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Effect of nicotinamide mononucleotide on brain mitochondrial respiratory deficits in an Alzheimer's disease-relevant murine model.

Long AN, Owens K, Schlappal AE, Kristian T, Fishman PS, Schuh RA - BMC Neurol (2015)

Bottom Line: Student t-test was used for direct comparison of two groups.Levels of SIRT1 and CD38 change with age and NMN treatment.This is the first study to directly examine amelioration of NAD(+) catabolism and changes in mitochondrial morphological dynamics in brain utilizing the immediate precursor NMN as a potential therapeutic compound.

View Article: PubMed Central - PubMed

Affiliation: Research Service, VAMHCS, 10 North Greene Street, Baltimore, MD, 21201, USA. Aaron.Long@va.gov.

ABSTRACT

Background: Mitochondrial dysfunction is a hallmark of neurodegenerative diseases including Alzheimer's disease (AD), with morphological and functional abnormalities limiting the electron transport chain and ATP production. A contributing factor of mitochondrial abnormalities is loss of nicotinamide adenine dinucleotide (NAD), an important cofactor in multiple metabolic reactions. Depletion of mitochondrial and consequently cellular NAD(H) levels by activated NAD glycohydrolases then culminates in bioenergetic failure and cell death. De Novo NAD(+) synthesis from tryptophan requires a multi-step enzymatic reaction. Thus, an alternative strategy to maintain cellular NAD(+) levels is to administer NAD(+) precursors facilitating generation via a salvage pathway. We administered nicotinamide mononucleotide (NMN), an NAD(+) precursor to APP(swe)/PS1(ΔE9) double transgenic (AD-Tg) mice to assess amelioration of mitochondrial respiratory deficits. In addition to mitochondrial respiratory function, we examined levels of full-length mutant APP, NAD(+)-dependent substrates (SIRT1 and CD38) in homogenates and fission/fusion proteins (DRP1, OPA1 and MFN2) in mitochondria isolated from brain. To examine changes in mitochondrial morphology, bigenic mice possessing a fluorescent protein targeted to neuronal mitochondria (CaMK2a-mito/eYFP), were administered NMN.

Methods: Mitochondrial oxygen consumption rates were examined in N2A neuroblastoma cells and non-synaptic brain mitochondria isolated from mice (3 months). Western blotting was utilized to assess APP, SIRT1, CD38, DRP1, OPA1 and MFN2 in brain of transgenic and non-transgenic mice (3-12 months). Mitochondrial morphology was assessed with confocal microscopy. One-way or two-way analysis of variance (ANOVA) and post-hoc Holm-Sidak method were used for statistical analyses of data. Student t-test was used for direct comparison of two groups.

Results: We now demonstrate that mitochondrial respiratory function was restored in NMN-treated AD-Tg mice. Levels of SIRT1 and CD38 change with age and NMN treatment. Furthermore, we found a shift in dynamics from fission to fusion proteins in the NMN-treated mice.

Conclusions: This is the first study to directly examine amelioration of NAD(+) catabolism and changes in mitochondrial morphological dynamics in brain utilizing the immediate precursor NMN as a potential therapeutic compound. This might lead to well-defined physiologic abnormalities that can serve an important role in the validation of promising agents such as NMN that target NAD(+) catabolism preserving mitochondrial function.

No MeSH data available.


Related in: MedlinePlus

Altered SIRT1 and CD38 immunoreactivity with age. Representative Western blot images for SIRT1 (A) and CD38 (C) in brain homogenates isolated from NTG and AD-Tg mice (3–12 months). (B) Significantly increased SIRT1 levels (ratio SIRT1:GAPDH) are observed in AD-Tg homogenates of 6 months compared to 3 months; and 9 months compared to both 3 and 6 months. (D) Significant increases in CD38 levels are observed at 3 and 12 months in AD-Tg homogenates compared to NTG mice. Significant decrease in CD38 levels are observed at 6 months in AD-Tg homogenates compared to 3 months AD-Tg mice. Data are presented as the average SIRT1 or CD38 ± SE. N = 3 separate animals per age group. **p < 0.05; *p < 0.01; a: AD-Tg significantly different from NTG; b: 6 months AD-Tg significantly different from 3 months AD-Tg.
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Fig5: Altered SIRT1 and CD38 immunoreactivity with age. Representative Western blot images for SIRT1 (A) and CD38 (C) in brain homogenates isolated from NTG and AD-Tg mice (3–12 months). (B) Significantly increased SIRT1 levels (ratio SIRT1:GAPDH) are observed in AD-Tg homogenates of 6 months compared to 3 months; and 9 months compared to both 3 and 6 months. (D) Significant increases in CD38 levels are observed at 3 and 12 months in AD-Tg homogenates compared to NTG mice. Significant decrease in CD38 levels are observed at 6 months in AD-Tg homogenates compared to 3 months AD-Tg mice. Data are presented as the average SIRT1 or CD38 ± SE. N = 3 separate animals per age group. **p < 0.05; *p < 0.01; a: AD-Tg significantly different from NTG; b: 6 months AD-Tg significantly different from 3 months AD-Tg.

