Mitochondrial Metabolic Competence Research Articles

Tipping the Balance in the Powerhouse of the Cell to “Protect” Colorectal Cancer

Tipping the Balance in the Powerhouse of the Cell to “Protect” Colorectal Cancer

Neuronal Apoptosis in Alzheimer's Disease

To assess the role of the mitochondrial metabolic competence (MMC) in the development of age-related changes, we measured the levels of immunohistochemically stained (IH) mitochondrial- and nuclear-encoded subunits of cytochrome oxidase (COX II and COX IV, respectively) and compared these data with mRNA in situ hybridization (ISH) of the same subunits and with cytochemically evidenced COX activity in the temporal (TC) and frontal (FC) cortex of adult and late-adult monkeys. Quantitative cytochemistry of COX activity was performed by calculating the ratio (R) of the area of the cytochemical precipitate to the area of the respective organelle. Although ISH studies showed reduced gene expression of both subunits in FC of late-adult monkeys, no significant age-related difference was found either in TC or FC when considering the IH data. R was significantly increased in FC of late-adult animals, and a quartile distribution of the mitochondrial area showed that R is higher in the FC of older animals independent of the organelle size. The assessment of COX genetic and phenotypic parameters reliably reports on MMC because this enzyme is the terminal complex of the electron transport chain. Taken together, the present IH, ISH, and R findings suggest that, with advancing age, compensating mechanisms are activated to preserve the mitochondrial functional metabolic capacities. Although significant mitochondrial defects are currently reported to occur in Alzheimer's disease pathogenesis, our data document that MMC is actively involved in the physiological rearrangement of the age-related neuronal network and may provide substantial metabolic support for the energy demand of neuronal apoptosis.

Selective Decline of the Metabolic Competence of Oversized Synaptic Mitochondria in the Old Monkey Cerebellum

The morphofunctional features of synaptic mitochondria, positive to the activity of cytochrome oxidase (COX), were investigated in the cerebellar cortex of adult and old monkeys (Macaca fascicularis) to assess the potential age-related changes in the energy metabolism occurring at the neuronal synaptic compartment. The following mitochondrial ultrastructural parameters-numeric density (Nv), volume density (Vv), average volume (V), and average length (Fmax)-were measured by computer-assisted morphometric methods. The ratio (R) area of the COX cytochemical precipitate/area of the mitochondrion was semi-automatically calculated and considered as an estimation of the mitochondrial metabolic competence (MMC), that is, the capacity of single organelles to provide adequate amounts of adenosinetriphosphate. No age-related significant differences were found in any of the ultrastructural parameters taken into account, whereas a significant decrease of R was observed in old animals. In these animals, the quartile distribution of the COX-positive organelles, according to their respective cross-sectional area, showed no significant difference of R when comparing small (I quartile), medium-sized (II quartile), and large (III quartile) mitochondria, while a significant decrease of R was evident in oversized mitochondria (IV quartile). Although our data document an age-related preservation of the morphological features of COX-positive mitochondria in the monkey cerebellum, the significant decrease of R in old animals needs to be considered from the functional standpoint. Since COX is the terminal enzyme of the mitochondrial respiratory chain, the estimation of its activity is regarded as a reliable MMC index; thus our findings, by matching preferential cytochemistry and morphometry, support the hypothesis that the specific functional impairment of enlarged synaptic mitochondria may seriously affect information processing and cell-to-cell communication at synaptic junctional areas with aging.

Brain aging: The zinc connection

At variance with other organs, where the functional and structural units are repeated, the brain is a composite assembly of groups of cells with different metabolic features and functional units. Deterioration of brain function occurs when the number of neurons or their connections decrease below a critical reserve level and coping with environmental stimulation is seriously hampered. Physiopathological alterations of the synaptic junctional areas are reported to play a central role in the process of brain aging. Current research is documenting an age-related numeric loss of synapses which is paired by a significant enlargement of the persisting contact zones: the final outcome of these balanced changes is a significant reduction of the overall synaptic junctional area per unit volume of neuropil. The progressive decline of the mitochondrial metabolic competence, i.e. the capacity of select pools of organelles to provide adequate amounts of adenosine triphosphate is supposed to represent a key determinant in synaptic aging. Cytochemical estimations of the activity of cytochrome oxidase confirm that mitochondrial dysfunctions play an early role in synaptic deterioration. Zinc ions act as physiological neuromodulators at glutamatergic synapses, however, in order to avoid neurotoxic damage, the intracellular free Zn++ concentration ([Zn++]i) must be tightly controlled by: (i) extrusion (Zn++ transporters); (ii) buffering (metallothioneins) and (iii) sequestration (mitochondria) systems. In physiological aging, if any of these systems is impaired and/or not adequately coordinated with the other two, the resulting significant rise of ([Zn++]i) may inhibit the cellular energy providing systems and affect mitochondria as primary target.

