Expression of aromatase P450(AROM) in the human fetal and early postnatal cerebral cortex.

Aromatase (P450(AROM)), the enzyme responsible for the conversion of testosterone (T) into 17-ß estradiol (E(2)), plays a crucial role in the sexual differentiation of specific hypothalamic nuclei. Moreover, recent findings indicate that local E(2) synthesis has an impact on other brain areas including hippocampus, temporal cortex and cerebellum, and may thus influence also cognitive functions. Numerous studies have described the expression and the distribution of P450(AROM) throughout ontogenesis and postnatal development of the central nervous system in several mammals, but data referring to humans are scarce. In the adult human brain, P450(AROM) has been detected in the hypothalamus, limbic areas, and in the basal forebrain, and described in glial cells of the cerebral cortex and hippocampus. In this study we report the expression, distribution and cellular localization of P450(AROM) in the human fetal and early postnatal cerebral cortex. In our series of fetal brains of the second trimester, P450(AROM) expression appeared at gestational week (GW) 17 and resulted limited to groups of cells localized close to the growing neuroepithelium in the ventricular and subventricular zones. At GWs 20-24, scattered P450(AROM) immunoreactive (-ir) neural cells were identified in the intermediate plate and subplate, and in the parietal cortical plate. In perinatal and early postnatal individuals the quantity of P450(AROM)-ir elements increased, and revealed the morphology typical of glial cells. Double labeling immunostaining with anti-GFAP and anti-P450(AROM) antisera, and subsequent confocal analysis, confirmed this observation. Our data show that the expression of P450(AROM) in the fetal cortex starts approx at the end of the fourth gestational month, but increases steadily only in the last trimester or in the early postnatal period. This temporal trend may suggest that P450(AROM) could act as a differentiation-promoting factor, based on timing of the steroid actions.

Physiological cholesterol concentration is a neuroprotective factor against ß-amyloid and ß-amyloid-metal complexes toxicity.

Alzheimer's disease is one of the most common forms of dementia in the elderly. One of its hallmarks is the abnormal aggregation and deposition of ß-amyloid (Aß). Endogenous and exogenous metal ions seem to influence ß-amyloid folding process, aggregation and deposition. Besides these variables other elements appear to affect ß-amyloid behavior, such as cholesterol. The physiological concentration of cholesterol in the cerebrospinal fluid (CSF) was used in order to determine the extent in which Aß and Aß-metal complexes in vitro aggregation and their toxicity on human neuroblastoma cell cultures is affected. Cholesterol did not appear to influence Aß and Aß-metal complexes aggregation, but it was effective in protecting neuroblastoma cells against Aß complexes' toxicity. The Aß-Al complex seemed to be the most effective in disrupting and damaging membrane external layer, and simultaneously it appears to increase its toxicity on cell cultures; both of these effects are preventable by cholesterol. The presence in physiological concentrations of cholesterol seemed to compensate membrane damage that occurred to neuroblastoma cells. These findings appear to contradict some data reported in literature. We believe that our results might shed some light on the role played by cholesterol at physiological concentrations in both cellular balance and membrane protection.

Role of metal ions in the abeta oligomerization in Alzheimer's disease and in other neurological disorders.

Neurodegeneration is a complex and multifaceted process leading to many chronic diseased states. Neurodegenerative disorders include a number of different pathological conditions, like Alzheimer's and Parkinson's diseases, which share similar critical metabolic processes, such as protein aggregation, which could be affected by some metal ions. A huge number of reports indicate that, among putative aggravating factors, metal ions (Al, Zn, Cu, Fe) could specifically impair protein aggregation of Abeta, prion protein, ataxin, huntingtin, etc. and their oligomeric toxicity. While studying the molecular basis of these diseases, it has become clear that protein conformation plays a critical role in the pathogenic process. In this review, we will focus on Alzheimer's disease and on the role of metal ions, specifically aluminium, in affecting amyloid aggregation, oligomerization and toxicity.

Manganese intoxication decreases the expression of manganoproteins in the rat basal ganglia: an immunohistochemical study.

