Ugo Caselli

Synaptic Structural Dynamics and Aging

Synaptic junctional areas are not immutable structures, on the contrary, they are remodelled throughout the individuals lifespan as a consequence of environmental stimulations. This adaptive capacity of the synapses is discussed from a morphological standpoint with reference to aging. In old subjects, the number of contacts and the total surface area of synaptic appositions per unit volume of tissue decrease significantly, while the average synaptic size increases at a different extent according to the CNS area taken into account. This increase in synaptic average area is due to a higher percent of a subpopulation of enlarged contacts supposed to represent either the degenerating junctional zones or a compensatory phenomenon counteracting the synaptic reduction in number. Recent studies on perforated synapses support that the enlarged junctions are possible intermediates in synaptic physiological restructuring, thus the higher percentage of this type of contacts in the old CNS may witness unaccomplished synaptic turnover cycles. Taking into account the high metabolic rate of nerve cells, an age-related impairment in energy provision at synaptic terminal regions may constitute an early and subtle alteration affecting synaptic dynamic morphology in 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 (Fmax) 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 Fmax 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.

Impaired succinic dehydrogenase activity of rat Purkinje cell mitochondria during aging

The perikaryal Purkinje cell mitochondria positive to the copper ferrocyanide histochemical reaction for succinic dehydrogenase (SDH) have been investigated by means of semiautomatic morphometric methods in rats of 3, 12 and 24 months of age. The number of organelles/microm3 of Purkinje cell cytoplasm (Numeric density: Nv), the average mitochondrial volume (V) and the mitochondrial volume fraction (Volume density: Vv) were the ultrastructural parameters taken into account. Nv was significantly higher at 12 than at 3 and 24 months of age. V was significantly decreased at 12 and 24 months of age, but no difference was envisaged between adult and old rats. Vv was significantly decreased in old animals vs. the other age groups. In young and old rats, the percentage of organelles larger than 0.32 microm3 was 13.5 and 11%, respectively, while these enlarged mitochondria accounted for less than 1% in the adult group. Since SDH activity is of critical importance when energy demand is high, the marked decrease of Vv supports an impaired capacity of the old Purkinje cells to match actual energy supply at sustained transmission of the nervous impulse. However, the high percentage of enlarged organelles found in old rats may witness a morphofunctional compensatory response.

Age-dependent decrease in the activity of succinic dehydrogenase in rat CA1 pyramidal cells: a quantitative cytochemical study

A computer-assisted morphometric study has been carried out on the ultrastructure of perikaryal CA1 pyramidal cell mitochondria positive to the copper ferricyanide cytochemical reaction for succinic dehydrogenase (SDH) in rats of 3, 12 and 23 months of age. The cytoplasmic volume fraction occupied by the positive mitochondria (Volume density: Vv), the number of organelles/micron 3 of CA1 pyramidal cell cytoplasm (Numerical density: Nv) and the average mitochondrial volume (V) were automatically calculated by means of computer-assisted morphometry. Vv was significantly decreased in 23-month-old animals versus the other age groups. Nv was unchanged between 3 and 12 months of age, but was decreased to a significant extent in old animals. V did not undergo significant changes in the three age groups taken into account. In the old animals the percent of organelles smaller than 0.16 micron 3 is above 20%, while in the young and adult groups the same size of mitochondria accounts for 7 and 3%, respectively. Thus, a reduction in the number of medium sized organelles appears to be responsible for the decrease in Vv due to age. Since SDH activity is known to support maximum rates of respiration, quantitative estimation of the active mitochondria provides information on the metabolic competence of the cells investigated when energy demand is high. In this context, our present findings document that a significant impairment in the efficiency to match actual energy provisions occurs in old CA1 pyramidal cells.

Succinic dehydrogenase activity in human muscle mitochondria during aging: a quantitative cytochemical investigation

A quantitative cytochemical study has been carried out on succinic dehydrogenase (SDH) activity in biopsy samples of vastus lateralis (VL) and anterior tibialis (AT) muscles from healthy men undergoing orthopaedic surgery. According to their age, the patients were divided into: young (25.0+/-4.4 years), middle-aged (50.4+/-7.5 years) and old (75.5+/-3.9 years) groups. Bioptically excised samples were processed for copper ferrocyanide preferential SDH cytochemistry. By a computer-assisted image analyser, we calculated the ratio (R): overall area of the precipitates due to the enzyme activity/area of each mitochondrion. No significant difference was found among the three age groups, despite an 8% increase of R in the adult vs. the other groups. R values are related to mitochondrial morphofunctional features since they may be modulated by enzyme activity and the physico-chemical conditions of the organelle membranes. Thus, R quantitation enables to estimate the mitochondrial capacities for adenosinetriphosphate provision. In this context, our present findings confirm previous data reporting a substantial age-related stability of muscle mitochondrial enzyme levels. In aging, energy-deficient sarcomeres are supported to be negatively selected and eliminated, while the surviving ones appear to maintain an adequate SDH activity.

