Mitochondrial encephalomyopathies and cytochrome c oxidase deficiency: muscle culture study

Summary The populations of cytochrome c oxidase (CCO)-positive and-negative mitochondria were analyzed in the elongated cells containing occasional multiple nuclei (myotubes) in primary muscle cultures derived from patients with various forms of mitochondrial encephalomyopathies with CCO deficiency. Even in control muscle cultures, CCO-positive (79.7%) and -negative (20.3%) mitochondria were distributed randomly, showing intracellular mosaicism. All mitochondria in all muscle cultures from two patients with clinical characteristics of Leigh's disease exhibited faint to negative CCO activity. In these patients no enzyme activity could be detected in any tissue including intrafusal fibers and fibroblasts in muscle biopsies. In patients with the fatal infantile and the encephalomyopathic forms of CCO deficiency, and myoclonic epilepsy with ragged-red fibers, two different types of myotubes containing mostly CCO-positive mitochondria and only negative mitochondria, respectively, representing intercellular mosaicism, were demonstrated. The intercellular mosaicism in biopsied and cultured muscles in the case of CCO deficiency supports the contention that both CCO-positive and -negative mitochondria coexist in the early myogenic cell and later randomly segregated during cell division (mitotic segregation), forming two different cells.Support in part by Grants-in-Aid for Scientific Research (No. 0267032), Scientific Research on Priority Areas (No. 62617523) and Developmental Scientific Research (No. 62870041) from the Ministry of Education, Science and Culture of Japan     

Fatal infantile mitochondrial myopathy due to cytochrome c oxidase deficiency

A case of cytochrome c oxidase deficiency primarily affecting skeletal muscle is described. The child was admitted at 4 weeks due to failure to thrive and examination at that time revealed weakness and hypotonia. His condition deteriorated until at 11 weeks respiratory arrest necessitated artificial ventilation and death occurred at 14 weeks. Biochemical investigation showed lactic acidaemia and generalised aminoaciduria. Histochemical examination of muscle obtained at biopsy showed strong reactions for some oxidative enzymes, but by contrast cytochrome c oxidase could not be detected. Cytochrome c oxidase activity was less than 5% of control values in an extract of fresh muscle. The reduced-minus oxidised absorption spectra of muscle mitochondrial fractions prepared from postmortem tissue showed an absence of cytochrome aa₃ and a partial deficiency of cytochrome b. Ultra-structural examination showed abnormal mitochondria with loss of cristae and an abnormal granular matrix. The family history suggests autosomal recessive inheritance.     

N-Acetylcysteine elicited increase in cytochrome c oxidase activity in mice synaptic mitochondria

It has been suggested that thiolic groups are essential for cytochrome c oxidase (COX) activity and other respiratory mitochondrial enzymes. Recent experiments showed that the thiolic antioxidant N-acetylcysteine (NAC) can protect against age-related impairment in COX activity in mice hepatic mitochondria. The present paper shows that NAC enhances COX activity in vitro in synaptic mitochondria isolated from young and old mice. The optimum NAC concentration for maximum COX activity was 5 mM in young and 10 mM in old synaptic preparations. Our data suggest that mitochondrial thiolic groups, which are essentials to oxidative phosphorylation, are impaired by aging.     

Cytochrome c oxidase deficient fibres in the limb muscle and diaphragm of man without muscular disease: An age-related alteration

Cytochromec oxidase (complex IV of the respiratory chain) was studied histochemically in human limb muscle (n = 109) and diaphragm (n = 115) obtained at autopsy revealing randomly distributed muscle fibres without enzyme activity. The defects were present both in normal type I and type II fibres and in ragged red like fibres with increased content of mitochondria. In both organs an age associated manifestation of the defect was observed. First defects occurred sporadically in the 3rd and 4th decade, but were present from the 6th to 9th decade in 66–83% of the limb muscles and 75–100% of the diaphragms. Also the number of defects/cm² (defect density) increased with age from approx. 5, and 7 in limb muscle and diaphragm below the 6th decade to 54 and 60 defects in the 8th–9th decade (P = 0,000). Between both muscles no statistically significant difference in defect density(P > 0.15) existed. Irrespective of the defect density the defect typically affected isolated fibres showing normal histochemical reactivity for succinate dehydrogenase (complex II). The results indicate that cytochromec oxidase deficient muscle fibres in normal skeletal muscle represent an age related phenomenon which probably results from cellular ageing and might be involved in the reduction of muscle mass and strength during senescence.     

