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Amit K Patel, Katy Mitchell, Baranda S. Hansen, Cindy Berlinicke, Risa Broyer, Donald J Zack, Derek Stuart Welsbie; MAP4K4 and GSK-3 pathways complement DLK/LZK signaling to control cell death and axon degeneration in retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2255.
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© ARVO (1962-2015); The Authors (2016-present)
Axonal injury induced death of retinal ganglion cells (RGCs) is a hallmark of glaucoma. We previously developed an RNA interference-based functional genomic screen in RGCs to discover neuroprotective targets. From this screen, we identified dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK) signaling as a key pathway that regulates RGC cell death. While inhibiting DLK/LZK results in robust protection of RGCs, there is room for improvement. Furthermore DLK/LZK inhibition does not prevent axon degeneration. Therefore, we performed a complementary small molecule screen using a library of highly-profiled protein kinase inhibitors (PKIs) to identify if there are complementary pathway targets which could improve RGC survival and protect axons.
We conducted a high-throughput small molecule screen in primary mouse RGCs using the Glaxo-Smith-Kline Protein Kinase Inhibitor Set (PKIS1). Using published kinase profiling of these compounds and the survival results from the screen, we can bioinformatically deconvolute which specific kinase(s) have a role in RGC death. We tested the positive hits for interactions with the DLK/LZK pathway and in their ability to prevent axon degeneration, using microtubule destabilization as a proxy for cell and axon injury in vitro.
In this screen, we identified glycogen synthase kinase three (GSK-3) and mitogen activated protein kinase kinase kinase kinase (MAP4K4) family members as targets whose inhibition leads to modest increases in RGC survival. Moreover, simultaneous inhibition of the GSK-3 and MAP4K4 pathways lead to a synergistic increase in RGC survival. As we already have identified the DLK/LZK pathway as a neuroprotective target, we wanted to see whether additionally targeting the MAP4K4/GSK-3 pathways could improve RGC survival. We found that the combined inhibition of the GSK-3/MAP4K4/DLK/LZK pathways resulted in near complete protection of RGCs. Furthermore, we found that MAP4K4/GSK-3 inhibition also protects against axonal degeneration in our model, in addition to increasing RGC survival.
MAP4K4/GSK-3 and DLK/LZK have complementary signaling pathways that interact to control cell death/survival and axon protection in RGCs. Furthermore, these results establish combinatorial kinase targeting as a robust neuroprotective strategy for optic neuropathies.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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