Soluble oligomers from a non-disease related protein mimic Abeta-induced tau hyperphosphorylation and neurodegeneration

Protein aggregation and amyloid accumulation in different tissues are associated with cellular dysfunction and toxicity in important human pathologies, including Alzheimer's disease and various forms of systemic amyloidosis. Soluble oligomers formed at the early stages of protein aggregation have been increasingly recognized as the main toxic species in amyloid diseases. To gain insight into the mechanisms of toxicity instigated by soluble protein oligomers, we have investigated the aggregation of hen egg white lysozyme (HEWL), a normally harmless protein. HEWL initially aggregates into beta-sheet rich, roughly spherical oligomers which appear to convert with time into protofibrils and mature amyloid fibrils. HEWL oligomers are potently neurotoxic to rat cortical neurons in culture, while mature amyloid fibrils are little or non-toxic. Interestingly, when added to cortical neuronal cultures HEWL oligomers induce tau hyperphosphorylation at epitopes that are characteristically phosphorylated in neurons exposed to soluble oligomers of the amyloid-beta peptide. Furthermore, injection of HEWL oligomers in the cerebral cortices of adult rats induces extensive neurodegeneration in different brain areas. These results show that soluble oligomers from a non-disease related protein can mimic specific neuronal pathologies thought to be induced by soluble amyloid-beta peptide oligomers in Alzheimer's disease and support the notion that amyloid oligomers from different proteins may share common structural determinants that would explain their generic cytotoxicities.     

Stabilization,Characterization, and Selective Removal of Cystatin C Amyloid Oligomers

The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers (dimers, trimers, tetramers, decamers, and high molecular weight oligomers). These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry, shape by electron and atomic force microscopy, and, function by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers.     

Binding with nucleic acids or glycosaminoglycans converts soluble protein oligomers to amyloid

Ample evidence suggests that almost all polypeptides can either adopt a native structure (folded or intrinsically disordered) or form misfolded amyloid fibrils. Soluble protein oligomers exist as an intermediate between these two states, and their cytotoxicity has been implicated in the pathology of multiple human diseases. However, the mechanism by which soluble protein oligomers develop into insoluble amyloid fibrils is not clear, and investigation of this important issue is hindered by the unavailability of stable protein oligomers. Here, we have obtained stabilized protein oligomers generated from common native proteins. These oligomers exert strong cytotoxicity and display a common conformational structure shared with known protein oligomers. They are soluble and remain stable in solution. Intriguingly, the stabilized protein oligomers interact preferentially with both nucleic acids and glycosaminoglycans (GAG), which facilitates their rapid conversion into insoluble amyloid. Concomitantly, binding with nucleic acids or GAG strongly diminished the cytotoxicity of the protein oligomers. EGCG, a small molecule that was previously shown to directly bind to protein oligomers, effectively inhibits the conversion to amyloid. These results indicate that stabilized oligomers of common proteins display characteristics similar to those of disease-associated protein oligomers and represent immediate precursors of less toxic amyloid fibrils. Amyloid conversion is potently expedited by certain physiological factors, such as nucleic acids and GAGs. These findings concur with reports of cofactor involvement with disease-associated amyloid and shed light on potential means to interfere with the pathogenic properties of misfolded proteins.     

Growth control of mouse mammary epithelial cells by keratin antibody-targeted liposome containing oligonucleotides antisense to epidermal growth factor receptor

NMuMG cells were incubated with 17beta-estradiol (E)+progesterone (P)+epidermal growth factor (EGF), with or without various types of oligomers (21-mers) to the EGF receptor activity domain (amino acid residues 718 to 724). Sense or antisense oligomers were encapsulated in protein A-bearing liposomes. Uncoupled protein A and unencapsulated sense or antisense oligomers were separated from liposomes on a Sepharose 4B column (the encapsulation efficiency of oligomers in liposome-protein A was 0.8%). The addition of various concentrations of EGF to E+P showed an interaction between them during DNA synthesis (P<0.05). Antisense oligomers (1 microM) decreased DNA synthesis induced by E+P+EGF (65.0% inhibition, P<0.05). Sense oligomers also inhibited DNA synthesis induced by E+P+EGF (P<0.05). However, random-sequence oligomers did not inhibit EGF-induced DNA synthesis. We cannot rule out the possibility that sense oligomers match an unknown functional gene mRNA involved in cell growth, which causes their inhibitory effect. Cells were incubated with a keratin monoclonal antibody and then with dilutions of protein A-bound liposomes containing sense or antisense oligomers in the presence of E+P+EGF. Dose dependent inhibition of DNA synthesis was observed. The encapsulated oligomers in protein A-bound liposomes inhibited DNA synthesis at a 100-fold lower concentration than that of unencapsulated oligomers or the oligomer+liposome mixture. The tyrosine kinase activity domain has an important role in EGF regulation of mammary growth. The effect of a cytokeratin-targeted antibody on DNA synthesis in normal mouse mammary epithelial cells was marginal.     

