Resolution of allelic and non-allelic variants of histone H1 by cation-exchange-hydrophilic-interaction chromatography.

A mixed-mode high-performance liquid chromatography (HPLC) method that resolves the six known non-allelic variants of chicken erythrocyte histone H1 is described. Common, but previously unknown, allelic variants of H1 that comigrate in polyacrylamide gel electrophoresis are also resolved. The resolution of H1 variants achieved by this method should be useful in determining the functional significance of H1 sequence heterogeneity and in analyses of post-translational modification of H1. Furthermore, the principles behind the separation should be applicable to analyses of polymorphism in other proteins.

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.

Desferrioxamine and Alzheimer's disease: video home behavior assessment of clinical course and measures of brain aluminum.

Drug trials designed to modify the progression of Alzheimer's disease (AD) have required the development of mental state and behavior evaluation instruments that are sensitive to cognitive decline and measure skills useful in everyday living. We describe a videotaped home behavior (VHB) assessment instrument with high construct validity and reliability and a strong relationship to criterion references. The VHB was employed to test the hypothesis that aluminum is an important pathogenic factor in AD. The trivalent chelating agent desferrioxamine (DFO), 125 mg i.m. twice daily five days per week, was used in a randomized single-blind, oral lecithin, placebo-controlled clinical trial in 48 patients with AD. Analysis showed that the treatment and no-treatment groups were closely matched at entry into the trial but that the rate of decline, as measured by the VHB over 2 years of observation, was twice as rapid in the no-treatment group compared with the DFO-treated group. Furthermore, trace-metal analysis of autopsied brain confirmed that extended treatment with DFO lowered neocortical brain aluminum concentrations to near control concentrations. Aluminum ion-specific chelation may be a useful palliative treatment for AD, and further clinical trials are indicated.

Rescue of axotomized immature rat facial motoneurons by R(-)-deprenyl: stereospecificity and independence from monoamine oxidase inhibition.

The role of monoamine oxidase B (MAO-B) in R(-)-deprenyl-mediated rescue of rat facial motoneurons axotomized at postnatal day 14 (P14) was investigated using the (+)- and (-)-enantiomers of deprenyl [S(+)-deprenyl and R(-)-deprenyl]. Previously, doses of R(-)-deprenyl sufficient to inhibit MAO-B were shown to increase the survival of motoneurons following an apparent loss of target-derived trophic support caused by axotomy in P14 rats. In the present experiments, motoneuronal survival was measured 21 d after unilateral facial nerve transection at P14. The animals were treated with saline or doses of R(-)- or S(+)-deprenyl ranging from 0.001 to 10 mg/kg every 2 days (/2d). Frontal serial 10 microns sections were taken through the length of the facial nuclei ipsilaterally and contralaterally to the facial nerve transections. Every third section was immunoreacted for an antibody against ChAT to identify the motoneuron somata, while every adjacent third section was Nissl stained to assess motoneuronal survival. A second series of P14 rats was treated with similar doses of the two deprenyl enantiomers or saline and the brainstems removed for measurement of MAO-A and MAO-B activity at 4 hr after the treatments. Averages of 24% of the facial motoneurons survived axotomy with either saline treatment or 0.001 mg/kg/2d doses of R(-)-deprenyl. Doses of R(-)-deprenyl of 0.005, 0.01, and 10.0 mg/kg/2d increased the surviving facial motoneuron to 38%, 51%, and 48%, respectively, indicating an ED50 of about 0.005 mg/kg/2d. Doses of S(+)-deprenyl as high as 10 mg/kg/2d did not increase motoneuronal survival, revealing a stereospecificity for the increased survival of at least 2000-fold. The ED50 for MAO-B inhibition in the P14 brainstem was approximately 0.1 mg/kg for the (-)-enantiomer and 2.0 mg/kg for the (+)-enantiomer, revealing a 20-fold higher sensitivity of the enzyme toward the (-)-enantiomer in the P14 rat brainstem. A dose of 10 mg/kg of S(+)-deprenyl inhibited about 65% of brainstem MAO-B activity without increasing motoneuronal survival, whereas 0.005 and 0.01 mg/kg of R(-)-deprenyl increased motoneuronal survival without significant inhibition of brainstem MAO-B activity.(ABSTRACT TRUNCATED AT 400 WORDS)

A predictor for side effects in patients with Alzheimer's disease treated with deferoxamine mesylate.

In a previously reported clinical trial, patients with Alzheimer's disease were treated with deferoxamine mesylate, which resulted in a 50% reduction in the average rate of deterioration over 2 years. There were five deaths in the untreated group during the trial and no deaths in the treated group, although five of 25 treated patients reported anorexia. Deferoxamine metabolite analysis of urine for 24 hours after deferoxamine injection from sensitive and nonsensitive patients showed marked differences. Occurrence of side effects correlated with increased formation of a monoamine oxidase catalyzed (major) metabolite, MFO1. The metabolite ratio, MFO1/total metabolites, plus parent drug (TOT) showed a bimodal distribution with a mean +/- SD value of 0.68 +/- 0.06 for the nonsensitive and 0.79 +/- 0.04 for sensitive patients. The MFO1/TOT ratio discriminates between sensitive and nonsensitive patients, and we suggest that the half difference mark between the two mean values (0.735) can be used as a predictor of side effects. Patients with a MFO1/TOT ratio of greater than 0.70 would be considered at risk and observed for onset of side effects. Patients with a MFO1/TOT ratio greater than 0.80 would be considered for immediate adjunct treatment with isoniazid or other monoamine oxidase inhibitors.

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.

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.

Would decreased aluminum ingestion reduce the incidence of Alzheimer's disease?

