Ryan T. Hamilton

Mitochondrial bioenergetic deficit precedes Alzheimer's pathology in female mouse model of Alzheimer's disease

Mitochondrial dysfunction has been proposed to play a pivotal role in neurodegenerative diseases, including Alzheimers disease (AD). To address whether mitochondrial dysfunction precedes the development of AD pathology, we conducted mitochondrial functional analyses in female triple transgenic Alzheimers mice (3xTg-AD) and age-matched nontransgenic (nonTg). Mitochondrial dysfunction in the 3xTg-AD brain was evidenced by decreased mitochondrial respiration and decreased pyruvate dehydrogenase (PDH) protein level and activity as early as 3 months of age. 3xTg-AD mice also exhibited increased oxidative stress as manifested by increased hydrogen peroxide production and lipid peroxidation. Mitochondrial amyloid beta (Aβ) level in the 3xTg-AD mice was significantly increased at 9 months and temporally correlated with increased level of Aβ binding to alcohol dehydrogenase (ABAD). Embryonic neurons derived from 3xTg-AD mouse hippocampus exhibited significantly decreased mitochondrial respiration and increased glycolysis. Results of these analyses indicate that compromised mitochondrial function is evident in embryonic hippocampal neurons, continues unabated in females throughout the reproductive period, and is exacerbated during reproductive senescence. In nontransgenic control mice, oxidative stress was coincident with reproductive senescence and accompanied by a significant decline in mitochondrial function. Reproductive senescence in the 3xTg-AD mouse brain markedly exacerbated mitochondrial dysfunction. Collectively, the data indicate significant mitochondrial dysfunction occurs early in AD pathogenesis in a female AD mouse model. Mitochondrial dysfunction provides a plausible mechanistic rationale for the hypometabolism in brain that precedes AD diagnosis and suggests therapeutic targets for prevention of AD.

Progesterone and Estrogen Regulate Oxidative Metabolism in Brain Mitochondria

The ovarian hormones progesterone and estrogen have well-established neurotrophic and neuroprotective effects supporting both reproductive function and cognitive health. More recently, it has been recognized that these steroids also regulate metabolic functions sustaining the energetic demands of this neuronal activation. Underlying this metabolic control is an interpretation of signals from diverse environmental sources integrated by receptor-mediated responses converging upon mitochondrial function. In this study, to determine the effects of progesterone (P4) and 17beta-estradiol (E2) on metabolic control via mitochondrial function, ovariectomized rats were treated with P4, E2, or E2 plus P4, and whole-brain mitochondria were isolated for functional assessment. Brain mitochondria from hormone-treated rats displayed enhanced functional efficiency and increased metabolic rates. The hormone-treated mitochondria exhibited increased respiratory function coupled to increased expression and activity of the electron transport chain complex IV (cytochrome c oxidase). This increased respiratory activity was coupled with a decreased rate of reactive oxygen leak and reduced lipid peroxidation representing a systematic enhancement of brain mitochondrial efficiency. As such, ovarian hormone replacement induces mitochondrial alterations in the central nervous system supporting efficient and balanced bioenergetics reducing oxidative stress and attenuating endogenous oxidative damage.

Decline in mitochondrial bioenergetics and shift to ketogenic profile in brain during reproductive senescence.

BACKGROUND We have previously demonstrated that mitochondrial bioenergetic deficits precede Alzheimers pathology in the female triple transgenic Alzheimers (3xTgAD) mouse model. Herein, we sought to determine the impact of reproductive senescence on mitochondrial function in the normal non-transgenic (nonTg) and 3xTgAD female mouse model of AD. METHODS Both nonTg and 3xTgAD female mice at 3, 6, 9, and 12 months of age were sacrificed and mitochondrial bioenergetic profile as well as oxidative stress markers were analyzed. RESULTS In both nonTg and 3xTgAD mice, reproductive senescence paralleled a significant decline in PDH, and Complex IV cytochrome c oxidase activity and mitochondrial respiration. During the reproductive senescence transition, both nonTg and 3xTgAD mice exhibited greater individual variability in bioenergetic parameters suggestive of divergent bioenergetic phenotypes. Following transition through reproductive senescence, enzymes required for long-chain fatty acid (HADHA) and ketone body (SCOT) metabolism were significantly increased and variability in cytochrome c oxidase (Complex IV) collapsed to cluster at a approximately 40% decline in both the nonTg and 3xTgAD brain which was indicative of alternative fuel generation with concomitant decline in ATP generation. CONCLUSIONS These data indicate that reproductive senescence in the normal nonTg female brain parallels the shift to ketogenic/fatty acid substrate phenotype with concomitant decline in mitochondrial function and exacerbation of bioenergetic deficits in the 3xTgAD brain. GENERAL SIGNIFICANCE These findings provide a plausible mechanism for increased life-time risk of AD in postmenopausal women and suggest an optimal window of opportunity to prevent or delay decline in bioenergetics during reproductive senescence.

