PMC

NAD⁺ deficiency impairs mitochondrial sirtuin function. (A–B) NAMPT inhibition caused selective mitochondrial protein hyperacetylation (n=4 biological replicates from two independent blots; 1-way ANOVA with Tukey post-hoc test). (C–D) NAMPT inhibition caused only modest changes in mitochondrial protein succinylation (n=6 biological replicates from three independent blots; 1-way ANOVA with Tukey post-hoc test). (E) NAD⁺ deficiency impaired SIRT3 activity (n=6/group; 2-tailed, unpaired t-test) but not SIRT5 activity (F; n=15–16/group; 2-tailed, unpaired t-test). Graphs depict mean + S.E.M. (B, D–F) (** p < .01; *** p < .001).

NAD⁺ deficiency disrupts retinal energy homeostasis and can be rescued with exogenous NMN. (A–C) Wild-type mice (129S1/SvImJ) receiving intraperitoneal injections of 300 mg/kg NMN were more resilient against light exposure (n=8/group; 2-way mixed ANOVA with Bonferroni post-hoc test) compared to vehicle-treated mice. (D) Representative electron microscopy images of retinas from 4-week old Nampt^{−rod/−rod} mice revealed mitochondria that are rounded, disorganized, and with loss of cristae compared to Nampt^F/F mice. (E) Nampt^{−rod/−rod} retinas also exhibited significant disruption of numerous metabolic pathways on metabolite set enrichment analysis. Graphs depict mean ± S.E.M. (A–C) (* p < .05).

Nampt^{−cone/−cone} mice exhibit cone-specific degeneration. (A) Cone-specific deletion of Nampt caused reduced intracellular NAMPT staining (red) in cone photoreceptors stained with cone-arrestin (green) and the nuclear DAPI stain (blue). (B) Representative fundus images from Nampt^{−cone/−cone} mice demonstrated retinal pigment epithelial cell mottling (arrows) and optic nerve atrophy (arrowhead) consistent with mild retinal degeneration. (C–E) Nampt^{−cone/−cone} mice also exhibited impaired retinal function on ERG (n=8–10 mice/group; 2-way mixed ANOVA with Bonferroni post-hoc test) and reduced photopic visual acuity (F; n=4 mice/group; Mann-Whitney U test). Graphs depict mean + S.E.M. (F) or mean ± S.E.M. (C–E) (* p < .05; ** p < .01; # p < .0001).

Exogenous NMN protects against retinal degeneration in mice lacking Nampt and may have efficacy against diverse retinal degenerative diseases. (A–C) Nampt^{−rod/−rod} mice receiving daily intraperitoneal injections of 150 mg/kg NMN beginning at P5 had improved retinal function on ERG (n=12 vehicle/5 NMN; 2-way mixed ANOVA with Bonferroni post-hoc test) compared to vehicle-treated mice, consistent with relative preservation of the outer nuclear layer on histology (D; see arrows). (E–G) Nampt^{−cone/−cone} mice receiving daily intraperitoneal injections of 150 mg/kg NMN beginning at P5 also had improved retinal function on ERG (n=9 vehicle/5 NMN; 2-way mixed ANOVA with Bonferroni post-hoc test). Retinal NAD⁺ deficiency is a feature of multiple mouse models of retinal dysfunction, including light-induced degeneration (H; n=10–12/group; 2-tailed, unpaired t-test), streptozotocin-induced diabetic retinopathy (I; n=5/group; 2-tailed, unpaired t-test), and age-associated retinal dysfunction (J; n=5/group; 2-tailed, unpaired t-test). Graphs depict mean + S.E.M. (H–J) or mean ± S.E.M. (A–C, E–G) (* p < .05; ** p < .01; *** p < .001; # p < .0001).

SIRT3 and SIRT5 are essential for photoreceptor survival. (A) Individual SIRT3 and SIRT5 knockdown but not SIRT4 knockdown caused significant loss of reductive capacity relative to negative control (NC); combined SIRT3/SIRT5 knockdown caused significantly more loss of reductive capacity than individual knockdowns, which could not be rescued with NMN (n=6/group from representative experiment; 1-way ANOVA with Tukey post-hoc test). (B) Individual SIRT3 and SIRT5 knockdown but not SIRT4 knockdown caused significant cell death relative to negative control (NC); combined SIRT3/SIRT5 knockdown caused significantly more cell death than individual knockdowns, which could not be rescued with NMN (n=18/group from three independent experiments; 1-way ANOVA with Tukey post-hoc test). (C) Individual and combined SIRT3/SIRT5 knockdowns recapitulated NAD-IDH dysfunction compared to negative control (NC; n=3/group from three independent experiments; 1-way ANOVA with Tukey post- hoc test). (D–F) Mice lacking SIRT3 and SIRT5 (SIRT3^KOSIRT5^KO) were significantly more vulnerable to light-induced degeneration (LID) compared to SIRT3^hetSIRT5^het mice, while SIRT3^KOSIRT5^het and SIRT3^hetSIRT5^KO mice exhibited intermediate vulnerability to LID (n=5–7 mice/group; 2-way mixed ANOVA with Bonferroni post-hoc test). Graphs depict mean + S.E.M. (A–C) or mean ± S.E.M. (D–F) (* p < .05; ** p < .01; *** p < .001; # p < .0001; ^ p < .05 relative to both SIRT3^KD and SIRT5^KD; red: SIRT3^hetSIRT5^het versus SIRT3^KOSIRT5^KO; brown: SIRT3^KOSIRT5^het versus SIRT3^KOSIRT5^KO; grey: SIRT3^hetSIRT5^KO versus SIRT3^KOSIRT5^KO).

