JP4876224B2 - Amyloid β oligomer and method for producing and using the same - Google Patents

Description

  The present invention relates to a soluble toxic assembly (oligomer) of amyloid β and methods for making and using the same.

  Amyloid β (hereinafter also referred to as “Aβ”) is a major protein component of plaque observed in Alzheimer's disease (hereinafter also referred to as “AD”) patients, and is also known to be a plaque component in Down's syndrome.

  Recently, soluble Aβ assemblies (also referred to as “Aβ oligomers”) (Non-Patent Document 1), protofibrils (Non-Patent Documents 2 and 3) or Aβ-derived diffusible ligands (also referred to as “ADDLs”) ( Non-Patent Document 4 and Patent Document 1) are attracting attention due to the strength of damage to neuronal function or the inductive strength of neurodegeneration (Non-Patent Documents 4 to 10).

  In addition to increasing soluble Aβ levels in the brain and cerebrospinal fluid of AD patients (Non-Patent Documents 11 to 13), disruption of cognitive function in vivo and long-term synergism by natural Aβ oligomers (Non-Patent Documents) 6, 14) suggest a pathological relevance of these soluble Aβ assemblies. How soluble Aβ assembly damages neurons is unclear, but is associated with specific binding to target molecules on neuronal membranes (Non-Patent Document 15) and perturbation of ionic homeostasis Several possibilities have been proposed, such as the ubiquitous destruction of the plasma membrane (Non-Patent Document 16).

  It has been reported that soluble Aβ assemblies are formed in vitro. For example, ADDL is contained in a supernatant after incubating an Aβ solution diluted with a medium at about 4 ° C. for about 2 hours to about 48 hours, and centrifuging at about 14,000 g at about 4 ° C. (Patent Document 1). ). ADDLs have molecular weights of about 26 to about 28 kD in non-denaturing gel electrophoresis, about 22 to about 24 kD or about 18 to about 19 kD in 15% polyacrylamide gel electrophoresis (PAGE), and are observed by atomic force microscopy. It is described that the size is about 4.7 to about 6.2 nm (Patent Document 1).

  In addition, it is described that a neurotoxic oligomer crosslinked with tyrosine can be obtained by incubating Aβ with hydrogen peroxide and peroxidase in a borate buffer solution at 37 ° C. for 5 days (Patent Document 2).

  Arctic Aβ, a hereditary variant, has a tendency to form a neurotoxic soluble Aβ assembly (Non-Patent Documents 10, 17, 18). The present inventors have recently observed that arctic Aβ forms a precipitable assembly in the presence of GM1 ganglioside in the same manner as wild-type Aβ (Non-patent Documents 19 and 20).

Nerve growth factor (NGF) plays an important role in the differentiation and function of a wide range of cell types and the low affinity NGF receptor (p75 ^NTR ) plays a dual role in cell survival and death. Evidence is accumulated. And it is suggested that the toxicity of insoluble Aβ assembly appears through the association with p75 ^NTR (Non-patent Documents 28 and 29), and that ADDLs can alter NGF-mediated signal transduction in cultured cells (Non-patent Document 8). Yes.

Special table 2001-501972 (WO98 / 33815) Special table 2004-501204 (WO2002 / 000245)

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  An object of the present invention is to provide a novel Aβ assembly useful for the study, prevention and treatment of amyloid-related diseases such as AD, and a method for producing the same. Furthermore, an object of the present invention is to provide methods, compounds and the like for the study, prevention and treatment of amyloid-related diseases using such Aβ assemblies.

  We have developed a new soluble toxic Aβ assembly (soluble toxic) that is more rapidly and more toxic from wild-type or hereditary variant Aβ, in particular arctic Aβ, in the presence of a specific concentration of GM1 ganglioside. The present invention was completed by finding that amyloid β (hereinafter also referred to as “TAβ”) was formed.

The present invention
[1] A soluble toxic amyloid β assembly formed in vitro in the presence of GM1 ganglioside,
(A) an apparent molecular weight of about 200 to about 300 kDa by size exclusion chromatography;
(B) containing spherical particles having a diameter of about 10 to about 20 nm as measured by atomic force microscopy;
(C) free of detectable GM1 ganglioside; and (d) inducing apoptosis-like cell death on cultured cells;
An assembly characterized by:
[2] The assembly according to [1], wherein amyloid β is a wild-type peptide or a peptide having an arctic mutation;
[3] A method for producing a soluble toxic amyloid β assembly, wherein the monomeric amyloid β is mixed with a liposome containing about 10 to about 15% GM1 ganglioside in a molar ratio of lipid components at about 35 to about 40 ° C. for about 1 Incubating for about 30 hours;
[4] The method according to [3] above, wherein the monomeric amyloid β is a wild-type peptide and the incubation time is 20 to 30 hours;
[5] The method according to [3] above, wherein the monomeric amyloid β is a peptide having an arctic mutation and the incubation time is 1 to 3 hours;
[6] A soluble toxic amyloid β assembly produced by the production method of any one of [3] to [5] above;
[7] Centrifuge the liquid containing the soluble toxic amyloid β assembly according to [1], [2] or [6] at 540,000 × g for 15 minutes or under the same conditions, and collect the supernatant. A method of purifying soluble toxic amyloid β assembly, characterized in that
[8] The method according to [7], wherein the collected supernatant is further subjected to size exclusion chromatography;
[9] A soluble toxic amyloid β assembly purified by the purification method of [7] or [8] above;
[10] A method for screening a substance that modulates neurotoxicity of soluble toxic amyloid β, comprising the following steps:
(A) a step of incubating cultured cells in the presence of the soluble toxic amyloid β assembly and the test substance according to any one of [1], [2], [6] or [9],
(B) after the step (a), measuring the life and death of the cultured cells;
(C) a method comprising a step of comparing the result obtained in the step (b) with a result obtained when the step (a) is performed in a state in which no test substance is contained,
I will provide a.

  The soluble toxic Aβ assembly of the present invention has a remarkably strong toxicity as compared with ADDL, insoluble assembly (fibril) and the like. Therefore, it is very sensitive in detecting compounds that affect toxicity.

  Further, TAβ of the present invention is known to induce apoptosis-like cell death in various neurons or non-neuronal cells through binding to NGF receptor, and is well characterized. Very useful in the study of apoptosis.

  The TAβ of the present invention is produced by incubating Aβ in the presence of liposomes containing a concentration of GM1 ganglioside appropriate for its genotype. The monomer Aβ used for the production of TAβ may be a wild type or a mutant type. However, from the viewpoint of the strength of toxicity and the like, a mutant type, particularly a mutation inside the Aβ domain is present. Existing types (eg, Italian type, Arctic type, Flemish type, etc.) are preferred, and arctic type is most preferred.