Manganese (Mn) is a cofactor for some metalloprotein enzymes, including Mn-superoxide dismutase (Mn-SOD), a mitochondrial enzyme predominantly localized in neurons, and glutamine synthetase (GS), which is selectively expressed in astroglial cells. The detoxifying effects of GS and Mn-SOD in the brain, involve catabolizing glutamate and scavenging superoxide anions, respectively. Mn intoxication is characterized by impaired function of the basal ganglia. However, it is unclear whether regional central nervous system expression of manganoproteins is also affected. Here, we use immunocytochemistry in the adult rat brain, to examine whether Mn overload selectively affects the expression of GS, Mn-SOD, Cu/Zn-SOD, another component of the SOD family, and glial fibrillary acid protein (GFAP), a specific marker of astrocytes. After chronic Mn overload in drinking water for 13 weeks, we found that the number and immunostaining intensity of GS- and Mn-SOD-positive cells was significantly decreased in the striatum and globus pallidus, but not in the cerebral frontal cortex. In addition, we found that GS enzymatic activity was decreased in the strio-pallidal regions but not in the cerebral cortex of Mn-treated animals. In contrast, Cu/Zn-SOD- and GFAP-immunoreactivity was unchanged in both the cerebral cortex and basal ganglia of Mn-treated rats. Thus, we conclude that in response to chronic Mn overload, a down-regulation of some manganoproteins occurs in neurons and astrocytes of the striatum and globus pallidus, probably reflecting the vulnerability of these regions to Mn toxicity.

Effect of aluminum consumption on the vestibulo-ocular reflex.

The effects of chronic exposure (90 days) to Aluminum chloride (AlCl(3)) were analyzed in 3, 10 and 24 month old male rats (n=270) by investigating the function of the VOR (vestibulo-ocular reflex) in correlation with Aluminum (Al) concentrations in blood and brain. The VOR was chosen and tested in basal conditions (pre-exposure measures) and during the continuous administration of three different concentrations of AlCl(3) (0.5, 1, 2 g/l in drinking solution): the control animals being exposed to NaCl (0.125, 0.25 and 5 g/l in drinking solution). Results showed that LOAEL (Lowest Observed Adverse Effect Level) was 43.1+/-11.4 mg Al/kg-day. At this concentration the Al induced significant VOR impairment in all exposed rats, regardless of animal age. Neuroanatomical analysis showed that VOR impairment was not dependent on astrocyte damage nor evidences of amyloid deposits in the exposed rats was found. Significant changes of Al concentrations were observed in nervous tissue, while its concentration in whole blood was unaffected. Furthermore, results show that it is possible to identify an individual neurotoxic threshold for each animal and therefore hypothesize the clinical use of the VOR test for the evaluation of individual risk toxicity to chronic Al exposure.

Metallothionein-I-II and GFAP positivity in the brains from frontotemporal dementia patients.

Frontotemporal dementia regards a group of presenile progressive neurodegenerative form of dementias which includes Pick's disease, corticobasal degeneration, frontotemporal dementia with motor neuron disease, frontal lobe degeneration, dementia-parkinsonism-amyotrophy complex, familial non-specific dementia mapping to chromosome 3, non-Alzheimer degenerative dementia lacking distinctive histological features as well as a number other infrequent syndromes with dementia and focal neurological signs. The aim of this study was to investigate the regional distribution of metallothionein-I-II, an ubiquitary group of buffering proteins, in cases of frontotemporal dementia. The aim of the present study was to study the metallothionein-I-II expression in relationship to the expression in astrocytes of glial fibrillary acidic protein (GFAP) as we have already done in previous studies of Alzheimer's and Binswanger's diseases [31,32]. Our findings indicate that metallothionein-I-II expression in the most affected areas is likely to be regionally distinct and layer-dependent, in that it is highest in the deep layers of the frontotemporal cortex and the allocortex (hippocampus) while insignificantly immunopositive in the occipital cortex. In addition, the potential use of metallothionein-I-II as a new pharmacological approach to contrast some deleterious aspects of this disease has been also discussed.

Aluminum-triggered structural modifications and aggregation of beta-amyloids.

