PROJECT SUMMARY Glaucoma is a neurodegenerative disease defined by the injury of retinal ganglion cell (RGC) axons at the optic nerve head, leading to cell death and irreversible vision loss. Current therapies center around lowering intraocular pressure (IOP) although this can be challenging in some patients. In order to advance towards a neuroprotective strategy that could complement IOP-lowering, we have been identifying potential neuroprotective targets in primary RGCs using high- throughput functional genomic screening. We initially identified dual leucine zipper kinase (DLK) and its paralog, leucine zipper kinase (LZK) as being key upstream activators of JUN N-terminal kinase (JNK) and axon injury signaling. We showed that DLK/LZK inhibition provides robust and durable protection to RGCs in multiple rodent models of optic neuropathy and, more recently, validated the survival effect in a nonhuman primate model of glaucoma. Despite their central role as neuronal messengers of axonal injury, little is known about the regulation of DLK/LZK. Moreover, inhibition of DLK/LZK prevents axon regeneration and has only modest effects on distal axon degeneration. Using chemical genetic- and clustered regularly interspaced short palindromic repeat (CRISPR)-based screens, we have identified two subgroups of Sterile 20 (STE20) kinases, including thousand and one amino acid (TAO) and germinal center kinase IV (GCK-IV) kinases which seem to regulate the interpretation and duration of the DLK/LZK axon injury signal. Moreover, inhibition increases rather than decreases axon regeneration and has a much greater impact on distal axonal degeneration. Based on insights from cancer biology, we recognized that similar STE20 kinases converge to regulate Hippo signaling, a developmental growth pathway not previously linked to axon injury signaling. We show that inhibition of the central kinases of Hippo signaling, large tumor suppressor (LATS) 1 and 2, robustly protects primary mouse RGCs and we attempt to demonstrate that the canonical Hippo transcription factor, transcriptional coactivator with PDZ-binding motif (TAZ) modulates DLK/LZK signaling by affecting JUN-dependent transcription. The central theme of this proposal is that combinatorial inhibition of STE20 kinases can be leveraged to generate robust somal and axonal protection combined with long-distance, sustained axon regeneration and that a key molecular mechanism involves Hippo signaling. In Specific Aim 1 (SA1), we focus on the role of STE20 kinases in axon degeneration and evaluate the potential for pancellular and functional RGC protection in a rat model of glaucoma. For this, we built a novel all-in-one adeno-associated virus (AAV)/CRISPR reagent capable of disrupting multiple kinases. In SA2, we test the role of combinatorial STE20 kinase inhibition in axon regeneration. We also use transcriptomic assays to dissect the mechanism by which DLK/LZK and STE20 kinase inhibition differs with respect to axon regeneration. Finally, in SA3, we explore the role of Hippo signaling in both mouse and human RGC survival and its link to STE20 and DLK/LZK signaling. Together, we anticipate this proposal will lead to a robust RGC neuroprotective strategy for combined axonal and somal preservation and a deeper understanding of the key axon injury pathway in RGCs.

NARRATIVE We previously identified dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK) as promising neuroprotective targets in retinal ganglion cell (RGC) degenerations/optic neuropathies like glaucoma. More recently, we have used chemical genetic and clustered regularly interspaced short palindromic repeat (CRISPR)-based screens in RGCs to identify a set of sterile 20 (STE20) kinases that regulate DLK/LZK. In this proposal, we explore novel mechanisms by which STE20 kinases regulate this key neuronal injury pathway and exploit this knowledge to develop robust neuroprotective and neuroregenerative strategies that we then test in rodent models of glaucoma.