RIP1 Inhibition Protects Retinal Ganglion Cells in Preclinical Glaucoma Models

Based on our findings, ONC induces retrograde axonal injury and mechanical stress, which likely activate multiple cell death pathways beyond the classical RIP1–RIP3–MLKL-mediated necroptosis. In contrast, the IRI model involves ischemia-reperfusion injury, where necroptosis plays a more prominent role. This difference in sensitivity suggests that RIP1 may mediate broader pathological responses in ONC, possibly through non-canonical, RIP1 kinase-dependent or scaffold-dependent mechanisms that are beyond the canonical necroptosis axis. As a result, RIP3 or MLKL deletion alone provides limited protection in ONC, while RIP1 inhibition yields more robust neuroprotection in both models.

These data also highlight the importance of aligning therapeutic strategies with the specific stress biology underlying the disease phenotype. If the targeted pathways do not play a central role in driving pathology in the chosen model, there is a significant risk of generating false-negative results, even if the therapeutic approach is valid in the context of human disease.

The development of RIP1-targeted therapies for glaucoma would require a comprehensive translational strategy, including therapeutic modality, route of administration, formulation optimization, and patient access planning. Given that glaucoma is a chronic, progressive, and typically asymptomatic neurodegenerative disease of aging, long-term management of the disease is essential to preserve vision. Therefore, oral administration would be preferable to intravitreal injections, as it reduces the treatment burden associated with frequent doctor visits and improves patient adherence and compliance. Depot formulations, such as sustained-release implants or port delivery systems, may also be considered to minimize injection frequency if local delivery is necessary to reduce systemic toxicity.

One key advantage of RIP1 inhibitors is their mechanism of action. The small molecules selectively inhibit RIP1 kinase activity when the cell is pathologically shifting toward death signaling, rather than in homeostatic states. This context-dependent action confers a favorable safety profile with low systemic toxicity, making oral delivery a viable and clinically attractive route.

Targeting RIP1 in glaucoma represents a neuroprotective and anti-inflammatory approach with translational potential, especially if formulated for long-term, patient-friendly administration. Further preclinical work is required to determine optimal dosing regimens, tissue exposure, and long-term safety to inform clinical development.

I used both technologies, PERG and OptoDrum, to address different aspects of vision. PERG captures RGC-intrinsic electrical activity (action potential) at the retinal level, while OptoDrum evaluates visual behavior, providing evidence that the brain actually perceives visual stimulation from the retina. Using both PERG and OptoDrum allowed us to validate not only structural protection, but also complete functional preservation at both the cellular and systemic levels in glaucoma animal models.

The OCT analysis we used in this study primarily measures changes in overall retinal thickness. In the ONC model, the damage is predominantly driven by mechanical axonal injury, which mainly affects the RGC layer. As a result, subtle changes confined to the RGC layer may not be detected when analyzing the retina as a whole. It is possible that we missed ONC-specific damage because we assessed total retinal thickness rather than isolating changes in the RGC layer. A more layer-specific OCT analysis might better capture the localized neurodegeneration caused by ONC. Alternatively, we may need a longer observation period, longer than 7 days to detect full-thickness retinal thinning, as structural degeneration can evolve slowly after initial axonal injury.

We believe that RIP1 inhibition contributes to both neuroprotection and the suppression of inflammation-driven damage.

First, RIP1 inhibition provides direct protection to RGCs by blocking RIP1-dependent necroptosis. This leads to preservation of both RGC somas and axons in injury models. Second, RIP1 inhibition also exerts a strong anti-inflammatory effect. We suggested that It may suppress a specific subtype of microglia known as RIP1-regulated inflammatory microglia, which are characterized by their high production of proinflammatory cytokines such as TNF. In our study, inhibiting RIP1 reduced microglial infiltration to the RGC layer and TNF expression in the retina, thereby lowering inflammatory toxicity around RGCs. Notably, this dual protective effect was more pronounced in the IRI model, where necroptosis plays a dominant role. RIP1 inhibition was more effective than targeting downstream mediators like RIP3 or MLKL, highlighting its central role in regulating both cell death and inflammation. Thus, RIP1 inhibition protects RGCs both intrinsically, by preventing necroptotic cell death, and extrinsically, by dampening harmful immune responses that lead to structural and functional preservation in glaucoma models.

