Administration of nicotinamide mononucleotide in the treatment of disease
11793824 · 2023-10-24
Assignee
Inventors
Cpc classification
A61K9/0053
HUMAN NECESSITIES
A61K31/706
HUMAN NECESSITIES
A61K31/7064
HUMAN NECESSITIES
A61K9/0014
HUMAN NECESSITIES
International classification
A61K31/455
HUMAN NECESSITIES
A61K31/706
HUMAN NECESSITIES
Abstract
Disclosed are methods and compositions related to methods of treating, ameliorating, mitigating, slowing, arresting, preventing or reversing various diseases and conditions, including age-related obesity, age-related increases in blood lipid levels, age-related decreases in insulin sensitivity, age-related decreases in memory function, and age-related changes in eye function such as macular degeneration. The methods comprise administering nicotinamide mononucleotide (NMN) to a subject. In some embodiments, the administration can be oral administration. Also disclosed are pharmaceutical compositions comprising NMN.
Claims
1. A method of treating age-associated impairment of memory function in the brain of a subject in need of treatment thereof, comprising administering to the subject in need of treatment an amount of nicotinamide mononucleotide (NMN) effective for the treatment of age-associated impairment of memory function in the brain of said subject.
2. A method in accordance with claim 1, wherein the age-associated impairment of memory function is hippocampus-dependent memory function.
3. A method in accordance with claim 1, wherein the NMN is administered at a dosage of 0.5 mg, about 0.5 mg, 1 mg, about 1 mg, 5 mg, about 5 mg, 10 mg, about 10 mg, 20 mg, about 20 mg, 30 mg, about 30 mg, 40 mg, about 40 mg, 50 mg, about 50 mg, 60 mg, about 60 mg, 70 mg, about 70 mg, 80 mg, about 80 mg, 90 mg, about 90 mg, 100 mg, about 100 mg, 150 mg, about 150 mg, 200 mg, about 200 mg, 250 mg, about 250 mg, 300 mg, about 300 mg, 400 mg, about 400 mg, 450 mg, about 450 mg, 500 mg, about 500 mg, 600 mg, about 600 mg, 700 mg, about 700 mg, 800 mg, about 800 mg, 900 mg, about 900 mg, 1000 mg, about 1000 mg, 1100 mg, about 1100 mg, 1200 mg, about 1200 mg, 1300 mg, about 1300 mg, 1400 mg, about 1400 mg, 1500 mg, about 1500 mg, 1600 mg, about 1600 mg, 1700 mg, about 1700 mg, 1800 mg, about 1800 mg, 1900 mg, about 1900 mg, 2000 mg, about 2000 mg, 2100 mg, about 2100 mg, 2200 mg, about 2200 mg, 2300 mg, about 2300 mg, 2400 mg, about 2400 mg, 2500 mg, about 2500 mg, 2600 mg, about 2600 mg, 2700 mg, about 2700 mg, 2800 mg, about 2800 mg, 2900 mg, about 2900 mg, 3000 mg, about 3000 mg, 3500 mg, about 3500 mg, 4000 mg, about 4000 mg, 4500 mg, about 4500 mg, 5000 mg, about 5000 mg, 5500 mg, about 5500 mg, 6000 mg, about 6000 mg, 6500 mg, about 6500 mg, 6800 mg, about 6800 mg, or from 0.5 mg per day to 6800 mg per day, and at a rate of once per day, twice per day, three times per day or four times per day.
4. A method in accordance with claim 1, wherein the NMN is administered orally in a formulation selected from the group consisting of a pill, a tablet, a caplet, a capsule, a chewable tablet, a quick dissolve tablet, a powder, a granule, an effervescent tablet, a hard gelatin capsule, a soft gelatin capsule, a non-aqueous liquid, an aqueous liquid, a suspension, a solution, an emulsion, a syrup, a sterilized aqueous suspension, a sterilized aqueous solution, a non-aqueous suspension, a non-aqueous solution, and a lyophilized formulation.
5. A method in accordance with any one of claims 1 to 4, wherein the NMN is administered at a dosage of 200 mg, about 200 mg, 250 mg, about 250 mg, 300 mg, about 300 mg, 400 mg, about 400 mg, 450 mg, about 450 mg, 500 mg, about 500 mg, 600 mg, about 600 mg, 700 mg, about 700 mg, 800 mg, about 800 mg, 900 mg, about 900 mg, 1000 mg, about 1000 mg, 1100 mg, about 1100 mg, 1200 mg, about 1200 mg, 1300 mg, about 1300 mg, 1400 mg, about 1400 mg, 1500 mg, or about 1500 mg.
6. A method in accordance with any one of claims 1 to 4, wherein the NMN is administered at a rate of once per day, or twice per day.
7. A method in accordance with any one of claims 1 to 4, wherein the NMN is administered orally in a formulation selected from the group consisting of a pill, a tablet, a caplet, a capsule, a chewable tablet, a quick dissolve tablet, a powder, granules, an effervescent tablet, a hard gelatin capsule, and a soft gelatin capsule.
8. A method in accordance with any one of claims 1 to 4, wherein the NMN is administered sublingually.
9. A method in accordance with any one of claims 1 to 4, wherein the subject is a human.
10. A method in accordance with any one of claims 1 to 4, wherein the NMN is administered to cross the blood-brain barrier.
11. A method in accordance with claim 5, wherein the NMN is administered orally in a formulation selected from the group consisting of a pill, a tablet, a caplet, a capsule, a chewable tablet, a quick dissolve tablet, a powder, granules, an effervescent tablet, a hard gelatin capsule, and a soft gelatin capsule, and wherein the subject is a human.
12. A method in accordance with claim 5, wherein the NMN is administered at a rate of once per day.
13. A method in accordance with claim 5, wherein the NMN is administered at a rate of twice per day.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
Abbreviations
(36) BMD: Bone mineral density CC: Corpus callosum DG: Dentate gyms DXA: Dual-energy X-ray absorptiometry EIR: Enhanced immediate release ERG: Electroretinography FFA: Free fatty acid HFD: High fat diet NMN: Nicotinamide mononucleotide OPC: Oligodendrocyte precursor cells PR: Photoreceptor SGZ: Subgranular zone SVZ: Subventricular zone
Methods
(37) The methods and compositions described herein utilize laboratory techniques well known to skilled artisans, and can be found in laboratory manuals such as Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; Spector, D. L. et al., Cells: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998; Nagy, A., Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition), Cold Spring Harbor, NY, 2003 and Harlow, E., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. Methods of administration of pharmaceuticals and dosage regimes, can be determined according to standard principles of pharmacology well known skilled artisans, using methods provided by standard reference texts such as Remington: the Science and Practice of Pharmacy (Alfonso R. Gennaro ed. 19th ed. 1995); Hardman, J. G., et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, 1996; and Rowe, R. C., et al., Handbook of Pharmaceutical Excipients, Fourth Edition, Pharmaceutical Press, 2003. As used in the present description and the appended claims, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context indicates otherwise.
