JP4469281B2 - Rapid induction of Alzheimer's disease amyloid plaque formation by sulfated glycosaminoglycans - Google Patents

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a method for the formation of specific amyloid plaques and the screening application of such plaques in the treatment of Alzheimer's disease and Parkinson's disease.

BACKGROUND OF THE INVENTION Alzheimer's disease is characterized by the accumulation of 39-43 amino acid peptides that are fibrillar, termed as extracellular amyloid plaques, and as amyloid in the cerebral vascular wall, termed beta-amyloid protein or Aβ. Attached. Fibrotic Aβ amyloid deposition in Alzheimer's disease is harmful to the patient and ultimately leads to toxicity and neuronal cell death that are hallmarks of Alzheimer's disease. Various morphologically distinct types of Aβ-containing plaques have been described in the brains of Alzheimer's disease patients, including diffuse plaques (showing Aβ immunoreactivity, such as Congo Red and Thioflavin S) Not stained for fibrillar amyloid), neuritic plaques (containing a central amyloid core stained with Congo Red and Thioflavin S and surrounded by dystrophic neurites) and dense Includes burned-out plaques or “amyloid star” plaques (stained with Congo red and usually show a Maltese cross pattern when viewed under polarized light). In vitro formation of dense plaques exhibiting a Maltese cross pattern when stained with Congo Red and observed under polarized light has not been achieved previously.

  The development of amyloid star plaques in the brain of human Alzheimer's disease is generally considered in the art to take years to form and accumulate. However, once they appear, these plaques are then resistant to natural proteases from the brain or other clearance mechanisms. Therefore, any agent that helps dissolve or modify these amyloid star plaques to make them more susceptible to protease digestion and clearance is a good candidate for treating Alzheimer's disease. Let's go.

  In the art, screening of such candidate agents against amyloid star plaques in vitro is achieved only by physically extracting and isolating amyloid plaques from Alzheimer's brain after death. The This process is not only technically cumbersome but also very time consuming and the amount of amyloid star plaques obtained is very small and as such is very variable. Furthermore, in the United States, only a few selected centers have access to the Alzheimer's brain bank and have their own banks.

Diffuse, neurite and dense (“amyloid star”) plaques Neuritic plaques are considered more mature and contain dystrophic neurites surrounding a spherical amyloid plaque core (Barcikowska et al., Acta). Neuropath.78: 225-231, 1989; Ikeda et al., Lab.Invest. 60: 113-122, 1989; Masliah et al., J. Neuropath. Exp. Neurol. 52: 619-632, 1993). The amyloid core within these plaques is Aβ immunopositive and stained with Congo Red and Thioflavin S. Furthermore, amyloid plaque cores within neurite plaques are usually spherical and resemble a Maltese cross pattern when stained with Congo red and viewed under polarized light (Ikeda et al., Lab. Invest. 60: 113-122, 1989; Wisniewski et al., Acta Neuropath. 78: 337-347, 1989; Schmidt et al., Am. J. Path. 147: 503-515, 1995). Amyloid cores in neurite plaques resemble amyloid stars when observed with an electron microscope (Wisnewski et al., Acta Neuropath. 78: 337-347, 1989). Dense amyloid core (also called burnout or core plaque) is also similar to amyloid star when observed with an electron microscope (Selkoe et al., J. Neurochem. 46: 1820-1834, 1986; Snow et al., Am). J. Path. 133: 456-463, 1988) and is generally considered to correspond to a more mature form of plaque formation (Wisniewski et al., Acta Neuropath. 78: 337-347, 1989; Schmidt et al., Am J. Path. 147: 503-515, 1995; Dickson, J. Neuropath. Exp. Neurol. 56: 321-339, 1997). These globular plaques are Aβ immunopositive and stained with Congo red (again resembling a Maltese cross when viewed under polarized light) and Thioflavin S.

  Previously, no one in the art had caused in vitro the formation of such plaque deposits similar to those found in the brains of Alzheimer's disease patients. Using such in vitro plaque formation, it is also possible to evaluate and identify agents that have unique anti-plaque treatment potential and can serve as novel approaches for the treatment of Alzheimer's disease. There is a need for compounds and assay techniques that can be used to screen and identify agents that may inhibit or disrupt the development of amyloid plaques. Such compounds and methods would be useful in assessing amyloid plaque formation associated with the onset and progression of Alzheimer's disease and Parkinson's disease.

  Thus, there is a need to generate amyloid star plaques in vitro, and that need has faced many technical problems, as previously disclosed in the parent application. There is still a need for a method of forming amyloid star plaques in higher yields to further test in vitro potential candidates that may serve as a treatment for Alzheimer's disease and Parkinson's disease. In vitro amyloid plaques, as such, ideally exhibit a Maltese cross pattern when observed under polarized light when stained with properties known to in vivo amyloid plaques, such as stability, protease resistance, and Congo red Maintain properties. Finally, in vitro amyloid plaque formation must occur more rapidly and in large quantities than the time it takes in the human brain.

DISCLOSURE OF THE INVENTION This application is a continuation-in-part of application 10 / 007,779, filed November 30, 2001, and provisional application 60 / 423,185, filed November 1, 2002. Requesting priority, both of which are hereby incorporated by reference as if fully set forth herein.

  Congophilic maltese-cross dense amyloid plaques (ie, “dense plaques” or “amyloid stars”) that are substantially identical to the congophilic maltese-cross dense plaques present in human Alzheimer's disease brain Disclosed is a method for rapid formation in vitro. Also disclosed are methods for consistently forming such Alzheimer's plaques for use in several different assay techniques and animal models that identify anti-plaque agents for Alzheimer's and Parkinson's disease. Beta-amyloid protein (Aβ) (residues 1-40 but not residues 1-42) and highly sulfated glycosaminoglycans (GAG) or related sulfated macromolecules As further disclosed in the specification and under the appropriate molar ratio / weight ratio of Aβ: sulfated proteoglycan / GAG, rapid and large quantities of dense amyloid plaques formed after these co-incubations The formation of is disclosed.

  Such dense Congo-affected maltese-cross amyloid plaques may not form after incubation with Aβ1-40 or Aβ1-42 alone (up to 1 week at 37 ° C.). Preferred in vitro conditions for inducing amyloid plaque formation are not Aβ42 but Aβ40 sulfated GAG (such as heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate) or GAG related macromolecules (dextran sulfate) ).

  4-65 μm amyloid plaques (mean diameter = 25-26 μm) formed by co-incubation of Aβ1-40 with highly sulfated GAG or related sulfated macromolecules are present in the brain of human Alzheimer's disease It has all the characteristics of dense amyloid plaques: 1) amyloid plaques showing a Maltese cross pattern when stained with Congo red and observed under polarized light, 2) when observed with a transmission electron microscope, Included are spherical “amyloid star” forms with radioid bundles of amyloid fibrils (each having a fibril diameter of 7-10 nm) that appear to radiate from the center of the plaque. Other characteristics of these plaques are: a) spherical or dense shape, b) Congo affinity Maltese cross pattern when stained with Congo red and observed under polarized light (ie against the green color of the plaque) Red), c) positive staining with thioflavin S when observed with a fluorescence microscope, d) spherical appearance and / or “amyloid star” appearance when observed with a transmission electron microscope E) Spherical or dense shape (spots with a diameter of 10-40 μm) and / or f) resistance to protease degradation when observed with a scanning electron microscope. Also disclosed is the use of such amyloid plaques formed in vitro as a screening tool to identify anti-plaque agents for Alzheimer's disease and Parkinson's disease.

  All highly sulfated glycosaminoglycans (GAGs), such as those disclosed herein, and related sulfated GAG macromolecules such as dextran sulfate are all beta-amyloid protein (Aβ) (residual Groups 1-40) were found to be induced in vitro to convert to amyloid plaque deposits substantially identical to the Congophilic maltese-cross dense amyloid plaques present in the brain of human Alzheimer's disease.

  Changing temperature (−80 ° C. to 45 ° C.), optimal initial concentration of Aβ40 (25, 37.5, 50, 62.5, 75, 87.5, 100, 112.5, 125, 250, and 500 μM) Various sulfated GAGs (heparin, heparan sulfate (from multiple sources), keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate) or sulfated GAG related macromolecules (dextran) for co-incubation Sulfuric acid), different ratios of Aβ40: sulfated GAG or macromolecules (1: 0.01, 1: 0.05, 1: 0.1, 1: 1, 1: 2, 1: 3, 1: 4, 1 : 5, 1: 6, 1: 7, 1: 8, 1: 9, 1:10, 1:12, 1:14, 1:16, 1:18, 1:20 wt / wt), sonication With or without sonication, different ranges different, including pH (3.0-9.5) and optimal incubation time (0, 1, 2, 3, 5, 7, 10, 12, 15, 17, 20, 24, 26, 28, 30 days) The conditions were examined to establish a consistent in vitro amyloid plaque formation method. Sonication is a term known to those skilled in the cell research industry. Disclosed herein are optimal conditions within these disclosed ranges that induce the formation of dense Congophilic maltese-cross “amyloid star” plaques, as observed in Alzheimer's disease brains.

