Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~092/06376 PCT/FI91/00300
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2~73
Method for detection of the ~-amvloid protein usina
unspecific bindina of oli~onucleotides
Technical field
The invention relates to the detection of proteins by
means of oligonucleotides. The invention may be utilized
especially in diagnostics.
Background
Alzheimer's disease is a disease resulting in a difficult
dementia, in which the risk of falling ill increases along
with aging. Symptoms of Alzheimer's disease are the
progressive deterioration of memory, perception and other
higher brain functions, which finally results in the
patient's complete feeblemindedness.
Although the reasons for the outbreak of Alzheimer's
disease are not known, the pathological changes caused by
it are fairly well-known. The most typical changes are the
extracellular accumulation of amyloid protein on the walls
of the cerebral membranes and blood vessels as well as as
senile plaques. In addition, the cyto-skelenton components
of neurons aggregate intracellularly as so-called neurofi-
brillary tangles. In both cases, the filamentary proteins
may completely change the structure of the neuron, which
finally results in the dystrophy and cell death of the
neurons. The number of amyloid plaques correlates fairly
well with the deterioration of cognitive functions, the
neuron loss and the decrease in neurotransmitters (Tanzi
et al. 1989).
Senile or neuritic plagues consist of an amyloid core,
which is surrounded by a periphery comprised of dystrophic
neurites and extracellular material. The plaques may be
dense, so-called classic plaques or diffuse concentrations
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W O 92/06376 P ~ tF191/00300
206~731 2
formed of loosely interconnected material. Diffuse plaques
have in fact been assumed to be pre-stages of denser
formations, which may form during several years without
first causing neurologic symptons (Joachim et al. 1989,
Tanzi 1989).
The amyloid in the senile plaques forms of 6-10 nm
stranded ~-amyloid protein, whose identical subunits are
arranged antiparallelly into a beta-structure. This
hydrofobic protein of ca. 4.2-4.5 kDa is synthesized most
likely in neurons as part of a longer ~-amyloid precursor
protein. The gene coding for the precursor protein is
localized in the chromosome 21 (Goldgaber et al. 1978, ~-
Tanzi 1987).
One reason for the cumulation of amyloid has been assumed
to be a defect in the gene locus, since a correspondiny
plaque formation also occurs on nearly all Down's syndrome
patients of over 40 years (trisomy in chromosome 21),
whereby the overproduction of amyloid can be explained by
means of an extra gene. An autosomally dominant gene
defect in the chromosome 21 has been observed in patients
with the hereditary Alzheimer's disease, but it has not
been possible to prove a connection between it and the ~-
amyloid gene located in the same chromosome (St. George-
Hyslop et al. 1978, Tanzi et al. 1987).
The ~-amyloid precursor is a receptor-like, transmembranal
glycosylated protein, in which two thirds are located in
the extracellular state (Kang et al. 1987). The ~-amyloid
dipeptide is in turn located partly in the transmembranal
area so that its proteolytic release from the precursor
occurs either before the precursor's loosening onto or
after loosening from cellular membrane (Tanzi 1989).
It has been observed that the ~-amyloid gene codes for at
least three transcripts acting as models for polypeptides
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YYO 92/06376 P ~ /FI91/~0300
~ ` 3 2~973~
with 695, 751 and 770 amino acids. The two longer polypep-
tides contain an extra sequence with 56 the amino acid,
which is 50% homogenous with the enzymes belonging to the
so-called Kuniz's serine protease inhibitor family. Two
contradictory hypotheses corcerning the possible share of
the protease inhibitor sequence in the aggregation of the
~-amyloid have been presented: 1) it may prevent the
cumulation of amyloid by inhibiting proteases which
release ~-peptide from the precursor, or 2) the protease
inhibitor may advance the cumulation of amyloid by
preventing specific proteinases from purifying the tissue
from ~-amyloid. The function of the protease inhibitor
sequence of the ~-amyloid precursor protein and also its
release from the precursor into an active enzyme are thus
not known for certain.