Mentions: SIRT1 immunoreactivity in brain homogenates was examined to assess potential additive effects of AD transgenes in older animals (3–9 months). There was significantly (p < 0.05) increased SIRT1 immunoreactivity in the 9 months AD-Tg mice as compared with both the 3 and 6 months animals (Figure 5A, B). Further, the 6 months AD-Tg SIRT1 levels were also significantly (p ≤ 0.05) elevated compared to the 3 months AD-Tg mice (Figure 5A, B). As described above, an alternative NAD+ consumer is the NAD+ glycohydrolase CD38 (CD38). We therefore also probed brain homogenates from both NTG and AD-Tg mice aged 3–12 months utilizing Western blots. There was a significant increase (p < 0.05) in CD38 immunoreactivity in the 3 and 12 months AD-Tg mice as compared to age-matched NTG animals (Figure 5C,D). There was a significant decrease (p < 0.05) in CD38 immunoreactivity between 3 and 6 months AD-Tg mice (Figure 5C,D). There was no significant difference in CD38 immunoreactivity in NTG mice across the age groups examined. Although there was an overall significant difference in CD38 levels comparing genotypes (p < 0.01) or age (p < 0.01), there was no genotype x age interaction (Figure 5C, D).Figure 5


Effect of nicotinamide mononucleotide on brain mitochondrial respiratory deficits in an Alzheimer's disease-relevant murine model.

Long AN, Owens K, Schlappal AE, Kristian T, Fishman PS, Schuh RA - BMC Neurol (2015)

Altered SIRT1 and CD38 immunoreactivity with age. Representative Western blot images for SIRT1 (A) and CD38 (C) in brain homogenates isolated from NTG and AD-Tg mice (3–12 months). (B) Significantly increased SIRT1 levels (ratio SIRT1:GAPDH) are observed in AD-Tg homogenates of 6 months compared to 3 months; and 9 months compared to both 3 and 6 months. (D) Significant increases in CD38 levels are observed at 3 and 12 months in AD-Tg homogenates compared to NTG mice. Significant decrease in CD38 levels are observed at 6 months in AD-Tg homogenates compared to 3 months AD-Tg mice. Data are presented as the average SIRT1 or CD38 ± SE. N = 3 separate animals per age group. **p < 0.05; *p < 0.01; a: AD-Tg significantly different from NTG; b: 6 months AD-Tg significantly different from 3 months AD-Tg.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4358858&req=5

