Loss of NAMPT in aging retinal pigment epithelium reduces NAD⁺ availability and promotes cellular senescence

NAD⁺ levels decline with increasing age in mouse RPE

NAD⁺ levels decline with age in many cell and tissue types [12,13]. Whether the same occurs in aging RPE is unclear. As such, our first step was to evaluate temporally the availability of NAD⁺ in the RPE of the living animal. To do so, we isolated RPE tissue from the eyes of C57BL/6J mice that per the established average lifespan of a normal lab mouse (~24 months) belonged to one of the following three age groups: young-adult (age 1.5 - 6 months), middle-aged adult (age 7 – 11 months) or adults of advanced age (12 months of age or greater). Levels of NAD⁺ in mouse RPE correlated inversely with age (Fig. 1A). NAD⁺ levels remained stable in this cellular layer up to six months of age but began to decline significantly shortly thereafter such that by 12 months of age, there was on average, a 40-50 percent decrease in RPE-specific NAD⁺ content. Further reductions in NAD⁺ were detected in RPE obtained from mice of advanced age.

Nicotinamide phosphoribosyltransferase (NAMPT) is essential to NAD⁺ and down-stream SIRT1 expression in RPE

To identify the specific mechanism(s) responsible for the age-dependent decline in levels of NAD⁺ that we observed in mouse RPE, we next evaluated the expression of enzymes pertinent to NAD⁺ biosynthesis. In mammals, NAD⁺ is generated via one of two principal routes, the de novo or the salvage pathway ([8,14]; Fig. 1B). Congruent with this, we monitored the expression of nicotinamide adenylyltransferase (NMNAT), quinolinate phosphoribosyltransferase (QPRT) and nicotinamide phosphoribosyltransferase (NAMPT), key enzymes relevant to one or both of the aforementioned major pathways to NAD⁺ generation. NMNAT and QPRT expression in RPE remained relatively stable across all ages tested despite the age-associated decline in NAD⁺ content (Figs. 1C and D, respectively). However, NAMPT expression (Fig. 1E) mirrored closely the age-dependent decline in NAD⁺, strongly implicating this enzyme as the principal enzyme driving NAD⁺ generation in RPE. This finding is consistent with findings in the existing scientific literature demonstrating that the bulk of NAD⁺ that is generated in mammals under physiological conditions is derived from the salvage pathway, also known as the deamidated route [14]. We evaluated also the expression of sirtuin family member 1 (SIRT1), a highly studied NAD⁺-dependent histone deacetylase touted as being a principal metabolic sensor and regulator of cellular energy metabolism and stress responses. Importantly, SIRT1 has also been linked to ocular aging and/or processes associated with the development and progression of age-related retinal disease [15]. Like NAD⁺ and NAMPT mRNA expression, levels of SIRT1 protein declined significantly in mouse RPE in association with increasing age (Fig. 1F). Further, some of the senescence-associated markers were increased in aged mouse RPE (Fig. S1).

FK866 inhibits NAMPT thereby decreasing NAD⁺ levels and promoting RPE senescence

Our data to this point confirm that NAD⁺ levels decline in RPE as age increases. They also demonstrate the critical importance of NAMPT to the generation and maintenance of NAD⁺ levels in RPE. Hence, therapies targeting NAMPT and NAD⁺ may be of benefit in preserving RPE health and viability in aging and age-related retinal diseases like AMD. However, the development and testing of new therapies in this field of research is impeded significantly by the lack of experimental model systems that recapitulate accurately characteristics of human aging and age-related disease and/or, the extended time periods often required to generate reliable models and conduct such studies. Thus, to further our present studies and evaluate the impact of reduced NAMPT and consequent NAD⁺ availability on RPE cell viability, we used the highly specific and non-competitive NAMPT-inhibitor FK866 [16] to mimic acutely the age-related effect of decreased NAMPT expression and NAD⁺ content in cultured RPE cells and intact mouse retina.

Confluent, well-differentiated cultures of human RPE (ARPE-19) cells were exposed to varying concentrations of FK866 for 72 h. As anticipated, FK866 induced a dose-dependent decline in levels of NAD⁺ (Fig. 2A). Cell viability assays confirmed the absence of robust cellular toxicity due to FK866 exposure across the range of doses of the compound that were tested (0.01 – 10 μM; Fig. 2B). There was a significant and dose-dependent increase in the number of RPE cells positive for the expression of β-galactosidase, an established marker of cellular senescence, in cells treated with FK866 (Fig. 2C-D), suggesting that while the compound did not induce robust cell death, it did on the other hand, stimulate cellular senescence. To confirm this, we evaluated also the mRNA and/or protein expression of cyclin-dependent kinase inhibitor 1 (p21^Waf/Cip1), cyclin-dependent kinase Inhibitor 2A (p16^INK4a), apolipoprotein J (ApoJ), and connective tissue growth factor (CTGF), key additional biomarkers of aging and cellular senescence [17–20]. Congruent with β-galactosidase staining, the expression of each of these markers was increased in association with increasing concentrations of FK866 and the associated decline in NAD⁺ (Fig. 3A-D). Additionally, the expression and activity of SIRT1 was dose-dependently decreased (Fig. 4A-B).

