U.S. patent application number 10/384172 was filed with the patent office on 2003-12-18 for perlecan transgenic animals and methods of identifying compounds for the treatment of amyloidoses.
Invention is credited to Fukuchi, Ken-Ichiro, Hassell, John, Snow, Alan D..
Application Number | 20030233669 10/384172 |
Document ID | / |
Family ID | 21784774 |
Filed Date | 2003-12-18 |
United States Patent
Application |
20030233669 |
Kind Code |
A1 |
Snow, Alan D. ; et
al. |
December 18, 2003 |
Perlecan transgenic animals and methods of identifying compounds
for the treatment of amyloidoses
Abstract
The invention provides a transgenic non-human animal expressing
a perlecan encoding transgene. Also provided is a double-transgenic
non-human animal expressing a perlecan and an amyloid encoding
transgene. A method of screening for a compound which alters the
rate or extent of amyloid deposition is additionally provided. The
method consists of: (a) constructing a perlecan transgenic animal;
(b) administering an effective amount of a test compound to said
perlecan transgenic animal; and (c) determining whether said test
compound alters the extent or rate of amyloid deposition. Finally,
the invention provides a method of screening for a compound which
alters the rate or extent of amyloid deposition. The method
consists of: (a) constructing a perlecan/amyloid double-transgenic
animal; (b) administering an effective amount of a test compound to
said perlecan/amyloid double-transgenic animal; and (c) determining
whether said test compound alters the extent o rate of amyloid
deposition.
Inventors: |
Snow, Alan D.; (Lynnwood,
WA) ; Fukuchi, Ken-Ichiro; (Birmingham, AL) ;
Hassell, John; (Tampa, FL) |
Correspondence
Address: |
PATRICK M. DWYER
PROTEOTECH, INC
SUITE 114
1818 WESTLAKE AVENUE N
SEATTLE
WA
98109
US
|
Family ID: |
21784774 |
Appl. No.: |
10/384172 |
Filed: |
March 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10384172 |
Mar 5, 2003 |
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09536231 |
Mar 27, 2000 |
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6563016 |
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09536231 |
Mar 27, 2000 |
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08870987 |
Jun 6, 1997 |
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60017830 |
Jun 6, 1996 |
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Current U.S.
Class: |
800/18 ;
800/21 |
Current CPC
Class: |
A01K 2267/0312 20130101;
A01K 2267/0356 20130101; C07K 14/4711 20130101; A01K 2217/05
20130101; A01K 2267/0362 20130101; A01K 67/0275 20130101; A01K
2267/0306 20130101; A01K 67/0278 20130101; A61K 49/0008 20130101;
A01K 2267/0318 20130101; C12N 15/8509 20130101; A01K 2207/15
20130101; A01K 2217/00 20130101; A01K 2227/105 20130101; C12N
2800/108 20130101; C12N 2830/008 20130101 |
Class at
Publication: |
800/18 ;
800/21 |
International
Class: |
A01K 067/027 |
Claims
We claim:
1. A method for making a transgenic non-human mammal, the method
comprising the steps of selecting appropriate restriction sites for
ligation of cDNA clones, and ligating together 7 overlapping cDNA
clones to produce a ligated cDNA clone that encodes for perlecan's
.about.400 kilodalton core protein.
2. The method of claim 1 wherein the non-human mammal is a
mouse.
