July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Identification of MAP4K4/GSK-3 as a parallel acting pathway with DLK/LZK signaling to control cell death programs in retinal ganglion cells
Author Affiliations & Notes
  • Amit K Patel
    Ophthalmology, University of California San Diego, La Jolla, California, United States
  • Katherine Mitchell
    Johns Hopkins University, Baltimore, Maryland, United States
  • Baranda Hansen
    Johns Hopkins University, Baltimore, Maryland, United States
  • Cindy Berlinicke
    Johns Hopkins University, Baltimore, Maryland, United States
  • Miranda Montgomery
    Ophthalmology, University of California San Diego, La Jolla, California, United States
  • Risa Broyer
    Ophthalmology, University of California San Diego, La Jolla, California, United States
  • Donald J Zack
    Johns Hopkins University, Baltimore, Maryland, United States
  • Derek Stuart Welsbie
    Ophthalmology, University of California San Diego, La Jolla, California, United States
  • Footnotes
    Commercial Relationships   Amit Patel, None; Katherine Mitchell, None; Baranda Hansen, None; Cindy Berlinicke, None; Miranda Montgomery, None; Risa Broyer, None; Donald Zack, None; Derek Welsbie, None
  • Footnotes
    Support  Ziegler Career Development Award
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 2612. doi:https://doi.org/
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      Amit K Patel, Katherine Mitchell, Baranda Hansen, Cindy Berlinicke, Miranda Montgomery, Risa Broyer, Donald J Zack, Derek Stuart Welsbie; Identification of MAP4K4/GSK-3 as a parallel acting pathway with DLK/LZK signaling to control cell death programs in retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2612. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Glaucoma is characterized by axonal injury leading to the death of retinal ganglion cells (RGCs). In order to identify potential neuroprotective drug targets, we previously developed an RNA interference-based functional genomic screen in primary RGCs. This work identified dual leucine zipper kinase (DLK)/JUN N-terminal (JNK) signaling as a key pathway that regulates RGC cell death. To elucidate other mediators, potentially overlooked by knockdown-based screens, here we describe a complementary small molecule screen using a library of highly-profiled protein kinase inhibitors (PKIs).

Methods : A high-throughput screening platform in primary RGCs was used to screen 366 PKIs from the Glaxo-Smith-Kline Protein Kinase Inhibitor Set (PKIS1). The PKIS1 library has been profiled in their ability to inhibit 220 purified protein kinase and survival-promoting activity of each compound was compared alongside the published kinase profiling to bioinformatically deconvolute kinase(s) that are responsible for RGC cell death. Positive hits from this screen were tested for interaction with the previously identified DLK pathway using genetic and pharmacologic methods.

Results : This screen identified that inhibition of glycogen synthase kinase three (GSK-3) and mitogen activated protein kinase kinase kinase kinase (MAP4K4) family members led to modest increases in survival. Moreover, simultaneous MAP4K4 and GSK-3 family inhibition increased RGC survival synergistically. As we have already identified DLK and leucine zipper kinase (LZK) inhibition as a neuroprotective mechanism, we tested the effect of combined GSK-3/MAP4K4/DLK/LZK inhibition and found that it resulted in almost complete protection of primary RGCs when challenged with microtubule destabilization, a pharmacologic model of axon injury. To explore the mechanism by which these kinase pathways might interact, we measured the activity of DLK/LZK/JNK signaling using JUN phosphorylation in the presence or absence of MAP4K4/GSK-3 family inhibition and found no difference, suggesting that MAP4K4/GSK-3 modulates RGC death in parallel to or downstream of DLK/LZK.

Conclusions : These findings suggest that MAP4K4/GSK-3 and DLK/LZK signaling interact to control cell death/survival decisions in RGCs. Moreover, these results establish kinase multitargeting as a robust neuroprotective strategy for optic neuropathies.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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