  Consistent amyloid plaque formation in vitro is optimized not only by co-incubation of highly sulfated GAG or GAG-related macromolecules with Aβ1-40, but also by the quality of Aβ1-40 used. Some properties of preferred Aβ1-40 are such that the thioflavin T fluorometric reading upon initial solubilization is between 400 and 4000 FU (excitation 444 nm / emission 485 nm), preferably FU to 2000. Is a low fibril; Aβ1-40 in 1 × TBS is at a relatively neutral pH (pH˜6.5-7.5); and the maximum concentration of Aβ1-40 is 1 mg / ml or 250 μM It is also preferred not to exceed.

  Light microscopy (as discussed further herein) shows that amyloid plaques formed by co-incubation of highly sulfated GAG or highly sulfated GAG-related macromolecules in addition to Aβ (1-40) Show substantially the same properties and morphology as those of the amyloid plaque core isolated from human Alzheimer's disease brain. With these findings, the congophilic Maltese cross and dense amyloid plaques observed in Alzheimer's disease brains are formed over time by the simultaneous deposition and accumulation of highly sulfated GAG and Aβ1-40 It is shown that there is a possibility.

  Disclosed are methods for forming congophilic maltese-cross dense amyloid plaques in vitro using Aβ and GAG and / or GAG-related macromolecules, or portions thereof. Such GAGs include heparan sulfate, heparin, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, keratan sulfate, hyaluronic acid, and dextran sulfate. In preferred embodiments, such dense amyloid plaque formation comprises Aβ1-40 and GAG sulfate at 25 ° C. to 40 ° C., and preferably at about 37 ° C., and an appropriate Aβ: GAG weight ratio as described herein and Achievable by co-incubation under a molar ratio.

Disclosed is a method for inducing amyloid plaques, a) immobilizing an amount of a selected sulfated glycosaminoglycan (SGAG) or GAG-related macromolecule on a selected medium;
b) Said method comprising the step of adding a certain amount of dissolved low fibrillar Aβ1-40 (LFAβ) to SGAG immobilized on the medium.

  In this method, LFAβ is Aβ: SGAG weight / weight (w / w) ratio in the range between 1: 0.01 and 1:20, or in the range between 1: 0.1 and 1:10. Aβ: SGAG w / w ratio, preferably an Aβ: SGAG w / w ratio in the range between 1: 0.5 and 1: 2, and preferably an Aβ: SGAG w / w ratio of about 1: 1. Is added. The medium chosen is either a slide, film, or titer well plate, and the preferred titer well plate is an 18-96 well Teflon split slide. SGAG is preferably selected from heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate and chondroitin-6-sulfate, and the GAG-related macromolecule is preferably dextran sulfate. LFAβ is preferably a small amount in wells demarcated with Teflon so that the Teflon partially repels the liquid and retains the liquid in the bubble on the support, in this case fixed SGAG. The liquid is added to the immobilized SGAG by a bubbling technique involving pipetting.

A method for screening selected amyloid therapeutic candidates:
a) immobilizing an amount of a selected sulfated glycosaminoglycan (SGAG) or GAG-related macromolecule on a selected medium;
b) To an amount of dissolved low fibrillar Aβ1-40 (LFAβ), a selected amount of a selected amyloid therapeutic agent candidate is added to produce a test solution;
c) adding a selected amount of the test solution to SGAG immobilized on the medium;
This allows the percent inhibition in amyloid plaque formation as compared to the test reference sample prepared as described above without the selected amyloid therapeutic candidate to be the percent effectiveness of the selected amyloid therapeutic candidate. The method is also disclosed, which is indicative.

  In this screening method, the details of the Aβ: SGAG weight / weight (w / w) ratio range are as disclosed above with respect to the plaque formation method, and the selection range of the selected medium and the preferred SGAG are also listed above. As disclosed.

Another method for screening selected amyloid therapy candidates is:
a) immobilizing an amount of a selected sulfated glycosaminoglycan (SGAG) or GAG-related macromolecule on a selected medium;
b) adding a selected amount of dissolved low fibrillar Aβ1-40 (LFAβ) to SGAG immobilized on the medium to form amyloid plaques on the medium;
c) adding to the amyloid plaques on the medium a selected amount of the test solution of the selected candidate amyloid therapeutic agent;
This allows the percent destruction of amyloid plaques on the media when compared to a test reference sample prepared as described above without the selected amyloid therapeutic agent candidate to be effective for the selected amyloid therapeutic agent candidate. Percentage indicator.

  A kit for screening selected amyloid therapeutic candidates, wherein a quantity of sulfated glycosaminoglycan (SGAG) immobilized on a medium and a quantity of low fibrillar Aβ1-40 (LFAβ) as described above A) a kit for inhibition testing with screening instructions, and a quantity of amyloid plaques pre-formed on media as disclosed above, and screening instructions as described above, Another kit for destruction is disclosed.

  Inhibition and destruction of amyloid plaque formation as discussed herein, as will be appreciated by those skilled in the art by comparing plaque morphology, number of plaques, or other measurements known to those skilled in the art It is also possible to quantitate these inhibitions and disruptions, and after testing with protease degradation or microglia degradation, the affected plaques will now have natural clearance. Includes measurements confirming that they are not subject to the specific amyloid-star plaques discussed herein, both in Alzheimer's disease brain and synthesized, which are subject to It is also possible.

  In one embodiment, Aβ1-40 is utilized with heparin (either low or high molecular weight) to form Congophilic maltese-cross dense amyloid plaques. In this embodiment, Aβ1-40 in the range of 10 μM to 250 μM is mixed with heparin in distilled water, PBS or Tris buffered saline (pH 7.4), and A0.0: heparin. Incubate at 37 ° C. within a molar ratio and preferably about 1: 0.5 μM.

  In another embodiment, Aβ1-40 is utilized with non-anticoagulant heparin to form a congophilic maltese-cross dense amyloid plaque. In this preferred embodiment, 10 μM to 250 μM Aβ1-40 is added to non-anticoagulant heparin, heparin-like molecule, or fragment thereof in distilled water, PBS or Tris buffered saline (pH 7.4) 1: 0. Incubate at 37 ° C. within a molar ratio of Aβ: non-anticoagulant heparin from 0.02 to 1: 100, and preferably about 1: 0.5 μM.

  In another embodiment, Aβ1-40 is utilized with heparan sulfate to form Congophilic maltese-cross dense amyloid plaques. In this embodiment, Aβ1-40 is combined with heparan sulfate in distilled water, PBS or Tris buffered saline (pH 7.4) within a range of Aβ: heparan sulfate weight ratio of 1: 0.1 to 1: 100. And preferably at about 1: 1 at 37 ° C. This same range and selection applies to other sulfated GAGs such as dermatan sulfate, keratan sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate.

  In another embodiment, Aβ1-40 is utilized with dextran sulfate to form Congophilic maltese-cross dense amyloid plaques. In this preferred embodiment, 10 μM to 250 μM Aβ1-40 is added to dextran sulfate and 1: 0.02 to 1: 100 Aβ: dextran sulfate in distilled water, PBS or Tris buffered saline (pH 7.4). Incubate at 37 ° C. within a molar ratio and preferably about 1: 0.5 μM.

  In another embodiment, Aβ1-40 is utilized with pentosan polysulfate to form a Congophilic maltese-cross dense amyloid plaque. In this preferred embodiment, 10 μM to 250 μM Aβ1-40 is added to pentosan polysulfate and 1: 0.02 to 1: 100 Aβ in distilled water, PBS or Tris buffered saline (pH 7.4): Incubate at 37 ° C. within the molar ratio of pentosan to polysulfate and preferably about 1: 0.5 μM.

  In another embodiment, Aβ1-40 is utilized with polyvinyl sulfonic acid to form Congophilic maltese-cross dense amyloid plaques. In this preferred embodiment, Aβ1-40 is combined with polyvinyl sulfonic acid in distilled water, PBS or Tris buffered saline (pH 7.4) and an Aβ: polyvinyl sulfonic acid weight ratio of 1: 0.1 to 1: 100. And at about 1: 1, preferably at 37 ° C.

