Microsecond Dynamics During the Binding-induced Folding of an Intrinsically Disordered Protein

Tau is an intrinsically disordered protein implicated in many neurodegenerative diseases. The repeat domain fragment of tau, tau-K18, is known to undergo a disorder to order transition in the presence of lipid micelles and vesicles, in which helices form in each of the repeat domains. Here, the mechanism of helical structure formation, induced by a phospholipid mimetic, sodium dodecyl sulfate (SDS) at sub-micellar concentrations, has been studied using multiple biophysical probes. A study of the conformational dynamics of the disordered state, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS) has indicated the presence of an intermediate state, I, in equilibrium with the unfolded state, U. The cooperative binding of the ligand (L), SDS, to I has been shown to induce the formation of a compact, helical intermediate (IL₅) within the dead time (∼37 µs) of a continuous flow mixer. Quantitative analysis of the PET-FCS data and the ensemble microsecond kinetic data, suggests that the mechanism of induction of helical structure can be described by a U ↔ I ↔ IL₅ ↔ FL₅ mechanism, in which the final helical state, FL₅, forms from IL₅ with a time constant of 50–200 µs. Finally, it has been shown that the helical conformation is an aggregation-competent state that can directly form amyloid fibrils.     

Collagen type I fibril packing in vivo and in vitro

Reviewed are works concerning the mechanisms of collagen (type I) fibril packing and the influence of macromolecular structure and physicochemical parameters of the medium on the process.     

Antibodies to Cholinergic Neurons in Alzheimer''s Disease

Alzheimer's disease (AD) is associated with degenerative changes in nuclei of the basal forebrain which provide most of the cholinergic input to the cortex and hippocampus and with a reduction in presynaptic cholinergic parameters in these areas. Although the etiology and pathogenesis of AD are not known, several reports indicate the involvement of immunological mechanisms. In the present work we examined the existence of antibodies in sera of AD patients that bind specifically to cholinergic neurons. As antigens we employed the purely cholinergic electromotor neurons of the electric fish Torpedo which are chemically homogeneous and cross-react antigenically with human and other mammalian cholinergic neurons. Our findings show that immunoglobulins from sera of AD patients bind to a specific antigen (molecular mass 200 kilodaltons) in the cell bodies and axons of Torpedo electromotor neurons and that the levels of such antibodies are significantly higher in AD patients than in controls. The possible role of these antibodies in the cholinergic dysfunction in AD and their diagnostic potential are discussed.     

Galanin-Like Immunoreactivity Is Unchanged in Alzheimer''s Disease and Parkinson''s Disease Dementia Cerebral Cortex

Galanin is a recently isolated neuropeptide that is of particular interest in dementing disorders because of its known colocalization with choline acetyltransferase in magnocellular neurons of the basal nucleus of Meynert. These neurons degenerate in Alzheimer's disease, and there is a corresponding deficiency of cortical choline acetyltransferase activity. In the present study, galanin-like immunoreactivity was measured in the postmortem cerebral cortex and hippocampus of 10 controls and 14 patients who had had Alzheimer's disease. Significant reductions of choline acetyltransferase activity (50-60%) were found in all regions examined; however, there was no significant effect on concentrations of galanin-like immunoreactivity. Similar measurements were made in postmortem tissues of 12 control and 13 demented Parkinsonian patients who had had Alzheimer-type cortical pathology. Choline acetyltransferase activity was again significantly decreased in all regions examined but there were no significant reductions in galanin-like immunoreactivity. Experimental lesions of the fornix in rats produced parallel significantly correlated reductions of both choline acetyltransferase activity and galanin-like immunoreactivity in the hippocampus. Galanin-like immunoreactivity in the human hypothalamus consisted of two molecular-weight species on gel-permeation chromatography, and two forms were resolved by reverse-phase HPLC. The paradoxical preservation of galanin-like immunoreactivity, despite depletion of the activity of choline acetyltransferase, with which it is colocalized, is as yet unexplained. Recent studies have shown that galanin inhibits both acetylcholine release in the hippocampus and memory acquisition; therefore, preserved galanin may exacerbate the cholinergic and cognitive deficits that accompany dementia.     

Glycosaminoglycans in Cortical Autopsy Samples from Alzheimer Brain

Each of the known classes of mammalian glycosaminoglycans, with the exception of keratan sulphate, was found in cerebral cortex samples from patients with Alzheimer-type dementia and age-matched controls. These molecules were quantitated, after electrophoresis and staining with Alcian Blue dye, by scanning densitometry. No significant differences were found between the mean levels of each of the above glycosaminoglycans in frontal cortex from patients with dementia compared with controls. An increase (26%; p less than 0.05) in the mean level of hyaluronate, but not of other glycosaminoglycans, was found in temporal cortex samples. On the other hand, the uronic acid content of hyaluronate degradation products following Streptomyces hyaluronidase treatment of brain glycosaminoglycans did not reveal any statistically significant changes in Alzheimer's disease. HPLC of disaccharide products from Arthrobacter chondroitinase AC digests did not reveal any significant changes in sulphate substitution of chondroitin sulphate in Alzheimer brain.     

