Epitaxial assembly dynamics of mutant amyloid ß25-35_N27C fibrils explored with time-resolved scanning force microscopy.

Amyloid ß25-35 (Aß25-35) is a toxic fragment of Alzheimer's beta peptide. We have previously shown that Aß25-35 fibrils form a trigonally oriented network on mica by epitaxial growth mechanisms. Chemical reactivity can be furnished to the fibril by introducing a cysteine residue (Aß25-35_N27C) while maintaining oriented assembly properties. Previously we have shown that fibril binding to mica is strongly influenced by KCl concentration. In the present work we explored the kinetics of epitaxial assembly of the mutant fibrils at different peptide and KCl concentrations by using in situ time-resolved AFM. We measured the length of Aß25-35_N27C fibrils as a function of time. Increasing free peptide concentration enhanced fibril growth rate, and the critical peptide concentration of fibril assembly was 3.92µM. Increasing KCl concentration decreased the number of fibrils bound to the mica surface, and above 20mM KCl fibril formation was completely abolished even at high peptide concentrations. By modulating peptide and KCl concentrations in the optimal ranges established here the complexity of the Aß25-35_N27C network can be finely tuned.

Effect of the beta-sheet-breaker peptide LPFFD on oriented network of amyloid ß25-35 fibrils.

Amyloid fibrils are self-associating filamentous structures deposited in extracellular tissue in various neurodegenerative and protein misfolding disorders. It has been shown that beta-sheet-breaker (BSB) peptides may interfere with amyloid fibril assembly. Although BSB peptides are prospective therapeutic agents in amyloidosis, there is ambiguity about the mechanisms and generality of their action. In the present work we analyzed the effect of the BSB peptide LPFFD on the growth kinetics, morphologic, and mechanical properties of amyloid ß25-35 (Aß25-35) fibrils assembled in an oriented array on mica surface. Aß25-35 is thought to represent the biologically active, toxic fragment of the full-length Aß peptide. Growth kinetics and morphologic features were analyzed using in situ atomic force microscopy in the presence of various concentrations of LPFFD. We found that the addition of LPFFD only slightly altered the assembly kinetics of Aß25-35 fibrils. Already formed fibrils did not disassemble in the presence of high concentrations of LPFFD. The mechanical stability of the fibrils was explored with force spectroscopy methods. The nanomechanical behavior of Aß25-35 fibrils is characterized by the appearance of force staircases which correspond to the force-driven unzipping and dissociation of several protofilaments. In the presence of LPFFD single-plateau force traces dominated. The effects of LPFFD on Aß25-35 fibril assembly and stability suggest that inter-protofilament interactions were slightly weakened. Complete disassembly of fibrils, however, was not observed. Thus, under the conditions explored here, LPFFD may not be considered as a BSB peptide with generalized beta-sheet breaking properties.

Controlled in situ preparation of A beta(1-42) oligomers from the isopeptide "iso-A beta(1-42)", physicochemical and biological characterization.

Beta-amyloid (A beta) peptides play a crucial role in the pathology of the neurodegeneration in Alzheimer's disease (AD). Biological experiments (both in vitro and animal model studies of AD) require synthetic A beta peptides of standard quality, aggregation grade, neurotoxicity and water solubility. The synthesis of A beta peptides has been difficult, owing to their hydrophobic character, poor solubility and high tendency for aggregation. Recently an isopeptide precursor (iso-A beta(1-42)) was synthesized by Fmoc-chemistry and transformed at neutral pH to A beta(1-42) by O-->N acyl migration in a short period of time. We prepared the same precursor peptide using Boc-chemistry and studied the transformation to A beta(1-42) by acyl migration. The peptide conformation and aggregation processes were studied by several methods (circular dichroism, atomic force and transmission electron microscopy, dynamic light scattering). The biological activity of the synthetic A beta(1-42) was measured by ex vivo (long-term potentiation studies in rat hippocampal slices) and in vivo experiments (spatial learning of rats). It was proven that O-->N acyl migration of the precursor isopeptide results in a water soluble oligomeric mixture of neurotoxic A beta(1-42). These oligomers are formed in situ just before the biological experiments and their aggregation grade could be standardized.

Differences between normal and alpha-synuclein overexpressing SH-SY5Y neuroblastoma cells after Abeta(1-42) and NAC treatment.