A common consequence of cellular senescence is the decreased ability of cells to handle the oxidative stress produced as a normal consequence of cellular metabolism [21]. This potentiates inflammatory processes and the further production of pro-oxidant byproducts. This vicious cycle of oxidative stress and inflammation has been identified as key factor in the development and progression of AMD and other degenerative retinal diseases. As such, we evaluated the effect of FK866 on the expression of interleukins 6 and 8 (IL-6 and IL-8, respectively), two pro-inflammatory cytokines that have been shown to be elevated commonly in degenerating RPE [17,22]. Levels of IL-6 and IL-8 were increased in association with increasing concentrations of FK866 in our experimental system (Fig. 4C-D).

Based upon our immediate prior findings, a dose of 10 µM FK866 was confirmed as the optimal standard dose. To confirm the optimal incubation period for FK866 treatment, additional cultures of ARPE-19 were treated with 10 µM FK866 for 24, 48 or 72 h and parameters identical to those described above (Figs. 2-4) were evaluated. FK866 was effective at reducing NAD⁺ levels at each of the time points tested, however the greatest efficacy was achieved in association with a 72 h incubation period (Fig. 5A), the time period that was used in our initial studies. Cell viability assays were repeated in these additional batches of cells and the absence of cellular toxicity in cells exposed to 10 µM FK866 at all-time points tested was confirmed (Fig. 5B). β-galactosidase staining and related quantification revealed a time-dependent increase in the number of β-gal-positive cells in FK866-treated cultures (Fig. 5C-D). Similarly, the expression of p21^Waf/Cip1, p16^INK4a, ApoJ and CTGF was increased (Fig. 6A-D) and SIRT1 expression and activity decreased (Fig. 7A-B) time-dependently, further supporting the optimality of the 72 h incubation period (Fig. 6A-D).

FK866 induces expression of inflammatory factors and ROS production in RPE

Senescent cells secret numerous kinds of pro-inflammatory cytokines, chemokines, growth factors and proteases, exemplifying what is referred to as a senescence-associated secretory phenotype (SASP) [23]. Further, oxidative stress also plays an important role in RPE senescence [24–26]. Congruent with this, we treated additional ARPE-19 cells with 10 µM FK866 for 72 h, the dose and time-point most effective at reducing NAD⁺ and promoting cellular senescence without robust toxicity, and monitored them for expression of the pro-inflammatory cytokines IL-6 and IL-8 (Fig. 7C-D), and CellROX and MitoSOX positivity, a general marker of oxidative stress and a mitochondrial-specific superoxide marker, respectively (Fig. 7E). Like IL-6 and IL-8 expression, the number of cells positively stained with CellROX or MitoSOX dyes (green fluorescence and red fluorescence, respectively) and therefore in which there was an increased amount of oxidative stress, was increased in ARPE-19 cells exposed to FK866 compared to control, non-exposed cell cultures (Fig. 7E).

Nicotinamide mononucleotide (NMN) treatment preserves NAD⁺ and prevents RPE senescence in vitro

Therapies that enhance NAD⁺ have been shown to reduce the incidence and/or severity of age-associated complications [27–31]. However, it is unclear, whether the latter is also of benefit in aged RPE and associated degenerative retinal disease. Our studies up to this point have established the decline in NAMPT and related NAD⁺ availability in aged RPE and validated the FK866-treated cell culture system as a suitable in vitro model system in which to study these factors. Therefore, we used this experimental model to determine whether augmenting NAD⁺ content using the NAD⁺ precursor nicotinamide mononucleotide (NMN) could preserve NAD⁺ and prevent FK866-induced RPE senescence. NMN dose-dependently enhanced NAD⁺ levels in cells exposed to FK866 (Fig. 8A). Additionally, the compound prevented FK866-induced RPE senescence as indicated by the suppression of p16^INK4a and p21^Waf/Cip1 expression and the reduction of SIRT1 expression (Fig. 8B). The number of β-galactosidase positive cells was also decreased in cultures exposed to NMN (Fig. 8C-D).

