Fig. 4: EGCG increases NAD and provides a robust neuroprotection following retinal ganglion cell injury.

A EGCG increases NAD in a dose-dependent manner in dissociated cortical neurons, with 50 nM the lowest dose to give a significant increase in NAD compared to untreated controls (each condition assessed in a sample from the same biological replicate; n = 4). B EGCG increases NAD in a time dependent manner in dissociated cortical neurons. EGCG was first added to the 6-h samples (and maintained throughout), 2 h later EGCG was added to the 4-h sample, etc. The 0-time sample was incubated for 6 h in media without EGCG. An increased cell viability in the samples treated with EGCG at an earlier time point may also contribute to the 5-fold increase in NAD (each condition assessed in a sample from the same biological replicate; n = 4). C EGCG significantly increased NAD in neuron-enriched tissue (cortex; n = 4 per condition) but not in neuron-low tissues (spleen, muscle, and liver; n = 4 per condition) suggesting a specificity towards Nmnat2 over Nmnat1. D EGCG dissolved in the culture media robustly protects against RGC death at 3 days ex vivo (3 DEV) following axotomy in comparison to untreated controls (n = 6, all conditions) (interventional treatment). E In the rat OHT model, prophylactic oral EGCG provided a modest neuroprotection relative to untreated controls at 40 mg/kg/d (n = 12), but not at 20 mg/kg/d (n = 7), although this was improved in combination with hNMNAT2 (n = 11). F In postmortem retinal punches (n = 10 donor retinas) maintained ex vivo for 7 days (7 DEV) significant RGC loss occurs which is significantly reduced by EGCG (or nicotinamide, NAM, the precursor for NAD through the NAD-salvage pathway) relative to uncultured controls (0 DEV), supporting the human utility of neuroprotection by EGCG (each condition assessed in a sample from the same biological replicate, n = 10). The prolonged postmortem time (24–48 h) results in significant RGC loss, and so in this context EGCG is able to provide interventional neuroprotection to an already degenerating system. Scale bars = 25 µm in D, E and 50 µm in F. *P < 0.05, **P < 0.01, ***P < 0.001, NS non-significant (P > 0.05); Student’s t-test to control for A, B, and C; One-way ANOVA with Tukey’s HSD for D, E, and F. For box plots, the centre hinge represents the median with upper and lower hinges representing the first and third quartiles; whiskers represent 1.5 times the interquartile range.