Human erythrocytes and neuroblastoma cells are affected in vitro by Au(III) ions.

Gold compounds are well known for their neurological and nephrotoxic implications. However, haematological toxicity is one of the most serious toxic and less studied effects. The lack of information on these aspects of Au(III) prompted us to study the structural effects induced on cell membranes, particularly that of human erythrocytes. AuCl(3) was incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of the erythrocyte membrane. The latter consisted of multibilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, phospholipids classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. This report presents evidence that Au(III) interacts with red cell membranes as follows: (a) in scanning electron microscopy studies on human erythrocytes it was observed that Au(III) induced shape changes at a concentration as low as 0.01 microM; (b) in isolated unsealed human erythrocyte membranes Au(III) induced a decrease in the molecular dynamics and/or water content at the glycerol backbone level of the lipid bilayer polar groups in a 5-50 microM concentration range, and (c) X-ray diffraction studies showed that Au(III) in the 10 microm-1mM range induced increasing structural perturbation only to dimyristoylphosphatidylcholine bilayers. Additional experiments were performed in human neuroblastoma cells SH-SY5Y. A statistically significant decrease of cell viability was observed with Au(III) ranging from 0.1 microM to 100 microM.

Chelation therapy for neurodegenerative diseases.

Mounting evidence suggests a central role for transition biometals in the etiopathogenesis of neurodegenerative diseases (ND). Indeed, while studying the molecular basis for this heterogeneous group of diseases, it has become increasingly evident that biometals and nonphysiological Al are often involved in pathology onset and progression, either by affecting the conformation of specific proteins or by exacerbating local oxidative stress. The apparently critical role played by metal dishomeostasis in ND makes chelation therapy an attractive pharmacological option. However, classical metal chelation approaches, relying on potent metal ligands, turned out to be successful only in those rare cases where exceptional brain metal accumulation occurs due to specific defects in metal metabolism. In contrast, metal-targeted approaches using ligand of intermediate strength seem to be more appropriate in fighting the major ND, although their benefits are still questioned. We report here a survey of recent evidences supporting the use of a variety of metal ligands, and even functionalized nanoparticles, for the treatment of the most common ND. The beneficial neuropharmacological actions of metal-targeted agents most likely arise from local metal redistribution rather than from massive metal removal. The perspectives for the development of new effective agents against ND are critically discussed.

Interaction between Alzheimer's amyloid-beta and amyloid-beta-metal complexes with cell membranes.

A number of observations indicate that the primary target of amyloid-beta (Abeta) peptide is the cellular membrane of neurons. In the context of these observations we investigated, using X-ray diffraction techniques, whether Abeta-metal complexes were able to affect lipid bilayers as a model of cell membranes. The binding of Al to Abeta gave particular conformational properties to the peptide that led to a marked alteration of the lipid bilayer representing phospholipids located in the outer monolayer of cell membranes. This effect was peculiar, since in our experimental conditions Abeta alone did not affect the lipid architecture, whereas the Al salt did, but only at concentrations several orders of magnitude higher than those of the Abeta-Al complex. In accordance with the effects observed with lipid bilayers, studies with human neuroblastoma cells demonstrated an impairment of cell functioning only in the presence of Abeta-Al complex. Our findings imply that Al, compared to the other Abeta-metal complexes tested, could have a specifically relevant effect in enhancing Abeta toxicity.

Acidic vesicles of the endo-exocytic pathways as targets for some anti-monoamine oxidase drugs.

Acidic vesicles are cytoplasmatic organelles delimited by a single lipoprotein membrane. They contain a large number of enzymes, mostly acidic hydrolases, catalysing various reactions at optimal acidic pH, capable of participating in intracellular digestion. In this paper, some anti-monoamine oxidase drugs (clorgyline, pargyline, amantadine and deprenyl), utilized as pharmacological treatment in some neurological disorders (e.g., Alzheimer's, Parkinson's etc. diseases), were tested for their ability to influence the pH of the acidic intracellular organelles with the aim of exploring their possible pharmacological action. Of the above mentioned drugs, clorgyline showed the most effective action in modifying the acidic vesicles' internal pH, followed by deprenyl, pargyline and amantadine. The effect was not ascribed to an increased proton conductance, but was most likely due to a weak base-like mechanism, in that they exhibit equilibria among species associated with H(+) ions and species lacking this association.

