Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1994-04-15
2001-12-11
Kunz, Gray L. (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007210, C435S069100, C436S815000
Reexamination Certificate
active
06329155
ABSTRACT:
DESCRIPTION
Background
Alzheimer's disease (AD) is a chronic progressive neurodegenerative disease. Clinically, it is characterized by progressive deficits in memory and other cognitive functions that occur in the face of an otherwise normal neurological examination. Postmortem examination reveals a variety of typical AD brain lesions, including deposition of amyloid plaques, formation of neurofibrillary tangles, and neuronal degeneration.
The amyloid deposits characteristic of AD pathology consist of aggregates of a 39-42 amino acid peptide termed &bgr;/A4 (G. G. Glenner and C. W. Wong,
Biochem. Biophys. Res. Comm.,
120:885 (1984); C. L. Masters et al.,
Proc. Natl. Acad. Sci. USA,
82:4245 (1985); D. J. Selkoe et al.,
J. Neurochem.,
46:1820 (1986)), which is an abnormal cleavage product of a larger amyloid precursor protein (APP) (J. Kang et al.,
Nature,
325:733 (1987)). APP is an integral membrane glycoprotein, existing as several distinct forms derived from alternative mRNA splicing (R. E. Tanzi et al., ibid, 331:528-530 (1988); P. Ponte et al. ibid, 331:525 (1988); N. Kitaguchi et a., ibid., 331:530 (1988)). Water-soluble APP fragments lacking the COOH terminus have been detected in conditioned cell culture media and in human cerebrospinal fluid (A. Weidemann et al.,
Cell,
57:115, (1989), M. R. Palmert et al.,
Proc. Natl. Acad. Sci. USA,
86:6338 (1989)), indicating that APP is a secretory protein. Normal secretion of water-soluble NH
2
-terminal APP fragments involves cleavage of full-length APP at an extracellular site located close to the transmembrane domain, and within the &bgr;/A4 domain (T. Oltersdorf et al.,
Nature,
341:144 (1989); F. S. Esch et al.,
Science,
248:1122 (1990); S. S. Sisodia et al., ibid., 248:492 (1990); J. P. Anderson et al.,
Neurosci. Lett.,
128:126 (1991); R. Wang et al.,
J. Biol. Chem.,
262:16960 (1991)). This cleavage event presumably precludes the formation of amyloidogenic APP fragments. Alternatively to secretion, APP can be processed by an internal lysosomal pathway (C. Haass, A. Y. Hung, D. J. Selkoe,
J. Neurosci.,
11:3783 (1991); C. Haass, E. H. Koo, A. Mellon, A. Y. Hung, D. J. Selkoe,
Nature,
357:500 (1992)) that may generate amyloidogenic cleavage products (S. Estus et al.,
Science,
255:726 (1992); T. E. Golde, S. Estus, L. H. Younkin, D. J. Selkoe, S. G. Younkin, ibid., 255:728 (1992)). It is therefore likely that aberrations of APP processing pathways contribute to amyloid formation. The mechanisms regulating cellular APP processing, however, are unknown.
SUMMARY OF THE INVENTION
This invention is based on the results of an assessment of control of APP processing in cells. As described herein, it has been discovered that cell-surface neurotransmitter receptors regulate APP processing. In particular, it has been shown that cellular APP release is controlled by cell-surface neurotransmitter receptor activity (i.e., APP release is “triggered” or signaled by activation of cell-surface neurotransmitter receptors), and that the regulatory effect on the release of APP derivatives is receptor-subtype specific (i.e., is controlled or signaled by activation of cell-surface neurotransmitter receptors which are linked to phospholipase and a protein kinase, such as protein kinase C (PKC)). This work has resulted in the description of a novel cell-surface neurotransmitter receptor-mediated mechanism for the stimulation of release of soluble NH
2
-terminal APP derivatives.
