Natascia Gracciotti

Distribution of MAP2 in Hippocampus and Cerebellum of Young and Old Rats by Quantitative Immunohistochemistry

The microtubule-associated protein MAP2 is a cytoskeletal protein that plays a regulatory role in neuronal plasticity and in maintaining the morphology of differentiated neurons. MAP2 distribution was assessed in hippocampus and cerebellum of young and old rats by quantitative immunohistochemistry. In old vs young rats, densitometric analysis showed a significant decrease of MAP2 immunoreactivity in the hippocampus CA1 field (-93%), whereas no difference was found in cerebellar MAP2 distribution. These preliminary data suggest that in areas of the brain involved in memory acquisition and consolidation, MAP2-dependent neuroplasticity and structural integrity are significantly decreased in aging. (J Histochem Cytochem 49:1065–1066, 2001)

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/μm3 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)

Quantitative Immunohistochemistry of Glucose Transport Protein (Glut3) Expression in the Rat Hippocampus During Aging

Immunohistochemistry of Glut3 (45 kD), an integral membrane peptide mediating the transport of glucose in neurons, was carried out in the hippocampus of 3- and 28-month-old rats to assess the effect of age on energy metabolism. Free-floating sections of fixed-frozen hippocampi were processed for quantitative immunohistochemistry of Glut3. A rabbit affinity-purified antibody identified Glut3 immunoreactivity. Glut3 staining was intense in neuropil, axons, and dendrites, whereas nerve cell bodies were unstained. With aging, Glut3 reactivity was significantly decreased in the inner molecular layer of the hip-pocampal dentate gyrus (–46%) and the mossy fibers of the CA3 sector (–34%), whereas the stratum radiatum of CA1 did not show any difference due to age. These data document an age-dependent decrease in Glut3 expression in discrete areas of rat hippocampus. Glut3 constitutes the predominant glucose transporter in neurons and is found abundantly in regions with high synaptic density characterized by frequent bursts of function-adequate metabolic activity. Our findings therefore lend further support to the critical role of an impaired metabolism in age-related brain dysfunctions and disease. (J Histochem Cytochem 49:671–672, 2001)

Age-related effects of moderate alcohol consumption on GAP-43 levels in rat hippocampus

The effects of moderate intake of ethanol and ageing were investigated on the levels of the growth-associated protein GAP-43, whose expression has been used as an indicator of axonal growth during development, regeneration and remodelling of synaptic connections. Groups of female Wistar rats (12 and 24 months of age), were alcohol-fed for one month while age-matched control groups received an isocaloric diet. A quantitative evaluation of GAP-43 was performed in hippocampus and in hippocampal selected areas in view of the vulnerability of this complex to alcohol aggression by means of two different methods, namely Western blot analysis and immunohistochemistry. While the former measures total extractable GAP-43, the latter allows visualisation of in situ changes in topographical distribution of GAP-43. Western blot analysis revealed an age-dependent reduction (-47%) and an ethanol-associated increase (81%) of GAP-43 demonstrated only in the old group. Conversely, quantitative immunohistochemistry of GAP-43 in the entire hippocampus showed a non-significant ethanol-related decrement in 24-month-old rats (-30%), although the age-dependent reduction was confirmed. Ageing was associated with a decrement of GAP-43 immunostaining in CA3 stratum radiatum (CA3) and in inner molecular layer of dentate gyrus (IML). Treatment determined a decrease of GAP-43 immunostaining in adult rat CA3 and IML and no change in CA1 stratum radiatum (CA1). Our results suggest that immunohistochemistry evaluation underestimates GAP-43 levels in ethanol-treated animals possibly as a consequence of conformational changes induced by alcohol, resulting in non-targeting of the specific antibody. Western blot analysis demonstrate that although there is a reduction of GAP-43 levels in hippocampus of aged rats, this structure retain a remarkable potential to compensate for ethanol toxicity during ageing.

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.

Cellular Distribution of GAP-43 mRNA in Hippocampus and Cerebellum of Adult Rat Brain by In Situ RT-PCR

The growth-associated protein GAP-43 is a presynaptic membrane phosphoprotein that plays a key role in guiding the growth of axons and in modulating the formation of new synapses. To identify the cells that synthesize GAP-43 mRNA, we applied direct in situ reverse transcription-polymerase chain reaction (in situ RT-PCR) in cerebellum and hippocampus of adult rat brain. In situ RT-PCR revealed GAP-43 mRNA in cerebellar granule cells, in Purkinje cells and in some interneurons of the molecular layer. Previous in situ hybridization studies had demonstrated a dense label throughout the granular layer of the cerebellar cortex but no labeling of other cerebellar neurons. Hippocampal cells showing distinct GAP-43 mRNA signal after in situ RT-PCR were CA1 and CA3 pyramidal neurons, CA4 hilar cells, and dentate gyrus granule cells, whereas in situ hybridization studies had detected GAP-43 mRNA only in CA3 and CA1 pyramidal neurons. Our data indicate that GAP-43 mRNA is widely distributed, suggesting that many cell types are potentially involved in synaptic plasticity events. (J Histochem Cytochem 49:1195–1196, 2001)

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

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/μm3 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 μm3 >) 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 μm3 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.