Potential pathogenic role of beta-amyloid(1-42)-aluminum complex in Alzheimer's disease.

The etiopathogenesis of Alzheimer's disease is far from being clearly understood. However, the involvement of metal ions as a potential key factor towards conformational modifications and aggregation of amyloid is widely recognized. The aim of the present study is to shed some light on the relationship between metal ions, amyloid conformation/aggregation, and their potential relationship with the conformational aspects of AD. We compare the effects of beta-amyloid(1-42) and its various metal complexes (beta-amyloid-Al, beta-amyloid-Zn, beta-amyloid-Cu, beta-amyloid-Fe) in human neuroblastoma cells in terms of cell viability, membrane structure properties, and cell morphology. No significant toxic effects were observed in neuroblastoma cells after 24h treatment both with beta-amyloid and beta-amyloid-metals (beta-amyloid-Zn, beta-amyloid-Cu, beta-amyloid-Fe); on the other hand, there was a marked reduction of cellular viability after treatment with beta-amyloid-Al complex. In addition, treatment with beta-amyloid-Al increased membrane fluidity much more than other beta-amyloid-metal complexes, whose contribution was negligible. Furthermore, the cellular morphology, as observed by electron microscopy, was deeply altered by beta-amyloid-Al. Importantly, beta-amyloid-Al toxicity is closely and significantly associated with a great difference in the structure/aggregation of this complex with respect to that of beta-amyloid alone and other beta-amyloid-metal complexes. In addition, beta-amyloid, as a consequence of Al binding, becomes strongly hydrophobic in character. These findings show a significant involvement of Al, compared to the other metal ions used in our experiments, in promoting a specific amyloid(1-42) aggregation, which is able to produce marked toxic effects on neuroblastoma cells, as clearly demonstrated for the first time in this study.

Accumulation of copper and other metal ions, and metallothionein I/II expression in the bovine brain as a function of aging.

Accumulation of metal ions in the brain contributes to heighten oxidative stress and neuronal damage as evidenced in aging and neurodegenerative diseases, both in humans and in animals. In the present paper we report the analysis of Cu, Zn and Mn in the brain of two series of respectively young (8-16 months) and adult (9-12 years) bovines. Our data indicate that the concentrations of Cu varied of one order of magnitude between 1.67 and 15.7microg/g wet tissue; the levels of Zn varied between 6.13 and 17.07microg/g wet tissue and the values of Mn resulted between 0.19 and 1.24microg/g wet tissue. We found relevant age-dependent differences in the distribution of Cu and Zn, whose concentrations were markedly higher in older animals. By contrast, Mn seemed to redistribute in the different cerebral areas rather than drastically change with age. Tissues from bovine brain were also analysed immunohistochemically for the presence and distribution of metallothionein I/II and also for the expression of glial fibrillary acidic protein. Metallothionein I/II immunoreactive elements included ependymal cells lining the lateral ventricles and neural cells in middle layer of the cerebellar cortex. No age differences were evident between calves and adult. The presence of liquor-contacting metallothionein I/II in cells confirms that their functions in the central nervous system are not yet completely established.

Aluminum modulates effects of beta amyloid(1-42) on neuronal calcium homeostasis and mitochondria functioning and is altered in a triple transgenic mouse model of Alzheimer's disease.