Alpha-synuclein (alphaSN) plays a major role in numerous neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease. Intracellular inclusions containing aggregated alphaSN have been reported in Alzheimer's and Parkinson's affected brains. Moreover, a proteolytic fragment of alphaSN, the so-called non-amyloid component of Alzheimer's disease amyloid (NAC) was found to be an integral part of Alzheimer's dementia related plaques. Despite the extensive research on this topic, the exact toxic mechanism of alphaSN remains elusive. We have taken the advantage of an alphaSN overexpressing SH-SY5Y cell line and investigated the effects of classical apoptotic factors (e.g. H(2)O(2), amphotericin B and ruthenium red) and aggregated disease-related peptides on cell viability compared to wild type neuroblastoma cells. It was found that alphaSN overexpressing cells are more sensitive to aggregated peptides treatment than normal expressing counterparts. In contrast, cells containing elevated amount of alphaSN were less vulnerable to classical apoptotic stressors than wild type cells. In addition, alphaSN overexpression is accompanied by altered phenotype, attenuated proliferation kinetics, increased neurite arborisation and decreased cell motility. Based on these results, the alphaSN overexpressing cell lines may represent a good and effective in vitro model for Alzheimer's and Parkinson's disease.

Ligand-induced flocculation of neurotoxic fibrillar Abeta(1-42) by noncovalent crosslinking.

Aggregation of the amyloid-beta (Abeta) peptides has a pivotal role in Alzheimer's disease (AD). Small molecules and short peptides/peptidomimetics can exert their full protective effects against Abeta within a short time-frame, but the exact mechanism of action is unclear. Time-dependent NMR spectroscopic binding and replacement experiments were carried out for peptide LPFFD and thioflavine T (ThT) on neurotoxic fibrillar Abeta(1-42), which revealed transient binding behavior for both compounds, and complex time-dependent features in the replacement experiments. The results of particle size measurements through the use of diffuse light-scattering and transmission electron microscopy support the conclusions that the studied ligands induced interfibrillar association on a short timescale, which explains the NMR spectroscopic binding and replacement results. zeta-Potential measurements revealed a slightly increased electrostatic stability of the Abeta fibrils upon ligand binding; this suggests that the interfibrillar assembly is driven by specific noncovalent cross-linking interactions. A specific surface and mobility decrease due to the ligand-induced flocculation of the Abeta fibrils can explain the neuroprotective effects.

Aggregation of Abeta(1-42) in the presence of short peptides: conformational studies.

CD and infrared spectroscopic studies were performed on (i) the inhibitory effects of equimolar quantities of LPFFD-OH and LPYFD-NH(2) on the time-dependent aggregation of amyloid beta-protein (Abeta) (1-42) and (ii) the beta-sheet-breaker effects of two-fold molar excess of the pentapeptides on aggregated Abeta(1-42) aged 1 week. The data obtained from the time-dependent studies demonstrated that LPFFD-OH did not significantly influence, whereas LPYFD-NH(2) exerted some inhibitory effect on the aggregation of Abeta(1-42). When added to a solution of Abeta(1-42) aged 1 week, LPFFD-OH accelerated, while LPYFD-NH(2) delayed, but did not prevent further fibrillogenesis. The difference in the effects of these two pentapeptides on the aggregational profile of Abeta(1-42) is probably due to the difference in their conformational preferences: LPFFD-OH adopts a beta-turn and extended structures, while LPYFD-NH(2) adopts a prevailing beta-turn conformation.

Oriented epitaxial growth of amyloid fibrils of the N27C mutant beta 25-35 peptide.

Amyloid fibrils are present in the extracellular space of various tissues in neurodegenerative and protein misfolding diseases. Amyloid fibrils may be used in nanotechnology applications, because of their self-assembly properties and stability, if their growth and orientation can be controlled. Recently, we have shown that amyloid beta 25-35 (A beta 25-35) forms a highly oriented, K(+)-dependent network on mica. Here, we analyzed the properties of A beta 25-35_N27C, the cysteine residue of which may be used for subsequent chemical modifications. We find that A beta 25-35_N27C forms epitaxially growing fibrils on mica, which evolve into a trigonally oriented branched network. The binding is apparently more sensitive to cation concentration than that of the wild-type peptide. By nanomanipulating A beta 25-35_N27C fibrils with a gold-coated AFM tip, we show that the sulfhydryl of Cys27 is reactive and accessible from the solution. The oriented network of A beta 25-35_N27C fibrils can therefore be specifically labeled and may be used for constructing nanobiotechnological devices.

Stepwise dynamics of epitaxially growing single amyloid fibrils.

The assembly mechanisms of amyloid fibrils, tissue deposits in a variety of degenerative diseases, is poorly understood. With a simply modified application of the atomic force microscope, we monitored the growth, on mica surface, of individual fibrils of the amyloid beta25-35 peptide with near-subunit spatial and subsecond temporal resolution. Fibril assembly was polarized and discontinuous. Bursts of rapid (up to 300-nm(-1)) growth phases that extended the fibril by approximately 7 nm or its integer multiples were interrupted with pauses. Stepwise dynamics were also observed for amyloid beta1-42 fibrils growing on graphite, suggesting that the discontinuous assembly mechanisms may be a general feature of epitaxial amyloid growth. Amyloid assembly may thus involve fluctuation between a fast-growing and a blocked state in which the fibril is kinetically trapped because of intrinsic structural features. The used scanning-force kymography method may be adapted to analyze the assembly dynamics of a wide range of linear biopolymers.

