Project description:The free energy landscape for folding of the Alzheimer's amyloid-beta(25-35) peptide is explored using replica exchange molecular dynamics in both pure water and in HFIP/water cosolvent. This amphiphilic peptide is a natural by-product of the Alzheimer's amyloid-beta(1-40) peptide and retains the toxicity of its full-length counterpart as well as the ability to aggregate into beta-sheet-rich fibrils. Our simulations reveal that the peptide preferentially populates a helical structure in apolar organic solvent, while in pure water, the peptide adopts collapsed coil conformations and to a lesser extent beta-hairpin conformations. The beta-hairpin is characterized by a type II' beta-turn involving residues G29 and A30 and two short beta-strands involving residues N27, K28, I31, and I32. The hairpin is stabilized by backbone hydrogen-bonding interactions between residues K28 and I31; S26 and G33; and by side-chain-to-side-chain interactions between N27 and I32. Implications regarding the mechanism of aggregation of this peptide into fibrils and the role of the environment in modulating secondary structure are discussed.
Project description:The amyloid-?(25-35) peptide plays a key role in the etiology of Alzheimer's disease due to its extreme toxicity even in the absence of aging. Because of its high tendency to aggregate and its low solubility in water, the structure of this peptide is still unknown. In this work, we sought to understand the early stages of aggregation of the amyloid-?(25-35) peptide by conducting simulations of oligomers ranging from monomers to tetramers. Our simulations show that although the monomer preferentially adopts a ?-hairpin conformation, larger aggregates have extended structures, and a clear transition from compact ?-hairpin conformations to extended ?-strand structures occurs between dimers and trimers. Even though ?-hairpins are not present in the final architecture of the fibril, our simulations indicate that they play a critical role in fibril growth. Our simulations also show that ?-sheet structures are stabilized when a ?-hairpin is present at the edge of the sheet. The binding of the hairpin to the sheet leads to a subsequent destabilization of the hairpin, with part of the hairpin backbone dangling in solution. This free section of the peptide can then recruit an extra monomer from solution, leading to further sheet extension. Our simulations indicate that the peptide must possess sufficient conformational flexibility to switch between a hairpin and an extended conformation in order for ?-sheet extension to occur, and offer a rationalization for the experimental observation that overstabilizing a hairpin conformation in the monomeric state (for example, through chemical cross-linking) significantly hampers the fibrillization process.
Project description:Amyloid nanostructures are originated from protein misfolding and aberrant aggregation, which is associated with the pathogenesis of many types of degenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease. The secondary conformation of peptides is of a fundamental importance for aggregation and toxicity of amyloid peptides. In this work, A?25-35, a fragment of amyloid ?(1-42) (A?42), was selected to investigate the correlation between secondary structures and toxicity of amyloid fibrils. Furthermore, each aggregation assemblies show different cell membrane disruption and cytotoxicity. The structural analysis of amyloid aggregates originated from different secondary structure motifs is helpful to understand the mechanism of peptides/cell interactions in the pathogenesis of amyloid diseases.
Project description:Beta-amyloid (Abeta) peptides may cause malfunction and death of neurons in Alzheimer's disease. We investigated the effect of Abeta on key transporters of amino acid neurotransmission in cells cultured from rat cerebral cortex. The cultures were treated with Abeta(25-35) at 3 and 10 microM for 12 and 24 h followed by quantitative analysis of immunofluorescence intensity. In mixed neuronal-glial cell cultures (from P1 rats), Abeta reduced the concentration of system A glutamine transporter 1 (SAT1), by up to 50% expressed relative to the neuronal marker microtubule-associated protein 2 (MAP2) in the same cell. No significant effects were detected on vesicular glutamate transporters VGLUT1 or VGLUT2 in neurons, or on glial system N glutamine transporter 1 (SN1). In neuronal cell cultures (from E18 rats), Abeta(25-35) did not reduce SAT1 immunoreactivity, suggesting that the observed effect depends on the presence of astroglia. The results indicate that Abeta may impair neuronal function and transmitter synthesis, and perhaps reduce excitotoxicity, through a reduction in neuronal glutamine uptake.
Project description:Short fragments of amyloidogenic proteins are widely used as model systems in studies of amyloid formation. Fragment 11-25 of the amyloid beta protein involved in Alzheimer's disease (Abeta11-25) was recently shown to form amyloid fibrils composed of anti-parallel beta-sheets. Interestingly, fibrils grown under neutral and acidic conditions were seen to possess different registries of their inter-beta-strand hydrogen bonds. In an effort to explain the microscopic origin of this pH dependence, we studied Abeta11-25 fibrils using methods of theoretical modeling. Several structural models were built for fibrils at low and neutral pH levels and these were examined in short molecular dynamics simulations in explicit water. The models that displayed the lowest free energy, as estimated using an implicit solvent model, were selected as representative of the true fibrillar structure. It was shown that the registry of these models agrees well with the experimental results. At neutral pH, the main contribution to the free energy difference between the two registries comes from the electrostatic interactions. The charge group of the carboxy terminus makes a large contribution to these interactions and thus appears to have a critical role in determining the registry.