We investigated the structural effects induced by Al3+ on different beta-amyloid (Abeta) fragments at pH 7.4 and T=25 degrees C, with particular attention given to the sequences 1-40 and 1-42. Al3+ caused peptide enrichment in beta sheet structure and formation of solvent-exposed hydrophobic clusters. These intermediates evolved to polymeric aggregates which organized in fibrillar forms in the case of the Al3+-Abeta(1-42) complex. Comparative studies showed that Zn2+ and Cu2+ were much less efficient than Al3+ in stimulating the spontaneous aggregation/fibrillogenesis of Abetas. Studies with liposomes as membrane models showed dramatic changes in the structural properties of the lipid bilayer in the presence of Al3+-Abeta complexes, suggesting a major role of Al3+ in Abeta-induced cell dysfunction. Al3+ effects were abolished by desferrioxamine mesylate (DFO) only in solution. We concluded that, in vivo, DFO may act as a protective agent by preventing or reverting Abeta aggregation in the extracellular spaces.

Copper and zinc dismetabolism in the mouse brain upon chronic cuprizone treatment.

Recent reports describe successful treatment using copper chelation therapy in neurodegenerative animal models. However, the success claimed for chelation therapy in neurodegenerative diseases is still rather controversial. To acquire new information on copper metabolism/homeostasis, we utilized cuprizone, a very sensitive and selective copper-chelating agent with well-known neurotoxic properties, as a relevant chemical model in mice. Upon cuprizone treatment, mice developed a pronounced astrocytosis, with brain oedema and spongiosis characterised by vacuolisations of the neuropil predominantly in the white matter. In addition, cuprizone treatment severely altered copper and zinc homeostasis in the central nervous system (CNS) as well as in all other tissues examined, with increasing metal ion concentrations particularly in the CNS. Concomitant with this increase in the Cu and Zn concentration in the brain, metallothionein-I and -II were also highly immunoreactive in astrocyte, consistent with the astrocytosis and demyelination observed in our and other laboratories.

Structural effects of titanium citrate on the human erythrocyte membrane.

The structural effects of titanium citrate on the human erythrocyte membrane were studied through its interaction with intact erythrocytes and isolated unsealed human erythrocyte membranes (IUM). The studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Titanium citrate induced shape changes in erythrocytes, which were damaged and ruptured leaving empty and retracted membranes. Fluorescence spectroscopy measurements in IUM indicated a disordering effect at both the polar head group and the acyl chain packing arrangements of the membrane phospholipid bilayer. Titanium citrate also interacted with molecular models of the erythrocyte membrane consisting in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representing classes of phospholipids located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction indicated that titanium citrate induced structural perturbation of the polar head group and of the hydrophobic acyl regions of DMPC, while the effects on DMPE bilayers were negligible. This conclusion is supported by fluorescence spectroscopy measurements on DMPC large unilamellar vesicles. All these findings indicate that the structural perturbations induced by titanium to human erythrocytes can be extended to other cells, thereby affecting their functions.

Aluminum fluoride affects the structure and functions of cell membranes.

No useful biological function for aluminum has been found. To the contrary, it might play an important role in several pathologies, which could be related to its interactions with cell membranes. On the other hand, fluoride is a normal component of body fluids, soft tissues, bones and teeth. Its sodium salt is frequently added to drinking water to prevent dental caries. However, large doses cause severe pathological alterations. In view of the toxicity of Al(3+) and F(-) ions, it was thought of interest to explore the damaging effects that AlF(3) might induce in cell membranes. With this aim, it was incubated with human erythrocytes, which were examined by phase contrast and scanning electron microscopy, and molecular models of biomembranes. The latter consisted of large unilamellar vesicles (LUV) of dimyristoylphosphatidylcholine (DMPC) and bilayers of DMPC and dimyristoylphosphatidylethanolamine (DMPE) which were studied by fluorescence spectroscopy and X-ray diffraction, respectively. In order to understand the effects of AlF(3) on ion transport (principally sodium and chloride) we used the isolated toad skin to which electrophysiological measurements were applied. It was found that AlF(3) altered the shape of erythrocytes inducing the formation of echinocytes. This effect was explained by X-ray diffraction which revealed that AlF(3) perturbed the structure of DMPC, class of lipids located in the outer monolayer of the erythrocyte membrane. This result was confirmed by fluorescence spectroscopy on DMPC LUV. The biphasic (stimulatory followed by inhibitory) effects on the isolated skin suggested changes in apical Cl(-) secretion and moderate ATPase inactivation.