A MORPHOMETRIC STUDY ON HUMAN MUSCLE MITOCHONDRIA IN AGING

Mitochondria are dynamic organelles capable of significant changes of their ultrastructural features according to the tissue-specific energy demands. In human biopsies of vastus lateralis and anterior tibialis muscles from young (25.0 ± 4.4 years), middle-aged (50.4 ± 7.5 years) and old (75.5±3.9 years) healthy volunteers, we carried out a morphometric study on subsarcolemmal and intermyofibrillar mitochondria to assess whether age-related alterations of the morphology of these organelles contribute to the muscle performance decay in aging. By computer-assisted methods, we measured: the average area (MAA), the longer diameter (Dmax) and the ratio perimeter to area (pleomorphic index: Plei) of mitochondria. No significant age-related ultrastructural differences were found either in subsarcolemmal or intermyofibrillar organelles. However, in middle-aged as well as in the old group of patients vs. the young one, MAA and Dmax showed a clear trend to decrease, while Plei showed a marked, age-related tendency to increase. Higher percentages of less pleomorphic organelles were found in the youngest group of patients and this was particularly evident in the subsarcolemmal mitochondrial population. In addition to reporting on discrete aspects of mitochondrial ultrastructure, MAA, Dmax and Plei are closely related to each other and provide a reliable index of the muscle mitochondria adaptive response to age. Thus, we interpret our results as indicating a substantial preservation of muscle mitochondrial ultrastructure during aging.

β-amyloid fragment 25-35 induces changes in cytosolic free calcium in human platelets

The beta-amyloid (βA) peptide has a central role in Alzheimer’s disease (AD). Indeed, the major histopathologic hallmarks of AD include βA deposits in brain parenchima (senile plaques) and also around and within the walls of blood vessels (cerebral amyloid angiopathy).1 βA deposits are the final result of an amyloidogenic process triggered by oxidative and conformational modifications and by crosslinking of βA.2–4 βA is a peptide produced by the proteolysis of the amyloid precursor protein (APP). It has been reported that abnormalities of brain APP metabolism may be reflected in platelets, which also possess all the machinery to generate the amyloid β (Aβ)-fragment from APP; moreover, platelets are the primary source of Aβ-peptide in human blood.5–7 In human cortical neurons β-amyloid peptides have been shown to destabilize calcium homeostasis and cause neurodegenerative effects.8,9 Alteration in calcium homeostasis induced by Aβ have been reported also for nonneuronal cells.10,11 Moreover, it has been demonstrated that Aβ25–35 increases cellular APP by inhibiting its secretory processing in human extraneuronal cells.12 The data reported above encourage further exploration about amyloid and platelets as a peripheral laboratory mirroring central amyloid metabolism and activity.1 In the present paper we investigate the effects of neurotoxic Aβ25–35 peptide on platelets and we show that this Aβ-fragment is able to induce dosedependent changes of calcium concentration and degenerative effects in normal human platelets. Blood samples were taken from male healthy donors aged between 31 and 50 years (mean age: 38 ± 7). Citrated blood was centrifuged for 10 min at 200 × g to obtain platelet-rich plasma (PRP). Platelets were separated from PRP by centrifugation at 2000 × g for 20 min and washed twice in phosphate-buffered saline (PBS) 0.1 M, pH 7.4. Platelets were resuspended at a density of 108/ml in Hepes buffer containing 145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, 10 mM glucose, adjusted to pH 7.4. Ten μM prostaglandin E1 (PGE1) was added to prevent aggregation. The platelet suspension was incubated in 1 μM Aβ25–35

Morphometry of Axon Cytoskeleton at Internodal Regions of Rat Sciatic Nerve during Aging