3. The method of claim 1 further comprising the following steps: a)
constructing a plasmid (p Front End) containing cDNA for perlecan
domains I, II and III from clone 19-J (-XbaI), clone 54 and clone
DIII, by isolating the 3.3 kilobase SalI/XbaI fragment isolated
from clone DIII, isolating the 1.4 kilobase Not I/Bcl I DNA
fragment from clone 19-J (XbaI) and cloning it into the NotI and
Bcl sites of clone 54 to produce clone 19-J/54, isolating the 1.8
kilobase NotI/Sal I DNA fragment from clone 19-J/54 and ligating it
onto the Sal I site of the 3.3 kilobase SalI/XbaI fragment isolated
from clone DIII, and cloning a resulting 5.1 kilobase Not I/Xba I
fragment into the Not I and Xba I site of pBluescript I to produce
clone pFront End; b) constructing a plasmid (p Back End) containing
cDNA for most of perlecan domain IV and all of domain V from clone
DV and clone 12, to introduce a Hind III site into clone DV by
replacing the XhoI site in clone DV with a Hind III site using Hind
III linkers to produce clone DV with a Hind III site, and isolating
the 3.2 kilobase Xba I/Cla I fragment from clone 12 and cloning it
into the Xba I/Cla I sites of clone DV with a Hind III site from
which a smaller Xba I/Cla I fragment is previously removed to
produce clone p Back End; c) constructing a clone pF+ B by
connecting together the p Front End and p Back End plasmids from
step a) and step b), and cloning the 5.1 kilobase NotI/Xba I
fragment from step a) into the same restriction enzyme sites of p
Back End so that clone pF+B contains cDNA for the entire core
protein of perlecan except for a small region in the N-terminal
part of domain IV; d) constructing a plasmid (p Missing Link)
containing cDNA for small region in the N-terminal part of perlecan
domain IV from clone 72 and clone 7 by isolating a 1.5 kilobase
NotI/Hind III fragment isolated from clone 72 and a 2.2 kilobase
Hind III/Bam HI fragment from clone 7 and cloning them respectively
into the NotI and Bam HI sites of pBluescript I to produce clone
72/7, and isolating a 190 base pair Ppu MI fragment from clone 7
and inserting it into a restriction enzyme site in clone 72/7 from
which smaller Ppu MI fragments have been previously removed; e)
inserting the cDNA of p Missing Link into pF+B, isolating a 2.8
kilobase Nru I/Bss HII fragment from p Missing Link and cloning it
into the NFu I and Bss HII sites of pF+ B from which a small Nru
I/Bss HII fragment has been removed to produce plasmid pBS
DI-V.
4. A transgenic non-human mammal expressing a perlecan transgene,
wherein the perlecan transgene comprises one or more sequences each
coding for one or more fragments of perlecan, and wherein the
perlecan fragments are each homologous to a portion of a perlecan
domain selected from the group of perlecan domains consisting of
domains I-V and splice variants of domains I-V.
5. The transgenic mammal of claim 4, wherein the non-human mammal
is a mouse.
6. A method for making a transfected cell, the method comprising
the steps of selecting appropriate restriction sites for ligation
of cDNA clones, and ligating together 7 overlapping cDNA clones to
produce a ligated cDNA clone in the transfected cell that encodes
for perlecan's .about.400 kilodalton core protein.
7. The method of claim 6 wherein the transfected cell encodes for a
perlecan selected from the group of perlecans consisting of mouse
perlecan, bovine perlecan and human perlecan.
8. The method of claim 6 wherein the cell type selected for
transfection is selected from the group of cell types consisting of
COS cells and P19 cells.
9. A transfected cell expressing a perlecan encoding transgene,
wherein the perlecan transgene comprises one or more sequences each
coding for one or more fragments of perlecan, and wherein the
perlecan fragments are each homologous to a portion of a perlecan
domain selected from the group of perlecan domains consisting of
domains I-V and splice variants of domains I-V.
10. A double-transgenic non-human animal expressing a perlecan and
an amyloid encoding transgene.
11. A method of screening for a compounds which alters the rate or
extent of amyloid deposition comprising: (a) constructing a
perlecan transgenic animals; (b) administering an effective amount
of a test compounds to said perlecan transgenic animal; and (c)
determining whether said test compound alters the extent or rate of
amyloid deposition.
12. A method of screening for a compound which alters the rate or
extent of amyloid deposition comprising: (a) constructing a
perlecan/amyloid double-transgenic animal; (b) administering an
effective amount of a test compound to said perlecan/amyloid
double-transgenic animal; and (c) determining whether said test
compound alters the extent or rate of amyloid deposition.
13. A method for making a transgenic non-human mammal using unique
restriction sites to ligate together 7 overlapping cDNA clones to
produce a single .about.12 kb cDNA clone that encodes for
perlecan's .about.400 kilodalton core protein.
14. The transgenic non-human mammal of claim 7, wherein said
neuropathological condition is selected from the group consisting
of an Alzheimer's disease, an Alzheimer's-like disease, a familial
Alzheimer's disease, and a hereditary cerebral hemorrhage with
amyloidosis-Dutch type.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 09/536,231 filed Mar. 27, 2000, which is a continuation of U.S.
application Ser. No. 08/870,987 filed Jun. 6, 1997, which claims
priority to U.S. provisional application No. 60/017,830, filed Jun.
6, 1996.
[0002] The invention relates to the overproduction of perlecan in
transgenic animals and transfected animal cells as screening tools
to identify lead therapeutics for the amyloid diseases.
