Michael G. Zagorski

Solution conformations and aggregational properties of synthetic amyloid β-peptides of Alzheimer's disease : analysis of circular dichroism spectra

The A4 or beta-peptide (39 to 43 amino acid residues) is the principal proteinaceous component of amyloid deposits in Alzheimers disease. Using circular dichroism (c.d.), we have studied the secondary structures and aggregational properties in solution of 4 synthetic amyloid beta-peptides: beta-(1-28), beta-(1-39), beta-(1-42) and beta-(29-42). The natural components of cerebrovascular deposits and extracellular amyloid plaques are beta-(1-39) and beta-(1-42), while beta-(1-28) and beta-(29-42) are unnatural fragments. The beta-(1-28), beta-(1-39) and beta-(1-42) peptides adopt mixtures of beta-sheet, alpha-helix and random coil structures, with the relative proportions of each secondary structure being strongly dependent upon the solution conditions. In aqueous solution, beta-sheet structure is favored for the beta-(1-39) and beta-(1-42) peptides, while in aqueous solution containing trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP), alpha-helical structure is favored for all 3 peptides. The alpha-helical structure unfolds with increasing temperature and is favored at pH 1 to 4 and pH 7 to 10; the beta-sheet conformation is temperature insensitive and is favored at pH 4 to 7. Peptide concentration studies showed that the beta-sheet conformation is oligomeric (intermolecular), whereas the alpha-helical conformation is monomeric (intramolecular). The rate of aggregation to the oligomeric beta-sheet structure (alpha-helix----random coil----beta-sheet) is also dependent upon the solution conditions such as the pH and peptide concentration; maximum beta-sheet formation occurs at pH 5.4. These results suggest that beta-peptide is not an intrinsically insoluble peptide. Thus, solution abnormalities, together with localized high peptide concentrations, which may occur in Alzheimers disease, may contribute to the formation of amyloid plaques. The hydrophobic beta-(29-42) peptide adopts exclusively an intermolecular beta-sheet conformation in aqueous solution despite changes in temperature or pH. Therefore, this segment may be the first region of the beta-peptide to aggregate and may direct the folding of the complete beta-peptide to produce the beta-pleated sheet structure found in amyloid deposits. Differences between the solution conformations of the beta-(1-39) and beta-(1-42) peptides suggests that the last 3 C-terminal amino acids are crucial to amyloid deposition.

Methionine 35 Oxidation Reduces Fibril Assembly of the Amyloid Aβ-(1–42) Peptide of Alzheimer's Disease

The major component of amyloid plaques in Alzheimers disease (AD) is Aβ, a small peptide that has high propensity to assemble as aggregated β-sheet structures. Using three well established techniques for studying amyloid structure, namely circular dichroism, thioflavin-T fluorescence, and atomic force microscopy, we demonstrate that oxidation of the Met-35 side chain to a methionine sulfoxide (Met-35ox) significantly hinders the rate of fibril formation for the 42-residue Aβ-(1–42) at physiological pH. Met-35ox also alters the characteristic Aβ fibril morphology and prevents formation of the protofibril, which is a key intermediate in β-amyloidosis and the associated neurotoxicity. The implications of these results for the biological function and role of Aβ with oxidative stress in AD are discussed.

Methodological and chemical factors affecting amyloid beta peptide amyloidogenicity.

Publisher Summary The immense difficulties and unexpected questions presented by hundreds of amyloid toxicity studies beg for a careful study of A β assembly processes, evaluating the kinetics and structures of different aggregation pathways that convert the soluble monomer into the higher order A β assemblies that harbor neurotoxic properties. A large array of sophisticated instrumental techniques, including analytical ultracentrifugation, atomic force microscopy, solid-state and solution NMR, and fluorescence tracer techniques, will help elucidate the bioactive A β structure(s), whereas specific cellular and biochemical techniques will elucidate the specific steps involved in the aftermath of A β cellular interactions. Nonetheless, all these potentially useful high technology methods are ineffective unless proper care regarding A β sample preparation is taken into account. This chapter briefly summarizes potential problems involved with A β sample preparation and has also described methods to deal with these issues. The most critical complications are related to the starting structures and aggregation states of the A β .