Background: Nerve endings undergo a lifespan morphofunctional modulation which is reported to be markedly impaired with aging. Neurone structural remodelling is in charge of processes occurring in the nerve cell soma, however the axonal transportation of organelles and molecules by cytoskeletal elements plays a very important role in the morphological rearrangements taking place at peripheral compartments. Objective: To assess the involvement of axonal ultrastructure in the reported age-related decline of slow axoplasm flow mechanisms, we carried out a morphometric study of axon cytoskeleton in aging. Methods: Female Wistar rats (3, 12 and 30 months of age) were anesthetized and perfused with saline followed by a fixation solution (glutaraldehyde 5% + formalin 2% in 0.1 cacodylate buffer pH 7.4). The excised sciatic nerves were processed according to conventional electron microsopic procedures. Axons sectioned perpendicularly to their longitudinal axis at the internodal region (mean axoplasm area: 18.25–26.5 μm2) were sampled by a systematic random procedure. The overall number of neurofilaments (No.Nfs) and microtubules (No.Mts) per total axoplasm area analysed, the numeric density (number/μm2 of axoplasm area) of neurofilaments (NaNfs) and microtubules (NaMts), the myelin thickness, the number of myelin lamellae and the R proportion [No.Nfs/(No.Nfs + No.Mts)] were the parameters measured by computer-assisted semiautomatic methods. Results: No.Nfs, NaNfs, myelin thickness and the number of myelin lamellae did not change between 12 and 30 months of age, while a significant increase of these parameters was found in a comparison with younger rats. No.Mts and NaMts were significantly increased at 12 vs. 3 as well as at 30 vs. 12 months of age, respectively. R proportion did not show any difference due to age. Conclusions: The present findings support that the dynamic condition of the axonal cytoskeleton appears to be preserved at a high extent in aging. Thus, the intra-axonal defective spacing of cytoskeletal elements (e.g. neurofilaments), rather than their number, is proposed to contribute to the decline of the slow axonal transport of organelles and molecules reported in aging.

Dietary restriction modulates synaptic structural dynamics in the aging hippocampus

A computer-assisted morphometric study has been carried out on the synaptic ultrastructural features in the hippocampus of 14-month old (DR14) and 27-month old (DR27) dietary restricted (−50% lipids and −35% carbohydrates) rats. Age-matched controls were maintained on an ad libitum (AL) feeding schedule. Synaptic numeric density (Nv), surface density (Sv) and average area (S) were the parameters measured. In old AL vs. adult AL animals, Nv decreased to a not significant extent, while S increased and Sv decreased significantly. In DR14 rats vs. AL littermates Nv increased significantly, but S and Sv were unchanged. DR27 rats vs. age-matched AL controls showed a significant increase of Nv and Sv while S was significantly decreased. Comparing DR14 vs. DR27, no significant difference due to age was documented. Both in DR14 and in DR27 groups the percent distribution of S showed a marked increase of smaller contact zones. Despite reporting on discrete aspects of synaptic ultrastructure, Nv and S are supported to be in an inverse relationship which aims at maintaining Sv constant. Thus, these three ultrastructural parameters when taken together per experimental group, appear to provide information on synaptic morphological rearrangements. In this context, the percent increase of smaller synapses in DR animals is consistent with the idea of a marked remodelling process. Considering previous data from the same groups of rats reporting significant changes in neuronal membrane lipid composition and fluidity, we interpret our findings to account for a positive modulation of dietary restriction on the synaptic structural dynamics.

Monovalent electrolyte content in vitamin E-deficient rats: Clues to understand brain aging

Aging has been defined as a time-related loss of the cellular capacity to maintain homeostasis. This is of critical importance in postmitotic cells, like neurons, where a prompt substitution of damaged molecules is required for proper function. Primary causes of aging, as defined above, have been claimed to be free radicals, originating physiologically from cellular oxidative phosphorylation (Harman, 1981). Vitamin E (á-tocopherol) is a well known biological antioxidant able to quench the free radical-mediated lipid peroxidation chain (Burton and Ingold, 1989) and to stabilize the molecular composition of cellular membranes (Kagan, 1989). Recently, an isolated vitamin E deficiency syndrome in the absence of fat malabsorption has been associated with neurological dysfunction (Sokol, 1989): in addition to the biochemical evidence of á-tocopherol deficiency, the affected patients show many hallmarks typical of aging. This similarity of alterations suggested to us that vitamin E deficiency in laboratory animals may be considered a model of precocious brain aging, the present paper reports the results of our X-ray microanalysis studies performed to assess the reliability of our assumption.