Aromatic small molecules remodel toxic soluble oligomers of amyloid beta through three independent pathways

In protein conformational disorders ranging from Alzheimer to Parkinson disease, proteins of unrelated sequence misfold into a similar array of aggregated conformers ranging from small oligomers to large amyloid fibrils. Substantial evidence suggests that small, prefibrillar oligomers are the most toxic species, yet to what extent they can be selectively targeted and remodeled into non-toxic conformers using small molecules is poorly understood. We have evaluated the conformational specificity and remodeling pathways of a diverse panel of aromatic small molecules against mature soluble oligomers of the Aβ42 peptide associated with Alzheimer disease. We find that small molecule antagonists can be grouped into three classes, which we herein define as Class I, II, and III molecules, based on the distinct pathways they utilize to remodel soluble oligomers into multiple conformers with reduced toxicity. Class I molecules remodel soluble oligomers into large, off-pathway aggregates that are non-toxic. Moreover, Class IA molecules also remodel amyloid fibrils into the same off-pathway structures, whereas Class IB molecules fail to remodel fibrils but accelerate aggregation of freshly disaggregated Aβ. In contrast, a Class II molecule converts soluble Aβ oligomers into fibrils, but is inactive against disaggregated and fibrillar Aβ. Class III molecules disassemble soluble oligomers (as well as fibrils) into low molecular weight species that are non-toxic. Strikingly, Aβ non-toxic oligomers (which are morphologically indistinguishable from toxic soluble oligomers) are significantly more resistant to being remodeled than Aβ soluble oligomers or amyloid fibrils. Our findings reveal that relatively subtle differences in small molecule structure encipher surprisingly large differences in the pathways they employ to remodel Aβ soluble oligomers and related aggregated conformers.     

Cross-linking of native myosin forms oligomers of myosin heavy chain dimers

Chemical cross-linking of native myosin in 0.6 M NaCl with p-phenylene bis maleimide or glutaraldehyde resulted in rapid formation of myosin heavy chain dimers and their oligomers. Monomers and odd-number oligomers disappeared after the prolonged treatment with these reagents. When denatured myosin was cross-linking, myosin heavy chain monomers and odd-number oligomers remained after the prolonged treatment, although dimers and their even-number oligomers were abundant. For high molecular weight markers, myosin heavy chain oligomers formed from denatured myosin with glutaraldehyde or p-phenylene bis maleimide are recommended.     

Uranyl ion-catalyzed synthesis of several 8-substituted 2'-5'-oligoadenylates, and their properties

We have synthesized 2'-5' linked oligomers from 8-substituted adenosine-5'-phosphorimidazolides using uranyl ion catalyst. 8-amino derivative, as highly susceptible to hydrolysis, gave short chained oligomers in a low yield, while the rest of 8-substituted or unsubstituted derivatives gave the corresponding oligomers in high yields. Properties of 8-substituted 2'-5' oligomers were studied applying spectrometer and through enzymatic digestion.     

Fibrillogenic oligomers of human cystatin C are formed by propagated domain swapping

Cystatin C and the prion protein have been shown to form dimers via three-dimensional domain swapping, and this process has also been hypothesized to be involved in amyloidogenesis. Production of oligomers of other amyloidogenic proteins has been reported to precede fibril formation, suggesting oligomers as intermediates in fibrillogenesis. A variant of cystatin C, with a Leu68-->Gln substitution, is highly amyloidogenic, and carriers of this mutation suffer from massive cerebral amyloidosis leading to brain hemorrhage and death in early adulthood. This work describes doughnut-shaped oligomers formed by wild type and L68Q cystatin C upon incubation of the monomeric proteins. Purified oligomers of cystatin C are shown to fibrillize faster and at a lower concentration than the monomeric protein, indicating a role of the oligomers as fibril-assembly intermediates. Moreover, the present work demonstrates that three-dimensional domain swapping is involved in the formation of the oligomers, because variants of monomeric cystatin C, stabilized against three-dimensional domain swapping by engineered disulfide bonds, do not produce oligomers upon incubation under non-reducing conditions. Redox experiments using wild type and stabilized cystatin C strongly suggest that the oligomers, and thus probably the fibrils as well, are formed by propagated domain swapping rather than by assembly of domain-swapped cystatin C dimers.     