Recent findings suggest that beta-amyloid (A beta) is more neurotoxic when present in its oligomeric configuration rather than as monomers or fibrils. Previous work from our laboratories has shown that A beta aggregation is strongly influenced by the conjugation of the peptide with metal ions (aluminum A, copper [Cu], zinc [Zn], and iron [Fe]) that are found in high concentrations in the core of senile plaques. Disruption of Ca++ signaling and mitochondrial dysfunction are potent triggers of neuronal death and have been implicated in the neuronal loss that is associated with Alzheimer's disease (AD). In this study, we explored whether A beta-metal complexes can have detrimental effects on intraneuronal Ca++ ([Ca++]i) homeostasis and mitochondrial function in vitro. Results from our experiments indicate that, when conjugated with Al, A beta perturbs neuronal [Ca++]i homeostasis and inhibits mitochondrial respiration. Finally, we analyzed the content of the four metals in the brain of a triple transgenic animal model of AD and found that Al is the only one to be increased in the cortex of these mice.

Human cells and cell membrane molecular models are affected in vitro by chlorpromazine.

This study presents evidence that chlorpromazine (CPZ) affects human cells and cell membrane molecular models. Human SH-SY5Y neuroblastoma cells incubated with 0.1 mM CPZ suffered a decrease of cell viability. On the other hand, phase contrast microscopy observations of human erythrocytes indicated that they underwent a morphological alteration as 1 microM CPZ changed their discoid normal shape to stomatocytes, and to hemolysis with 1 mM CPZ. X-ray diffraction experiments performed on dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) bilayers, classes of the major phospholipids present in the outer and inner sides of the erythrocyte membrane, respectively showed that CPZ disordered the polar head and acyl chain regions of both DMPC and DMPE, where these interactions were stronger with DMPC bilayers. Fluorescence spectroscopy on DMPC LUV at 18 degrees C confirmed these results. In fact, the assays showed that CPZ induced a significant reduction of their generalized polarization (GP) and anisotropy (r) values, indicative of enhanced disorder at the polar head and acyl chain regions of the DMPC lipid bilayer.

Copper deficiency and neurological disorders in man and animals.

Copper metabolism in the brain is far from being completely understood and further studies are needed on the role of copper in the CNS, starting with careful measurements, metal and biological speciation of metabolites on the molecular level, and combining copper concentration in different brain areas with morphological as well as biochemical alteration after Cu-depletion/deficiency. So far a pathological role for copper has been clearly demonstrated in some human genetic diseases (e.g., Menkes' and Wilson's diseases), but other pathological features connected with metal depletion are under investigation in several laboratories. The metabolic interaction between copper and other metal ions in some neurological disorders is also discussed in this contribution.

Destabilization of non-pathological variants of ataxin-3 by metal ions results in aggregation/fibrillogenesis.

Ataxin-3 (AT3), a protein that causes spinocerebellar ataxia type 3, has a C-terminus containing a polyglutamine stretch, the length of which can be expanded in its pathological variants. Here, we report on the role of Cu(2+), Mn(2+), Zn(2+) and Al(3+) in the induction of defective protein structures and subsequent aggregation/fibrillogenesis of three different non-pathological forms of AT3, i.e. murine (Q6), human non-expanded (Q26) and human moderately expanded (Q36). AT3 variants showed an intrinsic propensity to misfolding/aggregation; on the other hand, Zn(2+) and Al(3+) strongly stimulated the amplitude and kinetics of these conformational conversions. While both metal ions induced a time-dependent aggregation into amyloid-like fibrillar forms, only small oligomers and/or short protofibrillar species were detected for AT3s alone. The rate and extent of the metal-induced aggregation/fibrillogenesis processes increased with the size of the polyglutamine stretch. Mn(2+) and Cu(2+) had no effect on (Q6) or actually prevented (Q26 and Q36) the AT3 structural transitions. The observation that Zn(2+) and Al(3+) promote AT3 fibrillogenesis is consistent with similar results found for other amyloidogenic molecules, such as beta-amyloid and prion proteins. Plausibly, these metal ions are a major common factor/cofactor in the etiopathogenesis of neurodegenerative diseases. Studies of liposomes as membrane models showed dramatic changes in the structural properties of the lipid bilayer in the presence of AT3, which were enhanced after supplementing the protein with Zn(2+) and Al(3+). This suggests that cell membranes could be a potential primary target in the ataxin-3 pathogenesis and metals could be a biological factor capable of modulating their interaction with AT3.

