T.P.A. Kruck

New Evidence for an Active Role of Aluminum in Alzheimer's Disease

Application of molecular biological techniques and sensitive elemental analysis have produced new evidence implicating aluminum as an important factor in down regulation of neuronal protein metabolism. Aluminum in Alzheimers disease may act by electrostatically crosslinking proteins, particularly the methionine containing histone H1(0), and DNA. The consequence of such crosslinking is reduced transcription of at least one neuron specific gene, the low molecular weight component of neurofilaments. In the superior temporal gyrus in Alzheimers disease, down regulation of this gene occurs in approximately 86% of surviving neurons and, therefore, aluminum must be considered as having an active role in the pathogenesis. Epidemiological studies are reviewed that independently support the hypothesis that environmental aluminum is a significant risk factor. Preliminary evidence also suggests that a disorder in phosphorylation may be an important initiating factor.

Characterization of an NF-κB-regulated, miRNA-146a-mediated down-regulation of complement factor H (CFH) in metal-sulfate-stressed human brain cells

Micro RNAs (miRNAs) represent a family of small ribonucleic acids (RNAs) that are post-transcriptional regulators of messenger RNA (mRNA) complexity. Brain cells maintain distinct populations of miRNAs that support physiologically normal patterns of expression, however, certain miRNA abundances are significantly altered in neurodegenerative disorders such as Alzheimers disease (AD). Here we provide evidence in human neural (HN) cells of an aluminum-sulfate- and reactive oxygen species (ROS)-mediated up-regulation of an NF-kappaB-sensitive miRNA-146a that down-regulates the expression of complement factor H (CFH), an important repressor of inflammation. This NF-kappaB-miRNA-146a-CFH signaling circuit is known to be similarly affected by Abeta42 peptides and in AD brain. These aluminum-sulfate-inducible events were not observed in parallel experiments using iron-, magnesium-, or zinc-sulfate-stressed HN cells. An NF-kappaB-containing miRNA-146a-promoter-luciferase reporter construct transfected into HN cells showed significant up-regulation of miRNA-146a after aluminum-sulfate treatment that corresponded to decreased CFH gene expression. These data suggest that (1) as in AD brain, NF-kappaB-sensitive, miRNA-146a-mediated, modulation of CFH gene expression may contribute to inflammatory responses in aluminum-stressed HN cells, and (2) underscores the potential of nanomolar aluminum to drive genotoxic mechanisms characteristic of neurodegenerative disease processes.

Up-regulation of NF-kB-sensitive miRNA-125b and miRNA-146a in metal sulfate-stressed human astroglial (HAG) primary cell cultures

Micro RNAs (miRNAs) constitute a unique class of small, non-coding ribonucleic acids (RNAs) that regulate gene expression at the post-transcriptional level. The presence of two inducible miRNAs, miRNA-125b and miRNA-146a, involved in respectively, astroglial cell proliferation and in the innate immune and inflammatory response, is significantly up-regulated in human neurological disorders including Alzheimers disease (AD). In this study we analyzed abundances miRNA-125b and miRNA-146a in magnesium-, iron-, gallium, and aluminum-sulfate-stressed human-astroglial (HAG) cells, a structural and immune-responsive brain cell type. The combination of iron- plus aluminum-sulfate was found to be significantly synergistic in up-regulating reactive oxygen species (ROS) abundance, NF-кB-DNA binding and miRNA-125b and miRNA-146a expression. Treatment of metal-sulfate stressed HAG cells with the antioxidant phenyl butyl nitrone (PBN) or the NF-кB inhibitors curcumin, the metal chelator-anti-oxidant pyrollidine dithiocarbamate (PDTC), or the resveratrol analog CAY10512, abrogated both NF-кB signaling and induction of these miRNAs. Our observations further illustrate the potential of physiologically relevant amounts of aluminum and iron sulfates to synergistically up-regulate specific miRNAs known to contribute to AD-relevant pathogenetic mechanisms, and suggest that antioxidants or NF-кB inhibitors may be useful to quench metal-sulfate triggered genotoxicity.

