Carlo Bertoni-Freddari

Morphological adaptive response of the synaptic junctional zones in the human dentate gyrus during aging and Alzheimer's disease

A computer-assisted morphometric study has been carried out on ethanol phosphotungstic acid (E-PTA) stained synaptic junctions in the human dentate gyrus supragranular layer from adult, old and Alzheimers disease (AD)-affected patients. The number of synapses per unit volume of tissue (Nv = numerical density), the average area of the single junction (S) and the total area of the synaptic contact zones in a unit volume of tissue (Sv = surface density) were the 3 parameters taken into account. The synapse to neurone ratio was also calculated for each patient. During physiological aging, Nv and Sv significantly decreased and S increased, respectively. In the AD hippocampi, Nv and Sv underwent a further decrease which was in the range of more than 40% with reference to the adult values. S was the same as the old control group. In comparison with the adult values, the number of synapse/neurone decreased by 15.6 and 48% in old and AD patients, respectively. Nv, S and Sv, while reporting on discrete ultrastructural features of the synaptic junctional zones, are closely related to each other and, taken together per group of patients, may represent a reliable index of the morphological adaptive changes taking place at the synapses. Thus, the significant increase of S both in old and AD hippocampi may be regarded as a CNS plastic response to aging and disease, although the marked decrease of Nv and Sv supports that in AD synaptic ultrastructural alterations proceed beyond a critical threshold for functional recovery.

Morphological plasticity of synaptic mitochondria during aging

A morphometric investigation has been carried out on the synaptic mitochondria of cerebellar glomeruli in young, adult and old rats by means of a computer-assisted image analysis technique. Mitochondrial volume density (Vv), numerical density (Nv), average volume (V) and average length (Skeleton = Sk) were investigated in tissue samples fixed, embedded and sectioned according to conventional electron microscopic methods. Vv was unchanged in the three groups of age taken into account. Nv was significantly increased in adult vs. young animals, whereas it was decreased in the old group as compared to both the other two groups investigated. V and Sk showed the same age-dependent changes: they significantly decreased in the adult vs. the young and the old groups of rats while increased significantly in the old rats vs. both the adult and young animals. A percentage distribution of Sk demonstrated that in the old group 20.6% of the population of synaptic mitochondria accounts for elongated organelles (> 5 microns) as compared to 8.6% and 5.3% in young and adult animals, respectively. The present findings match the changes previously reported by us on the ultrastructure of synaptic contact zones both in rats and human beings, and support the idea of an age-dependent dynamic adaptation in the morphology of synaptic mitochondria to cope with the metabolic needs of the pattern of synaptic connectivity they subserve.

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.

Quantitative investigation of the morphological plasticity of synaptic junctions in rat dentate gyrus during aging

The morphological plasticity of synaptic junctions was investigated by means of quantitative stereology in the dentate gyrus supragranular layer of young (3 months), adult (12 months) and old (30 months) female Fisher 344 rats. The numerical (Nv) and surface (Sv) density and the average surface area (S) of EPTA-stained synaptic junctions were calculated on 100 EM pictures per age group. We found that Nv significantly increased between the young and adult group and decreased in old animals when compared both to 3 and 12 months of age. Sv was unchanged in young and adult rats, whereas it underwent a significant reduction in the old group. The percentage distribution of S showed that at 3 months of age the majority of the synaptic population had an average size ranging between 0.12 and 0.20 micron 2, whereas during adulthood and aging there was a shift towards smaller and larger synaptic contacts, respectively. Taking into account Nv, Sv and S all together per age group, the present findings demonstrate that the morphological aspect of synaptic plasticity is markedly influenced by time and that during aging it undergoes a serious impairment. The possible involvement in memory and learning processes of these synaptic age-related morphological adaptations and the fact that the investigated junctions are supposed to be cholinergic is discussed.

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 melatonins ability to preserve the content and structural integrity of cardiolipin molecules, which play a pivotal role in mitochondrial bioenergetics. The melatonins 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.

Zinc-bound metallothioneins as potential biological markers of ageing

Metallothioneins (MTs) (I+II) play pivotal roles in metal-related cell homeostasis because of their high affinity for metals forming clusters. The main functional role of MTs is to sequester and/or dispense zinc participating in zinc homeostasis. Consistent with this role, MT gene expression is transcriptionally induced by a variety of stressing agents to protect cells from reactive oxygen species. In order to accomplish this task, MTs induce the secretion of pro-inflammatory cytokines by immune and brain cells, such as astrocytes, for a prompt response against oxidative stress. These cytokines are in turn involved in new synthesis of MTs in the liver and brain. Such protective mechanism occurs in the young-adult age, when stresses are transient. Stress-like condition is instead constant in the old age, and this causes continuous stealing of intracellular zinc by MTs and consequent low bioavailability of zinc ions for immune, endocrine, and cerebral functions. Therefore, a protective role of zinc-bound MTs (I+II) during ageing can be questioned. Because free zinc ions are required for optimal efficiency of the immune-endocrine-nervous network, zinc-bound MTs (I+II) may play a different role during ageing, switching from a protective to a deleterious one in immune, endocrine, and cerebral activities. Physiological zinc supply, performed cautiously, can correct deficiencies in the immune-neuroendocrine network and can improve cognitive performances during ageing and accelerated ageing. Altogether these data indicate that zinc-bound MTs (I+II) can be considered as novel potential markers of ageing.