Synthesis of Abeta[1-42] and its derivatives with improved efficiency.

It has been proved that the principal component of senile plaques is aggregates of beta-amyloid peptide (Abeta) in cases of one of the most common forms of age-related neurodegenerative disorders, Alzheimer's disease (AD). Although the synthetic methods for the synthesis of Abeta peptides have been developed since their first syntheses, Abeta[1-42] is still problematic to prepare. The highly hydrophobic composition of Abeta[1-42] results in aggregation between resin-bound peptide chains or intrachain aggregation which leads to a decrease in the rates of deprotection and repetitive incomplete coupling reactions during 9-flurenylmethoxycarbonyl (Fmoc) synthesis. In order to avoid aggregation and/or disrupt internal aggregation during stepwise Fmoc solid phase synthesis and to improve the quality of crude products, several attempts have been made. Since highly pure Abeta peptides in large quantities are used in biological experiments, we wanted to develop a method for a rational synthesis of human Abeta[1-42] with high purity and adequate yield. This paper reports a convenient methodology with a novel solvent system for the synthesis of Abeta[1-42], its N-terminally truncated derivatives Abeta[4-42] and Abeta[5-42], and Abeta[1-42] labeled with 7-amino-4-methyl-3-coumarinylacetic acid (AMCA) at the N-terminus using Fmoc strategy. The use of 10% anisole in Dimethylformamide/Dichloromethane (DMF/DCM) can substantially improve the purity and yield of crude Abeta[1-42] and has been shown to be an optimal coupling condition for the synthesis of Abeta[1-42]. Anisole is a cheap and simple aid in the synthesis of 'difficult sequences' where other solvents are less successful in the prevention of aggregation during the synthesis.

Endomorphin-2, an endogenous tetrapeptide, protects against Abeta1-42 in vitro and in vivo.

The underlying cause of Alzheimer's disease (AD) is thought to be the beta-amyloid aggregates formed mainly by Abeta1-42 peptide. Protective pentapeptides [e.g., Leu-Pro-Phe-Phe-Asp (LPFFD)] have been shown to prevent neuronal toxicity of Abeta1-42 by arresting and reversing fibril formation. Here we report that an endogenous tetrapeptide, endomorphin-2 (End-2, amino acid sequence: YPFF), defends against Abeta1-42 induced neuromodulatory effects at the cellular level. Although End-2 does not interfere with the kinetics of Abeta fibrillogenesis according to transmission electron microscopic studies and quasielastic light scattering measurements, it binds to Abeta1-42 during aggregation, as revealed by tritium-labeled End-2 binding assay and circular dichroism measurements. The tetrapeptide attenuates the inhibitory effect on cellular redox activity of Abeta1-42 in a dose-dependent manner, as measured by 3-(4,5-dimethylthiazolyl-2)-2,-5-diphenyltetrazolium bromide (MTT) assay. In vitro and in vivo electrophysiological experiments show that End-2 also protects against the field excitatory postsynaptic potential attenuating and the NMDA-evoked response-enhancing effect of Abeta1-42. Studies using [D-Ala (2), N-Me-Phe (4), Gly (5)-ol]-enkephalin (DAMGO), a mu-opioid receptor agonist, show that the protective effects of the tetrapeptide are not mu-receptor modulated. The endogenous tetrapeptide End-2 may serve as a lead compound for the drug development in the treatment of AD.

In vitro model of neurotoxicity of Abeta 1-42 and neuroprotection by a pentapeptide: irreversible events during the first hour.

The cell biology of Alzheimer's disease (AD) is characterized mainly by the neurodegeneration caused by the beta-amyloid (Abeta) peptides and by the formation of neurofibrillary tangles. The initial events of neurodegeneration in the brain tissue include synaptic dysfunction and axonopathy. Abeta-induced axonopathy and neurite degeneration were studied in vitro on differentiated human-derived neurotypic SH-SY5Y cells. Different methods were used to investigate the mechanism of action of aggregated Abeta on neuroblastoma cells. Abeta 1-42 aggregated for 1 h induced irreversible changes in the neurite morphology. Change of tau hyperphosphorylation and cell viability (cytoplasmic redox state and active membrane uptake) was irreversible during the first hour after the addition of Abeta 1-42 to the cells. These rapid events indicate that Abeta might induce neurodegeneration even at an early stage of Abeta-cell contact. A novel pentapeptide LPYFD-amide, an analog of Soto's LPFFD, significantly decreased neurite degeneration, tau aggregation, and cell viability reduction induced by Abeta 1-42.