Monoamine neurotransmitters and their metabolites in brain regions in alzheimer's disease: A postmortem study

1. Concentrations of the neurotransmitter amines noradrenaline (NA), dopamine (DA), and 5-hydroxytryptamine (5-HT) and the acid metabolites homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were determined in four regions of postmortem brains of demented patients with or without Alzheimer's disease (AD). 2. NA was deficient in the temporal cortex (BA 21) of AD, but not of non-AD, patients. 3. Caudate, in particular, had an impaired dopaminergic system in AD patients, with low HVA levels. 4. In all regions investigated [amygdala, caudate, putamen, temporal cortex (BA 21)] 5-HT was significantly depleted in AD patients, and 5-HIAA was also depleted in amygdala and caudate. 5. These results indicate that neurotransmitter systems other than cholinergic systems are also widely affected in AD and suggest that these deficits may also play an important role in determining the symptomatology of AD.     

Reduced Glycine Stimulation of [3H]MK-801 Binding in Alzheimer''s Disease

The novel N-methyl-D-aspartate receptor channel ligand (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5, 10-imine maleate ([3H]MK-801) has been utilized to label this receptor in human brain tissue. Characteristics of [3H]MK-801 binding to well-washed membranes from 17 control subjects and 16 patients with Alzheimer's disease were determined in frontal, parietal, and temporal cerebral cortex and cerebellar cortex. In control tissue the pharmacological specificity of the binding of this substance is entirely consistent with the profile previously reported for rat brain. Binding could be stimulated by the addition of glutamic acid to the incubation medium; addition of glycine produced further enhancement which was not prevented by strychnine. The specificity of the effects of these and other amino acids on the binding was the same as in the rat. In Alzheimer's disease significantly less binding was observed in the frontal cortex under glutamate- and glycine-stimulated conditions. This appears to be associated with a reduced affinity of the site whereas the pharmacological specificity of the site remained unchanged. The effect did not appear to be due to differences in mode of death between Alzheimer's disease and control subjects and is unlikely to be related to factors for which the groups were matched. In contrast, binding was not altered in the absence of added amino acids and presence of glutamate alone. These results imply that in the cerebral cortex the agonist site and a site in the cation channel of the receptor are not selectively altered, but that their coupling to a strychnine-insensitive glycine recognition site is impaired.     

Down''s Syndrome Individuals Begin Life with Normal Levels of Brain Cholinergic Markers

We measured the activities of the cholinergic marker enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in autopsied brains of seven infants (age range 3 months to 1 year) with Down's syndrome (DS), a disorder in which virtually all individuals will develop by middle age the neuropathological changes of Alzheimer's disease accompanied by a marked brain cholinergic reduction. When compared with age-matched controls cholinergic enzyme activity was normal in all brain regions of the individuals with infant DS with the exception of above-normal activity in the putamen (ChAT) and the occipital cortex (AChE). Our neurochemical observations suggest that DS individuals begin life with a normal complement of brain cholinergic neurons. This opens the possibility of early therapeutic intervention to prevent the development of brain cholinergic changes in patients with DS.     

Activation of m1 Muscarinic Acetylcholine Receptor Regulates τ Phosphorylation in Transfected PC12 Cells

Abstract: Hyperphosphorylated τ proteins are the principal fibrous component of the neurofibrillary tangle pathology in Alzheimer's disease. The possibility that τ phosphorylation is controlled by cell surface neurotransmitter receptors was examined in PC12 cells transfected with the gene for the rat m₁ muscarinic acetylcholine receptor. Stimulation of m₁ receptor in these cells with two acetylcholine agonists, carbachol and AF102B, decreased τ phosphorylation, as indicated by specific τ monoclonal antibodies that recognize phosphorylation-dependent epitopes and by alkaline phosphatase treatment. The muscarinic effect was both time and dose dependent. In addition, a synergistic effect on τ phosphorylation was found between treatments with muscarinic agonists and nerve growth factor. These studies provide the first evidence for a link between the cholinergic signal transduction system and the neuronal cytoskeleton that can be mediated by regulated phosphorylation of τ microtubule-associated protein.     

Aggregation and Metal-Binding Properties of Mutant Forms of the Amyloid Aβ Peptide of Alzheimer's Disease

Abstract: The fibrillogenic properties of Alzheimer's Aβ peptides corresponding to residues 1–40 of the normal human sequence and to two mutant forms containing the replacement Ala²¹ to Gly or Glu²² to Gln were compared. At pH 7.4 and 37°C the Gln²² peptide was found to aggregate and precipitate from solution faster than the normal Aβ, whereas the Gly²¹ peptide aggregated much more slowly. Electron microscopy showed that the aggregates all had fibrillar structures. Circular dichroism spectra of these peptides revealed that aggregation of the normal and Gln²² sequences was associated with spectral changes consistent with a transformation from random coil to β sheet, whereas the spectrum of the Gly²¹ peptide remained almost unchanged during a period in which little or no aggregation occurred. When immobilised by spotting onto nitrocellulose membranes the peptides bound similar amounts of the radioisotope ⁶⁵Zn²⁺. Of several competing metal ions, tested at 20× the concentration of Zn²⁺, Cu²⁺ displaced >95% of the radioactivity from all three peptides and Ni²⁺ produced >50% displacement in each case. Some other metal ions tested caused lesser displacement, but Fe²⁺ and Al³⁺ were without effect. In a saturation binding assay, a value of 3.2 µM was obtained for the binding of Zn²⁺ to Aβ but our data provided no evidence for a reported higher affinity site (107 nM). The results suggest that the neuropathology associated with the Gly²¹ mutation is not due to enhanced fibrillogenic or different metal-binding properties of the peptide and that the binding of zinc to amyloid peptides is not a specific phenomenon.