Tubular aggregates and partial cytochrome c oxidase deficiency in skeletal muscle of patients with AIDS treated with zidovudine

Summary We report on two patients, who had myalgias while receiving long-term zidovudine treatment for an HIV infection, in whom muscle biopsy findings included a partial cytochrome c oxidase (CCO) deficiency, a feature of zidovudine myopathy, and tubular aggregates, a finding hitherto unreported in HIV-infected patients. The CCO deficit was observed in 28% and 24% of muscle fibers, respectively. Tubular aggregates were the prominent histopathological feature in patient 1, and were detected by systematic electron microscopy in patient 2. Inflammation and myonecrosis were not detected. In patient 1, the typical mitochondrial and myofibrillar changes of zidovudine myopathy were present and 12% of fibers showed tubular aggregates. The aggregates were not stained at CCO reaction, and 96% of myofibers enclosing tubular aggregates showed a decreased CCO activity. This suggested more than a chance association between mitochondrial dysfunction and the formation of tubular aggregates. We conclude that tubular aggregates are detected in some patients treated by zidovudine, and that the finding could be related to the long-term administration of the drug.     

Alterations in cytochrome c oxidase activity and energy metabolites in response to kainic acid-induced status epilepticus

The effects of kainic acid (KA)-induced limbic seizures have been investigated on cytochrome c oxidase (COx) activity, COx subunit IV mRNA abundance, ATP and phosphocreatine (PCr) levels in amygdala, hippocampus and frontal cortex of rat brain. Rats were killed either 1 h, three days or seven days after the onset of status epilepticus (SE) by CO₂ and decapitation for the assay of COx activity and by head-focused microwave for the determination of ATP and PCr. Within 1 h COx activity and COx subunit IV mRNA increased in all brain areas tested between 120% and 130% of control activity, followed by a significant reduction from control, in amygdala and hippocampus on day three and seven, respectively. In amygdala, ATP and PCr levels were reduced to 44% and 49% of control 1 h after seizures. No significant recovery was seen on day three or seven. Pretreatment of rats with the spin trapping agent N-tert-butyl-α-phenylnitrone (PBN, 200 mg kg⁻¹, i.p.) 30 min before KA administration had no effect on SE, but protected COx activity and attenuated changes in energy metabolites. Pretreatment for three days with the endogenous antioxidant vitamin E (Vit-E, 100 mg/kg, i.p.) had an even greater protective effect than PBN. Both pretreatment regimens attenuated KA-induced neurodegenerative changes, as assessed by histology and prevention of the decrease of COx subunit IV mRNA and COx activity in hippocampus and amygdala, otherwise seen following KA-treatment alone. These findings suggest a close relationship between SE-induced neuronal injury and deficits in energy metabolism due to mitochondrial dysfunction.     