ARPE-19, though of human origin, is a “transformed” retinal cell line. Therefore, to validate findings obtained using ARPE-19 cell cultures, key experiments were repeated using cultures of primary mouse RPE (Fig. 9). FK866 was effective at reducing NAD⁺ expression in primary RPE though cell viability data indicate increased sensitivity in the form of cellular toxicity (Fig. 9A). Therefore, in subsequent studies using these cells, we refrained from using higher doses of the compound (Fig. 9B-G). As observed in ARPE-19 cells, exposure of RPE cells to FK866 potentiated cellular senescence (Fig. 9C), and these phenomena were inhibited by co-exposure of cells to NMN (Fig. 9D-F). Importantly, NMN also prevented the FK866-induced decrease in SIRT1 expression (Fig. 9G).

Nicotinamide mononucleotide (NMN)treatment preserves NAD⁺ and prevents RPE senescence in vivo

To determine whether our findings in the ARPE-19 and primary RPE cell culture model systems could be extrapolated to the living animal, we injected FK866 sub-retinally into the eyes and/or NMN intraperitoneally to adult C57BL/6J mice. NAD⁺ levels were significantly lower in RPE tissue isolated from mice injected with FK866 than in vehicle (PBS)-injected controls (Fig. 10A). On the other hand, NAD⁺ levels more than doubled in association with NMN injection only, and in animals that received NMN in conjunction with FK866, NAD⁺ levels were significantly preserved. SIRT1 expression was additionally preserved in conjunction with NMN injection (Fig. 10B).

Animals

All experiments involving animals adhered to the Public Health Service Policy on the Humane Care and Use of Laboratory Animals (2015 Department of Health, Education and Welfare publication, NIH 80-23), the Association for Research in Vision and Ophthalmology Statement for use of Animals in Ophthalmic and Vision Research and were approved by the Augusta University Institutional Animal Care and Use Committee. Male C57BL/6J mice of different age groups obtained from a commercial vendor (Jackson Laboratories, Bar Harbor, ME, USA) and the National Institute of Aging (National Institutes of Health, Bethesda, MD, USA) were housed under identical conditions in a pathogen-free environment with a 12:12 h light/dark cycle and free access to laboratory chow and water. Mice were acclimatized for at least 1 week before the experiments.

Cell culture

Human retinal pigment epithelial (ARPE-19) cells were cultured in Dulbecco’s modified Eagle medium DMEM/F12 medium (supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin) and maintained at 37 °C in a humidified chamber with 5% CO₂. The culture medium was replaced with fresh medium every other day. Cultures were passaged by dissociation in 0.05% (w/v) trypsin in phosphate-buffered saline (PBS; 0.01 M phosphate buffer, 0.0027 M KCl, 0.137 M NaCl, pH 7.4). Completely confluent, well-differentiated cultures were used for experimentation.

Primary mouse RPE cells were prepared from C57BL/6J mouse eyes and the purity of these cultures was confirmed per our published method [50–53]. Primary mouse RPE cells were then maintained at 37 °C in a humidified chamber with 5% CO₂ and sub-cultured using trypsin–EDTA solution. All experiments were carried out using primary RPE cells in passages 1 and 2.

Reverse transcription–quantitative polymerase chain reaction

Total RNA was isolated from RPE/eyecup of mice (2, 12 and 18 months old) or ARPE-19 cells using RNAeasy mini kit (Qiagen, USA). cDNA was prepared from total RNA using the iScript cDNA Synthesis Kit (Bio-Rad) and subjected to qPCR assay. Assays were performed in 96-well PCR plates using All-in-One™ qPCR Mix (Genecopia, USA). The reaction volume of 20 μl contained 10.0 μl SYBR green master mix (2X), 1 μl cDNA, 1 μl of each primer and 7 μl nuclease-free water. Primer sequences are listed in Table 1. The following two-step thermal cycling profile was used (StepOnePlus Real-Time PCR, Life Technologies, Grand Island, NY): Step I (cycling): 95 °C for 5 min, 95 °C for 15 s, 60 °C for 30 s and 72 °C for 15s for 40 cycles. Step II (melting curve): 60 °C for 15 s, 60 °C 1 min and 95 °C for 30 s. The template amplification was confirmed by melting curve analysis. mRNA expression of genes were normalized to 18s expression and fold change in expression was calculated by 2^–∆∆Ct method.