Preparation and characterization of neurotoxic tau oligomers

Tau aggregation is a pathological hallmark of Alzheimer's disease, Parkinson's disease, and many other neurodegenerative disorders known as tauopathies. Tau aggregates take on many forms, and their formation is a multistage process with intermediate stages. Recently, tau oligomers have emerged as the pathogenic species in tauopathies and a possible mediator of amyloid-β toxicity in Alzheimer's disease. Here, we use a novel, physiologically relevant method (oligomer cross-seeding) to prepare homogeneous populations of tau oligomers and characterize these oligomers in vitro. We show that both Aβ and α-synuclein oligomers induce tau aggregation and the formation of β-sheet-rich neurotoxic tau oligomers.     

Oligodeoxynucleotide hybridization inhibition of HIV and influenza virus.

Synthetic oligodeoxyribonucleotides have been found to inhibit the replication and expression of HIV and of Influenza virus in growing tissue cultures. Nuclease resistant oligomers have been more effective than unmodified oligomers. In mouse and rat studies, labeled oligomers, on intravenous or intraperitoneal injection, leave the blood stream rapidly and enter numerous organs of the body. The half life in these organs is 24-48 hours in the case of the phosphorothioate oligomers.     

Optimum conditions for the precipitation of chitosan oligomers with DP 5–7 in concentrated hydrochloric acid at low temperature

Chitosan was partially hydrolysed with 35% hydrochloric acid for 2 h at 80°C and the hydrolysate stored at −20°C after dilution with water to precipitate higher molecular weight (MW) chitosan oligomers. When the hydrolysate was not diluted with water, no precipitate was formed but 7.3% chitosan oligomers were precipitated at a dilution ratio of 1.0 (ml water/ml hydrolysate). The time for precipitation was not significantly changed after storing the hydrolysate at −20°C for 1 day. In addition, the precipitation yield was not significantly influenced by the concentration of HCl used for the hydrolysis except at less than 5.0 (ml HCl/g chitosan). However, the yield of precipitated oligomers changed with partial hydrolysis time. For 0.5 and 2 h hydrolysis, 10.1 and 7.3% of the oligomers were precipitated, respectively, but only 3.1% of the oligomers were obtained after a 4 h reaction. When methanol was added to the hydrolysate, the precipitation yield increased up to 70% but the amounts of lower MW chitosan oligomers in the precipitated oligomers also increased with the increase of higher MW. The precipitated oligomers were mainly composed of pentamers and hexamers.     

Natural and Phosphorothioate-Modified Oligodeoxyribonucleotides Exhibit a Non-Random Cellular Distribution

Abstract

Addition of specific antisense oligomers to LTK- cells infected with HSV-1 has been shown to decrease viral production (1,8). We have investigated the cellular components that contain these oligomers and a phosphorothioate derivative by Normarski light microscopy and gel analysis of sucrose gradient cell fractions. Fractionation analysis suggests that these oligomers are distributed throughout the cell in a non-random manner and gel analysis suggest that intact oligomers are not equally distributed in the cytosol, nuclear or membrane component. Information about the cellular location of antisense oligomers should aid in the understanding of their antiviral effect and in the design of more effective oligonucleotide derivatives as potential antiviral agents.     

Biological activity of oligonucleotide-poly(L-lysine) conjugates: mechanism of cell uptake

We have previously shown that antisense oligomers linked to poly(L-lysine) (PLL) exhibit antiviral properties against vesicular stomatitis virus (VSV) at concentrations lower than 1 microM. The conjugation to PLL provides an interesting alternative to natural or neutral oligomers to increase the biological effects of antisense oligomers. The internalization pathway of oligomer-PLL conjugates as compared to unconjugated oligomers has been studied in L929 cells. In parallel to their enhanced antiviral activity, PLL increases greatly the uptake of fluorescently tagged oligomers. This internalization follows a classical endocytic pathway and the oligomer has to be cleaved from PLL in the cell to exhibit an antiviral effect.     

