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.

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.

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.

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.

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.

Synaptic pathology in the brain cortex of old monkeys as an early alteration in senile plaque formation.

Synaptic numeric density (Nv), average size (area: S), surface density (Sv) and number of synapses/neurone (Syn/Neur) were morphometrically measured in frontal (FC) and temporal (TC) cortex of adult and old monkeys. Sv was constant, a clear age-related trend to decrease by Nv and increase by S were observed in both areas investigated. Syn/Neur significantly decreased in TC of aged animals (-21.1%), whereas FC showed a not significant reduction (-2.6%). The present data support the hypothesis of an increased sensitivity to deterioration of TC synapses in aged monkeys, which might constitute a predisposing condition to the development of senile plaques.

The effect of chronic physical exercise on succinic dehydrogenase activity in the heart muscle of old rats.

Succinic dehydrogenase (SDH) activity, preferentially evidenced by cytochemical methods, has been measured by computer-assisted morphometry in the heart muscle of old sedentary and age-matched animals chronically undergone physical exercise (20 min, twice a day, 5 days a week). The area of the SDH-positive mitochondria (MA) and the overall area of the cytochemical precipitates due to SDH activity (PA) were semiautomatically measured and the ratios PA/MA as well as MA/overall myocardial tissue area analysed (MA/TA) were the parameters taken into account. No significant difference was found between the two groups investigated as regards PA/MA, whereas the MA/TA value is significantly increased in the animals undergone physical training. The present findings document that chronic physical exercise significantly increases the overall mitochondrial area involved in energy provision in the old myocardial tissue. Considering that myocardial function highly relies on mitochondrial metabolism, our results support a beneficial effect of chronic physical exercise on the old heart muscle.