Yes, RIP1 inhibition can affect non-neuronal cell types as RIP1 is not exclusively expressed in neurons. It is broadly present in non-neuronal retinal cells, including microglia, astrocytes, and vascular endothelial cells.

In our study, we observed a significant reduction in IBA1-positive microglial infiltration following RIP1 inhibition, which indicates that the protective effects are not limited to neuron-intrinsic pathways. Importantly, RIP1 plays a regulatory role in shaping innate immune responses through controlling a specialized subpopulation of microglia known as RIP1-regulated inflammatory microglia, which secrete high levels of TNF and other proinflammatory cytokines. Suppressing this population may alleviate the inflammatory environment that exacerbates retinal damage in glaucoma. Moreover, given RIP1’s known role in endothelial cell necroptosis and inflammation in other ischemia-reperfusion models, it is possible that RIP1 inhibition may help preserve the neurovascular unit and prevent vascular dysfunction in the retina. Although our study focused on RGCs and microglia, the impact on astrocytes, Müller glia, and retinal vasculature remains an open area for future research. Overall, our data support the idea that RIP1 inhibition mediates neuroprotection not only through direct RGC survival but also by modulating the broader cell types in the retinal microenvironment, suggesting a multi-cellular protective mechanism.

We used an AAV2.7m8 vector encoding the OPTN-E50K construct. Intravitreal injection was employed to achieve targeted gene delivery to the retina, enabling efficient transduction in RGC and other inner retinal layers.

We used 661W cells throughout this study for several reasons.

First, we needed a cell model that is easily genetically modifiable, as we used CRISPR and phiC31 technology to generate OPTN variants. Second, there is currently no fully reliable in vitro RGC model. 661W cells, a murine neuroretinal cell line, served as a surrogate for RGCs in this study. Third, since our study focuses on programmed cell death pathways, especially necroptosis, it was critical to use a model that expresses the necessary molecular machinery. Many immortalized or stem-cell-derived cell lines often lack the full machinery required for executing cell death. In contrast, 661W cells, which express a comprehensive set of cell death components, provide a robust in vitro system to study RIP1-mediated necroptosis.

In our study, we primarily focused on evaluating RGC soma survival and functional preservation. While we did not directly quantify axonal regeneration or perform detailed axonal tracing in RIPK1-KO, RIPK3-KO, or MLKL-KO animals, we assessed axonal integrity indirectly through structural (RGC number) and functional endpoints (PERG and OMR). Given that the ONC model primarily induces retrograde axonal injury, direct evaluation of axonal preservation would be informative. Future studies incorporating optic nerve tracing techniques, such as electron microscopy imaging or fluorophore-conjugated tetrodotoxin, could offer a more comprehensive understanding of the protective effects.

That said, it is important to note that RIP1 inhibition is more likely to exert protective effects by preventing degeneration, rather than promoting active axonal regeneration. Therefore, while it is effective in preserving existing neuronal structures, it may not be sufficient as a regenerative therapy and requires further investigation.

Thank you for the suggestion. While MEA systems are indeed powerful tools for assessing functional activity at the cellular level, I strategically decided not to use it in this study.

MEA provides high-resolution ex vivo recordings of RGC activity, which is valuable for mechanistic insight, but it comes with several limitations that do not suit our purpose of the study. It is time-limited due to short tissue viability, technically demanding, low-throughput, and lacks longitudinal tracking capability. Moreover, it cannot be combined with subsequent OMR (OptoDrum) evaluation, which is particularly important as functional deficits of RGCs are expected to precede observable behavioral changes. Especially in injury-based models like ONC or IRI, retinal tissue becomes extremely fragile, making MEA technically challenging and prone to variability, with low reproducibility across animals or experimental batches. This variability makes it difficult to normalize results across animals, genotypes, or injury types.

In contrast, PERG is non-invasive, better suited for assessing functional visual output over time, and allows repeated in vivo measurements with relatively good throughput, which is critical for monitoring disease progression and therapeutic response in glaucoma models. By combining PERG and OptoDrum evaluations, I was able to assess not only RGC firing activity in response to visual stimulation but also visually driven behavioral performance, providing functional evidence that visual signals are successfully transmitted to and processed in the brain.

For our study’s goals in capturing dynamic RGC function across time points and evaluating neuroprotective effects together with OMR measurements, PERG provided a more translational and practical solution.