Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death

Abstract

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.G laucoma is the leading cause of irreversible blindness worldwide (1). It is a neurodegenerative disease in which vision loss is caused by the axonal injury and death of retinal ganglion cells (RGCs) (2), the projection neurons that process and transmit vision from the retina to the brain. Current therapies (i.e., surgery, laser, and eye drops) all act by lowering intraocular pressure (IOP). However, pressure reduction can be difficult to achieve, and even with significant pressure lowering, RGC loss can continue. Efforts have therefore been made to develop neuroprotective agents that would complement IOP-lowering therapies by directly inhibiting the RGC cell death process (3, 4). However, no neuroprotective agent has yet been approved for clinical use.Protein kinases provide attractive targets for the development of neuroprotective agents. A number of kinases, including cyclindependent kinases, death-associated protein kinases, JNK1-3, MAPKs, and glycogen synthase kinase-3β, are involved in neuronal cell death (5-12). An additional attraction is that protein kinases are readily druggable. The pharmacology and medicinal chemistry of kinase inhibitors are well-developed, with kinases now being the most important class of drug targets after G protein-coupled receptors (13). Although the primary clinical use of kinase inhibitors continues to be as antineoplastic agents, increasing attention is being paid to their use in other areas (14,15).To identify, in a comprehensive and unbiased manner, kinases that could serve as targets for neuroprotective glaucoma therapy, we screened the entire mouse kinome for kinases whose inhibition promotes RGC survival. For this screen, we develope...

DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury

“…These observations suggest that RGCs do not undergo a rapid degeneration after insult but, rather, enter a prolonged period in which stress signals are elevated before their eventual apoptosis. The type of slow, progressive degeneration may more accurately reflect the mechanism of RGC loss that occurs in the context of neurodegenerative diseases such as glaucoma, and this study and others suggest that DLK inhibition may represent an attractive therapeutic target in this indication (34).…”

Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A

“…RGCs were purified as previously described (54). HRECs (Cell Systems) were maintained in endothelial cell growth medium-2-MV (Lonza), as previously described (53), and used before passage 9.…”

SkpA Restrains Synaptic Terminal Growth during Development and Promotes Axonal Degeneration following Injury

“…DLK/Wnd is required for axonal regeneration in Drosophila, C. elegans, and mice (Hammarlund et al, 2009;Yan et al, 2009;Xiong et al, 2010;Shin et al, 2012;. In addition, mutants in dlk or inhibitors of Jnk result in a delay in axonal degeneration in both fly and mouse models of Wallerian degeneration (Miller et al, 2009;Yoshimura et al, 2011), as well as models of glaucoma Welsbie et al, 2013). Finally, hiw and phr1 mutants maintain synaptic connections and block axon loss in Drosophila and mouse models of Wallerian degeneration (Massaro et al, 2009;Xiong et al, 2012;Babetto et al, 2013).…”