  Arctic mutation is a substitution of the amino acid located at codon 693 of human amyloid precursor protein (APP) (glutamic acid is replaced by glycine; E693G), and this mutation is a substitution of the 22nd amino acid in Aβ. It corresponds to. In the context of the present invention, the arctic type includes those having mutations corresponding to the above mutations in sequences derived from animals other than humans. The same applies to other mutant forms, including mutations in other animals corresponding to the respective mutations in humans.

  Aβ used in the present invention can be preliminarily removed of insoluble peptides that can become seeds of Aβ polymerization by subjecting it to ultracentrifugation after dissolution.

  Liposomes containing GM1 ganglioside, as used in the present invention, have a molar ratio in total lipid components of about 10/100 to about 15/100 (about 10 to about 15 moles) lower than required for amyloid fibril formation. %) Of GM1 ganglioside, and can be prepared by a known general method. The reason why TAβ is favorably formed in the presence of this particular concentration of GM1 ganglioside has not been elucidated, but suggests that the lateral distribution of GM1 ganglioside affects the spatial arrangement of the oligosaccharide chains of the molecule. (Non-patent Document 26), it is considered that a specific concentration of GM1 ganglioside may regulate the conformation of GM1 ganglioside and provide a favorable microenvironment for TAβ formation.

  Components other than GM1 ganglioside in the lipid mixture may be cholesterol or phospholipid, and the phospholipid is preferably phospholipid present in the brain. Examples of such include cholesterol, sphingomyelin, phosphatidylcholine and the like, and cholesterol and sphingomyelin are preferable. A phospholipid may be used individually by 1 type, and may be used in combination of 2 or more type. Components other than GM1 ganglioside in the lipid mixture most preferably include cholesterol and sphingomyelin. In particular, the molar ratio of cholesterol: sphingomyelin is desirably 0.5: 1.5 to 1.5: 0.5. Among them, a mixture in which the molar ratio of cholesterol: sphingomyelin is about 0.5: 1 to 1: 0.5, and further about 0.8: 1.2 to 1.2 to 0.8 is obtained as GM1 ganglioside. It is likely to give a fine environment to provide a favorable conformation for TAβ formation and is considered preferred, with a 1: 1 molar ratio mixture being most preferred.

  Specifically, suitable liposomes containing GM1 ganglioside have a molar ratio of cholesterol: sphingomyelin: GM1 ganglioside of 45: 45: 10-42.5: 42.5: 15 (ie, as described below, for example) The lipid mixture is prepared so that the ratio of GM1 ganglioside in the lipid mixture is 10 to 15% in molar ratio, and this is suspended in an appropriate buffer (for example, Tris buffered saline (TBS)) immediately before use. Can be prepared.

Incubation of liposomes containing GM1 ganglioside with Aβ can be initiated by bringing each together in solution at an appropriate concentration. For example, when Aβ is 10 to 200 μM, preferably about 25 to 100 μM, as a final concentration in such an incubation mixture (a solution containing a liposome containing GM1 ganglioside and Aβ), GM1 ganglioside is 30 to 500 μM, Preferably about 62 to 375 μM, particularly preferably about 100 to 200 μM, and the total lipid constituting the liposome is 0.3 to 5 mM, preferably 0.62 to 3.75 mM, and particularly preferably about 1 to 2 mM. Coexist in
Incubation for TAβ formation is generally performed at about 20 to about 40 ° C. (preferably about 37 ° C., ie, about 35 to about 40 ° C.) for about 1 to about 30 hours. The optimum conditions can be appropriately selected according to the conditions. For example, when using a wild-type peptide, about 35 to about 40 ° C. (especially about 37 ° C.) for about 20 to 30 hours; when using arctic Aβ, about 1 to about 35 to about 40 ° C. (particularly about 37 ° C.) Incubations of up to about 5 hours (especially about 1 to about 3 hours) are each optimal.

  The choice of incubation time is important. TAβ tends to appear early in the incubation period and disappear through subsequent incubations. This suggests that TAβ may also be an intermediate in the process of forming larger Aβ assemblies. In addition, conventional evidence, in particular, that soluble Aβ assemblies are formed at the beginning of the incubation period in vitro, and then mature fibrils often appear (Non-patent Documents 2 and 10), and certain inhibitors of Aβ fibril production are present. Strongly blocking Aβ oligomer production (Non-Patent Document 24) suggests that soluble Aβ assembly and protofibrils are formed as intermediates in amyloid fibril formation.

  However, as described above, the TAβ of the present invention is preferably formed in the presence of GM1 ganglioside at a concentration lower than the GM1 ganglioside concentration generally required for amyloid fibril formation, and TAβ formation is TAβ has a different pathway from that which causes amyloid fibril formation linearly because it is not inhibited by an antibody specific to amyloid fibril-forming species (GM1 ganglioside and Aβ bound to each molecule). It is shown that it is formed through.

  The TAβ of the present invention is suitably formed after incubation under the conditions as described above and is included in the incubation mixture. Although it is believed that little or no fibrils (insoluble assemblies) are formed under the preferred conditions for the formation of TAβ of the present invention, this incubation mixture can be treated, for example, at 540,000 × g for 15 minutes or equivalent. The ββ of the present invention can be concentrated or partially purified by centrifuging under the above conditions and collecting the supernatant. As used herein, “equivalent conditions” refers to conditions that result in substantially the same separation as centrifugation under specified conditions. Further, TAβ can be further purified by subjecting the recovered supernatant to further size exclusion chromatography and recovering a fraction containing TAβ.

  As will be described later, TAβ of the present invention comprises at least rat primary culture neurons; PC12 cells natively or differentiated by NGF treatment, human brain microvascular endothelial (hBME) cells, human brain astrocytes ( hAST), human neuroblastoma SH-SY5Y (SY5Y) cells, human embryonic kidney 293 (HEK293) cells, cultured cells containing mouse monocytes RAW264.7 (RAW264) cells (including non-neuronal cells), Cell death exhibiting apoptotic-like features can be induced. Therefore, the presence of TAβ of the present invention can be confirmed by, for example, adding such an incubation mixture or its centrifugal supernatant or fraction to these TAβ-sensitive cultured cells to determine whether cell death is induced. Can be confirmed. The determination of cell viability can be easily performed by a known method, for example, staining with a known dye as will be described later, lactate dehydrogenase (LDH) release assay, or the like.

  The TAβ of the present invention has the greatest feature in its toxic strength. As an example, when PC12N cells described later are incubated with TAβ of the present invention (Aβ concentration of 25 μM) at 37 ° C. for 48 hours, LDH release rate (ie, cells are lysed with Triton-X100 to release LDH in all cells. The percentage of LDH released when the sample is treated with respect to the amount of LDH released when the sample is treated), showing an LDH release rate of at least 30%, preferably 50% or more, more preferably 60% or more, almost completely Can cause cells to die. In addition, in the cytotoxicity test by the LDH release rate, the amount of LDH released from the inside of the cell due to the cell membrane being broken is used as an index. In the case of cell death due to apoptosis, the cell membrane is not broken for a while even if the cell is dead. Some do not release LDH (eg, cell atrophy). Therefore, when the LDH release rate is about 70% or more, almost all cells are dead, and the LDH release rate by TAβ of the present invention as described above is very high.

