Katalin Soós

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.

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 β25–35 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 ≈7 nm or its integer multiples were interrupted with pauses. Stepwise dynamics were also observed for amyloid β1–42 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.

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.

Controlled in situ preparation of Aβ(1–42) oligomers from the isopeptide “iso-Aβ(1–42)”, physicochemical and biological characterization

Beta-amyloid (A beta) peptides play a crucial role in the pathology of the neurodegeneration in Alzheimers 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.

Endomorphin-2, an endogenous tetrapeptide, protects against Aβ1–42 in vitro and in vivo

The underlying cause of Alzheimers disease (AD) is thought to be the β‐amyloid aggregates formed mainly by Aβ1–42 peptide. Protective pentapeptides [e.g., Leu‐Pro‐Phe‐Phe‐Asp (LPFFD)] have been shown to prevent neuronal toxicity of Aβ1–42 by arresting and reversing fibril formation. Here we report that an endogenous tetrapeptide, endomorphin‐2 (End‐2, amino acid sequence: YPFF), defends against Aβ 1–42 induced neuromodulatory effects at the cellular level. Although End‐2 does not interfere with the kinetics of Aβ fibrillogenesis according to transmission electron microscopic studies and quasielastic light scattering measurements, it binds to Aβ1–42 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 Aβ1–42 in a dose‐dependent manner, as measured by 3‐(4,5‐dimethylthiazolyl‐2)‐2,‐5‐diphenyltetrazolium bromide (MIT) 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 responseenhancing effect of Aβ1–42. Studies using [d‐Ala (2), N‐Me‐Phe (4), Gly (5)‐ol]‐enkephalin (DAMGO), a µ‐opioid receptor agonist, show that the protective effects of the tetrapeptide are not µ‐receptor modulated. The endogenous tetrapeptide End‐2 mayserve as a lead compound for the drug development in the treatment of AD.—Szegedi, V., Juhász, G., Rózsa, E., Juhász‐Vedres, G., Datki, Z., Fülöp, L., Bozsó, Z., Lakatos, A., Laczkó, I., Farkas, T., Kis, Z., Tóth, G., Soós, K., Zarándi, M., Budai, D., Toldi, J., Penke, B. Endomorphin‐2, an endogenous tetrapeptide, protects against Aβ1–42 in vitro and in vivo. FASEB J. 20, E324–E333 (2006)

Oriented epitaxial growth of amyloid fibrils of the N27C mutant β25–35 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 β25–35 (Aβ25–35) forms a highly oriented, K+-dependent network on mica. Here, we analyzed the properties of Aβ25–35_N27C, the cysteine residue of which may be used for subsequent chemical modifications. We find that Aβ25–35_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 Aβ25–35_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 Aβ25–35_N27C fibrils can therefore be specifically labeled and may be used for constructing nanobiotechnological devices.

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

Alpha-synuclein (alphaSN) plays a major role in numerous neurodegenerative disorders, such as Alzheimers disease and Parkinsons disease. Intracellular inclusions containing aggregated alphaSN have been reported in Alzheimers and Parkinsons affected brains. Moreover, a proteolytic fragment of alphaSN, the so-called non-amyloid component of Alzheimers disease amyloid (NAC) was found to be an integral part of Alzheimers 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 Alzheimers and Parkinsons disease.

Ligand-Induced Flocculation of Neurotoxic Fibrillar Aβ(1-42) by Noncovalent Crosslinking

Aggregation of the amyloid‐β (Aβ) peptides has a pivotal role in Alzheimer’s disease (AD). Small molecules and short peptides/peptidomimetics can exert their full protective effects against Aβ 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 Aβ(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. ζ‐Potential measurements revealed a slightly increased electrostatic stability of the Aβ 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 Aβ fibrils can explain the neuroprotective effects.

Enhanced G-protein activation by a mixture of Aβ(25-35), Aβ(1-40/42) and zinc

β‐Amyloid peptides (Aβs) bind to several G‐protein coupled receptor proteins and stimulate GTPase activity in neurons. In this study we determined the effects of Aβ(1–42), Aβ(1–40), Aβ(25–35) and their mixtures on [35S]GTP binding in rat brain cortical membranes in the absence and presence of zinc. We found that the Aβs alone induced a concentration‐dependent activation of G‐proteins (IC50∼ 10−6 m), while aggregated Aβ fibrils only affected GTP binding at concentrations above 10−5 m. Mixing Aβ(25–35) with Aβ(1–42) or Aβ(1–40) induced a several‐fold increase in GTP‐binding. This potentiation followed a bell shaped curve with a maximum at 50 : 50 ratios. No potentiating effect could be seen by mixing Aβ(1–40) and Aβ(1–42) or highly aggregated Aβs. Zinc had no effect on Aβ(1–40/42) but strongly potentiated the Aβ(25–35) or the mixed peptides‐induced GTP‐binding. Changes in secondary structure accompanied the mixed peptides or the peptide/zinc complexes induced potentiation, revealing that structural alterations are behind the increased biological action. These concentration dependent potentiating effects of zinc and the peptide mixtures could be physiologically important at brain regions where peptide fragments and/or zinc are present at elevated concentrations.

Effect of the beta-sheet-breaker peptide LPFFD on oriented network of amyloid β25-35 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 β25‐35 (Aβ25‐35) fibrils assembled in an oriented array on mica surface. Aβ25‐35 is thought to represent the biologically active, toxic fragment of the full‐length Aβ 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 Aβ25‐35 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 Aβ25‐35 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 Aβ25‐35 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. Copyright