Comparative effects of Abeta(1-42)-Al complex from rat and human amyloid on rat endothelial cell cultures.

Metal ions are widely recognized as a key factor for the conformational changes and aggregation of the Alzheimer's disease amyloid (Abeta). So far Al(3+) has received much less attention than other biometals in terms of interaction with Abeta. Brain endothelial cells have been identified as important regulators of the neuronal microenvironment, including Abeta levels. The purpose of this study is to compare the effects of the complex amyloid (Abeta(1-42))-Al, from human and rat, with the effects produced by metal-free Abeta on rat neuroendothelial cells (NECs). To establish Abeta and Abeta-Al toxicity on NECs, survival, vitality, and angiogenesis are evaluated. Cell survival is reduced by human and rat Abeta in a time-dependent manner. This toxic effect is remarkably pronounced in the presence of human Abeta-Al. Moreover, rat Abeta has anti-angiogenic properties on NECs, and this effect is aggravated dramatically by using both human and rat Abeta-Al complexes. The data and arguments presented herein clearly demonstrate the involvement of Al(3+) in Abeta aggregation and, consequently, increasing endothelial cell toxicity.

The C2 variant of human serum transferrin retains the iron binding properties of the native protein.

The tryptic digests of blood samples obtained from transferrin C1 and C2 (TfC 1 and TfC2 hereafter) genotypes were analysed by Liquid Chromatography coupled to Electrospray Mass Spectrometry (LC/ESI--MS/MS). The analytical results confirmed the single base change in exon 15 of the Tf gene. The solution behaviour and the iron binding properties of the two Tf variants were studied by UV-visible spectrophotometry and by circular dichroism. It appears that TfC2 globally manifests the same spectral features as the native protein. The local conformation of the two iron binding sites is conserved in the two Tf variants as evidenced by the visible absorption and CD spectra. Also, the iron binding capacities and their pH-dependent profiles are essentially the same. Overall, our investigation points out that the single amino acid substitution in TfC2 (Pro 570 Ser) does not affect the general conformation of the protein nor the local structure of the iron binding sites. The implications of these results for the etiopathogenesis of Alzheimer's disease are discussed.

Metallothionein-I-II expression in young and adult bovine pineal gland.

Aging is characterized, among other features, by an increased concentration of metal ions in the brain that may contribute to a greater increase in free radicals production. The present paper reports data regarding the concentration of some relevant metal ions (Cu, Fe, Mn, Zn), as well as the immunopositivity of metallothionein-I-II and GFAP in the bovine pineal gland with respect to animal aging. The pineal gland of young bovines displays several immunoreactive metallothionein-I-II positive elements in the parenchyma, whose number decreases with age. We also report that a well defined group of neurons bordering the third ventricle and located close to the subcommissural organ shows an intense metallothionein-I-II immunopositivity. The presence of metallothionein-I-II was confirmed by means of liquid chromatography coupled to tandem mass spectrometry. In particular, it proved possible to identify the amino acid sequences of the unique tryptic peptide not containing cysteine and two pepsin fragments containing cysteines. In conclusion, our data suggest the presence of a metallothionein-I-II expressing system in the pineal gland and ventricle-adjacent areas of the bovine epithalamus might possibly be related to the anti-aging effects of melatonin.

The effect of Zn(II) and streptozotocin administration in the mouse brain.

Streptozotocin is a natural antibiotic produced by Streptomyces achromogenes able to induce diabetes in experimental animals. Among various toxic properties, streptozotocin is a potent source for reactive oxygen species. In this paper, we report the biological response of brain, upon treatment with streptozotocin in terms of metal ions dismetabolism and metallothionein expression. In addition, important information on the preventive effect of zinc in eliciting the pharmacological effect of the drug are reported, in relation to the effective role of the metal ions in inducing metallothionein synthesis. In the brain, streptozotocin treatment affects mostly the hippocampus and cerebellum as shown by a high GAFP and MT-I-II immunopositivity of glial cells. The Zn pre-treatment reduces significantly, as a general effect, the occurrence of hyperglycaemic status. At the brain level, the observed astrocytosis is strongly reduced. The high inducibility of MT represents a rapid and convenient response able to prevent the deleterious effects consequent to the oxidative stress. All together these results support the efficacy of the Zn treatment in order to prevent streptozotocin effects, including brain tissues.