General description of invention
The invention and certain preferred applications thereof
are defined in the patent claims.
The invention is based on the surprising discovery that
oligonucleotides bind to the ~-amyloid protein or its
precursor. The ~-amyloid protein or its precursor thus
binds DNA. The binding occurs especially in the ~-amyloid
protein or its precursor present in a peripheral tissue
(e.g. skin, mucous membrane). The phenomenon may be
utilized especially in diagnostic tests (e.g. Alzheimer's
disease, Down's syndrome, dementias, old persons).
Detailed description of invention
The object of our examination was the expression of ~-
amyloid on the frontal cortex and hippocampus of Alzheimer
patients. As a research method was used the RNA in situ
hybridization and as a specific tester was a cocktail
formed by three short oligonucleotides corresponding to
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W092/06376 :2 ~ ~ ~7 31 PCT/F191/00300
different areas of the ~-amyloid sequence. The oligonuc-
leotides were labelled at the 3'-end with biotin. For
specific antisense-RNA-oligonucleotides were synthesized
complementary sense-RNA-oligonucleotides, which were
labelled in the same way as the antisense probes. The in
situ hybridization was performed by using the routine
methods of our laboratory (a hybridized biotinylated
intron is allowed to bind to a streptavidine-alkaline
phosphatase complex, which is in turn detected by means of
a substrate reaction). The brain samples of 3 different
Alzheimer patients as well as two normal samples were
examined. As a result could be observed strong signals in
the area of the cortex and hippocampus corresponding both
by their location and shape to senile plaques. In additi-
on, a positive colouring was seen in the walls of blood
vessels, as described in connection with the ~-amyloid
protein. When we used the sense probe, we could to our
surprise see the same result as when using the antisense
probe. (When a specific mRNA expression is examined, the
sense probe should produce a negative result.) A negative
result was by contrast obtained, when water was used
instead of the gene probe; the reactions were otherwise
performed in the same way as when using the senselantisen-
se probe. All colouring results were negative, when the
normal samples were subjected to examination.
The in situ hybridization was repeated several times to
preclude a possible false positive reaction caused by
endogenic biotin or alkaline phosphatase.
Furthermore, the in situ hybridization was repeated by
using a radioactive label or other enzymes for the
detection of a biotinylized hybrid (e.g. peroxidase).
However, the results were always the same. Owing to the
results, also other oligonucleotides were tested, which
deviated from the published sequence of ~-amyloid (e.g.
erb cancer gene or 18 and 16 of human papilloma virus
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W092/06376 ~ PCT/FI9l/00300
5 20~973~
(HPV). The results remained the same.
The location of senile plaques and amyloid was additional-
ly ensured by using conventional stainings: kongo,
Bielschowski. The samples were additionally stained also
immunohistochemically by using as a primary antibody an
antibody made against a commercial ~-amyloid protein.
These stainings gave considerably weaker signals, but the
positive areas were the same.
Our results indicated that oligonucleotides (independently
of the sequence) bind to the ~-amyloid protein or its
precursor.
It has previously been postulated (Pepys and Butler, 1987
Breathnach et al. 1989) that serum's amyloid P binds DNA.
This binding is dependent on calcium. It has generally
been believed that serum's P amyloid cannot be seen in the
amyloid cumulations in connection with Alzheimer's
disease. However, it has recently been shown that serum
P's antibodies could provide reactivity also in the brain
area (Kalaria and Grahovac, 1990).
Joachim et al., 1989, indicated that ~-amyloid is also
found in the skin samples of Alzheimer patients (the
method used was immunohistology). We have been able to
show that oligonucleotides bind to ~-amyloid or its
precursor also in the skin sample of an Alzheimer patient.
Our results show that the cumulation in amyloid is a
secondary phenomenon and not only related to the brain
area.
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