Stéphane Mélik Parsadaniantz

Wolfram-like syndrome leads to retinal ganglion cell degeneration and vision loss. Wolfram-like syndrome is primarily caused by variants in the WFS1 gene, which encodes an endoplasmic reticulum resident transmembrane protein, Wolframin. To date, the disease mechanism remains unclear, and no therapies are available. Here, we generated a mouse model carrying the pathogenic WFS1 ^E864K allele that recapitulated key features of human Wolfram-like syndrome, including bilateral optic atrophy, retinal nerve fiber thinning and lamination of the outer plexiform layer. We demonstrated, using the Wfs1 ^E864K mouse model, that alteration of the protein leads to impairments of retinal ganglion cell function, associated with a thinning of the inner retina layer and nerve fibers. These alterations are associated with myelin disorganization, axonal death, mitochondrial alterations in the axons, and impairment of endoplasmic reticulum-mitochondria communication in the soma. Our data showed that primary deficits are localized in the optic nerve before progressing towards the retinal ganglion cell soma. RNAseq analysis identified several altered signaling pathways such as in lipid metabolism, glia activation response, metabolic stress, organelle transport and quality control. These findings highlighted the critical role of Wolframin in optic nerve mitochondrial physiology, providing us with a pertinent model to develop novel innovative therapeutic strategies.

Glaucoma is a leading cause of blindness, affecting retinal ganglion cells (RGCs) and their axons. By 2040, it is likely to affect 110 million people. Neuroinflammation, specifically through the release of proinflammatory cytokines by M1 microglial cells, plays a crucial role in glaucoma progression. Indeed, in post-mortem human studies, pre-clinical models, and ex-vivo models, RGC degeneration has been consistently shown to be linked to inflammation in response to cell death and tissue damage. Recently, Rho kinase inhibitors (ROCKis) have emerged as potential therapies for neuroinflammatory and neurodegenerative diseases. This study aimed to investigate the potential effects of three ROCKis (Y-27632, Y-33075, and H-1152) on retinal ganglion cell (RGC) loss and retinal neuroinflammation using an ex-vivo retinal explant model. Rat retinal explants underwent optic nerve axotomy and were treated with Y-27632, Y-33075, or H-1152. The neuroprotective effects on RGCs were evaluated using immunofluorescence and Brn3a-specific markers. Reactive glia and microglial activation were studied by GFAP, CD68, and Iba1 staining. Flow cytometry was used to quantify day ex-vivo 4 (DEV 4) microglial proliferation and M1 activation by measuring the number of CD11b+, CD68+, and CD11b+/CD68+ cells after treatment with control solvent or Y-33075. The modulation of gene expression was measured by RNA-seq analysis on control and Y-33075-treated explants and glial and pro-inflammatory cytokine gene expression was validated by RT-qPCR. Y-27632 and H-1152 did not significantly protect RGCs. By contrast, at DEV 4, 50 µM Y-33075 significantly increased RGC survival. Immunohistology showed a reduced number of Iba1+/CD68+ cells and limited astrogliosis with Y-33075 treatment. Flow cytometry confirmed lower CD11b+, CD68+, and CD11b+/CD68+ cell numbers in the Y-33075 group. RNA-seq showed Y-33075 inhibited the expression of M1 microglial markers (Tnfα, Il-1β, Nos2) and glial markers (Gfap, Itgam, Cd68) and to reduce apoptosis, ferroptosis, inflammasome formation, complement activation, TLR pathway activation, and P2rx7 and Gpr84 gene expression. Conversely, Y-33075 upregulated RGC-specific markers, neurofilament formation, and neurotransmitter regulator expression, consistent with its neuroprotective effects. Y-33075 demonstrates marked neuroprotective and anti-inflammatory effects, surpassing the other tested ROCKis (Y-27632 and H-1152) in preventing RGC death and reducing microglial inflammatory responses. These findings highlight its potential as a therapeutic option for glaucoma.

Dry eye disease (DED) is a multifactorial disorder in which the eyes respond to minor stimuli with abnormal sensations, such as dryness, blurring, foreign body sensation, discomfort, irritation, and pain. Corneal pain, as one of DED’s main symptoms, has gained recognition due to its increasing prevalence, morbidity, and the resulting social burden. The cornea is the most innervated tissue in the body, and the maintenance of corneal integrity relies on a rich density of nociceptors, such as polymodal nociceptor neurons, cold thermoreceptor neurons, and mechano-nociceptor neurons. Their sensory responses to different stimulating forces are linked to the specific expression of transient receptor potential (TRP) channels. TRP channels are a group of unique ion channels that play important roles as cellular sensors for various stimuli. These channels are nonselective cation channels with variable Ca²⁺ selectivity. TRP homologs are a superfamily of 28 different members that are subdivided into 7 different subfamilies based on differences in sequence homology. Many of these subtypes are expressed in the eye on both neuronal and non-neuronal cells, where they affect various stress-induced regulatory responses essential for normal vision maintenance. This article reviews the current knowledge about the expression, function, and regulation of TRPs in ocular surface tissues. We also describe their implication in DED and ocular pain. These findings contribute to evidence suggesting that drug-targeting TRP channels may be of therapeutic benefit in the clinical setting of ocular pain.

