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.

Anticholinesterasic drugs: tacrine but not physostigmine, accumulates in acidic compartments of the cells.

Tacrine (THA) and physostigmine (PHS) have been used in Alzheimer's disease therapy for their anticholinesterasic activity. Here we show that THA is taken up in rat lysosomes in an energy-dependent manner, and that it is also accumulated in acidic compartments of rat thymocytes and neuroblastoma cells. A concentration of THA less than 1 mM dissipated the pH gradient (delta pH) in all the above mentioned in vitro systems. On the contrary higher concentrations of PHS (1-2 mM) were ineffective. The accumulation of THA in acidic organelles of the cell may be relevant for the pharmacological action of THA in Alzheimer's treatment.

The lipid intermediates arising during glycoprotein biosynthesis in liver microsomes.

Incubation of liver microsomes with GDP [14C] mannose leads to the formation of lipid-linked derivatives of [14C] mannose, a dolichol phosphate monosaccharide and dolichol pyrophosphate oligosaccharides. Standard procedures for separating these two types of compounds from each other were found to be deficient in that fractions thought to contain only dolichol pyrophosphate oligosaccharides are contaminated with dolichol phosphate mannose. This paper presents a column chromatographic procedure which conveniently separates the products of an 8 min labeling experiment into two components; dolichol phosphate [14C]mannose and a [14C]-mannose containing oligosaccharide which is also lipid bound. When this oligosaccharide is released from the lipid by hydrolysis and chromatographed on Sephadex G-50 or G-15 it gives a single peak with an indicated molecular weight of 1100. However, when this released oligosaccharide is chromatographed on concanavalin A Sepharose it is resolved into two peaks suggesting that there may be 2 oligosaccharide of approximately the same size but different structures. After brief periods of labeling with GDP [14C]mannose (5 s) an additional oligosaccharide of 3 to 4 sugar residues can be found in the dolichol pyrophosphate oligosaccharides fraction. Incubation of liver microsomes with UDP [14C]glucose or UDP[14C]galactose produces oligosaccharide components containing 7--8 sugar residues. Labeling of microsomes with UDP[14C]acetylglucosamine gives rise to three different components, including a lipid bound oligosaccharide containing 3- 5 sugar residues.

The transfer of galactose from UDP-galactose to endogenous lipid acceptors in liver microsomes.

When the microsomal fraction of beef liver is incubated with UDP-[14-C]-galactose in the presence of an inhibitor of nucleotide pyrophosphatase, there is an incorporation of the [14-C]galactose into glycoprotein and into two lipid components, one soluble in chloroform and the other in chloroform/methanol/water (1:1:0.3). Chromatography of the chloroform fraction on DEAE-cellulose or Kieselguhr G gives a single peak with behavior identical to that of dolichol phosphate mannose. Hydrolysis of the chloroform fraction released free galactose. It seems, therefore, that galactose, like glucose, mannose, and N-acetylglucosamine, can be transferred from its respective sugar nucleotide to glycoprotein via dolichol intermediates.

Aluminum alters intracellular calcium homeostasis in vitro.

The present paper reports data regarding the influence of aluminum, at micromolar concentrations, on intracellular calcium homeostasis. Al3+ modifies Ca2+ uptake in the endoplasmic reticulum (ER), accelerates Ca2+ release from mitochondria and strongly inhibits Ca2+-ATPase activity with a consequent high-level calcium accumulation inside the cell. These results suggest that Al3+ neurotoxicity may be related to an alteration of the intracellular calcium regulatory system.

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.

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.

Formation of titanium(IV) transferrin by reaction of human serum apotransferrin with titanium complexes.