Intracellular water and dry mass content as measured in bulk specimens by energy-dispersive X-ray microanalysis

Intracellular water content (IWC) was measured in freeze-fractured biological bulk specimens by means of energy-dispersive X-ray microanalysis. The method is based on the concentration differences of certain elements (potassium and phosphorus) between frozen-hydrated and frozen-dried states of the tissues as applied formerly to sectioned material by others. A new mathematical formula has been derived giving rather precise figures for IWC. No elemental standards are necessary for the measurement: one has to obtain only the peak to background ratios in wet and dry states of the cells. the method is sensitive enough to reveal age-dependent as well as drug-induced changes of IWC in liver and brain cells. The values obtained are quite comparable with the theoretically expected one. Technical problems of the application of this method are discussed in detail.

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

Abstract:  The main component of Alzheimers disease (AD) senile plaques in the brain is amyloid‐β peptide (Aβ), a proteolytic fragment of the amyloid precursor protein (APP). Platelets contain both APP and Aβ 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 Aβ ending at residue 40. To verify whether APP β‐processing could be observed also in an in vitro system of highly concentrated platelets, we measured the Aβ released in the incubation media of 5 × 109 platelets/mL by enzyme‐linked immunosorbent assay (ELISA). The activation status of platelets was investigated by ultrastructural analysis. We found that Aβ40 levels were significantly higher in incubation media of 5 × 109/mL platelets in comparison with 108/mL platelets (normalized values), while Aβ42 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 β‐processing with consequent release of Aβ40. Investigating the biochemical pathways triggering Aβ secretion in platelets might provide important information for developing tools to modulate this phenomenon in AD brains.

Energy dispersive X-ray microanalysis of the electrolytes in biological bulk specimen: II. Age-dependent alterations in the monovalent ion contents of cell nucleus and cytoplasm in rat liver and brain cells

Energy dispersive X-ray microanalysis was carried out on the nucleus and cytoplasm of large brain cortical and liver cells of young, adult and old (1, 12 and 24 months of age, respectively) female Wistar rats, using a new method for preparation of freeze-dried bulk specimens. It was found that the K + , Na + and Cl − contents per unit dry mass in the nucleus in both tissues significantly increased except the K + content of the hepatocyte nucleus between 1 and 24 months of age. At the same time, in the cytoplasm some ions increased, others decreased or remained unchanged. There is an age-dependent intracellular water loss the extent of which is not known for the tissues studied, however, in human organism it averages about 10–14% between 20 and 99 years. Therefore, intranuclear and intracytoplasmic ionic strengths of the rat tissues studied were calculated assuming 2–6% water loss between 1 and 24 months of age. Already a water loss of 2% results in considerable increases of the ionic strength within the cell water even in those cases where the dry mass fraction values slightly decreased or remained unchanged. The values obtained in the old cell nuclei reach ranges of ionic strength where nonhistonic regulatory proteins are separated from the chromatin in vitro . The results are discussed in the light of recent biochemical observations.

Neuronal plasticity in aging: A quantitative immunohistochemical study of GAP-43 distribution in discrete regions of the rat brain

Age-related changes in neuroplasticity have been investigated considering the neuronal growth-associated protein GAP-43 as a marker of nerve cell structural adaptive capabilities. We carried out a quantitative immunohistochemical study on the distribution of GAP-43 in the molecular layer of the cerebellar cortex, in the inner molecular layer of the hippocampal dentate gyrus, in the stratum radiatum of the CA1 region, in layer 1 of the cingulate cortex and in the nerve fiber layer of the main olfactory bulb of 3-, 18- and 31-month-old Wistar rats. A decrease of GAP-43 immunoreactivity was observed in the old rats in comparison with the adult animals in all the 5 areas analyzed, although these variations were only statistically significant in the dentate gyrus, cingulate cortex and olfactory bulb. In these latter zones, GAP-43 immunolabeling is reduced by 54, 42 and 38%, respectively, in the old versus the adult group. Comparing these data with the age-dependent decrease of neuron density innervating the areas investigated, we support the hypothesis that the decline of GAP-43 observed in old animals documents a consistent reduction of axon plasticity in the inner molecular layer of the dentate gyrus and in layer 1 of the cingulate cortex. These results suggest an important role of GAP-43 as a marker of age-dependent deterioration of synaptic plasticity, especially in those areas of the brain involved in memory and emotional behavior.