BACKGROUND OF THE INVENTION
[0003] The Amyloid Diseases
[0004] The "amyloid diseases" consist of a group of clinically and
generally unrelated human diseases which all demonstrate a marked
accumulation in tissues of an insoluble extracellular substance
known as "amyloid", and usually in an amount sufficient to impair
normal organ function. Rokitansky in 1842 (Rokitansky, "Handbuch
der pathologischen Anatomie", Vol. 3, Braumuller and Seidel,
Vienna) was the first to observe waxy and amorphous looking tissue
deposits in a number of tissues from different patients. However,
it wasn't until 1854 when Virchow (Virchow, Arch. Path. Anat. 8:416
(1854)) termed these deposits as "amyloid" meaning "starch-like"
since they gave a positive staining with the sulfuric acid-iodine
reaction, which was used in the 1850's for demonstrating cellulose.
Although cellulose is not a constituent of amyloid, nonetheless,
the staining that Virchow observed was probably due to the presence
of proteoglycans (PGs) which appear to be associated with all types
of amyloid deposits. The name amyloid has remained despite the fact
that Friederich and Kekule in 1859 discovered the protein nature of
amyloid (Friedrich and Kekule, Arch. Path. Anat. Physiol. 16:50
(1859)). For many years, based on the fact that all amyloids have
the same staining and structural properties, lead to the postulate
that a single pathogenetic mechanism was involved in amyloid
deposition, and that amyloid deposits were thought to be composed
of a single set of constituents. Current research has clearly shown
that amyloid is not a uniform deposit and that amyloids may consist
of different proteins which are totally unrelated (Glenner, N.
England J. Med. 302:1283-1292 (1980)).
[0005] Although the nature of the amyloid itself has been found to
consist of completely different and unrelated proteins, all
amyloids appear similar when viewed under the microscope due to
amyloid's underlying protein structure to adapt into a fibrillar
structure. All amyloids regardless of the nature of the underlying
protein 1) stain characteristically with the Congo red dye and
display a classic red/green birefringence when viewed under
polarized light (Puchtler et al, J. Histochem. Cytochem. 10:355-364
(1962)), 2) ultrastructurally consists of fibrils with a diameter
of 7-10 nanometers and of indefinite length, 3) adopt a predominant
beta-pleated sheet secondary structure. Thus, amyloid fibrils
viewed under an electron microscope (30,000 times magnification)
from the post-mortem brain of an Alzheimer's disease patient would
look nearly identical to the appearance of amyloid present in a
biopsied kidney from a rheumatoid arthritic patient. Both these
amyloids would demonstrate a similar fibril diameter of 7-10
nanometers.
[0006] In the mid to late 1970's amyloid was clinically classified
into 4 groups, primary amyloid, secondary amyloid, familial amyloid
and isolated amyloid. Primary amyloid, is amyloid appearing de
novo, without any preceding disorder. In 25-40% of these cases,
primary amyloid was the antecedent of plasma cell dysfunction such
as the development of multiple myeloma or other B-cell type
malignancies. Here the amyloid appears before rather than after the
overt malignancy. Secondary amyloid, appeared as a complication of
a previously existing disorder. 10-15% of patients with multiple
myeloma eventually develop amyloid (Hanada et al., J. Histochem.
Cytochem. 19:1-15 (1971)). Patients with rheumatoid arthritis,
osteoarthritis, ankylosing spondylitis can develop secondary
amyloidosis as with patients with tuberculosis, lung abscesses and
osteomyelitis (Benson and Cohen, Arth. Rheum. 22:36-42 (1979);
Kamei et al., Acta Path. Jpn. 32:123-133 (1982); McAdam et al.,
Lancet 2:572-575 (1975)). Intravenous drug users who
self-administer and who then develop chronic skin abscesses can
also develop secondary amyloid (Novick, Mt. Sin. J. Med. 46:163-167
(1979)). Secondary amyloid is also seen in patients with specific
malignancies such as Hodgkin's disease and renal cell carcinoma
(Husby et al., Cancer Res. 42:1600-1603 (1982)). Although these
were all initially classified as secondary amyloid, once the
amyloid proteins were isolated and sequenced many of these turned
out to contain different amyloid proteins.
[0007] The familial forms of amyloid also showed no uniformity in
terms of the peptide responsible for the amyloid fibril deposited.