  In addition, when the rat primary cultured neurons were incubated with the TAβ of the present invention (Aβ concentration 25 μM) for 48 hours at 37 ° C. by the method described later and the viability of the cells was determined by dye staining, the number of surviving neurons was determined by the TAβ of the present invention. 75% or less, preferably 60% or less, more preferably 40% or less, and most preferably 20% or less compared to a target culture containing no sucrose.

  This toxicity of TAβ of the present invention is suppressed by A11 anti-oligomer antibodies (anti-Oligo). The A11 antibody is an antibody specific to an oligomer of amyloidogenic protein, and reacts with, for example, a soluble Aβ40 oligomer, but does not react with a soluble monomeric Aβ40 or Aβ40 fibril.

  The TAβ of the present invention has a completely different form from amyloid fibrils that are insoluble or other known soluble assemblies. For example, the TAβ of the present invention includes structures that are not observed with a normal electron microscope, but appear as spherical particles with a diameter of about 10 to about 20 nm by an atomic force microscope. The diameter referred to here is the length based on the height difference obtained from the sample in accordance with the principle of an atomic force microscope (AFM), and is measured by a cross-sectional analysis method.

  Further, TAβ of the present invention does not form a band by SDS polyacrylamide gel electrophoresis (PAGE), and has a peak at about 200 to about 300 kDa in molecular weight measurement by size exclusion chromatography. Furthermore, no GM1 ganglioside is detected in TAβ. An antibody against amyloid fibril-forming species (4396C, Non-Patent Document 25) inhibits amyloid fibril formation, but the production of TAβ of the present invention is not inhibited by this antibody.

  Therefore, TAβ of the present invention is clearly different from known oligomers such as ADDLs and known fibrils.

TAβ is also formed in vivo and is thought to have a pathological role. TAβ of the present invention is significantly formed in the presence of synaptosomes, particularly synaptosomes derived from the hippocampus of aged mouse brain. This is in conjunction with our recent observation that the density of GM1 gangliosides in synaptosomes increases with age and apolipoprotein E4 expression (27), in vivo, especially for the risk factors for developing AD. It suggests that TAβ can be formed in certain areas of the brain, such as the hippocampus associated with. Soluble Aβ assemblies such as TAβ are also thought to cause loss of NGF-dependent neurons in cholinergic basal forebrain in AD in vivo.
Therefore, substances that reduce or prevent the toxicity of TAβ are important in the prevention or treatment of amyloid-related diseases such as AD.

  The TAβ of the present invention can be used to screen for substances that affect its properties (eg, toxicity). For example, after incubating cultured cells sensitive to TAβ of the present invention in the presence of TAβ of the present invention and a test substance, the test substance affects the characteristics of TAβ by measuring the viability of the cultured cells. It is possible to identify whether or not a substance having a desired influence is identified.

  The cultured cells used here may be any cells that are sensitive to TAβ, and may be primary cells or established cell lines. Examples include, but are not limited to, cells in which cell death is induced by TAβ as described above.

  The test substance is not particularly limited, and is generally a low molecular compound, peptide, protein (including antibodies and modifications thereof), nucleic acid (single-stranded or double-stranded polymer or low-molecular DNA or RNA), mixtures of extracts such as plants and animals, microorganisms such as viruses and bacteria, and the like.

  Cell viability can be measured by known methods as described above. If cell death is increased when the test substance is included compared to the result of incubation without the test substance, the test substance can enhance the toxicity of TAβ. If the cell death is reduced, the test substance can be determined to have an action of suppressing the toxicity of TAβ. In the former case, whether or not the test substance has intrinsic toxicity can be examined by separately conducting the same test in the presence of the test substance and in the absence of TAβ.

  A substance determined to have an action of suppressing the toxicity of TAβ by the above screening can be selected as a drug candidate substance.

TAβ of the present invention binds competitively with NGF for the NGF receptor. That is, cell death induced by TAβ of the present invention is significantly suppressed in the presence of NGF. Moreover, cell death induced by TAβ is also remarkably suppressed by knockdown of either of the NGF receptors p75 ^NTR and TrkA. This can be used to screen for compounds that suppress the toxicity of TAβ.

  In this method, in the same manner as described above, the cultured cells sensitive to TAβ of the present invention are incubated in the presence of TAβ of the present invention and a test substance, and then the viability of the cultured cells is measured. Compounds that suppress the toxicity of can be identified. In this case, if the cell death is reduced when the test substance is contained as compared to the result of incubation without the test substance, the test substance is not bound to the NGF receptor. It can be determined that there is a possibility of suppressing the binding of TAβ.

I. Production of soluble toxic Aβ assembly (TAβ)
1. Aβ solution synthesis Aβ (Aβ1-40) (both wild type and arctic type synthesized at Peptide Institute, Osaka, Japan) was dissolved in 0.02% ammonia solution to a concentration of 500 μM. This solution was centrifuged at 540,000 × g for 3 hours using an “Optima TL” (trade name, Beckman, USA) ultracentrifuge to remove insoluble peptides that could act as seeds. The obtained supernatant (hereinafter also referred to as “seed-free Aβ solution”) was collected, divided into aliquots, and stored at −80 ° C. until use.
Immediately before use, aliquots were thawed and diluted with Tris-buffered saline (TBS: 150 mM NaCl and 10 mM Tris-HCl, pH 7.4).

2. Preparation of liposomes For the preparation of liposomes, cholesterol (Sigma, catalog number: C8667), sphingomyelin (Sigma, catalog number: S7004), and GM1 ganglioside (Matreya, catalog number: 1061) were mixed with 50: At a molar ratio of 50: 0, 45:45:10, 42.5: 42.5: 15, or 40:40:20, the total lipid concentration is 15 mM (ie, the GM1 ganglioside concentration is 0 mM, Dissolved in 1.5 mM, 2.25 mM, or 3.0 mM) chloroform / methanol (1: 1, volume ratio) solution. Therefore, the ratio of GM1 ganglioside in the total lipid molecules constituting the liposome was 0%, 10%, 15% and 20%, respectively. 86 μL of this lipid mixture solution was dispensed into a glass tube, and nitrogen gas (40 ° C.) was blown for 1 hour to evaporate the solvent. This lipid mixture was stored at −80 ° C. until use.

  Immediately before use, 100 μL of TBS (pH 7.4) was added to each glass tube to resuspend the lipid mixture in TBS, and this suspension was freeze-thawed 10 times before sonicator (TOMY UD- 201, output = 2, continuous mode) for 5 minutes and subjected to ultrasonic treatment three times. By this operation, the lipid mixture became uniform spherical liposomes having a diameter of about 500 μm or less.

