Platelet as a physiological model to investigate apoptotic mechanisms in Alzheimer beta-amyloid peptide production.

Although neuronal apoptosis in Alzheimer's disease is generally interpreted as the consequence of the toxicity of extracellular beta-amyloid (Abeta) peptide aggregates, some experimental results provide evidence that the Abeta overproduction can be the result of a primary neuronal degeneration. As platelets are considered a good model where to study proteolytic processing of the amyloid precursor protein (APP), we exposed platelets to the proapoptotic agent ionomycin and analyzed Abeta40 and Abeta42 levels in the intracellular and extracellular compartments. The activation of apoptotic pathways in platelets has been verified by mitochondrial membrane depolarization, exposure of phosphatidylserine, protease activation and morphological changes. A significative increase in intraplatelet Abeta40, but not Abeta42, was observed after 10 min treatment with ionomycin. Thus, the activation of apoptotic pathways in platelets determines an altered processing of APP leading to elevated levels of intracellular Abeta40. The specific intracellular production of Abeta40 represents a potential threat to the cells since very high local Abeta40 concentration increases the risk of its aggregation and toxicity. As a result, Abeta40 might be dangerous even before it becomes secreted rendering neurons highly vulnerable.

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.

Long-term visual object recognition memory in aged rats.

Aging is associated with memory impairments, but the neural bases of this process need to be clarified. To this end, behavioral protocols for memory testing may be applied to aged animals to compare memory performances with functional and structural characteristics of specific brain regions. Visual object recognition memory can be investigated in the rat using a behavioral task based on its spontaneous preference for exploring novel rather than familiar objects. We found that a behavioral task able to elicit long-term visual object recognition memory in adult Long-Evans rats failed in aged (25-27 months old) Wistar rats. Since no tasks effective in aged rats are reported in the literature, we changed the experimental conditions to improve consolidation processes to assess whether this form of memory can still be maintained for long term at this age: the learning trials were performed in a smaller box, identical to the home cage, and the inter-trial delays were shortened. We observed a reduction in anxiety in this box (as indicated by the lower number of fecal boli produced during habituation), and we developed a learning protocol able to elicit a visual object recognition memory that was maintained after 24 h in these aged rats. When we applied the same protocol to adult rats, we obtained similar results. This experimental approach can be useful to study functional and structural changes associated with age-related memory impairments, and may help to identify new behavioral strategies and molecular targets that can be addressed to ameliorate memory performances during aging.

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.

Synaptic remodeling in hippocampal CA1 region of aged rats correlates with better memory performance in passive avoidance test.

Aging is associated with deficits in long-term declarative memory formation, and wide differences in performance can be observed among aged individuals. The cellular substrates of these deficits and the reasons for such marked individual differences are not yet fully understood. In the present study, morphologic parameters of synapses and synaptic mitochondria in stratum molecolare of CA1 hippocampal region were investigated in aged (26- to 27-month-old) female rats after a single trial inhibitory avoidance task. In this memory protocol animals learn to avoid a dark compartment in which they received a mild, inescapable foot shock. Rats were tested 3 and 6 or 9 hours after the training, divided into good and bad responders according to their performance (retention times above or below 100 seconds, respectively) and immediately sacrificed. The number of synapses and synaptic mitochondria per cubic micrometer of tissue (numeric density), the average area of synapses and volume of synaptic mitochondria, the total area of synapses per cubic micrometer of tissue, the percentage of perforated synapses and the overall volume of mitochondria per cubic micrometer of tissue were evaluated. In the good responder group, the numeric density of synapses and mitochondria was significantly higher and the average mitochondrial volume was significantly smaller 9 hours versus 6 hours after the training. No significant differences were observed among bad responders. Thus, better performances in passive avoidance memory task are correlated with more efficient plastic remodeling of synaptic contacts and mitochondria in hippocampal CA1. Present findings indicate that maintenance of synaptic plastic reactivity during aging is a critical requirement for preserving long-term memory consolidation.

Immunoproteasome in Macaca fascicularis: no age-dependent modification of abundance and activity in the brain and insight into an in silico structural model.

