The Molecular Regulation of Astroglia-Driven Neuroinflammation in Experimental Glaucoma

Research Background

Glaucoma is a major cause of blindness, characterized by complex neurodegenerative conditions that include extensive inflammatory responses in glial cells, accompanied by the progressive loss of Retinal Ganglion Cells (RGCs), optic nerve axons, and synaptic connections. Although there are variations in subtypes, topologies, and temporal progressions, the inflammatory response of glial cells is ubiquitous throughout the visual pathways from the retina to the brain. Initially, the response of glial cells may be beneficial, aiding in tissue clearance and healing, but once it transitions to a state of chronic activation, it exacerbates harmful neuroinflammation and promotes feedback loops leading to neuronal loss. At this stage, beyond increasing the production of neurotoxic pro-inflammatory molecules, the withdrawal of mechanical, nutritional, and bioenergetic support from glial cells to RGCs also contributes to glial cell dysfunction. Additionally, glial cell-driven attacks by autoreactive T cells, autoantibodies, and complements may amplify the damage during glaucomatous neurodegenerative processes.

Recent studies have particularly focused on the role of nuclear factor κB (NF-κB) in various inflammatory signaling pathways, including tumor necrosis factor receptor (TNFR) and Toll-like receptor (TLR) signaling, as well as inflammasomes. These inflammatory pathways regulate glial cell-driven neuroinflammation and neurodegeneration. TNFα, a transcriptional target of NF-κB, is a major pro-inflammatory cytokine upregulated in glaucomatous glial cells. Under the influence of TNFR and TLR signaling, caspase-8 acts as an initial caspase, capable of triggering RGC apoptosis, oligodendrocyte death, and axon degeneration. Concurrently, caspase-8 is also involved in neuroinflammation by regulating the proteolytic release of mature inflammatory cytokines through the modulation of inflammasomes.

cFLIP (FLICE-like inhibitory protein), a caspase-8 homolog, functions as a molecular switch regulating the programs of inflammation, apoptosis, or necrosis mediated by caspase-8. This study aims to determine the role of cFLIP in regulating glial cell-driven neuroinflammation in experimental glaucoma. Therefore, the objective of this research is to evaluate this regulatory mechanism by analyzing the deletion of cFLIP or cFLIPL in glial cells.

Research Sources

This paper is a collaborative effort by Yang Xiangjun, Zeng Qun, Inam Maide Gözde, Inam Onur, Lin Chyuan-sheng, and Tezel Gülgün, with the authors primarily affiliated with the Department of Ophthalmology and the Department of Pathology and Cell Biology at the Vagelos College of Physicians and Surgeons, Columbia University. The study was published in 2024 in the Journal of Neuroinflammation.

Research Process

Research Subjects and Experimental Design

The study utilized a mouse model, inducing elevated intraocular pressure in mice through anterior chamber microbead injection to simulate glaucoma. The study was conducted in mice with or without the conditional deletion of cFLIP or cFLIPL.

Mouse Strains

The research included mouse strains with neuron-specific conditional deletion of cFLIP. The first strain was generated by crossing GFAP-CreERT mice with cFLIP floxed mice. The second strain involved mice with a specific conditional deletion of cFLIPL, generated by crossing cFLIP latch flox/flox mice with GFAP-CreERT mice. Additionally, the experiment included control groups (wild-type and Cre-only controls) and an additional oil vehicle injection group.

Induction of Ocular Hypertension

Intraocular hypertension in mice was induced via anterior chamber injection of microbeads, ensuring experimental intraocular pressure levels stabilized at 28.06 ± 4.12 mmHg. The experiment continued with similar injections repeated over four weeks to ensure a 12-week duration.

Research Methods

Morphological Analysis

The morphological analysis involved examining retinal samples with immunolabeling for GFAP and TNFα, and additional Tunel staining to detect responses in specific marker-labeling glial cells. Imaging data were collected using confocal microscopy and analyzed quantitatively using ImageJ/Fiji software.

Molecular Analysis

A comprehensive molecular analysis of cytokine, chemokine, and protein levels in the retina and optic nerve was conducted using methods including a 36-plex immunoassay, NanoString analysis, and Western blot analysis.

Research Findings

Primary Results from Morphological and Molecular Analysis

1. Morphological Analysis: Compared to controls, the expression of GFAP- or TNFα-labeled glial cells increased significantly in the retinas and optic nerves of samples exhibiting glaucomatous glial cell reactivity.
2. Molecular Analysis: The 36-plex immunoassay results showed significantly lower levels of pro-inflammatory cytokines, such as IL1, IL2, IFNγ, and TNFα, in GFAP/cFLIP and GFAP/cFLIPL samples under experimental intraocular hypertension compared to the control group (p < 0.001).
3. Gene Profiling: NanoString-based molecular analysis revealed significant upregulation and downregulation of inflammation-related genes in GFAP/cFLIP mice, showing trends similar to those observed in the immunoassay.

Specific Changes and Self-Regulation Mechanisms

In samples with cFLIP-deficient glial cells induced by glaucoma, there was a decline in certain transcriptional components such as RelA, while RelB activation increased (evidenced by increases in RNA counts). The data from this experiment reveal a transcriptional self-regulatory mechanism that may enhance cell survival while simultaneously reducing inflammatory responses.

Conclusion

Summary of Main Results

1. Significant Immunomodulatory Effects: Mice with cFLIP deficiency showed significantly reduced inflammatory responses and decreased labeling of cytokines such as TNFα.
2. Molecular Mechanisms: Changes in RelA and RelB suggest a cross-pathway response mechanism, potentially enhancing the model’s survival while limiting pro-inflammatory responses.
3. Neuroprotection: Approximately 36% and 39% of RGC in ocular hypertensive gfap/cflip and gfap/cflipl mice were protected compared to controls, supporting the potential for immunomodulation in glaucoma treatment.

Significance and Value of the Study

Exploring the interaction between cFLIP and caspase-8 is of great significance for developing therapeutic approaches targeting neuroinflammation and neurodegenerative damage. This research could pave the way for more promising immunomodulatory treatment pathways through more in-depth molecular studies and clinical trials in glaucoma and other neuroinflammatory neurodegenerative diseases.

Highlights

1. Novel Mechanism: The study discovered the regulatory role of cFLIP in glial cell-driven neuroinflammation, demonstrating a unique regulatory response mechanism.
2. Practical Value: It provides a novel potential treatment for glaucoma through immunomodulation to protect neurons.
3. Supportive Data for Important Conclusions: The experimental data are comprehensive, with morphology, gene expression, and protein analysis mutually supporting the study’s clear logic and credible conclusions.