Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-05-09
2003-06-17
Ulm, John (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S183000, C435S195000, C435S219000, C530S350000
Reexamination Certificate
active
06579689
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to proteolytic processing of the &bgr;-amyloid precursor protein, and more particularly to assays for determining factors that affect such processing.
BACKGROUND OF THE INVENTION
A number of important neurological diseases including Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), and prion-mediated diseases are characterized by the deposition of aggregated proteins, referred to as amyloid, in the central nervous system (CNS) (for reviews, see Glenner et al. (1989)
J Neurol. Sci.
94:1-28; Haan et al. (1990)
Clin. Neurol. Neurosurg.
92(4):305-310. These highly insoluble aggregates are composed of nonbranching, fibrillar proteins with the common characteristic of a &bgr;-pleated sheet conformation. In the CNS, amyloid can be present in cerebral and meningeal blood vessels (cerebrovascular deposits) and in brain parenchyma (plaques). Neuropathological studies in human and animal models indicate that cells proximal to amyloid deposits are disturbed in their normal functions (Mandybur (1989)
Acta Neuropathol.
78:329-331; Kawai et al. (1993)
Brain Res.
623:142-6; Martin et al. (1994)
Am. J. Pathol.
145:1348-1381; Kalaria et al. (1995)
Neuroreport
6:477-80; Masliah et al. (1996)
J. Neurosci.
16:5795-5811). AD studies additionally indicate that amyloid fibrils may actually initiate neurodegeneration (Lendon et al. (1997)
J. Am. Med. Assoc.
277:825-31; Yankner (1996)
Nat. Med.
2:850-2; Selkoe (1996)
J. Biol. Chem.
271:18295-8; Hardy (1997)
Trends Neurosci.
20:154-9).
AD and CAA share biochemical and neuropathological markers, but differ somewhat in the extent and location of amyloid deposits as well as in the symptoms exhibited by affected individuals. The neurodegenerative process of AD, the most common cause of progressive intellectual failure in aged humans, is characterized by the progressive and irreversible deafferentation of the limbic system, association neocortex, and basal forebrain accompanied by neuritic plaque and tangle formation (for a review see Terry et al. (1994) “
Structural alteration in Alzheimer's disease.
” In: Alzheimer's disease (Terry et al. eds.), pp. 179-196. Raven Press, New York). Dystrophic neurites, as well as reactive astrocytes and microglia, are associated with these amyloid-associated neurite plaques. Although, the neuritic population in any given plaque is mixed, the plaques generally are composed of spherical neurites that contain synaptic proteins, APP (type I), and fusiform neurites containing cytoskeletal proteins and paired helical filaments (PHF; type II).
CAA patients display various vascular syndromes, of which the most documented is cerebral parenchymal hemorrhage. Cerebral parenchymal hemorrhage is the result of extensive amyloid deposition within cerebral vessels (Hardy (1997)
Trends Neurosci.
20:154-9; Haan et al. (1990)
Clin. Neurol. Neurosurg.
92:305-10; Terry et al., supra; Vinters (1987)
Stroke
18:211-24; Itoh et al. (1993)
J. Neurological Sci.
1 16:135-41; Yamada et al. (1993)
J Neurol. Neurosurg. Psychiatry
56:543-7; Greenberg et al. (1993)
Neurology
43:2073-9; Levy et al. (1990)
Science
248:1124-6). In some familial CAA cases, dementia was noted before the onset of hemorrhages, suggesting the possibility that cerebrovascular amyloid deposits may also interfere with cognitive functions.
In both AD and CAA, the main amyloid component is the amyloid protein (A&bgr;). The A&bgr; peptide, which is generated from the amyloid precursor protein (APP) by two putative secretases, is present at low levels in the normal CNS and blood. Two major variants, A&bgr;
1-40
and A&bgr;
1-42
, are produced by alternative carboxy-terminal truncation of APP (Selkoe et al.(1988)
Proc. Natl. Acad. Sci. USA
85:7341-7345; Selkoe, (1993)
Trends Neurosci
16:403-409). A&bgr;
1-42
is the more fibrillogenic and more abundant of the two peptides in amyloid deposits of both AD and CAA. In addition to the amyloid deposits in AD cases described above, most AD cases are also associated with amyloid deposition in the vascular walls (Hardy (1997), supra; Haan et al. (1990), supra; Terry et al., supra; Vinters (1987), supra; Itoh et al. (1993), supra; Yamada et al. (1993), supra; Greenberg et al. (1993), supra; Levy et al. (1990), supra). These vascular lesions are the hallmark of CAA, which can exist in the absence of AD.