(38) The Following Methods are Applicable to Examples 1-7
(39) Administration of NMN Through Drinking Water and Determination of NMN Stability and Toxicity
(40) A 12 month-long NMN administration study using wild-type mice under a regular chow-fed condition was conducted. NMN was administered through drinking water, and two doses of NMN, 100 and 300 mg/kg body weight/day, were tested. The stability of NMN was examined in drinking water and found that NMN was stable in solution. No significant degradation was observed at room temperature. Water intake was also monitored very carefully, and the water intake did not change significantly through the experimental period. To assess beneficial and possible adverse effects of NMN, a variety of physiological parameters were periodically monitored, including body weight, body temperature, food and water intake, fed and fasted blood glucose levels, fed and fasted plasma lipid panels, and glucose and insulin tolerance, in NMN-administered and control mice. Blood chemistry, blood cell counts, urine strip test, and other physiological tests including physical activity test were also checked. Based on all these assessments, no adverse effects, such as malnutrition, or signs of toxicity were observed in either of the 100 mg/kg or 300 mg/kg groups.
(41) Memory Function Study
(42) Two groups of wild-type C57BL/6 mice at −2 months of age were fed a high fat diet (HFD) containing 42% of the total calories from fat (TD88137; Harlan Taklad). NMN at a dose of 300 mg/kg/day began to be administered through drinking water to one of the HFD-fed groups after 4 months of HFD feeding. The control group was fed a regular chow. After 8 months of HFD feeding with or without 4 months of NMN treatment, the contextual fear conditioning test, a sensitive test to examine the memory function that involves the hippocampus, was conducted for mice in these three groups.
(43) The Following Methods are Applicable to Examples 8-15
(44) Mice
(45) Mice were maintained on a regular chow ad libitum on a 12 hr light/dark cycle (lights on from 6 am to 6 pm). Namptflox/flox mice (Rongvaux et al, 2008), in which exons 5 and 6 of the Nampt gene are flanked by loxP sites, were crossed to Nestin-CreERT2 mice (Lagace et al, 2007) to generate Nampt flox/+; Cre double heterozygous mice. Double heterozygous mice were bred to Namptflox/flox mice to obtain Namptflox/flox; Cre mutant mice (iNSPC-Nampt-KO mice) in the expected Mendelian ratio. To trace the progeny of adult NSPCs and to confirm the specificity and magnitude of the recombination induced by tamoxifen injection, iNSPC-Nampt-KO and Nestin-CreERT2 mice were crossed to a reporter mouse strain that expresses a loxP-flanked STOP cassette that prevents transcription of the downstream enhanced green fluorescent protein [ZsGreen1; Jackson laboratories #7906 (Madisen et al, 2010)]. Recombination PCR on hippocampal extracts of tamoxifen or vehicle treated mice showed successful deletion upon treatment with tamoxifen (
(46) Induction of Nampt Deletion
(47) Tamoxifen injections were performed as described previously (Lagace et al, 2007). Briefly, iNSPC-Nampt-KO mice (5-7 weeks old) were administered tamoxifen (TAM, Sigma T5648) at 180 mg/kg/d for 5 days (d, intraperitoneally; dissolved in 10% EtOH/90% sunflower oil), a protocol that produces maximal recombination with minimal lethality (5%) (Lagace et al, 2007).
(48) BrdU Incorporation
(49) 5′-bromodeoxyuridine (BrdU, Sigma, B9285) was diluted in sterile saline and administered by intraperitoneal injections (100 mg/kg body weight). For analysis of the cumulative effects of loss of Nampt, mice were given BrdU twice a day for 2 days and sacrificed the following day or 28 days later. For analysis of the effect of loss of Nampt on adult NSC differentiation and postnatal oligodendrocyte differentiation, mice were given BrdU twice a day for 1 day and sacrificed 2 days later.
Cuprizone
(50) Demyelination was induced by feeding 6 to 8-week-old mice a diet containing 0.2% cuprizone (bis-cyclohexanone oxaldihydrazone; Sigma C9012) mixed into a ground standard rodent chow for 4 to 5 weeks (Harlan Laboratories, TD.01453). To allow recovery from cuprizone treatment, food was replaced with standard chow for an additional 1 week. This protocol has been shown to successfully demyelinate and remyelinate the hippocampus (Skripuletz et al, 2011).
(51) Immunofluorescence
(52) All tissue sections were and cells incubated in blocking/permeabilization solution containing 10% normal goat serum, 1% BSA, and 0.3% Triton-X in PBS for 45 to 60 min prior to 24 or 48 h of incubation with primary antibodies in 5% normal goat serum and 0.1% Triton-X in PBS at 4° C. at the concentrations listed below. Alexa627, Alexa488, or Cy3 conjugated-secondary antibodies diluted in 2% normal goat serum, 1% BSA, and 0.1% Triton-X in PBS were added for 2 h at room temperature. Nuclei were stained with 4,6-diamidino-2-phenylindole (Sigma) for 10 min at room temperature.
(53) Cells were harvested by fixation with 4% paraformaldehyde in PBS (15 min). Mice were anesthetized by i.p. injection of ketamine and xylazine, and perfused transcardially through left ventricle with cold 0.1 M phosphate buffer at pH 7.4 followed by a phosphate-buffered solution of 4% paraformaldehyde (PFA). Brains were postfixed with 4% PFA overnight and placed into 15% sucrose followed by 30% sucrose, frozen, and stored at −80° C. until use. Coronal sections (30 μm) were made by cryostat in a 1 in 8 series and stored at −30° C. in cryoprotectant until use. To remove any endogenous peroxidase activity, all sections were incubated with 3% H.sub.2O.sub.2 for 10 min. Tissue sections used to assess BrdU incorporation were treated before the immunostaining procedure with 50% formamide in 2× saline/sodium citrate (SSC) at 65° C. for 2 h, 2N HCl for 30 min at 37° C., 0.1 M borate pH 8.5, and then washed twice with PBS before proceeding with the staining protocol. Tissue sections not used to assess BrdU incorporation were either incubated in 50% formamide in 2× saline/sodium citrate (SSC) at 65° C. for 2 h or 10 mM citrate buffer at 65° C. for 1 h before proceeding with the staining protocol. Detection of Dcx, Nestin, Nampt, and APC was performed using the TSA-Plus fluorescein kit (PerkinElmer).