Aluminum complexing enhances amyloid beta protein penetration of blood-brain barrier.

A significant co-morbidity of Alzheimer's disease and cerebrovascular impairment suggests that cerebrovascular dysregulation is an important feature of dementia. Amyloid beta protein (Abeta), a relevant risk factor in Alzheimer's disease, has neurotoxic properties and is thought to play a critical role in the cognitive impairments. Previously, we demonstrated that the 42mer of Abeta (Abeta42) complexed with aluminum (Al-Abeta42) is much more cytotoxic than non-complexed Abeta42. The level of Abeta in the brain is a balance between synthesis, degradation, and fluxes across the blood-brain barrier (BBB). In the present paper, we determined whether complexing with aluminum affected the ability of radioactively iodinated Abeta to cross the in vivo BBB. We found that the rates of uptake of Al-Abeta42 and Abeta42 were similar, but that Al-Abeta42 was sequestered by brain endothelial cells much less than Abeta42 and so more readily entered the parenchymal space of the brain. Al-Abeta42 also had a longer half-life in blood and had increased permeation at the striatum and thalamus. Brain-to-blood transport was similar for Al-Abeta42 and Abeta42. In conclusion, complexing with aluminum affects some aspects of blood-to-brain permeability so that Al-Abeta42 would have more ready access to brain cells than Abeta42.

Metallothionein I-II immunocytochemical reactivity in Binswanger's encephalopathy.

Binswanger's disease is a subacute form of hypertensive encephalopathy characterized by patchy-confluent myelin loss of the deep hemispheric white matter, associated with marked regressive changes of the oligodendrocytes and variable astroglial reaction. To understand the distribution and the specificity of astrocyte pathology in Binswanger's disease we quantified reactive and degenerating astrocytes in different regions of the deep and subcortical white matter and of the cerebral cortex. Sections of frontal, temporal, parietal, and occipital lobes of 12 histologically proven cases of Binswanger's disease were immunostained with antibodies to glial fibrillary protein (GFAP) and to metallothionein I and II (MT-I-II), markers which specifically identify normal and reactive astrocytes. Control tissues were from 6 elderly patients without neurological diseases. The brains of Binswanger's disease were characterized by few and lightly immunostained astrocytes in the deep white matter, but normal and reactive astrocytes, strongly immunoreactive for GFAP and MT-I-II, were prominent in the subcortical white matter and the cerebral cortex. However, the relative distribution of GFAP positive and MT-I-II positive astrocytes was significantly different between the cerebral cortex and the subcortical white matter, the MT-I-II positive astrocytes being more frequent in the cerebral cortex, and the GFAP positive astrocytes in the subcortical white matter (p < 0.02). The GFAP and MT-I-II expressions in subsets of reactive astrocytes in the cortico-subcortical layers together with regressive astroglial changes in the deep white matter suggest that the dynamic plasticity of astroglia is topographically and biochemically differentiated in vascular dementia of Binswanger type.

Astrocytosis, microgliosis, metallothionein-I-II and amyloid expression in high cholesterol-fed rabbits.

Cholesterol is considered a risk factor in vascular dementia as well as in Alzheimer's disease. Several biochemical, epidemiological and genetic aspects established a correlation between cholesterol concentration and Alzheimer's disease. Microglia activation, astrocytosis with metallothionein-I-II overexpression, amyloid beta intraneuronal accumulation and a rare formation of amyloid beta extracellular positive deposits were the major immunohistochemical features observed in the brain of high cholesterol-fed animals. The relevance on the cholesterol metabolism in Alzheimer's disease pathogenesis is also discussed.

