Nicotinamide and WLDS Act Together to Prevent Neurodegeneration in Glaucoma

Pete A Williams, Jeffrey M Harder, Nicole E Foxworth, Brynn H Cardozo, Kelly E Cochran, Simon W M John, Pete A Williams, Jeffrey M Harder, Nicole E Foxworth, Brynn H Cardozo, Kelly E Cochran, Simon W M John

Abstract

Glaucoma is a complex neurodegenerative disease characterized by progressive visual dysfunction leading to vision loss. Retinal ganglion cells are the primary affected neuronal population, with a critical insult damaging their axons in the optic nerve head. This insult is typically secondary to harmfully high levels of intraocular pressure (IOP). We have previously determined that early mitochondrial abnormalities within retinal ganglion cells lead to neuronal dysfunction, with age-related declines in NAD (NAD+ and NADH) rendering retinal ganglion cell mitochondria vulnerable to IOP-dependent stresses. The Wallerian degeneration slow allele, WldS , decreases the vulnerability of retinal ganglion cells in eyes with elevated IOP, but the exact mechanism(s) of protection from glaucoma are not determined. Here, we demonstrate that WldS increases retinal NAD levels. Coupled with nicotinamide administration (an NAD precursor), it robustly protects from glaucomatous neurodegeneration in a mouse model of glaucoma (94% of eyes having no glaucoma, more than WldS or nicotinamide alone). Importantly, nicotinamide and WldS protect somal, synaptic, and axonal compartments, prevent loss of anterograde axoplasmic transport, and protect from visual dysfunction as assessed by pattern electroretinogram. Boosting NAD production generally benefits major compartments of retinal ganglion cells, and may be of value in other complex, age-related, axonopathies where multiple neuronal compartments are ultimately affected.

Keywords: NAD+; WldS; axon degeneration; glaucoma; retinal ganglion cell.