(54) Quantification
(55) For tissue sections, high-magnification (20×, 0.8DICII or 40× oil 1.3DICII) microscopic imaging was performed using a Zeiss Axioimage.Z1. Images were taken in z-stacks of 1 μm steps through the range of tissue section immunoreactivity. For the dorsolateral corner of the SVZ, images were taken from bregma 1.10 to −0.10 mm. For the corpus callosum, images were taken from bregma −1.06 to −2.54 mm. For the dentate gyrus, images were taken from bregma −1.34 to −3.64 mm. Quantification was performed blinded to genotype on 3-8 tissue sections per animal. Cell densities were estimated by the number of immunoreactive cells divided by the area of the structure, measured with ImageJ. Verification of colocalization was achieved by importing stacks of Z images into ImageJ and performing 3D rendering. For cells, 10 or 20× microscopic imaging was performed using a Zeiss Axioimage.Z1. Quantification was performed blinded to genotype on 2 to 3 fields of view per sample and treatment, from 3 to 9 independent samples.
(56) NAD+ Measurement
(57) NAD+ levels were determined using an HPLC system (Shimadzu) with a Supelco LC-18-T column (15 cm×4.6 cm; Sigma), as described previously (Yoshino et al, 2011).
(58) Microarrays and Bioinformatic Analyses
(59) For individual genes, raw microarray data were subjected to Z score transformation, and Z ratios were calculated as described previously (Cheadle et al, 2003). Subsequent analysis and Parametric Analysis of Gene Set Enrichment (PAGE) analysis was performed as previously described (Yoshino et al, 2011). The microarray data used in this study has been deposited into the NCBI GEO database (GEO accession number GSE49784).
(60) Western Blotting
(61) Protein extracts (15-50 μg) from mouse hippocampi or neurospheres were prepared as previously described (Yoshino et al, 2011).
(62) Quantitative Real-Time RT-PCR
(63) Total RNA was extracted from the hippocampus using an RNeasy kit (Qiagen) and reverse-transcribed into cDNA with a High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Quantitative real-time RT-PCR was conducted with the TaqMan Fast Universal PCR Master mix and appropriate TaqMan primers for each gene with the GeneAmp 7500 fast sequence detection system (Applied Biosystems). Relative expression levels were calculated for each gene by normalizing to Gapdh levels and then to a control.
(64) Reagents
(65) The following primary and secondary antibodies were used:
(66) Primary antibodies and their uses or cell type specificities (See von Bohlen and Halbach, 2011): Actin: normalization, WB 1:4000 CPO1, Sigma; Gapdh: normalization, WB 1:4000 6C5 Millipore CB1001; Nampt: IHC 1:1000; WB 1:3000 Alexis Biochemicals ALX-804-717-C100; Pdgfrα: oligodendrocyte precursor cells, IF 1:500 APAS BD Biosciences; Olig2: all oligodendrocyte lineage cells, IHC 1:500, IF 1:1000; Millipore; O4: immature oligodendrocytes, IF 1:1000 Millipore, MAB345; APC: oligodendrocytes, IHC 1:1000 Millipore CC-1 OP80; MBP: mature oligodendrocytes, IHC 1:1000 Millipore MAB386; Ki67: proliferating cells, IHC, IF 1:500; WB 1:3000 Abcam ab66155; Pcna: proliferating cells, WB 1:2000; PC10 Cell signaling #2586; 5-bromo-2′-deoxyuridine (BrdU): a thymine analog that incorporates into the DNA of cells in S phase, IHC 1:500; OBT0030 Accurate; Activated caspase 3: apoptosis, IHC, IF 1:500; Cell Signaling #9661; LC3B: autophagy, WB 1:1000; Novus NB600-1384; TUNEL: cell death, Roche In Situ Cell Death Detection Kit 11 684 795 910; Dcx: newly born neurons, IHC 1:1000; Cell Signaling #4604; NeuN: mature neurons, IHC, 1:500, Millipore, MAB377; Nestin: NSPCs, IHC, IF 1:1000, Millipore MAB353; Sox2: NSPCs, IHC, IF 1:500; WB 1:2000; Millipore AB5603; Gfap: NSPCs and astrocytes, IHC, IF 1:1000; Millipore MAB360;
(67) Secondary antibodies: Jackson ImmunoResearch anti-rat, anti-rabbit, anti-mouse Cy3 (1:400), Alexa Fluor488 (1:200), and Alexa Fluor647 (1:200). Anti-rabbit, anti-mouse horseradish peroxidase (Invitrogen).
(68) FK866 (Hasmann & Schemainda, 2003) (Sigma F8557), EX527 (Peck et al, 2010) (Cayman Chemical 10009798), and AGK2 (Outeiro et al, 2007) (Sigma A8231) were dissolved in DMSO and used to inhibit Nampt, Sirt1, and Sirt2 respectively.
(69) Neurosphere Culture
(70) Neurosphere cultures and culture media were prepared as described by Dasgupta & Gutmann, 2005 and Lu & Ramanan, 2012 with the following minor modifications. Briefly, postnatal hippocampi were dissected in HibernateA (Invitrogen, A12475-01) and trypsinized at 37° C. for 7 m. Cells were mechanically dissociated by pipetting and pelleted by centrifugation (1700 rpm, 7 min). Dissociation medium (0.1% sodium bicarbonate, 15 mM HEPES, 0.5% glucose in HBSS) was used to wash the cells before they were resuspended in growth medium. Growth medium consisted of DMEM:F12 (1:1, Invitrogen 11966-025 and 21700-075, respectively), B27 (Invitrogen, 17504-044), N2 (Invitrogen, 17502-048), Pen/Strep (Invitrogen), epidermal growth factor (EGF, 20 ng/ml, Sigma, E4127), fibroblast growth factor (FGF, 10 ng/mL, R&D Systems, 233-fb), and heparin (Sigma). Cultures were maintained at 37° C. with 5% CO2, and passaged twice before use in experiments. Three to nine independent samples, each in 1 to 3 replicates, from at least two different litters, were used in all experiments. Neurospheres were cultured in the physiological glucose level of 5 mM (Dienel & Cruz, 2006), which has been previously shown to have no negative consequences on NSPC proliferation, differentiation, or death (Fu et al, 2006; Gao & Gao, 2007).
(71) Neurosphere Infection
(72) Neurospheres derived from Nampt.sup.flox/flox mice were infected with Ad5 Cre recombinase- or b-galactosidase-expressing (LacZ, control) adenoviruses at an MOI of 100. All assessments were performed at least 6 days post infection.
(73) Neurosphere Proliferation Analysis
(74) Neurospheres derived from Nampt.sup.flox/flox mice were dissociated by trypsin digestion and seeded at similar cell densities in 24-well plates with fresh growth medium. Every 24 hours, neurospheres from triplicate wells were collected, dissociated, and counted on a hemocytometer using 0.2% trypan blue exclusion to distinguish viable cells. For analysis of neurosphere diameter, the largest neurosphere in each well was imaged (20× objective) and the diameter was calculated using ImageJ. For secondary neurosphere analysis, the total number of neurospheres in each well was counted at 7 days post-plating.