Although the cause of Alzheimer's disease (AD) remains unknown there is mounting evidence that implicates aluminum as a toxic environmental factor of considerable importance. Four independent lines of evidence--laboratory studies of the effects of intracerebral aluminum on the cognitive and memory performance of animals, biochemical studies, epidemiologic studies and the slowing of the progress of the disease with the use of an agent that removes aluminum from the body--now support the concept that aluminum is one of the pathogenic factors in AD. The evidence warrants serious consideration of reducing human exposure to aluminum. We hypothesize that a public health effort to restrict human ingestion of aluminum would reduce the incidence of this common chronic illness in the elderly.

Intramuscular desferrioxamine in patients with Alzheimer's disease.

Although epidemiological and biochemical evidence suggests that aluminium may be associated with Alzheimer's disease (AD), there is no convincing proof of a causal link for aluminium in disease progression. We have completed a two year, single-blind study to investigate whether the progression of dementia could be slowed by the trivalent ion chelator, desferrioxamine. 48 patients with probable AD were randomly assigned to receive desferrioxamine (125 mg intramuscularly twice daily, 5 days per week, for 24 months), oral placebo (lecithin), or no treatment. No significant differences in baseline measures of intelligence, memory, or speech ability existed between groups. Activities of daily living were assessed and videorecorded at 6, 12, 18, and 24 month intervals. There were no differences in the rate of deterioration of patients receiving either placebo or no treatment. Desferrioxamine treatment led to significant reduction in the rate of decline of daily living skills as assessed by both group means (p = 0.03) and variances (p less than 0.04). The mean rate of decline was twice as rapid for the no-treatment group. Appetite (n = 4) and weight (n = 1) loss were the only reported side-effects. We conclude that sustained administration of desferrioxamine may slow the clinical progression of the dementia associated with AD.

Suppression of deferoxamine mesylate treatment-induced side effects by coadministration of isoniazid in a patient with Alzheimer's disease subject to aluminum removal by ionspecific chelation.

Deferoxamine treatment may produce serious side effects that can be eliminated by modification of treatment and by control of deferoxamine metabolism. A patient suffering from dementia of the Alzheimer type with normal liver and kidney function who was treated with deferoxamine initially tolerated a dose of 7 mg/kg deferoxamine mesylate injected intramuscularly twice a day for a total of 5 days a week. After several months nausea and weight loss gradually developed in the patient that could be controlled initially by dose reduction, leading to levels inappropriate for aluminum chelation. HPLC analysis of blood and urine revealed several metabolites including, as a major component, a plasma monoamine oxidase (MAO) catalyzed end product MFO1. Coadministration of isoniazid, a plasma MAO inhibitor, with deferoxamine resulted in reduction of MFO1 from 81% to 8% accompanied by increases in the amounts of metabolite 2 (MFO2) from 2% to 24% and unmetabolized deferoxamine from 17% to 68% after 6 months of treatment. The side effects subsided, the patient regained weight, and treatment could be continued.

Development of an intravenous desferrioxamine mesylate treatment protocol for swine: monitoring of desferrioxamine and metabolites by high-performance liquid chromatography.

Desferrioxamine (DFO) metabolism and its pharmacokinetics were studied in a swine model using high-performance liquid chromatography. DFO and three iron-binding metabolites occurred in plasma. Interindividual differences in pharmacokinetics and metabolism were observed. Urine analysis in 4 pigs showed three iron-binding metabolites. The mean percent dose excreted in urine in the form of the parent drug was 45 +/- 10% and 10 +/- 2% (means +/- SD) in the form of metabolites. Of the total amount of the parent drug infused, 3 h after initiation, 87% was in the form of DFO, whereas 13% was present as the DFO-iron III complex which represented 45 mg of urinary iron elimination. The described DFO infusion protocol provides for sufficient DFO to chelate significant amounts of ferric iron in excess of normal levels, thus allowing experimental studies of iron chelation in a variety of disease states.

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

The etiology of some, if not all, cases of Alzheimer's 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 Alzheimer's 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 Hl(o) content on dinucleosomes released by light (3-5% ASN) micrococcal nuclease digestion, and an increase in the affinity of histone Hl(o) 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 Alzheimer's disease neocortex is to increase the binding of histones, particularly Hl(o), 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 Alzheimer's 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 Hl(o) 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 Hl(o) 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 Hl(o) 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 Alzheimer's disease. Further understanding of the role of aluminum in Alzheimer's disease requires a detailed investigation of the precise sites of co-ordination of this trivalent metal within chromatin.

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 Alzheimer's 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 Alzheimer's 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.

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(0) 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 Alzheimer's disease where aluminum preferentially associates with DNA containing structures of the nucleus.

High-performance liquid chromatographic analysis of desferrioxamine. Pharmacokinetic and metabolic studies.

A high-performance liquid chromatographic method has been developed that permits determination and quantitation of desferrioxamine and metabolites as their iron (III) complexes in small samples of mammalian plasma at levels encountered with ion-specific chelation treatments. The technique permits measurement of desferrioxamine and metabolite concentrations which can be used in pharmacokinetic studies. A human study is presented as an example.

Acetylcholine receptor density and binding in murine dystrophy.

The concentration and binding characteristics of acetylcholine receptors (AChR) in sarcolemmal fractions from normal and dystrophic mice have been measured. Unlike the results following denervation, AChR concentration (per g wet weight) is unchanged and AChR affinity for two different alpha-neurotoxins is also unchanged. In contrast sarcolemmal ATPase is significantly reduced in dystrophic tissues. These data reaffirm that denervation is a poor model of dystrophy.