The formation of A&bgr; is considered to be a key pathogenic process in Alzheimer's disease and related neurodegenerative disorders (reviewed by Selkoe in Nature Suppl.399: A23, 1999). The precise mechanisms by which neuritic plaques are formed and the relationship of plaque formation to the AD-associated, and CAA-associated neurodegenerative processes are not well-defined. However, evidence indicates that dysregulated expression and/or processing of APP gene products or derivatives of these gene products derivatives are involved in the pathophysiological process leading to neurodegeneration and plaque formation. For example, missense mutations in APP are tightly linked to autosomal dominant forms of AD (Hardy (1994)
Clin. Geriatr. Med.
10:239-247; Mann et al. (1992)
Neurodegeneration
1:201-215). The role of APP in neurodegenerative disease is further implicated by the observation that persons with Down's syndrome who carry an additional copy of the human APP (hAPP) gene on their third chromosome 21 show an overexpression of hAPP (Goodison et al. (1993)
J. Neuropathol. Exp. Neurol.
52:192-198; Oyama et al. (1994)
J. Neurochem.
62:1062-1066) as well as a prominent tendency to develop AD-type pathology early in life (Wisniewski et al. (1985)
Ann. Neurol.
17:278-282). Mutations in A&bgr; are linked to CAA associated with hereditary cerebral hemorrhage with amyloidosis (Dutch (HCHWA-D)(Levy et al. (1990), supra), in which amyloid deposits preferentially occur in the cerebrovascular wall with some occurrence of diffuse plaques (Maat-Schieman et al. (1994)
Acta Neuropathol.
88:371-8; Wattendorff et al. (1995)
J. Neurol. Neurosurg. Psychiatry
58:699-705). A number of hAPP point mutations that are tightly associated with the development of familial AD encode amino acid changes close to either side of the A&bgr; peptide (for a review, see, e.g., Lannfelt et al. (1994)
Biochem. Soc Trans.
22:176-179; Clark et al. (1993)
Arch. Neurol.
50:1164-1172). Finally, in vitro studies indicate that aggregated A&bgr; can induce neurodegeneration (see, e.g., Pike et al. (1995)
J. Neurochem.
64:253-265).
APP is a glycosylated, single-membrane-spanning protein expressed in a wide variety of cells in many mammalian tissues. Examples of specific isotypes of APP which are currently known to exist in humans are the 695-amino acid polypeptide described by Kang et al. (1987)
Nature
325:733-736, which is designated as the “normal” APP. A 751-amino acid polypeptide has been described by Ponte et al. (1988) Nature 331:525-527 and Tanzi et al. (1988) Nature 331:528-530. A 770-amino acid isotype of APP is described in Kitaguchi et al. (1988) Nature 331 :530-532. A number of specific variants of APP have also been described having point mutations which can differ in both position and phenotype. A general review of such mutations is provided in Hardy (1992) Nature Genet. 1:233-234. A mutation of particular interest is designated the “Swedish” mutation where the normal Lys-Met residues at positions 595 and 596 are replaced by Asn-Leu. This mutation is located directly upstream of the normal &bgr;-secretase cleavage site of APP, which occurs between residues 596 and 597 of the 695 isotype.
APP is post-translationally processed by several proteolytic pathways resulting in the secretion of various fragments or intracellular fragmentation and degradation. F. Checler,
J. Neurochem.
65:1431-1444 (1995). The combined activity of &bgr;-secretase and &ggr;-secretase on APP releases an intact &bgr;-amyloid peptide (A&bgr;), which is a major constituent of amyloid plaques. A&bgr; is an approximately 43 amino acid peptide which comprises residues 597-640 o
Cordell Barbara
Higaki Jeffrey N.
Mutz Mitchell
Bozicevic Karl
Bozicevic Field & Francis LLP
Chernyshev Olga N.
Scios Inc.
Ulm John
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