Nicolas Belforte

The glial cell modulator ibudilast attenuates neuroinflammation and enhances retinal ganglion cell viability in glaucoma through protein kinase A signaling

Glaucoma is a neurodegenerative disease and the leading cause of irreversible blindness worldwide. Vision deficits in glaucoma result from the selective loss of retinal ganglion cells (RGC). Glial cell-mediated neuroinflammation has been proposed to contribute to disease pathophysiology, but whether this response is harmful or beneficial for RGC survival is not well understood. To test this, we characterized the role of ibudilast, a clinically approved cAMP phosphodiesterase (PDE) inhibitor with preferential affinity for PDE type 4 (PDE4). Here, we demonstrate that intraocular administration of ibudilast dampened macroglia and microglia reactivity in the retina and optic nerve hence decreasing production of proinflammatory cytokines in a rat model of ocular hypertension. Importantly, ibudilast promoted robust RGC soma survival, prevented axonal degeneration, and improved anterograde axonal transport in glaucomatous eyes without altering intraocular pressure. Intriguingly, ocular hypertension triggered upregulation of PDE4 subtype A in Müller glia, and ibudilast stimulated cAMP accumulation in these cells. Co-administration of ibudilast with Rp-cAMPS, a cell-permeable and non-hydrolysable cAMP analog that inhibits protein kinase A (PKA), completely blocked ibudilast-induced neuroprotection. Collectively, these data demonstrate that ibudilast, a safe and well-tolerated glial cell modulator, attenuates gliosis, decreases levels of proinflammatory mediators, and enhances neuronal viability in glaucoma through activation of the cAMP/PKA pathway. This study provides insight into PDE4 signaling as a potential target to counter the harmful effects associated with chronic gliosis and neuroinflammation in glaucoma.

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension- Dependent Glaucoma in Mice

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial neurodegenerative disease. With the advent of numerous transgenic mouse lines, there is increasing interest in inducible murine models of ocular hypertension. Here, we present an occlusion model of glaucoma based on the injection of magnetic microbeads into the anterior chamber of the eye using a modified microneedle with a facetted bevel. The magnetic microbeads are attracted to the iridocorneal angle using a handheld magnet to block the drainage of aqueous humour from the anterior chamber. This disruption in aqueous dynamics results in a steady elevation of intraocular pressure, which subsequently leads to the loss of retinal ganglion cells, as observed in human glaucoma patients. The microbead occlusion model presented in this manuscript is simple compared to other inducible models of glaucoma and also highly effective and reproducible. Importantly, the modifications presented here minimize common issues that often arise in occlusion models. First, the use of a bevelled glass microneedle prevents backflow of microbeads and ensures that minimal damage occurs to the cornea during the injection, thus reducing injury-related effects. Second, the use of magnetic microbeads ensures the ability to attract most beads to the iridocorneal angle, effectively reducing the number of beads floating in the anterior chamber avoiding contact with other structures (e.g., iris, lens). Lastly, the use of a handheld magnet allows flexibility when handling the small mouse eye to efficiently direct the magnetic microbeads and ensure that there is little reflux of the microbeads from the eye when the microneedle is withdrawn. In summary, the microbead occlusion mouse model presented here is a powerful investigative tool to study neurodegenerative changes that occur during the onset and progression of glaucoma.

AB015. Metabolic stress in glaucoma engages early activation of the energy biosensor adenosine monophosphate-activated protein kinase leading to neuronal dysfunction

Background: Metabolic stress has been proposed to contribute to neuronal damage in glaucoma, but the mechanism driving this response is not understood. The adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy homeostasis that becomes active at the onset of energy stress. AMPK is a potent inhibitor of the mammalian target of rapamycin complex 1 (mTORC1), which we showed is essential for the maintenance of retinal ganglion cell (RGC) dendrites, synapses, and survival. Here, we tested the hypothesis that AMPK is an early mediator of metabolic stress in glaucoma. Methods: Unilateral elevation of intraocular pressure was induced by injection of magnetic microbeads into the anterior chamber of mice expressing yellow fluorescent protein in RGCs. Inhibition of AMPK was achieved by administration of siRNA or compound C. RGC dendritic trees were 3D-reconstructed and analyzed with Imaris (Bitplane), and survival was assessed by counting Brn3a or RBPMS-labeled soma and axons in the optic nerve. RGC function was examined by quantification of anterograde axonal transport after intraocular administration of cholera toxin β-subunit. Retinas from glaucoma patients were analyzed for expression of active AMPK. Results: Ocular hypertension triggered rapid upregulation of AMPK activity in RGCs concomitant with loss of mTORC1 function. AMPK inhibition with compound C or siRNA effectively restored mTORC1 activity and promoted an increase in total dendritic length, surface and complexity relative to control retinas. Attenuation of AMPK activity led to robust RGC soma and axon survival. For example, 95% of RGCs (2,983±258 RGCs/mm, mean ± S.E.M.) survived with compound C compared to 77% in vehicle-treated eyes (2,430±233 RGCs/mm) (ANOVA, P<0.001) at three weeks after glaucoma induction (n=8–10/group). Importantly, blockade of AMPK activity effectively restored anterograde axonal transport. Lastly, RGC-specific upregulation of AMPK activity was detected in human glaucomatous retinas relative to age-matched controls (n=10/group). Conclusions: Metabolic stress in glaucoma involves AMPK activation and mTORC1 inhibition promoting early RGC dendritic pathology, dysfunction and neurodegeneration.