Acetylcholinesterase inhibitors in Alzheimer's disease influence Zinc and Copper homeostasis.

BACKGROUND: Alzheimer's disease (AD) is the most common age-related neurodegenerative disease. An altered homeostasis of Zinc (Zn) and Copper (Cu), as well as a dysregulated expression of Zn-regulatory proteins have been previously described in AD. Acetylcholinesterase inhibitors (AChEI) are commonly used as AD treatment to improve cognitive function, but their effect on Zn homeostasis is still unexplored. OBJECTIVES: The aims of this study were to define the metal dyshomeostasis in AD patients, to investigate AChEI influence on Zn homeostasis and inflammation, and to analyze the relationship between cognitive impairment at two-year follow-up and metal concentrations, considering AChEI use. METHODS AND RESULTS: 84 Healthy Elderly (HE) and 95 AD patients were enrolled (62 AchEI user and 33 AchEI naïve). HE showed similar plasma Zn and Cu concentrations and Cu/Zn ratio in comparison to AChEI users, but significantly higher Zn level, as well as lower Cu amount and Cu/Zn ratio than AChEI naïve patients. Moreover, AChEI users had increased Zn plasma level, reduced Cu amount, Cu/Zn ratio, and IL1ß concentration and lower Zip2 lymphocytic expression vs. naïve patients. A multiple linear regression analysis showed that the MMSE score decline after two-year follow-up was reduced by AChEI therapy and was positively associated with plasma Zn decrease over time. CONCLUSION: Our data revealed that AChEI use may affect peripheral Zn and Cu homeostasis in AD patients, decrease Cu/Zn ratio demonstrating a general reduction of inflammatory status in patients under AChEI treatment. Finally, AChEI influence on circulating Zn could be implicated in the drug-related slowdown of cognitive decline.

My Mind Project: the effects of cognitive training for elderly-the study protocol of a prospective randomized intervention study.

BACKGROUND: Cognitive decline and dementia represent a key problem for public health as they heavily impair social functioning and independent living. The development of new strategies to support recommendations for patients and their caregivers may represent an outstanding step forward. AIMS: To describe the study protocol and methods of "My Mind Project: the effect of cognitive training for elderly" (Grant No. 154/GR-2009-1584108), which investigates, by the use of a multidisciplinary approach, the effects of a comprehensive cognitive training programme on performances in aged subjects with mild-moderate Alzheimer's disease, mild cognitive impairment and normal cognitive functioning. METHODS: The study is a prospective randomized intervention for the assessment of cognitive training effects in three groups of elderly subjects with different cognitive status. A total of 321 elderly people were enrolled in Marche Region, Italy. Each subject was randomly assigned to an experimental group or to a control group. Cognitive performances and biochemical blood markers have also been analysed before cognitive training (baseline), immediately after termination (follow-up 1), after 6 months (follow-up 2) and after 2 years (follow-up 3). DISCUSSION: The results will be useful to identify some efficient programmes for the enhancement of cognitive performance in elderly with and without cognitive decline. CONCLUSION: The application of a non-pharmacological approach in the treatment of elderly with cognitive disorders could have a profound impact on National Health Service.

Forced mild physical training improves blood volume in the motor and hippocampal cortex of old mice.

OBJECTIVES: To assess the effect of mild forced physical training on cerebral blood volume (CBV) and other brain parameters in old mice. SETTING: Treadmill in the animal house. PARTICIPANTS: Thirty old (>25 mo) male mice were randomly assigned to one of three groups, exercise (E), exercise plus testosterone (T) (ET), and rest (C). INTERVENTION: Mild physical training on treadmill (30 min a day at belt speed = 8 m/min, five days a week) with or without one weekly injection of testosterone. MEASUREMENTS: CBV, quantitative transverse relaxation time (T2) maps, and cortical thickness were measured by magnetic resonance imaging. RESULTS: A significant increase of CBV was found in the motor and hippocampal cortex of E and ET mice; cortical thickness was not affected. T2 maps analysis suggested that water distribution did not change. T administration did not add to the effect of physical training. CONCLUSION: This work provides first quantitative evidence that exercise initiated at old age is able to improve the hemodynamic status of the brain cortex in key regions for movement and cognition without inducing edema.

