Abstract
Purpose :
Inhibition of dual leucine zipper kinase (DLK) and leucine zipper-bearing kinase (LZK) pathways promotes retinal ganglion cell (RGC) survival both in vitro and in vivo, but has limited effect on, and may even inhibit axonal regeneration. With the goal of finding ways to promote axonal regeneration of RGCs treated with DLK/LZK inhibitors, we investigated the effect of histone deacetylase inhibition and myosin inhibition on the neurite outgrowth of injured RGCs.
Methods :
Human pluripotent stem cell-derived RGCs (hRGCs) were challenged with the microtubule-destabilizing agent colchicine and treated with a DLK/LZK inhibitor (tozasertib), an HDAC inhibitor (trichostatin A, TSA), and/or a myosin inhibitor (blebbistatin). After seventy-two hours, the viability of hRGCs was evaluated using a live cell stain (Calcein-AM), a dead cell stain (ethidium homodimer), and the ATP-based CellTiter-Glo assay. Neurite length and branchpoint number were quantified by high content screening. In addition to testing a wild-type stem cell line, a CRISPR-generated DLK/LZK knock-out hRGC line was also tested. The expression levels of genes of interest were determined by quantitative PCR.
Results :
Although colchicine-induced hRGC cell death was inhibited by DLK/LZK inhibitor, tozasertib, the neurite length and the number of branchpoint were greatly reduced. The addition of the HDAC inhibitor TSA or the myosin inhibitor blebbistatin to colchicine/tozasertib treated hRGCs led to increases in neurite length and branchpoint number compared with colchicine/ tozasertib treatment alone. Combined treatment with TSA and blebbistatin led to further increases in neurite length and the number of branchpoint. Similar results were obtained in an hRGC line in which both DLK and LZK were knocked out. Furthermore, colchicine-induced reduction of expression of the RGC-specific genes BRN3A, RBN3B, THY1, and SOX11 was partially reversed by treatment with tozasertib, tozasertib/TSA, or tozasertib/blebbistatin.
Conclusions :
In this study, we found that treatment with HDAC and myosin inhibitors increased neurite outgrowth in injured hRGCs which were concurrently treated with a DLK/LZK inhibitor. The effect of TSA and blebbistatin was additive. The combined inhibition of DLK/LZK, HDAC, and myosin suggests a possible therapeutic approach to repair injured RGCs.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.