In this study, we investigated proteasome composition and activity in the brain of Macaca fascicularis, in order to test whether this nonhuman primate species might be a suitable animal model for anti-aging therapies in the central nervous system, addressed to the ubiquitin-proteasome system. We detected the catalytic beta subunits of constitutive proteasome, as well as the PA28 regulator and a subunit of immunoproteasome (i.e., beta1i [LMP2]), in seven adult, six old, and one young nonhuman primate brains. Subunit expression and proteasome activity were not influenced by the age of the animal in any of the brain regions (temporal and frontal cortex and cerebellum) we studied. However, an area-specific susceptibility to aged-related oxidative stress emerged. On the whole, the results suggest that, compared to humans, Macaca fascicularis primates may have a different age-dependent regulation of the ubiquitin-proteasome system and, possibly, of neuroinflammation in the brain. An in silico model of the 20S immunoproteasome containing the Macaca fascicularis alpha and beta subunits, present in database or identified by our group (i.e., LMP2), has been developed. Additional information was obtained by de novo sequencing of the beta1 (delta) subunit of Macaca fascicularis. A comparison with humans suggests that in multiprotein complexes some functional subunits, such as alpha subunits, appear to be preferentially conserved during evolution.

Melatonin prevents age-related mitochondrial dysfunction in rat brain via cardiolipin protection.

Reactive oxygen species (ROS) are considered a key factor in brain aging process. Complex I of the mitochondrial respiration chain is an important site of ROS production and hence a potential contributor to brain functional changes with aging. Appropriate antioxidant strategies could be particularly useful to limit this ROS production and associated mitochondrial dysfunction. Melatonin has been shown to possess antioxidant properties and to reduce oxidant events in brain aging. The mechanism underlying this protective effect of melatonin is not well established. In the present study, we examined the effects of long-term treatment of aged rats with melatonin on various parameters related to mitochondrial bioenergetics in brain tissue. After isolation of mitochondria from control, aged, and melatonin-treated young and aged rats, various bioenergetic parameters were evaluated such as complex I activity, rates of state 3 respiration, mitochondrial hydrogen peroxide (H2O2) production, and membrane potential. The mitochondrial content of normal and oxidized cardiolipin was also evaluated. We found that all these mitochondrial parameters were significantly altered with aging, and that melatonin treatment completely prevented these age-related alterations. These effects appear to be due, at least in part, to melatonin's ability to preserve the content and structural integrity of cardiolipin molecules, which play a pivotal role in mitochondrial bioenergetics. The melatonin's ability to prevent complex I dysfunction and cardiolipin peroxidation was also demonstrated by in vitro experiments on brain mitochondria treated with tert-butyl hydroperoxide. In summary, this study documents a decline of mitochondrial bioenergetic functions in brain with aging and the beneficial effect of melatonin.

Decreased presence of perforated synapses in a triple-transgenic mouse model of Alzheimer's disease.

Transgenic mouse models of Alzheimer's disease (AD) are useful tools to further our understanding of AD genotype-phenotype interaction. The triple transgenic mice harboring mutant forms of APP/PS1/Tau (3xTg-AD) exhibit beta-amyloid (Abeta) plaques (by 6 months of age) as well as neurofibrillary tangles (by 10-12 months of age). In this study, we characterized morphological alterations of hippocampal synapses obtained from 13-month-old 3xTg-AD and age-matched control (PS1-KI) mice. Numeric density of synapses (Nv, number of junctions/microm(3) of tissue), average synaptic contact area (S), and synaptic surface density (Sv, total synaptic contact area/microm(3) of tissue) were investigated by morphometric methods in the AD vulnerable CA1 pyramidal cell layer. Comparisons between 3xTg-AD and control mice showed no statistically significant differences in any of the three parameters; however, a significant decrease (by 28.5%) in the fraction of perforated junctional areas (PS) was observed in the 3xTg-AD mice. As PS is a reliably indirect index of synaptic plasticity, a decreased PS number might represent a subtle and early sign of synaptic dysfunction occurring in the 3xTg-AD mice, and lend support to the hypothesis that altered synaptic function is a critical feature of AD.

Ketogenic diets cause opposing changes in synaptic morphology in CA1 hippocampus and dentate gyrus of late-adult rats.