The cornea, an anterior ocular tissue that notably serves to protect the eye from external insults and refract light, requires constant epithelium renewal and efficient healing following injury to maintain ocular homeostasis. Although several key cell populations and molecular pathways implicated in corneal wound healing have already been thoroughly investigated, insufficient/impaired or excessive corneal wound healing remains a major clinical issue in ophthalmology, and new avenues of research are still needed to further improve corneal wound healing. Because of its implication in numerous cellular/tissular homeostatic processes and oxidative stress, there is growing evidence of the role of Hedgehog signaling pathway in physiological and pathological corneal wound healing. Reviewing current scientific evidence, Hedgehog signaling and its effectors participate in corneal wound healing mainly at the level of the corneal and limbal epithelium, where Sonic Hedgehog-mediated signaling promotes limbal stem cell proliferation and corneal epithelial cell proliferation and migration following corneal injury. Hedgehog signaling could also participate in corneal epithelial barrier homeostasis and in pathological corneal healing such as corneal injury-related neovascularization. By gaining a better understanding of the role of this double-edged sword in physiological and pathological corneal wound healing, fascinating new research avenues and therapeutic strategies will undoubtedly emerge.

Background: Glaucoma is a blinding degenerative neuropathy in which the death of retinal ganglion cells (RGCs) causes progressive loss of visual field and eventually vision. Neuroinflammation appears to be a key event in the progression and spread of this disease. Thus, microglial immunomodulation represents a promising therapeutic approach in which mesenchymal stem cells (MSCs) might play a crucial role. Their neuroprotective and regenerative potentials have already raised hope in animal models. Yet no definitive treatment has been developed, and some safety concerns have been reported in human trials. In the present study, we investigated the neuroprotective and immunomodulatory properties as well as the safety of MSCs in an ex vivo neuroretina explant model. Methods: Labeled rat bone marrow MSCs were placed in coculture with rat retinal explants after optic nerve axotomy. We analyzed the neuroprotective effect of MSCs on RGC survival by immunofluorescence using RBPMS, Brn3a, and NeuN markers. Gliosis and retinal microglial activation were measured by using GFAP, CD68, and ITGAM mRNA quantification and GFAP, CD68, and Iba1 immunofluorescence stainings. We also analyzed the mRNA expression of both ‘M1’ or classically activated state inflammatory cytokines (TNFα, IL1β, and IL6), and ‘M2’ or alternatively activated state microglial markers (Arginase 1, IL10, CD163, and TNFAIP6). Results: The number of RGCs was significantly higher in retinal explants cultured with MSCs compared to the control group at Day 7 following the optic nerve axotomy. Retinal explants cultured with MSCs showed a decrease in mRNA markers of gliosis and microglial activations, and immunostainings revealed that GFAP, Iba1, and CD68 were limited to the inner layers of the retina compared to controls in which microglial activation was observed throughout the retina. In addition, MSCs inhibited the M1 phenotype of the microglia. However, edema of the explants was observed in presence of MSCs, with an increase in fibronectin labeling at the surface of the explant corresponding to an epiretinal membrane-like phenotype. Conclusion: Using an ex vivo neuroretina model, we demonstrated a neuroprotective and immunomodulatory effect of MSCs on RGCs. Unfortunately, the presence of MSCs also led to explant edema and epiretinal membrane formation, as described in human trials. Using the MSC secretome might offer the beneficial effects of MSCs without their potential adverse effects, through paracrine signaling.

The trabecular meshwork (TM) is the main site of drainage of the aqueous humor, and its dysfunction leads to intraocular pressure elevation, which is one of the main risk factors of glaucoma. We aimed to compare the effects on cytoskeleton organization and extracellular matrix (ECM) of latanoprost (LT) and a Rho-kinase inhibitor (ROCKi) on a transforming growth factor beta2 (TGF-β2)-induced glaucoma-like model developed from primary culture of human TM cells (pHTMC). The TGF-β2 stimulated pHTMC were grown and incubated with LT or a ROCKi (Y-27632) for 24 h. The expression of alpha-smooth muscle actin (αSMA) and fibronectin (FN), and phosphorylation of the myosin light chain (MLC-P) and Cofilin (Cofilin-P) were evaluated using immunofluorescence and Western blot. The architectural modifications were studied in a Matrigel^TM 3D culture. TGF-β2 increased the expression of αSMA and FN in pHTMC and modified the cytoskeleton with cross-linked actin network formation. LT did not alter the expression of αSMA but decreased FN deposition. The ROCKi decreased TGF-β2-induced αSMA and FN expression, as well as MLC-P and Cofilin-P, and stimulated the cells to recover a basal cytoskeletal arrangement. In the preliminary 3D study, pHTMC organized in a mesh conformation showed the widening of the TM under the effect of Y-27632. By simultaneously modifying the organization of the cytoskeleton and the ECM, with fibronectin deposition and overexpression, TGF-β2 reproduced the trabecular degeneration described in glaucoma. The ROCKi was able to reverse the TGF-β2-induced cytoskeletal and ECM rearrangements. LT loosened the extracellular matrix but had no action on the stress fibers.