Retinal ganglion cell rescue in glaucoma: target-derived support

One of our most valuable senses, vision, is the end result of an outstanding interplay between our eyes and our brain. Damage to the tie that connects both structures—i.e., the optic nerve—leads to vision loss. Diseases caused by optic nerve injury are collectively grouped as optic neuropathies, of which glaucoma is a well-known example. Glaucoma is the most common cause of irreversible blindness and, unfortunately, there is still an unmet medical need for novel therapies. The efficacy of current therapies is rather low as they aim at symptomatic relief but do not impede the underlying cause, i.e. retinal ganglion cell (RGC) death. Despite years of extensive research, clinical translation of novel glaucoma therapies is still hampered. The reason for this is, at least, twofold: we lack a powerful pre-clinical glaucoma model and secondly, the pathophysiology of glaucoma is far from completely understood. Glaucoma is currently categorized as a multifactorial disease, in which various pathological mechanisms are running in parallel, or co-operate. Yet, the instigating mechanism as well as the sequence and interplay between ensuing factors are still up for debate. In this PhD dissertation, we aimed to tackle both of these hurdles witnessed in the glaucoma research field by (i) optimizing and characterizing a widely employed experimental glaucoma model—i.e., the microbead occlusion model—and (ii) by exploring the molecular signature underlying a well-known theory that tries to explain the glaucoma pathology—i.e., the neurotrophic factor deprivation theory. This theory states that an insufficient retrograde transport of target-derived neurotrophic factors fails to protect RGCs from apoptosis in glaucomatous conditions. We previously evidenced that RGC neuroprotection could be achieved by solely interfering with the activity of target neurons via optogenetics in an acute glaucoma model. To increase the translational value of these findings, we optimized a more chronic experimental toolbox by replacing optogenetics by chemogenetics and the acute and rather drastic glaucoma model by a more chronic, and mild model. The murine microbead occlusion model induces a mild phenotype that highly mimics the human pathology, yet also complicates proof-of-concept drug studies. We evaluated various approaches to lever the use of the microbead occlusion model in the glaucoma research domain. In summary, neither protocol adaptations, nor the use of different mouse strains improved the severity of the model but a refinement of evaluation tools was shown to facilitate the assessment of RGC injury and future neuroprotection. We propose anterior chamber depth measurements and scotopic threshold recordings as two easy-to-measure, in vivo and effective tools to assess model induction and disease progression. Besides, we showed that dendritic arbor analysis or quantifying RGC densities with our newly developed automated deep-learning tool (RGCode) are two valuable post-mortem read-outs in the glaucoma field. Since glaucoma is a chronic disease, we decided to optimize the chemogenetic DREADD (Designer Receptors Exclusively Activated by Designer Drugs) platform for chronical, in vivo usage and, performed a bulk RNA-sequencing study on long-term DREADD-stimulated superior colliculus lysates and the unstimulated isolated RGCs of the same glaucomatous mice to evaluate the molecular underpinning of our experimental set-up. The transcriptome analysis confirmed the predicted pro-survival stimulus by chemogenetic activation in the superior colliculus, as well as in the RGCs. Surprisingly, this pro-survival stimulus did—despite matching to previous research findings and predictions in relation to neuroprotection—not result in improved RGC health. This serves as a valuable warning that chronic chemogenetics and growth factor signaling is very complex, with many angles to be considered. In summary, we propose methodological advances towards the improved use of the murine microbead occlusion model and molecular clues regarding the neurotrophic factor deprivation theory. These results can, at least in part, help towards bridging the gap between preclinical and clinical glaucoma research and contribute to insights in the neurodegenerative field.

Publication year:2022

Accessibility:Open