3. Incubation of Aβ with liposomes (TAβ preparation)
Above 1. 1. Dilute the seed-free Aβ solution prepared in step 2 above. Each of the liposome-containing TBS solutions was diluted 10-fold and incubated at 37 ° C. for 2 hours (also performed for 24 hours for comparison). The final concentration in this incubation mixture was Aβ 50 μM and liposome concentration (total lipid concentration) 1250 μM (of which the GM1 ganglioside concentration was 0 μM, 125 μM, 187.5 μM, or 250 μM).

II. Characterization of TAβ Using various incubation mixtures of the seed-free Aβ (Aβ1-40) solution (wild type or arctic type) prepared above and GM1 ganglioside-containing liposomes, features of TAβ neurotoxicity, morphology, etc. I did it.

1. Neurotoxicity test for primary cultured neurons As primary cultured neurons, cerebral cortical neurons were prepared from 17-day embryos of Sprague-Dawley rats, and Dulbecco's modified Eagle medium nutrient mixture (trade name; Invitrogen, catalog number: 12800-017). And N2 supplement (“N2-supplement” (trade name; Invitrogen, catalog number: 17502-048)) (5% CO ₂ , 37 ° C., under humidification).

The primary cultured neurons (number of cells: 1.5 × 10 ⁴ cells / well) were treated with each of the above incubation mixtures (incubation mixture: medium = 1: 1, Aβ concentration 25 μM, treatment time was 48 hours). Neurons were stained with calcein AM (Invitrogen, Carlsbad, Calif.) Ethidium homodimer. As a result, live cells are stained green and dead cells are stained red.

The results are shown in FIG. 1, panels (A) and (B).
Panel (A) shows liposomes with GM1 ganglioside content 0% (top), 10% (middle), 20% (bottom), no Aβ (left column) or wild type (“wild”) Aβ (middle Columns) or light micrographs of cells treated with incubation mixtures (37 ° C., 2 hours) containing Arctic (“Arctic”) Aβ (right column). The lower right bar is 50 μm.

  Panel (B) treated with wild-type or arctic-type Aβ preincubated for 2 hours at 37 ° C. at 50 μM (final concentration in incubation mixture) in the presence or absence of GM1 ganglioside under the indicated conditions. Shown is the number of primary cultured neurons that survived. Each column is the average (± SD) of the three values of the percentage relative to a control culture (GM10%, Aβ (−)) containing neither Aβ nor GM1 ganglioside, * represents P <0.0001.

  From these results, very significant neuronal death was observed in cultures treated with arctic Aβ preincubated for 2 hours at 37 ° C. in the presence of liposomes containing 10% GM1 ganglioside. Therefore, in the subsequent experiments, TAβ formed under this condition was basically used (that is, TAβ formed under this condition unless otherwise indicated).

2. Toxicity test on PC12 cells differentiated with nerve growth factor (NGF) Rat pheochromocytoma PC12 cells were treated with 10% heat-inactivated horse serum (Invitrogen) and 5% fetal bovine serum (FBS) (Invitrogen). ) Was added in Dulbecco's modified Eagle's medium (DMEM) (trade name, Invitrogen, Carlsbad, CA).

PC12 cells were seeded at a density of 20,000 cells / cm ² in 2 cm ² dishes coated with poly-L-lysine (10 mg / mL) and 100 ng / mL nerve growth factor (NGF; Alomone Labs, Jerusalem, Israel) The cells were cultured for 6 days in the added DMEM and differentiated (hereinafter also referred to as “PC12N” cells).

  The cells were used in place of primary cultured neurons and treated with the incubation mixture as described above, and the cells were observed.

  The results are shown in FIG. 1, panel (C). In this figure, the right end (Aβ (+) / GM1 (+)) is treated with an incubation mixture containing arctic Aβ preincubated for 2 hours at 37 ° C. in the presence of liposomes containing GM1 ganglioside (10%). Cells. Similarly, the center left (Aβ (+) / GM1 (−)) is Aβ only, the center right (Aβ (−) / GM1 (+)) is only GM1 ganglioside, and the left end (Aβ (−) / GM1 (−)). Are cells treated with incubation mixtures each containing only TBS.

  NGF-treated PC12 cells were also confirmed to be sensitive to toxic Aβ assemblies formed from arctic Aβ in the presence of GM1 ganglioside. In the following experiments, PC12N cells were mainly used.

3. ThT assay Thioflavin T (ThT) specifically recognizes amyloid fibril structure. In order to examine the presence or amount of amyloid fibril structure in the incubation mixture, the ThT fluorescence intensity of each incubation mixture was measured using a spectrofluorometer (RF-5300PC) (Shimadzu, Kyoto, Japan). Optimal fluorescence intensity of amyloid fibrils was determined using 1.0 mL of reaction mixture (including 10 μl of incubation mixture) containing 5 μM ThT and 50 mM glycine-NaOH (pH 8.5) with excitation and emission wavelengths of 446 nm and 490 nm, respectively. It was measured. The fluorescence intensity was measured immediately after preparing the reaction mixture.

  The results are shown in FIG. 2, panel (A). This figure shows no Aβ containing liposomes with GM1 ganglioside content of 0%, 10%, 20% (“Aβ (−)”; white), wild type Aβ (“wild”; hatched), arctic type (“Arctic” "; Black)) for the incubation mixture.

  The fluorescence intensity of ThT did not coincide with the strength of neurotoxicity and remained at a low level in the incubation mixture containing arctic Aβ and GM1 ganglioside (10%) that showed strong neurotoxicity. Therefore, this result showed that the Aβ assembly showing high neurotoxicity is not amyloid fibril.

4). Lactate dehydrogenase (LDH) release assay 2. PC12N cells were prepared in the same manner as in the above experiment.
PC12N cells were treated with the I. The cells were treated with each incubation mixture prepared in the same manner as described above (number of cells: 1.5 × 10 ⁴ cells / well, 37 ° C., treatment time: 48 hours, incubation mixture: medium = 1: 1).

  Using the culture medium of these cells as a sample, LDH assay was performed using “LDH assay toxicity kit” (trade name, Promega, Madison, Wis.). The extent of LDH release in each sample was assessed by measuring absorbance at 490 nm using an “Emax precision” (trade name, Molecular Devices Corp., Sunnyvale, Calif.) Microplate reader. Background absorbance was measured using wells without cells and subtracted from the absorbance of each test sample. According to the instructions attached to the kit, the absorbance measured in the three wells is averaged and divided by the measured value for each test sample treated with 1% Triton X-100 to induce intracellular LDH release, LDH release rate (%) was calculated.