Fig5: Altered SIRT1 and CD38 immunoreactivity with age. Representative Western blot images for SIRT1 (A) and CD38 (C) in brain homogenates isolated from NTG and AD-Tg mice (3–12 months). (B) Significantly increased SIRT1 levels (ratio SIRT1:GAPDH) are observed in AD-Tg homogenates of 6 months compared to 3 months; and 9 months compared to both 3 and 6 months. (D) Significant increases in CD38 levels are observed at 3 and 12 months in AD-Tg homogenates compared to NTG mice. Significant decrease in CD38 levels are observed at 6 months in AD-Tg homogenates compared to 3 months AD-Tg mice. Data are presented as the average SIRT1 or CD38 ± SE. N = 3 separate animals per age group. **p < 0.05; *p < 0.01; a: AD-Tg significantly different from NTG; b: 6 months AD-Tg significantly different from 3 months AD-Tg.
Mentions: SIRT1 immunoreactivity in brain homogenates was examined to assess potential additive effects of AD transgenes in older animals (3–9 months). There was significantly (p < 0.05) increased SIRT1 immunoreactivity in the 9 months AD-Tg mice as compared with both the 3 and 6 months animals (Figure 5A, B). Further, the 6 months AD-Tg SIRT1 levels were also significantly (p ≤ 0.05) elevated compared to the 3 months AD-Tg mice (Figure 5A, B). As described above, an alternative NAD+ consumer is the NAD+ glycohydrolase CD38 (CD38). We therefore also probed brain homogenates from both NTG and AD-Tg mice aged 3–12 months utilizing Western blots. There was a significant increase (p < 0.05) in CD38 immunoreactivity in the 3 and 12 months AD-Tg mice as compared to age-matched NTG animals (Figure 5C,D). There was a significant decrease (p < 0.05) in CD38 immunoreactivity between 3 and 6 months AD-Tg mice (Figure 5C,D). There was no significant difference in CD38 immunoreactivity in NTG mice across the age groups examined. Although there was an overall significant difference in CD38 levels comparing genotypes (p < 0.01) or age (p < 0.01), there was no genotype x age interaction (Figure 5C, D).Figure 5

Bottom Line: Student t-test was used for direct comparison of two groups.Levels of SIRT1 and CD38 change with age and NMN treatment.This is the first study to directly examine amelioration of NAD(+) catabolism and changes in mitochondrial morphological dynamics in brain utilizing the immediate precursor NMN as a potential therapeutic compound.

View Article: PubMed Central - PubMed

Affiliation: Research Service, VAMHCS, 10 North Greene Street, Baltimore, MD, 21201, USA. Aaron.Long@va.gov.

ABSTRACT

Background: Mitochondrial dysfunction is a hallmark of neurodegenerative diseases including Alzheimer's disease (AD), with morphological and functional abnormalities limiting the electron transport chain and ATP production. A contributing factor of mitochondrial abnormalities is loss of nicotinamide adenine dinucleotide (NAD), an important cofactor in multiple metabolic reactions. Depletion of mitochondrial and consequently cellular NAD(H) levels by activated NAD glycohydrolases then culminates in bioenergetic failure and cell death. De Novo NAD(+) synthesis from tryptophan requires a multi-step enzymatic reaction. Thus, an alternative strategy to maintain cellular NAD(+) levels is to administer NAD(+) precursors facilitating generation via a salvage pathway. We administered nicotinamide mononucleotide (NMN), an NAD(+) precursor to APP(swe)/PS1(ΔE9) double transgenic (AD-Tg) mice to assess amelioration of mitochondrial respiratory deficits. In addition to mitochondrial respiratory function, we examined levels of full-length mutant APP, NAD(+)-dependent substrates (SIRT1 and CD38) in homogenates and fission/fusion proteins (DRP1, OPA1 and MFN2) in mitochondria isolated from brain. To examine changes in mitochondrial morphology, bigenic mice possessing a fluorescent protein targeted to neuronal mitochondria (CaMK2a-mito/eYFP), were administered NMN.

Methods: Mitochondrial oxygen consumption rates were examined in N2A neuroblastoma cells and non-synaptic brain mitochondria isolated from mice (3 months). Western blotting was utilized to assess APP, SIRT1, CD38, DRP1, OPA1 and MFN2 in brain of transgenic and non-transgenic mice (3-12 months). Mitochondrial morphology was assessed with confocal microscopy. One-way or two-way analysis of variance (ANOVA) and post-hoc Holm-Sidak method were used for statistical analyses of data. Student t-test was used for direct comparison of two groups.

Results: We now demonstrate that mitochondrial respiratory function was restored in NMN-treated AD-Tg mice. Levels of SIRT1 and CD38 change with age and NMN treatment. Furthermore, we found a shift in dynamics from fission to fusion proteins in the NMN-treated mice.

Conclusions: This is the first study to directly examine amelioration of NAD(+) catabolism and changes in mitochondrial morphological dynamics in brain utilizing the immediate precursor NMN as a potential therapeutic compound. This might lead to well-defined physiologic abnormalities that can serve an important role in the validation of promising agents such as NMN that target NAD(+) catabolism preserving mitochondrial function.

No MeSH data available.


Related in: MedlinePlus