Effects of lead on the human erythrocyte membrane and molecular models.

Lead has no biological function; however, low, and particularly, high levels of exposure have a number of negative consequences for human health. Despite the number of reports about lead toxicity, very little information has been obtained regarding its effects on cell membranes. For this reason, the structural effects of lead on the human erythrocyte membranes were investigated. This aim was attained by making lead ions interact with intact erythrocytes, isolated unsealed erythrocyte membranes (IUM) and molecular models. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representing phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane. The results, obtained by electron microscopy, fluorescence spectroscopy and X-ray diffraction, indicated that (a) lead particles adhered to the external and internal surfaces of the human erythrocyte membrane; (b) lead ions disturbed the lamellar organization of IUM and DMPC large unilamellar vesicles (LUV) and (c) induced considerable molecular disorder in both lipid multilayers, the effects being much more pronounced in DMPC.

In vivo and in vitro effects of aluminum on the activity of mouse brain acetylcholinesterase.

Cholinesterases are a large family of enzymatic proteins widely distributed throughout both neuronal and non-neuronal tissues. In Alzheimer's disease (AD), analytical as well as epidemiological studies suggest an implication of an abnormal focal accumulation of aluminum in the brain. In this devastating disease, aluminum may interfere with various biochemical processes including acetylcholine metabolism, and can thus act as a possible etiopathogenic cofactor. Acetylcholinesterase (AChE) exists in several molecular forms that differ in solubility and mode of membrane attachment rather than in catalytic activity. Mice were treated orally with aluminum chloride or aluminum lactate (Al(lac)(3)), and AChE activity in their brain homogenates was then assayed. Results showed that this in vivo treatment augmented the activity of the enzyme. An activating effect was also observed in vitro, when the aluminum compounds were added directly to mouse brain homogenates. However, the activating effect observed in vivo was much more marked than that observed in vitro. In addition, the activation produced by Al(lac)(3) was higher than that obtained after aluminum chloride treatment. Kinetics measurements of AChE activity in the absence and presence of treatment with aluminum both in vivo and in vitro are reported. The influence of the metal speciation on enzymatic activity is discussed in relation to a possible implication of aluminum in some neurodegenerative diseases.

Roles of the metallothionein family of proteins in the central nervous system.

Metallothioneins (MTs) constitute a family of proteins characterized by a high heavy metal [Zn(II), Cu(I)] content and also by an unusual cysteine abundance. Mammalian MTs are comprised of four major isoforms designated MT-1 trough MT-4. MT-1 and MT-2 are expressed in most tissues including the brain, whereas MT-3 (also called growth inhibitory factor) and MT-4 are expressed predominantly in the central nervous system and in keratinizing epithelia, respectively. All MT isoforms have been implicated in disparate physiological functions, such as zinc and copper metabolism, protection against reactive oxygen species, or adaptation to stress. In the case of MT-3, an additional involvement of this isoform in neuromodulatory events and in the pathogenesis of Alzheimer's disease has also been suggested. It is essential to gain insight into how MTs are regulated in the brain in order to characterize MT functions, both in normal brain physiology, as well as in pathophysiological states. The focus of this review concerns the biology of the MT family in the context of their expression and functional roles in the central nervous system.

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.

Effects of AlCl3 on toad skin, human erythrocytes, and model cell membranes.

Aluminum, a very abundant metal, could play a toxic role in several pathological processes, including neurodegeneration. Although the effects of Al(III) on biological membranes have been extensively described, direct information concerning the molecular basis of its biological activity is rather scanty. To examine aluminum challenges on cell membranes, various concentrations of AlCl3 in aqueous solutions were incubated with human erythrocytes, isolated toad skin, and molecular models of biomembranes. The latter consisted of multilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, representing phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane. These specimens were studied by scanning electron microscopy, electrophysiological measurements, and x-ray diffraction. The results indicate that Al(III) in the concentration range of 10-100 microM induced the following structural and functional effects: (i) change in the normal discoid shape of human erythrocytes to echinocytes due to the accumulation of Al(III) ions in the outer moiety of the red cell membrane; (ii) perturbation of dimyristoylphosphatidylcholine, and to a lesser extent of dimyristoylphosphatidylethanolamine bilayers, and (iii) decrease in the short-circuit current and in the potential difference of the isolated toad skin, effects that are in accordance with a time-dependent modulation of ion transport in response to changes in the molecular structure of the lipid bilayer.