Proline reduces brain cytochrome c oxidase: prevention by antioxidants

In the present study, we initially investigated the in vivo (acute and chronic) and in vitro effects of proline on cytochrome c oxidase (complex IV) activity in rat cerebral cortex to test the hypothesis that proline might alter energy metabolism and that this alteration could be provoked by oxidative stress. The action of alpha-tocopherol and ascorbic acid on the effects produced by proline was also evaluated. For acute administration, 29- and 60-day-old rats received one subcutaneous injection of proline (18.2 micromol/g body weight) or an equivalent volume of 0.9% saline solution (control) and were sacrificed 1h later. For chronic treatment, proline was injected subcutaneously twice a day at 10h intervals from the 6(th) to the 28(th) day of age. Rats were sacrificed 12h (29(th)) or 31 days (60(th)) after the last injection. Results showed that acute administration of proline significantly diminished the activity of cytochrome c oxidase in the cerebral cortex of 29- and 60-day-old rats. On the other hand, chronic hyperprolinemia reduced this complex activity only on day 29, but not on the 60(th) day of life. In another set of experiments, 22-day-old rats or 53-day-old rats were pretreated for 1 week with daily intraperitoneal administration of alpha-tocopherol (40 mg/kg) and ascorbic acid (100mg/kg) or saline. Twelve hours after the last antioxidant injection, rats received a single injection of proline or saline and were killed 1h later. In parallel to chronic treatment, rats received a daily intraperitoneal injection of alpha-tocopherol and ascorbic acid from the 6(th) to the 28(th) day of life and were killed 12h after the last injection. Results showed that the pretreatment with alpha-tocopherol and ascorbic acid before acute proline administration or concomitant to chronic proline administration significantly prevented these effects. We also observed that proline (3.0 microM-1.0 mM) when added to the incubation medium (in vitro studies) did not alter cytochrome c oxidase activity. Data suggest that the inhibitory effect of proline on cytochrome c oxidase activity is possibly associated with oxidative stress and that this parameter may be involved in the brain dysfunction observed in hyperprolinemia.     

Mosaicism of mitochondria in mitochondrial myopathy: an electronmicroscopic analysis of cytochrome c oxidase

Summary Electron microscopic histochemistry was applied to the study of cytochrome c oxidase activity in each mitochondrion of biopsied muscles from four patients with mitochondrial myopathy [one case of fatal infantile mitochondrial myopathy, one case of myoclonus epilepsy associated with ragged-red fibers (MERRF), and two cases of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS)]. In the patient with fatal infantile mitochondrial myopathy, intercellular heterogeneity of mitochondria was recognized. In the three patients with either MERRF or MELAS, cytochrome c oxidase activity was segmentally changed from positive to negative within single muscle fibers. In the two patients with MELAS, small groups of positive-stained mitochondria were located among negative-stained mitochondria in the negative segment of a few muscle fibers. These findings revealed that there were heterogeneous populations of normal and abnormal mitochondria intracellularly or intercellularly within the muscles of these patients.Supported in part by Grant-in-Aid for Scientific Research 63570422 from the Ministry of Education, Science and Culture, and Grant 62A-5-08 from the National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare, Japan     

In situ hybridization of muscle mitochondrial mRNA in mitochondrial myopathies

Summary To determine whether a mitochondrial mRNA deficiency exists in mitochondrial myopathies, muscle biopsies from a patient with chronic progressive external ophthalmoplegia (CPEO) and a patient with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) were studied using in situ hybridization. Histochemistry and immunohistochemistry were performed along with hybridization. Hybridization reactions were widely distributed over the sarcoplasm of all muscle fibers in the patient with MELAS. In the patient with CPEO, 80% of the fibers showed a marked decrease in density of autoradiographic grains. This marked decrease corresponded to the histochemical and immunohistochemical findings of a very weak staining of cytochromec oxidase (CCO). The isotope-labeled cDNA probe used in in situ hybridization in this study complements a part of subunit I of CCO and a part of subunit II of complex I in the mitochondrial gene. Our results suggest a defect in the mRNA in this CPEO patient.Supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education and by a grant (62-2-05) from the National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare, Japan     

Mitochondrial myopathy: correlation between oxidative defect and mitochondrial DNA deletions at single fiber level