Nuclear compartmentalization of aluminum in Alzheimer's disease (AD)

Senile dementia of the Alzheimer type (AD) is a fatal encephalopathy of uncertain etiology. Whether the neurotoxin aluminum plays any role in the AD process in unknown. Here we report an increased amount of aluminum in a chromatin subcompartment, the micrococcal nuclease (MN; EC 3.1.31.1) accessible dinucleosome fraction, in neocortical nuclei isolated from 17 control and 21 AD-affected brains. At these MN-accessible loci we also observe an increase in H1 zero linker histone proteins, DNA-binding proteins which are thought to act as regulators of chromatin compaction. These data support the hypothesis that one deleterious effect of aluminum upon nuclear structure in AD-afflicted brain may be to condense brain chromatin nonrandomly through an interaction with H1 zero linker protein and thereby alter the ability of brain DNA to be effectively transcribed.

Selective accumulation of aluminum in cerebral arteries in Alzheimer's disease (AD).

Once biologically available aluminum bypasses gastrointestinal and blood-brain barriers, this environmentally-abundant neurotoxin has an exceedingly high affinity for the large pyramidal neurons of the human brain hippocampus. This same anatomical region of the brain is also targeted by the earliest evidence of Alzheimers disease (AD) neuropathology. The mechanism for the selective targeting and transport of aluminum into the hippocampus of the human brain is not well understood. In an effort to improve our understanding of a pathological aluminum entry system into the brain, this study examined the aluminum content of 8 arteries that supply blood to the hippocampus, including the aorta and several cerebral arteries. In contrast to age-matched controls, in AD patients we found a gradient of increasing aluminum concentration from the aorta to the posterior cerebral artery that supplies blood to the hippocampus. Primary cultures of human brain endothelial cells were found to have an extremely high affinity for aluminum when compared to other types of brain cells. Together, these results suggest for the first time that endothelial cells that line the cerebral vasculature may have biochemical attributes conducive to binding and targeting aluminum to selective anatomical regions of the brain, such as the hippocampus, with potential downstream pro-inflammatory and pathogenic consequences.

Aluminum, altered transcription, and the pathogenesis of Alzheimer's disease

AbstractThe etiology of some, if not all, cases of Alzheimers disease is linked to a mutation in the proximal portion of the long arm of chromosome 21∶21q11.2 → 21q22.2. While the functional consequences of the mutation are unknown, we speculate that one consequence of the mutation is loss of the natural barriers and intracellular ligands for aluminum. As a result, aluminum gains access to several brain sites including the nuclear compartment in certain neurons of the central nervous system.Both sporadic and familial Alzheimers disease are associated with an increased compaction of DNA within chromatin as measured by physical shearing and resistance to digestion by micrococcal nuclease and DNase I. There is also an increase in linker histone Hlo content on dinucleosomes released by light (3–5% ASN) micrococcal nuclease digestion, and an increase in the affinity of histone Hlo for DNA as measured by a salt elution technique. The change in enzyme accessibility to chromatin also involves the 5′ promoter region of at least one physiologically important gene: the gene which codes for the low molecular weight moiety of neurofilament (NF-L). The conformation change involving the 5′ regulator region probably reduces transcription because the pool size of the mRNA coding for NF-L is reduced to 14% of age matched control in cerebral grey matter. Reduced transcription may account for many disorders in cellular metabolic processes including the regulation of phosphorylation, calcium homeostasis, free radical metabolism, proteolysis and neurotransmitter metabolism.The experimental evidence indicates that one important toxic action of aluminum in Alzheimers disease neocortex is to increase the binding of histones, particularly Hlo, to DNA which results in increased compaction of chromatin and reduced transcription. The supporting evidence includes:(1)A statistically reliable correlation between the aluminum to DNA ratio on intermediate euchromatin and the amount of highly condensed heterochromatin found in a given preparation from Alzheimer affected neocortex (Crapperet al., 1980).(2)A nine-fold increase in aluminum content in Alzheimers disease in the di- and tri- nucleosome fraction released by light micrococcal nuclease digestion of nuclei from cerebral grey matter compared to age matched controls. Compared to age matched control dinucleosomes, the Alzheimer affected dinucleosomes contain an increased abundance of the linker histone Hlo and an increased proportion of DNA containing the promoter region of the gene coding for NF-L.(3)A reduction in abundance to 14% of control mRNA coding for NF-L in Alzheimer affected neocortex (Crapper McLachlanet al., 1988).(4)In vitro evidence that Alzheimer linker histones bind more tightly to DNA than control and that aluminum added to nuclei,in vitro, extracted from normal control brain, enhances DNA-protein binding of Hl and Hlo at concentrations found in the Alzheimer affected chromatin (Lukiwet al., 1987).(5)Application of a band retardation assay indicates that aluminum,in vitro, selectively binds human Hlo to a 300 bp human ALU DNA fragment from a crude extract of 5% per chloric acid soluble proteins.(6)Aluminum experimentally applied to rabbit CNS induces a marked reduction in NF-L mRNA in anterior horn cells (Mumaet al., 1988). We therefore conclude that aluminum plays a major role in the pathogenesis of Alzheimers disease. Further understanding of the role of aluminum in Alzheimers disease requires a detailed investigation of the precise sites of co-ordination of this trivalent metal within chromatin.