Preparation of oligodeoxyribonucleoside methylphosphonates on a polystyrene support.

An efficient procedure is described for synthesizing deoxyribonucleoside methylphosphonates on polystyrene polymer supports which involves condensing 5'-dimethoxytrityldeoxynucleoside 3'-methylphosphonates. The oligomers are removed from the support and the base protecting groups hydrolyzed by treatment with ethylenediamine in ethanol, which avoids hydrolysis of the methylphosphonate linkages. Two types of oligomers were synthesized: those containing only methylphosphonate linkages, d-Np(Np)nN, and those which terminate with a 5' nucleotide residue, dNp (Np)nN. The latter oligomers can be phosphorylated by polynucleotide kinase, and are separated by polyacrylamide gel electrophoresis according to their chain length. Piperdine randomly cleaves the oligomer methylphosphonate linkages and generates a series of shorter oligomers whose number corresponds to the length of the original oligomer. Apurinic sites introduced by acid treatment spontaneously hydrolyze to give oligomers which terminate with free 3' and 5' OH groups. These reactions may be used to characterize the oligomers.     

Preparation of trimers and tetramers of mixed sequence oligodeoxynucleoside methylphosphonates and assignment of configurations at the chiral phosphorus.

Synthesis of stereoregular DNA methylphosphonates has been accomplished for homo-oligomers, but remains a formidable problem for oligomers of a defined antisense target sequence. In this work, four trimer and tetramer deoxynucleoside methylphosphonates of mixed sequence (dACA, dCCAA, dAGGG, and dGCAT) were prepared by block coupling of diastereomerically pure dimers with either monomers or other diastereomerically pure dimers. These oligomers were separated chromatographically into individual diastereomers, and the configurations of the chiral methylphosphonate linkages were assigned. Three types of methods were used to assign configuration of a new methylphosphonate linkage: preparation of the same diastereomer through multiple synthetic pathways, base hydrolysis, and acid hydrolysis. Hydrolysis of the diastereomerically pure oligomers into component dimers and monomers was followed by chromatographic comparison with control dimers of known configuration. In all cases studied, oligomers with R configurations displayed faster elution from silica gel than did oligomers with the respective S configuration. NMR spectra of individual diastereomers of dACA were studied, revealing characteristic differences in chemical shifts which may prove useful in configurational assignments of longer oligomers. Thus, these data provide a methodological basis for synthesis and configurational assignment of longer methylphosphonate oligomers to use as antisense probes.     

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bovine serum albumin oligomers produced by lipid peroxidation.

The oligomers of bovine serum albumin were produced by controlled reaction with peroxidizing linoleic acid to examine their possible utility as calibration proteins insodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polymerization was effected in reaction mixtures containing linoleic acid undergoing peroxidation in the presence of ascorbic acid, and conditions that yield soluble oligomers with a wide molecular weight distribution were established. The interaction of these soluble oligomers with sodium dodecyl sulfate exhibited a binding isotherm indistinguishable from that obtained with bovine serum albumin. Furthermore, sodium dodecy sulfate-polyacrylamide gel electrophoresis of the albumin oligomers conformed to the empirical relation of molecular weight to mobility that pertains to the use of these oligomers as standard molecular weight markers.     

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. I. Properties of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) monomers.

Oligomers and monomers of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers were 14.8 +/- 1.7 and 19.2 +/- 1.4 nm, respectively. Twenty-four-hour incubation with Emulgen 913 at 4 degrees C at a molar ratio of 1:100 led to the monomerization of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers, the particle sizes diminishing to 6.1 +/- 1.3 and 5.2 +/- 0.4 nm, respectively. The thermal stability of NADPH-cytochrome P450 reductase monomers was the same as that of oligomers, whereas cytochrome P450 LM2 monomers were less thermostable than oligomers and cytochrome P450 in microsomes. Similar to cytochrome P450 LM2 oligomers and the microsomal hemoprotein, cytochrome P450 LM2 monomers formed complexes with type I and II substrates, but with Kd values higher than those of microsomes and cytochrome P450 LM2 oligomers. Kinetic parameters (Vmax and Km) of H2O2- and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of cytochrome P450 LM2 oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in cytochrome P450 LM2 oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein.     