  An anti-plaque therapeutic agent that inhibits, destroys or removes Congophilic maltese-cross spherical amyloid plaques in vitro, and a screening method for identifying the agent that can be identified using polarized light microscopy For the use of the amyloid plaques produced herein. In such preferred embodiments, amyloid plaque cores that initially exhibit a typical Maltese cross pattern when stained with Congo Red and viewed under polarized light are first formed in vitro. After incubation with the test compound (with appropriate dosage and incubation time determined experimentally), the amyloid plaque core is observed under a polarizing microscope so that there is a loss of Congo affinity and / or Maltese cross formation Determine whether a given compound or agent is capable of inhibiting, destroying or removing the amyloid plaque structure. First, these compounds identified by such polarization microscopy techniques are further analyzed in transmission and / or scanning electron microscopy and / or secondary or tertiary assays utilizing protease digestion to detect plaque inhibition. It is also possible to confirm destruction or removal.

  In yet another preferred embodiment, anti-plaque that inhibits and destroys the structure (ie size and / or diameter) of globular amyloid plaques in vitro using the dense amyloid plaques produced herein for screening methods A therapeutic agent is identified that is identifiable using a methodology that involves a cell sorter. In such assays, dense spherical amyloid plaques formed in vitro can be passed through a cell sorter to determine the average diameter (and diameter range) of these plaques. These plaques are then incubated with various compounds or agents (with a predetermined dosage and incubation time determined experimentally), and then again through a cell sorter, the predetermined compound shatters these plaques. It is also possible to determine whether it was effective to disturb the size (and thus the diameter).

BEST MODE FOR CARRYING OUT THE INVENTION
Definitions As used herein, refers to amyloid plaques in the brain of human Alzheimer's disease that are immunoreactive with a variety of different anti-Aβ antibodies but generally are not stained for fibrillar amyloid (ie, Congo Red, Thioflavin S) The term “diffuse plaques” is used (Ikeda et al., Lab. Invest. 60: 113-122, 1989; Verga et al., Neurosc. Lett. 105: 294-299, 1989).

  As used herein, the term “neurite plaque” is used to refer to a plaque in a human Alzheimer's disease brain that contains a dystrophic neurite surrounding a spherical amyloid plaque core (Barcikovska et al., Acta Neuropath. 78: 225). 23, 1989; Ikeda et al., Lab. Invest. 60: 113-122, 1989; Masliah et al., J. Neuropath. Exp. Neurol. 52: 619-632, 1993). The amyloid core within these plaques is Aβ immunopositive and stained with Congo Red and Thioflavin S. Furthermore, amyloid plaque cores within neurite plaques are usually spherical and resemble a Maltese cross when stained with Congo red and viewed under polarized light (Ikeda et al., Lab. Invest. 60: 113). -122, 1989; Wisniewski et al., Acta Neuropath. 78: 337-347, 1989; Schmidt et al., Am. J. Path. 147: 503-515, 1995).

  As used herein, the term “dense” or “burn-out” plaques refers to plaques in the brain of human Alzheimer's disease or prion disease, which are generally considered to correspond to more mature forms of plaque formation. (Wisniewski et al., Acta Neuropath. 78: 337-347, 1989; Schmidt et al., Am. J. Path. 147: 503-515, 1995; Dickson, J. Neuropath. Ol. 1997). These globular plaques are immunopositive for Aβ or prion protein, and are stained with Congo red (also similar to the Maltese cross when viewed under polarized light) and Thioflavin S. “Dense” or “burn-out” plaques also exhibit a Maltese cross pattern when stained with Congo Red and observed under polarized light.

  As used herein, the term “congo affinity” is used to describe fibrillar amyloid deposits that exhibit red / green apple birefringence when stained with Congo Red and viewed under polarized light. Congophilic deposits do not necessarily exhibit the Maltese cross pattern (see below for definition).

  The term “Malta cross” is a spherical and dense amyloid that stains with Congo red and exhibits a Maltese cross pattern (ie, red is 90 degrees to the green apple color) when viewed under polarized light. Refers to spots. As the polarization device rotates, a shift in the color of the plaque occurs so that the red turns green apple and the green apple turns red (ie, red / green birefringence). Amyloid plaques formed in vitro as described in the present invention, amyloid cores of neurite plaques in the brain of human Alzheimer's disease, “dense” or “burn-out” plaques in the brain of human Alzheimer's disease, and humans All amyloid plaques in the cerebellum of Creutzfeldt-Jakob disease, Gerstmann-Stroisler syndrome and Kourou exhibit a “Maltese cross” pattern when stained with Congo red and observed under polarized light.

  As used herein, the term “amyloid star” is used to refer to “dense” amyloid plaques or “burn-out” amyloid plaques that resemble star deposits of amyloid when viewed with an electron microscope (Selkoe et al., J. Neurochem. 46: 1820-1834, 1986; Snow et al., Am. J. Path. 133: 456-463, 1988). The appearance of the “amyloid star” of the plaque is due to the radial bundle structure of amyloid fibrils that appear to be emitted from the center of the plaque.

  As used herein, the term “induction” or “formation” is used to refer to dense amyloid plaques formed in vitro when incubated under appropriate conditions. When discussing with respect to derivation or formation, within the scope of the disclosed method optionally includes gentle mixing of the incubation components.

  As used herein, a) directly lysing, inhibiting or destroying the structure, staining properties, or structure of the dense plaque, and / or b) the dense plaque is in other cells (ie, neurons). The term “anti-plaque agent” is used to refer to a compound or agent that is effective in inhibiting a deleterious effect (ie, neurotoxicity) that may have on a tissue or organ.

  The term “beta-amyloid protein (Aβ1-40, also referred to herein as Aβ40)” refers to SEQ ID NO: 1 and is known for human disease (eg, a single amino acid substitution in Aβ1-40) Including all single amino acid substitutions or multiple amino acid substitutions occurring in Alzheimer's disease), or species mutations (rodent Aβ1-40, known to have 3 amino acid differences compared to human Aβ1-40) Is possible.

The following examples, figures and discussion are illustrative of embodiments of the invention and are not meant to limit the scope of the invention.
FIG. 1 shows that perlecan forms a congophilic maltese-cross spherical amyloid plaque in vitro, but other amyloid plaque-related macromolecules known to exist in the brain of human Alzheimer's disease may cause amyloid plaques in vitro. It is not formed by. In these studies, 25 μM Aβ (1-40) was used alone (FIG. 1C), or 100 nM P component (FIG. 1D), alpha 1-antichymotrypsin (FIG. 1E), apoE (FIG. 1F), Double distilled water or Tris buffered saline in the presence of C1q (FIG. 1G), laminin (FIG. 1H), fibronectin (FIG. 1I), collagen type IV (FIG. 1J) or perlecan (FIGS. 1K and 1L) And incubated at 37 ° C. On a gelatin-coated slide, a 5 fl aliquot of the incubation mixture was air dried, stained with Congo Red, and observed under polarized light. Preincubation of Aβ1-40 and perlecan at 37 ° C. with a preferred Aβ: perlecan molar ratio of 250: 1 (ie, a weight ratio of 1: 0.8) results in the formation of Congophilic maltese-cross spherical amyloid plaque-like deposits. Induced (FIGS. 1K and 1L). Using 125 μM Aβ (1-40) and 0.625 μM perlecan (ie 200: 1 Aβ: perlecan molar ratio; 1: 1 Aβ: perlecan weight ratio), similar amyloid plaque formation was observed, With other amyloid plaque co-components listed above with 125 μM Aβ (1-40) and 0.625 μM, amyloid plaque formation was not observed (not shown). The amyloid plaques induced by perlecan were substantially identical in morphology and staining characteristics (ie Maltese cross after Congo red staining) to dense amyloid plaques in human Alzheimer's disease brains (FIGS. 1K and 1L). Compare to 1A). Bars in Figures A, B and K = 25 μm. Figures A, C and H are taken at the same magnification, and Figures B, D-G and I-J are also at the same magnification.