Testing Mitochondrial Metabolic Competence by Cytochrome Oxidase Preferential Cytochemistry Versus Immunoreactivity of Subunits I and IV

Cytochemically evidenced COX activity was compared with levels of immunohistochemically stained mitochondrial- and nuclear-encoded subunits (CO I and CO IV) in the dentate gyrus outer molecular layer (OML) and cerebellar granular layer (GL) of adult and old rats. COX activity decreased significantly in aging, whereas CO I and CO IV levels were significantly increased both in GL and OML of old animals. These findings suggest that the age-related decay of the mitochondrial metabolic competence is not caused by a reduction of COX subunits levels, but causal events affecting mitochondria as discrete morphofunctional units of the cellular bioenergetic machinery.

Synaptic and mitochondrial physiopathologic changes in the aging nervous system and the role of zinc ion homeostasis

Brain performances, e.g. learning and memory, decay during aging. Deterioration of synaptic junctions, as structural correlates of these key functions of the central nervous system, may play a central role in this impairment. Current research on the age-related changes of synapses is documenting that the numeric loss of contacts appears to trigger a compensatory reaction by the old CNS, i.e. the surviving junctional areas in old individuals are larger than in adult subjects. The final outcome of the balanced changes in synaptic number and size is that the overall synaptic junctional area per cubic micron of neuropil is also reduced in aging and this may account for the age-associated functional decay of CNS performances. Among the suggested determinants of synaptic deterioration in aging, a considerable number of recent studies support an early and pivotal role of the progressive decline of the mitochondrial metabolic competence, i.e. the capacity of select pools of organelles to provide adequate amounts of adenosine triphosphate. Quantitative ultrastructural studies together with cytochemistry of key enzymes of the respiratory chain (cytochrome oxidase and succinic dehydrogenase) have shown that mitochondrial dysfunctions play an early and central role in synaptic deterioration events associated with aging and neurodegenerative diseases. Among the various causes, the multiple mechanisms and molecules involved in zinc ion homeostasis have been supposed to be less efficient in the aging brain. Thus, a transient imbalance of free zinc ion concentration in the cytosol ([Zn 2+] i ) can be considered an unfavourable trigger of subtle mitochondrial damage and synaptic pathology.

Age-related decline in metabolic competence of small and medium-sized synaptic mitochondria

A computer-assisted morphometric investigation of cytochrome oxidase (COX) activity, selectively evidenced by preferential diaminobenzidine cytochemistry, has been carried out on synaptic mitochondria in the cerebellar cortex of adult and old rats. The ratio (R) of the area of the cytochemical precipitate (CPA) to the overall area of each mitochondrion (MA) was calculated. R refers to the fraction of the inner mitochondrial membrane actively involved in cellular respiration, thus its quantitative estimation constitutes a reliable index of the mitochondrial metabolic competence (MMC). In adult rats a significant negative correlation between MA and R values was found, while in old animals there was just a positive trend. Paired-quartile comparisons of R values showed a significant age-related decrease in small and medium-sized mitochondria, whereas the lowest and not significant age-related reduction was found in oversized organelles. A paired decrease in number and increase in size is reported to be a general trend for mitochondria during aging, but oversized organelles, according to their low R value, constitute a scanty, though functional, compensating reaction. Thus, the present findings support the argument that the currently reported age-related cellular metabolic decay appears to rely both on the decline in MMC of the small and medium-sized mitochondria, and on their specific reduction in number. This novel result is of biological relevance since it is largely the small and medium-sized mitochondria that are required for the provision of adequate amounts of ATP for actual cellular performance, while the significantly enlarged organelles are thought to represent an intermediate ultrastructural feature in mitochondrial genesis and/or remodelling.