Conformational studies of oligomeric oxetane-based dipeptide isosteres derived from L-rhamnose or D-xylose.

Conformational investigations have been undertaken on oligomers (dimers, tetramers, hexamers) of five closely related oxetane-based dipeptide isosteres. All the oligomers were subjected to a range of studies by NMR, FT-IR and CD spectroscopy. The oligomers derived from methyl 2,4-anhydro-5-azido-3-O-tert-butyldimethylsilyl-5-deoxy-L-rhamnonate 'monomer' all exhibited evidence of ordered conformations in chloroform and 2,2,2-trifluoroethanol (TFE) solution. 5-Acetamido and N-methylamide derivatives of the L-rhamnonate 'monomer', along with a 'dimer' lacking silyl protection at C-3, were synthesized to ascertain the role of intramolecular interactions. This led to the conclusion that, for the L-rhamnonate oligomers, steric interactions govern the conformational preference observed. The equivalent silyl-protected D-lyxonate oligomers gave ordered CD spectra in TFE solution, but NMR and FT-IR spectroscopy in chloroform solution suggested an irregular, non-hydrogen bonded system. The remaining silyl-protected 6-deoxy-L-altronate, 6-deoxy-D-gulonate and D-fuconate oligomers appear to be characterized by their lack of ordered conformation in TFE and chloroform solution.     

Exosomal cell-to-cell transmission of alpha synuclein oligomers

ABSTRACT: BACKGROUND: Aggregation of alpha-synuclein (alphasyn) and resulting cytotoxicity is a hallmark of sporadic and familial Parkinson's disease (PD) as well as dementia with Lewy bodies, with recent evidence implicating oligomeric and pre-fibrillar forms of alphasyn as the pathogenic species. Recent in vitro studies support the idea of transcellular spread of extracellular, secreted alphasyn across membranes. The aim of this study is to characterize the transcellular spread of alphasyn oligomers and determine their extracellular location. RESULTS: Using a novel protein fragment complementation assay where alphasyn is fused to non-bioluminescent amino-or carboxy-terminus fragments of humanized Gaussia Luciferase we demonstrate here that alphasyn oligomers can be found in at least two extracellular fractions: either associated with exosomes or free. Exosome-associated alphasyn oligomers are more likely to be taken up by recipient cells and can induce more toxicity compared to free alphasyn oligomers. Specifically, we determine that alphasyn oligomers are present on both the outside as well as inside of exosomes. Notably, the pathway of secretion of alphasyn oligomers is strongly influenced by autophagic activity. CONCLUSIONS: Our data suggest that alphasyn may be secreted via different secretory pathways. We hypothesize that exosome-mediated release of alphasyn oligomers is a mechanism whereby cells clear toxic alphasyn oligomers when autophagic mechanisms fail to be sufficient. Preventing the early events in alphasyn exosomal release and uptake by inducing autophagy may be a novel approach to halt disease spreading in PD and other synucleinopathies.     

Human cofilin forms oligomers exhibiting actin bundling activity.

Human cofilin possesses the tendency for self-association, as indicated by the rapid formation of dimers and oligomers when reacted with water-soluble carbodiimide, Ellman's reagent, or glutathione disulfide. Intermolecular disulfide bonds involve Cys(39) and probably Cys(147) of two adjacent cofilin units. The disulfide-linked dimers and oligomers exhibit a biological activity distinct from the monomer. While monomeric cofilin decreased viscosity and light-scattering of F-actin solutions, dimers and oligomers caused an increase in viscosity and light scattering. Electron microscopy revealed that cofilin oligomers induce the formation of highly ordered actin bundles with occasionally blunt ends similar to actin-cofilin rods observed in cells under oxidative stress. Bundling activity of the disulfide-linked oligomers could be completely reversed into severing activity by dithiothreitol. Formation of cofilin oligomers occurred also in the presence of actin at pH 8, but not at pH 6.6, and was significantly enhanced in the presence of phosphatidylinositol 4,5-bisphosphate. Our data are consistent with the idea that cofilin exists in two forms in vivo also: as monomers exhibiting the known severing activity and as oligomers exhibiting actin bundling activity. However, stabilization of cofilin oligomers in cytoplasm is probably achieved not by disulfide bonds but by a local increase in cofilin concentration and/or binding of regulatory proteins.