Maire E. Percy

Neurofilament Light and Polyadenylated mRNA Levels Are Decreased in Amyotrophic Lateral Sclerosis Motor Neurons

The presence of large neurofilamentous accumulations in the perikaryon and proximal axon of motor neurons in amyotrophic lateral sclerosis (ALS) suggests that the expression of this abundant cytoskeletal protein may be altered. We performed quantitative in situ hybridization for the low molecular weight neurofilament subunit (NF-L) messenger RNA in six cases of sporadic ALS and six controls. We found a 41% decrease (p < 0.02) in the NF-L mRNA levels in anterior horn cells in ALS, with a 60% decrease (p > 0.01) in alpha motor neurons. This alteration may represent a non-specific response to axonal or neuronal injury or, alternatively, reflect the regenerative activity of residual normal motor neurons. NF-L mRNA levels were consistently low (in the third and fourth quartiles) in spheroid-bearing motor neurons, indicating that the neurofilamentous accumulations observed in ALS are not likely the result of overexpression of the NF-L gene. Total neuronal polyadenylated mRNA levels were also 50% lower (p = 0.02) in anterior horn cells and 48% lower (p > 0.05) in alpha motor neurons in ALS, possibly reflecting a decrease in selected mRNA species in diseased motor neurons.

Are hereditary hemochromatosis mutations involved in Alzheimer disease

Mutations in the class I-like major histocompatibility complex gene called HFE are associated with hereditary hemochromatosis (HHC), a disorder of excessive iron uptake. We screened DNA samples from patients with familial Alzheimer disease (FAD) (n = 26), adults with Down syndrome (DS) (n = 50), and older (n = 41) and younger (n = 52) healthy normal individuals, for two HHC point mutations-C282Y and H63D. Because the apolipoprotein E (ApoE) E4 allele is a risk factor for AD and possibly also for dementia of the AD type in DS, DNA samples were also ApoE genotyped. Chi-squared analyses were interpreted at the 0.05 level of significance without Bonferroni corrections. In the pooled healthy normal individuals, C282Y was negatively associated with ApoE E4, an effect also apparent in individuals with DS but not with FAD. Relative to older normals, ApoE E4 was overrepresented in both males and females with FAD, consistent with ApoE E4 being a risk factor for AD; HFE mutations were overrepresented in males and underrepresented in females with FAD. Strong gender effects on the distribution of HFE mutations were apparent in comparisons among ApoE E4 negative individuals in the FAD and healthy normal groups (P < 0.002). Our findings are consistent with the proposition that among ApoE E4 negative individuals HFE mutations are predisposing to FAD in males but are somewhat protective in females. Further, ApoE E4 effects in our FAD group are strongest in females lacking HFE mutations. Relative to younger normals there was a tendency for ApoE E4 and H63D to be overrepresented in males and underrepresented in females with DS. The possibility that HFE mutations are important new genetic risk factors for AD should be pursued further.

Molecular and prospective phenotypic characterization of a pedigree with familial Alzheimer's disease and a missense mutation in codon 717 of the β‐amyloid precursor protein gene

We present prospective clinical and neuropathologic details of a pedigree segregating familial Alzheimers disease (FAD) associated with a mutation (G→A substitution) at nucleotide 2149 in exon 17 of the amyloid precursor protein (APP) gene. This mutation, which is predicted to cause the missense substitution of isoleucine for valine at codon 717 of APP, cosegregated perfectly with the FAD trait (lod score = 3.49 at = 0.00). The earliest clinical manifestations of the disease relate to deficits in memory function, cognitive processing speed, and attention to complex cognitive sets. These changes occurred in the absence of changes in nonmemory language and visuospatial functions. The neuropathologic features of FAD associated with the APP717 mutation in this family include severe neuronal loss, abundant neurofibrillary tangles, amyloid plaques, and amyloid angiopathy. These results provide independent confirmation that mutations in the APP gene are linked to the FAD trait in some families.

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.

Is hemochromatosis a risk factor for Alzheimer's Disease?

Excess iron accumulation in the brain is a consistent observation in Alzheimers Disease. Iron affects amyloid precursor protein (AbetaPP) processing and promotes deposition of Abeta. Iron is also among the most potent biological toxins because of its ability to react with oxygen to form reactive oxygen species. Consequently, elucidation of the mechanisms associated with maintaining brain iron homeostasis is fundamentally important to understanding the underlying pathogenesis in AD. The iron overload disorder, Hemochromatosis, is the most common genetic disorder (1:200) so a significant percentage of AD patients can be expected to carry this mutation. Heterozygotes for this mutation also have an increased, but sub-clinical iron burden. Given the high percentage of the population who are at significant risk for iron overload, we propose that the hemochromatosis mutation be considered as a confounding factor when evaluating the contribution of genetic associations with AD and treatment strategies and efficacy. Two recent papers and new evidence presented here that the protein associated with hemochromatosis is expressed on blood vessels, choroid plexus and the ependymal cells in the brain are offered as support for this proposal.