(75) Neurosphere Differentiation
(76) Three to five days after their first passage, neurospheres were trypsinized, washed with dissociation medium, and plated at 150,000 cells per well in 24-well plates in differentiation medium [growth medium without FGF and EGF and with BDNF (5 ng/mL, Peprotech, 450-02) on glass coverslips coated with poly-D-lysine (50 ug/mL; Sigma) and laminin (20 ug/mL; BD Biosciences)]. 6-well plates were coated with poly-D-lysine (20 ug/mL) and laminin (10 ug/mL). To enrich for oligodendrocytes, PDGFαα (10 ng/ml, Peprotech 100-13A) was added to neurospheres at passage 2 and PDGFαα (2.5 ng/ml) and 3,3-,5-triiodo-L-thyronine (T3, 40 ng/ml, Sigma T4397) were added to differentiation medium. The percentage of oligodendrocyte precursor cells (OPCs) generated was analyzed after 2 d of differentiation, and the percentage of differentiated oligodendrocytes was analyzed after 6-7 d of differentiation.
(77) Statistical Analyses
(78) Differences between two groups were assessed using the Student's unpaired t-test. Comparisons among several groups were performed using one-way ANOVA with the Tukey-Kramer post hoc test except for
(79) The Following Methods are Applicable to Example 15
(80) Administration of NMN Through Drinking Water and Determination of NMN Stability and Toxicity
(81) A 12 month-long NMN administration study using wild-type mice under a regular chow-fed condition was conducted. NMN was administered through drinking water, and two doses of NMN, 100 and 300 mg/kg body weight/day, were tested. The stability of NMN was examined in drinking water and found that NMN was stable in solution. No significant degradation was observed at room temperature. Water intake was also monitored very carefully, and the water intake did not change significantly through the experimental period. To assess beneficial and possible adverse effects of NMN, a variety of physiological parameters were periodically monitored, including body weight, body temperature, food and water intake, fed and fasted blood glucose levels, fed and fasted plasma lipid panels, and glucose and insulin tolerance, in NMN-administered and control mice. Blood chemistry, blood cell counts, urine strip test, and other physiological tests including physical activity test were also checked. Based on all these assessments, no adverse effects, such as malnutrition, or signs of toxicity were observed in either of the 100 mg/kg or 300 mg/kg groups.
EXAMPLES
(82) The present teachings including descriptions provided in the Examples that are not intended to limit the scope of any claim or aspect. Unless specifically presented in the past tense, an example can be a prophetic or an actual example. The following non-limiting examples are provided to further illustrate the present teachings. Those of skill in the art, in light of the present disclosure, will appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present teachings.
Example 1
(83) This example illustrates a suppressive effect of NMN on age-associated body weight increase.
(84) In these experiments, NMN was administered to mice at a dosage rate of 100 mg/kg per day or 300 mg/kg per day. NMN demonstrated a suppressive effect on age-associated body weight increase in a 12 month-long NMN administration study. (See Methods; Administration of NMN through Drinking Water and Determination of NMN Stability and Toxicity) In these experiments, NMN demonstrated a suppressive effect on age-associated body weight increase (
(85) Average body weights in each group are shown through 0-12 months. There was a statistically highly significant interaction between time and group (P<0.001 from the two-way RANOVA), and linear dose-dependent effects were statistically significant at all time points through 4-12 months (P<0.05 from one-way RANOVA with the unweighted linear term). The average percent body weight reduction normalized to control mice were 4% and 9% in 100 and 300 mg/kg groups, respectively.
(86) This suppressive effect of NMN on age-associated body weight increase was further recognized by calculating body weight gains in each group (
(87) Taken together, these results from this 12 month-long NMN administration study demonstrate that NMN can suppress age-associated body weight increase in a dose-dependent manner, without showing any serious side effects during the entire experimental period. These results demonstrate that NMN can be used for the treatment, reduction or prevention of age-associated obesity.
Example 2
(88) This example illustrates an enhancement of energy metabolism over age with NMN administration.
(89) In this long-term NMN administration study (See Methods; Administration of NMN through Drinking Water and Determination of NMN Stability and Toxicity), the inventors measured oxygen consumption, energy expenditure, and respiratory quotient for control, mice administered 100 mg/kg NMN per day and mice administered 300 mg/kg NMN per day at the 12 month time point by using the Oxymax Lab Animal Monitoring System (Columbus Instruments, Columbus, OH).
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(91) Energy expenditure measurements also showed significant increases in both 100 mg/kg and 300 mg/kg groups through 24 hours (P<0.001, Wilcoxon signed ranks test) (
(92) Respiratory quotient in control, 100 and 300 mg/kg NMN-administered mice are presented in
Example 3
(93) This example illustrates a suppressive effect of NMN on age-associated increases in blood lipid levels.
(94) Blood levels of cholesterol, triglycerides, and free fatty acids are shown over 12 months in the control and the 100 and 300 mg/kg NMN-administered cohorts in
(95) Blood levels of cholesterol, triglycerides, and free fatty acids are shown over 12 months in the control and the 100 and 300 mg/kg NMN-administered cohorts. The results were analyzed with two-way RANOVA and one-way RANOVA. All values are presented as mean±SEM (n=25 for each group).
(96) In the control cohort of a long-term NMN study (See Methods; Administration of NMN through Drinking Water and Determination of NMN Stability and Toxicity), blood levels of cholesterol showed steady increases over time, whereas blood levels of triglycerides and free fatty acids (FFAs) peaked at the 6-month time point and then decreased. However, in both 100 and 300 mg/kg groups, these age-associated increases in cholesterol and free fatty acids tended to be suppressed (
(97) Without being limited by theory, since NMN has an effect of suppressing age-associated body weight increase, it was hypothesized that NMN's effect on blood lipid levels could be due to the reduction in body weight. To address this possibility, lipid levels were compared among individual mice whose average body weights were matched through control and experimental cohorts. Body weight-matched blood levels of cholesterol, triglycerides, and free fatty acids are shown over 12 months in the control and the 100 and 300 mg/kg NMN-administered cohorts in
(98) It has been reported that chronic treatment with nicotinic acid tends to increase blood FFA levels, whereas it lowers total cholesterol and triglyceride levels (Wang, W., et al. Am. J. Phyisol. Endocrinol. Metab. 279: E50-E59, 2000). As shown herein, NMN has a capability of suppressing the age-associated increase in blood FFA levels, which distinguishes NMN from nicotinic acid. NMN is also able to reduce cholesterol levels through the suppression of age-associated body weight increase. Whereas chronic administration of nicotinic acid can cause skeletal muscle insulin resistance (Fraterrigo, G., et al. Cardiorenal. Med. 2: 211-7, 2012), NMN does not show any adverse effect on glucose metabolism. Therefore, NMN administration can be an effective intervention to suppress age-associated increases in blood lipid levels.