  FIG. 2 is congophilic and Maltese cruciform with highly sulfated glycosaminoglycans (ie heparin and heparan sulfate) and related sulfated macromolecules (ie dextran sulfate, pentosan polysulfate) Figure 2 shows in vitro formation of amyloid plaques. In these studies, 25 μM Aβ1-40 alone (FIG. 2B) or various amounts of heparin (FIG. 2C), heparan sulfate (FIG. 2D), dermatan sulfate (FIG. 2E), Congo red (FIG. 2F). , Pentosan polysulfate (FIG. 2G), or in the presence of dextran sulfate (FIG. 2I) in double distilled water or Tris buffered saline (pH 7.4) at 37 ° C. On a gelatin-coated slide, a 5 fl aliquot of the incubation mixture was air dried, stained with Congo Red, and observed under polarized light. Preliminary experiments show that the optimal Aβ: GAG / sulfated macromolecular ratio for dense amyloid plaque formation is a 1: 5 molar ratio for heparin, dextran sulfate and pentosan polysulfate, with Aβ1-40 maintained at 25 μM, And for heparan sulfate it was determined that the weight ratio was 1: 8. Similar results were obtained when 125 μM Aβ1-40 was used in double distilled water. Preincubation of Aβ1-40 with heparin, heparan sulfate, pentosan sulfate, or dextran sulfate at 37 ° C at the same molar ratio / weight ratio induced the formation of congophilic maltese-cross sphere amyloid plaque-like deposits (Fig. 2C, 2D, 2G-2I). The amyloid plaques induced by these highly sulfated GAGs and related sulfated macromolecules were substantially identical to the dense amyloid plaques of human Alzheimer's disease brain (compare with FIG. 2A). . Bars in Figures A, B and I = 25 μm. Figures A, C, D and H are taken at the same magnification, and Figures B and E are also at the same magnification.

  FIG. 3 shows in vitro formation of amyloid plaques that are congophilic and maltese cruciform with polyvinyl sulfonic acid (PVS), and the change in the weight ratio of Aβ: PVS shows the potential of dense amyloid plaque formation How it affects. In these studies, 50 fg of Aβ1-40 in double-distilled water was added to 25 fg PVS (2: 1 Aβ: PVS weight ratio) (FIG. 3A), 50 fg PVS (1: 1 Aβ: PVS weight) in a total volume of 100 fl. Ratio) (FIG. 3B), 200 fg PVS (1: 4 Aβ: PVS weight ratio) (FIG. 3C), 250 fg PVS (1: 5 Aβ: PVS weight ratio) (FIG. 3D), 400 fg PVS (1: 8 Aβ: PVS weight ratio) (FIG. 3E), 500 fg PVS (1:10 Aβ: PVS weight ratio) (FIG. 3F), 800 fg PVS (1:16 Aβ: PVS weight ratio) (FIG. 3G), 2000 fg PVS ( At 37 ° C. in the presence of increasing amounts of PVS, including 1:40 Aβ: PVS weight ratio (FIG. 3H), and 4000 fg PVS (1:80 Aβ: PVS weight ratio) (FIG. 3I). It was down incubation. On slides coated with gelatin, 5 fl or 10 fl aliquots of the incubation mixture were air-dried, stained with Congo Red, and observed under polarized light. Congophilic maltese-cross spherical amyloid plaque formation was induced by PVS, but only when the Aβ: PVS weight ratio was 1: 5 or more. Optimal amyloid plaque core formation was observed at an Aβ: PVS weight ratio of 1:40 (FIG. 3H). Bar in Figures A and C = 25 μm. Figures A, B, H and I are taken at the same magnification, and Figures C-G are also at the same magnification.

  FIG. 4 shows congophilic maltese-cross dense amyloid plaque formation induced by a ~ 220 kDa heparan sulfate proteoglycan (HSPG) isolated from an Engelbreth-Form-Sworm tumor. 50 fg Aβ (1-40) in 100 fl Tris buffered saline (pH 7.4) either alone or in the presence of 10 fg of ~ 220 kDa HSPB (5: 1 Aβ: HSPB weight ratio). And incubated at 37 ° C. FIG. 4A shows irregular Congophilic amyloid deposits (arrows) formed after 1 week incubation of Aβ alone, in which no obvious Congophilic Maltese cross amyloid plaques are formed. FIG. 4B shows Congophilic maltese-cross amyloid plaques (arrowheads) formed after 1 week incubation of Aβ1-40 with ˜220 kDa HSPG. The amyloid plaques formed were identical to the dense plaques present in the human Alzheimer's disease brain (see FIGS. 1A and 2A). Figures A and B are taken at the same magnification. Bar = 25 μm.

  FIG. 5 shows in vitro formation of spherical amyloid plaques induced by perlecan as appearing to be fixed in plastic. In this study, 125 μM Aβ1-40 was incubated at 37 ° C. in double distilled water in the presence of 0.625 μM perlecan (200: 1 Aβ: perlecan molar ratio; 1: 1 Aβ: perlecan weight ratio). did. A 10 fl aliquot of the incubation mixture was then air dried for 1 hour on a plastic petri dish and then fixed in situ with 3% glutaraldehyde in 0.1 M NaPO4 buffer (pH 7.3) for 10 minutes. After rinsing three times with filtered distilled water, it was post-fixed with 1% osmium tetroxide in distilled water for 10 minutes, rinsed as before, and air dried overnight. This figure shows amyloid plaque-like deposits as seen in plastics, induced in perlecan and observed with a phase contrast optical microscope. Perlecan induced Aβ to form globular amyloid plaque deposits (FIGS. 5A and 5B, arrowheads), which corresponded to a radial bundle of amyloid fibrils that appeared to be emitted from the central source. These plaques formed resemble amyloid plaque cores isolated from human Alzheimer's disease brain. Bar = 25 μm.

  FIG. 6 shows perlecan-induced spherical “amyloid star” formation, substantially identical to an isolated amyloid plaque core from human Alzheimer's disease brain, as observed by transmission electron microscopy. Show. In this study, 125 μM Aβ1-40 was incubated at 37 ° C. in double distilled water in the presence of 0.625 μM perlecan (200: 1 Aβ: perlecan molar ratio; 1: 1 Aβ: perlecan weight ratio). did. Perlecan-induced amyloid plaque cores (FIGS. 6C and 6D) formed amyloid stars with a radial bundle of amyloid fibrils that appeared to be emitted from a central source. The diameter of individual amyloid fibrils was determined to be 7-10 nm. These amyloid plaques produced in vitro were substantially identical to the amyloid plaque core isolated from human Alzheimer's disease brain (FIGS. 6A and 6C). Bar = 2 μm. Figures A and B have the same magnification, and Figures C and D have the same magnification.

  FIG. 7 shows amyloid plaque core formation induced by perlecan or dextran sulfate and observed by scanning electron microscopy. In this study, 125 μM Aβ1-40 was used alone (FIG. 7B) or 0.625 μM perlecan (200: 1 Aβ: perlecan molar ratio) (FIGS. 7D and 7E) or dextran sulfate (1: 5 Incubate at 37 ° C. in double distilled water either in the presence of Aβ: dextran sulfate molar ratio). Furthermore, only 0.625 μM perlecan was incubated at 37 ° C. (FIG. 7C). No amyloid plaque core formation was observed after 1 week incubation with Aβ (FIG. 7B) or perlecan (FIG. 7C) alone. However, dense amyloid plaque formation was induced by Aβ in the presence of perlecan (FIGS. 7D and 7E) or dextran sulfate (FIG. 7F). The shape and general morphology of amyloid plaques induced by perlecan or dextran sulfate are similar to the shape and general morphology of amyloid plaque cores isolated from human Alzheimer's disease brain when observed by scanning electron microscopy It was. The magnification is shown below each figure.

Examples The following examples are provided to disclose in detail preferred embodiments of in vitro formation of amyloid plaque cores induced by highly sulfated GAGs and related sulfated macromolecules. However, it should not be construed that the present invention is limited to these specific embodiments, since variations in timing, volumetric measurements and concentrations can be achieved by those skilled in the art without departing from the scope of the present invention. . In general, variations of these amounts by as much as 50%, and especially as much as 10-20%, will still yield good results if not optimal.

Rapid induction of plaques Method 1: In vitro plaque formation in solution with sonication:
1) Thaw a 1 mg bin of lyophilized Aβ40 from Recombinant Peptides (Catalog No. A-1052-2, 245 University Circle, Athens, GA 30605, USA www.rpeptideide.com) 2) Final concentration of 5 mg / ml in 1 × TBS 3) Vortex and mix for 5 seconds 4) Add 2 ml of stock GAG solution (5 mg / ml) to 1 mg lyophilized bottle of Aβ40 5) Vortex and mix for 5 seconds 6) Transfer 500 μl / tube to 1.5 ml polypropylene microcentrifuge tube 7) Vortex for 5 seconds to mix 8) Level 7 at 5 ° C. for 5 minutes sonicate 9) Cover the tube with parafilm To prevent evaporation 10) Dark place without vibration Between 1 to 14 days, preferably 5 days incubation at 37 ° C..