Analysis of the functional effects of a mutation in SOD1 associated with familial amyotrophic lateral sclerosis

Mutations in the Cu, Zn superoxide dismutase (SOD1) gene have been reported in some pedigrees with Familial Amyotrophic Lateral Sclerosis (FALS). We have investigated the functional and structural effects of a Gly-->Ser mutation at codon 41 of SOD1 in a pedigree with FALS and the topography of SOD1 expression in the mammalian CNS. These analyses show that the 41Gly-->Ser mutation causes a 27% reduction in Cu, Zn SOD activity. SOD1 is transcribed at high levels in rat motoneurons and four other types of neurons homologous to upper motoneurons that degenerate in human ALS. However, SOD1 is transcribed at lower levels in other types of neurons, such as cerebellar Purkinje cells, which are not usually involved significantly in human ALS. On the other hand, immunocytochemical studies indicate that most types of rat neurons contain similar levels of Cu, Zn SOD immunoreactive protein. Nevertheless, these results suggest that the essential feature causing this subtype of ALS is either a reduction in Cu, Zn SOD activity in cell types that presumably critically require Cu, Zn SOD for protection against oxidative damage or the fact that the mutation in SOD1 associated with FALS results in a novel gain of function that is particularly deleterious to those cell types expressing SOD1 at high levels.

Hemochromatosis and the enigma of misplaced iron: Implications for infectious disease and survival

The mystery surrounding the apparent lack of iron within the macrophages of individuals with hereditary hemochromatosis, a condition of excessive uptake of dietary iron, has yet to be fully explained. We have suggested that iron deficiency of macrophages in people with hereditary hemochromatosis mutations is associated with increased resistance to infection by Yersinia and other intracellular pathogens, a selection pressure resulting in unusually high current population frequencies of hereditary hemochromatosis mutations. Such selection pressure has been called Epidemic Pathogenic Selection (EPS). In support of the theory of EPS, a considerable number of virulent species of bacteria multiply mainly in iron-rich macrophages of their mammalian hosts. Among these fastidious pathogens are strains of Chlamydia, Coxiella, Francisella, Legionella, Mycobacterium, Salmonella and Yersinia. Iron deficiency of macrophages of persons with hereditary hemochromatosis gene mutations may result in increased resistance to members of these bacterial pathogens. People with genes that result in hereditary hemochromatosis may be protected against coronary artery disease associated with Chlamydia and Coxiella infection in the absence of iron overload. In the clinical setting, when a patient appears to be iron deficient, the reason for this should be carefully evaluated. Iron supplementation may adversely affect the health of individuals who have mounted an acute phase response to infection, injury or stress, or who carry genes predisposing them to iron overload disorders.

Towards the prevention of potential aluminum toxic effects and an effective treatment for Alzheimer’s disease

In 1991, treatment with low dose intramuscular desferrioxamine (DFO), a trivalent chelator that can remove excessive iron and/or aluminum from the body, was reported to slow the progression of Alzheimers disease (AD) by a factor of two. Twenty years later this promising trial has not been followed up and why this treatment worked still is not clear. In this critical interdisciplinary review, we provide an overview of the complexities of AD and involvement of metal ions, and revisit the neglected DFO trial. We discuss research done by us and others that is helping to explain involvement of metal ion catalyzed production of reactive oxygen species in the pathogenesis of AD, and emerging strategies for inhibition of metal-ion toxicity. Highlighted are insights to be considered in the quests to prevent potentially toxic effects of aluminum toxicity and prevention and intervention in AD.

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.

Molecular shuttle chelation: the use of ascorbate, desferrioxamine and Feralex-G in combination to remove nuclear bound aluminum.

Abstract1. Abundant data suggest that aluminum (Al(III)) exposure may be an environmental risk factor contributing to the development, progression and/or neuropathology of several human neurodegenerative disorders, including Alzheimers disease (AD).2. Nuclei appear to be one directed target for Al(III) binding, accumulation, and Al(III)-mediated dysfunction due in part to their high content of polyphosphorylated nucleic acids, nucleotides, and nucleoproteins.3. The design of chelation therapies dealing with the removal of Al(III) from these genetic compartments therefore represents an attractive strategy to alleviate the development and/or progression of central nervous system dysfunction that may arise from excessive Al(III) exposure.4. In this study we have investigated the potential application of 10 natural and synthetic Al(III) chelators, including ascorbate (AS), desferrioxamine (DF), and Feralex-G (FG), used either alone or in combination, to remove Al(III) preincubated with intact human brain cell nuclei.5. Although nuclear bound Al(III) was found to be highly refractory to removal, the combination of AS+FG was found to be particularly effective in removing Al(III) from the nuclear matrix.6. Our data suggest that chelators carrying cis-hydroxy ketone groups, such as FG, are particularly suited to the removal of Al(III) from complex biological systems.7. We further suggest a mechanism whereby small chelating molecules may penetrate the nucleus, bind Al(III), diffuse to regions accessible by the larger DF or FG molecules and transfer their Al(III) to DF or FG.8. The proposed mechanism, called molecular shuttle chelation may provide a useful pharmacotherapy in the potential treatment of Al(III) overload disease.