Transferrin C2 variant does confer a risk for Alzheimer's disease in caucasians.

Several gene mutations are associated with an increased risk of Alzheimer's disease. Previous studies reported higher transferrin C2 allele frequencies in Alzheimer's disease compared with normal controls. However, potential interactions between transferrin C2 and APOE (epsilon 4), have not been extensively investigated and have been the subject of controversial reports from several laboratories. We have carried out a case-control study on the association between Alzheimer's disease and transferrin C2 and APOE epsilon 4 alleles. epsilon 4 allele was associated with a four fold increase in the risk of disease, and transferrin C2 allele was significantly associated with Alzheimer's disease only in epsilon 4 negative subjects. These results suggest that apoE and transferrin may be part of a complex mechanism in the pathogenesis of Alzheimer's disease.

The effect of chronic aluminum(III) administration on the nervous system of aged rats: clues to understand its suggested role in Alzheimer's disease.

The effect of chronic aluminum intake has been investigated in the brain of aged male Wistar rats to assess the potential role of the accumulation of this metal ion on the development of neurodegenerative features observed in Alzheimer's disease. AlCl3 x 6 H2O (2g/L) was administered to experimental animals for 6 months in the drinking water. The total content of Al (microg/g fresh tissue) was measured by inductively coupled plasma atomic emission spectrometry (ICP-AES), while the content of Cu, Zn and Mn was determined by flame AAS in the prosencephalon + mesencephalon, pons-medulla and cerebellum of control and Al(III)-treated animals. The area occupied by mossy fibres in the CA3 field of the hippocampus was estimated by a computer-assisted morphometric method following Timm's preferential staining. In Al(III)-treated rats the concentration of Cu, Zn and Mn did not increase significantly (p < 0.5) in prosencephalon + mesencephalon, nor in pons-medulla (p < 0.5) except for Cu (p < 0.05) in pons-medulla. In the cerebellum the only significant increase was seen for Zn (p < 0.01) while no change was observed for Cu and Mn. The area occupied by the mossy fibres in the hippocampal CA3 field was significantly increased (+32%) in aged Al(III)-treated rats. Since Cu, Zn and Mn are essential components of the cytosolic and mitochondrial superoxide dismutases, it is possible that the increased content of these ions in aged Al(III)-treated rats represents an increased amount of genetic expression of these antioxidant enzymes. Considering that the positivity to Timm's reaction is based on the presence of free or loosely bound Zn2+ ions within synaptic terminals and that Zn2+ ions are reported to be accumulated by hippocampal neurons when tissue injury occurs, the increased area of the mossy fibres in CA3 field of Al(III)-treated rats could indicate increased hippocampal damage in these animals. Taken together, the present findings indicate that the aging CNS is particularly susceptible to Al(III) toxic effects which may increase the cell load of oxidative stress and may contribute, as an aggravating factor, to the development of neurodegenerative events as observed in Alzheimer's disease.

Chronic aluminum administration to old rats results in increased levels of brain metal ions and enlarged hippocampal mossy fibers.

The effect of chronic aluminium administration (2 g/L/6 months) was investigated in the central nervous system (CNS) of old rats. The content of Al(3+), Cu(2+), Zn(2+), and Mn(2+) was measured in prosencephalon + mesencephalon, pons-medulla, and cerebellum. The area occupied by the mossy fibers in the hippocampal CA3 zone was also measured. In Al-treated rats the contents of Al(3+), Cu(2+), Zn(2+), and Mn(2+) were significantly increased in prosencephalon + mesencephalon and pons-medulla, while no change was observed in the cerebellum except a Cu(2+) decrease. The area occupied by the mossy fibers in the CA3 field was significantly increased (+32%) in Al-treated rats. Taken together, the present findings document that the aging CNS is particularly susceptible to aluminum toxic effects that may be responsible for a consistent rise in the cell load of oxidative stress. This may contribute, as an aggravating factor, to the development of neurodegenerative events, as observed in Alzheimer disease.