Method 2: In vitro plaque formation on immobilized sulfated GAG:
1) Make a sulfated GAG stock solution in water at a concentration of 1-10 mg / ml, preferably 1 mg / ml 2) Pipette 50 μl GAG / well in triplicate onto a glass slide coated with blue Teflon 3) Air-dry the spots at 25 ° C. to make a thin film 4) Make an Aβ40 solution by adding 1 ml 1 × TBS directly to 1 mg lyophilized Aβ40 to a final concentration of 1 mg / ml 5) Vortex for 5 seconds to mix and check the initial thio T reading. Continue if initial thio-T reading is 4000 FU (excitation 444 nm, issue 485 nm) or less 6) Pipette 50 μl of 1 mg / ml Aβ40 / well bubble or spot onto immobilized GAG 7) Sealed humidified chamber (This can simply consist of a Kimwipe saturated with water in a Tupperware container that is sealed when the lid is closed) 8) Put the slide in 8) 8-37 hours at 37 ° C without shaking. Incubate preferably for about 24 hours 9) Congo red stain according to the described staining protocol 10) Observe under polarized light In vitro amyloid plaques Congo red staining method:
1) Aliquot 10 μl in vitro plaque solution / well onto blue Teflon coated glass slide containing 18 wells 2) Add 5 μl Congo Red / well (125 μM stock) to in vitro plaque solution, and Stain for 10 minutes at 25 ° C. 3) Add 5 μl of 50% glycerol / well 4) Air dry slides to a total volume of approximately 5-10 μl / well 5) Each stained well with 5 mm coverslip (Belco 6) Observation under polarized light (Example 1)
In vitro plaque formation using different sulfated GAGs or GAG-related macromolecules FIG. 8 shows several different sulfations at various weight ratios tested, including 1: 0.1, 1: 1, and 1:10. Figure 5 shows in vitro formation of GAG-induced Congophilic maltese-cross spherical amyloid plaques. In these studies, 50 microliters of GAG per well (10 mg / ml stock at 1:10 w / w, 1 mg / ml stock at 1: 1 w / w, 0 at 1: 0.1 w / w) .1 mg / ml stock) were spotted onto Teflon-coated 18-well glass slides and air dried to film. Once GAG was immobilized on the glass surface, 50 microliters of Aβ1-40 solubilized in 1 × TBS (pH 7.4) at a stock concentration of 1 mg / ml was spotted onto each well. The assay slide was placed in a humidified chamber for 18 hours at 37 ° C. without shaking. The wells were then stained with 5 μl of 125 μM Congo Red solution for 15 minutes, after which 10 microliters of 50% glycerol solution was added per well. The glass slide wells were then air dried to a volume of approximately 5 microliters per well and then a 5 mm cover slip was placed. Congophilic maltese-cross amyloid plaques were observed under a polarizing microscope. The investigated sulfated GAGs include heparin, heparan sulfate (pig mucosa intestinal tract; HS (pim)), dermatan sulfate (DermS), chondroitin-4-sulfate (C-4-S), chondroitin-6-sulfate (C- 6-S), and keratan sulfate (keratan S). Sulfated GAG-related macromolecules tested include dextran sulfate (DexS) and polyvinyl sulfonic acid (not shown).

(Example 2)
Amyloid star plaques and corresponding electron micrographs (EM) in the brain of human Alzheimer's disease compared to Maltese cross in vitro plaques
FIG. 9 shows the similarity in physical properties between amyloid plaques formed in vitro and amyloid star plaques in Alzheimer's disease brain. A) Congophilic maltese cross plaques stained with Congo red, which are embedded in postmortem human Alzheimer's disease brain tissue. B) Congo-affected maltese-cross plaques formed in vitro, showing the same Maltese cross properties as observed under polarized light. C) Scanning electron microscope observation of single amyloid star plaques isolated from human Alzheimer's disease brain. D) Scanning electron microscope observation of single amyloid star plaques formed in vitro. Note that the spherical shapes are similar and close in size.

(Example 3)
In Vitro Maltese Cross Plaque Formation with and without Sonication FIG. 10 shows a rapid sonication method for in vitro plaque formation in Tris-buffered saline (pH 7.4) solution. In these studies, 200 microliters of 250 μM Aβ40 was solubilized in 1 × Tris buffered saline (pH 7.4) and 200 microliters was used to give a final molar ratio of 1: 5 or a weight to weight ratio of 1:10. Mixed with or without 10 liters of 10 mg / ml dextran sulfate. The mixture was then subjected to a simple 5 minute sonication or no sonication and was then incubated at 37 ° C. for 3-7 days, preferably 5 days. A) Aβ40 alone in TBS without sonication showed congophilic material but no red / green birefringence or Maltese cross formation. B) In the case of Aβ40 alone in TBS which was sonicated for 5 minutes, maltese cruciform formation still does not occur and only a Congo-affinity stained material is seen. C) Aβ40 in the presence of dextran sulfate without sonication produces very small amounts of Maltese cross plaques in vitro. Instead, evidence of red / green birefringence when stained with Congo Red and observed under polarized light shows highly aggregated amyloid formation. D) Aβ40 in the presence of dextran sulfate, subjected to a simple 5 minute sonication, produced large amounts of Congophilic maltese-cross plaques.

Example 4
In Vitro Maltese Cross Spot Formation in Immobilized GAG Method FIG. 11 shows a rapid method of in vitro plaque formation in Tris-buffered saline (pH 7.4) on immobilized GAG or GAG-related macromolecules . For this study, 18-well glass slides coated with Teflon were coated with either TBS, distilled deionized water, or 50 microliters of dextran sulfate per well (10 mg / ml stock in deionized water) and air dried. And made a thin transparent film. Then 50 microliters of 250 μM Aβ40 in 1 × TBS was added per well and the slides were placed in a humidified chamber and incubated at 37 ° C. for 12-24 hours. The incubation slides were then stained with 5 microliters of 125 μM Congo red solution per well and viewed under polarized light with a 2 × objective, indicating that a large amount of Maltese cross plaque formation was seen. A) TBS alone; B) Aβ40 alone in TBS; C) Aβ40 on immobilized dextran sulfate in TBS. Using this method, more than 1,000 plaques / ml can be formed in less than 24 hours.

  FIG. 12 shows Aβ40 on immobilized GAG or GAG related macromolecules such as heparin (low molecular weight form), heparin (high molecular weight form), dextran sulfate (low molecular weight form), and dextran sulfate (high molecular weight form). Figure 2 shows the congophilic maltese-cross plaque formation in vitro. In this experiment, 18 well glass slides coated with Teflon were transferred to TBS or 50 microliters per well of dextran sulfate (10 mg / ml stock in deionized water; high or low molecular weight form as indicated), or heparin. (10 mg / ml stock in deionized water; high or low molecular weight form) either coated with air and air dried to a thin transparent film. Then 50 microliters of 250 μM Aβ40 in 1 × TBS was added per well and the slides were placed in a humidified chamber and incubated at 37 ° C. for 12-24 hours. The incubation slides were then stained with 5 microliters of 125 μM Congo red solution per well and viewed under polarized light with a 2 × objective, indicating that a large amount of Maltese cross plaque formation was seen. A) Aβ40 alone in TBS; B) Aβ40 on immobilized dextran sulfate (low molecular weight type) in TBS; C) Aβ40 on immobilized dextran sulfate (high molecular weight type) in TBS; D) Immobilization in TBS Aβ40 on immobilized heparin (low molecular weight form); E) Aβ40 on immobilized heparin (high molecular weight form) in TBS; F) 1/10 dilution of sample from (C) in distilled deionized water, 125 μM Of Congo red solution at 5 μl / well. Using this method, more than 10,000 plaques / ml can be formed in less than 24 hours.

(Example 5)
Image analysis of maltese cruciform plaques stained with Congo red to determine plaque size FIG. 13 shows in vitro congo-affected maltese cruciform plaques and the amount of maltese cruciform plaques formed in vitro The plaques subjected to image analysis using ImageProPlus 4.1, which has the ability to arrange the plaques according to size, as well as calculate the plaques are shown. In this example, FIG. 12F shows maltese-cross plaques formed in vitro using Aβ40 on immobilized dextran sulfate (high molecular weight form) in a 1:10 weight ratio or 1: 5 molar ratio. This is shown and used for further image analysis. The amount of plaque in this 5 microliter aliquot was 63, and it was estimated that a total of 12,600 plaques were formed per ml used. The plaque diameter ranges from 1-4 microns to 60-100 microns, according to which the average plaque diameter ranges between 10-45 microns.

