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Pingwu Zhang, Yukan Duan, Jie Cheng, Cynthia Berlinicke, Amit K Patel, Derek Stuart Welsbie, Donald J Zack; Genetic deletion of DLK and LZK reduce colchicine-induced cell death in human stem cell-derived retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):6152. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma, one of the leading cause of irreversible blindness, is characterized by progressive degeneration of retinal ganglion cells (RGCs). The cause and mechanism of glaucoma remain incompletely understood. Inhibition of dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK), by both RNAi and pharmacologic inhibitors, attenuates death of mouse and human RGCs. As an independent and more-specific approach to achieve DLK and LZK inhibition, here we describe the specific effect of genetic deletion of DLK/LZK on RGC survival after axonal injury induced by the microtubule polymerization inhibitor colchicine.
We generated DLK and LZK knock-out (KO) human embryonic stem cell (hESC) lines based on a previously generated BRN3B::tdTomato ES line using CRISPR/Cas9 genome engineering and differentiated the resulting cells into BRN3B positive RGCs. These cells were purified by MACS method as previously described (Sluch et al, 2017, Stem Cells Translational Medicine) and cultured in 96-well plates. RGCs were challenged with colchicine as a pharmacologic model of axonal injury and survival, neurite length, neurite number were measured 48 hours later (Thermo Fisher’s Live/Dead kit and Cellomics HCS Reader).
There was no overall difference in the ability of DLK or LZK KO lines to be differentiated into RGCs. Colchicine treatment induced significant RGC cell death (26.8 ±3.7% survival), and DLK-KO was highly neuroprotective (70.5± 13.9% survival, p<0.001). LZK-KO RGCs showed a modest increase of 36.7 ± 4.1% survival (P<0.05). At baseline, the number of neurites per neuron was similar among wild type, DLK-KO, and LZK-KO RGCs. However, after colchicine treatment, neurite counts were 34% higher (P<0.001) in DLK-KO RGCs compared to either LZK-KO or WT RGCs. Similarly, when we measured the average length of the remaining neurites, DLK-KO RGCs were 36% higher than either LZK KO or WT RGCs (P < 0.001).
DLK and LZK deletion both promote survival of hESC-derived RGCs, with DLK KO being significantly more neuroprotective. DLK, but not LZK, disruption was also able to partially rescue neurite degeneration. Ongoing studies are looking at the effect of combined DLK/LZK KO. These findings indicate that RGC protection through DLK/LZK inhibition is a promising therapeutic strategy for human RGC degenerations like glaucoma.
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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