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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

OCR deficits in non-synaptic brain mitochondria from mutant APP expressing mice following NMN treatment. Average OCR (pMoles/min) in non-synaptic mitochondria isolated from APP(swe)/PS1(ΔE9) (AD-Tg) and non-transgenic (NTG) mouse (3 months) brain. Solid black bar (NTG vehicle control), white bar (AD-Tg vehicle control) and striped bar (AD-Tg NMN). Data are presented as the average OCR ± SE. N = 3–5 separate animals per group. *p < 0.01.
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Fig3: OCR deficits in non-synaptic brain mitochondria from mutant APP expressing mice following NMN treatment. Average OCR (pMoles/min) in non-synaptic mitochondria isolated from APP(swe)/PS1(ΔE9) (AD-Tg) and non-transgenic (NTG) mouse (3 months) brain. Solid black bar (NTG vehicle control), white bar (AD-Tg vehicle control) and striped bar (AD-Tg NMN). Data are presented as the average OCR ± SE. N = 3–5 separate animals per group. *p < 0.01.

Mentions: In a previous study, we determined that brain mitochondrial ADP-stimulated OCR were significantly deficient in 3 months old male AD-Tg mice as compared to their non-transgenic littermates [14]. NAD+ as a cofactor for tricarboxylic acid (TCA) cycle and Complex I enzymatic reactions and deficiencies give rise to bioenergetic dysfunction. The NAD+ precursor nicotinamide mononucleotide (NMN) inhibits NAD+ degradation as well as enhancing its synthesis [27]. Therefore, to examine the potential relationship between mitochondrial respiratory dysfunction and NAD+ catabolism, AD-Tg and non-transgenic (NTG) mice (2 months) were given NMN or phosphate buffered saline (PBS, vehicle). Non-synaptic mitochondria were then isolated from the forebrains of male and female (3 months) AD-Tg mice and their NTG littermates. There were no significant differences in basal OCR between the transgenic and non-transgenic mice regardless of NMN treatment (Figure 3, Basal). However, following addition of ADP to initiate State 3 respiration, the AD-Tg vehicle-treated mice had significantly lower OCR (p < 0.01) compared to the NTG animals (Figure 3, ADP) recapitulating our previous study [14]. The AD-Tg vehicle-treated mice were significantly deficient (p < 0.01) in State 3 respiration as compared to the transgenic mice that received NMN treatment (Figure 3, ADP). Interestingly, the AD-Tg NMN-treated mice had significantly increased (p < 0.01) OCR as compared to the non-transgenic animals (Figure 3, ADP). Following oligomycin addition reducing the rate of O2 consumption to that of State 4° respiration, there was no significant difference in OCR between the transgenic and non-transgenic mice regardless of treatment group (Figure 3, Oligo). Although, following addition of FCCP to assess maximal OCR there was no significant difference between NTG vehicle and AD-Tg vehicle-treated mice, the AD-Tg NMN-treated mice had significantly increased (p < 0.01) OCR as compared to both the transgenic vehicle-treated and non-transgenic animals (Figure 3, FCCP).Figure 3


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)

OCR deficits in non-synaptic brain mitochondria from mutant APP expressing mice following NMN treatment. Average OCR (pMoles/min) in non-synaptic mitochondria isolated from APP(swe)/PS1(ΔE9) (AD-Tg) and non-transgenic (NTG) mouse (3 months) brain. Solid black bar (NTG vehicle control), white bar (AD-Tg vehicle control) and striped bar (AD-Tg NMN). Data are presented as the average OCR ± SE. N = 3–5 separate animals per group. *p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4358858&req=5

Fig3: OCR deficits in non-synaptic brain mitochondria from mutant APP expressing mice following NMN treatment. Average OCR (pMoles/min) in non-synaptic mitochondria isolated from APP(swe)/PS1(ΔE9) (AD-Tg) and non-transgenic (NTG) mouse (3 months) brain. Solid black bar (NTG vehicle control), white bar (AD-Tg vehicle control) and striped bar (AD-Tg NMN). Data are presented as the average OCR ± SE. N = 3–5 separate animals per group. *p < 0.01.
Mentions: In a previous study, we determined that brain mitochondrial ADP-stimulated OCR were significantly deficient in 3 months old male AD-Tg mice as compared to their non-transgenic littermates [14]. NAD+ as a cofactor for tricarboxylic acid (TCA) cycle and Complex I enzymatic reactions and deficiencies give rise to bioenergetic dysfunction. The NAD+ precursor nicotinamide mononucleotide (NMN) inhibits NAD+ degradation as well as enhancing its synthesis [27]. Therefore, to examine the potential relationship between mitochondrial respiratory dysfunction and NAD+ catabolism, AD-Tg and non-transgenic (NTG) mice (2 months) were given NMN or phosphate buffered saline (PBS, vehicle). Non-synaptic mitochondria were then isolated from the forebrains of male and female (3 months) AD-Tg mice and their NTG littermates. There were no significant differences in basal OCR between the transgenic and non-transgenic mice regardless of NMN treatment (Figure 3, Basal). However, following addition of ADP to initiate State 3 respiration, the AD-Tg vehicle-treated mice had significantly lower OCR (p < 0.01) compared to the NTG animals (Figure 3, ADP) recapitulating our previous study [14]. The AD-Tg vehicle-treated mice were significantly deficient (p < 0.01) in State 3 respiration as compared to the transgenic mice that received NMN treatment (Figure 3, ADP). Interestingly, the AD-Tg NMN-treated mice had significantly increased (p < 0.01) OCR as compared to the non-transgenic animals (Figure 3, ADP). Following oligomycin addition reducing the rate of O2 consumption to that of State 4° respiration, there was no significant difference in OCR between the transgenic and non-transgenic mice regardless of treatment group (Figure 3, Oligo). Although, following addition of FCCP to assess maximal OCR there was no significant difference between NTG vehicle and AD-Tg vehicle-treated mice, the AD-Tg NMN-treated mice had significantly increased (p < 0.01) OCR as compared to both the transgenic vehicle-treated and non-transgenic animals (Figure 3, FCCP).Figure 3

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