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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 fission/fusion proteins in brain mitochondria isolated from mouse brain following NMN treatment. (A) Representative Western blot images of fission (DRP1 and phosphorylated form P616-DRP1) and fusion (OPA1 and MFN2) proteins. (B-F) Graphs showing quantification of the Western blots in (A). The OPA1 oligomeric complex comprises the long (L) fusogenic form and the short (S) form [32]. Voltage-dependent anion channel (VDAC) was utilized as a loading control. Data are presented as the average DRP, P616-DRP1, OPA1L OPA1S, MFN2 ± SE. N = 5–7 separate animals per group. **p < 0.05.
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Fig7: Immunoreactivity of fission/fusion proteins in brain mitochondria isolated from mouse brain following NMN treatment. (A) Representative Western blot images of fission (DRP1 and phosphorylated form P616-DRP1) and fusion (OPA1 and MFN2) proteins. (B-F) Graphs showing quantification of the Western blots in (A). The OPA1 oligomeric complex comprises the long (L) fusogenic form and the short (S) form [32]. Voltage-dependent anion channel (VDAC) was utilized as a loading control. Data are presented as the average DRP, P616-DRP1, OPA1L OPA1S, MFN2 ± SE. N = 5–7 separate animals per group. **p < 0.05.

Mentions: The dynamin-related protein 1 (DRP1) with mitochondrial fission 1 protein (Fis1) control mitochondrial fragmentation. When DRP1 immunoreactivity was assessed in isolated brain mitochondria, there was a trend toward an increase in DRP1 immunoreactivity in AD-Tg mice as compared to NTG animals (p = 0.072) that was not changed in the NMN-treated cohort (Figure 7A, B). When DRP1 is phosphorylated at a specific serine residue (Ser616, P616-DRP1), stabilization of the outer membrane of cytoplasmic DRP1 occurs [31]. There was a significant (p < 0.05) increase in P616-DRP1 immunoreactivity in mitochondria isolated from AD-Tg mice as compared to NTG animals (Figure 7A, C). The AD-Tg mice treated with NMN had significantly (p < 0.05) decreased P616-DRP1 immunoreactivity as compared to AD-Tg vehicle-treated animals (Figure 7A, C). There was no significant difference in mitochondrial P616-DRP1 immunoreactivity between NTG animals and AD-Tg mice treated with NMN (Figure 7A, C).Figure 7


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 fission/fusion proteins in brain mitochondria isolated from mouse brain following NMN treatment. (A) Representative Western blot images of fission (DRP1 and phosphorylated form P616-DRP1) and fusion (OPA1 and MFN2) proteins. (B-F) Graphs showing quantification of the Western blots in (A). The OPA1 oligomeric complex comprises the long (L) fusogenic form and the short (S) form [32]. Voltage-dependent anion channel (VDAC) was utilized as a loading control. Data are presented as the average DRP, P616-DRP1, OPA1L OPA1S, MFN2 ± SE. N = 5–7 separate animals per group. **p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4358858&req=5

Fig7: Immunoreactivity of fission/fusion proteins in brain mitochondria isolated from mouse brain following NMN treatment. (A) Representative Western blot images of fission (DRP1 and phosphorylated form P616-DRP1) and fusion (OPA1 and MFN2) proteins. (B-F) Graphs showing quantification of the Western blots in (A). The OPA1 oligomeric complex comprises the long (L) fusogenic form and the short (S) form [32]. Voltage-dependent anion channel (VDAC) was utilized as a loading control. Data are presented as the average DRP, P616-DRP1, OPA1L OPA1S, MFN2 ± SE. N = 5–7 separate animals per group. **p < 0.05.
Mentions: The dynamin-related protein 1 (DRP1) with mitochondrial fission 1 protein (Fis1) control mitochondrial fragmentation. When DRP1 immunoreactivity was assessed in isolated brain mitochondria, there was a trend toward an increase in DRP1 immunoreactivity in AD-Tg mice as compared to NTG animals (p = 0.072) that was not changed in the NMN-treated cohort (Figure 7A, B). When DRP1 is phosphorylated at a specific serine residue (Ser616, P616-DRP1), stabilization of the outer membrane of cytoplasmic DRP1 occurs [31]. There was a significant (p < 0.05) increase in P616-DRP1 immunoreactivity in mitochondria isolated from AD-Tg mice as compared to NTG animals (Figure 7A, C). The AD-Tg mice treated with NMN had significantly (p < 0.05) decreased P616-DRP1 immunoreactivity as compared to AD-Tg vehicle-treated animals (Figure 7A, C). There was no significant difference in mitochondrial P616-DRP1 immunoreactivity between NTG animals and AD-Tg mice treated with NMN (Figure 7A, C).Figure 7

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