In situ hybridization combined with immunohistochemical techniques has been applied to study patients affected by mitochondrial myopathies with large mitochondrial (mt)DNA deletions. All patients' muscle biopsies showed ragged red fibers (RRFs) and cytochrome oxidase (COX) deficiency. Two digoxygenin-labeled, polymerase chain reaction (PCR)-amplifed DNAs were used as probes. One probe was designed to hybridize only with wild-type mtDNAs, while the other recognized both wild-type and deleted mtDNAs. Concomitant immunocytochemical analysis using antibodies against subunits II, III, (encoded by mtDNA) and IV (encoded by nuclear DNA) of COX was carried out. In our patients deleted mtDNAs are overexpressed in COX-negative RRFs, while wild-type mtDNAs are decreased in the same fibers. Immunohistochemistry studies show that COX IV is overexpressed in RRFs and that COX II and COX III subunits are still present. Deleted mtDNAs are spatially segregated in muscle fibers, where they interfere with the local population of normal mitochondrial genomes, causing a regional deficiency of the mitochondrial respiratory activity.This work was supported by the Associazione Amici del Centro Dino Ferrari     

Chronic progressive external ophthalmoplegia: A correlative study of mitochondrial DNA deletions and their phenotypic expression in muscle biopsies

Deleted mitochondrial DNA (mtDNA) has been shown to coexist with normal mtDNA (heteroplasmy) in muscles from chronic progressive external ophthalmoplegia, including Kearns-Sayre syndrome. In this study, we correlated heteroplasmic mtDNA abnormality with clinical, biochemical and histological findings with the following results: (1) large deletions ranging from 1.8 to 8.8 kb in 22 muscle specimens from 28 patients who had ophthalmoplegia clinically and focal cytochromec oxidase (CCO) deficiency by histochemistry, (2) no difference in clinical and biochemical findings between patients with and without mtDNA deletions, (3) no relationship between the size, site or populations of deleted mtDNA and respiratory chain enzyme activities in muscles, (4) positive correlation between the number of CCO-deficient fibers and the populations of deleted mtDNA, and (5) higher incidence of CCO-negative fibers in patients with deleted mtDNA than in those with no deletion of mtDNA. These results suggest that deleted mtDNA is, at least in part, responsible for focal CCO deficiency as a phenotypic expression and that the investigation on pathogenetic mechanism of focal CCO deficiency may provide a clue to understanding the underlying pathophysiology in this disorder.     

Quantitative evaluation of electron transport system proteins in mitochondrial encephalomyopathy

Summary The levels of mitochondrial electron transport system proteins cytochrome c oxidase (COX) and complex III were measured in muscle fibers of patients with mitochondrial encephalomyopathy using quantitative immunoelectron microscopy. In a patient with Leigh's encephalopathy, immunoreactive COX protein was decreased to 20% of the normal mean value in all muscle fibers examined, while the amount of complex III was within the normal range. In a patient with fatal infantile COX deficiency, the level of COX protein was found to be decreased to 27–40% of the normal value in all muscle fibers examined. In patients with mitochondrial myopathy, encephalopathy, lactic acidosis associated with stroke-like episodes (MELAS) and chronic progressive external ophthalmoplegia (CPEO), COX protein levels were decreased to 20% of normal in muscle fibers lacking COX activity. In normal fibers, however, COX protein levels were also normal. The amount of complex III protein was normal in COX-deficient muscle fibers. In two patients, in situ hybridization was performed for detection of mitochondrial mRNA. Mitochondrial mRNAs were found to be abundant in muscle fibers with decreased COX protein, suggesting a defect at the mitochondrial protein-synthesis level in a COX-deficient muscle fiber.Supported in part by a Grant-in-Aid for Scientic Research No. 63570422 from the Ministry of Education, Science and Culture, and Grant No. 32A-5-08 from the National Center of Neurology and Psychiatry of the Ministry of Health and Welfare, Japan     

