Neuroprotective effects and mechanisms of action of nicotinamide mononucleotide (NMN) in a photoreceptor degenerative model of retinal detachment

Results

NMN supplementation reduces photoreceptor cell death in the early phase of RD

The in vivo experimental procedures of RD were successfully induced in mouse eyes (Figure 1A, 1B), resulting in photoreceptor cell death which was attenuated after NMN administration at 24 hours after RD (Figure 1C, 1E, 1F). NMN at 250 mg/kg reduced cell death numbers by 52.7% (2292 ± 690 cells/mm², p<0.001) whereas NMN at 500 mg/kg decreased cell death by 71.0% (1405 ± 290 cells/mm², p<0.001).

Figure 1. Protective effects of NMN administration in the early phase of retinal detachment (RD). (A) A flow chart for the in vivo experiments. (B) schematic anatomy of a mouse eye by hematoxylin and eosin (HE) staining showing a bullous after successful RD surgery. Scale bar: 50 μm. (C, E, F) TUNEL⁺ cells (green) were seen with the highest numbers in vehicle-treated retinas and lowest in the 500 mg/kg NMN-treated retinas at both 24h (E) and three days (F) post RD. Nuclei were counterstained with DAPI (blue). N = 4 to 8 eyes per group. Scale bar: 50 μm. (D, G, H) CD11b⁺ macrophages (red) infiltrated in the subretinal space after RD, NMN administration significantly reduced the number as soon as 24h post RD (G), and in a dose-dependent manner after three days of RD (H). Nuclei were counterstained with DAPI (blue). N = 5 to 8 eyes per group. Scale bar: 100μm. Statistical significance was analyzed with one-way ANOVA followed by Tukey-Kramer adjustments. *p<0.05, **p<0.01, ***p<0.001. Data are mean ± SEM.

Similar effects were seen 3 days after RD with NMN reducing the number of TUNEL⁺ cells by 57.8% in the 250 mg/kg NMN group (984 ± 274 cells/mm² in vehicle vs. 415 ± 179 cells/mm² in NMN; p<0.01) and 70.8% in the 500 mg/kg group (287 ± 147 cells/mm²; p<0.001).

These results show that daily supplementation of the NAD⁺ precursor NMN is effective in inhibiting cell death at the acute phase of photoreceptor injury, with the most protective results achieved at the dose of 500 mg/kg.

NMN supplementation suppresses retinal inflammation

Our results suggested that 24 hours after RD induction, there was an accumulation of CD11b⁺ cells in the subretinal space in the vehicle RD group (9 ± 4 cell/mm²) (Figure 1D–1G). The number was reduced by an increasing dose of NMN from 250 mg/kg (7 ± 4 cell/mm²) to 500 mg/kg (3 ± 3 cells/mm²; p<0.05 compared to vehicle group).

The number of CD11b⁺ cells peaked three days post RD with the highest number in the vehicle group (17 ± 4 cells/mm²) followed by fewer cells in the 250 mg/kg NMN group (10 ± 4 cells/mm²; p<0.05 compared to vehicle group) and the lowest number in the 500 mg/kg NMN group (3 ± 2 cell/mm², p< 0.001 compared to vehicle group) (Figure 1D–1H). Taken together, these results suggest that NMN supplementation is associated with suppressed inflammation after RD in a dose-dependent manner.

NMN normalizes oxidative stress and upregulates antioxidant HO-1 after RD

We have previously identified elevated reactive oxygen species (ROS) as major pathophysiological changes after RD [13, 16]. We, therefore, investigated the effect of NMN supplementation on oxidative stress in the retina. Consistent with our previous results, there was a significant increase in oxidative stress as measured by protein carbonyl content (PCC) in the detached retinas (0.81 ± 0.1 nmol/mg) compared to the attached retinas (0.55 ± 0.15 nmol/mg; p<0.01) three days after RD (Figure 2A). NMN administration completely abolished this consequence (0.55 ± 0.1 nmol/mg, p<0.01 compared to vehicle) and maintained the tissue PCC comparable to the normal level as in the attached retina.