  The result is shown in FIG. 2, panel (B). Each column represents an average value (± SD) of three values, and * represents p <0.0001 (one-way ANOVA combined with Scheffe's test). The column contains no Aβ containing liposomes with GM1 ganglioside content 0%, 10%, 20% (“Aβ (−)”, ie TBS only control; open), wild type Aβ (“wild”; hatched), arc Tick type (“Arctic”; painted black). Arctic Aβ was found to be toxic to cell death in the presence of liposomes containing 10% or 15% GM1 when incubated for 2 hours. On the other hand, wild-type Aβ showed little toxicity after 2 hours of incubation, and was more toxic in the presence of higher concentrations (15% or 20%) of GM1 ganglioside when incubated for 24 hours.

  An incubation mixture containing arctic Aβ that was incubated for 2 hours at 37 ° C. in the presence of liposomes containing GM1 ganglioside (10%), which showed the strongest toxicity, was 540,000 × g, 15 minutes at 4 ° C. The supernatant and the precipitate were collected by ultracentrifugation. Using each of these supernatants, precipitates and pre-centrifugation incubation mixtures, LDH release assays were performed as described above. For comparison, incubation mixtures were similarly prepared for Aβ alone (Aβ + / GM1-), GM1 ganglioside alone (Aβ− / GM1 +), and TBS alone (Aβ− / GM1-), and LDH assay was performed.

  The results are shown in FIG. 2, panel (C). Each column shows the average of three values (± SD), * represents p <0.0001. Toxicity exhibited by the pre-centrifugation Aβ incubation mixture (“whole”) incubated in the presence of GM1 ganglioside was recovered only in the supernatant (“sup”) obtained by ultracentrifugation and the precipitate (“ppt”) ) Showed little toxicity.

  These results indicated that the assembly formed from arctic and wild-type Aβ in the presence of GM1 ganglioside at appropriate concentrations, respectively, is a toxic soluble Aβ assembly.

5. SDS gel electrophoresis (SDS-PAGE)
To determine the biophysical and structural characteristics of TAβ, SDS-PAGE of the incubation mixture containing TAβ was performed. Based on the method described in Schagger et al., Analytical Biochemistry Vol. 166, 368-379, 1987, the anode buffer is 0.2 M Tris (pH 8.9), the cathode buffer is 0.1 M Tris-0.1 M Tricine- As a 0.1% SDS buffer, 4 to 20% polyacrylamide gel (first chemical minigel) was used, and electrophoresis was performed at 40 mA (constant flow) for 30 minutes. The gel was blotted on nitrocellulose paper at 60 mA for 1 hour, and Western blotting was performed using a monoclonal antibody that recognizes Aβ (6E10, Signet Laboratories (Dedham, Mass.)) For detection.

  However, no band was detected. This is because soluble Aβ assemblies are disaggregated by denaturing gel electrophoresis when they are not cross-linked (Non-patent Documents 21 and 22) except for certain oligomers (Non-patent Document 1) in cell culture ( This is consistent with the conventional knowledge of Non-Patent Document 3).

6). Dot blot analysis Next, conditions similar to those described above for incubation mixtures containing arctic Aβ only, liposomes containing GM1 ganglioside (10%) only, or liposomes containing arctic Aβ and GM1 ganglioside (10%), respectively. Using the supernatant ("sup") obtained by ultracentrifugation in, dot blot analysis was performed by the method of Non-Patent Document 23. For comparison, the pre-centrifugation incubation mixture (“whole”) was used as well.

  The nitrocellulose paper (blot) on which each sample was doted was added to the anti-oligomer antibody “anti-Oligo” (Biosource Inc., Camarillo, Calif.) Or HRP-conjugated cholera toxin subunit B “CTX” (Sigma, St. Louis, MO). And reacted. The reaction conditions were overnight at 4 ° C., and ECL (chemiluminescence) was used as the detection method. CTX is a natural ligand that specifically reacts with GM1 ganglioside.

  The results are shown in FIG. 3, panel (A). TAβ in the incubation mixture containing arctic Aβ and GM1 ganglioside (10%) containing liposomes was readily recognized by anti-Oligo, but no reactivity with CTX was recovered in the supernatant. Therefore, TAβ is considered not to contain GM1 ganglioside.

7). Inhibition of TAβ toxicity by anti-oligomer antibody In order to examine the influence of the antibody on TAβ toxicity, the above 4. In the same manner as in the experiment of I. above. LDH release in PC12N cells after preincubation for 120 minutes at 37 ° C. with an anti-Oligo antibody at a concentration of 0.3 μM using an incubation mixture prepared similarly to (10% GM1 ganglioside (125 μM) +50 μM Arctic Aβ) The assay was performed. As a control, IgG was used instead of anti-Oligo.

  The results are shown in FIG. 3, panel (B). Each column shows the average of three values (± SD), * represents p <0.0001. TAβ toxicity, ie, TAβ-induced LDH release from PC12N cells, was significantly suppressed by preincubating the incubation mixture with anti-Oligo.

8). Morphological observation with an electron microscope Observation with an electron microscope was performed as follows. Samples were diluted with water and spread on a carbon coated grid. This grid was negatively stained with 2% uranyl acetate and observed with a transmission electron microscope (JEM-2000EX, JEOL, Tokyo, Japan) at an acceleration voltage of 100 kV (magnification: 50,000 times). For comparison, protofibrils were prepared according to the method described in Non-Patent Document 17 and observed in the same manner.

  The results are shown in FIG. 4, panel (A). In panel (A), the left side (“A”) is an incubation mixture containing arctic Aβ incubated by the method of Non-Patent Document 17 so that protofibril formation is possible, and the right side (“B”) is TAβ. Electron micrograph (lower right bar is 100 μm) of the incubation mixture contained (GM1 (10%), Arctic, 37 ° C., 2 hours).

  A typical structure of protofibrils was observed in “A”. On the other hand, no reliable structure was observed in “B” (it is liposomes that look round). Therefore, TAβ of the present invention was not observed by electron microscopy under conditions where protofibrils prepared as previously reported (Non-patent Document 17) were readily detectable.

9. Evaluation by morphological observation AFM using an atomic force microscope (AFM) was performed as described in Non-Patent Document 1. The supernatant after ultracentrifugation of the incubation mixture containing GM1 ganglioside alone or Aβ alone, or both containing 540,000 × g, 15 minutes at 4 ° C. (“Aβ + GM1”; GM1 (10%), Arctic , 37 ° C., 2 hours) was dropped onto the newly cut mica. After standing for 3 minutes, it was washed with water, and the sample was evaluated in solution using “Nanoscope IIIa” (trade name, Digital Instruments, Santa Barbara, CA, USA) set to tapping mode (Non-patent Document 2). did. “OMCL-TR400PSA” (trade name, Olympus, Japan) was used as a cantilever. The resonant frequency was about 9 kHz. The amplitude range was 0.1V.

