Identifying substances using cells that express p75NTR

Details Identifying substances using cells that express p75NTR Identifying substances using cells that express p75NTR

2001-05-29

2004-02-24

Ulm, John (Department: 1646)

Chemistry: analytical and immunological testing

Involving antibody fragments

C436S501000, C435S007100, C435S007210, C435S007800, C530S300000, C514S002600

Reexamination Certificate

active

06696303

ABSTRACT:

BACKGROUND OF THE INVENTION
Dementia is a condition of deteriorating mentality that is characterized by marked decline in the individual's former intellectual level, including memory loss, impaired judgment, speech and orientation, and is often accompanied by emotional apathy. (WEBSTER'S MEDICAL DESK DICTIONARY, Merriam-Webster, Inc., Springfield, Mass., p.169 (1986)).
A leading cause of dementia is Alzheimer's disease,(AD), a neurodegenerative disorder affecting 17 to 20 million people worldwide (Yamazaki, T., et al.,
J. Cell. Biol.,
129;431-442 (1995); Brinaga, M.,
Science,
269:917-918 (1995); Lavy-Lahad, E., et al.,
Science,
269:970-972 (1995); Lavy-Lahad, E., et al,
Science,
269:973-977 (1995)). AD is characterized by progressive dementia together with neuropathological findings of “senile plaques” in the brain formed by deposits of &bgr;-amyloid protein, surrounded by clusters of degenerating neurons. &bgr;-amyloid protein itself is a fragment of the 770 amino acid membrane bound &bgr;-amyloid precursor protein (&bgr;APP) that is expressed in both neuronal and non-neuronal tissues.
Muteins of &bgr;APP have been produced for the purpose of developing a &bgr;APP substrate system wherein &bgr;APP is cleavable or not cleavable such that &bgr;AP producing enzymes and inhibitors thereof may be isolated.
The specific cause of Alzheimer's disease has not yet been determined. A mutation in the &bgr;APP gene in families with one form of autosomal dominant AD was found to be associated with increased &bgr;-amyloid synthesis and aggregation in the brain. A receptor for &bgr;APP has been identified as the low density lipoprotein receptor-related protein, ApoE, and it has been postulated that this receptor protein, the enzyme responsible for &bgr;APP cleavage in the cell membrane, production of &bgr;APP and/or production of extracellular matrix molecules may be abnormal individually or in combination in AD patients, resulting in excess &bgr;-amyloid deposition and the observed neurotoxicity. However, the mechanism by which other known &bgr;APP gene mutations cause AD, as well as the pathophysiology of non-familial AD in which &bgr;APP gene mutations have not been recognized, is not understood.
Therefore, diagnosing Alzheimer's disease as the cause of an individual's dementia, as well as treating AD and developing drug therapies is very difficult. Although recent reports of using Positron-emission tomography (PET) (Reiman, E. M., et al.,
New Eng. J Med.,
334:752-758 (1996), determining the genotype of an individual's ApoE, or measuring the levels of &bgr;-amyloid protein in cerebral spinal fluid may be promising, diagnosis of Alzheimer's is currently confirmed only upon autopsy to determine the presence of &bgr;-amyloid senile plaques.
In vitro systems employed to study Alzheimer's disease to date consist of malignant, or transformed cells that are not of neural crest origin, or lower vertebrate neuronal cultures. It would be of great advantage to have an Alzheimer's disease model system using normal human neural crest-derived cells. However, to date, no such model system has been developed.
Moreover, recent studies have shown that damage to CNS neurons due to Alzheimer's disease begins years before clinical symptoms are evident. (Reiman, E. M., et al.,
New Eng. J Med.,
334:752-758 (1996)), suggesting that therapy could begin in the pre-symptomatic phase of the disease if a sensitive diagnostic test and targeted therapies were available. There exists a great need to determine the physiological mechanisms involved with the disease and for an accurate and easy to perform assay to evaluate the risk of developing Alzheimer's disease.
SUMMARY OF THE INVENTION