Nampt^{−rod/−rod} mice exhibit severe retinal degeneration. (A) Rod-specific deletion of Nampt caused significant reduction in Nampt mRNA expression from rod-enriched retinal isolates (n=7 isolates/group; Mann-Whitney U test). (B) Nampt^{−rod/−rod} retinas had lower NAD⁺ levels than those from Nampt^F/F mice (n=5–8 retinas/group; 2-tailed, unpaired t-test). (C) Rod-enriched retinal isolates from Nampt^{−rod/−rod} mice had lower NAD⁺ levels than those from Nampt^F/F retinas (n=9–10 pooled samples/group; 1-tailed, unpaired t-test). (D) Representative fundus images from Nampt^{−rod/−rod} mice demonstrated severe signs of retinal degeneration including vascular attenuation and optic nerve atrophy. (E) Representative retinal sections from Nampt^{−rod/−rod} mice at 6 weeks stained with hematoxylin & eosin showed significantly reduced outer nuclear layer thickness (see arrows) with secondary retinal degeneration. (F–H) Nampt^{−rod/−rod} mice exhibited impaired retinal function on ERG (n=5 Nampt^F/F mice/10 Nampt^{−rod/−rod} mice; 2-way mixed ANOVA with Bonferroni post-hoc test), and reduced photopic visual acuity (I; n=5–6 mice/group; 2-tailed, unpaired t-test). Graphs depict mean + S.E.M. (A–C, I) or mean ± S.E.M. (F–H) (* p < .05; ** p < .01; *** p < .001; # p < .0001).

NAMPT inhibition causes metabolic dysfunction and photoreceptor death. (A–B) NAMPT inhibition with 20 μM FK866 caused loss of reductive capacity in 661W cone photoreceptor-like cells by 24 hours and 48 hours (n=15/group from three independent experiments; 1-way ANOVA with Tukey post-hoc test). (C–D) This metabolic dysfunction caused cell death by 48 hours (n=14/group from three independent experiments; 1-way ANOVA with Tukey post-hoc test). The effects of NAMPT inhibition were rescued with 100 μM NMN (A–B, D). (E) NAMPT inhibition with 20 μM FK866 led to a significant reduction in total NAD⁺ in photoreceptor cells by 6 hours, which was restored to near-normal levels with 100 μM NMN (n=3/group; 1-way ANOVA with Tukey post-hoc test). (F) 24 hours of 20 μM FK866 treatment led to undetectable levels of NAD⁺ (N.D. = not detected), which once again was restored with 100 μM NMN (n=5–7/group). (G) NAMPT inhibition also caused ATP depletion but in a delayed time frame relative to NAD⁺ depletion (n=4/group). (H–I) FK866-treated photoreceptor cells had reduced oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) at baseline, impaired ECAR acceleration after oligomycin treatment, and impaired OCR acceleration after FCCP treatment (n=15–16/group from representative experiment; 2-way mixed ANOVA with Bonferroni post-hoc test). 100 μM NMN restored normal metabolic responses (H–I). (J) NAD⁺-dependent isocitrate dehydrogenase (NAD-IDH/IDH3) activity was reduced in rods isolated from Nampt^{−rod/−rod} mice compared to those isolated from Nampt^F/F mice, even when sufficient NAD⁺ was added to the reaction mixture (n=6/group from three independent experiments; 2-tailed, unpaired t-test) and despite similar Idh3a expression levels (K; n=11–13/group from three independent experiments; 2-tailed, unpaired t-test). (L–M) The activities of other NAD⁺-dependent enzymes alpha-ketoglutarate dehydrogenase (AGDH; n=5/group from three independent experiments; Mann-Whitney U test) and malate dehydrogenase (MDH; n=7/group from three independent experiments; 2-tailed, unpaired t-test) in rods from Nampt^{−rod/−rod} mice were restored with exogenous NAD⁺. Graphs depict mean + S.E.M. (A–F, J–M) or mean ± S.E.M. (G–I) (* p < .05; ** p < .01; *** p < .001; # p < .0001; red: vehicle versus FK; blue: FK versus FK+NMN).