Example 4
(99) This example illustrates that administration of NMN enhances insulin sensitivity in old individuals.
(100) In these investigations, insulin sensitivity, assessed by the insulin tolerance test, showed significant differences among the control and the 100 mg/kg and 300 mg/kg NMN-administered groups after the 12-month time point. (See Methods; Administration of NMN through Drinking Water and Determination of NMN Stability and Toxicity). As illustrated in
(101) In these experiments, the last measured time point when mice reached 17 month-old, the NMN-administered, body weight-matched mice showed significantly enhanced insulin sensitivity compared to the body weight-matched control group (
Example 5
(102) This example illustrates improvement of memory function under a high-fat diet (HFD) by administration of NMN. (See Methods; Memory Function Study)
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Example 6
(104) This example illustrates the improvement of retinal photoreceptor cell function over age.
(105) In the long-term NMN administration study (See Methods; Administration of NMN through Drinking Water and Determination of NMN Stability and Toxicity), retinal function was evaluated by fundus biomicroscopy and electroretinography (ERG).
(106) Intraretinal whitish deposits were reduced dramatically in NMN-administered mice. On fundus biomicroscopy, all five control mice at 18 months of age showed many intraretinal whitish deposits, whereas two and four each out of five mice at 100 mg/kg and 300 mg/kg doses, respectively, showed dramatic reductions in these deposits, suggesting that age-associated pathological changes in the retina are suppressed by NMN (
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(108) Although there were no significant interactions between stimulus and group for the scotopic b and photopic b waves, there appeared to be a trend of improvement for the photopic b wave, which represents cone cell function, through an entire range of stimulus in both 100 and 300 mg/kg groups (
(109) The present inventors assessed the physiological importance of NAMPT-mediated NAD biosynthesis in the retina by generating rod cell- and cone cell-specific NAMPT knockout mice. On fundus biomicroscopy, cone cell-specific NAMPT knockout mice had an atrophic appearance at the optic nerve head, intraretinal whitish deposits, and perivascular sheathing, while littermate control animals were normal. ERG demonstrated a significant and dramatic decrease in the scotopic b and photopic b wave amplitudes as compared to the littermate control mice. The scotopic a wave amplitudes in the cone cell-specific NAMPT knockout mice were significantly decreased but to a lesser extent than the photopic b wave responses. Furthermore, rod cell-specific NAMPT knockout mice exhibited total retinal degeneration. Both a and b wave ERG responses were completely depressed as characterized by a total lack of response to all stimuli. Additionally, the treatment of the mouse photoreceptor-derived 661W cone cell line with FK866, a potent NAMPT inhibitor, led them to apoptotic cell death. Adding NMN to the culture media successfully rescued 661W cells from FK866-mediated cell death, suggesting that NAD deficiency causes the observed cell death. These results indicate that inhibition of NAMPT-mediated NAD biosynthesis by genetic and pharmacologic means leads to photoreceptor cell death and eventually retinal degeneration. NMN administration is an effective intervention to treat/prevent retinal degeneration.
Example 7
(110) This example illustrates the improvement of tear production over age using modified Schirmer testing.
(111) In this long-term NMN administration study (See Methods; Administration of NMN through Drinking Water and Determination of NMN Stability and Toxicity), tear production was assessed in control and NMN-administered mice with modified Schirmer's test. All values are presented as mean±SEM. **p<0.01. NMN increased tear production in a dose-dependent manner in 18 month-old mice (
Example 8
(112) This example illustrates that hippocampal NAD+ levels and Nampt expression decline with age.
(113) The inventors hypothesized that aging may reduce Nampt-mediated NAD+ biosynthesis in the brain, particularly in the hippocampus, affecting the function of NSPCs. The inventors first measured NAD+ levels in hippocampi isolated from 1, 3-4, 6, and 10-12 month-old C57B16 mice.
(114) NAD+ levels gradually decreased with age, reaching 63% in 10-12 month-old mice compared to that of 1 month-old mice (
Example 9
(115) This example illustrates that Nampt is expressed in a subpopulation of SGZ NSPCs.
(116) Nampt has been reported as predominantly expressed in hippocampal neurons but not in stellate astrocytes (Wang et al, 2011a; Zhang et al, 2010). Consistent with this finding, immunohistochemistry for Nampt and cell type specific markers revealed almost all NeuN+ neurons in the granule layer of the DG expressed Nampt, while almost no S100β+ glial cells did (
(117) However, the inventors also noticed that many Nampt immunoreactive cells along the SGZ of the DG did not express NeuN (
(118) To assess in vivo colocalization between Nampt and Nestin, the inventors crossed mice expressing Cre recombinase under the Nestin promoter (Nestin-CreERT2) to a GFP reporter mouse strain that expresses a loxP-flanked STOP cassette that prevents transcription of the downstream enhanced GFP (see Methods), generating iNSPC-GFP mice. Nampt also colocalized with GFP driven by the Nestin promoter (NestinGFP,
