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Assay for evaluating the therapeutic effectiveness of agents in reducing Alzheimer's disease pathology

Inactive Publication Date: 2005-04-28
ROSKAMP RESEARCH LLC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010] According to the present invention, there is provided an assay method for determining the effect of an agent on Alzheimer's Disease pathology by treating microglial cells with Aβ peptides, adding CD40 ligand to the microglial cells, adding a therapeutic agent to the microglial cells, and measuring Alzheimer's Disease pathology. Also provided is a method of determining therapeutic effectiveness of an agent for Alzheimer's Disease by measuring the inhibition of CD40-CD40L binding and/or its functional outcomes in the presence of the agent. A method of testing the eff

Problems solved by technology

The symptoms of AD include gradual loss of short-term memory, declined ability to perform routine tasks such as eating, confusion, disorientation, the inability of the patient to care for him or herself, and eventually death.
However, treatment strategies aimed at lessening the negative effects of inflammation in AD are only available to a very limited extent.
Furthermore, rather than targeting AD-associated neuro-inflammation, these drugs tend to be general inhibitors of inflammation (such as non-steroidal anti-inflammatory agents like aspirin) which only provide partial therapeutic benefit (Rich, J. B., Rasmusson, D. X., Folstein, M. F., Carson, K. A., Kawas, C.
However, such doses of Aβ1-42 rapidly produce large amounts of Aβ fibrils and loss of Aβ solubility in vitro [Castillo et al., 1997; Genis et al., 1999; Schneider et al., 1999; Sturchler-Pierrat et al., 1997; James et al., 1996; Higgins et al., 1995].
However, the mechanisms of Aβ-induced microglial activation remain speculative, and often require a co-stimulatory factor such as the pro-inflammatory cytokine interferon-γ.
However, it has not previously been established what, if any, role CD40 plays in the AD pathogenic process, nor how to utilize this knowledge in potential treatments of AD.

Method used

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  • Assay for evaluating the therapeutic effectiveness of agents in reducing Alzheimer's disease pathology
  • Assay for evaluating the therapeutic effectiveness of agents in reducing Alzheimer's disease pathology
  • Assay for evaluating the therapeutic effectiveness of agents in reducing Alzheimer's disease pathology

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0079] To determine whether Aβ could induce CD40 expression in cultured N9 and N60 microglial cells, cells were treated with 250 nM of freshly solublized Aβ1-40 or Aβ1-42. As shown in both FIG. 2A, both Aβ1-40 and Aβ1-42 significantly induce CD40 expression on cultured microglial cells when compared to Aβ-free, reverse Aβ (Aβ40-1) or the 695 isoform of soluble amyloid precursor protein (sAPPα-695) when compared to control peptide (human thyrocalcitonin) or Aβ free conditions. In addition, Aβ1-42 induced microglial CD40 expression in a dose dependent manner (from 50 to 1250 nM, FIG. 2B).

[0080] To determine whether endogenous overexpression of Aβ could lead to microglial CD40 expression, CD40 expression on microglia was measured from a transgenic mouse model of AD (Tg APPSW, overexpressing Aβ1-40 and Aβ1-42), (11) and control (wild-type) littermates (12). Microglia from Tg APPSW newborn mice had markedly increased levels of soluble Aβ1-40 compared to control littermates (13), and the...

example 2

Materials and Methods

Endothelial Cell Culture and Reagents

[0088] HAEC and HAEC medium were purchased from Clonetics (San Diego, Calif.). HAEC were cultured and expression assays were performed as previously described [29]. Aβ1-40, Aβ1-42, and reverse Aβ40-1, (control) peptides were obtained from QCB (Hopkinton, Mass.). Reverse transcriptase polymerase chain reaction (RT-PCR) kits and RNA reagents were obtained from Invitrogen Inc. (San Diego, Calif.). Human IFN-γ recombinant protein was purchased from Genzyme (Cambridge, Mass.).

RT-PCR

[0089] Cultured HAEC were plated at 1×105 cells / well in 6-well culture plates (Falcon, Becton Dickinson Inc. N.J.). HAEC were treated with freshly solubilized Aβ1-40, or Aβ1-42, (500 nM, in dH20), control peptide (500 nM) or IFN-γ (10 U / mL) for 48 hours after plating. Total RNA was isolated, and cDNA was prepared as previously described [30]. PCR was performed for 30 cycles, with each cycle consisting of 94° C. for one minute, 55° C. for two minut...

example 3

Materials and Methods

Materials

[0102] Cell culture media, fetal bovine serum (FBS) and other culture reagents were supplied by Clonetics, GibcoBRL and Sigma. Aβ1-40, Aβ1-42 and Aβ25-35 were supplied by RBI and / or M.D. Enterprise. All Aβ peptides used were freshly dissolved in Sigma H2O and aliquots were promptly stored at −20° C. The ABC-based enzyme-linked immunoassay (ELISA) kit was obtained from Sigma. The monoclonal antibodies (mAbs) against human CD40, CD45, CD40L, IFN-γR , IL-1β, IFN-γ ELISA kit was ordered from R&D Systems or Endogen, respectively.

Cell Cultures

[0103] Human aortic endothelial cell (HAEC, 3rd passage) line was obtained from Clonetics, and grown in endothelial cell growth medium (EGM, Clonetics) containing endothelial cell basal medium, supplemented with 10 ng / ml human recombinant epidermal growth factor, 1 μg / ml hydrocortisone, 12 μg / ml bovine brain extract, 2% FBS, 50 μg / ml Gentamicin and 50 ng / ml Amphotericin B. As described by the manufacturer, this HAE...

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Abstract

The present invention provides a method of treating Alzheimer's Disease by blocking CD-40 receptor-mediated, Aβ-induced microglial cell activation with an agent. Also provided is a method of determining the therapeutic effectiveness of an agent for Alzheimer's Disease by measuring the inhibition of the interaction between the CD40 receptor and its ligand (CD40L) in the presence of the agent. The present invention further provides a method of testing the efficacy of a therapeutic agent by producing a TgAppSW / CD40L deficient mouse and administering to the mouse the therapeutic agent to be tested and determining the efficacy of the drug in suppressing neurodegeneration of Alzheimer's Disease.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This is a conversion of U.S. Provisional Patent Application No. 60 / 137,016, filed Jun. 1,1999, incorporated herein by reference.TECHNICAL FIELD [0002] The present invention relates to methods for treating Alzheimer's Disease (AD). More specifically, the present invention relates to methods of treating AD by inhibiting Aβ-induced microglial activation. BACKGROUND ART [0003] Alzheimer's disease (AD) is a neurodegenerative disease characterized by the presence of extracellular amyloid deposits (composed mainly of Aβ) and intraneuronal tangles (consisting of the cytoskeletal protein tau) in specific brain regions. Increased phosphorylation of tau is thought to result in neurofibrillary tangles in AD brains and with AD-like pathology in transgenic models of the disease [Genis et al., 1999; Schneider et al., 1999; Sturchler-Pierrat et al., 1997; James et al., 1996; Higgins et al., 1995]. [0004] The symptoms of AD include gradual loss of short...

Claims

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Application Information

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IPC IPC(8): G01N33/50G01N33/68
CPCG01N33/5008G01N33/502G01N33/5044G01N33/5058Y10S435/975G01N2500/10G01N2800/2821G01N2800/52G01N33/6896
Inventor TAN, JUNTOWN, TERRENCEMULLAN, MICHAEL
Owner ROSKAMP RESEARCH LLC
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