Human cells expressing the m1 or m3 subtypes of the human brain muscarinic acetylcholine receptor (mAChR) have been shown to be activated by cholinergic agonists, and, as a result, to release a water-soluble NH2-terminal cleavage product of APP which lacks the COOH terminal portion. This stimulation and associated APP processing is blocked by muscarinic antagonists. In contrast, basal release of APP fragments from wild-type cells and from cells expressing subtypes m2 or m4 mAChR receptors is not stimulated. These results indicate that neurotransmitter-control of the release of APP derivatives is receptor subtype-specific, presumably due to the differential coupling of the receptor subtypes to distinct second messenger systems (E. G. Peralta, A. Ashkenazi, J. W. inslow, J. Ramachandran, D. J. Capon,
Nature,
334:434 (1988); J. Sandmann, E. G. Peralta, R. J. Wurtman,
J. Biol. Chem.
266, 6031 (1991)): activation of the phospholipase C-linked mAChR subtypes m1 and m3 stimulated APP release, whereas the adenylyl cyclase-linked subtypes m2 and m4 did not mediate this response.
In addition to mAChR subtypes m1 and m3, bradykinin receptors and serotonin receptor subtypes 5HT
2
and 5HT
1C
demonstrate specific control of the release of APP derivatives. Therefore, these and other receptors linked to phospholipase and a protein kinase, such as protein kinase C, can be used to identify substances which cause release of non-amyloidogenic APP fragments and therefore may be useful for treatment of AD, as well as for preventing the formation of amyloidal plaques.
An assay based upon the above findings may be used to determine whether compounds which activate cell-surface receptors increase the proteolytic processing of APP along with increased release of NH2-terminal derivatives. In the assay of the present invention, the cells employed may be any mammalian or human cells which process and secrete APP normally, and which express an endogenous gene or an exogenous gene (i.e., a gene introduced into the cell, such as by transection) encoding a surface-expressed neurotransmitter receptor linked to phospholipase C and protein kinase C (PKC), such as AChR subtypes m1 and m3, bradykinin receptors, or serotonin receptors subtypes 5HT
2
and 5HT
1C
. Such cells are referred to herein as test cells. A sample of the agent to be tested is contacted with cultured test cells; after incubation, the level of secretion of APP derivatives is measured. The amount of APP derivative released is compared to the level released by a control sample of the test cells under the same conditions but in the absence of the test agent. If the level of APP secretion in the presence of the test agent is less than the level of APP derivatives by the control, the test agent may have inhibited the receptor, and, thereby, decreased the secretion of APP derivatives. If the level of APP secretion in the presence of the test agent is the same as the level of APP derivatives by the control (i.e., in the absence of the agent), the test agent did not exhibit receptor-mediated regulatory activity on APP processing. If the level of secretion of APP derivatives in the presence of the agent is greater than the level of APP by the control, the test agent may have activated the receptors, and thereby increased secretion of APP derivatives.
To verify that the test agent stimulates the neurotransmitter receptors, the effect of an antagonist of the neurotransmitter receptors upon stimulation by the test agent can also be examined. If the receptor antagonist inhibits the stimulating effect of the test agent, then it is even clearer that the test agent is an activator of the receptors. Finally, the size and cleavage pattern of the secreted APP are determined by known methods (e.g., gel electrophoresis, western blot, or enzyme-linked immunosorbent assay (ELISA)), to determine whether the secreted fragment is a non-amyloidogenic fragment or an amyloidogenic fragment generated from pre-existing APP molecules in the cell. The agents identified through this assay may be useful in the treatment and prevention of AD: agents which alter APP processing in individuals by stimulating non-amyloidogenic APP processing, can thereby inhibit the amyloidogenic APP processing and formation of amyloidal plaques.
REFERENCES:
patent: 5242932 (1993-09-01), Gandy et al.
patent: 5348963 (1994-09-01), Gandy et al.
patent: 5385915 (1995-01-01), Buxbaum et al.
patent: 0 457 295 A2 (1991-11-01), None
Buxbaum et al, “Processing of Alzheimer B/A4 amyloid precursor . . . ”PNAS 87:6003-6006 (Aug. 1990).*
Weidemann et al, “Identification, Biogen
Growdon John H.
Nitsch Roger M.
Slack Barbara E.
Wurtman Richard J.
Kunz Gray L.
Massachusetts Institute of Technology
Pepper Hamilton LLP
Pouliquen Corinne M.
Villacorta Gilberto M.
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