(Example 6)
Thio S staining plaques formed in vitro on fixed GAGs show thio S staining plaques formed in vitro and observed under fluorescence. For this study, Teflon-coated 18-well glass slides were prepared from TBS or 50 microliters per well of dextran sulfate (10 mg / ml stock in deionized water; high molecular weight form as shown), polyvinyl sulfonic acid (de-ionized). Coated with either 10 mg / ml stock in ionic water) or dermatan sulfate (10 mg / ml stock in deionized water) and air dried to a thin clear film. Then 50 microliters of 250 μM Aβ40 in 1 × TBS was added per well and the slides were placed in a humidified chamber and incubated at 37 ° C. for 12-24 hours. Then, 30 microliters, 20 microliters, or 8 microliters aliquots per well of the incubation slide were stained with 5 microliters per well of 125 μM Thioflavin S solution and viewed with a 2 × objective under fluorescent FITC light. As a spheroid, a large amount of thio S positive plaque was observed.

(Example 7)
Image analysis of thio S-stained plaques formed in vitro on fixed GAG FIG. 15 shows the amount of thioflavin S positive plaques formed in vitro and spherical thio S positive plaques formed in vitro The plaques subjected to image analysis using ImageProPlus 4.1, which has the ability to arrange the plaques according to size, as well as calculate the plaques are shown. In this example, Aβ40 on immobilized dextran sulfate (high molecular weight type) in a 1:10 weight ratio or 1: 5 molar ratio was used for image analysis. The count range for image analysis was set as an area ranging between 30-3500 square microns and a diameter ranging between 4-100 microns. Anything below or above this set threshold was not included in the data analysis. Also, visually verify objects that fall outside this set range, and these are either particulates or junk / fiber clumps on the glass and as seen by Congo red staining, It was confirmed that it does not show a typical Congo affinity Maltese cross pattern (not shown). The amount of plaque in this 8 microliter aliquot was 323, and it was estimated that a total of 40,375 plaques were formed per ml used. The plaque diameters range from 1-4 microns to 60-100 microns, according to which the average plaque diameter ranges between 10-25 microns.

(Example 8)
Figure 16 shows plaque destruction upon application of various lead compounds, which inhibits, reduces or eliminates the Congo affinity maltese-cross pattern of plaques identified using polarizing microscopy after Congo red staining. Compounds or agents incubated with in vitro preformed amyloid plaques to screen for the ability to For these studies, 30 microliters per well of dextran sulfate (1 mg / ml stock DexS in deionized water), dermatan sulfate (1 mg / ml stock DermS in deionized water), heparin (1 mg / ml in deionized water) ml stock), or TBS alone, was spotted onto an 18-well glass slide coated with Teflon and air dried to a thin transparent film. Then 30 μl of 250 μM Aβ40 in 1 × TBS was added per well and the slides were placed in a humidified chamber and incubated at 37 ° C. for 12-24 hours. Subsequently, 5 microliters per well was subjected to Congo red staining to confirm the formation of Maltese cross plaques. The remaining unstained incubation slides were then further subjected to a 3-day treatment with selected anti-amyloid compounds containing DC-0004, DC-0021, and DC-0051 at final concentrations of 0.5 mg / ml. After treatment with compound for 3 days, the wells were then subjected to normal Congo red staining and observed under polarized light. As can be seen in this example, Maltese cross plaques are formed in vitro under all conditions. Treatment with certain compounds shows, to varying degrees, the destruction of the Maltese cruciform pattern. For example, DC-0004 was able to break Maltese cross plaques under all conditions, while DC-0021 showed only mild destruction against these preformed Maltese cross plaques. DC-0051 is not as robust as DC-0004, but could affect the Maltese cruciform plaques of Aβ40 alone, Aβ40 + dextran sulfate and Aβ40 + dermatan sulfate. Aβ40 + heparin plaques were less affected by DC-0051 than other plaques formed in vitro by other GAGs or GAG-related molecules. This type of screening can also be performed on 96-well assay plates using Thioflavin T as a marker for amyloid plaque mass for higher throughput screening.

Example 9
Destruction of plaques after protease digestion with lead compounds FIG. 17 shows compounds or agents that are pre-formed in vitro for amyloid plaques to screen for their ability to sensitize plaques to protease digestion. The compound or agent is incubated with and then treated with proteinase K for 24 hours. For these studies, 30 microliters per well of dextran sulfate (1 mg / ml stock DexS in deionized water), dermatan sulfate (1 mg / ml stock DermS in deionized water), heparin (1 mg / ml in deionized water) ml stock), or TBS alone, was spotted onto an 18-well glass slide coated with Teflon and air dried to a thin transparent film. Then 30 microliters of 250 μM Aβ40 in 1 × TBS was added per well and the slides were placed in a humidified chamber and incubated at 37 ° C. for 12-24 hours. Subsequently, 5 microliters per well was subjected to Congo red staining to confirm the formation of Maltese cross plaques. The remaining unstained incubation slide was then further subjected to a 3-day treatment with selected anti-amyloid compounds containing final concentrations of 0.5 mg / ml DC-0004, DC-0021, and DC-0051. After treatment with compound for 3 days, the wells are then subjected to proteinase K treatment (1 U / ml) at 37 ° C. for 24 hours. Incubation slides were then stained with 5 microliters / well of 125 μM Congo Red and viewed under polarized light. As can be seen in this example, Maltese cross plaques are formed in vitro under all conditions. Treatment with proteinase K after treatment with certain compounds shows the disappearance of the Maltese cross plaque pattern to varying degrees. For example, DC-0004 enabled plaque removal by protease treatment under all conditions except Aβ40 + dextran sulfate plaques. DC-0021 treatment gave protease sensitivity to plaques formed in vitro with the combination of Aβ40 + dermatan sulfate and heparan sulfate, but not Aβ40 + dextran sulfate or Aβ40 + heparin plaques. Treatment with DC-0051 was only moderately successful in making plaques pre-formed with Aβ40 + dermatan sulfate and Aβ40 + heparan sulfate susceptible to protease treatment, while pre-formed with Aβ40 + dextran sulfate and Aβ40 + heparin. It was ineffective in plaques.

Application for identifying anti-plaque therapeutics As described herein, the in vitro formed congophilic maltese-cross dense amyloid plaques are used in screening methods as anti-lead compounds in the treatment of Alzheimer's disease. It is also possible to identify plaque therapy agents. In preferred embodiments, such screening methods will utilize radiolabeled amyloid protein (Aβ), sulfated GAG, sulfated or anionic macromolecules or fragments thereof. In a preferred embodiment, Aβ1-40 is conjugated to a radiolabel such as radioiodine (ie 125I). However, other suitable labeling agents and techniques can be used and include, but are not limited to, enzyme labels, fluorescent labels, chemiluminescent labels, or antigen labels. Within the isotope, any radioactive material that can be incorporated into the Aβ protein or fragment thereof can be used. Preferred isotopes include, but are not limited to, 125I, 123I, and 131I. 131I has a shorter half-life and higher energy level. It is also possible to incorporate iodine radioisotopes into proteins or protein fragments by oxidative iodination. Alternatively, by using a Bolton-Hunter reagent to add a 3-iodo-4-hydroxyphenylproprionyl group or a 3,5-diiodo-4-hydroxypropionyl group to the nucleophilic group of the peptide, It is also possible to incorporate radioactive iodine.

  Other isotopes can also be incorporated by reacting with nucleophilic groups or peptides. For example, tritium (3H) can be incorporated by reacting with propionyl-N-hydroxysuccinimide, and radioactive sulfur (35S) can be incorporated by a similar reagent. Labeling of GAG or sulfated macromolecules with 35S by methods known to those skilled in the art also allows amyloid plaque cores formed in vitro to be labeled and monitored as described below. Will do. It is also possible to incorporate radioactive phosphorus (32P) by enzymatic methods. In addition, various radioactive metal ions such as 99m technetium can be incorporated into Aβ or fragments thereof if appropriate chelating groups are added first.

  Enzyme labels are also useful for detection using in vitro assays according to the present invention. Among preferred enzyme labels are peroxidases such as horseradish peroxidase (HRP), or phosphatases such as alkaline phosphatase.

  Modification of a peptide or peptide fragment by adding an antigenic group that binds to the antibody allows direct detection of the peptide or peptide fragment itself. For example, the antigen digoxigenin can be linked to a peptide and then visualized with a labeled digoxigenin-specific antibody, or a labeled anti-antibody.

  Although less sensitive than radioisotopes, fluorophores can also be incorporated into Aβ peptides and detected according to known fluorescence detection techniques. Examples of suitable phosphors include fluorescein, Texas red, and the like.

