Towards an Effective Neuroprotective Strategy for Glaucoma: Identification of Protein Kinase Inhibitors That Promote RGC Survival and Neurite Outgrowth

Abstract

Purpose: : We have been performing a high-content screen (HCS) of small molecule libraries to identify novel compounds/molecules that could (1) act as molecular probes to help investigate the mechanism of retinal ganglion cell (RGC) injury, survival, and axon regeneration; and (2) serve as candidate leads for the development of neuroprotective treatment approaches for glaucoma and other optic neuropathies.

Methods: : A HCS was performed with primary mouse RGCs to identify compounds that could stimulate RGC survival and/or neurite outgrowth. We then validated the neuroprotective and axon-stimulating activities of identified hits with various in vitro assays and a particularly promising "hit" was further tested for activity in a mouse optic nerve crush model. Signaling pathways were explored by western blot and microarray analysis.

Results: : We identified a number of protein kinase inhibitors (PKIs) that can facilitate RGC survival and/or neurite outgrowth in vitro. Among the identified PKIs, one showed particularly strong activity in promoting RGC survival and neurite growth, activity that in many of our assays was stronger than that of brain derived neurotrophic factor (BDNF). We have begun preliminary in vivo experiments with this compound, and in these studies both systemic and intravitreal administration of the molecule promotes RGC survival and axon regeneration following mechanical optic nerve injury. In addition, the PKI can partially block glutamate-induced excitotoxicity of RGCs both in vitro and in vivo. Ongoing microarray and phosphoprotein western blot studies are exploring the signaling mechanisms by which this PKI exerts its effects on RGCs.

Conclusions: : Our findings indicate that HCS offers an effective approach for identifying small molecule molecular probes that modulate RGC survival and function. In addition, some of the PKIs that we have identified may have the potential to aid in the development of novel neuroprotective approaches for the treatment of RGC neurodegenerative diseases such as glaucoma.

Keywords: neuroprotection • retinal culture • ganglion cells