Vitamin B3 modulates mitochondrial vulnerability and prevents glaucoma in aged mice

Abstract

Glaucomas are neurodegenerative diseases that cause vision loss, especially in the elderly. The mechanisms initiating glaucoma and driving neuronal vulnerability during normal aging are unknown. Studying glaucoma-prone mice, we show that mitochondrial abnormalities are an early driver of neuronal dysfunction, occurring before detectable degeneration. Retinal levels of nicotinamide adenine dinucleotide (NAD⁺, a key molecule in energy and redox metabolism) decrease with age and render aging neurons vulnerable to disease-related insults. Oral administration of the NAD⁺ precursor nicotinamide (vitamin B₃), and/or gene therapy (driving expression of Nmnat1, a key NAD⁺-producing enzyme), was protective both prophylactically and as an intervention. At the highest dose tested, 93% of eyes did not develop glaucoma. This supports therapeutic use of vitamin B₃ in glaucoma and potentially other age-related neurodegenerations.

Fig. 1. Mitochondrial dysfunction is associated with progressive RGC damage in glaucoma

(A) RGC samples were divided into molecularly distinct groups by HC of RNA-seq determined gene expression. Control (D2-Gpnmb⁺) and young samples were molecularly similar (Spearman’s rho; n = 63 samples). Circles = samples from D2 RGCs, triangles = samples from D2-Gpnmb⁺ RGCS. Inset: number of DE genes (q < 0.05) between D2-Gpnmb⁺ and each group. D2 Group 1 and D2-Gpnmb⁺ represent no glaucoma at a molecular/transcriptomic level. (B) Mitochondrial: nuclear read total ratio increased with increasing HC distance from controls. Colors match key in A. (C) Top significantly enriched pathways between clusters and control based on IPA analysis (there are no differentially expressed pathways in D2 Group 1) (see also Table S3). (D) Transcript expression primarily increased for nuclear encoded mitochondrial proteins with increasing HC distance from controls. Dots represent individual genes, grey = not differentially expressed, red = differentially expressed at q < 0.05. Genes taken from mouse MitoCarta2.0 (28) (E) OXPHOS genes were differentially expressed across all groups. Red = highest expression, blue = lowest expression, I–V = mitochondrial complexes I–V (tabulated in Table S1), G = D2-Gpnmb⁺, 1–4 = D2 Groups 1–4. (F) RNA-seq identified increased mitochondrial fission gene transcripts early in glaucoma and (G) suggests an early mitochondrial unfolded protein response compared to controls. Data shown is for D2 Group 4. (H and I) Mitochondria of D2 mice have decreased cristae volume (and cristae : total volume ratio, not shown) in RGC somal and dendritic mitochondria (there was no significant difference in total mitochondrial size/volume) (n > 400 mitochondria from 6 retinas/group). Scale bar = 350 nm. All data is at 9 mo unless otherwise stated. ** = P < 0.01, * = P < 0.001 (Student’s t test). See also Table 1, Table 2.

Fig. 2. Vitamin B₃/NAM supplementation protects against glaucoma development in mice

(A) NAD(t) levels were increased in Vitamin B₃/NAM treated D2 retinas as measured by colorimetric assay (n = 22/group). (B and E) NAM intervention protected from optic nerve degeneration as assessed by PPD staining (a sensitive stain for damaged axons). Green = no or early damage (<5% axon loss; NOE), yellow = moderate damage (~30% axon loss; MOD), red = severe (>50% axon loss; SEV) damage. Early start indicates mice that started treatment at 6 mo (prior to IOP elevation in most eyes in our colony, thus prophylactic). Late start indicates mice that started treatment at 9 mo (when the majority of eyes have had continuing IOP elevation, thus interventional). Fisher’s exact test: ** = P < 0.01, *** = P < 0.001. (C and E) NAM protected from RGC soma loss (number of RBPMS+ cells, n = 8/group, the density drop between D2 and D2-Gpnmb⁺ is due to pressure induced stretching). (D) NAM protected from early loss in PERG amplitude (n > 20/group). (E) NAM protected from RGC soma loss (n = 8/group), retinal NFL and IPL thinning (n = 8/group), optic nerve degeneration (n > 50/group), and loss of anterograde axoplasmic transport (n = 20/group). Corresponding markers and color keys are beneath each column. Scale bars; RBPMS (a specific marker of RGCs; immunofluorescence) = 20 μm, Nissl (a pan-neuronal stain; light microscopy) = 20 μm, PPD (light microscopy) = 20 μm, CT-β = 100 μm (retina; immunofluorescence), 200 μm (LGN, Sup. Col.). ONH = optic nerve head, LGN = lateral geniculate nucleus, Sup. Col. = superior colliculus. White asterisk denotes loss of axonal transport at the site of the ONH. (F) Heatmap of gene expression (all expressed genes) shows that NAM treated RCGs were molecularly similar to controls. (G) Individual gene expression plots show metabolic and DNA damage pathways were returned to normal in NAM treated RGCs. Dots represent individual genes, grey = not differentially expressed, red = differentially expressed at q < 0.05 compared to D2-Gpnmb⁺ 9 mo control. (B, D, and E) * = P < 0.05, ** = P < 0.01, *** = P < 0.001 (Student’s t test). For boxplots, center hinge represents the mean, and the upper and lower hinges represent the first and third quartiles, whiskers represent 1.5 * interquartile range, values beyond the whiskers are plotted as outliers. See also Fig. S4, Tables 1–3.

Fig. 3. Gene therapy protected eyes from glaucomatous neurodegeneration

D2 eyes were intravitreally injected at 5.5 mo with AAV2.2 carrying a plasmid to overexpress murine Nmnat1 under a CMV promoter. (A) Nmnat1 overexpression prevented RGC soma loss (red, scale bar = 50 μm) and loss of anterograde axoplasmic transport (n = 10/group) (as demonstrated in Fig. 2.) (B; red, scale bar = 100 μm). Nmnat1 gene therapy also protected D2 eyes with elevated IOP against optic nerve degeneration (n > 40/group; *** = P < 0.001, Fisher’s exact test) (C), soma loss (n = 6/group) (D), and PERG amplitude (n > 20/group) (E). Addition of NAM^Lo in drinking water afforded additional protection against optic nerve degeneration (Nmnat1 compared to Nmnat1 + NAM^Lo = P < 0.001, Fisher’s exact test) (C). (D and E) ** = P < 0.01, *** = P < 0.001 (Student’s t test).