Inhibitory effect of aluminum on dopamine beta-hydroxylase from bovine adrenal gland.

Aluminum is a well known neurotoxic agent that is overaccumulated in the substantia nigra of patients affected by Parkinson's disease as well as in certain cerebral areas of other neurodegenerative pathologies such as Alzheimer's disease. Although the role of aluminum in neurodegenerative diseases is yet to be clearly understood, the metal ion is known to substantially alter the activity of several key enzymes in the central nervous system. The present paper reports data on the effect of aluminum on the activity of dopamine-beta-hydroxylase from bovine adrenaL gland utiLized as a model study. The metal ion inhibited the activity of this enzyme with a mixed type mechanism following the Michaelis-Menten equation. In the absence of Al, the enzyme exhibited a Km and Vmax of 2.56 mM of 4.12 pmol/min respectively, while in the presence of Al its Km and Vmax were 3.85 mM and 2.86 pmol/min respectively. The potential implications of aluminum in the etiopathogenesis of neurological disorders are discussed.

Aluminum as an inducer of the mitochondrial permeability transition.

Treatment of rat liver mitochondria with aluminum in the presence of Ca2+ results in large amplitude swelling accompanied by loss of endogenous Mg2+ and K+ and oxidation of endogenous pyridine nucleotides. The presence of cyclosporin A, ADP, bongkrekic acid, N-ethylmaleimide and dithioerythritol prevent these effects, indicating that binding of aluminum to the inner mitochondrial membrane, most likely at the level of adenine nucleotide translocase, correlates with the induction of the membrane permeability transition (MPT). Indeed, aluminum binding promotes such a perturbation at the level of ubiquinol-cytochrome c reductase, which favors the production of reactive oxygen species. These metabolites generate an oxidative stress involving two previously defined sites in equilibrium with the glutathione and pyridine nucleotides pools, the levels of which correlate with the increase in MPT induction. Although the above-described phenomena are typical of MPT, they are not paralleled by other events normally observed in response to treatment with inducers of MPT (e.g., phosphate), such as the collapse of the electrochemical gradient and the release of accumulated Ca2+ and oxidized pyridine nucleotides. Biochemical and ultrastructural observations demonstrate that aluminum induces a pore opening having a conformation intermediate between fully open and closed in a subpopulation of mitochondria. While inorganic phosphate enhances the MPT induced by ruthenium red plus a deenergizing agent, aluminum instead inhibits this phenomenon. This finding suggests the presence of a distinct binding site for aluminum differing from that involved in MPT induction.

Aluminum inhibits the lysosomal proton pump from rat liver.

Lysosomes are cytoplasmatic organelles, delimitated by a single lipoprotein membrane, that contain several enzymes mostly belonging to the hydrolases in that they function mainly for intracellular digestion. Lysosomal internal pH is characteristically acidic and it is maintained around pH 4.5 by a proton pump, an ATPase, that uses energy from ATP hydrolysis to translocate H+ ions into lysosomes. In the presence of Al3+ the proton pump activity is markedly reduced compromising acidic vesicles functionality. Among different species utilized, Al2(SO4)3 and AlF3 were the most effective. Aluminum effect was not observed when the delta pH was produced artificially by nigericin.

Effects of aluminum on activity of krebs cycle enzymes and glutamate dehydrogenase in rat brain homogenate.

Aluminum is a neurotoxic agent for animals and humans that has been implicated as an etiological factor in several neurodegenerative diseases and as a destabilizer of cell membranes. Due to its high reactivity, Al3+ is able to interfere with several biological functions, including enzymatic activities in key metabolic pathways. In this paper we report that, among the enzymes that constitute the Krebs cycle, only two are activated by aluminum: alpha-ketoglutarate dehydrogenase and succinate dehydrogenase. In contrast, aconitase, shows decreased activity in the presence of the metal ion. Al3+ also inhibits glutamate dehydrogenase, an allosteric enzyme that is closely linked to the Krebs cycle. A possible correlation between aluminum, the Krebs cycle and aging processes is discussed.