Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-10-21
2004-01-06
Chen, Shin-Lin (Department: 1632)
Chemistry: molecular biology and microbiology
Vector, per se
C536S023100, C536S023500, C536S024100
Reexamination Certificate
active
06673600
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a transgenic
C. elegans
which expresses an amyloid precursor protein (APP) or a part thereof, to the transgene itself, to the protein encoded by the transgene, and also to a process for preparing the transgenic
C. elegans
and to its use.
2. Description of Related Art
Several publications are referenced in the application. These references describe the state of the art to which this invention pertains, and are incorporated herein by reference.
Alzheimer's disease (morbus Alzheimer) is a neurodegenerative disorder of the brain which, at the cellular level, is accompanied by a massive loss of neurons in the limbic system and in the cerebral cortex. At the molecular level, it is possible to detect protein depositions, so-called plaques, in the affected areas of the brain, which depositions constitute an important feature of Alzheimer's disease. The protein which most frequently occurs in these plaques is a peptide of from 40 to 42 amino acids in size which is termed the A&bgr; peptide. This peptide is a cleavage product of a substantially larger protein of from 695 to 751 amino acids, the so-called amyloid precursor protein (APP).
APP is an integral transmembrane protein which traverses the lipid double layer once. By far the largest part of the protein is located extracellularly, while the shorter C-terminal domain is directed into the cytosol (FIG.
1
). The A&bgr; peptide is shown in dark gray in FIG.
1
. About two thirds of the A&bgr; peptide are derived from the extracellular domain of APP and about one third from the transmembrane domain.
In addition to the APP which is located in the membrane, it is also possible to detect a secreted form of the amyloid precursor protein, which form comprises the large ectodomain of the APP and is termed APPsec (“secreted APP”). APPsec is formed from APP by proteolytic cleavage which is effected by &agr;-secretase. The proteolytic cleavage takes place at a site in the amino acid sequence of APP which lies within the amino acid sequence of the A&bgr; peptide (after amino acid residue 16 of the A&bgr; peptide). Proteolysis of APP by the &agr;-secretase consequently rules out the possibility of the A&bgr; peptide being formed.
The A&bgr; peptide can consequently only be formed from APP by an alternative processing route. It is postulated that two further proteases are involved in this processing route, with one of the proteases, which is termed &bgr;-secretase, cutting the APP at the N terminus of the A&bgr; peptide and the second protease, which is termed &ggr;-secretase, releasing the C terminus of the A&bgr; peptide (Kang, J. et al., Nature, 325, 733) (FIG.
1
).
It has not as yet been possible to identify any of the three secretases or proteases (&agr;-secretase, &bgr;-secretase and &ggr;-secretase). However, knowledge of the secretases is of great interest, in particular within the context of investigations with regard to Alzheimer's disease and with regard to identifying the proteins involved, which proteins can then in turn be employed as targets in follow-up studies since, on the one hand, inhibition of the &bgr;-secretase, and in particular of the &ggr;-secretase, could lead to a decrease in A&bgr; production and, on the other hand, activation of the &agr;-secretase would increase the processing of APP into APPsec and thereby simultaneously reduce formation of the A&bgr; peptide.
There is a large amount of evidence that the A&bgr; peptide is a crucial factor in the development of Alzheimer's disease. Inter alia, A&bgr; fibrils are postulated to be neurotoxic in cell culture (Yankner, B. A. et al., (1990) Proc Natl Acad Sci USA,87, 9020). Furthermore, the neuropathology which is characteristic of Alzheimer's disease already appears at the age of 30 in Down's syndrome patients, who have an additional copy of APP. In this case, it is assumed that overexpression of APP is followed by an increased conversion into the A&bgr; peptide (Rumble, B. et al., (1989), N. Engl. J. Med., 320,1446).
