June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Role for dual leucine zipper kinase in human embryonic stem cell-derived retinal ganglion cell death signaling
Author Affiliations & Notes
  • Derek Stuart Welsbie
    Ophthalmology, University of California San Diego, La Jolla , California, United States
  • Katherine L. Mitchell
    Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Valentin Sluch
    Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Pingwu Zhang
    Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Amit Patel
    Ophthalmology, University of California San Diego, La Jolla , California, United States
  • Donald J Zack
    Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Derek Welsbie, None; Katherine Mitchell, None; Valentin Sluch, None; Pingwu Zhang, None; Amit Patel, None; Donald Zack, None
  • Footnotes
    Support  NIH K08EY022078, RPB CDA, Ziegler Foundation CDA
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 2952. doi:
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      Derek Stuart Welsbie, Katherine L. Mitchell, Valentin Sluch, Pingwu Zhang, Amit Patel, Donald J Zack; Role for dual leucine zipper kinase in human embryonic stem cell-derived retinal ganglion cell death signaling. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2952.

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

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Abstract

Purpose : Glaucoma is a neurodegenerative disease characterized by the axonal injury and loss of retinal ganglion cells (RGCs). In order to identify a novel neuroprotective strategy, we previously developed a high-throughput, RNA interference-based screen in primary mouse RGCs and identified dual leucine zipper kinase (DLK) inhibition as being highly neuroprotective, including in multiple rodent models of optic neuropathy. Although DLK inhibition has many attractive neuroprotective properties, there is no data yet that DLK plays a role in human neurodegenerations. Here, we describe the use of human embryonic stem cell (hESC)-derived RGCs and a pharmacologic model of axonal injury signaling in order to test the role of DLK in human RGCs cell death signaling.

Methods : Genome editing was used to modify H7 ESCs with a P2A-tdTomato-P2A-Thy1.2 cassette fused in frame to the C-terminus of the RGC-specific BRN3B gene. After retinal differentiation, BRN3B+ RGCs express the mouse cell surface antigen, Thy1.2, thereby allowing for immunopurification. After five days in culture, dissociated hESC-derived RGCs were treated with the microtubule-destabilizing drug, colchicine, in order to trigger axonal injury signaling and produce cell death.

Results : In order to test the role of DLK in injury signaling, hESC-derived RGCs were challenged with colchicine in the presence of four structurally distinct protein kinase inhibitors (PKIs), each with activity against DLK, or the vehicle control. While colchicine was able to kill over 95% of cells, each of the four DLK inhibitors was able to produce a near-total rescue cell death. Given the lack of specificity with the PKIs, we took two orthogonal genetic approaches to validate the findings, including the knockdown and knockout of DLK. Confirming the PKI findings, genetic disruption of DLK was also able to mitigate the cell death associated with microtubule disruption. Surprisingly, although pathway members downstream of DLK are activated in response to microtubule disruption, DLK protein is not increased, contrasting with the robust post-translational upregulation seen in mouse P0-P3 primary RGCs.

Conclusions : Pharmacologic and genetic disruption of DLK can reduce cell death in an in vitro model of human RGC neurodegeneration. Future work will test the function of DLK in other injury paradigms as well as the role for other recently-identified DLK pathway members.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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