The results are shown in FIG. 4, panel (B). The lower right bar is 200 nm. Unlike when using an electron microscope, when AFM is used, spherical particles having a diameter of about 10 to about 20 nm, together with rod-shaped structures, are obtained by ultracentrifugation of an incubation mixture containing TAβ (“Aβ + GM1 )). No sure structure was observed in the supernatant of incubation mixtures containing only Arctic Aβ (upper left) or GM1 ganglioside (10%) only (lower left).
The diameter of the spherical particles was measured using the above-mentioned “Nanoscope IIIa” and software incorporated therein.

10. Measurement of molecular weight of TAβ by size exclusion chromatography The molecular size of TAβ was determined by size exclusion chromatography (SEC) and dot blot analysis using anti-Oligo. Aβ alone or “Aβ + GM1” sample (10% GM1 ganglioside and 50 μM Arctic Aβ were incubated at 37 ° C. for 2 hours, and then the supernatant obtained by ultracentrifugation at 540,000 × g, 15 minutes, 4 ° C. And applied to a “Superose 12” (trade name) size exclusion column (1 cm × 30 cm, Pharmacia Biotech., Uppsala, Sweden) equilibrated with PBS (pH 7.4) at a flow rate of 0.5 mL / min. Fractions were collected and analyzed by dot blot using anti-Oligo as described above 6. Elution samples of 35 fractions were doted onto a nitrocellulose membrane. And reacted.

The results are shown in FIG. 4, panel (C). The result on the left is the result of using the supernatant after centrifugation of Aβ alone and the right is “Aβ + GM1”. Fractions 2 and 4 correspond to fractions containing TAβ and monomer Aβ, respectively.
Immunoreactivity with anti-Oligo was recovered as a single peak with a relative molecular weight of about 200 to about 300 kDa in the “Aβ + GM1” sample. This was similar to certain soluble Aβ assemblies called amyloid pores (18) formed from arctic Aβ. However, the amyloid pore has a hole-like structure in the center and looks like a donut on the photograph, whereas no pore was observed in TAβ.

11. Inhibition of TAβ formation by antibodies against amyloid fibril-forming species It was investigated whether TAβ formation was inhibited by a monoclonal antibody (4396C) specific for the species for amyloid fibril formation (Non-patent Document 25).

  Incubated for 2 hours at 37 ° C. at 50 μM (final concentration in the incubation mixture) in the presence of liposomes containing GM1 ganglioside (10%) with 0, 0.3, 1 or 3 μM 4396C or anti-Aβ monoclonal antibody (4G8). The incubation mixture containing the arctic Aβ was examined for ThT fluorescence intensity in the same manner as described above.

  The results are shown in FIG. 5, panel (A). The anti-Aβ monoclonal antibody (4G8) did not affect the ThT fluorescence intensity, but when the 4396C antibody coexists, the ThT fluorescence intensity decreased in a concentration-dependent manner, and it was confirmed that the 4396C antibody inhibits amyloid fibril formation.

  PC12N treated with arctic Aβ pre-incubated in the presence of liposomes containing GM1 ganglioside (10%) (37 ° C., 2 hours) in the presence or absence of 0.3 μM 4396C antibody The cells were used for LDH release assay as described above. IgG 2a (Sigma, catalog number: M9144) was used as a control.

  The results are shown in FIG. 5, panel (B). The 4396C antibody did not inhibit TAβ production.

12 Formation of TAβ in the presence of synaptosomes We tested whether TAβ could be formed in the presence of natural neuronal membranes.
Synaptosomes were prepared as previously reported (Non-Patent Document 3). The entire brain, excluding the hippocampus or hippocampus of mouse brains from three different age groups (1 month, 1 year, 2 years), was homogenized in 0.32 M sucrose buffer containing 0.25 mM EDTA. The homogenate was centrifuged at 580 × g for 8 minutes. The supernatant was centrifuged at 145,000 × g for 20 minutes. The obtained pellet was suspended in EDTA-free 0.32M sucrose buffer and overlaid on Ficoll (trade name, Sigma) in sucrose buffer. After centrifuging at 87,000 × g for 30 minutes, the interface enriched with synaptosomes was collected and centrifuged again to remove the remaining Ficoll.

  Arctic Aβ was incubated in the presence of synaptosomes prepared from mouse brain. Arctic Aβ is specific for GM1 ganglioside in the presence or absence of each prepared synaptosome (100 μg / mL protein) at 50 μM (final concentration in the incubation mixture) at 37 ° C. for 2 hours. Pre-incubation (37 ° C., 2 hours) with or without antibody (“anti-GM1”) or anti-Oligo antibody (0.3 μM). IgG was used as a control.

  TAβ formation was assessed by a similar LDH release assay in PC12N cell cultures treated with these incubation mixtures.

  The results are shown in FIG. “Wh” is the whole brain excluding the hippocampus, and “Hp” is the synaptosome derived from the hippocampus. Each column shows the average (± SD) of the four values. * Represents p <0.0001, and ** represents p <0.005. The level of TAβ formation was observed in all other incubation mixtures (excluding younger (1 month and 1 year) mouse brain hippocampus or hippocampus) in incubation mixtures containing synaptosomes prepared from the hippocampus of aged (2 year old) mice. Significantly higher than those containing synaptosomes from the whole brain).

13. Observation by nuclear staining The surprising feature of TAβ is its strong toxicity. To characterize cell death induced by TAβ, an incubation mixture containing 50βM Arctic Aβ and 10% GM1 ganglioside (37 ° C, 2 ° C) using Hoechst 33258 (trade name), a nuclear penetrating dye. Time) for 12 hours (cell number 1.5 × 10 ⁴ cells / well, incubation mixture: medium = 1: 1) nuclear staining of PC12N cells was performed.

  A photograph of PC12N cells after treatment is shown in FIG. 7, panel (A) (bar is 50 μm). a and b are phase contrast microscope images, and c and d are nuclear stained images. PC12N cells (b and d) treated with the above incubation mixture containing TAβ for 12 hours are apoptosis including retracted neurites, atrophyed cell bodies, nuclear condensation and fragmentation, etc. The characteristics of such changes (indicated by arrows in the figure) are shown.

14 TAβ toxicity to various types of cultured cells Various types of cultured cells were treated with TAβ and the sensitivity of the cells to TAβ was examined.
In addition to the above PC12 cells and PC12N cells, the following cells were used. Human brain microvascular endothelial (hBME) cells and human brain astrocytes (hAST) (both Dainippon Pharmaceutical Inc., Osaka, Japan) were prepared in CS-C medium (Dainippon Pharmaceutical Inc.) in flasks coated with collagen. In culture. Rat astrocytes (rAST) were cultured in DMEM medium containing 10% fetal calf serum. Human neuroblastoma SH-SY5Y (SY5Y) cells were cultured in DMEM / Ham's F-12 medium supplemented with 10% FBS. Human embryonic kidney 293 (HEK293) cells were cultured in DMEM supplemented with 10% FBS. Mouse monocyte RAW264.7 (RAW264) cells were purchased from Dainippon Pharmaceutical Inc. RAW264 cells were cultured in DMEM supplemented with 10% FBS. Mouse fibroblasts immortalized with large T antigen (“fibroblast”) were cultured in DMEM supplemented with 10% FBS. All cells were cultured in humidified 5% CO ₂ at 37 ° C.