Discovering pathways of sarcopenia in older adults: a role for insulin resistance on mitochondria dysfunction.

The precise cause of sarcopenia, skeletal muscle loss and strength, in older persons is unknown. However, there is a strong evidence for muscle loss due to insulin resistance as well as mitochondrial dysfunction over aging. Considering that epidemiological studies have underlined that insulin resistance may have a specific role on skeletal muscle fibre atrophy and mitochondrial dysfunction has also been extensively shown to have a pivotal role on muscle loss in older persons, a combined pathway may not be ruled out. Considering that there is growing evidence for an insulin-related pathway on mitochondrial signaling, we hypothesize that a high degree of insulin resistance will be associated with the development of sarcopenia through specific alterations on mitochondrial functioning. This paper will highlight recent reviews regarding the link between skeletal muscle mitochondrial dysfunction and insulin resistance. We will specifically emphasize possible steps involved in sarcopenia over aging, including potential biomolecular mechanisms of insulin resistance on mitochondrial functioning.

Decreased numeric density of succinic dehydrogenase-positive mitochondria in CA1 pyramidal neurons of 3xTg-AD mice.

Alzheimer disease (AD) is associated with mitochondrial dysfunction. In this study, we investigated succinic dehydrogenase (SDH) activity in mitochondria of hippocampal CA1 pyramidal neurons obtained from 10-month-old 3xTg-AD mice, an animal model of AD, as well as from age-matched control mice PS1-KI. In SDH-positive mitochondria, we measured numeric density (Nv, number of mitochondria/microm(3) of cytoplasm), average organelle volume (V), volume density (Vv, volume fraction of mitochondria/microm(3) of cytoplasm), average length (Fmax), and the ratio (R) between the total area of the cytochemical precipitate due to SDH activity and the total mitochondrial area. Our results indicate that 3xTg-AD mitochondria show a significant decrease of Nv, increase in V and Fmax, as well as a trend toward a reduction of R, whereas Vv is unchanged. Our findings further support the idea that mitochondrial dysfunction is involved in AD and are in line with studies indicating that both amyloid precursor protein (APP) and amyloid-beta (Abeta) localize to mitochondria.

Structural synaptic remodeling in the perirhinal cortex of adult and old rats following object-recognition visual training.

The ultrastructural features of layer II synapses in the perirhinal cortex of adult (4- to 6-month-old) and old (25- to 27-month-old) rats exposed to a six-session object recognition visual training were investigated by morphometric methods. The comparative analysis showed a higher synaptic numeric density, a lower synaptic average area, and a lower percentage of megasynapses (S > 0.5 microm2) in old trained rats versus controls, and a higher percentage of small (S < 0.15 microm2) junctions in adult trained rats versus controls. The more marked synaptic remodeling underlying memory consolidation in the perirhinal cortex of old rats might reflect a pre-existing lower dynamic status.

Transthyretin inhibition of amyloid beta aggregation and toxicity.

OBJECTIVES: The aim of this study was to investigate transthyretin (prealbumin) effects on Abeta25-35-induced cytotoxicity. DESIGN AND METHODS: In view of the well-recognized literature data demonstrating that Abeta25-35 fibrillar aggregates cause in vitro cytotoxicity to human red blood cells and apoptotic changes to SK-N-BE neuroblastoma cells in cultures (ultrastructural evidence), we tested transthyretin effects on these two experimental models. RESULTS: Incubation of Abeta25-35 with transthyretin (at transthyretin concentrations equal to CSF physiological levels) demonstrated both inhibition of red blood cells lysis and neutralization of SK-N-BE neuroblastoma cells ultrastructural apoptotic changes. Moreover, transthyretin was shown to be able to inhibit the formation of fibrillar macroaggregates of Abeta25-35. CONCLUSIONS: The findings imply that experimental systems investigating Abeta-induced cytotoxicity consider the protective interaction of transthyretin with Abeta; an interaction to be considered also in vivo in view of the fact that transthyretin immunoreactivity has been previously demonstrated in amyloid plaques of brains from Alzheimer's disease patients.

