilience factors that can be reengineered to counter age-related functional decline across tissues.

Background - Glaucoma is a complex multifactorial disease where apoptosis and inflammation represent two key pathogenic mechanisms. However, the relative contribution of apoptosis versus inflammation in axon degeneration and death of retinal ganglion cells (RGCs) is not well understood. In glaucoma, caspase-8 is linked to RGC apoptosis, as well as glial activation and neuroinflammation. To uncouple these two pathways and determine the extent to which caspase-8-mediated inflammation and/or apoptosis contributes to the death of RGCs, we used the caspase-8 D387A mutant mouse (Casp8^DA/DA) in which a point mutation in the auto-cleavage site blocks caspase-8-mediated apoptosis but does not block caspase-8-mediated inflammation. Methods- Intracameral injection of magnetic microbeads was used to elevate the intraocular pressure (IOP) in wild-type, Fas deficient Fas^lpr, and Casp8^DA/DA mice. IOP was monitored by rebound tonometry. Two weeks post microbead injection, retinas were collected for microglia activation analysis. Five weeks post microbead injection, visual acuity and RGC function were assessed by optometer reflex (OMR) and pattern electroretinogram (pERG), respectively. Retina and optic nerves were processed for RGC and axon quantification. Two- and five-weeks post microbead injection, expression of the necrosis marker, RIPK3, was assessed by qPCR. Results- Wild-type, Fas^lpr, and Casp8^DA/DA mice showed similar IOP elevation as compared to saline controls. A significant reduction in both visual acuity and pERG that correlated with a significant loss of RGCs and axons was observed in wild-type but not in Fas^lpr mice. The Casp8^DA/DA mice displayed a significant reduction in visual acuity and pERG amplitude and loss of RGCs and axons similar to that in wild-type mice. Immunostaining revealed equal numbers of activated microglia, double positive for P2ry12 and IB4, in the retinas from microbead-injected wild-type and Casp8^DA/DA mutant mice. qPCR analysis revealed no induction of RIPK3 in wild-type or Casp8^DA/DA mice at two- or five-weeks post microbead injection. Conclusions- Our results demonstrate that caspase-8-mediated extrinsic apoptosis is not involved in the death of RGCs in the microbead-induced mouse model of glaucoma implicating caspase-8-mediated inflammation, but not apoptosis, as the driving force in glaucoma progression. Taken together, these results identify the caspase-8-mediated inflammatory pathway as a potential target for neuroprotection in glaucoma.

Neovascular age-related macular degeneration (AMD), results from choroidal neovascularization (CNV), retinal edema and loss of photoreceptors. Previous studies suggested that Fas Ligand (FasL) on retinal pigment epithelial cells inhibited CNV by inducing apoptosis of infiltrating Fas + vascular endothelial cells. However, induction of apoptosis depends on membrane-bound (mFasL) while the FasL cleavage product (sFasL) is neuroprotective. To better understand how FasL regulates the development of CNV, we used a mouse model of laser CNV to evaluate the development of CNV in mice with a FasL cleavage site mutation (ΔCS) and can only express the membrane-bound form of FasL. There was no significant difference in CNV size and area of vascular leakage in homozygous FasL ^ΔCS/ΔCS mice when compared to wild type mice. Unexpectedly, heterozygous FasL ^ΔCS/WT mice developed significantly less vascular leakage and showed accelerated neovessel maturation. However, CNV was not prevented in heterozygous FasL ^ΔCS/WT mice if the Fas receptor was deleted in myeloid cells (FasL ^ΔCS/+ Fas ^flox/flox Cre ^LysM ). Thus, FasL-mediated CNV inhibition depends on the extent of FasL cleavage, and on FasL engagement of Fas + myeloid cells. Moreover, accelerated neovessel maturation prevents vascular leakage in AMD.

Glaucoma is a progressive neurodegenerative disease characterized by damage to the optic nerve that results in irreversible vision loss. While the exact pathology of glaucoma is not well understood, emerging evidence suggests that dysregulation of the complement system, a key component of innate immunity, plays a crucial role. In glaucoma, dysregulation of the complement cascade and impaired regulation of complement factors contribute to chronic inflammation and neurodegeneration. Complement components such as C1Q, C3, and the membrane attack complex have been implicated in glaucomatous neuroinflammation and retinal ganglion cell death. This review will provide a summary of human and experimental studies that document the dysregulation of the complement system observed in glaucoma patients and animal models of glaucoma driving chronic inflammation and neurodegeneration. Understanding how complement-mediated damage contributes to glaucoma will provide opportunities for new therapies.

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.

Glaucoma is a group of optic neuropathies characterised by the degeneration of retinal ganglion cells, resulting in damage to the optic nerve head (ONH) and loss of vision in one or both eyes. Increased intraocular pressure (IOP) is one of the major aetiological risk factors in glaucoma, and is currently the only modifiable risk factor. However, 30–40% of glaucoma patients do not present with elevated IOP and still proceed to lose vision. The pathophysiology of glaucoma is therefore not completely understood, and there is a need for the development of IOP-independent neuroprotective therapies to preserve vision. Neuroinflammation has been shown to play a key role in glaucoma and, specifically, the NLRP3 inflammasome, a key driver of inflammation, has recently been implicated. The NLRP3 inflammasome is expressed in the eye and its activation is reported in pre-clinical studies of glaucoma. Activation of the NLRP3 inflammasome results in IL-1β processing. This pro inflammatory cytokine is elevated in the blood of glaucoma patients and is believed to drive neurotoxic inflammation, resulting in axon degeneration and the death of retinal ganglion cells (RGCs). This review discusses glaucoma as an inflammatory disease and evaluates targeting the NLRP3 inflammasome as a therapeutic strategy. A hypothetical mechanism for the action of the NLRP3 inflammasome in glaucoma is presented.

Fas ligand (FasL) is best known for its ability to induce cell death in a wide range of Fas-expressing targets and to limit inflammation in immunoprivileged sites such as the eye. In addition, the ability of FasL to induce a much more extensive list of outcomes is being increasingly explored and accepted. These outcomes include the induction of proinflammatory cytokine production, T cell activation, and cell motility. However, the distinct and opposing functions of membrane-associated FasL (mFasL) and the C-terminal soluble FasL fragment (sFasL) released by metalloproteinase cleavage is less well documented and understood. Both mFasL and sFasL can form trimers that engage the trimeric Fas receptor, but only mFasL can form a multimeric complex in lipid rafts to trigger apoptosis and inflammation. By contrast, a number of reports have now documented the anti-apoptotic and anti-inflammatory activity of sFasL, pointing to a critical regulatory function of the soluble molecule. The immunomodulatory activity of FasL is particularly evident in ocular pathology where elimination of the metalloproteinase cleavage site and the ensuing increased expression of mFasL can severely exacerbate the extent of inflammation and cell death. By contrast, both homeostatic and increased expression of sFasL can limit inflammation and cell death. The mechanism(s) responsible for the protective activity of sFasL are discussed but remain controversial. Nevertheless, it will be important to consider therapeutic applications of sFasL for the treatment of ocular diseases such as 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 (Fas^lpr) 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 Fas^lprmice 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-1β, IL-6, IL-18, MIP-1α, MIP-1β, 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.