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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

Immunoreactivity of SIRT1 in homogenates isolated from NTG and AD-Tg mouse brain following NMN treatment. Each lane represents a different animal. (A) Representative Western blot images. (B) Significantly increased SIRT1 levels (ratio SIRT1:GAPDH) are observed in AD-Tg (vehicle) homogenates compared to NTG vehicle-treated animals. These levels are significantly reduced (~49%) by the addition of NMN in AD-Tg mice although they remain significantly elevated. NTG SIRT1 levels normalized to GAPDH = 0.004 as presented in (B). Data are presented as the average SIRT1 ± SE. N = 6 separate animals per group. **p ≤ 0.05.
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Fig4: Immunoreactivity of SIRT1 in homogenates isolated from NTG and AD-Tg mouse brain following NMN treatment. Each lane represents a different animal. (A) Representative Western blot images. (B) Significantly increased SIRT1 levels (ratio SIRT1:GAPDH) are observed in AD-Tg (vehicle) homogenates compared to NTG vehicle-treated animals. These levels are significantly reduced (~49%) by the addition of NMN in AD-Tg mice although they remain significantly elevated. NTG SIRT1 levels normalized to GAPDH = 0.004 as presented in (B). Data are presented as the average SIRT1 ± SE. N = 6 separate animals per group. **p ≤ 0.05.

Mentions: NAD+ is utilized as an important cofactor in many metabolic reactions [28] including oxidative phosphorylation and enzymatic reactions of the TCA cycle [5], and as a substrate for enzymes including histone deacetylase sirtuin 1 (SIRT1), poly(ADP-ribose) polymerase 1 (PARP1) and NAD+ glycohydrolase CD38. We assessed brain homogenates from AD-Tg and non-transgenic NMN- and vehicle-treated mice to determine if NAD+ catabolism was due to increased SIRT1 levels. There was a significant increase (p < 0.05) in SIRT1 immunoreactivity in brain homogenates from AD-Tg mice when compared to both NTG animals as well as AD-Tg animals pre-treated with NMN (Figure 4). Although the AD-Tg mice pre-treated with NMN had lower SIRT1 immunoreactivity compared to non-NMN treated AD-Tg mice, these levels were still significantly elevated (p ≤ 0.05) as compared to NTG animals (Figure 4).Figure 4


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)

Immunoreactivity of SIRT1 in homogenates isolated from NTG and AD-Tg mouse brain following NMN treatment. Each lane represents a different animal. (A) Representative Western blot images. (B) Significantly increased SIRT1 levels (ratio SIRT1:GAPDH) are observed in AD-Tg (vehicle) homogenates compared to NTG vehicle-treated animals. These levels are significantly reduced (~49%) by the addition of NMN in AD-Tg mice although they remain significantly elevated. NTG SIRT1 levels normalized to GAPDH = 0.004 as presented in (B). Data are presented as the average SIRT1 ± SE. N = 6 separate animals per group. **p ≤ 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4358858&req=5

Fig4: Immunoreactivity of SIRT1 in homogenates isolated from NTG and AD-Tg mouse brain following NMN treatment. Each lane represents a different animal. (A) Representative Western blot images. (B) Significantly increased SIRT1 levels (ratio SIRT1:GAPDH) are observed in AD-Tg (vehicle) homogenates compared to NTG vehicle-treated animals. These levels are significantly reduced (~49%) by the addition of NMN in AD-Tg mice although they remain significantly elevated. NTG SIRT1 levels normalized to GAPDH = 0.004 as presented in (B). Data are presented as the average SIRT1 ± SE. N = 6 separate animals per group. **p ≤ 0.05.
Mentions: NAD+ is utilized as an important cofactor in many metabolic reactions [28] including oxidative phosphorylation and enzymatic reactions of the TCA cycle [5], and as a substrate for enzymes including histone deacetylase sirtuin 1 (SIRT1), poly(ADP-ribose) polymerase 1 (PARP1) and NAD+ glycohydrolase CD38. We assessed brain homogenates from AD-Tg and non-transgenic NMN- and vehicle-treated mice to determine if NAD+ catabolism was due to increased SIRT1 levels. There was a significant increase (p < 0.05) in SIRT1 immunoreactivity in brain homogenates from AD-Tg mice when compared to both NTG animals as well as AD-Tg animals pre-treated with NMN (Figure 4). Although the AD-Tg mice pre-treated with NMN had lower SIRT1 immunoreactivity compared to non-NMN treated AD-Tg mice, these levels were still significantly elevated (p ≤ 0.05) as compared to NTG animals (Figure 4).Figure 4

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