Ethanol-induced decrease of the expression of glucose transport protein (Glut3) in the central nervous system as a predisposing condition to apoptosis: the effect of age.

We measured the effect of chronic ethanol administration on the expression of Glut3 in the cerebellum and hippocampus of adult and old rats. Glut3 expression significantly decreased in aging, in ethanol-treated rats vs. age-matched controls, and in adult- vs. old ethanol-treated rats. These findings lend consistent support to the hypothesis that disturbances of glucose metabolism due to ethanol may constitute an unfavorable condition predisposing to neuronal death.

In situ hybridization analysis of preprotachykinin-A mRNA levels in young and old rats.

Preprotachykinin-A (PPT-A) mRNA levels in discrete rat brain regions were examined. Analysis of silver grains revealed a 19.2% and 31.5% statistically significant decrease in PPT-A mRNA in the dorsal and ventral caudate putamen (d-CPu and v-CPu), respectively, a 30% lower expression of PPT-A mRNA in the bed nucleus of the stria terminalis (BNST), a 33.7% decrease in PPT-A mRNA in the habenula (Hb), and a 30% decrease of PPT-A mRNA levels in the posterodorsal part of the medial amygdala (MePD). Results show that aging of the CNS is associated with widespread changes in tachykinin gene expression, suggesting that alterations in the tachykinergic system may have implications in the physiopathology of the elderly.

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.

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/microm(3) 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)

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)

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.

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.

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 hippocampal 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)

Quantitative cytochemical mapping of mitochondrial enzymes in rat cerebella.

Mitochondrial metabolic competence, defined as the organelle's capacity to provide adequate amounts of ATP in due time, appears to constitute an important determinant in several biological processes and pathological conditions. Thus, the assessment of the metabolic efficiency of the mitochondrial population in a given tissue area or cellular compartment may provide clues to identifying alterations of the cellular bioenergetic machinery, which may constitute a predisposing condition leading to impaired organ and system functions. In the cerebellar cortex of adult rats, the activities of the enzymes cytochrome oxidase (COX) and succinic dehydrogenase (SDH) were, respectively, evidenced by means of the diaminobenzidine and copper ferrocyanide preferential cytochemical techniques. At the electron microscope, the activities of these two key molecules of the respiratory chain were clearly visualised as dark precipitates at the inner mitochondrial membrane sites where COX and SDH are located. By means of the disector method, unbiased mitochondrial samplings were carried out to measure: the number of mitochondria/microm(3) of tissue (numeric density: Nv); the mitochondrial volume fraction/microm(3) of tissue (volume density: Vv) and the average mitochondrial volume (V) both on COX- and SDH-positive organelles in the cerebellar glomeruli and Purkinje cells, respectively. The ratio R (total area of the precipitates due either to COX or SDH activity within the single mitochondrion/area of the same organelle) was also evaluated to get information on the enzyme activity related to mitochondrial size.The documented accumulation of mutant mitochondrial DNA particularly in postmitotic cells results in a marked heteroplasmy (mixtures of normal and mutated genomes) at mitochondrial and cellular levels, thus the cellular potential for energy production is demanded to a mosaic of organelles with different functional capabilities. Assessment of the mitochondrial mosaic outline by means of quantitative cytochemistry of key enzymes of the respiratory chain, such as COX and SDH, may allow for the morphofunctional metabolic mapping of mitochondrial efficiency in discrete cellular or tissue compartments.

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/µm(3) 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 µm(3) >) 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 µm(3) 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.