The familial forms of Alzheimer's disease constitute what is probably the most powerful evidence of the central role of the A&bgr; peptide. In these forms, there are mutations in the APP gene around the region of the &bgr;-secretase and &ggr;-secretase cleavage sites or in two further AD-associated genes (presenilins) which, in cell culture, lead to a substantial increase in A&bgr; production (Scheuner, D. et al., (1996), Nature Medicine, 2, 864).
While
C. elegans
has already been used as a model organism in Alzheimer's disease, these studies do not relate to the processing of APP into the A&bgr; peptide. Some of the studies are concerned with two other Alzheimer-associated proteins, i.e. the presenilins. The presenilins are transmembrane proteins which traverse the membrane 6-8 times. They are of great importance in familial cases of Alzheimers since specific mutations in the presenilin genes lead to Alzheimer's disease. In this connection, it was shown that homologs to the human presenilins (sel-12, spe-4 and hop-1) are present in
C. elegans
, with the function of the presenilins being conserved in humans and worm (Levitan D, Greenwald I (1995) Nature 377, 351; Levitan et al.(1996) Proc Natl Acad Sci USA, 93, 14940; Baumeister R (1997) Genes & Function 1, 149; Xiajun Li and Iva Greenwald (1997) Proc Natl Acad Sci USA, 94, 12204).
Other studies deal with the APP homolog in
C. elegans
, which is termed Apl-1, and with expression of the A&bgr; peptide in
C. elegans
. However, Apl-1 does not possess any region which is homologous with the amino acid sequence of the A&bgr; peptide;
C. elegans
does not therefore possess any endogenous A&bgr; peptide (Daigle I, Li C (1993) Proc Natl Acad Sci USA, 90 (24), 12045).
C. D. Link, Proc Natl Acad Sci USA (1995) 92, 9368 described the expression of A&bgr; peptide (but not that of an A&bgr; precursor protein) in
C. elegans
. These studies involve preparing transgenic worms which express an A&bgr;1-42 peptide (i.e. the A&bgr; peptide which consists of 42 amino acids) as a fusion protein together with a synthetic signal peptide and under the control of the muscle-specific promoter unc 54. Muscle-specific protein depositions which reacted with anti-&bgr;-amyloid antibodies were detected in the studies.
Other studies (e.g. C. Link et al. personal communication) relate to investigations of the aggregation and toxicity of the A&bgr; peptide in the
C. elegans
model system.
Transgenic
C. elegans
lines were established in the present study in order to investigate the existence of a processing machinery in
C. elegans
which is involved in the formation of A&bgr; peptide and to identify potential secretases in this worm.
SUMMARY OF THE INVENTION
In this invention, APP genes have been transferred into
C. elegans
to create a transgenic
C. elegans
organism. This transgenic
C. elegans
can then be used to investigate the processing machinery involved in the formation of the A&bgr; peptide and to identify potential secretases.
The present invention relates to a transgene (a gene that has been transferred from one species to another by genetic engineering) which contains
a) a nucleotide sequence encoding an amyloid precursor protein (APP) or a part thereof, wherein the nucleotide sequence comprising the APP peptide or part thereof, contains, as part of the sequence, a nucleotide sequence comprising a complete A&bgr; peptide or a part of the A&bgr; peptide, and
b) where appropriate, one or more further coding and/or non-coding nucleotide sequences, and
c) a promoter for expression in a cell of the nematode
Caenorhabditis elegans
(
C. elegans
).
The nucleotide sequence preferably encodes the 100 carboxyterminal amino acids of APP, beginning with the sequence of the A&bgr; peptide and ending with the carboxyterminal amino acid of APP (C100 fragment). The APP is preferably one of the isoforms APP695 (695 amino acids), APP751 (751 amino acids), APP770 (770 amino acids) and L-APP. All th
Baumeister Ralf
Hoppe Edmund
Peraus Gisela
Aventis Pharma Deutschland GmbH
Chen Shin-Lin
Frommer Lawrence & Haug
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