FK866 and Nicotinamide mononucleotide (NMN) treatments

ARPE-19 cells were serum starved overnight and treated with varying doses (0.01-10μM) of FK866 (Sigma-Aldrich) for different time intervals (24, 48 or 72 h). Following FK866 treatment, cells were collected for one or more of the following analyses: MTT assay, NAD⁺ measurement, SIRT1 activity assay, RNA or protein studies. Primary mouse RPE cells were treated and analyzed similarly except that lower doses of FK866 (0.01, 0.1 and 0.5 μM) were employed. The above experiments were used to establish the optimal dose and time for use of FK866 to adequately inhibit NAMPT without inducing robust cellular toxicity in ARPE-19 and primary RPE cells. Congruent with these studies, ARPE-19 and primary mouse RPE cell cultures were exposed to FK866 (10 μM) for 72 h in the presence or absence of varying concentrations of the NAD⁺ precursor, nicotinamide mononucleotide (NMN; 0.05 – 1.0 mM).

For in vivo studies, adult male C57BL/6J mice were deeply anesthetized via a single intraperitoneal injection of ketamine (80 mg/kg) and xylazine (12 mg/kg; Sigma-Aldrich, St. Louis, MO, USA). Upon confirmation of anesthetic depth via toe pinch, FK866 (10μM) was delivered sub-retinally in a total volume of 2 μL using a 33-gauge Hamilton Syringe (Hamilton, Reno, NV, USA) at day 0 and again at day 7. Sham control eyes (left eyes) were injected in the same manner except with 2 μL of 0.01M PBS. A group of FK866 injected mice were treated with 150 mg/kg NMN (i.p.) once daily for 2 weeks [36]. Mice were euthanized one-week post delivery of the second FK866 injection and RPE tissue harvested for measurement of NAD⁺ and protein analyses.

Western blot analysis

Protein was extracted from RPE/eyecup of mice (2, 12 and 18 months old) or ARPE-19 cells or primary RPE cells using RIPA cell lysis buffer (Thermo Scientific) containing protease and phosphatase inhibitors and concentration was determined using the coomassie protein assay reagent (Sigma-Aldrich, USA). Equivalent amount of protein samples were subjected to SDS–PAGE, transferred to PVDF membranes, and then incubated with primary antibodies: SIRT-1, p21^Waf/Cip1 (1:1000; Cell signaling) and p16^INK4a (1:500; Abcam) overnight at 4°C. Next day, blots were washed with TBST and incubated with horseradish peroxidase conjugated secondary antibody (1:3000; Sigma-Aldrich, USA) for 60 min with gentle shaking at room temperature. Blots were then washed (with TBST) and developed with chemiluminescence reagent (Bio-Rad, Hercules, CA) using autoradiography films (Genesee Scientific, San Diego, CA). β-actin (1:3000; Sigma-Aldrich, USA) expression was evaluated to determine equivalent loading. Scanned images of blots were used to quantify protein expression using NIH ImageJ software (http://rsb.info.nih.gov/ij/).

NAD⁺ assay

NAD⁺ levels in cells and tissue were quantified with a commercially available kit (Sigma, MO, United States) according to the manufacturer's instructions.

SIRT1 activity assay

The SIRT-1 deacetylase assay in ARPE-19 cells was performed using a fluorometric SIRT1 assay kit (Sigma-Aldrich, USA), according to the manufacturer’s protocol. Briefly, the reaction was carried out at 37°C for 30 min and deacetylase activity was detected as a fluorescence emission at 450 nm, using an excitation wavelength of 360 nm. The intensity of the fluorescence emitted by the compounds at 450 nm was subtracted from initially determined assay values.

β-galactosidase assay

Senescence-associated beta-galactosidase (β-Gal) staining was performed according to the manufacturer's protocols (Cell Signaling Technology). At the end of treatments, cells were fixed for 15 minutes in 1x fixative solution (formaldehyde-glutaraldehyde mix), followed by PBS washes, and incubated overnight with β-Gal staining solution in a dry incubator at 37°C. Cells were viewed under brightfield microscopy (Leica BM5500B Microscope; Leica Biosystems, Wetzlar, Germany) for blue color development and were photographed using a Photometrics CoolSNAP HQ² camera and associated Leica Application Suite software v4.1.

CellROX and MitoSOX staining

ARPE-19 cells were treated with 10 μM FK866 for 72 hr. as mentioned above. At the end of treatments, cells were incubated for 5 μM CellROX for 30 min or 5 μM MitoSOX for 10 min. At the end of incubation, cells were washed with PBS, mounted using fluoroshield mounting medium with DAPI and images were captured using Zeiss Axioplan-2 imaging florescence microscope.

Statistical analysis

Results are presented as mean ± S.E.M for minimum of three independent experiments. Statistical significance was defined as p < 0.05 and determined using student’s t-test (normally-distributed data). Graphs were prepared using GraphPad Prism 7 software.