Several geographic populations have now been identified with
genetically inherited forms of amyloid. One group is found in
Israel and this disorder is called Familial Mediterranean Fever and
is characterized by amyloid deposition, along with recurrent
inflammation and high fever (Mataxas, Kidney 20:676-685 (1981)).
Another form of inherited amyloid is Familial Amyloidotic
Polyneuropathy, and has been found in Swedish (Skinner and Cohen,
Biochem. Biophys. Res. Comm. 99:1326-1332 (1981)), Portuguese
(Saraiva et al., J. Lab. Clin. Med. 102:590-603 (1983); J. Clin.
Invest. 74:104-119 (1984)) and Japanese (Tawara et al., J. Lab.
Clin. Med. 98:811-822 (1981)) nationalities. Amyloid deposition in
this disease occurs predominantly in the peripheral and autonomic
nerves. Hereditary amyloid angiopathy of Icelandic origin is an
autosomal dominant form of amyloid deposition primarily affecting
the vessels in the brain, and has been identified in a group of
families found in Western Iceland (Jennson et al., Clin. Genet.
36:368-377 (1989)). These patients clinically have massive cerebral
hemorrhages in early life which usually causes death before the age
of 40.
[0008] The primary, secondary and familial forms of amyloid
described above tend to involve many organs of the body including
heart, kidney, liver, spleen, gastrointestinal tract, skin,
pancreas, and adrenal glands. These amyloid diseases are also
referred to as "systemic amyloids" since so many organs within the
body demonstrate amyloid accumulation. For most of these
amyloidoses, there is no apparent cure or effective treatment and
the consequences of amyloid deposition can be detrimental to the
patient. For example, amyloid deposition in kidney may lead to
renal failure, whereas amyloid deposition in heart may lead to
heart failure. For these patients, amyloid accumulation in systemic
organs leads to eventual death generally within 3 to 5 years.
[0009] Isolated forms of amyloid, on the other hand, tend to
involve a single organ system. Isolated amyloid deposits have been
found in the lung, and heart (Wright et al., Lab. Invest.
30:767-773 (1974); Pitkanen et al., Am. J. Path. 117:391-399
(1984)). Up to 90% of type II diabetic patients (non-insulin
dependent form of diabetes) have isolated amyloid deposits in the
pancreas restricted to the beta cells in the islets of Langerhans
(Johnson et al., New Engl. J. Med. 321:513-518 (1989); Lab Invest
66:522-535 (1992)). Isolated forms of amyloid have also been found
in endocrine tumors which secrete polypeptide hormones such as in
medullary carcinoma of the thyroid (Butler and Khan, Arch. Path.
Lab. Med. 110:647-649 (1986); Berger et al., Virch. Arch. A Path.
Anat. Hist. 412:543-551 (1988)). A serious complication of long
term hemodialysis is amyloid deposited in the medial nerve and
clinically associated with carpal tunnel syndrome (Gejyo et al.,
Biochem. Biopys. Res, Comm. 129:701-706 (1985); Kidney Int.
30:385-390 (1986)). By far, the most common type and clinically
relevant type of organ-specific amyloid, and amyloid in general, is
that found in the brains of patients with Alzheimer's disease (see
U.S. Pat. No. 4,666,829 and Glenner and Wong, Biochem. Biophys.
Res. Comm. 120:885-890 (1984); Masters et al., Proc. Natl. Acad,
Sci. USA 82:4245-4249 (1985)). In this disorder, amyloid is
predominantly restricted to the central nervous system. Similar
deposition of amyloid in the brain occurs in Down's syndrome
patients once they reach the age of 35 years (Rumble et al., New
England J. Med. 320:1446-1452 (1989); Mann et al., Neurobiol. Aging
10:397-399 (1989)). Other types of central nervous system amyloid
deposition include rare but highly infectious disorders known as
the prion diseases which include Creutzfeldt-Jakob disease,
Gerstmann-Straussler syndrome, and kuru (Gajdusek et al., Science
197:943-960 (1977); Prusiner et al., Cell 38:127-134 (1984);
Prusiner, Scientific American 251:50-59 (1984); Prusiner et al.,
Micr. Sc. 2:33-39 (1985); Tateishi et al., Ann. Neurol. 24:35-40
(1988)).
[0010] It was misleading to group the various amyloidotic disorders
strictly on the basis of their clinical features, since when the
major proteins involved were isolated and sequenced, they turned
out to be different. For example, amyloid seen in rheumatoid
arthritis and osteoarthritis, now known as AA amyloid, was the same
amyloid protein identified in patients with the familial form of
amyloid known as Familial Mediterranean Fever. Not to confuse the
issue, it was decided that the best classification of amyloid
should be according to the major protein found, once it was
isolated, sequenced and identified.