Figures

Figure 1
Figure 1
Dysregulation of synaptic and axonal transcripts occurs early in glaucoma. (A) Ingenuity pathway analysis of RNA transcripts from retinal ganglion cells from 9 mo D2 eyes compared to no glaucoma, age-, and sex-matched D2-Gpnmb+ controls. Pathway analysis identifies highly significant early transcriptomic changes to synaptic and axonal pathways, which become more enriched with increasing disease progression at a transcriptomic level (all samples came from mice with no glaucoma, thus these changes occur before detectable neurodegeneration). These pathways contain differentially expressed genes encoding multiple proteins that are known to be associated axon and synapse dysfunction or degeneration in other neurodegenerations. These include multiple members of the ephrin family of receptors (Epha2, Epha4, Epha5, Epha6, Ephb2, Ephb3, Ephb4 Ephb6; Chen et al., 2012), metabotropic glutamate receptors (Grm3, Grm5, Grm6, Grm7; Ribeiro et al., 2017), Ryr3 (Balschun et al., ; Del Prete et al., 2014), and semaphorins (Sema3a, Sema3b, Sema3d, Sema4a, Sema5a, Sema6a, Sema6c, Sema7a; Shirvan et al., ; Good et al., ; Pasterkamp and Giger, ; Smith et al., ; Gutiérrez-Franco et al., 2016). There are no significantly enriched pathways in D2 Group 1 or D2 + NAM samples compared to controls. (B–D) Individual gene expression plots show gene sets representing proteins in the post-synaptic density (B), the synaptosome (C), and the synaptic mitochondrial proteome (D) as previously defined (Collins et al., 2006). Dots represent individual genes, gray, not differentially expressed; red, differentially expressed [at FDR (q) < 0.05 compared to D2-Gpnmb+ control]. For all gene sets, number of differentially expressed genes increases with increased disease progression, this implies early transcriptomic dysfunction within synaptic compartments. These differentially expressed gene changes are absent from D2 Group 1 (i.e., no glaucoma at transcriptomic level), as well as from D2 + NAM samples, suggesting that NAM prevents early synaptic transcriptomic changes.
Figure 2
Figure 2
NAM supplementation and/or WLDS robustly restores NAD(t) levels through to 12 months. NAD(t) levels decrease with age in D2 retinas. The presence of the WldS allele or a diet supplemented in NAM robustly increases NAD(t) levels through to 12 months (a typical end-time in this model where the majority of eyes have severe glaucoma; n = 22/group). *P < 0.05, **P < 0.01, ***P < 0.001; Student's t-test compared to age-matched D2 retinas.
Figure 3
Figure 3
NAM supplementation or WLDS robustly protects from synapse elimination and neuronal stress. (A) Presence of the WldS allele or NAM prevents decreases in TOM20 staining (red). TOM20 is a central component of an essential complex responsible for recognition and translocation of mitochondrial preproteins (Neupert, 1997). We have previously demonstrated decreased mtRNA content in glaucomatous RGCs (Williams et al., 2017). These changes are correlated with a decrease TOM20 staining within the dendrite/synapses of D2 retinas. Retinas from NAM supplemented mice or mice carrying the WldS allele have TOM20 immunoreactivity levels similar to non-glaucomatous controls. 12 mo shown for all. Quantitative data are presented in (D). (B) Retinal ganglion cell synapses elimination begins early in glaucoma and progresses to widespread synapse loss by 12 mo. Presence of the WldS allele or NAM protects retinal ganglion cell synapses (as assessed by synaptophysin staining, green), even at late time points when degeneration is almost complete in untreated D2 eyes. 12 mo shown for all. Quantitative data are presented in (E). (C) GAP-43 levels increase in the retinal ganglion cell following axon injury at the proximal end of the axon (i.e., close to the optic nerve head; Doster et al., 1991). Presence of the WldS allele or NAM prevents increases in GAP-43 (red), reflecting dampened axon injury. Quantitative data are presented in (F). Images are shown from representative samples corresponding to nerve grades at 12 mo. n = 8/group. **P < 0.01, ***P < 0.001; Student's t-test compared to D2. Scale bars = 20 μm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer.
Figure 4
Figure 4
NAM supplementation in combination with WLDS robustly protects from glaucomatous neurodegeneration. (A) Presence of the WldS allele or NAM significantly protect D2 eyes from optic nerve degeneration as assessed by PPD staining (examples in F). Combination of the WldS allele and NAM significantly protects a greater proportion of optic nerves than either treatment alone (94% of eyes have no detectable glaucoma). Green, NOE; no or early glaucoma, yellow, MOD; moderate glaucoma, red, SEV; severe glaucoma. n > 60/group. (B) Presence of the WldS allele and/or NAM protects from retinal ganglion cell soma loss (D), red, RBPMS, a specific marker of retinal ganglion cells, n > 6/group. The density drop between D2 NOE and D2-Gpnmb+ is due to pressure induced stretching of the retina, inner plexiform layer thinning and nerve fiber layer thinning (examples in E). (C) Presence of the WldS allele or NAM significantly protect from a loss of visual function as assessed by pattern electroretinography (PERG, n > 20/group). PERG is a sensitive measure of retinal ganglion cell function, and thus the WldS allele and/or NAM protect even the earliest changes in glaucoma. ***P < 0.001 between 12 mo treatment groups and D2 12 mo. (G) Presence of the WldS allele and/or NAM protects retinal ganglion cells from axon loss. These axons terminate in key brain regions and have functional axoplasmic transport as assessed by CT-β labeling (green, n = 10/group). Retinal ganglion cell counts and axoplasmic transport images are from representative samples corresponding to NOE nerve grades at 12 mo. Representative images are shown in D–G. (C,D)**P < 0.01, ***P < 0.001, ns, not significant. Scale bars = 20 μm (D–F), 200 μm (G). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer.
Figure 5
Figure 5
Presence of the WldS allele or a diet supplemented in NAM do not change clinical disease progression/presentation in treated eyes. IOP profiles (A) and clinical presentation of iris disease (B). IOP is not significantly different between cohorts within the same age-group. Iris disease (iris pigment dispersion resulting in asynchronous ocular hypertension) progressed at a similar rate and reached a severe state in all groups within the same time-frame. For boxplots, the upper and lower hinges represent the upper and lower quartiles. The centerline of each diamond (red) represents the mean, and the upper and lower diamond points represent 95% confidence intervals of the mean (n > 45 for 8–9 and 10 month groups, n > 25 for 12 month groups).

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