Abstract
Purpose :
Optic nerve crush (ONC) injury leads to increased histochemical staining for zinc in synaptic vesicles of amacrine cells (AC) over the first 24 hours, and importantly, injection of chelators into the vitreous promotes RGC survival and axon regeneration [Li et al., PNAS 2017]. Here, we tested the hypothesis that the negative effects of zinc on RGC survival and axon regeneration require exocytosis from AC terminals.
Methods :
We inhibited exocytosis in amacrine cell terminals using Clostridium tetani neurotoxin (TeNT), which selectively targets inhibitory neurons, or Clostridium botulinum type A light chain (BoNT). We injected TeNT (2, 20, 200 nM) or BoNT (0.2, 2, 10 µM) into the eye immediately after ONC (129S1 male mice) and, 2 weeks later, assayed RGC survival by beta-III tubulin and axon regeneration by GAP-43 immunostaining. In additional mice, we used selenite autometallography (AMG) to visualize accumulation of vesicular zinc.
Results :
In intact mice, TeNT and BoNT induced a dose-dependent increase in AMG staining in the inner plexiform layer (IPL) after 3 days, reflecting an accumulation of metal ions in synaptic vesicles. Deletion of the vesicular zinc transporter ZnT3 suppressed this accumulation. After ONC and vehicle injection, AMG labeling in the IPL was elevated on day 1 and diminished to near normal levels by day 3. Injection of TeNT immediately after ONC caused AMG staining to remain elevated at day 3, suggesting that dissipation of ONC-induced zinc accumulation in the IPL occurs between day 1 and day 3 by exocytosis. Blockage of this dissipation by TeNT or BoNT improved RGC survival (160-180%) and axon regeneration (380 - 420%).
Conclusions :
These results show that inhibition of vesicular release from AC terminals is sufficient to protect RGCs and promote optic nerve regeneration, and suggest that zinc must be exocytosed from AC terminals to suppress RGC survival and regeneration. TeNT and BoNT cause mobile zinc to accumulate in synaptic vesicles of AC terminals even in normal mice further suggesting that the elevation of zinc after ONC could in part reflect an inhibition of the synaptic vesicle cycle in zinc-containing terminals. Further studies will investigate whether the effects of TeNT on RGC survival and axon regeneration after ONC also require blocking the release of inhibitory neurotransmitters.
This is a 2020 ARVO Annual Meeting abstract.