[0011] Thus, amyloid today is classified according to the specific
amyloid protein deposited. The amyloid diseases include, but are
not limited to, the amyloid associated with Alzheimer's disease,
Down's syndrome and hereditary cerebral hemorrhage with amyloidosis
of the Dutch type (wherein the specific amyloid is now known as the
beta-amyloid protein or A.beta.), the amyloid associated with
chronic inflammation, various forms of malignancy and Familial
Mediterranean Fever (AA amyloid or inflammation-associated
amyloidosis), the amyloid associated with multiple myeloma and
other B-cell abnormalities (AL amyloid), the amyloid associated
with type II diabetes (amylin or islet amyloid), the amyloid
associated with the prion diseases including Creutzfeldt-Jakob
disease, Gerstmann-Straussler syndrome, kuru and animal scrapie
(PrP amyloid), the amyloid associated with long-term hemodialysis
and carpal tunnel syndrome (beta.sub.2-microglobulin amyloid), the
amyloid associated with senile cardiac amyloid and Familial
Amyloidotic Polyneuropathy (prealbumin or transthyretin amyloid),
and the amyloid associated with endocrine tumors such as medullary
carcinoma of the thyroid (variants of procalcitonin).
[0012] Although there are many different types of amyloid diseases
as described above, there are only a few animal models for these
diseases to develop new therapeutic agents. Inflammation-associated
or AA amyloidosis has a well-defined experimental mouse model which
is used to induce amyloid deposition in systemic organs such as
spleen, liver and kidney (Snow and Kisilevsky, Lab. Invest.
53:37-44 (1985)). In addition, only some of the Alzheimer's disease
neuropathology is observed in current animal models of the disease
(described below). Thus, there is a need for the development of new
transgenic animal models for the amyloid diseases including
Alzheimer's disease. In addition, there is a need for new cell
culture models to rapidly screen and identify new lead therapeutics
for each of the amyloid diseases.
SUMMARY OF THE INVENTION
[0013] The invention provides a transgenic non-human animal
expressing a perlecan encoding transgene. Also provided is a
double-transgenic non-human animal expressing a perlecan and a
amyloid encoding transgene. A method of screening for a compound
which alters the rate or extent of amyloid deposition is
additionally provided. The method consists of: (a) constructing a
perlecan transgenic animal; (b) administering an effective amount
of a test compound to said perlecan transgenic animal; and (c)
determining whether said test compound alters the extent or rate of
amyloid deposition. Finally, the invention provides a method of
screening for a compound which alters the rate or extent of amyloid
deposition. The method consists of: (a) constructing a
perlecan/amyloid double-transgenic animal; (b) administering an
effective amount of a test compound to said perlecan/amyloid
double-transgenic animal; and (c) determining whether said test
compound alters the extent or rate of amyloid deposition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a schematic demonstrating the five structural
domains of perlecan.
[0015] FIGS. 2A-2G are schematics showing the construction strategy
of the full-length cDNA for perlecan core protein using plasmid
clones containing cDNAs for overlapping parts of perlecan.
[0016] FIGS. 3A-C shows the construction strategy for the
cytomegalovirus enhancer/chick .beta.-actin promoter linked to the
expression vector pCA-DI-V used to overexpress mouse perlecan in
transgenic mice and in transfected cells.
[0017] FIG. 4 is a black and white photograph of Western blots from
the cell lysates of non-transfected versus transfected COS cells.
Overexpression of perlecan in the cell layer of transfected COS
cells is demonstrated.
[0018] FIG. 5 is a black and white photograph of Western blots from
the media of non-transfected versus transfected COS cells.
Overexpression of perlecan in the media of transfected COS cells is
demonstrated.
[0019] FIG. 6 is a black and white photograph of Western blots from
the media and cell lysates of non-transfected versus transfected
P19 cells. Overexpression of perlecan in the cell layer and media
is demonstrated.
[0020] FIG. 7 is a black and white photograph of Western blots
detecting .beta.PP from the media and cell lysates of P19 cells
overexpressing both perlecan and .beta.PP-695, versus only
.beta.PP-695.
[0021] FIG. 8 is a black and white photograph of Western blots
detecting secreted A.beta. in the media from P19 cells
overexpressing both perlecan and .beta.PP-695, versus only
.beta.PP-695.