Ovarian hormone loss induces bioenergetic deficits and mitochondrial β-amyloid

Previously, we demonstrated that reproductive senescence was associated with mitochondrial deficits comparable to those of female triple-transgenic Alzheimers mice (3xTgAD). Herein, we investigated the impact of chronic ovarian hormone deprivation and 17β-estradiol (E2) replacement on mitochondrial function in nontransgenic (nonTg) and 3xTgAD female mouse brain. Depletion of ovarian hormones by ovariectomy (OVX) in nontransgenic mice significantly decreased brain bioenergetics, and induced mitochondrial dysfunction and oxidative stress. In 3xTgAD mice, OVX significantly exacerbated mitochondrial dysfunction and induced mitochondrial β-amyloid and β-amyloid (Aβ)-binding-alcohol-dehydrogenase (ABAD) expression. Treatment with E2 at OVX prevented OVX-induced mitochondrial deficits, sustained mitochondrial bioenergetic function, decreased oxidative stress, and prevented mitochondrial β-amyloid and ABAD accumulation. In vitro, E2 increased maximal mitochondrial respiration in neurons and basal and maximal respiration in glia. Collectively, these data demonstrate that ovarian hormone loss induced a mitochondrial phenotype comparable to a transgenic female model of Alzheimers disease (AD), which was prevented by E2. These findings provide a plausible mechanism for increased risk of Alzheimers disease in premenopausally oophorectomized women while also suggesting a therapeutic strategy for prevention.

SELECTIVE OESTROGEN RECEPTOR MODULATORS DIFFERENTIALLY POTENTIATE BRAIN MITOCHONDRIAL FUNCTION

The mitochondrial energy‐transducing capacity of the brain is important for long‐term neurological health and is influenced by endocrine hormone responsiveness. The present study aimed to determine the role of oestrogen receptor (ER) subtypes in regulating mitochondrial function using selective agonists for ERα (propylpyrazoletriol; PPT) and ERβ (diarylpropionitrile; DPN). Ovariectomised female rats were treated with 17β‐oestradiol (E2), PPT, DPN or vehicle control. Both ER selective agonists significantly increased the mitochondrial respiratory control ratio and cytochrome oxidase (COX) activity relative to vehicle. Western blots of purified whole brain mitochondria detected ERα and, to a greater extent, ERβ localisation. Pre‐treatment with DPN, an ERβ agonist, significantly increased ERβ association with mitochondria. In the hippocampus, DPN activated mitochondrial DNA‐encoded COX I expression, whereas PPT was ineffective, indicating that mechanistically ERβ, and not ERα, activated mitochondrial transcriptional machinery. Both selective ER agonists increased protein expression of nuclear DNA‐encoded COX IV, suggesting that activation of ERβ or ERα is sufficient. Selective ER agonists up‐regulated a panel of bioenergetic enzymes and antioxidant defence proteins. Up‐regulated proteins included pyruvate dehydrogenase, ATP synthase, manganese superoxide dismutase and peroxiredoxin V. In vitro, whole cell metabolism was assessed in live primary cultured hippocampal neurones and mixed glia. The results of analyses conducted in vitro were consistent with data obtained in vivo. Furthermore, lipid peroxides, accumulated as a result of hormone deprivation, were significantly reduced by E2, PPT and DPN. These findings suggest that the activation of both ERα and ERβ is differentially required to potentiate mitochondrial function in brain. As active components in hormone therapy, synthetically designed oestrogens as well as natural phyto‐oestrogen cocktails can be tailored to improve brain mitochondrial endpoints.

Medroxyprogesterone Acetate Antagonizes Estrogen Up-Regulation of Brain Mitochondrial Function

The impact of clinical progestins used in contraception and hormone therapies on the metabolic capacity of the brain has long-term implications for neurological health in pre- and postmenopausal women. Previous analyses indicated that progesterone and 17β-estradiol (E2) sustain and enhance brain mitochondrial energy-transducing capacity. Herein we determined the impact of the clinical progestin, medroxyprogesterone acetate (MPA), on glycolysis, oxidative stress, and mitochondrial function in brain. Ovariectomized female rats were treated with MPA, E2, E2+MPA, or vehicle with ovary-intact rats serving as a positive control. MPA alone and MPA plus E2 resulted in diminished mitochondrial protein levels for pyruvate dehydrogenase, cytochrome oxidase, ATP synthase, manganese-superoxide dismutase, and peroxiredoxin V. MPA alone did not rescue the ovariectomy-induced decrease in mitochondrial bioenergetic function, whereas the coadministration of E2 and MPA exhibited moderate efficacy. However, the coadministration of MPA was detrimental to antioxidant defense, including manganese-superoxide dismutase activity/expression and peroxiredoxin V expression. Accumulated lipid peroxides were cleared by E2 treatment alone but not in combination with MPA. Furthermore, MPA abolished E2-induced enhancement of mitochondrial respiration in primary cultures of the hippocampal neurons and glia. Collectively these findings indicate that the effects of MPA differ significantly from the bioenergetic profile induced by progesterone and that, overall, MPA induced a decline in glycolytic and oxidative phosphorylation protein and activity. These preclinical findings on the basis of acute exposure to MPA raise concerns regarding neurological health after chronic use of MPA in contraceptive and hormone therapy.