The PI3K-Akt-mTOR pathway regulates Aβ oligomer induced neuronal cell cycle events

Accumulating evidence suggests that neurons prone to degeneration in Alzheimers Disease (AD) exhibit evidence of re-entry into an aberrant mitotic cell cycle. Our laboratory recently demonstrated that, in a genomic amyloid precursor protein (APP) mouse model of AD (R1.40), neuronal cell cycle events (CCEs) occur in the absence of beta-amyloid (Aβ) deposition and are still dependent upon the amyloidogenic processing of the amyloid precursor protein (APP). These data suggested that soluble Aβ species might play a direct role in the induction of neuronal CCEs. Here, we show that exposure of non-transgenic primary cortical neurons to Aβ oligomers, but not monomers or fibrils, results in the retraction of neuronal processes, and induction of CCEs in a concentration dependent manner. Retraction of neuronal processes correlated with the induction of CCEs and the Aβ monomer or Aβ fibrils showed only minimal effects. In addition, we provide evidence that induction of neuronal CCEs are autonomous to primary neurons cultured from the R1.40 mice. Finally, our results also demonstrate that Aβ oligomer treated neurons exhibit elevated levels of activated Akt and mTOR (mammalian Target Of Rapamycin) and that PI3K, Akt or mTOR inhibitors blocked Aβ oligomer-induced neuronal CCEs. Taken together, these results demonstrate that Aβ oligomer-based induction of neuronal CCEs involve the PI3K-Akt-mTOR pathway.

Solution structure of the E200K variant of human prion protein. Implications for the mechanism of pathogenesis in familial prion diseases.

Prion propagation in transmissible spongiform encephalopathies involves the conversion of cellular prion protein, PrPC, into a pathogenic conformer, PrPSc. Hereditary forms of the disease are linked to specific mutations in the gene coding for the prion protein. To gain insight into the molecular basis of these disorders, the solution structure of the familial Creutzfeldt-Jakob disease-related E200K variant of human prion protein was determined by multi-dimensional nuclear magnetic resonance spectroscopy. Remarkably, apart from minor differences in flexible regions, the backbone tertiary structure of the E200K variant is nearly identical to that reported for the wild-type human prion protein. The only major consequence of the mutation is the perturbation of surface electrostatic potential. The present structural data strongly suggest that protein surface defects leading to abnormalities in the interaction of prion protein with auxiliary proteins/chaperones or cellular membranes should be considered key determinants of a spontaneous PrPC → PrPSc conversion in the E200K form of hereditary prion disease.

Phenolic compounds prevent amyloid β-protein oligomerization and synaptic dysfunction by site-specific binding

Background: Epidemiological evidence suggests that consumption of phenolic compounds reduce the incidence of Alzheimer disease (AD). Results: Myricetin and rosmarinic acid reduced cellular and synaptic toxicities by inhibition of amyloid β-protein (Aβ) oligomerization. Myricetin promoted NMR changes of Aβ. Conclusion: Phenolic compounds are worthy therapeutic candidates for AD. Significance: Phenolic compounds blocked early assembly processes of Aβ through differently binding. Cerebral deposition of amyloid β protein (Aβ) is an invariant feature of Alzheimer disease (AD), and epidemiological evidence suggests that moderate consumption of foods enriched with phenolic compounds reduce the incidence of AD. We reported previously that the phenolic compounds myricetin (Myr) and rosmarinic acid (RA) inhibited Aβ aggregation in vitro and in vivo. To elucidate a mechanistic basis for these results, we analyzed the effects of five phenolic compounds in the Aβ aggregation process and in oligomer-induced synaptic toxicities. We now report that the phenolic compounds blocked Aβ oligomerization, and Myr promoted significant NMR chemical shift changes of monomeric Aβ. Both Myr and RA reduced cellular toxicity and synaptic dysfunction of the Aβ oligomers. These results suggest that Myr and RA may play key roles in blocking the toxicity and early assembly processes associated with Aβ through different binding.

