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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 in APP(swe)-overexpressing N2A hippocampal neuroblastoma cells (blue lines) and control transfected cells (red lines). Baseline-normalized oxygen consumption rates (OCR) of N2A neuroblastoma cells exposed to successive additions of mitochondrial respiratory modulators (arrows) are shown. Cells received 1 μg/ml oligomycin (Oligo), 1 μM FCCP, 10 mM pyruvate (Pyr), and 1 μM antimycin A (Ant A). Subsets of the cells (solid lines) were pre-incubated with 10 mM nicotinamide adenine dinucleotide (NAD+) prior to measurements while control cells (dashed lines) had no exogenous NAD+. Rates are normalized to the third baseline measurement point for n = 3–5 replicates per group from 2 separate cultures.
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Fig1: OCR in APP(swe)-overexpressing N2A hippocampal neuroblastoma cells (blue lines) and control transfected cells (red lines). Baseline-normalized oxygen consumption rates (OCR) of N2A neuroblastoma cells exposed to successive additions of mitochondrial respiratory modulators (arrows) are shown. Cells received 1 μg/ml oligomycin (Oligo), 1 μM FCCP, 10 mM pyruvate (Pyr), and 1 μM antimycin A (Ant A). Subsets of the cells (solid lines) were pre-incubated with 10 mM nicotinamide adenine dinucleotide (NAD+) prior to measurements while control cells (dashed lines) had no exogenous NAD+. Rates are normalized to the third baseline measurement point for n = 3–5 replicates per group from 2 separate cultures.

Mentions: N2A hippocampal neuroblastoma cells were transiently co-transfected with constructs containing mutant APP(swe)/PS1(ΔE9) and a tetracycline transactivator (TTA) as a cell-based model of effects of mutant amyloid toxicity. Co-transfected (transfected) or TTA-transfected (control) N2A cells were pre-incubated ± nicotinamide adenine dinucleotide (NAD+) and oxygen consumption rates (OCR) measured. Transfected N2A cells (no exogenous NAD+; blue dashed line) had decreased maximal OCR when challenged with uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) and pyruvate, compared to control cells + NAD+ (24% decrease; Figure 1, red solid line); or control cells without exogenous NAD+ addition (18% decrease; Figure 1, red dashed line). This OCR deficit (Figure 1, blue dashed line) could be ameliorated if exogenous NAD+ was present (27% increase; Figure 1, blue solid line). Control N2A cultures had similar OCR regardless of NAD+ addition (Figure 1, red solid versus red dashed line). These experiments suggest that deficiencies in NAD+ levels could play a role in mitochondrial respiratory dysfunction in transgenic mice expressing this mutation in vivo [14] and could be corrected with added NAD+.Figure 1


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 in APP(swe)-overexpressing N2A hippocampal neuroblastoma cells (blue lines) and control transfected cells (red lines). Baseline-normalized oxygen consumption rates (OCR) of N2A neuroblastoma cells exposed to successive additions of mitochondrial respiratory modulators (arrows) are shown. Cells received 1 μg/ml oligomycin (Oligo), 1 μM FCCP, 10 mM pyruvate (Pyr), and 1 μM antimycin A (Ant A). Subsets of the cells (solid lines) were pre-incubated with 10 mM nicotinamide adenine dinucleotide (NAD+) prior to measurements while control cells (dashed lines) had no exogenous NAD+. Rates are normalized to the third baseline measurement point for n = 3–5 replicates per group from 2 separate cultures.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: OCR in APP(swe)-overexpressing N2A hippocampal neuroblastoma cells (blue lines) and control transfected cells (red lines). Baseline-normalized oxygen consumption rates (OCR) of N2A neuroblastoma cells exposed to successive additions of mitochondrial respiratory modulators (arrows) are shown. Cells received 1 μg/ml oligomycin (Oligo), 1 μM FCCP, 10 mM pyruvate (Pyr), and 1 μM antimycin A (Ant A). Subsets of the cells (solid lines) were pre-incubated with 10 mM nicotinamide adenine dinucleotide (NAD+) prior to measurements while control cells (dashed lines) had no exogenous NAD+. Rates are normalized to the third baseline measurement point for n = 3–5 replicates per group from 2 separate cultures.
Mentions: N2A hippocampal neuroblastoma cells were transiently co-transfected with constructs containing mutant APP(swe)/PS1(ΔE9) and a tetracycline transactivator (TTA) as a cell-based model of effects of mutant amyloid toxicity. Co-transfected (transfected) or TTA-transfected (control) N2A cells were pre-incubated ± nicotinamide adenine dinucleotide (NAD+) and oxygen consumption rates (OCR) measured. Transfected N2A cells (no exogenous NAD+; blue dashed line) had decreased maximal OCR when challenged with uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) and pyruvate, compared to control cells + NAD+ (24% decrease; Figure 1, red solid line); or control cells without exogenous NAD+ addition (18% decrease; Figure 1, red dashed line). This OCR deficit (Figure 1, blue dashed line) could be ameliorated if exogenous NAD+ was present (27% increase; Figure 1, blue solid line). Control N2A cultures had similar OCR regardless of NAD+ addition (Figure 1, red solid versus red dashed line). These experiments suggest that deficiencies in NAD+ levels could play a role in mitochondrial respiratory dysfunction in transgenic mice expressing this mutation in vivo [14] and could be corrected with added NAD+.Figure 1

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