Treat each cell with an incubation mixture containing TAβ (50 μM Arctic Aβ and 10% GM1 ganglioside, 37 ° C., 2 hours) as above (cell number 1.5 × 10 ⁴ cells / well, 37 ° C., 48 hours). Incubation mixture: medium = 1: 1) and LDH release assay was performed.

  The results are shown in FIG. 7, panel (B). Each column represents the average of three values (± SD), * represents P <0.0001 relative to the value when treated with an incubation mixture containing only Aβ. Non-neuronal cells, including native PC12 cells, were also sensitive to TAβ.

15. Binding of TAβ to NGF receptor To examine whether TAβ induces apoptosis through the NGF receptor, PC12N cells and native PC12 cells were treated with TAβ in the presence of exogenous NGF. 100 ng / mL NGF was added to PC12N cells and PC12 cells (cell number 1.5 × 10 ⁴ cells / well), and incubation mixture containing TAβ (50 μM Arctic Aβ and 10% GM1 ganglioside, 37 Each cell was treated at 2 ° C. for 2 hours and an LDH release assay was performed.

  The results are shown in FIG. 8, panel (A). In both cultures, the degree of cell death was significantly suppressed when NGF was present. Thus, TAβ was shown to bind competitively to the NGF receptor.

16. Suppression of TAβ toxicity by siRNA-mediated knockdown of TrkA or p75 ^NTR TrkA and p75 ^NTR were knocked down using specific small interfering RNA (siRNA).

“Stealth” ® small interfering RNA (siRNA) double-stranded oligonucleotides for PC12 cells TrkA (GenBank no. NM — 021589) and p75 (GenBank no. NM — 012610) were synthesized by Invitrogen (Carlsbad, Calif.). . The siRNA sequences were as follows:
rTrkA-siRNA (position 1370)
Sense = 5′-GCCCUCCUCUCUUGUGCUCAACAAAU-3 ′;
Antisense = 5′-AUUUGUUGAGCACUAGGAGGAGGGGC-3 ′;
rTrkA-siRNA-control sense = 5'-GCCCUCCCGAUCUCGUCAACAUCAAU-3 ';
Antisense = 5′-AUUGAUGUUGACGAGAUCGGAGGGGC-3 ′;
rp75-siRNA (position 1212)
Sense = 5'-CAGCCUGAACAUAUAGACUCCUCUUA-3 ';
Antisense = 5′-UAAAAGGAGUCUUAUAUGUGUCAGCGCUG-3 ′;
rp75-siRNA-control sense = 5'-CAGGUAAAAUAUAUGUCCCUCCUUA-3 ';
Antisense = 5′-UAAGGAGGGACUAUAUUGUUACCUCG-3 ′.

These siRNA oligonucleotides (RNA amount 20 pmol) were transfected into PC12 cells (cell number 1.5 × 10 ⁴ cells / well) using “LipofectAMINE 2000” (trade name, Invitrogen) according to the protocol attached to the product. .

After treatment with siRNA against TrkA or p75 ^NTR , these PC12 cells were treated with an incubation mixture containing TAβ (50 μM Arctic Aβ and 10% GM1 ganglioside, 37 ° C., 2 hours) as described above, and LDH release. The assay was performed.

The reduction of TrkA and p75 ^NTR expression levels in each cell was determined by Western blot analysis (7.5% and 4-20%) of cell lysates using anti-TrkA (Santa Cruz) and anti-p75 ^NTR (NeoMarker) antibodies, respectively. Polyacrylamide gel (1st chemical minigel) was used, run at 40 mA for 1 hour and then blotted at 120 mA for 2 hours).

The results are shown in FIG. 8, panel (B). Each column shows the average of three values (± SD), * represents p <0.0001. Not only p75 ^NTR but also TrkA knockdown significantly suppressed cell death induced by TAβ.
From these results, TAβ disrupts the function of the heteromeric TrkA / p75 ^NTR complex by binding to p75 ^NTR or TrkA, resulting in apoptosis through activation of domains involved in the death of the cytoplasmic portion of the p75 ^NTR molecule. It was suggested that