Determination of desferoxamine and a major metabolite by high-performance liquid chromatography : Application to the treatment of aluminium-related disorders

A high-performance liquid chromatography method is described that permits separation and quantification of desferoxamine, a major metabolite, the iron(III) and the aluminum(III) chelates of desferoxamine. This method now facilitates pharmacokinetic studies on desferoxamine and derivatives designed to study side-effects and metabolite patterns in patients undergoing treatment.

Linker histone-DNA complexes: enhanced stability in the presence of aluminum lactate and implications for Alzheimer's disease.

The binding of human brain linker histone proteins to a radiolabelled human Alu repetitive element was examined by mobility shift assay.. Analysis of the complexes formed from protein extracts of whole neocortical nuclei, under physiological conditions in vitro revealed that linker histone H1° has the highest affinity for the Alu DNA sequence. The linker histone‐DNA complexes assembled in the presence of aluminum lactate were more resistant to sodium chloride‐induced dissociation than those formed in the presence of sodium lactate. The enhanced stability of deoxyribonucleoprotein (DNP) complexes in the presence of the aluminum cation may be of significance in neurodegenerative conditions such as Alzheimers disease where aluminum preferentially associates with DNA containing structures of the nucleus.

Aluminum-binding serum proteins: desferrioxamine alters serum aluminum speciation

The aluminum content of four size classes of protein (high and low molecular weight, transferrin/albumin and a fraction provisionally termed albindin) in sera from healthy volunteers (group I) and from aluminum workers with normal (group II) and high (group III) total serum aluminum was compared using size exclusion chromatography and electrothermal atomic absorption spectroscopy. In the absence of any drug treatment the transferrin/albumin fraction was the major carrier, containing 29% to 33% of the aluminum recovered, in all three subject groups. Desferrioxamine treatment of groups II and III significantly decreased the proportion of aluminum bound by albumin/transferrin (P less than 0.05 in group III) and increased that bound by albindin (P less than 0.05 in groups II and III). The albindin fraction contained over 40% of the aluminum recovered from sera of group III subjects during desferrioxamine treatment. We conclude that the albindin fraction contains a protein or proteins that can form stable complexes with aluminum which may be important in preventing aluminum toxicity.

Metal sulfate-mediated induction of pathogenic genes and repression by phenyl butyl nitrone and Feralex-G.

Neurotoxic metal-induced oxidative damage to nervous tissue has been implicated in several progressive neurodegenerative disorders including Alzheimers disease. In this study, using human brain cells in primary culture, the quenching of metal sulfate-induced reactive oxygen species (ROS) and ROS-sensitive gene expression was studied using the antioxidants ascorbate, folic acid, phenyl butyl nitrone and the chelators desferrioxamine and Feralex-G. Antioxidants ascorbate, folic acid, phenyl butyl nitrone, desferrioxamine or Feralex-G were found to quench ROS and cPLA2 and COX-2 gene induction to various degrees, and a synergism was observed when certain combinations of them were used. These findings support the idea that specific antioxidants and metal ion chelators when used together can effectively and synergistically quench ROS-mediated induction of pathogenic gene expression.