Ketogenic diets (KDs) have beneficial effects on several diseases, such as epilepsy, mitochondriopathies, cancer, and neurodegeneration. However, little is known about their effects on aging individuals. In the present study, late-adult (19-month-old) rats were fed for 8 weeks with two medium chain triglycerides (MCT)-KDs, and the following morphologic parameters reflecting synaptic plasticity were evaluated in stratum moleculare of hippocampal CA1 region (SM CA1) and outer molecular layer of hippocampal dentate gyrus (OML DG): average area (S), numeric density (Nv(s)), and surface density (Sv) of synapses, and average volume (V), numeric density (Nv(m)), and volume density (Vv) of synaptic mitochondria. In SM CA1, MCT-KDs induced the early appearance of the morphologic patterns typical of old animals (higher S and V, and lower Nv(s) and Nv(m)). On the contrary, in OML DG, Sv and Vv of MCT-KDs-fed rats were higher (as a result of higher Nv(s) and Nv(m)) versus controls; these modifications are known to improve synaptic function and metabolic supply. The opposite effects of MCT-KDs might reflect the different susceptibility to aging processes: OML DG is less vulnerable than SM CA1, and the reactivation of ketone bodies uptake and catabolism might occur more efficiently in this region, allowing the exploitation of their peculiar metabolic properties. Present findings provide the first evidence that MCT-KDs may cause opposite morphologic modifications, being potentially harmful for SM CA1 and potentially advantageous for OML DG. This implies risks but also promising potentialities for their therapeutic use during aging.

Alterations of synaptic turnover rate in aging may trigger senile plaque formation and neurodegeneration.

The changes of synaptic ultrastructure were investigated by morphometry in the frontal (FC) and temporal (TC) cortex of adult and aged monkeys, to assess the potential role of age-related synaptic deterioration in neurodegeneration. The average synaptic size (S), the synaptic numeric density (Nv: number of synapses/microm(3) of tissue), the synaptic surface density (Sv: overall area of synaptic junctional zones/microm(3) of tissue), and the number of synapses/neuron (Syn/Neur) were calculated. In FC, significant differences of Nv and Sv due to age were not revealed, while the S value was significantly increased in the aged animals. In TC, Sv did not change in relation to age, whereas Nv was significantly decreased and S significantly increased in aged monkeys. A percent distribution of S showed that the fraction of enlarged synapses (>0.20 microm(2)) was higher in TC than in FC, regardless of the age of the animals (21.3% versus 16.9% in adult and 33.9% versus 26.0% in aged monkeys, respectively). In aged animals, Syn/Neur was not significantly decreased in TC and not significantly increased in FC (4.4%). The above morphometric parameters account for the ongoing rearrangements of synaptic ultrastructure, reacting to the environmental stimuli. Our findings provide evidence of an age-related decline of synaptic plasticity in the brain of aged monkeys that is statistically significant in TC. According to current literature data on synaptic structural dynamics, this decay may represent an early and subtle alteration able to trigger the development of senile plaques and neurodegenerative events.

Preservation of mitochondrial volume homeostasis at the early stages of age-related synaptic deterioration.

A morphometric study on synaptic mitochondria was performed in the frontal (FC) and temporal (TC) cortex of adult and aged monkeys to seek ultrastructural alterations due to age. The overall volume covered by mitochondria (volume density: Vv), the number of mitochondria/microm(3) of tissue (numeric density: Nv), the average mitochondrial size (average volume: V), and the average mitochondrial shape (average length: Fmax) were calculated. Either in FC and TC, no significant age-related differences were revealed for any of the above-mentioned morphometric parameters. Namely, in FC of aged monkeys, a decrease of Vv (2%) and Nv (6%) was observed, whereas V and Fmax were increased by 5% and 2%, respectively. In TC of aged animals, both Vv and Nv increased by 7%, V decreased by 2%, and Fmax increased by 1%. The above morphometric parameters account for changes in single aspects of mitochondrial ultrastructure; nonetheless, when considered together per experimental group, they provide information regarding the structural rearrangements occurring on discrete populations of organelles. Considering these assumptions, the present findings document a preservation of the mitochondrial volume homeostasis in the brain of aged monkeys. Because our data from a previous investigation on the same animals showed early signs of synaptic deterioration in FC and TC during aging, this seems to be in contrast with the results of the present study. However, the clear age-related preservation of the mitochondrial potential for structural dynamics may be interpreted as a reactive response to early signs of synaptic deterioration.