(119) Quantification of these observations revealed that along the SGZ, 32% of Sox2+ cells, 55% of radial Gfap+ cells, and 78% of NestinGFP+ cells expressed Nampt (
(120) TABLE-US-00001 TABLE 1 Top 50 Downregulated Pathways Z P Pathway Members Changed Ratio Value NUCLEAR_PART 579 468 −7.07 0 DNA_METABOLIC_PROCESS 257 226 −6.49 0 DNA_REPLICATION 102 85 −6.2 0 DNA_DEPENDENT_DNA_ 56 46 −6.07 0 REPLICATION DNA_REPAIR 125 111 −5.98 0 NUCLEUS 1433 1169 −5.95 0 INTRACELLULAR_ 1192 988 −5.85 0 ORGANELLE_PART RESPONSE_TO_DNA_ 162 138 −5.8 0 DAMAGE_STIMULUS ORGANELLE_PART 1197 992 −5.72 0 NUCLEOBASE_ 1246 1051 −5.34 0 NUCLEOSIDE_NUCLEOTIDE_ AND_NUCLEIC_ACID ORGANELLE_LUMEN 458 372 −5.04 0 MEMBRANE_ENCLOSED_ 458 372 −5.04 0 LUMEN CHROMOSOME 124 106 −4.75 0 CELL_CYCLE_PROCESS 193 167 −4.75 0 NUCLEAR_LUMEN 387 310 −4.74 0 CELL_CYCLE_GO_0007049 315 268 −4.63 0 RNA_PROCESSING 174 145 −4.32 0 BASE_EXCISION_REPAIR 17 15 −4.3 0 CELL_CYCLE_PHASE 170 147 −4.23 0 RESPONSE_TO_ 200 168 −4.18 0 ENDOGENOUS_STIMULUS MACROMOLECULAR_ 945 793 −4.14 0 COMPLEX NUCLEAR_CHROMOSOME 54 45 −4.11 0 STRUCTURE_SPECIFIC_ 56 45 −4.08 0 DNA_BINDING DOUBLE_STRANDED_ 32 26 −3.95 0 DNA_BINDING CELL_CYCLE_ 48 38 −3.92 0 CHECKPOINT_GO_0000075 TRNA_METABOLIC_PROCESS 19 18 −3.76 0 DNA_RECOMBINATION 47 45 −3.69 0 M_PHASE 114 100 −3.67 0 HYDROLASE_ACTIVITY_ 10 10 −3.63 0 HYDROLYZING_ N_GLYCOSYL_COMPOUNDS NUCLEOPLASM 279 230 −3.62 0 REPLICATION_FORK 18 16 −3.6 0 CONDENSED_CHROMOSOME 34 27 −3.57 0 TRNA_PROCESSING 10 9 −3.51 0 RIBONUCLEOPROTEIN_ 143 116 −3.49 0 COMPLEX MITOTIC_CELL_CYCLE 153 133 −3.48 0.001 CHROMOSOMAL_PART 96 83 −3.48 0.001 NUCLEOLUS 126 97 −3.47 0.001 NON_MEMBRANE_ 632 513 −3.46 0.001 BOUND_ORGANELLE INTRACELLULAR_NON_ 632 513 −3.46 0.001 MEMBRANE_BOUND_ ORGANELLE RNA_BINDING 259 211 −3.44 0.001 TRANSFERASE_ACTIVITY_ 37 34 −3.37 0.001 TRANSFERRING_ONE_ CARBON_GROUPS CONDENSED_NUCLEAR_ 18 15 −3.36 0.001 CHROMOSOME NUCLEOBASE_NUCLEOSIDE_ 52 45 −3.35 0.001 AND_NUCLEOTIDE_ METABOLIC_PROCESS METHYLTRANSFERASE_ 36 33 −3.3 0.001 ACTIVITY INTERPHASE 68 57 −3.28 0.001 NUCLEOTIDE_METABOLIC_ 42 36 −3.21 0.001 PROCESS DNA_INTEGRITY_ 24 16 −3.2 0.001 CHECKPOINT SINGLE_STRANDED_ 35 27 −3.17 0.002 DNA_BINDING PROTEIN_COMPLEX 816 689 −3.08 0.002 INTERPHASE_OF_MITOTIC_ 62 52 −3.07 0.002 CELL_CYCLE
(121) TABLE-US-00002 TABLE 2 Top 50 Upregulated Pathways Z P Pathway Members Changed Ratio Value SYSTEM_DEVELOPMENT 863 759 4.69 0.000 MULTICELLULAR_ 1051 910 4.64 0.000 ORGANISMAL_ DEVELOPMENT SIALYLTRANSFERASE_ 10 10 1.61 0.000 ACTIVITY PHOSPHOINOSITIDE_ 20 13 4.49 0.000 BINDING ANATOMICAL_ 1017 891 4.47 0.000 STRUCTURE_ DEVELOPMENT ORGAN_DEVELOPMENT 572 499 4.28 0.000 RECEPTOR_BINDING 378 321 4.25 0.000 EXTRACELLULAR_REGION 448 374 4.10 0.000 ENZYME_LINKED_ 140 126 4.05 0.000 RECEPTOR_ PROTEIN_SIGNALING_ PATHWAY Growth 77 63 3.85 0.000 NEGATIVE_REGULATION_ 40 34 3.84 0.000 OF_GROWTH FOCAL_ADHESION_ 10 9 3.83 0.000 FORMATION OLIGOSACCHARIDE_ 11 11 3.82 0.000 METABOLIC_PROCESS EXTRACELLULAR_ 339 283 3.82 0.000 REGION_PART REGULATION_OF_SIGNAL_ 223 189 3.77 0.000 TRANSDUCTION SYSTEM_PROCESS 563 489 3.74 0.000 EXTRACELLULAR_SPACE 246 204 3.66 0.000 NEURON_PROJECTION 21 18 3.62 0.000 INTERMEDIATE_FILAMENT_ 24 22 3.61 0.000 CYTOSKELETON INTERMEDIATE_FILAMENT 24 22 3.61 0.000 SENSORY_PERCEPTION 190 159 3.57 0.000 MEMBRANE 1998 1678 3.55 0.000 POSITIVE_REGULATION_ 20 15 3.51 0.000 OF_SECRETION REGULATION_OF_ 46 38 3.49 0.000 CELL_GROWTH REGULATION_OF_GROWTH 58 49 3.48 0.000 SIGNAL_TRANSDUCTION 1637 1394 3.47 0.001 PLASMA_MEMBRANE 1429 1199 3.46 0.001 MESODERM_DEVELOPMENT 22 17 3.45 0.001 CELL_DEVELOPMENT 579 512 3.44 0.001 FOCAL_ADHESION 13 10 3.44 0.001 ANATOMICAL_STRUCTURE_ 379 335 3.39 0.001 MORPHOGENESIS NERVOUS_SYSTEM_ 386 342 3.39 0.001 DEVELOPMENT EARLY_ENDOSOME 18 15 3.37 0.001 CYTOSKELETAL_PROTEIN_ 159 137 3.36 0.001 BINDING TASTE_RECEPTOR_ 15 3 3.36 0.001 ACTIVITY AXON_GUIDANCE 22 19 3.34 0.001 GENERATION_OF_ 83 76 3.34 0.001 NEURONS CELL_JUNCTION 83 67 3.29 0.001 LIPID_HOMEOSTASIS 16 12 3.28 0.001 PDZ_DOMAIN_BINDING 14 13 3.26 0.001 LIGAND_DEPENDENT_ 25 23 3.26 0.001 NUCLEAR_RECEPTOR_ ACTIVITY NEUROGENESIS 93 84 3.21 0.001 CELL_MATRIX_JUNCTION 18 14 3.20 0.001 VACUOLE 69 56 3.19 0.001 IDENTICAL_PROTEIN_ 305 253 3.19 0.001 BINDING CELL_MATRIX_ADHESION 38 34 3.18 0.001 TRANSMEMBRANE_ 83 75 3.18 0.001 RECEPTOR_PROTEIN_ TYROSINE_KINASE_ SIGNALING_PATH ACTIN_FILAMENT_ 116 96 3.18 0.001 BASED_PROCESS CELL_SURFACE_RECEPTOR_ 642 547 3.17 0.002 LINKED_SIGNAL_TRANS- DUCTION_GO_000166 CELL_SUBSTRATE_ 16 13 3.17 0.002 ADHERENS_JUNCTION
(122) To confirm that Nampt is highly expressed in NSPCs, the inventors cultured NSPCs from the hippocampi of postnatal pups as neurospheres. Neurospheres showed 22 or 32% higher expression levels of Nampt than did whole hippocampal extracts taken from postnatal (P12) or adult mice (2.5-4.5 months), respectively (
(123) The inventors thresholded Nampt immunoreactivity, and assessed the thresholded Nampt+ cells for colocalization with the neuronal marker NeuN and the NSPC marker Sox2 to determine which cell populations lose Nampt expression with age. With age, the percentage of intensely Nampt immunoreactive cells that colocalized with NeuN increased slightly, whereas the percentage of intensely Nampt immunoreactive cells that colocalized with Sox2 decreased from 21% to 4% (
Example 10
(124) This example illustrates that adult NSPC-specific deletion of Nampt impairs NSPC self-renewal in vivo.