Increased Mitochondrial and Nuclear Gene Expression of Cytochrome Oxidase Subunits I and IV in Neuronal Aging

To assess the role of mitochondrial metabolic competence (MMC) in neuronal aging, quantitative immunohistochemistry of cytochrome oxidase (COX) subunits I (mitochondrial-encoded) and IV (nuclear-encoded) was carried out in the cerebellar cortex of adult and old rats. The optical density (OD) values of the immunostained COX subunits I and IV were measured on an overall area of 75,000 microm(2) in the granular and molecular layers of the cerebellar cortex of each animal. In old animals, OD values of subunit I were increased by 35.5 and 34.2% in the molecular and granular layers, respectively, but only the difference found in the latter cerebellar zone was statistically significant (p < 0.05%). As regards subunit IV, old animals showed higher, not significant, densitometric values in the molecular (120.6%) and granular (126.8%) layers. The present findings sustain that gene expression of COX subunits I and IV appears not to be involved in the well-documented time-related mitochondrial decay. The proper functioning of COX depends on several factors that can affect MMC in the aging cell. In the fully assembled holoenzyme, both the subunits I and IV span the inner mitochondrial membrane. On the basis of these molecular biology data, it is reasonable to suppose that any alteration of the physicochemical features and chemical composition of the mitochondrial membranes reported to occur in aging (e.g., decreased membrane fluidity and cardiolipin content, increased cholesterol/phospholipid molar ratio and free-radical damage, etc.) may significantly affect the proper assembling of the enzyme and, in turn, its activity. Considering the reported significant decline of COX activity with advancing age, our findings further support that an adequate mitochondrial metabolic competence, while including proper nuclear and mitochondrial gene expression of subunits of the respiratory chain, relies on the overall balance among various determinants that can be differently damaged by aging and represent critical causative events responsible for the age-related functional decline of selected mitochondrial populations.

Decay of mitochondrial metabolic competence in the aging cerebellum.

Cytochemically evidenced cytochrome oxidase activity was morphometrically measured in the cerebellar cortex of adult and old rats. The ratio (R) between the area of the precipitate due to the cytochemical reaction and the overall area of each mitochondrion was calculated. While in adult rats an inverse correlation between mitochondrial size and R values (r = -.905) was envisaged, in old animals increasing values of R were paired by increases in mitochondrial area (r =.561). Paired-quartile comparisons of the R values from adult and old animals documented a marked age-related impairment of the mitochondrial metabolic competence in small (I quartile: -31.6%) and medium-sized (II quartile: -26.4; III quartile: -16.4) mitochondria, while large organelles showed the lowest age-related decrease (IV quartile: -3.0%). The present findings support that a marked dysfunction of small and medium-sized mitochondria contributes to the significant decay of energy metabolism currently reported in physiological aging.

Inverse correlation between mitochondrial size and metabolic competence: a quantitative cytochemical study of cytochrome oxidase activity.

Mitochondria are topologically closed bilayered systems where the synthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate occurs via oxidative phosphorylation. The ordered architecture (and its extension) of the mitochondria (i.e. inner membrane, outer membrane and cristae) constitutes a critical topographic arrangement for their energy-providing mechanisms. Thus, quantitative estimations of the ultrastructural features of organelles preferentially stained by means of function-related cytochemical reactions reliably report on their potential to supply adequate amounts of ATP. On the basis of this rationale, we carried out a computer-assisted cytochemical study of cytochrome oxidase (COX) activity on mitochondria of different size in the cerebellar cortex of adult rats. The total intra-mitochondrial area of the cytochemical precipitates (CPA)/mitochondrion, the area (MA) and the longer diameter (F(max)) of COX-positive organelles were measured. The ratio (R): CPA/MA was also calculated and referred to as the percentage of mitochondrial inner membrane area involved in COX activity. The regression analysis of R vs MA showed a significant inverse correlation (r=-0.905). The fourfold increase in MA from quartiles I to IV was matched by increases in F(max) and CPA, respectively, but it was also related to a 25% decrease in R. By matching quantitative cytochemical estimations of COX activity within mitochondria with the morphometric assessment of their ultrastructural features, the present study correlates size to the metabolic competence of COX-positive organelles. Quantitative cytochemistry of COX activity is currently regarded as a reliable marker of cellular metabolism; thus our findings support the hypothesis that enlargements in size are inversely correlated with the mitochondrial metabolic competence.

Mapping of mitochondrial metabolic competence by cytochrome oxidase and succinic dehydrogenase cytochemistry.