Release of beta-amyloid from high-density platelets: implications for Alzheimer's disease pathology.

The main component of Alzheimer's disease (AD) senile plaques in the brain is amyloid-beta peptide (Abeta), a proteolytic fragment of the amyloid precursor protein (APP). Platelets contain both APP and Abeta and much evidence suggests that these cells may represent a useful tool to study both amyloidogenic and nonamyloidogenic pathways of APP processing. It has been demonstrated that platelets activated by physiological agonists, such as thrombin and collagen, specifically secrete Abeta ending at residue 40. To verify whether APP beta-processing could be observed also in an in vitro system of highly concentrated platelets, we measured the Abeta released in the incubation media of 5 x 10(9) platelets/mL by enzyme-linked immunosorbent assay (ELISA). The activation status of platelets was investigated by ultrastructural analysis. We found that Abeta(40) levels were significantly higher in incubation media of 5 x 10(9)/mL platelets in comparison with 10(8)/mL platelets (normalized values), while Abeta(42) levels were not affected by cell density. The ultrastructural analysis showed platelets at different phases of activation: some platelets were at earlier stage, characterized by granule swelling and dilution, others had granules concentrated in a compact mass in the cell centers within constricted rings of circumferential microtubules (later stage). Normally concentrated cells had the characteristic morphology of resting platelets. Our data suggest that high-density platelets undergo activation likely by increased frequency of platelet-platelet collisions. This, in turn, determines the activation of APP beta-processing with consequent release of Abeta(40). Investigating the biochemical pathways triggering Abeta secretion in platelets might provide important information for developing tools to modulate this phenomenon in AD brains.

Synaptic and mitochondrial morphometry provides structural correlates of successful brain aging.

Average synaptic size (S), synaptic numeric density (Nv) and surface density (Sv), average mitochondrial volume, mitochondrial numeric density, and mitochondrial volume density were measured by morphometry in the frontal (FC) and temporal (TC) cortex from adult and old monkeys (Macaca fascicularis). In relation to aging, Sv did not change, while Nv was significantly decreased in TC, but not in FC. S was significantly increased in FC and TC. No significant difference due to age was found with regard to mitochondrial ultrastructure. Considering the functional significance of the above parameters, their substantial age-related constancy suggests that they may reasonably represent structural correlates of successful brain aging.

Brain, aging and neurodegeneration: role of zinc ion availability.

Actual fields of research in neurobiology are not only aimed at understanding the different aspects of brain aging but also at developing strategies useful to preserve brain compensatory capacity and to prevent the onset of neurodegenerative diseases. Consistent with this trend much attention has been addressed to zinc metabolism. In fact, zinc acts as a neuromodulator at excitatory synapses and has a considerable role in the stress response and in the functionality of zinc-dependent enzymes contributing to maintaining brain compensatory capacity. In particular, the mechanisms that modulate the free zinc pool are pivotal for safeguarding brain health and performance. Alterations in zinc homeostasis have been reported in Parkinson's and Alzheimer's disease as well as in transient forebrain ischemia, seizures and traumatic brain injury, but little is known regarding aged brain. There is much evidence that that age-related changes, frequently associated to a decline in brain functions and impaired cognitive performances, could be related to dysfunctions affecting the intracellular zinc ion availability. A general agreement emerges from studies of humans' and rodents' old brains about an increased expression of metallothionein (MT) isoforms I and II, but dyshomogenous results are reported for MT-III, and it is still uncertain whether these proteins maintain in aging the protective role, as it occurs in adult/young age. At the same time, there is considerable evidence that amyloid-beta deposition in Alzheimer's disease is induced by zinc, but the pathological significance and the causes of this phenomenon are still an open question. The scientific debate on the role of zinc and of some zinc-binding proteins in aging and neurodegenerative disorders, as well as on the beneficial effect of zinc supplementation in aged brain and neurodegeneration, is extensively discussed in this review.

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 ([Zn2+]i) can be considered an unfavourable trigger of subtle mitochondrial damage and synaptic pathology.

Reactive structural dynamics of synaptic mitochondria in ischemic delayed neuronal death.

