Ca²⁺/calmodulin-dependent protein kinase II enhances retinal ganglion cell survival but suppresses axon regeneration after optic nerve injury

Abstract

Purpose : Retinal ganglion cells (RGCs), the projection neurons of the eye, degenerate in optic neuropathies after axon injury, and there are no clinical therapies to prevent their loss or restore their connectivity to targets in the brain. Here we demonstrate a profound neuroprotective effect of exogenous expression of various Ca²⁺/calmodulin-dependent protein kinase type 2 (CaMKII) isoforms in mice.

Methods : Wild type 129X1/SvJ and wild type 129S1/SvlmJ were used for experiments. New AAV transfer plasmids were constructed that express Flag-tagged constitutively active CaMKII mutant proteins under mouse γ-synuclein (Sncg) promoter. AAVs and other reagents included Zymosan, oncomodulin, stromal-derived factor 1 (SDF-1) and the non-hydrolyzable cAMP analog CPT-cAMP were injected intravitreally at 2-3 weeks before optic nerve injury. Retinas and optic nerves were collected at 2 weeks post injury. Retinal flat-mount were immunostained with anti-RBPMS or anti-Tuj1 to observe RGC survival and cholera toxin subunit B (CTB) were injected 2 days before perfusion to trace regenerated axons.

Results : A dramatic increase in RGC survival resulting from optic nerve trauma was elicited by expression of constitutively active variants of multiple CaMKII isoforms in RGCs using adeno-associated viral (AAV) vectors across a 100-fold range of AAV dosing in vivo. Despite this neuroprotection, however, short-distance RGC axon sprouting was suppressed by CaMKII, and long-distance axon regeneration elicited by several pro-axon-growth treatments was likewise inhibited even as CaMKII further enhanced RGC survival in these models. Notably, in a dose-escalation study, AAV-expressing CaMKII was more potent for axon growth suppression than promotion of survival.

Conclusions : That diffuse overexpression of constitutively active CaMKII strongly promotes RGC survival after axon injury may be clinically valuable for neuroprotection per se. However, the associated strong suppression of optic nerve axon regeneration demonstrates the need for understanding intracellular domain- and target-specific CaMKII activities to the development of CaMKII signaling pathway-directed strategies for the treatment of optic neuropathies.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.