FIG. 1 shows the toxicity of soluble Aβ assembly formed in the presence of GM1 ganglioside. Panel (A): Primary cerebral cortical neurons cultured in serum-free N2 supplemented medium (N2-supplemented meddium) in the presence or absence of GM1 ganglioside (0, 10 or 20 mol%) in liposomes. Treated with incubation mixture containing seed-free wild-type or arctic-type Aβ (Aβ1-40) pre-incubated for 2 hours at 0 ° C. It is the photograph which dye | stained the neuron with calcein AM (Invitrogen, Carlsbad, CA) ethidium homodimer. Green represents live cells and red represents dead cells. The bar is 50 μm. Panel (B): Surviving primary cultured neurons treated with wild-type or arctic-type Aβ preincubated for 2 hours at 37 ° C. in the presence or absence of GM1 ganglioside under the indicated conditions. It is a figure which shows a number. Neurons were stained with calcein AM / ethidium homodimer and propidium iodine. Each column represents the mean (± SD) of the three percentages relative to the control culture without Aβ or GM1 ganglioside. * P <0.0001 Panel (C): Representative of PC12N cells treated with incubation mixture containing arctic Aβ pre-incubated for 2 hours at 37 ° C. in the presence or absence of GM1 ganglioside (10%) It is a typical image. The bar is 50 μm. FIG. 2 shows that what is toxic in the incubation mixture containing GM1 ganglioside and Aβ is a soluble assembly. Panel (A): ThT in the culture of primary cultured neurons treated with an incubation mixture containing wild type or arctic Aβ preincubated at 37 ° C. in the presence or absence of GM1 ganglioside under the indicated conditions. It is a figure which shows fluorescence intensity. Columns are white = TBS (control), hatched = wild type, black = arctic type. Panel (B): LDH release rate from PC12N cells treated with incubation mixtures containing wild type or arctic Aβ preincubated in the presence or absence of GM1 ganglioside under the indicated conditions. is there. Each column represents the% LDH released after treatment with the incubation mixture /% LDH released after treatment with Triton-X100. Each column shows the average (± SD) of the three values. * Represents p <0.0001 (one-way ANOVA combined with Scheffe's test). Columns are white = TBS (control), hatched = wild type, black = arctic type. Panel (C): treated with supernatant (sup) and precipitate (ppt) obtained by ultracentrifugation of incubation mixture (whole) containing arctic Aβ pre-incubated in the presence of GM1 ganglioside (10%) It is a figure which shows the LDH release rate from the made PC12N cell. Each column shows the average (± SD) of the three values. * Represents p <0.0001. FIG. 3 is a diagram showing the characteristics of TAβ (reactivity with antibodies). Panel (A): Dot of supernatant (sup) obtained by ultracentrifugation of incubation mixture (whole) containing arctic Aβ alone, GM1 ganglioside (10%) alone, or arctic Aβ + GM1 ganglioside (10%) The result of blot analysis is shown. Blots were reacted with anti-Oligo antibody (Biosource Inc., Camarillo, CA) or HRP-conjugated cholera toxin subunit B (CTX (Sigma, St. Louis, MO). Panel (B): with anti-Oligo Fig. 6 shows suppression of TAβ-induced LDH release from PC12N cells by co-incubation, where each column shows the mean (± SD) of three values, * represents p <0.0001. FIG. 4 is a diagram showing biophysical and structural analysis of TAβ. Panel (A): An electron micrograph of an incubation mixture (A) containing arctic Aβ pre-incubated to form protofibrils or an incubation mixture (B) containing TAβ. The bar is 100 μm. Panel (B): AFM image of the fraction containing TAβ. The supernatant obtained by ultracentrifugation of the incubation mixture containing TAβ was analyzed by AFM. Incubation mixtures contained arctic Aβ alone (Aβ), GM1 ganglioside (10%) alone (GM1), or both (Aβ + GM1). The bar is 200nm. Panel (C): It is a figure which shows the result of the size exclusion chromatography of the TA (beta) containing fraction using Superose12. 35 fractions of elution sample were doted onto a nitrocellulose membrane. The blot was reacted with anti-Oligo antibody. Five representative fractions are shown. Fractions 2 and 4 correspond to fractions containing TAβ and monomer Aβ, respectively. The immunoreactivity was collected as a single peak (2) with an apparent molecular weight of about 200 to about 300 kDa. FIG. 5 shows the inability to suppress TAβ formation by the 4396C antibody. Panel (A): ThT fluorescence intensity of an incubation mixture containing arctic Aβ incubated at 37 ° C. in the presence of GM1 ganglioside with 4396C or anti-Aβ monoclonal antibody (4G8). Panel (B): LDH release rate from arctic Aβ-treated PC12N cells pre-incubated in the presence of GM1 ganglioside (10%) in the presence or absence of 4396C antibody. Each column shows the average (± SD) of the three values. * P <0.0001, ns: not significant. FIG. 6 shows TAβ formation by arctic Aβ incubation in the presence of synaptosomes. TAβ formation was assessed by LDH release assay in PC12N cell cultures treated with arctic Aβ. Wh = whole brain excluding hippocampus, Hp = hippocampus. The mouse age is shown below (1 mo = 1 month, 1 yr = 1 year, 2 yrs = 2 years). Each column shows the average (± SD) of the four values. * Represents p <0.0001, and ** represents p <0.005. FIG. 7 shows the characteristics of TAβ toxicity to PC12 or PC12N cells. Panel (A): PC12N cells treated with an incubation mixture containing TAβ for 12 hours. a and b are phase contrast microscopic images, and c and d are Hoechst 33258 nuclear stained images. Arrows indicate nuclear condensation and fragmentation, cell body contraction and neurite retraction. The bar is 50 μm. Panel (B): shows TAβ toxicity to various types of cultured cells. Cell viability was assessed by LDH release assay in these cultures. PC12N = NGF-treated P12 cells, PC12 = native PC12 cells, SH-SY5Y = SH-SY5Y neuroblastoma, hBME = human brain microvascular endothelial cells, HEK293 = HEK293 cells, hAST = human astrocytes, rAST = Rat astrocytes, RAW 264.7 = mouse monocytes, and fibroblast = mouse fibroblasts immortalized with large T antigen. Each column shows the average (± SD) of the three values. * P <0.0001, value when treated with incubation mixture containing only Aβ. FIG. 8 shows that TAβ induces cell death via the NGF receptor. Panel (A): It is a figure which shows suppression of TA (beta) toxicity with respect to native PC12 cell and PC12N cell by addition of exogenous NGF (100 ng / mL). TAβ toxicity was assessed by LDH release assay in these cell cultures. Each column shows the average (± SD) of the three values. * P <0.0001 Panel (B): Inhibition of TAβ toxicity to native PC12 cells by siRNA-mediated knockdown of NGF receptor, TrkA or p75 ^NTR . PC12 cells were exposed to an incubation mixture containing TAβ after treatment with siRNA against TrkA or p75 ^NTR . The graph shows the results of the LDH release assay. Each column shows the average (± SD) of the three values. * Represents p <0.0001. The figure below the graph shows the results of Western blot analysis of cell lysates using anti-TrkA and anti-p75 ^NTR antibodies that confirmed the reduction of TrkA and p75 ^NTR expression levels.

Claims (10)

A soluble toxic amyloid β assembly formed in vitro in the presence of GM1 ganglioside,
(A) an apparent molecular weight of about 200 to about 300 kDa by size exclusion chromatography;
(B) containing spherical particles having a diameter of about 10 to about 20 nm as measured by atomic force microscopy;
(C) free of detectable GM1 ganglioside; and (d) inducing apoptosis-like cell death on cultured cells;
Assembly characterized by.   The assembly according to claim 1, wherein the amyloid β is a wild-type peptide or a peptide having an arctic mutation.   A method for producing a soluble toxic amyloid β assembly, wherein the monomeric amyloid β is combined with a liposome containing about 10 to about 15% GM1 ganglioside by mole ratio of lipid component at about 35 to about 40 ° C. Incubating for a period of time.   The method according to claim 3, wherein the monomeric amyloid β is a wild-type peptide, and the incubation time is 20 to 30 hours.   The method according to claim 3, wherein the monomeric amyloid β is a peptide having an arctic mutation and the incubation time is 1 to 3 hours.   A soluble toxic amyloid β assembly produced by the production method according to claim 3.   The liquid containing the soluble toxic amyloid β assembly according to claim 1, 2 or 6 is centrifuged at 540,000 × g for 15 minutes or under equivalent conditions, and the supernatant is recovered, Purification method of amyloid β assembly.   The method according to claim 7, wherein the recovered supernatant is further subjected to size exclusion chromatography.   A soluble toxic amyloid β assembly purified by the purification method according to claim 7 or 8. A method for screening a substance that modulates the toxicity of soluble toxic amyloid β, comprising the following steps:
(A) a step of incubating cultured cells in the presence of the soluble toxic amyloid β assembly and the test substance according to any one of claims 1, 2, 6 or 9.
(B) after the step (a), measuring the life and death of the cultured cells;
(C) A method comprising a step of comparing the result obtained in the step (b) with a result obtained when the step (a) is carried out without containing a test substance.

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