Harry LeVine

Quantification of beta-sheet amyloid fibril structures with thioflavin T.

Publisher Summary Despite the presence of a significant amount of carbohydrate in these fibrils, the staining reaction was eventually shown to be because of the protein component. The histologic benzothiazole dyes thioflavin S (ThS) and thioflavin T (ThT) under the appropriate conditions selectively stain amyloid structures in a number of pathological settings, as does the diazobenzidine sulfonate dye, Congo red, which is also birefringent when bound to fibrils. Phorwhite BBU, Sirius Red, and several other fluorescent and nonfluorescent aromatic molecules also show this property. Investigation of the amyloid fibril formation process requires not only the ability to distinguish the characteristic amyloid B-sheet structure from amorphous aggregates of the monomer or nonamyloid fibril forms of the precursor protein, but quantitation of the amyloid form as well. Congo red and thioflavin T undergo characteristic spectral alterations on binding to a variety of amyloid fibrils that do not occur on binding to the precursor polypeptides, monomers, or amorphous aggregates of peptide. Both dyes have been adapted to in vitro measurements of amyloid fibril formation.

Alzheimer's Disease and the Amyloid-β Peptide

Alzheimers disease (AD) pathogenesis is widely believed to be driven by the production and deposition of the amyloid-beta peptide (Abeta). For many years, investigators have been puzzled by the weak to nonexistent correlation between the amount of neuritic plaque pathology in the human brain and the degree of clinical dementia. Recent advances in our understanding of the development of amyloid pathology have helped solve this mystery. Substantial evidence now indicates that the solubility of Abeta, and the quantity of Abeta in different pools, may be more closely related to disease state. The composition of these pools of Abeta reflects different populations of amyloid deposits and has definite correlates with the clinical status of the patient. Imaging technologies, including new amyloid imaging agents based on the chemical structure of histologic dyes, are now making it possible to track amyloid pathology along with disease progression in the living patient. Interestingly, these approaches indicate that the Abeta deposited in AD is different from that found in animal models. In general, deposited Abeta is more easily cleared from the brain in animal models and does not show the same physical and biochemical characteristics as the amyloid found in AD. This raises important issues regarding the development and testing of future therapeutic agents.

Cognitive Decline in Alzheimer's Disease Is Associated with Selective Changes in Calcineurin/NFAT Signaling

Upon activation by calcineurin, the nuclear factor of activated T-cells (NFAT) translocates to the nucleus and guides the transcription of numerous molecules involved in inflammation and Ca2+ dysregulation, both of which are prominent features of Alzheimers disease (AD). However, NFAT signaling in AD remains relatively uninvestigated. Using isolated cytosolic and nuclear fractions prepared from rapid-autopsy postmortem human brain tissue, we show that NFATs 1 and 3 shifted to nuclear compartments in the hippocampus at different stages of neuropathology and cognitive decline, whereas NFAT2 remained unchanged. NFAT1 exhibited greater association with isolated nuclear fractions in subjects with mild cognitive impairment (MCI), whereas NFAT3 showed a strong nuclear bias in subjects with severe dementia and AD. Similar to NFAT1, calcineurin-Aα also exhibited a nuclear bias in the early stages of cognitive decline. But, unlike NFAT1 and similar to NFAT3, the nuclear bias for calcineurin became more pronounced as cognition worsened. Changes in calcineurin/NFAT3 were directly correlated to soluble amyloid-β (Aβ(1-42)) levels in postmortem hippocampus, and oligomeric Aβ, in particular, robustly stimulated NFAT activation in primary rat astrocyte cultures. Oligomeric Aβ also caused a significant reduction in excitatory amino acid transporter 2 (EAAT2) protein levels in astrocyte cultures, which was blocked by NFAT inhibition. Moreover, inhibition of astrocytic NFAT activity in mixed cultures ameliorated Aβ-dependent elevations in glutamate and neuronal death. The results suggest that NFAT signaling is selectively altered in AD and may play an important role in driving Aβ-mediated neurodegeneration.

Thioflavine T interaction with amyloid β-sheet structures

Thioflavine T (ThT) interacts in tissue sections with amyloid deposits comprised of a variety of protein species giving a characteristic fluorescent complex. Binding of the dye to amyloid fibrils in suspension generates an amyloid-specific fluorescent signal. This interaction with amyloid fibrils formed from different polypeptides and proteins containing antiparallel β-pleated sheet secondary structure is selective, occurring strongly with amyloid fibrils formed from insulin, transthyretin, polyglycine(I), Aβ (1–40), and weakly with β2–microglobulin. No fluorescence changes were seen with β-sheet fibrillar poly-L-lysine, islet amyloid peptide (20–29), or poly-L-serine. Native forms of transthyretin, insulin, β2-microglobulin, poly-L-lysine, Aβ(1–40), or several proteins containing high percentages of β-sheet were also unreactive. The affinity of the amyloid fibrils for ThT varied: (apparent Kds 0.033 – 10 μ Amyloid A protein

The role of microglial cells and astrocytes in fibrillar plaque evolution in transgenic APPSW mice