The reaction of human serum apotransferrin with titanium(IV) citrate under physiological conditions results in the formation of a specific bis-titanium(IV) transferrin adduct (Ti2Tf hereafter) with two titanium(IV) ions loaded at the iron binding sites. The same specific Ti2Tf complex is formed by reacting apotransferrin with titanium(III) chloride and exposing the sample to air. The derivative thus obtained was characterized by spectroscopic techniques, including absorption, UV difference, circular dichroism and 13C NMR spectroscopies, and shown to be stable within the pH range 5.5-9.0. Surprisingly, the reaction of apoTf with titanium(IV) nitrilotriacetate (NTA) does not lead to formation of appreciable amounts of Ti2Tf, even after long incubation times, although some weak interactions of Ti(IV)-NTA with apoTf are spectroscopically detected. Implications of the present results for a role of transferrin in the uptake, transport and delivery of soluble titanium(IV) compounds under physiological conditions are discussed.

Experimental aluminum pathology in rabbits: effects of hydrophilic and lipophilic compounds.

Aluminum lactate [Al(lact)3] (hydrophilic, hydrolytically unstable) and aluminum acetylacetonate [Al(acae)3] (lipophilic, hydrolytically stable) were tested as potential toxicants to rabbits upon IV administration both as aqueous solutions and as liposome suspensions. Both chemicals behaved as cardiotoxic agents when administered as aqueous solutions, but Al(acae)3 was at least two orders of magnitude more active than Al(lact)3. Al(acae)3, but not Al(lact)3, caused myocardial infarcts resembling those in humans (with contraction bands) at doses as low as 0.24 mg/kg body weight, as well as a prominent acanthocytosis. Al(lact)3, when administered as a liposome suspension, was about 300 times more toxic than in aqueous solution, although cardiac damage was not infarctual in character. Both chemical and physical speciation of aluminum(III) thus play an essential role in determining the toxicity of the metal.

Metallothioneins are highly expressed in astrocytes and microcapillaries in Alzheimer's disease.

One of the neuropathological characteristics of Alzheimer's disease is the presence of a large number of reactive astrocytes, often, but not always, associated with senile plaques. The factors responsible for such an activation are as yet totally unknown. Other characteristic features of this disease such as betaA4 amyloid accumulation, senile plaques and neurofibrillary tangles represent well known pathological phenomena. Some studies suggest that betaA4 plays a major role in the reactive astrocytosis characteristic of Alzheimer's disease. In the normal human brain, metallothionein isoforms I and II are expressed in astrocytes but not in neurons. In the present study, we used anti-metallothionein antibodies to detect cells expressing metallothioneins isoforms I and II in normal and Alzheimer's disease (AD) brain sections. Results showed that expression of these proteins in the cortex, cerebral white matter and cerebellum is a relevant anatomopathological characteristic of Alzheimer's disease. Analysis of Alzheimer's disease brain sections revealed high expression of metallothioneins I/II in astrocytes and microcapillaries, and in the granular but not the molecular layer of the cerebellum. Furthermore, metallothionein expression can be used as a marker to identify subtypes of astrocytes.

Evaluation of MAO activities on murine neuroblastoma cells upon acute or chronic treatment with aluminium (III) or tacrine.

Monoamine-oxidase catalyses the oxidative deamination of various primary amines such as norepinephrine, serotonin, dopamine and others. Such an enzyme exists under two well known isotypes, A and B. In the brain monoamine-oxidase activity increases with ageing. The present paper reports data regarding the evaluation on the activity of monoamine-oxidase A and B in murine neuroblastoma cells carried out by a radioassay upon acute or chronic treatment of cells by aluminium or tacrine. Our data show that aluminium has a pronounced activatory effect on both enzymatic isoforms either in acute or long-term adaptation treatment. Conversely, tacrine produces an inhibitory effect of both monoamine oxidase isotypes after an acute treatment, while an inhibitory effect on the isoform A and an activation of the isoform B in long-term adaptation was observed. Such effects are discussed in terms of aluminium neurotoxicity and therapeutic implications for tacrine.

Activation of monoamine oxidase type-B by aluminum in rat brain homogenate.