Muscle pathology in cytochromec oxidase deficiency

Summary Muscle biopsies from 16 patients with cytochromec oxidase (CCO) deficiency were examined morphologically. Two siblings had the fatal infantile form. The muscle of the older sister at the age of 5 months had numerous ragged-red fibers (RRF) and increased numbers of lipid droplets; at 28 days the brother had no RRF suggesting that the RRF formed later than 28 days. The muscle pathology in two patients with the benign infantile form improved as they grew older; numbers of RRF, lipid droplets and glycogen particles decreased and CCO activity increased in the second biopsy. In the encephalomyopathic form, RRF were seen in 5 of 12 muscles mostly in patients more than 6 years of age. Muscle spindles and blood vessel walls in the biopsies from three patients with rapid clinical aggravation had no CCO activity, suggesting that enzyme activity differed from tissue to tissue (tissue specificity).Supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (No. 62617523) from the Ministry of Education, Science and Culture of Japan     

Multiple mitochondrial DNA deletions in hereditary inclusion body myopathy

We have recently described an autosomal dominant hereditary inclusion body myopathy (h-IBM). Clinically it is is characterized by congenital joint contractures and slowly progressive, proximal muscle weakness and ophthalmoplegia. There is deterioration of muscle function between 30 and 50 years of age. While young patients show minor pathological changes in muscle, the middle-aged and old patients show rimmed vacuoles and inclusions of filaments measuring 15–18 nm in diameter. Except for the absence of significant inflammation the histopathology is similar to that found in sporadic inclusion body myositis (s-IBM). In s-IBM mitochondrial alterations including cytochrome c oxidase (COX) -deficient muscle fibers are common. These are due to multiple mitochondrial DNA (mtDNA) deletions. In this study we investigated the occurrence of mitochondrial alterations in autosomal dominant h-IBM. Young affected individuals showed no mitochondrial changes but three patients aged 38, 51 and 59 years, respectively, showed ragged red fibers and COX-deficient muscle fibers. Polymerase chain reaction analysis showed multiple mtDNA deletions. By in situ hybridization clonal expansions of mtDNA with deletions were demonstrated in COX-deficient muscle fibers. Most of the analyzed deletion breakpoints showed nucleotide repeats flanking the deletions. The results show that COX-deficient muscle fibers and somatic mtDNA deletions are present in this family with h-IBM. The same factors may be involved in the development of mtDNA deletions in s-IBM and this family with h-IBM. Received: 13 July 1999 / Revised: 6 October 1999 · Accepted: 12 October 1999     

Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia

The neuroprotective effect of Ginkgo biloba extract (EGb 761) against ischemic injury has been demonstrated in animal models. In this study, we compared the protective effect of bilobalide, a purified terpene lactone from EGb 761, and EGb 761 against ischemic injury. We measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global forebrain ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected CA1 neurons from death and from ischemia-induced reductions in COX III mRNA. In addition, both bilobalide and EGb 761 protected against ischemia-induced reductions in COX III mRNA in CA1 neurons prior to their death, at 1 day of reperfusion. These results suggest that oral administration of bilobalide and EGb 761 protect against ischemia-induced neuron death and reductions in mitochondrial gene expression.     

Regional expression of Bcl-2 mRNA and mitochondrial cytochrome c release after experimental brain injury in the rat

Regional levels of anti-apoptotic Bcl-2 mRNA and the cytosolic cytochrome c protein were measured after lateral fluid percussion (FP) brain injury in rats. Levels of Bcl-2 mRNA were significantly decreased in the injured left cortex (IC) and ipsilateral hippocampus (IH), but not in the contralateral right cortex (CC) and hippocampus (CH) after brain injury. Levels of Bcl-2 mRNA were significantly decreased as early as 2 h and stayed decreased as long as 48 h in the IC and IH after injury. Levels of the cytosolic cytochrome c protein were significantly increased in the IC and IH, but not in the CC and CH after brain injury. Levels of cytosolic cytochrome c were significantly increased in the IC at 30 min, 48 and 72 h, and in the IH at 2 h and as long as 72 h after injury. The increase of cytosolic cytochrome c suggests that the mitochondrial release of cytochrome is increased in the IC and IH after lateral FP brain injury. These data show that the reduction of anti-apoptotic Bcl-2 and increases of mitochondrial release of cytochrome c protein occur only in the IC and IH, regions which have been observed to undergo apoptosis and neuronal cell loss after lateral FP brain injury. Therefore, it is likely that the reduction of Bcl-2 and the increased cytochrome c protein in the cytosol contribute to the observed apoptosis and neuronal cell death in the IC and IH after lateral FP brain injury in rats.     