  Direct or indirect chemiluminescent labels such as dioxetane can also be used according to the present invention. For example, the Aβ peptide is modified with a group capable of emitting light as it degrades.

  Furthermore, it is also possible to detect Aβ peptides or peptide fragments in an in vitro assay using the avidin-biotin system. For example, it is possible to functionalize a peptide or fragment with biotin and add avidin or streptavidin to detect the protein or fragment.

Once Aβ is properly labeled as described above, it is combined with certain GAGs, sulfated or anionic macromolecules as described herein, and incubated at 37 ° C. to produce a Congophilic Maltese cross compact. Forms amyloid plaques. The labeled plaques are first tested to confirm that the staining and structural characteristics of the formed amyloid plaques are the same as those formed in the absence of the label. After proper labeling techniques, parameters that support plaque stability include:
a) Spherical or dense shape of the formed plaques, b) Congo-affected Maltese cross pattern when stained with Congo red and observed under polarized light (ie, a 90 degree plaque relative to the green color of the plaque) Red), c) positive staining with thioflavin S, d) spherical appearance when viewed with an electron microscope and / or “amyloid star” appearance, and e) spherical or dense when observed with an optical microscope. Shape (with spots of 10-40 μm diameter) is included. If the labeled amyloid plaques exhibit one or more of the staining and structural features as described above, they can also be utilized in a variety of in vitro methods for identifying anti-spot therapeutic agents.

In one such preferred method, labeled plaque cores are seeded into 96 well plates and allowed to bind overnight. Different methods known in the art are utilized to determine the optimal conditions for such labeled plaque binding to the wells. Once such binding is achieved, several compounds or agents in various solutions / buffers (determined experimentally) contain labeled plaques with varying incubation times (determined experimentally). Add to wells. Staining properties or structure of dense amyloid plaques by comparing staining properties and structural properties with wells that do not contain compounds or agents, or wells that contain compounds or agents that may not be effective in modifying plaque architecture. Identify agents or compounds that can be shattered, broken down or removed as described below. Agents or compounds that can shatter, destroy, or remove the staining or structural composition of dense amyloid plaques can be identified by a variety of means, including the following:
1) Increased radiolabeling of supernatant (ie, liquid phase) in plaque wells treated with compound or agent compared to plaque wells not treated with compound or agent. Methods for detecting labels such as radioactive isotopes vary depending on the isotope and its corresponding energy level. For example, a gamma counter can detect 125I but not 3H (tritium) or 35S-sulfate, in which case a scintillation counter would be required. An increase in label in the supernatant indicates that the plaque has been destroyed or shattered, and the given compound or agent is effective in shattering or destroying the plaque construction, and therefore , Identified as a potential anti-plaque agent. Such identified agents or compounds can be further identified by secondary or tertiary screening, including but not limited to: 1) stained with Congo Red and observed under polarized light Reduction or elimination of the Congophilic Maltese cross pattern, which is useful for a given compound or agent to reduce or alter amyloid fibril structure and thus potential anti-plaque therapy 2) reduction or elimination of positive staining with thioflavin S, which indicates that the given compound or agent is effective to reduce or alter amyloid fibril structure, and Therefore, it shows that it is identified as a potential anti-plaque therapeutic agent, 3) spherical appearance and / or when observed with an electron microscope Reduction, modification or elimination of the appearance of “amyloid star”, which is that a given compound or agent is effective in modifying the construction of amyloid plaques and is thus identified as a potential anti-plaque therapeutic agent And / or 4) reduction, modification or removal of the spherical or dense shape of amyloid plaques (with plaques 10-40 μm in diameter) when observed with a scanning electron microscope, It is shown that these compounds or agents are effective in modifying the construction of amyloid plaques and are therefore identified as potential anti-plaque therapeutic agents.

  WM Hunter and FC Greenwood, Nature 194: 495, 1962; AE Bolton and WM Hunter, Biochem., The disclosures of which are incorporated herein by reference. J. et al. 133: 529, 1973; and HP Too and JE Maggio, Meth. Neurosc. 6; 232, 1991. Radiolabeling of peptides containing aromatic amino acids by oxidative radioiodination with Na 125I and chloramine-T and separation from free iodine by reverse phase absorption using the method of 232, 1991. Is also possible.

  Another method of in vitro screening to identify anti-plaque agents is the formation in vitro as described herein, showing a Maltese cross pattern when stained with Congo red and viewed under polarized light. Will utilize unlabeled dense amyloid plaques. A compound or agent capable of inhibiting, reducing or eliminating the congophilic Maltese cross pattern of plaques after incubation with dense amyloid plaques for an appropriate time (determined experimentally) Observations are used to identify potential anti-malarial agents. In such a method, dense amyloid plaques are first formed (as described herein) that exhibit a typical Maltese cross pattern when stained with Congo Red and viewed under polarized light. Following incubation with the test compound (with appropriate dosage and incubation time determined experimentally), air-tight dense amyloid plaques (as described herein) on gelatin-coated slides and stained with Congo Red And a predetermined compound or agent inhibits, destroys or removes amyloid plaque structure so that there is a loss of Congo affinity and / or Maltese cross formation, as observed under a polarizing microscope Determine whether is possible. Secondary or tertiary screening will include analysis of these plaques by transmission and scanning electron microscopy after incubation with a given agent or compound.

  Another method of in vitro screening to identify anti-plaque therapeutics is a compact formed in vitro as described herein that shows positive staining when stained with thioflavin S and viewed under a fluorescence microscope. Will utilize amyloid plaques. A compound or agent capable of reducing or eliminating plaque positive thioflavin S fluorescence after incubation with dense amyloid plaques for an appropriate time (determined experimentally) is considered to be a potential anti-plaque therapeutic agent. Identified. Secondary and tertiary screening will include analysis of these plaques by transmission and scanning electron microscopy after incubation with a given agent or compound.

  Yet another method of in vitro screening to identify anti-plaque agents is in vitro as described herein, which exhibits a spherical appearance or “amyloid star” appearance when viewed with a transmission electron microscope. Will utilize the dense amyloid plaques formed in A compound or agent capable of destroying or modifying the globular plaque shape or “amyloid star” appearance after incubation with dense amyloid plaques for an appropriate time (determined experimentally) is potentially Identified as an anti-plaque agent.

  Yet another method of in vitro screening to identify anti-plaque therapeutic agents is that the present specification shows a spherical shape with an amyloid plaque diameter of 10-40 μm (average plaque diameter of 25 μm) when observed with a scanning electron microscope It will utilize dense amyloid plaques formed in vitro as described in the literature. A compound or agent capable of destroying or modifying the globular plaque shape, or substantially reducing the diameter of the amyloid plaque after incubation with the dense amyloid plaque for an appropriate time (determined experimentally) , Identified as a potential anti-malarial agent.

  Yet another method of in vitro screening to identify anti-plaque therapeutic agents will utilize the size and shape of dense amyloid plaques formed as described herein. Agents or compounds that inhibit, destroy or remove the structure (ie, size and / or diameter) of spherical amyloid plaques can be identified using methodologies involving cell sorters. In such assays, dense spherical amyloid plaques formed in vitro can be passed through a cell sorter to determine the average diameter (and diameter range) of these plaques. In one preferred embodiment, the amyloid plaque cores that are formed are loaded into a Coulter EPICS Elite ESP cell sorter (Coulter Corporation, Hialeah, FL) and flowed through a 100 Mm 3x chip at a flow rate of 1428 particles / second. For sorting based on side scattered light, an argon laser with maximum excitation at 488 nm is used. The selected plaque core will be based on size (10-50 Mm). Based on observations with an electron microscope, amyloid plaques formed in vitro by the methods described herein usually have a diameter range of 10-40 μm, with an average diameter of 25 μm. Under the appropriate conditions and incubation time (determined experimentally), after incubation with a given compound or agent, the agent or agent can be evaluated by evaluation using a cell sorter to determine the mean plaque diameter (ie size). The plaques formed in the absence of the compound are compared to the plaques formed upon incubation with the agent or compound. In another method, plaques formed in vitro are treated with a compound or agent for a specific time (determined experimentally) using methods such as those described herein, and then cells Using a sorter, the average diameter of the plaques thus treated is determined and compared with the average diameter of the untreated plaques. If the given compound or agent is effective in shattering or destroying the size (and thus the diameter) of the dense plaque, the smaller diameter (ie the smaller plaque or its shattered component) An increase in the ratio will be observed. A compound or agent capable of disturbing or substantially reducing the diameter of amyloid plaques after incubation with dense amyloid plaques for an appropriate time (determined experimentally) is a potential anti-plaque therapeutic agent Identified.