Figure 2. NMN administration attenuates oxidative stress and is protective in the late phase of retinal detachment (RD). (A) Protein carbonyl content (PCC) was significantly higher after RD while NMN treatment normalized PCC level comparable to the attached retinas at three days post RD. N = 6 eyes per group. (B) Heme oxygenase 1 (HO-1) was significantly up-regulated in the NMN groups as soon as 24h and remained highly expressed throughout the experimental period. N = 4 to 5 eyes per group. (C–F) Preservation of outer nuclear layer (ONL) thickness after NMN administration was seen 7 days after RD. N = 4 to 5 eyes per group. Scale bar=50 μm. The thickness of the layers was measured as the yellow lines indicated. (D–G) Inhibition of neuroinflammation seen by a reduced number of CD11b⁺ infiltrating macrophages (red) in NMN-treated retinas compared to vehicle-treated retinas. N = 4 to 5 eyes per group. Scale bar: 100 μm. (E–H) Reduced reactive gliosis represented by GFAP (green) staining in NMN-treated retinas compared to vehicle-treated retinas. N = 3 to 4 eyes per group. Scale bar: 50 μm. Statistical significance was analyzed with one-way ANOVA followed by Tukey-Kramer adjustments or the unpaired Student's t-test. *p<0.05. **p<0.01. ***p<0.001. Data are mean ± SEM.

To elucidate the antioxidant effect of NMN after RD, we further found that HO-1, an inducible isoform of heme oxygenase, was significantly upregulated in the NMN-treated retinas (Figure 2B). HO-1 expression was barely detectable in the attached retinas. In contrast, under stress induced by RD, HO-1 expressions were detectable in protein level, and NMN administration highly upregulated HO-1 levels as soon as 24 hours and throughout the whole experimental period. Our results demonstrate that NMN administration can counteract excessive oxidative stress in the detached retina, and this effect is possibly executed through the upregulation of HO-1.

NMN supplementation preserves ONL thickness and has an overall protection to the retina

To evaluate the long-term effect of NMN administration on photoreceptors after RD, we examined the overall impact on photoreceptor cell survival by measuring ONL thickness on Day 7 post RD. Administration of NMN at 500 mg/kg significantly preserved the retinal thickness seen by normalized ONL ratio (0.83) as compared to the vehicle-treated group (0.71, p<0.001) (Figure 2C–2F). Along with the increased survival of photoreceptors, NMN had a continued inhibitory effect of infiltrating CD11b⁺ macrophages (3 ± 1 cell/mm²) compared to the vehicle-treated retina (6 ± 1 cell/mm², p<0.05) (Figure 2D–2G), and a significant suppression of gliosis as measured by GFAP immunofluorescence (p<0.01) (Figure 2E–2H). These results further demonstrated the beneficial effect of NAD⁺-boosting molecules on regulating retinal homeostasis after injury.

NMN increases NAD⁺ levels and SIRT1 expression after RD

NAD⁺ deficiency has been identified among the primary causes leading to many neurodegenerative diseases [20, 22, 23], and the NAD⁺ precursors have beneficial effects in protecting neurons by replenishing the NAD⁺ pool in the majority of studies. However, we did not observe any change of NAD⁺ level in the vehicle-treated detached retina at neither 24h (NAD ratio of vehicle group 101.5% vs. attached retina 100%, p>0.05) or three days (NAD ratio of vehicle group 97.5% vs. attached retina 100%, p>0.05) post RD compared to the attached normal retina (Figure 3A, 3B). Whereas NMN administration significantly increased the NAD⁺ above normal levels in both time points (24h: 119.5%, p=0.005 compare to attached; 3 days: 119.7%, p=0.008 compare to attached). Similar to the results from the retina, NMN administration significantly increased NAD⁺ levels compared to both vehicle and the attached RPE/choroid (Supplementary Figure 1).

Figure 3. Increased NAD⁺ and SIRT1 levels after retinal detachment (RD) with NMN supplementation. Significant increase of NAD⁺ levels in the NMN-treated detached retinas compared to the vehicle and the attached retinas at 24h (A) and three days (B) after RD. NAD⁺ levels in vehicle-treated detached retinas were not lower compared to untreated controls. N = 8 to 11 eyes per group. (C) Upregulation of SIRT1 protein levels in NMN-treated retinas seen at 24h and 7 days post RD. SIRT1 levels were comparable among groups at three days post RD. N = 3 to 7 eyes per group. Statistical significance was analyzed with one-way ANOVA followed by Tukey-Kramer adjustments. *p<0.05. **p<0.01. ***p<0.001. Data are mean ± SEM.

To further elucidate the mechanism of NMN protection in the retina after increasing NAD⁺ levels, we examined the sirtuins, and found an upregulation of SIRT1 expression in the NMN-treated retinas at 24h and seven days after RD (Figure 3C). Retinal detachment alone did not induce the upregulation of SIRT1. This expression pattern was very similar to NAD⁺ content. SIRT1 expression did not differ significantly among groups three days after RD. Thus, SIRT1 may be necessary at different time points to confer protection after NMN administration, or they may have distinct and differing roles in photoreceptor survival.

