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Ablation of Cytochrome c in Adult Forebrain Neurons Impairs Oxidative Phosphorylation Without Detectable Apoptosis

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Abstract

Cytochrome c (Cyt c), a heme-containing mitochondrial protein, has a critical function in both respiration and apoptosis. Consistent with these vital functions, somatic Cyt c mouse knockout is embryonic lethal. In order to investigate the sensitivity of postnatal neurons to Cyt c depletion, we developed a neuron-specific conditional knockout model. Neuron-specific Cyt c KO mouse (nCytcKO) was created by crossing the floxed Cyt c mouse with a CamKIIα-cre transgenic mouse, which deletes the floxed alleles postnatally. nCytcKO mice were normal at birth but developed an abnormal phenotype starting at 8 weeks of age with weight loss, tremor, decreased sensorimotor coordination, and sudden death between 12 and 16 weeks. Histological analysis did not show major neuronal degeneration. Analyses of oxidative phosphorylation showed a specific reduction in complex IV levels. Markers of oxidative stress were also increased. This novel model showed that neuronal complex IV is destabilized in the absence of Cyt c. It also showed that ablation of Cyt c in neurons leads to severe behavioral abnormalities and premature death without detectable neuronal loss, suggesting that neurons have the potential to survive for extended periods of time without a functional OXPHOS.

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Abbreviations

3-MT:

3-Methoxytyramine/3-methoxy-4-hydroxyphenethylamine

Apaf1:

Apoptotic protease activating factor 1

BAD:

Bcl-2-associated death promoter

BAX:

Bcl-2-associated X protein

Bcl2:

B cell lymphoma 2

BN-PAGE:

Blue native polyacrylamide gel electrophoresis

CamKIIα:

Calcium/calmodulin-dependent protein kinase II-α

Cyt c :

Cytochrome c

CytcTG :

Transgene

CytcHET :

Heterozygous

nCytcKO :

Neuronal KO

COX:

Cytochrome c oxidase, complex IV

DAPI:

4′,6-Diamidino-2-phenylindole

DOPAC:

3,4-Dihydroxyphenylacetic acid

ETC:

Electron transport chain

GFAP:

Glial fibrillary acidic protein

GPX1:

Glutathione peroxidase 1

H&E:

Hematoxylin-eosin

HVA:

Homovanillic acid

mtDNA:

Mitochondrial DNA

NADH:

Nicotinamide adenine dinucleotide

NDUFB8:

NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8

OXPHOS:

Oxidative phosphorylation

ROS:

Reactive oxygen species

SDHA:

Succinate dehydrogenase complex, subunit A

SOD1, SOD2:

Superoxide dismutase 1/2

TUJ1:

Neuron-specific Class III β-tubulin

TUNEL:

Terminal deoxynucleotidyl transferase dUTP nick end labeling

UQCRFS1:

Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1

VDAC1:

Voltage-dependent anion channels

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Funding

This work was supported by the National Institutes of Health Grants 1R01NS079965, 1R01AG036871, and 5R01EY010804 (CTM). This study was supported by the NEI center grant P30-EY014801 from the National Institutes of Health (NIH).

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Authors

Contributions

M.P. designed, performed experiments, and wrote the manuscript; U.V. designed and performed experiments and edited the manuscript; F.D. and S.P.M. performed experiments and edited the manuscript; C.T.M. designed experiments and edited manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Milena Pinto or Carlos T. Moraes.

Ethics declarations

All experiments and animal husbandry were performed according to a protocol approved by the University of Miami Institutional Animal Care and Use Committee.

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The authors declare that they have no competing interests.

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All data generated or analyzed during this study are included in this published article [and its supplementary information files].

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The authors declare no competing interests.

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Pinto, M., Vempati, U.D., Diaz, F. et al. Ablation of Cytochrome c in Adult Forebrain Neurons Impairs Oxidative Phosphorylation Without Detectable Apoptosis. Mol Neurobiol 56, 3722–3735 (2019). https://doi.org/10.1007/s12035-018-1335-y

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  • DOI: https://doi.org/10.1007/s12035-018-1335-y

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