Drug Treatment Attenuates Retinal Ganglion Cell Death by Inhibiting Collapsin Response Mediator Protein 2 Phosphorylation in Mouse Models of Normal Tension Glaucoma

Neurology Biochemistry Basic Medical Sciences Life Sciences Ophthalmology Medicine Cell Biology
CRMP2 Normal Tension Glaucoma Retinal Ganglion Cell Phosphorylation

Scientific Research Report: Drug Treatment Alleviates Retinal Ganglion Cell Death in Normal Tension Glaucoma Mouse Model by Inhibiting Collapsin Response Mediator Protein 2 Phosphorylation

Background

Normal Tension Glaucoma (NTG) is a progressive neurodegenerative disease in the glaucoma family. Typically, glaucoma is associated with elevated intraocular pressure (IOP), but NTG presents retinal ganglion cell (RGC) degeneration, axon loss, and optic nerve damage even under normal IOP. As a subtype of open-angle glaucoma, the pathological mechanism of NTG has attracted widespread attention, especially glutamate excitotoxicity and oxidative stress. In NTG research, exploring the relationship between these pathogenic factors and RGC apoptosis is crucial for developing effective treatments.

Recent studies have shown that inhibiting the phosphorylation of Collapsin Response Mediator Protein 2 (CRMP2) can significantly reduce RGC death in NTG mouse models. This study aims to evaluate the inhibitory effects of two natural compounds, Huperzine A (HupA) and Naringenin (Nar), on CRMP2 phosphorylation, and explore whether they can alleviate excitotoxicity induced by N-Methyl-D-Aspartate (NMDA) and RGC death in Glast mutant mice.

Source and Author Information

This paper was jointly written by scientists including Yuebing Wang, Musukha Mala Brahma, Kazuya Takahashi, Alessandra Nolia Blanco Hernandez, Koki Ichikawa, Syuntaro Minami, Yoshio Goshima, Takayuki Harada, and Toshio Ohshima, from research institutions such as Waseda University, Nara Medical University, Yokohama City University Graduate School of Medicine, and Tokyo Metropolitan Institute of Medical Science. The paper was published in the journal “Neuromolecular Medicine” in 2024.

Research Workflow

Research Model and Experimental Design

  1. Experimental Animals: Mouse experiments were approved by the Animal Care and Use Committee of Waseda University. Mice were raised under 12-hour light/dark conditions with free access to food and water. The experiments included wild-type and genetically modified GLAST+/− and CRMP2 KI/KI mice.

  2. Intraocular Injection: Mice were anesthetized with diethyl ether at 10-16 weeks and injected intraocularly with 20 nmol NMDA to induce excitotoxicity in vivo. The control group was injected with the same volume of PBS.

  3. Drug Treatment Strategy: HupA (0.7 mg/kg) and Nar (200 mg/kg) were administered orally through mixed food. Experimental group mice started receiving HupA and Nar treatment one day before intraocular injection, with treatment times varying in different experiments.

  4. Histological Analysis:

    • Immunohistochemistry: Flat-mounted retinal samples were fixed by 4% PFA perfusion for immunostaining analysis.
    • H&E Staining: Mice were anesthetized with diethyl ether, and fixed retinal samples were taken for H&E staining to analyze RGC loss and Inner Retinal Layer (IRL) thickness.

  5. Statistical Analysis: Data are presented as mean ± standard error. Student’s t-test was used for comparisons between two groups, while ANOVA and Tukey’s post hoc test were used for multiple group comparisons. GraphPad Prism software was used as the analysis tool.

Experimental Results and Data Analysis

  1. Drug Treatment Inhibits NMDA-Induced CRMP2 Phosphorylation in Mouse Retina

    • Immunostaining observed weakened pCRMP2 signals in the retina of drug-treated mice, with HupA and Nar significantly inhibiting NMDA-induced CRMP2 phosphorylation.
    • Quantitative analysis showed a significant reduction in pCRMP2 positive cells in RGCs of mice treated with HupA and Nar compared to the NMDA-treated group.

  2. Reduction in RGC Degeneration and IRL Thinning in NMDA-Induced Model

    • Mice treated with HupA and Nar showed increased RGC survival rates and inhibition of IRL thickness reduction. For example, H&E staining results showed that in NMDA-treated mice, those receiving Nar treatment had higher RGC survival rates and IRL thickness.

  3. Drug Treatment Inhibits CRMP2 Phosphorylation in Glast Mutant Mouse Model

    • Immunostaining of 10-week-old Glast+/− mouse retinas revealed significantly elevated CRMP2 phosphorylation levels in RGCs compared to wild-type mice. However, after treatment with HupA and Nar, CRMP2 phosphorylation signals in Glast+/− mice were significantly reduced.

  4. HupA and Nar Inhibit RGC Degeneration in Glast Mutant Mice

    • Through HupA and Nar treatment, both 5-week-old and 10-week-old Glast+/− mice showed reduced RGC loss and improved IRL structural integrity. For example, H&E staining analysis showed significantly increased RGC numbers and thickened IRL in drug-treated mice compared to the control group.

  5. HupA and Nar Reduce Oxidative Stress in Glast Mutant Mice

    • Immunostaining showed significantly elevated 4-HNE (an oxidative stress marker) expression levels in the RGC layer of untreated Glast+/− mice, while 4-HNE levels were significantly reduced after HupA and Nar treatment.
    • These results suggest that HupA and Nar protect RGCs from degeneration in Glast mutant mice not only by inhibiting CRMP2 phosphorylation but also by reducing oxidative stress.

Conclusion

The study demonstrates that HupA and Nar significantly reduce RGC death and retinal degeneration in both NMDA injection models and Glast mutant mouse models by inhibiting CRMP2 phosphorylation. Additionally, both drugs showed significant reduction in oxidative stress. These findings provide new insights for NTG treatment, suggesting that HupA and Nar could be potential drugs for future NTG treatment.

Research Highlights

  1. Discovery of Multiple Drug Mechanisms: HupA and Nar protect RGCs not only by inhibiting CRMP2 phosphorylation but also by reducing oxidative stress.
  2. Validation in Multiple Experimental Models: The study used two different NTG mouse models, proving the effectiveness of HupA and Nar in various situations.
  3. Providing Treatment Clues: This study provides new clues for NTG treatment, offering basic data support for future development of effective neuroprotective agents.

Future Prospects

Although this study has achieved some positive results, the specific mechanisms of drug treatment for NTG still need further exploration. Additionally, future research is needed to improve the formulation, efficacy, safety, and bioavailability of HupA and Nar to achieve clinical application.