[0022] FIG. 9 shows bar graphs indicating normalized levels of
secreted A.beta. in the media from P19 cells overexpressing both
perlecan and .beta.PP-695, versus only .beta.PP-695. A marked 8-10
fold increase in the levels of A.beta. in cells overexpressing both
perlecan and .beta.PP-695 is demonstrated.
[0023] FIG. 10 shows bar graphs comparing the number of surviving
neurons in P19 cells overexpressing perlecan only, .beta.PP-695
plus perlecan, .beta.PP-695 only, and control parental cells (P19
only). Overexpression of perlecan only or perlecan plus
.beta.PP-695 leads to decreased neuronal survival.
[0024] FIG. 11 is a black and white photograph of Southern blots of
DNA taken from transgenic mice overexpressing mouse perlecan
(domains I-V). Chromosomal integration of the perlecan transgene is
demonstrated in Southern blots of DNA taken from transgenic mice,
and four perlecan transgenic founder mice are shown.
[0025] FIG. 12 is a black and white photograph of Northern blots of
RNA isolated from tissues of perlecan transgenic mice versus
control litter-mates. Overexpression of perlecan mRNA in several
tissues is demonstrated.
[0026] FIG. 13 is a black and white photograph of Western blots of
protein isolated from tissues of perlecan transgenic mice versus
control litter-mates. Overproduction of perlecan proteoglycan in
various organs of transgenic mice is demonstrated.
[0027] FIG. 14 are black and white microphotographs from brain and
kidney tissues of 5-month old perlecan transgenic mice versus
control litter-mates. The tissues were immunostained with an
antibody specific to perlecan, followed by counterstaining with
hematoxylin. Accumulation of overproduced perlecan in tissues of
transgenic mice is demonstrated.
[0028] FIGS. 15A and 15B demonstrate the construction strategy for
the cytomegalovirus enhancer/chick .beta.-actin promoter linked to
the signal peptide and to the C-terminal 99 amino acids of the
beta-amyloid precursor protein as an example to produce
beta-amyloid precursor protein transgenic mice (CA-S.beta.C).
[0029] FIG. 16 is a black and white photograph of Southern blots
taken from mouse tail DNA of double transgenic mice produced by
mating perlecan transgenic mice with transgenic mice that
overproduce the C-terminal 99 amino acids of the beta-amyloid
precursor protein. Pup #552 was found to incorporate the genes for
both perlecan and the C-terminal 99 amino acids of the beta-amyloid
precursor protein.
DETAILED DESCRITION OF THE INVENTION
[0030] As stated previously, perlecan is a specific heparan sulfate
proteoglycan and a common constituent of all amyloid deposits
regardless of the specific amyloid protein involved. Perlecan is
believed to play a primary role in the pathogenesis of amyloidosis
and contributes to the formation, deposition, accumulation and/or
persistence of amyloid in a variety of tissues and different
clinical settings. Previous animal models overexpressing a specific
amyloid protein only rarely produce some of the pathology
associated with different amyloid diseases, or produce fibrillar
amyloid in a different location than that observed clinically in
humans, making it extremely difficult to screen in vivo for
potential therapeutics for the various amyloid diseases.
Additionally, perlecan is an extremely difficult macromolecule to
isolate in pure and substantial quantities, and a transfected cell
line that overexpresses perlecan would enable one to isolate
perlecan for purification in sufficient quantities for use in a
number of different biological assays. Since perlecan is a
proteoglycan which contains a large core protein (.about.400
kilodaltons), previous attempts to successfully produce transgenic
mice which overexpress the entire perlecan core protein have not
been achieved. It appears that for the successful overexpression of
the entire perlecan core protein the strategies for ligating
overlapping cDNA clones which cover the entire 12 kb message of
murine perlecan core protein (or the entire 14 kb message of human
perlecan core protein), as well as the choice of specific promoter
and enhancers for use in the construction strategy of perlecan
transgenic mice is essential. In the present invention, unique
restriction sites were used to ligate together 7 overlapping cDNA
clones to produce a single 12 kb cDNA clone that encodes for mouse
perlecan's .sup..about.400 kDa core protein. A novel construct,
designated pCA-DI-V, which utilizes a cytomegalovirus enhancer and
chick .beta.-actin promoter, has led to successful overexpression
of mouse perlecan (domains I-V) in transfected cells and in
transgenic mice. Overproduction of perlecan was achieved in both
COS cells and P19 cells (embryonic carcinoma cells which
differentiate into neuron-like cells following retinoic acid
treatment). Overexpression of perlecan in P19 cells led to an
8-10-fold increase in secreted beta-amyloid protein (A.beta.)
levels, and contributed to a marked decrease in neuronal survival.