Trifluoroacetic acid pretreatment reproducibly disaggregates the amyloid β-peptide

Major problems exist with pharmacological and biophysical studies of the synthetic β(1-42) peptide, in that the results often lack reproducibility. The starting aggregation states and structures of the various synthetic commercial lots appear to vary, resulting in signijicant lot-to-lot variability. We describe here an easy, efficient trifluoroacetic acid (TFA) pretreatment method that renders the Aβ easily soluble both in aqueous, buffered solutions and in organic solvents such as hexafluoroisopropanol (HFIP) or dimethylsulfoxide (DMSO). The TFA treated Aβ exhibits the properties of monomeric, random coil structures and lacks pre-aggregated material that can act as seeds in fibrilization assays, thus reducing the batch to batch variation. In addition, nuclear magnetic resonance (NMR) spectra recorded in deuterated TFA allow measurement of the peptide purity that is not obtainable with other analytical methods.

Biosynthetic origins and assignments of carbon 13 NMR peaks of brevetoxin B.

remains similar to those in the other P-(ET),X salts. In conclusion, the high-T, superconducting phase, P*-(ET),I,, is completely ordered although it is not isostructural with other known p-( ET)2X materials. Furthermore, there appears to be a correlation between the degree of structural ordering and T, in these compounds. That is, P-(ET),I,Br does not become superconducting apparently because of the random I/Br occupancy of the terminal anion position. The ambient-pressure modulated P-(ET),I, structure is ordered, but the periodicity of the modulation is incommensurate with the average lattice. This may result in suppression of T, to 1.4 K. Finally, the pressure-induced structural phase transition from the modulated P-(ET),I, to the completely ordered P*-(ET),13 produces a dramatic rise in T, to 8 K. This value is now in agreement with band calculation predictions of increasing T:s for the IBrc, AuIT, and 1,salts, in that order,Ia which is also the order of increasing anion size.

Nicotine and amyloid formation

The major protein constituents of amyloid deposits in Alzheimers disease (AD) are the 40-residue beta-amyloid (Abeta) (1-40) peptide and the 42-residue Abeta(1-42) peptide. The Abeta(1-42) is more pathogenic and produced in greater quantities in familial forms of AD. A major goal of research is to uncover a suitable inhibitor that either slows down or inhibits Abeta formation (beta-amyloidosis). During beta-amyloidosis, structural changes associated with the conversion of monomeric Abeta peptide building blocks into the aggregated fibrillar beta-sheet structures occur (alpha-helix-->beta-sheet or random, extended chain-->beta-sheet). In previous work, we and others established that nicotine, a major component of cigarette smoke, inhibits beta-amyloidosis of the Abeta(1-42), which may result from nicotine binding to the alpha-helical structure. These conclusions were based on solution nuclear magnetic resonance (NMR) spectroscopic studies with the nonnative 28-residue Abeta(1-28). This information suggests that, when administered therapeutically to AD patients, nicotine may not only affect cholinergic activation, but could also conceivably alter amyloid deposition. In this report, NMR studies were augmented with the naturally occurring Abeta(1-42), under conditions where the peptide folds into a predominantly alpha-helical or random, extended chain structure. The major result is that nicotine shows only modest binding to these conformations, indicating that the nicotine inhibition to beta-amyloidosis probably results from binding to a small, soluble beta-sheet aggregate that is NMR invisible.

An NMR spectroscopic study of azadirachtin and its trimethyl ether

Abstract The neem tree Azadirachta indica A. Juss and the related Chinaberry tree Melia azedarach L. are attracting considerable Interest, particularly because of their insect-repelling properties.1,2 The most potent constituent is azadirachtin, a limonoid 3 which exerts strong physiological4 and phagorepellent activities.5 Incorporating the earlier chemical and spectroscopic studies carried out by Morgan and coworkers 6 and applying the technique of continuous wave proton decoupling in partially relaxed Fourier transform 13C NMR, we proposed structure 1 for azadirachtin in 1975.7 Since evidence for this complex structure was not convincing, we have been using this molecule as a test sample for the application of modern NMR methods, however, without arriving at a conclusive structure.8 After proposals of revised structures by Kraus and coworkers. Ley and coworkers, and ourselves starting In mid-1985,9 its structure was finally established as 2 by Kraus, et al.10 and by Ley, et al.11 In the following we summarize the results of our NMR studies and some of the unique difficulties encountered during the investigation of this compound and its 7,11,20-trimethyl ether.