(125) The inventors investigated whether inactivating Nampt specifically in adult NSPCs could recapitulate age-associated phenotypic changes in NSPC functionality in vivo. The inventors generated adult NSPC-specific inducible Nampt knockout mice by crossing Nampt.sup.flox/flox mice (Rongvaux et al, 2008) with Nestin-CreERT2 mice (iNSPC-Nampt-KO mice). To trace the progeny of adult NSPCs in which Nampt was inactivated and to confirm the specificity and magnitude of the deletion induced by tamoxifen, the inventors also crossed iNSPC-Nampt-KO mice to the aforementioned iNSPC-GFP mice. After tamoxifen injection, these mice expressed NestinGFP in the SGZ and SVZ but not in non-neurogenic regions of the brain such as the corpus callosum or cortex
(126) Immunohistochemistry and recombination PCR for NestinGFP confirmed that there was undetectable recombination present in vehicle injected mice. The ˜350 base pair band confirms the deletion of exons 5 and 6. The 1,800 base pair band corresponds to a Nampt gene with a full-length exon 5 to 6 sequence. (
(127) To assess the cumulative effect of loss of Nampt on NSPC proliferation, the inventors deleted Nampt in iNSPC-Nampt-KO mice at 6 weeks of age with 3 rounds of 5 consecutive days of tamoxifen injections, separated by 6 weeks (
(128) To assess the acute effect of loss of Nampt on NSPC fate decisions, the inventors induced deletion of Nampt at 6 weeks of age with 4 total tamoxifen injections followed by sacrifice 72 hours after the first injection (
(129) The inventors hypothesized that systemic administration of NMN may be able to correct age-associated defects in NSPC functionality. Intraperitoneal injection of NMN (500 mg/kg body weight) increased hippocampal NAD+ levels 34 to 39% within 15 minutes, suggesting that NMN can cross the blood-brain barrier (
Example 11
(130) This example illustrates that inhibition of Nampt in NSPCs in vitro impairs NAD+ biosynthesis and proliferation.
(131) The inventors hypothesized whether Nampt mediates NSPC-specific NAD+ biosynthesis by using hippocampal neurospheres as the in vitro NSPC culture model. Neurospheres were treated with a highly specific Nampt inhibitor, FK866, at a dosage and duration (10 nM, 48 hours) that has little to no effect on cellular viability (Hasmann & Schemainda, 2003). FK866 reduced NAD+ levels in neurospheres to 4% of controls, a decrease completely rescued by concurrent NMN treatment (
(132) The inventors investigated how inhibition of Nampt affects neurosphere proliferation. Consistent with the decreases in the NSPC pool and in NSPC proliferation in iNSPC-Nampt-KO mice, FK866 reduced NSPC number by 61% after 48 hours, but not 24 hours, of treatment (
(133) TABLE-US-00003 TABLE 3 Z P Rank Cell Cycle Total Implicated Ration Value 3 DNA_REPLICATION 102 85 −6.20 0.000 4 DNA_DEPENDENT_ 56 46 −6.07 0.000 DNA_REPLICATION 14 CELL_CYCLE_ 193 167 −4.75 0.000 PROCESS 15 NUCLEAR_LUMEN 387 310 −4.74 0.000 16 CELL_CYCLE_ 315 268 −4.63 0.000 GO_0007049 19 CELL_CYCLE_PHASE 170 147 −4.23 0.000 25 CELL_CYCLE_ 48 38 −3.92 0.000 CHECKPOINT_ GO_0000075 28 M_PHASE 114 100 −3.67 0.000 31 REPLICATION_FORK 18 16 −3.60 0.000 32 CONDENSED_ 34 27 −3.57 0.000 CHROMOSOME 35 MITOTIC_ 153 133 −3.48 0.001 CELL_CYCLE 45 INTERPHASE 68 57 −3.28 0.001 50 INTERPHASE_ 62 52 −3.07 0.002 OF_MITOTIC_ CELL_CYCLE
Example 12
(134) This example illustrates that genetic ablation of Nampt in NSPCs in vitro impairs NAD+ biosynthesis, proliferation, and differentiation.
(135) To assess the effect of chronic Nampt ablation on NSPC functionality, the inventors genetically ablated Nampt by infecting neurospheres from Nampt.sup.flox/flox mice with Cre recombinase- or LacZ-expressing (control) adenoviruses. Neurospheres infected with Cre recombinase (Nampt Ad-Cre) at passage 1 exhibited a 94% reduction in Nampt mRNA expression 3 days post deletion, and the corresponding decreases in Nampt protein expression and NAD+ levels appeared 6 days post deletion (
(136) Like FK866-treated cultures, proliferating Nampt Ad-Cre infected NSPCs displayed reduced cell number (
(137) Whereas the inventors did not observe a difference in NSPC fate decisions in the neurogenic environment of the SGZ in vivo, the inventors detected a decrease in self-renewal decisions. To see if this would occur in the absence of the influences of the SGZ niche, the inventors differentiated dissociated neurospheres and assessed the proportion of resulting cell types by immunofluorescence after 6 to 7 days of differentiation induced by removal of growth factors (
Example 13
(138) This example illustrates that genetic knockdown of Nampt impairs OPC formation in vitro.
(139) Since differentiation using a nonspecific lineage differentiation protocol (by removal of growth factors) revealed a specific requirement for Nampt in the successful generation of O4+ immature oligodendrocytes, the inventors next asked which stage(s) of NSPC differentiation into oligodendrocytes depends on Nampt by employing a differentiation protocol that promotes the oligodendrocyte lineage (
(140) The inventors observed that Sirt2 was upregulated during oligodendrocyte differentiation in vitro and expressed in the SGZ in Nampt+ cells and NestinGFP+ NSPCs (
Example 14
(141) This example illustrates adult NSPC-specific deletion of Nampt impairs NSPC differentiation in response to insult in vivo.
