Critical age-related loss of cofactors of neuron cytochrome C oxidase reversed by estrogen

Abstract

The mechanistic basis for the correlation between mitochondrial dysfunction and neurodegenerative disease is unclear, but evidence supports involvement of cytochrome C oxidase (CCO) deficits with age. Neurons isolated from the brains of 24 month and 9 month rats and cultured in common conditions provide a model of intrinsic neuronal aging. In situ CCO activity was decreased in 24 month neurons relative to 9 month neurons. Possible CCO-related deficits include holoenzyme activity, cofactor, and substrate. No difference was found between neurons from 24 month and 9 month rats in mitochondrial counts per neuron, CCO activity in submitochondrial particles, or basal respiration. Immunostaining for cytochrome C in individual mitochondria revealed an age-related deficit of this electron donor. 24 month neurons did not have adequate respiratory capacity to upregulate respiration after a glutamate stimulus, in spite of a two-fold upregulation of respiration seen in 9 month neurons. Respiration in 24 month neurons was inhibited by lower concentrations of potassium cyanide, suggesting a 50% deficit in functional enzyme in 24 month compared to 9 month neurons. In addition to cytochrome C, CCO requires cardiolipin to function. Staining with nonylacridine orange revealed an age-related deficit in cardiolipin. Treatment of 24 month neurons with 17-β-estradiol restored cardiolipin levels (10 ng/mL) and upregulated respiration under glutamate stress (1 pg/mL). Attempts to induce mitochondrial turnover by neuronal multiplication also rejuvenated CCO activity in 24 month neurons. These data suggest cytochrome C and cardiolipin levels are deficient in 24 month neurons, preventing normal upregulation of respiration needed for oxidative phosphorylation in response to stress. Furthermore, the data suggest this deficit can be corrected with estrogen treatment.

Decreased cytochrome c mediates an age-related decline of oxidative phosphorylation in rat kidney mitochondria

“…Further, fibroblasts isolated from individuals with early onset Parkinson’s disease and mutations in PINK1 were demonstrated to have decreased respiratory capacity related to a decrease in cytochrome c content [29]. Cultured neurons derived from 24 month rat brains appear to have reduced cytochrome c content and complex IV activity when compared with 9 month controls [30]. …”

Estrogen Receptors, the Hippocampus, and Memory

“…Furthermore, several transcriptional and post-translational feedback mechanisms control E2 activity, which likely contributes to the complex dose–response pattern. While the mechanism(s) underlying the reduced ability of ERT to provide cognitive protection with advancing age is unknown, it is known that E2 effects on several biological processes decline with age, including the ability to preserve the blood-brain barrier (Bake and Sohrabji 2004), protect the brain from stroke (Suzuki and others 2009) and neuroinflammation (Benedusi and others 2012), promote growth of glutamatergic synapses (Adams and others 2001; Aenlle and Foster 2010), influence mitochondrial function (Aenlle and Foster 2010; Jones and Brewer 2009), and activate rapid signaling cascades (Fan and others 2010; Foster 2005; Wu and others 2013). …”

Age-related deficiencies in complex I endogenous substrate availability and reserve capacity of complex IV in cortical neuron electron transport

“…Many potential pathways of energetic failure have been considered [2]. Among these mechanisms are oxidation of nucleic acids [3, 4, 5, 6], calcium dysregulation [7, 8, 9, 10], redox imbalance [11, 12, 13], reactive oxygen species (ROS) attacks [14, 15, 16], and oxidative phosphorylation deficits [17, 18, 19, 20]. Because the availability of energy from oxidative phosphorylation is so critical to neuron function, here, we investigated further the loss of oxidative phosphorylation by controlling the substrate availability to neurons in situ.…”

“…Brewer [36] showed that neurons cultured from different ages of rats demonstrate distinct age-related susceptibility to lactate, glutamate, and beta-amyloid. Live neurons isolated from the aging brain environment can be monitored in their endogenous state [17], or permeabilized to allow substrate control and pharmacologic isolation of complexes of the electron transport chain [33]. …”

“…In a previous study, we found that an age-related deficit in cytochrome C oxidase (complex IV) in whole cells at endogenous levels of cytochrome c was not apparent in substrate-supplemented submitochondrial particles, and that deficits in cardiolipin and upregulation of respiration in response to stress were corrected by estrogen treatment [17]. In this study, we expanded our methods to include substrate supplementation in whole cells, and we studied the three upstream respiratory complexes, NADH-ubiquinone oxidoreductase (complex I), succinate dehydrogenase (complex II), and cytochrome bc1 oxidoreductase (complex III).…”