Beta-amyloid-derived pentapeptide RIIGLa inhibits Abeta(1-42) aggregation and toxicity.

Pr-IIGL(a), a derivative of the tetrapeptide beta-amyloid 31-34 (Abeta(31-34)), exerts controversial effects: it is toxic in a neuroblastoma culture, but it protects glial cells from the cytotoxic action of Abeta(1-42). For an understanding of this phenomenon, a new pentapeptide, RIIGL(a) was synthetized, and both compounds were studied by different physicochemical and biological methods. Transmission electron microscopic (TEM) studies revealed that Pr-IIGL(a) forms fibrillar aggregates, whereas RIIGL(a) does not form fibrils. Congo red binding studies furnished the same results. Aggregated Pr-IIGL(a) acts as a cytotoxic agent in neuroblastoma cultures, but RIIGL(a) does not display inherent toxicity. RIIGL(a) co-incubated with Abeta(1-42) inhibits the formation of mature amyloid fibres (TEM studies) and reduces the cytotoxic effect of fibrillar Abeta(1-42). These results indicate that RIIGL(a) is an effective inhibitor of both the aggregation and the toxic effects of Abeta(1-42) and can serve as a lead compound for the design of novel neuroprotective peptidomimetics.

Enhancement of NMDA responses by beta-amyloid peptides in the hippocampus in vivo.

The effects of Alzheimer's disease-related beta-amyloid (Abeta) peptides on the N-methyl-D-aspartate (NMDA)-evoked cell firing rate were studied in hippocampal CA1 neurons of the rat. Extracellular single-unit recordings were combined with iontophoretic applications that allowed quantitative analyses of the interactions between Abeta peptides and NMDA receptor-mediated events in vivo. The NMDA responses were significantly increased both by the full length Abeta1-42 and by its model fragment Abeta25-35. Enhancements of the NMDA responses by the Abeta peptides lasted about 15 min and were irreversible. The effects of Abeta25-35 were prevented by the pentapeptide Lys-Leu-Val-Gly-Phe-amide (KLVGF) and were not evoked when its reversed sequence (Abeta35-25) was applied.

Method for measuring neurotoxicity of aggregating polypeptides with the MTT assay on differentiated neuroblastoma cells.

Reliable in vitro assays are essential for study of the effects of neurotoxic compounds such as beta-amyloid peptides (Abeta). The MTT assay has been used in cultures of different cells, e.g. SH-SY5Y neuroblastoma cells, for the quantitative measurement of Abeta toxicity. In our laboratory differentiated SH-SY5Y cells were used in the MTT assay. Cell differentiation with 10 microM all-trans-retinoic acid resulted in a constant cell number. The cells possess highly developed neurites and exhibit high sensitivity against Abeta. Owing to the constant cell number in differentiated SH-SY5Y cultures the decrease of the redox activity is directly proportional to the neurotoxicity of the substances, no correction is needed. The results of the MTT assay of Abeta peptides on differentiated SH-SY5Y cells displayed a good correlation also with the in vivo results. The present experiments reveal an effective assay for the study of potentially neurotoxic compounds.

Beta-amyloid peptide-induced blood-brain barrier disruption facilitates T-cell entry into the rat brain.

Activated T-lymphocytes can migrate through the blood-brain barrier (BBB) and are able to invade the central nervous system (CNS). In the present study, we investigated whether disruption of the BBB leads to enhanced T-cell migration into the CNS. Amyloid-beta peptide 25-35 (A beta) or tumor necrosis factor-alpha (TNFalpha) were administered into the right common carotid artery of adult male Wistar rats. The agents were administered either alone, or were followed by a cell suspension of exogenously activated T-cells. Rats of other groups received activated or non-stimulated T-lymphocytes only. Sagittal brain sections were analyzed with immunohistochemistry of CD3 to reveal the presence of T-lymphocytes within the CNS parenchyma. Administration of activated T-cells alone led to T-cell migration into the brain. Infusion of either substances (A beta or TNFalpha) resulted in T-cell invasion of the CNS even when no exogenous T-cells were added. Infusion of either of the agents together with T-lymphocytes generated a more intense T-lymphocyte migration than in the other groups. Electron microscopic analysis and Evans-blue extravasation studies confirmed parallel disruption of the BBB. Our study demonstrates that A beta and TNFalpha induce enhanced T-lymphocyte migration towards the brain. This effect may be attributed at least partly to dysfunctioning of the BBB, but other mechanisms are also possible.