The effect of transient global ischemia on the ultrastructural features of synaptic mitochondria at the distal dendrites of CA1 hippocampal neurons was investigated in 3-month-old rats. Sham surgery was performed on age-matched controls. The number of mitochondria/microm3 of neurophils (Nv: numeric density), the mitochondrial average size (average volume: V), and longer diameter (Fmax) as well as the overall fraction of neurophils occupied by mitochondria (volume density: Vv) were measured by computer-assisted morphometry. In ischemic rats, a 10% nonsignificant decrease of Nv was found, V increased nonsignificantly by 11%, and Fmax increased nonsignificantly by 5% versus controls. As a final outcome of these balanced changes, Vv remained unchanged between the two experimental groups investigated. In ischemic animals, the percentage distribution of V showed that the population of CA1 synaptic mitochondria was composed by an increased fraction of oversized organelles, while the Fmax distribution revealed that this enlargement was due to an increased percentage of elongated organelles. Thus, the observed increase in size should not be considered as a swelling phenomenon; on the contrary, it may represent a physiological and well-documented step in mitochondrial biogenesis. The above parameters are currently supposed to provide information on the adaptive structural reorganization of mitochondrial morphology under different environmental stimulations. Conceivably, these findings document a positive reactive response to ischemia of the mitochondrial structural dynamics at CA1 synaptic terminals and suggest consideration of these organelles as reliable targets in the development of neuroprotective therapeutic interventions to treat vascular brain diseases, for example, stroke.

Experimental apoptosis provides clues about the role of mitochondrial changes in neuronal death.

A quantitative morphometric study has been carried out in human neuroblastoma SK-N-BE cells to evaluate the ultrastructural features and the metabolic efficiency of mitochondria involved in the early steps of apoptosis. In mitochondria from control and apoptotic cells cytochrome oxidase (COX) activity was estimated by preferential cytochemistry. Number of mitochondria (numeric density: Nv), volume fraction occupied by mitochondria/microm3 of cytoplasm (volume density: Vv), and average mitochondrial volume (V) were calculated for both COX-positive and -negative organelles. The ratio (R) of the cytochemical precipitate area to the overall area of each mitochondrion was evaluated on COX-positive organelles to estimate the inner mitochondrial membrane fraction actively involved in cellular respiration. Following apoptotic stimulus, the whole mitochondrial population showed a significant increase of Nv and Vv, while V was significantly decreased. In COX-positive organelles higher values of Nv were found, V appeared significantly reduced, and Vv was unchanged. R was increased at a nonsignificant extent in apoptotic cells. COX-positive mitochondria accounted for 21% and 35% of the whole population in control and in apoptotic cells, respectively. These findings document that in the early stages of apoptosis the increased fraction of small mitochondria provides an adequate amount of ATP for progression of the programmed cell death and these more efficient organelles appear to represent a reactive response to the loss of metabolically impaired mitochondria. A better understanding of the mitochondrial role in neuronal apoptosis may suggest potential interventions to prevent the extensive nerve cell death typical of neurodegenerative diseases.

Level and distribution of microtubule-associated protein-2 (MAP2) as an index of dendritic structural dynamics.

Optical density of MAP2 immunoreactivity (OD), the ratio between the MAP2 stained area/total test area (area fraction: AF), the total length of MAP2 labeled profiles (TL) and the ratio perimeter/area of the immunostained profiles (pleomorphism index [PI]) were measured by quantitative immunohistochemistry in the brain of rats of different ages. In old rats versus young and adult animals, OD and AF were significantly lower, whereas PI was significantly higher, in dentate gyrus molecular layer, CA1 stratum radiatum and olfactory bulb. These findings lend support to the many converging results on the higher vulnerability to aging of the CNS areas featuring higher plasticity.

Cytochemical estimation of cytochrome oxidase activity as a morphofunctional mitochondrial check-up.

Cytochemical estimation of COX activity and morphometric measurement of mitochondrial ultrastructure were carried out in organelles from adult and old rats. Although no age-related difference was found in cytochemical precipitate (CPA) and mitochondrial area (MA), the ratio CPA:MA (R) decreased by 25.7% in aging. R was the same in oversized mitochondria, but in smaller organelles it was significantly decreased during aging. R reports on the functional mitochondrial surface involved in energy providing mechanisms; thus, by revealing age-related differences, these data suggest that the COX preferential cytochemistry associated with morphometry may serve as a reliable and sensitive mitochondrial morphofunctional checkup procedure.

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.