Project description:The interactions of the Alzheimer's ?-amyloid peptide, A?(25-35), with 18:1 (?9-Cis) PC 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), L-?-phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DOPG), and L-?-phosphatidylglycerol (EPG) phospholipid vesicles with and without cholesterol (Ch) are studied by the nitroxide spin probe electron paramagnetic resonance (EPR) method. Two nitroxide spin probes, 2,2,6,6-tetramethyl-piperidin-1-oxyl-4-yl hexadecanoate (TP, TEMPO-Palmitate) and 2-Ethyl-2-(15-methoxy-15-oxopentadecyl)-4,4-dimethyl-3-oxazolidinyloxy (16-DSE), are utilized in the study. TEMPO-Palmitate has the reporting EPR moiety located at the top of this spin probe, while 16-DSE has the reporting EPR moiety located at the tail of the spin probe. These two probes enable us to sample the surface and the middle of the phospholipid bilayer, respectively. All EPR measurements are done above the melting points of all four phospholipids when the bilayer is in the liquid crystal phase, the physiologically relevant phase. Due to non-linear spectral line fitting, the EPR spectral parameters are extracted with high precision. The results show that there are two populations of A?(25-35) and that one of them is located in the hydrophobic phospholipid layer below the hydrophilic headgroup region. The second population appears to be weakly coupled to the surface of the bilayer. Both hydrophobic and electrostatic interactions affect the insertion of A?(25-35) in the bilayer. Also, there is strong evidence for an interaction between cholesterol and A?(25-35), which affects the dielectric and dynamic properties of the bilayer.
Project description:Alzheimer's disease (AD) is a neurodegenerative disease characterized by extracellular deposits of amyloid ? protein (A?) in the brain. The conversion of soluble monomers to amyloid A? fibrils is a complicated process and involves several transient oligomeric species, which are widely believed to be highly toxic and play a crucial role in the etiology of AD. The development of inhibitors to prevent formation of small and midsized oligomers is a promising strategy for AD treatment. In this work, we employ ion mobility spectrometry (IMS), transmission electron microscopy (TEM), and molecular dynamics (MD) simulations to elucidate the structural modulation promoted by two potential inhibitors of A? oligomerization, cucurbituril (CB) and 1,2,3,4,6-penta-O-galloyl-?-d-glucopyranose (PGG), on early oligomer and fibril formation of the A?25-35 fragment. One and two CB molecules bind to A?25-35 monomers and dimers, respectively, and suppress aggregation by remodeling early oligomer structures and inhibiting the formation of higher-order oligomers. On the other hand, nonselective binding was observed between PGG and A?25-35. The interactions between PGG and A?25-35, surprisingly, enhanced the formation of A? aggregates by promoting extended A?25-35 conformations in both homo- and hetero-oligomers. When both ligands were present, the inhibitory effect of CB overrode the stimulatory effect of PGG on A?25-35 aggregation, suppressing the formation of large amyloid oligomers and eliminating the structural conversion from isotropic to ?-rich topologies induced by PGG. Our results provide mechanistic insights into CB and PGG action on A? oligomerization. They also demonstrate the power of the IMS technique to investigate mechanisms of multiple small-molecule agents on the amyloid formation process.
Project description:Previously, we have studied the minimal oligomer size of an aggregate amyloid seed and the mechanism of seed growth with a multilayer beta-sheet model. Under high temperature simulation conditions, our approach can test the stability of possible amyloid forms. Here, we report our study of oligomers of Alzheimer's amyloid beta-peptide (Abeta) fragments 16-22, 16-35, and 10-35 (abbreviated Abeta(16-22), Abeta(16-35), and Abeta(10-35), respectively). Our simulations indicate that an antiparallel beta-sheet orientation is the most stable for the Abeta(16-22), in agreement with a solid state NMR-based model [Balbach, J. J., Ishii, Y., Antzutkin, O. N., Leapman, R. D., Rizzo, N. W., et al. (2000) Biochemistry 39, 13748-13759]. A model with twenty-four Abeta(16-22) strands indicates a highly twisted fibril. Whereas the short Abeta(16-22) and Abeta(24-36) may exist in fully extended form, the linear parallel beta-sheets for Abeta(16-35) appear impossible, mainly because of the polar region in the middle of the 16-35 sequence. However, a bent double-layered hairpin-like structure (called hook) with the polar region at the turn forms parallel beta-sheets with higher stability. An intra-strand salt-bridge (D23-K28) stabilizes the bent hairpin-like hook structure. The bent double-beta-sheet model for the Abeta(10-35) similarly offers oligomer stability.
Project description:BACKGROUND AND PURPOSE: Cilostazol may be effective in dementia associated with a cerebral ischaemia. In this study, we examined whether it exerts beneficial effects on learning and/or memory impairment induced by A?(25-35) in mice, and compared its effects with those of aspirin. EXPERIMENTAL APPROACH: A?(25-35) (9?nmol) was administered to mice i.c.v. Learning and memory behaviour were evaluated by measuring spontaneous alternation in a Y-maze and a step-down type passive avoidance test, on the 5th and 8th days after injection respectively. Levels of lipid peroxidation (malondialdehyde) and cytokines in the frontal cortex and hippocampus were measured 2, 3, 5 and 7 days after the A?(25-35) injection. The effects of repeated administration of cilostazol and aspirin (both at 30 and 100?mg·kg(-1), p.o.) on any changes induced by A?(25-35) were evaluated. KEY RESULTS: Repeated administration of cilostazol significantly attenuated the impairment of spontaneous alternation and the shortened step-down latency induced by A?(25-35) . Aspirin did not show any beneficial effect. A significant increase in the levels of malondialdehyde (MDA) and IL-1? (only measured in hippocampus) was observed 2, 3 and 5 days after the A?(25-35) injection in the frontal cortex and hippocampus. Repeated administration of cilostazol (100?mg·kg(-1)) completely prevented the increase in MDA levels but failed to antagonize the increase in the expression of IL-1? induced by A?(25-35). CONCLUSIONS AND IMPLICATIONS: These results suggest that the protective effect of cilostazol on A?(25-35)-induced memory impairment may be related to oxidative stress in the frontal cortex and the hippocampus.