These latter studies suggest that perlecan overexpression in animal
cells or non-human transgenic animals may be sufficient to cause
the accumulation of Alzheimer's-type amyloid leading to
neuropathological and behavioral consequences. Production of
perlecan transgenic mice, and the mating of these mice with
transgenic animals which overexpress a given amyloid protein or its
precursor protein, and/or other components implicated in amyloid
pathogenesis (i.e. double transgenic mice) will produce progeny
that develop much or all of the phenotypic pathology of a given
amyloid disease. As an example for screening methods for
Alzheimer's disease, perlecan transgenic animals, cells derived
from perlecan transgenic animals, or perlecan-transfected animal
cells, either alone or in combination with other amyloid disease
implicated components, are used to screen for compounds altering
the pathological course of Alzheimer's disease as measured by their
effect on beta-amyloid precursor proteins (.beta.PPs), A.beta., and
numerous other Alzheimer's disease markers in animals, the
neuropathology of the animals, as well as behavioral alterations in
the animals. As an example of production of double transgenic mice,
successful mating of perlecan transgenic animals with transgenic
mice which overproduce the C-terminal 99 amino acids of .beta.PP,
has been achieved and led to the birth of progeny which carry both
the perlecan and .beta.PP genes. The production of new transgenic
animal models, and animal cells of amyloid diseases may be used as
in vivo and in vitro screening tools to aid in the identification
of lead therapeutics for the amyloidoses and for the treatment of
clinical manifestations associated with these diseases. The
successful overproduction of perlecan in transfected cells also
serves as a new means to isolate perlecan which will meet the
increasing demands for use of perlecan for a variety of in vitro
and in vivo assays.
[0031] "Amyloid" as a Therapeutic Target in Alzheimer's Disease
[0032] The most common form of amyloidosis is found in the brains
of patients with Alzheimer's disease. Alzheimer's disease is the
most common cause of dementia in middle and late life, and is
manifested by progressive impairment of memory, language,
visuospatial perceptions and behavior (A Guide to the Understanding
of Alzheimer's Disease and Related Disorders, edited by Jorm, New
York University Press, New York (1987)). A diagnosis of probable
Alzheimer's disease can be made on clinical criteria (usually by
the exclusion of other diseases, memory tests etc.), but a definite
diagnosis requires the histological examination of specific
abnormalities in the brain tissue usually obtained at autopsy.
[0033] In Alzheimer's disease, the parts of the brain essential for
cognitive processes such as memory, attention, language, and
reasoning degenerate, robbing victims of much that makes us human,
including independence. In some inherited forms of Alzheimer's
disease, onset is in middle age, but more commonly, symptoms appear
from the mid-60's onward. Alzheimer's disease is characterized by
the deposition and accumulation of a 39-43 amino acid peptide
termed the beta-amyloid protein, A.beta. or .beta./A4 (Glenner and
Wong, Biochem. Biophys. Res. Comm. 120:885-890 (1984); Masters et
al., Proc. Natl. Acad. Sci. USA 82:4245-4249 (1985); Husby et al.,
Bull. WHO 71:105-108 (1993)). This small peptide is a major
component which makes up the amyloid deposits of neuritic "plaques"
and in the walls of blood vessels (known as cerebrovascular amyloid
deposits) in the brains of patients with Alzheimer's disease. In
addition, Alzheimer's disease is characterized by the presence of
numerous neurofibrillary "tangles", consisting of paired helical
filaments which abnormally accumulate in the neuronal cytoplasm
(Grundke-Iqbal et al., Proc. Natl. Acad. Sci. USA 83:4913-4917
(1986); Kosik et al., Proc. Natl. Acad. Sci. USA 83:4044-4048
(1986); Lee et al., Science 251:675-678 (1991)). The pathological
hallmarks of Alzheimer's disease is therefore the presence of
"plaques" and "tangles", with amyloid being deposited in the
central core of plaques and within the blood vessel walls. It is
important to note that a so-called "normal aged brain" has some
amyloid plaques and neurofibrillary tangles present. However, in
comparison, an Alzheimer's disease brain shows an over abundance of
plaques and tangles. Therefore, differentiation of an Alzheimer's
disease brain from a normal brain from a diagnostic point of view
is primarily based on quantitative assessment of "plaques" and
"tangles".