Cytochromec oxidase deficiency in infancy

Summary Five children with early onset of muscle weakness, lactic acidosis and deficient cytochromec oxidase staining in the muscle biopsy were studied. By oximetric assay of the respiratory chain of isolated mitochondria, cytochromec oxidase deficiency was confirmed in four of the cases, while one case showed only a slight decrease of cytochromec oxidase activity but considerably reduced activity when assayed spectrophotometrically. The muscle biopsies exhibited mitochondrial structural abnormalities and lipid storage in the four cases with oximetrically confirmed cytochromec oxidase deficiency, while the biopsy of the case with markedly reduced activity of cytochromec oxidase only in the enzyme-histochemical and spectrophotometrical assays had normal morphology. The light microscopical staining of cytochromec oxidase in the four cases with oximetrically confirmed deficiency showed deficient staining of the enzyme in all extrafusal fibres in three cases but one of the cases had normal enzyme-histochemical activity of cytochromec oxidase in about 25% of the fibres. In two cases muscle spindles were included in the biopsy. The intrafusal fibres showed normal enzyme-histochemical activity of cytochromec oxidase. Ultrastructural examination of the enzyme distribution in two of the cases revealed great heterogeneity of the mitochondria. The structurally abnormal mitochondria were usually deficient of enzyme activity. The mitochondria of endothelial cells appeared to have normal activity. Immunohistochemical staining with polyclonal antibodies to cytochromec oxidase revealed presence of immunoreactive material corresponding to the localisation of mitochondria in all cases. The results show that enzyme-histochemical staining of cytochromec oxidase is a useful technique to reveal deficiency of the enzyme and to study the distribution of the deficiency within the tissue both at the light microscopical and ultrastructural levels. However, the results of one of the cases show that deficiency revealed by the enzyme-histochemical technique is not completely reliable. Oximetric studies on isolated mitochondria are necessary to confirm the suspected deficiency and to reveal combined defects of the respiratory chain.Supported by grants from the Swedish Medical Research Council (proj. 03X 585 and 07122), Barnhusfonden Göteborg L 174/87 and The First of May Flower Annual Campaign for Childrens Health     

Decreased platelet cytochrome c oxidase activity is accompanied by increased blood lactate concentration during exercise in patients with Alzheimer disease

Increasing evidence indicates that mitochondrial dysfunction occurs in the central nervous system as well as in the peripheral tissues from Alzheimer's disease (AD) patients. We have recently shown that mitochondrial cytochrome c oxidase (COX) activity is significantly reduced in brain and platelets from AD patients compared to controls. In the present study we investigated whether impaired COX activity could have functional consequences on energy metabolism. Blood lactate concentration was monitored at rest and during incremental exercise in 22 AD patients in whom COX activity in platelets was decreased compared to controls (35.7 +/- 11.4 vs 48.4 +/- 1.4 nmol/min/mg, P < 0.01). In both resting and exercising conditions, blood lactate was significantly higher in AD patients than in controls. Although the magnitude of exercise-related lactate accumulation was not different between the two groups, an anticipated anaerobic lactate threshold during the incremental forearm exercise was found in AD patients (50% of maximal voluntary contraction MVC compared to 60% in controls). COX activity was inversely related to lactate at a significant level for resting condition (r = -0.65) and borderline for anaerobic threshold exercise level. These results support the hypothesis of a systemic impairment of the mitochondrial function in AD and indicate that decreased COX activity could have functional consequences on metabolism.     

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

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.