To map the mitochondrial capacity to provide adenosine triphosphate (ATP), the activities of cytochrome oxidase (COX) and succinic dehydrogenase (SDH) were respectively evidenced by diaminobenzidine (DAB) and copper ferrocyanide cytochemical techniques in the cerebellar cortex of adult rats. Sampling of the positive mitochondria was carried out by the disector procedure. The ratio (R) overall area of the precipitates due to COX activity within the single mitochondrion/area of the same organelle was automatically calculated to estimate enzyme activity vs mitochondrial size. The number of SDH-positive mitochondria/microm(3) of tissue (numeric density, Nv) was morphometrically calculated. Cytochemistry of key enzymes of the respiratory chain enables measurement of the actual capacity of individual mitochondria to provide ATP. This quantitative estimation allows morphofunctional mapping of the mitochondrial metabolic competence in discrete tissue and/or cellular compartments. (J Histochem Cytochem 49:1191-1192, 2001)

Aging-like alterations of SDH activity in Purkinje cell mitochondria of adult vitamin-E deficient rats

The ultrastructural features of perikaryal mitochondria positive to the copper ferrocyanide cytochemical reaction due to SDH activity were investigated in Purkinje cells of adult rats fed a vitamin E (α-tocopherol) deficient diet (AVED) for 11 months. The mitochondrial volume fraction (volume density: Vv), the number of organelles/μm(3) of tissue (numeric density: Nv) and their average volume (V) were estimated by computer-assisted morphometry. The data obtained were compared with our previous results on 3, 12 and 24 month-old normally fed rats. In a comparison with age-matched controls, AVED animals showed significant decreases of the three morphometric parameters taken into account. These reductions were also observed in old, normally fed rats vs. the young and adult groups, but in AVED rats Vv and V decreased to a higher extent. In adult control animals, the percent of larger organelles (0.32 μm(3) >) decreases to less than 1%. Vitamin E deficiency resulted in a steeper reduction of this fraction of organelles, i.e. only 0.5% in the 0.24-0.32 μm(3) size range accounted for the largest mitochondria in the AVED group. Taken together, these data document a significant impairment of mitochondrial efficiency in old and AVED rats. We interpret these findings to support that the underlying processes of aging and vitamin E deficiency may share common mechanisms. Considering the antioxidant action of α-tocopherol and the SDH role in cellular bioenergetics, inadequate protection from free radical attacks appears to represent an important determinant in the age-related decline of the mitochondrial metabolic competence.

Quantitative cytochemical mapping of mitochondrial enzymes in rat cerebella

Mitochondrial metabolic competence, defined as the organelle's capacity to provide adequate amounts of ATP in due time, appears to constitute an important determinant in several biological processes and pathological conditions. Thus, the assessment of the metabolic efficiency of the mitochondrial population in a given tissue area or cellular compartment may provide clues to identifying alterations of the cellular bioenergetic machinery, which may constitute a predisposing condition leading to impaired organ and system functions.In the cerebellar cortex of adult rats, the activities of the enzymes cytochrome oxidase (COX) and succinic dehydrogenase (SDH) were, respectively, evidenced by means of the diaminobenzidine and copper ferrocyanide preferential cytochemical techniques. At the electron microscope, the activities of these two key molecules of the respiratory chain were clearly visualised as dark precipitates at the inner mitochondrial membrane sites where COX and SDH are located. By means of the disector method, unbiased mitochondrial samplings were carried out to measure: the number of mitochondria/μm3 of tissue (numeric density: Nv); the mitochondrial volume fraction/μm3 of tissue (volume density: Vv) and the average mitochondrial volume (V) both on COX- and SDH-positive organelles in the cerebellar glomeruli and Purkinje cells, respectively. The ratio R (total area of the precipitates due either to COX or SDH activity within the single mitochondrion/area of the same organelle) was also evaluated to get information on the enzyme activity related to mitochondrial size.The documented accumulation of mutant mitochondrial DNA particularly in postmitotic cells results in a marked heteroplasmy (mixtures of normal and mutated genomes) at mitochondrial and cellular levels, thus the cellular potential for energy production is demanded to a mosaic of organelles with different functional capabilities. Assessment of the mitochondrial mosaic outline by means of quantitative cytochemistry of key enzymes of the respiratory chain, such as COX and SDH, may allow for the morphofunctional metabolic mapping of mitochondrial efficiency in discrete cellular or tissue compartments.