(142) The inventors observed Nampt ablation on NSPC differentiation into OPCs in vitro, but not oligodendrogenesis in the SGZ of iNSPC-Nampt-KO mice in vivo (
(143) The inventors employed the cuprizone model of demyelination and remyelination. Specifically, the inventors fed 6- to 9-week-old iNSPC-Nampt-KO and littermate control mice (iNSPC-GFP) a diet containing 0.2% cuprizone for 4-5 weeks, inducing deletion of Nampt in the adult Nestin+ population the week before starting the cuprizone diet (
(144) Cuprizone feeding did not alter the total number of NestinGFP+ cells present in the iNSPC-GFP DG (
(145) The inventors assessed the fate decisions of migratory cells derived from the adult Nestin+ population in the subcallosal zone of the corpus callosum (
(146) In contrast, the NestinGFP+ cells in the iNSPC-Nampt-KO CC showed significantly less colocalization with Nestin, Sox10, and Apc and more colocalization with Gfap (
Example 15
(147) This example illustrates a model for the role of Nampt-mediated NAD biosynthesis in NSPCs without being limited by theory.
(148) Nampt-mediated NAD+ biosynthesis promotes NSPC self-renewal, proliferation and differentiation into oligodendrocytes. While the mechanism by which Nampt promotes self-renewal and proliferation remains unidentified, Nampt-mediated NAD+ biosynthesis activates Sirt1 and Sirt2 to promote NSPC oligodendrocyte lineage fate decisions by a mechanism involving transcriptional downregulation of Pdgfrα, Sox10, and Nkx2.2 and transcriptional upregulation of p21 (cdkn1a). Sirt1 and Sirt2 may act via an effect on Olig2 activity. (
(149) The inventors showed that the NSPC pool decreased with age and that long-term NMN administration was able to maintain the NSPC pool. The inventors assert that a higher dosage of NMN can be used to promote NSPC proliferation. Intraperitoneal injection of NMN substantially increases hippocampal NAD+ levels within 15 minutes (
(150) As E2F1-deficient mice have significantly reduced hippocampal NSPC death (Cooper-Kuhn et al, 2002), without being limited by theory, the observed decrease in E2F1 upon inhibition of Nampt may explain this phenomenon (
(151) The present inventors revealed that ablation of Nampt specifically reduced the proportion of NSPC-generated Pdgfrα+ OPCs as well as the transcription of Pdgfrα, Sox10, and Nkx2.2 but upregulated the expression of p21. The results showed that in neurospheres, treatment with NMN rescued defects in oligodendrogenesis caused by a reduction in NAD+ levels. Furthermore, systemic NMN administration was able to substantially augment hippocampal NAD+ levels and increase the NSPC pool. Thus, NMN administration could be an efficient intervention to enhance the NSPC pool and promote remyelination by activating endogenous NSPCs during the aging process and/or in neurodegenerative diseases that cause demyelination. The results provide evidence of the therapeutic potential of Nampt-mediated NSPC self-renewal, proliferation, and differentiation into oligodendrocytes.
Example 16
(152) This example illustrates an increase in bone density in aged individuals by NMN administration.
(153) The inventors measured the bone mineral density (BMD) of control and NMN-treated mice at the 12-month time point of a 12 month long NMN administration experiment by dual-energy X-ray absorptiometry (DXA). At this time point, mice were 17-18 month old. The inventors found that NMN-treated mice showed increases in the BMD in a dose-dependent manner, and the difference between control and 300 mg/kg groups is statistically significant (P=0.037, ANOVA, Tukey HSD post hoc test). Mice from 100 and 300 mg/kg groups showed 2.8% and 5.9% increases in the BMD, respectively. Although age-associated BMD loss is extensively varied among mouse strains (http://phenome.jax.org), the extent of these observed BMD increases is significant, indicating that NMN is able to enhance the BMD in aged individuals. These data indicate that NMN administration can be used to treat age-associated osteoporosis in humans.
Example 17
(154) This example illustrates characterization of loss of NAMPT-mediated NAD biosynthesis on PR neuron survival.
(155) The inventors examined the effect of selectively disrupting NAD biosynthesis within PR. The inventors utilized the cre-lox strategy to generate mice that had NAMPT conditionally deleted from either rods (NAMPT rod-CKO) or cones (NAMPT cone-CKO). The NAMPT fl/fl mice as well as the rhodopsin-cre and cone opsin-cre mice have been previously characterized. Both rod and cone cko mice are generated with normal Mendelian frequencies and are born normal with no observable systemic abnormalities (data not shown). All structural and functional analyses performed in CKO mice are analyzed in comparison to littermate controls. Rods constitute a majority of the PR neurons (97% of all photoreceptors). Retinas from NAMPT rod-CKO mice showed a significant reduction of NAMPT within rods by PCR, immunohistochemistry and immunoblotting (
Example 18
(156) This example illustrates electroretinography (ERG) performed to measure PR neuron and retinal function.
(157) NAMPT rod-CKO mice demonstrated a dramatic reduction in scotopic (rod-associated) and photopic (cone-associated) responses compared to littermate control animals (
(158) The present inventors determined that exogenous supplementation with NMN is able to rescue PR neurons from cell death in CKO mice. Intraperitoneal (i.p.) delivery was chosen to obtain early and sustained levels of NMN. In these experiments, NAMPT rod-CKO mice were given NMN (150 mg/kg) or PBS i.p. daily starting at day P5. ERG at 4 weeks in CKO mice treated with NMN showed significant rescue of photopic and scotopic function compared to PBS treatment (
Example 19
(159) This example illustrates NMN rescue in NAMPT cone-CKO mice.
(160) In these experiments, NAMPT cone-CKO mice (without NMN treatment) demonstrated similar but milder changes on biomicroscopy consistent with neuroretinal degeneration as seen in the rod-CKO mice (
(161) The inventors used a 661W cone PR cell line and treated the cells with the specific pharmacological NAMPT inhibitor FK866 (200 nM). FK866 treatment of cone cells in vitro causes decrease in intracellular NAD levels and significant cell death after the 4 hours of treatment (
Example 20
(162) This example illustrates that NAD-regulated PR survival is independent of individual sirtuins.
(163) The inventors examined the effect of deletion of several sirtuins on PR survival. Sirt 1 PR CKO mice and Sirt 2-5−/− mice had normal retinal and PR neuron structure and function when examined by fundus biomicroscopy and ERG (
Example 21
(164) This example illustrates that photoreceptor loss and blindness is associated with mitochondrial dysfunction.
(165) Electron microscopic examination demonstrated dysmorphic changes in the retinal inner segments along with disruption of the outer segments in rod CKO mice but showed normal cellular organization and sub-cellular structures in littermate controls at 4 weeks of age (
(166) A non-biased metabolomic analysis using mass spectrophotometry (GC-MS and LC-MS) was performed on retinas isolated from NAMPT rod CKO mice and compared to littermate control retinas. Significant differences were identified in mitochondrial metabolites involved in the TCA cycle.
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