Elevated neuronal nitric oxide synthase expression during ageing and mitochondrial energy production

This study evaluated the effect of ageing on brain mitochondrial function mediated through protein post-translational modifications. Neuronal nitric oxide synthase increased with age and this led to a discreet pattern of nitration of mitochondrial proteins. LC/MS/MS analyses identified the nitrated mitochondrial proteins as succinyl-CoA-transferase and F1-ATPase; the latter was nitrated at Tyr269, suggesting deficient ADP binding to the active site. Activities of succinyl-CoA-transferase, F1-ATPase and cytochrome oxidase decreased with age. The decreased activity of the latter cannot be ascribed to protein modifications and is most likely due to a decreased expression and assembly of complex IV. Mitochondrial protein post-translational modifications were associated with a moderately impaired mitochondrial function, as indicated by the decreased respiratory control ratios as a function of age and by the release of mitochondrial cytochrome c to the cytosol, thus supporting the amplification of apoptotic cascades.

Oscillatory Shear Stress Induces Mitochondrial Superoxide Production: Implication of NADPH Oxidase and c-Jun NH2-Terminal Kinase Signaling

Fluid shear stress is intimately linked with vascular oxidative stress and atherosclerosis. We posited that atherogenic oscillatory shear stress (OSS) induced mitochondrial superoxide (mtO2•-) production via NADPH oxidase and c-Jun NH(2)-terminal kinase (JNK-1 and JNK-2) signaling. In bovine aortic endothelial cells, OSS (±3 dyn/cm2) induced JNK activation, which peaked at 1 h, accompanied by an increase in fluorescein isothiocyanate-conjugated JNK fluorescent and MitoSOX Red (specific for mtO2•- production) intensities. Pretreatment with apocynin (NADPH oxidase inhibitor) or N-acetyl cysteine (antioxidant) significantly attenuated OSS-induced JNK activation. Apocynin further reduced OSS-mediated dihydroethidium and MitoSOX Red intensities specific for cytosolic O2•- and mtO2•- production, respectively. As a corollary, transfecting bovine aortic endothelial cells with JNK siRNA (siJNK) and pretreating with SP600125 (JNK inhibitor) significantly attenuated OSS-mediated mtO2•- production. Immunohistochemistry on explants of human coronary arteries further revealed prominent phosphorylated JNK staining in OSS-exposed regions. These findings indicate that OSS induces mtO2•- production via NADPH oxidase and JNK activation relevant for vascular oxidative stress.

LDL protein nitration : Implication for LDL protein unfolding

Oxidatively- or enzymatically-modified low-density lipoprotein (LDL) is intimately involved in the initiation and progression of atherosclerosis. The in vivo modified LDL is electro-negative (LDL(-)) and consists of peroxidized lipid and unfolded apoB-100 protein. This study was aimed at establishing specific protein modifications and conformational changes in LDL(-) assessed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and circular dichroism analyses, respectively. The functional significance of these chemical modifications and structural changes were validated with binding and uptake experiments to- and by bovine aortic endothelial cells (BAEC). The plasma LDL(-) fraction showed increased nitrotyrosine and lipid peroxide content as well as a greater cysteine oxidation as compared with native- and total-LDL. LC/MS/MS analyses of LDL(-) revealed specific modifications in the apoB-100 moiety, largely involving nitration of tyrosines in the alpha-helical structures and beta(2) sheet as well as cysteine oxidation to cysteic acid in beta(1) sheet. Circular dichroism analyses showed that the alpha-helical content of LDL(-) was substantially lower ( approximately 25%) than that of native LDL ( approximately 90%); conversely, LDL(-) showed greater content of beta-sheet and random coil structure, in agreement with unfolding of the protein. These results were mimicked by treatment of LDL subfractions with peroxynitrite (ONOO(-)) or SIN-1: similar amino acid modifications as well as conformational changes (loss of alpha-helical structure and gain in beta-sheet structure) were observed. Both LDL(-) and ONOO(-)-treated LDL showed a statistically significant increase in binding and uptake to- and by BAEC compared to native LDL. We further found that most binding and uptake in control-LDL was through LDL-R with minimal oxLDL-R-dependent uptake. ONOO(-)-treated LDL was significantly bound and endocytosed by LOX-1, CD36, and SR-A with minimal contribution from LDL-R. It is suggested that lipid peroxidation and protein nitration may account for the mechanisms leading to apoB-100 protein unfolding and consequential increase in modified LDL binding and uptake to and by endothelial cells that is dependent on oxLDL scavenger receptors.