In vitro study replicated the protection of NMN and revealed an association to be in part with the SIRT1/HO-1 signaling

Since we observed a very high oxidative stress status in the detached retina, we next tested the NMN mechanism of action in vitro using the photoreceptor-like cell line 661W in an oxidative stress model. A significant increase in cell survival was seen in the NMN administration group after ROS insult (Figure 4A). SRT2104, a direct SIRT1 activator, was also found protective to 661W cells after ROS insult (Figure 4B). However, the baseline level of NAD⁺ was also declined in the oxidative stress model (Supplementary Figure 2A). To examine if SIRT1 plays a role in mediating the effects of NMN supplementation, we transfected the 661W cells with SIRT1 siRNAs (Figure 4C). Our results showed a significantly lower level of cell survival in the SIRT1 siRNA transfection groups compared to the scramble siRNA control groups under NMN administration after ROS (Figure 4D). These results suggested that SIRT1 is at least partially responsible for the antioxidant property of NMN.

Figure 4. Protected effects of NMN on 661W cells under oxidative stress are partially SIRT1 dependent. (A) NMN administration significantly increased 661W cell viability after ROS insult. ***p<0.001 to tBuOOH only group. N = 4 to 5 per group. (B) SRT2104, a SIRT1 direct activator, had a similar effect as NMN in protecting 661W cells from ROS insult. *p<0.05 to tBuOOH only group. N = 3 to 4 per group. (C) SIRT1 was knocked down by siRNAs. (D) Silencing SIRT1 by siRNAs attenuated the protective effect of NMN after ROS insult compared to the scrambled (SC) siRNAs control group. $p<0.001 to tBuOOH only group in SC siRNA condition. #p<0.001 to tBuOOH and NMN treated group in SC siRNA condition. N = 4 to 8 per group. Statistical significance was analyzed with one-way ANOVA followed by Tukey-Kramer adjustments. Data are mean ± SEM.

Using western blot analysis, we found an upregulation of HO-1 protein expression after both NMN (Figure 5A) and SRT2104 treatment (Figure 5B) in the oxidative stress model. Knocking down of SIRT1 almost totally inhibited the induction of HO-1 with NMN administration (Figure 5C), whereas knocking down HO-1 didn’t alter the expression of SIRT1 under the NMN treated conditions (Figure 5D) suggesting that HO-1 upregulation after NMN was the downstream of SIRT1 upregulation.

Figure 5. A potential SIRT1/HO-1 signaling under the NMN-treated oxidative stress model. Both NMN (A) and SRT2104 (B) administration significantly increased HO-1 protein levels after ROS insult. N = 4 per group. Statistical significance was analyzed with the unpaired Student's t-test. *p<0.05. **p<0.01. ***p<0.001. Data are mean ± SEM. (C) Silencing SIRT1 by siRNA almost abolished the induced HO-1 expression by NMN after ROS insult. (D) HO-1 siRNA didn’t alter the expression of SIRT1 by NMN after ROS insult.

NMN and SIRT1 exerted protective effects directly on 661W cells not through action on immune cells in an in vitro model of immune mediated toxicity to photoreceptor cells

Our in vivo results have shown a significant reduction of infiltrating macrophages in the subretinal space after the NMN supplement. To determine whether this result is a direct anti-inflammatory effect of NMN or indirectly from the reduced photoreceptor cell death, we examined the role of NMN and SIRT1 in a RAW264.7 macrophage conditioned media (CM) toxicity model. CM samples from RAW264.7 cells treated or without the LPS were both toxic to 661W cells. NMN administration significantly increased the viability of 661W cells in both the LPS treated and untreated RAW264.7 CM (Figure 6A). The direct SIRT1 activator SRT1720 also showed a protective effect on 661W cells in the LPS treated RAW264.7 CM (Figure 6B).

Figure 6. Protective effects of NMN and SIRT1 activation to 661W cells in RAW264.7 conditioned media (CM). (A) NMN administration significantly increased the cell viability of 661W cells in both the LPS treated and untreated RAW264.7 CM. N = 3 to 4 per group. (B) SRT1720 also showed a protective effect to 661W cells in the LPS treated RAW264.7 CM. N = 4 per group. The CM from neither NMN (C) or SRT1720 (D) treated RAW264.7 cells had protective effects to 661W cells. N = 4 to 6 per group. LPS can induce significant increases in both TNF-α (E) and IL-1β (F) expressions, which were not altered by NMN administration. N = 3 per group. (G) SIRT1 siRNA in 661W cells significantly reduced cell viability in the LPS stimulated RAW264.7 CM. #p<0.05 to LPS CM treated group in SC siRNA condition. N = 3 to 4 per group. Statistical significance was analyzed with the unpaired Student's t-test between each treatment and its control, or with one-way ANOVA followed by Tukey-Kramer adjustments. *p<0.05. **p<0.01. ***p<0.001. Data are mean ± SEM.