The present invention relates to the discovery that neurons and epidermal melanocytes are neural crest-derived cells that undergo &bgr;-amyloid mediated apoptosis mediated by &bgr;-amyloid binding to the same receptor, the 75 kD neurotrophin receptor (p75
NTR
). As used herein, the term &bgr;-amyloid protein is intended to encompass &bgr;-amyloid protein (a 4.2 kD polypeptide (Selkoe, D. J.,
Neuron,
6:487-498 (1991); Glenner G. G. and Wong, C. W.,
Biochem. Biophys. Res. Commun.,
120:885-890 (1993), the teachings of which are herein incorporated by reference), &bgr;-amyloid precursor protein (&bgr;APP), and fragments of &bgr;-amyloid and &bgr;-amyloid precursor protein referred to herein as &bgr;-amyloid peptides, including &bgr;-amyloid 1-40 peptide, &bgr;-amyloid 1-42 peptide, &bgr;-amyloid 25-36 peptide or &bgr;-amyloid 28-30 peptide. (&bgr;-amyloid protein is also referred to herein as &bgr;-amyloid).
More specifically, it is demonstrated herein that &bgr;-amyloid protein or peptide binds to the p75 nerve growth factor receptor (p75
NTR
) of neural crest-derived cells, e.g. melanocytes, resulting in apoptosis of the cell. It is further demonstrated herein that inhibiting the binding of &bgr;-amyloid protein or peptide to the p75 nerve growth factor receptor, results in inhibiting the activation of the p75 nerve growth factor receptor, which in turn inhibits apoptosis.
The present invention relates to methods of inhibiting &bgr;-amyloid-mediated activation of the p75 nerve growth factor receptor of a cell that expresses the p75 nerve growth factor receptor, methods of inhibiting the binding of &bgr;-amyloid protein and &bgr;-amyloid peptides to the p75 nerve growth factor receptor, and methods of inhibiting &bgr;-amyloid-mediated apoptosis of neural crest-derived cells. The methods comprise contacting the cell with a substance, comprising, for example, the amino acid sequence lysine-glycine-lysine (KGK) or lysine-glycine-alanine (KGA), wherein the substance binds to the p75 nerve growth factor receptor, resulting in the inhibition of &bgr;-amyloid protein or &bgr;-amyloid peptide binding to and/or activation of the p75 nerve growth factor receptor, or wherein the substance inhibits &bgr;-amyloid protein or &bgr;-amyloid peptide mediated apoptosis of the cell which expresses the p75 nerve growth factor receptor.
It is has been reported that cell death receptors mediate apoptosis by aggregation resulting from ligand binding or membrane perturbation. Applicants have demonstrated that &bgr;-amyloid aggregates the p75
NTR
thereby inducing cell death. Peptides, specifically cyclic peptides, bind the receptors individually and block aggregation of the p75
NTR
, thereby inhibiting apoptosis. Specifically encompassed by the present invention are cyclic peptides (e.g., peptides in a &bgr;-loop conformation) which comprise lysine-glycine-lysine or lysine-glycine-alanine, or other sequences capable of binding to the p75
NTR.
Activation of the p75 nerve growth factor receptor can be determined by measuring the &bgr;-amyloid activation of the p75 nerve growth factor receptor of neural crest-derived cells, in culture or in a tissue sample, in the presence of the test substance and comparing the results with the &bgr;-amyloid activation of the p75 nerve growth factor receptor of neural crest-derived cells in a control culture or sample without the test-substance. A decrease of &bgr;-amyloid activation of the p75 nerve growth factor receptor of neural crest-derived cells in the test sample compared to &bgr;-amyloid activation of the p75 nerve growth factor receptor of neural crest-derived cells in the control sample is indicative of a substance that inhibits &bgr;-amyloid-mediated apoptosis in neural crest-derived cells.
The present invention further relates to in vitro methods of screening substances and identifying those substances capable of inhibiting, or decreasing cell apoptosis mediated by &bgr;-amyloid, or activation of the p75
NTR
, and to substances identified by these methods.
The method of identifying substances that inhibit &bgr;-amyloid-mediated apoptosis of cells that express the p75 nerve growth factor receptor comprises contacting the cells, in culture or in a tissue sample, with &bgr;-amyloid protein, or peptide and with the substance to be tested, wherein

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