amyloid-beta-peptides and sarkosyl

Accumulation of neurotoxic amyloid-β (Aβ) is a major hallmark of Alzheimer's disease (AD) pathology and an important player in its clinical manifestations. Formation of Aβ is controlled by the availability of an enzyme called γ-secretase. Despite its blockers being attractive therapeutic tools for lowering Aβ, this approach has failed because of their serious toxic side-effects. The discovery of the γ-secretase activating protein (GSAP), a co-factor for this protease which facilitates Aβ production without affecting other pathways responsible for the toxicity, is giving us the opportunity to develop a safer anti-Aβ therapy. In this study we have characterized the effect of Imatinib, an inhibitor of GSAP, in the 3×Tg mice, a mouse model of AD with plaques and tangles. Compared with controls, mice receiving the drug had a significant reduction in brain Aβ levels and deposition, but no changes in the steady state levels of AβPP, BACE-1, ADAM-10, or the four components of the γ-secretase complex. By contrast, Imatinib-treated animals had a significant increase in CTF-β and a significant reduction in GSAP expression levels. Additionally, we observed that tau phosphorylation was reduced at specific epitopes together with its insoluble fraction. In vitro studies confirmed that Imatinib prevents Aβ formation by modulating γ-secretase activity and GSAP levels. Our findings represent the first in vivo demonstration of the biological role that GSAP plays in the development of the AD-like neuropathologies. They establish this protein as a viable target for a safer anti-Aβ therapeutic approach in AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Benzamides; Cell Line, Tumor; Disease Models, Animal; Enzyme Inhibitors; Humans; Imatinib Mesylate; Mice; Mice, Transgenic; Mutation; Neuroblastoma; Peptide Fragments; Phosphorylation; Piperazines; Plaque, Amyloid; Presenilin-1; Proteins; Pyrimidines; Sarcosine; Statistics, Nonparametric; tau Proteins

Amyloid fibrils of Alzheimer's β-amyloid peptide (Aβ) are a primary component of amyloid plaques, a hallmark of Alzheimer's disease (AD). Enormous attention has been given to the structural features and functions of Aβ in amyloid fibrils and other type of aggregates in associated with development of AD. This report describes an efficient protocol to express and purify high-quality 40-residue Aβ(1-40), the most abundant Aβ in brains, for structural studies by NMR spectroscopy. Over-expression of Aβ(1-40) with glutathione S-transferase (GST) tag connected by a Factor Xa recognition site (IEGR(▾)) in Escherichia coli resulted in the formation of insoluble inclusion bodies even with the soluble GST tag. This problem was resolved by efficient recovery of the GST-Aβ fusion protein from the inclusion bodies using 0.5% (w/v) sodium lauroyl sarcosinate as solubilizing agent and subsequent purification by affinity chromatography using a glutathione agarose column. The removal of the GST tag by Factor Xa enzymatic cleavage and purification by HPLC yielded as much as ∼7 mg and ∼1.5mg of unlabeled Aβ(1-40) and uniformly (15)N- and/or (13)C-protein Aβ(1-40) from 1L of the cell culture, respectively. Mass spectroscopy of unlabeled and labeled Aβ and (1)H/(15)N HSQC solution NMR spectrum of the obtained (15)N-labeled Aβ in the monomeric form confirmed the expression of native Aβ(1-40). It was also confirmed by electron micrography and solid-state NMR analysis that the purified Aβ(1-40) self-assembles into β-sheet rich amyloid fibrils. To the best of our knowledge, our protocol offers the highest yields among published protocols for production of recombinant Aβ(1-40) samples that are amendable for an NMR-based structural analysis. The protocol may be applied to efficient preparation of other amyloid-forming proteins and peptides that are (13)C- and (15)N-labeled for NMR experiments.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Peptides; Carbon Isotopes; Escherichia coli; Gene Expression; Glutathione Transferase; Humans; Molecular Sequence Data; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments; Recombinant Fusion Proteins; Sarcosine; Solubility