Next, we administrated NMN or SRT1720 directly to RAW264.7 cells (stimulated with or without LPS), and subsequently, CM was collected and used to treat 661W cells. Neither NMN- nor SRT1720-treated RAW264.7 cells resulted in any protective effects on 661W cells, in contrast to those 661W cells that were directly treated with either molecules (Figure 6C, 6D). These results indicated that the protective effects of NMN and SRT2104 were probably acting directly on 661W cells, rather than by altering the toxicity of RAW264.7 CM. In order to see whether NMN administration has effect on the inflammatory cytokines, we treated RAW264.7 cells with LPS, in the presence or absence of NMN. The CM samples were collected, and the expression of neurotoxic cytokines TNF-α and IL-1β were assessed by ELISA. These results showed that LPS can induce significant increases in both TNF-α (Figure 6E) and IL-1β (Figure 6F). However, NMN administration at 1 mM didn’t significantly alter the levels of both cytokines.

Lastly, we used SIRT1 siRNAs to knockdown SIRT1 in 661W cells before treating them with RAW264.7 CM with or without LPS. Knocking down SIRT1 subjected 661W cells to a higher sensitivity to the neurotoxic effects of LPS stimulated CM from RAW264.7 cells, as can be seen by the significantly lower cell viability compared to the control group (Figure 6G).

Delayed NMN supplementation continues to exert protection to the retina after RD

To be more relevant to clinical settings, we administrated NMN at Day 3 post RD and evaluated the effects on the retina at Day 7 post RD (Figure 7A). Our results showed a preserved ONL thickness in comparison to the vehicle group (Figure 7B–7E), along with an inhibitory effect of infiltrating CD11b⁺ macrophages compared to the vehicle group (Figure 7C–7F). GFAP was unaltered (Figure 7D–7G). These results showed that delayed NMN administration can still exert protective effect after RD.

Figure 7. Protective effects of delayed NMN administration to retinal detachment (RD). (A) A flow chart for the in vivo experiments. (B–E) Delayed NMN administration still have protective effect to outer nuclear layer (ONL) thickness 7 days post RD. N = 3 to 4 eyes per group. Scale bar=50 μm. (C–F) Reduced number of CD11b⁺ infiltrating macrophages (red) in delayed NMN-treated groups compared to vehicle. N = 3 eyes per group. Scale bar: 100 μm. (D–G) No significance was found between NMN and vehicle treated retinas of the GFAP (green). N = 3 eyes per group. Scale bar: 50 μm. Statistical significance was analyzed with the unpaired Student's t-test. *p<0.05. **p<0.01. ***p<0.001. Data are mean ± SEM.

Author Contributions

X.C., J.A., D.G.V. and D.A.S. designed the experiments, X.C., G.A.M., J.L. Z.Y., and K.I. conducted the experiments, X.C., J.A., G.A.M., and J.L. performed data analysis, X.C. and J.A. wrote the manuscript. All authors contributed to the edits of the manuscript.

Acknowledgments

The authors would like to thank the help of the animal facility of Massachusetts Eye and Ear.

Conflicts of Interest

D. A. S. is a founder, equity owner, advisor, director, consultant, investor, and inventor on patents licensed to MetroBiotech, Liberty Biosecurity, Jumpstart Fertility, and Animal Biosciences, working on NAD-based therapeutics. D.A.S. is an inventor on a patent application filed by Harvard Medical School licensed to Elysium Health. https://genetics.med.harvard.edu/sinclair/people/sinclair-other.php. All other authors declare no competing interests.

Funding

This work was supported by the Oversea Study Program of Guangzhou Elite Project for scholarship (to X.C), the Yeatts Family Foundation (to D.G.V.); Monte J. Wallace (to D.G.V.); 2013 Macula Society Research Grant Award (to D.G.V.); a Physician Scientist Award (to D.G.V.); unrestricted grant from the Research to Prevent Blindness Foundation (to J.W.M. and D.G.V.); National Eye Institute (NEI) R21EY023079-01/A1 R01EY025362-01 (to D.G.V.); NEI Core Grant EY014104, P30EY003790 (Massachusetts Eye and Ear Infirmary Core Grant.); R01-DK- 100263-01A1 and R37AG028730- 08 (to D.A.S.); Loeffler Family Fund (to D.G.V.); ARI Young Investigator Award (to D.G.V.).

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