  Another potential utility of amyloid plaques formed in vitro as described herein is by identifying agents or compounds that are effective in reducing or eliminating the neurotoxic effects of Aβ. is there. In the first set of experiments, as described herein, it may be determined whether dense amyloid plaques formed in vitro cause toxicity to neurons in culture and / or in animal models. It will be important (described below). For such cell culture experiments, dense amyloid plaques are first formed in vitro as described herein, and primary neurons (standard and isolated using techniques known to those skilled in the art). Or put in a Petri dish containing neuronal cell lines. After prolonged incubation of neuronal cultures with amyloid plaques (ie 48 or 72 hours), neurotoxicity levels (using standard assays known in the art) are measured and compared to cultures that do not contain amyloid plaques To do. If dense amyloid plaques can show neurotoxic effects in cell culture, these amyloid plaques can be used to screen and identify potential anti-neurotoxic therapeutic agents or compounds. It can also be used. In such methods, dense amyloid plaques formed in vitro are cultured in primary neuronal cultures or in neuronal cell lines for extended periods (ie, 48 hours or 72 hours) and in the presence of a predetermined test compound or agent. Alternatively, incubate in the absence. Agents or compounds that can inhibit or reduce neurotoxicity caused by incubation of amyloid plaques are then identified as anti-neurotoxic agents.

Research applications It is expected that dense amyloid plaques formed in vitro will be useful in a variety of different research applications. In one example, preformed dense amyloid plaques are placed in a culture containing other cells (eg, neurons, microglia, astrocytes, oligodendrocytes) and the response of the cells to such amyloid plaques in culture ( That is, phagocytosis and decomposition) can be measured. In another example, individual macromolecules against these dense amyloid plaques in culture (ie other components associated with amyloidosis such as apolipoprotein E, amyloid P component, using standard techniques known to those skilled in the art) It is also possible to assess the response of complement factors, cytokines, inflammatory factors).

  In addition, the effects of dense amyloid plaque deposition, accumulation and persistence on cell architecture and / or metabolism of various macromolecules (ie beta-amyloid precursor protein, specific proteoglycans) can also be studied in vivo. is there. Such use of dense amyloid plaques in vitro and in vivo will create new tools for research in the future that are both practical and involve untapped applications.

  Another potential application of the present invention is to provide preformed dense amyloid plaques or the ability to produce such dense amyloid plaques in the form of a kit. Such kits may also be useful for screening and identification of compounds or agents that have potential as anti-plaque therapeutic agents. Such kits are: a) low fibrillar Aβ1-40 (preferably in solution or lyophilized Recombinant Peptides (catalog) in an amount or concentration required for dense amyloid plaque formation (described herein). A first container having Aβ40) of number A-1052-2), b) in solution or lyophilized in an amount or concentration required for dense amyloid plaque formation (described herein) It is also possible to consist of a second container containing a specific GAG (eg heparin or heparan sulfate) or a specific sulfated macromolecule (eg dextran sulfate, pentosan polysulfate or polyvinyl sulfonic acid). Such Congophilic maltese-cross amyloid plaque formation will occur after mixing the appropriate amount from each of the two containers.

  In another kit, dense amyloid plaques can be pre-formed and then frozen or lyophilized for distribution. Once received by a researcher or individual, dense amyloid plaques can be reshaped by placing in an appropriate solution, such as distilled water or Tris-buffered saline (pH 7.4), and in an appropriate volume of solution. is there. Such kits can also be used for research and / or commercial applications.

  In accordance with the law, the present invention has been described more or less specifically with respect to structural features. However, it is to be understood that the means and construction shown are not limited to the specific features shown, as they include preferred forms of accomplishing the invention. Accordingly, the invention is claimed in any type or modification that is properly construed in accordance with the doctrine of equivalence and within the scope of the appended claims.

1A-1L are photomicrographs of in vitro formation of Congophilic maltese-cross spherical amyloid plaques in one embodiment. 2A-2I are photomicrographs of in vitro formation of amyloid plaques that are congophilic and Maltese cruciform according to another embodiment. 3A-3I are photomicrographs of in vitro formation of amyloid plaques that are congophilic and Maltese cruciform according to another embodiment. 4A-4B are photomicrographs of in vitro formation of Congophilic maltese-cross spherical amyloid plaques according to another embodiment. 5A-5B are photomicrographs of in vitro formation of spherical amyloid plaques in another embodiment of the method of the present invention. FIGS. 6A-6D show the formation of spherical “amyloid star” induced by perlecan, substantially identical to an isolated amyloid plaque core from human Alzheimer's disease brain when observed with a transmission electron microscope. It is a micrograph. 7A-7F are photomicrographs of amyloid plaque core formation induced by perlecan or dextran sulfate and observed with a scanning electron microscope. FIG. 8 shows in vitro plaque formation using different sulfated GAGs or GAG-related macromolecules. FIG. 9 shows an example of amyloid star plaques in the brain of human Alzheimer's disease compared to Maltese cross in vitro plaques and corresponding electron micrographs (EM). FIG. 10 shows in vitro maltese cross plaque formation with and without sonication. FIG. 11 shows in vitro maltese cross plaque formation in the immobilized GAG method (2 × objective lens). FIG. 12 shows the Maltese cross plaque formation of FIG. 4, but using a 10 × objective lens. FIG. 13 shows image analysis of Maltese cross plaques stained with Congo Red to determine plaque size (micron diameter-μm). FIG. 14 shows plaques formed in vitro and stained with thio S on immobilized GAG (and / or GAG-related macromolecules). FIG. 15 shows image analysis of plaques formed in vitro and stained with thio S on immobilized GAG (and / or GAG-related macromolecules). FIG. 16 shows plaque destruction upon application of various lead compounds. FIG. 17 shows plaque destruction with lead compounds after protease digestion.

Claims (10)

A method for screening selected amyloid therapeutic candidates:
a) Immobilizing an amount of a selected sulfated glycosaminoglycan (SGAG) or GAG-related macromolecule selected from the group consisting of dextran sulfate, pentosan polysulfate and polyvinyl sulfonic acid on a selected medium. And one of the following:
b1) adding a selected amount of the selected amyloid therapeutic agent candidate to an amount of dissolved low fibrillar Aβ1-40 (LFAβ) to produce a test solution; and
c1) Adjust the Aβ: SGAG weight / weight (w / w) ratio by adding a selected amount of test solution to SGAG immobilized on the medium;
Including steps,
This allows the percent inhibition in amyloid plaque formation as compared to the test reference sample prepared as described above without the selected amyloid therapeutic candidate to be the percent effectiveness of the selected amyloid therapeutic candidate. Or b2) adjusting the Aβ: SGAG w / w ratio by adding a selected amount of dissolved low fibrillar Aβ1-40 (LFAβ) to SGAG immobilized on the medium. Forming amyloid plaques on the medium; and
c2) adding a selected amount of a test solution of the selected candidate amyloid therapeutic agent to the amyloid plaques fixed on the medium;
Including steps,
This allows the percent destruction of amyloid plaques on the media when compared to a test reference sample prepared as described above without the selected amyloid therapeutic agent candidate to be effective for the selected amyloid therapeutic agent candidate. The method, which is a percentage indicator.   The Aβ: SGAG weight / weight (w / w) ratio is 1: 0.01 to 1:20, 1: 0.1 to 1:10, or 1: 0.5 to 1: 2. the method of.   The method of claim 2 wherein the Aβ: SGAG w / w ratio is 1: 1.   4. The method of any one of claims 1-3, wherein the selected medium is either a slide, film, or titer well plate.   The method of claim 4 wherein the titer well plate is an 18-96 well Teflon split slide.   The method of claim 5, wherein LFAβ in step b2 of claim 1 is added to the immobilized SGAG by a bubbling technique.   6. The method of claim 5, wherein the test solution in step c1 of claim 1 is added to the immobilized SGAG by a bubbling technique.   The SGAG is selected from the group of SGAGs consisting of heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate and chondroitin-6-sulfate, and the GAG-related macromolecule is dextran sulfate. The method according to any one of the above.   A kit for screening selected amyloid therapeutic candidates: an amount of sulfated glycosaminoglycan (SGAG) immobilized on a medium; an amount of hypofibrillar Aβ1-40 (LFAβ); and claims The kit comprising the instructions for screening for each of steps a to c1 in Item 1.   A kit for screening selected amyloid therapeutic candidates: an amount of amyloid plaques formed on a medium according to claim 1, steps ab2; and the screening instructions for step c2 of claim 1 The kit.

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