Ultrastructural reconstruction of 27 fibrillar plaques in different stages of formation and maturation was undertaken to characterize the development of fibrillar plaques in the brains of human APP(SW) transgenic mice (Tg2576). The study suggests that microglial cells are not engaged in Abeta removal and plaque degradation, but in contrast, are a driving force in plaque formation and development. Fibrillar Abeta deposition at the amyloid pole of microglial cells appears to initiate three types of neuropil response: degeneration of neurons, protective activation of astrocytes, and attraction and activation of microglial cells sustaining plaque growth. Enlargement of neuronal processes and synapses with accumulation of degenerated mitochondria, dense bodies, and Hirano-type bodies is the marker of toxic injury of neurons by fibrillar Abeta. Separation of amyloid cores from neurons and degradation of amyloid cores by cytoplasmic processes of hypertrophic astrocytes suggest the protective and defensive character of astrocytic response to fibrillar Abeta. The growth of cored plaque from a small plaque with one microglial cell with an amyloid star and a few dystrophic neurites to a large plaque formed by several dozen microglial cells seen in old mice is the effect of attraction and activation of microglial cells residing outside of the plaque perimeter. This mechanism of growth of plaques appears to be characteristic of cored plaques in transgenic mice. Other features in mouse microglial cells that are absent in human brain are clusters of vacuoles, probably of lysosomal origin. They evolve into circular cisternae and finally into large vacuoles filled with osmiophilic, amorphous material and bundles of fibrils that are poorly labeled with antibody to Abeta. Microglial cells appear to release large amounts of fibrillar Abeta and accumulate traces of fibrillar Abeta in a lysosomal pathway.

Soluble multimeric Alzheimer β(1–40) pre-amyloid complexes in dilute solution

Abstract Aqueous solutions of β(1–40) peptide spontaneously associate to form pentameric/hexameric complexes that can be demonstrated by SDS-PAGE following treatment with glutaraldehyde and borohydride reduction. Under amyloidogenic conditions of pH and high peptide concentration these aggregates can further associate to form sedimentable and filterable structures with β-sheet amyloid characteristics of Thioflavine T fluorescence. The presence of such preamyloid structures at low peptide concentration suggests a mechanism by which amyloid plaques can accrete additional material by a cooperative rather than monomeric growth. The existence of a monomer ↭ multimer equilibrium may partly explain the divergence of biological consequences with respect to neurotoxicity.

Exogenous seeding of cerebral β-amyloid deposition in βAPP-transgenic rats

J. Neurochem. (2012) 120, 660–666.

The cerebral proteopathies: neurodegenerative disorders of protein conformation and assembly.

The abnormal assembly and deposition of specific proteins in the brain is the probable cause of most neurodegenerative disease afflicting the elderly. These “cerebral proteopathies” include Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), prion diseases, and a variety of other disorders. Evidence is accumulating that the anomalous aggregation of the proteins, and not a loss of protein function, is central to the pathogenesis of these diseases. Thus, therapeutic strategies that reduce the production, accumulation, or polymerization of pathogenic proteins might be applicable to a wide range of some of the most devastating diseases of old age.

Amino acid sequence determination of Ancrod, the thrombin‐like α‐fibrinogenase from the venom of Akistrodon rhodostoma

The thrombin‐like serine protease and antithrombotic agent. Ancrod, was rapidly purified from the crude venom of Akistradon rhodostoma by agmatine‐Sepharose affinity chromatography followed by MonoQ anion exchange chromatography. N‐Terminal sequencing and analysis of overlapping proteolytic fragments of purified Ancrod by automated Edman degradation in combination with tandem mass spectroscopy allowed the determination of the 234 amino acid sequence of the protease. Glycosylation sites at all five canonical N‐linked glycosylation sites were inferred from the appearance of blank sequencer cycles in the amino acid sequence and were confirmed by mass spectroscopic analysis of the N‐glycanase‐treated peptides. Monoclonal antibodies raised against the denatured protein and HF‐deglycosylated protein recognized Ancrod on Western blots. Sequence comparison to other thrombin‐like serine proteases and reptilian fibrinogenases revealed a number of similarities, most notably the catalytic triad and many conserved cysteine positions.

Multiple ligand binding sites on Aβ(1–40) fibrils

Although the structures of Thioflavin T and another benzothiazole, BTA-1, are similar, they bind to A beta non-competitively, probably to different sites on the A beta(1-40) fibrils. The amyloid fibril-induced fluorescence of ThT that corresponds to a fraction of total ThT binding is not displaced by high concentrations of (S)-naproxen or (R)-ibuprofen, which are reported to potently block high affinity binding of the radiolabeled malononitrile FDDNP and derivatives. The binding of the benzothiazole ligands is significantly substoichiometric with respect to A beta(1-40) monomer peptide, unlike Congo Red, which binds to A beta(1-40) fibrils on a 1:1 basis with monomer peptide. These results indicate that there are multiple domains for ligand binding to amyloid fibrils and suggest that it may be possible to design ligands that bind selectively to particular forms of fibrils that are connected with the pathogenesis of Alzheimers disease and potentially other protein misfolding diseases.Although the structures of Thioflavin T and another benzothiazole, BTA-1, are similar, they bind to Aß non-competitively, probably to different sites on the Aß (1–40) fibrils. The amyloid fibril-induced fluorescence of ThT that corresponds to a fraction of total ThT binding is not displaced by high concentrations of (S)-naproxen or (R)-ibuprofen, which are reported to potently block high affinity binding of the radiolabeled malononitrile FDDNP and derivatives. The binding of the benzothiazole ligands is significantly substoichiometric with respect to Aß (1–40) monomer peptide, unlike Congo Red, which binds to Aß (1–40) fibrils on a 1:1 basis with monomer peptide. These results indicate that there are multiple domains for ligand binding to amyloid fibrils and suggest that it may be possible to design ligands that bind selectively to particular forms of fibrils that are connected with the pathogenesis of Alzheimers disease and potentially other protein misfolding diseases.