Abstract
Purpose :
Our limited understanding of the signals that regulate retinal ganglion cell (RGC) death and axon regeneration after axon injury has prevented the development of curative therapies. Inhibiting DLK (MAP3K12) and LZK (MAP3K13) signaling after axonal injury is highly neuroprotective but blocks axonal regenerative capacity even in the presence of a strongly pro-regenerative treatment. The objective of our studies is to investigate the role of transcription factors downstream of DLK and LZK to identify gene regulatory networks (GRNs) that may independently regulate RGC cell death and axon regeneration.
Methods :
Conditional deletion of DLK/LZK in RGCs was achieved using intraocular delivery of AAV2-CRE in DLKfl/fl;LZKfl/fl mice. Selective knockout of the transcription factors (TFs) known to lie downstream of DLK/LZK was achieved by intraocular delivery of AAV2-gRNAs into mice with RGC-specific Cas9 expression (Vglut2-CRE;ROSA26-LSL-Cas9). Gene manipulation was conducted 2-3 weeks prior to mouse optic nerve injury (ONI). DLK/LZK signaling was assessed via immunofluorescent staining at 1 and 5 days post-injury (dpi), the onset and peak of signaling, respectively. RGC survival and axon regeneration was assessed at 14 dpi.
Results :
We found that deletion of both DLK and LZK in RGCs dramatically increased RGC survival at 14 dpi (63.4±6.5% survival in DKO vs 21.5±2.4% in wildtype), but blocked axon regeneration (89±18% reduction) and induction of regeneration associated genes (RAGs), including Gap43, even in the presence of strongly pro-regenerative treatments (incl. Zymosan + CPT-cAMP). We next investigated the roles of the downstream TFs. Independent knockout of Jun, Sox11, Mef2a, and Atf2 in RGCs resulted in significantly increased RGC survival at 14 days after ONI with no improvement of endogenous axon regeneration. However, when axon regeneration was stimulated using pro-regenerative treatments, we identified particular TFs that either enabled or suppressed axon regeneration.
Conclusions :
These results show that there are discrete GRNs that independently regulate cell death versus axon regeneration, pointing to novel gene therapies that can function in combination with other well-known treatments to both improve RGC survival while also improving axon regeneration after optic nerve injury.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.