Monoamine oxidase type B (MAO-B) activity is elevated in certain neurological diseases such as Alzheimer's and Parkinson's disease with respect to age-matched controls; the cause of this elevation is unknown. The documented accumulation of aluminum in certain neurodegenerative diseases prompted us to test the effect of Al3+ on the activity of MAO-B in rat brain homogenate. Results showed that the metal ion significantly increased MAO-B enzymatic activity in a dose-dependent manner, yielding a K(M) of 5.69 microM compared with 34.45 microM in the absence of the metal ion. The Vmax of 45.34 micromol/min was unchanged in the presence of the metal ion.

Deposition of aluminum in brain tissues of rats exposed to inhalation of aluminum acetylacetonate.

The present paper reports data concerning aluminum accumulation and compartmentation in the central nervous system from rats exposed by inhalation to aluminum acetylacetonate [Al(acac)3] for two weeks. The complex Al(aca)3 was chosen for being neutral, hydrolytically stable and lipophilic. After animals treatment, Al(III) was identified fluorimetrically by using morin (3,5,7',2',4'-pentahydroxyflavone) that gives a specific green-yellow fluorescence when complexed to the metal. Al(III) was observed to be accumulated in the brain cortex, hippocampus, enthorinal area, olfactory bulb as well as in the Purkinje cells of cerebellum, and in the white matter.

Activation of acetylcholinesterase by aluminium(III): the relevance of the metal species.

The present paper reports how aluminium [Al(III)] at a concentration of 3.7 microM can activate the bovine erythrocytic enzyme acetylcholinesterase (AChE) by about 38% in vitro. This same activating effect was observed on AChE form human as well as from rat erythrocyte ghosts and murine neuroblastoma cells. The interaction between Al3+ and gamma-peripheral sites of the enzyme produces AChE structural modifications as evidenced by circular dichroism measurements. This may provide a molecular explanation of the raised enzymatic activity.

Aluminum(III) influences the permeability of the blood-brain barrier to [14C]sucrose in rats.

To determine the influence of the metal coordination sphere on the permeability of the blood-brain barrier (BBB), rats were injected intraperitoneally with aluminum lactate (Al(lact)3), aluminum acetylacetonate (Al(acac)3), aluminum maltolate (Al(malt)3) at pH 7.5, or with physiological saline. Two h after each treatment, [14C]sucrose physiological saline solution was injected in animals, and the radioactivity was measured in 5 brain regions (cerebral cortex, mesencephalon, diencephalon, medulla-pons, cerebellum). Radioactivity was significantly elevated in brains from animals treated with Al(malt)3 (hydrolytically stable and hydrophilic), and with Al(acac)3 (hydrolytically stable and lipophilic) but not with Al(lact)3. Time-course study carried out at 2, 4 and 24 h with different aluminum compounds showed a persistent radioactivity 24 h after treatment only in the brain from animals treated with Al(acac)3. Morin stain localized AlIII only in neurons from animals treated with Al(acac)3. These findings indicate that AlIII alters the BBB function in the rat either permanently or transiently depending on the physiochemical properties of the metal coordination sphere. Implications of these results, in terms of AlIII as a potential toxic factor in humans, are considered and discussed.

Different effects of aluminum upon carbonic anhydrases and Na+/K+-ATPase activities in rat.

Aluminum (Al(III)) is a well established toxicant implicated as an etiological factor in several neuropathies. In this paper we report results regarding opposite effects produced by Al(III) on the activity of two enzymes utilized as models. While sodium-potassium ATP-ase (Na/K-ATPase) is strongly activated by Al(III) in a dose-effect dependent way, on the contrary, carbonic anhydrase (CA) is remarkably inhibited. The relevance of the metal speciation together with the enzymatic structural modification demonstrated by circular dichroism measurements could explain the observed modified enzymatic activities. In addition, a new experimental protocol for the preparation of Al(III) solutions at physiological pH useful in the standardization of Al(III) experimental toxicology is also proposed and discussed.

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.

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.

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.