[0034] In an Alzheimer's disease brain, are usually thousands of
neuritic plaques. The neuritic plaques are made up of extracellular
deposits consisting of an amyloid core usually surrounded by
enlarged axons and synaptic terminals, known as neurites, and
abnormal dendritic processes, as well as variable numbers of
infiltrating microglia and surrounding astrocytes. The
neurofibrillary tangles present in the Alzheimer's disease brain
mainly consist of tau protein, which is a microtubule-associated
protein (Grundke-Iqbal et al., Proc. Natl. Acad. Sci. USA
83:4913-4917 (1986); Kosik et al., Proc. Natl. Acad. Sci. USA
83:4044-4048 (1986); Lee et al., Science 251:675-678 (1991)). At
the ultrastructural level, the tangle consists of paired helical
filaments twisting like a ribbon, with a specific crossing over
periodicity of 80 nanometers. In many instances within a
neurofibrillary tangle, there are both paired helical filaments and
straight filaments. In addition, many times the nerve cell will
die, leaving the filaments behind. These tangles are known as
"ghost tangles" since they are the filamentous remnants of the dead
neuron.
[0035] The other major type of lesion found in the brain of an
Alzheimer's disease patient is the accumulation of amyloid in the
walls of blood vessels, both within the brain parenchyma and in the
walls of the larger meningeal vessels which lie outside the brain.
The amyloid deposits localized to the walls of blood vessels are
referred to as cerebrovascular amyloid or congophilic angiopathy
(Mandybur, J. Neuropath. Exp. Neurol. 45:79-90 (1986); Pardridge et
al., J. Neurochem. 49:1394-1401 (1987)).
[0036] In addition, Alzheimer's disease patients demonstrate
neuronal loss and synaptic loss. Furthermore, these patients also
exhibit loss of neurotransmitters such as acetylcholine. Tacrine,
the first FDA approved drug for Alzheimer's disease is a
cholinesterase inhibitor (Cutler and Sramek, New Enal. J. Med.
328:808-810 (1993)). However, this drug has showed limited success,
if any, in the cognitive improvement in Alzheimer's disease
patients and initially had major side effects such as liver
toxicity.
[0037] For many years there has been an ongoing scientific debate
as to the importance of "amyloid" in Alzheimer's disease and
whether the "plaques" and "tangles" characteristic of this disease,
were a cause or merely the consequences of the disease. Recent
studies during the last few years have now implicated that amyloid
is indeed a causative factor for Alzheimer's disease and not merely
an innocent bystander. The Alzheimer's disease major protein known
as beta-amyloid protein (A.beta.), in cell culture has been shown
to cause degeneration of nerve cells within short periods of time
(Pike et al., Br. Res. 563:311-314 (1991); J. Neurochem. 64:253-265
(1994)). Studies suggest that it is the fibrillar structure, a
characteristic of all amyloids, that is responsible for the
neurotoxic effects. The A.beta. has also been found to be
neurotoxic in slice cultures of hippocampus (the major memory
region affected in Alzheimer's) (Harrigan et al., Neurobiol. Aging
16:779-789 (1995)) and induces nerve cell death in transgenic mice
(Games et al., Nature 373:523-527 (1995); Hsiao et al., Neuron
15:1203-1218 (1995)). In addition, injection of the Alzheimer's
A.beta. into rat brain causes memory impairment and neuronal
dysfunction (Flood-et al., Proc. Natl. Acad. Sci. 88:3363-3366
(1991); Br. Res. 663:271-276 (1994)), two hallmarks of Alzheimer's
disease. Probably, the most convincing evidence that amyloid (i.e.
beta-amyloid protein) is directly involved in the pathogenesis of
Alzheimer's disease comes from recent genetic studies. It has been
discovered that the production of A.beta. can result from mutations
in the gene encoding, its precursor, known as the beta-amyloid
precursor protein (Van Broeckhoven et al., Science 248:1120-1122
(1990); Europ. Neurol. 35:8-19 (1995); Murrell et al., Science
254:97-99 (1991); Haass et al., Nature Med. 1:1291-1296 (1995)).
This precursor protein when normally processed only usually
produces very little of the toxic A.beta.. The identification of
mutations in the amyloid precursor protein gene which causes
familial, early onset Alzheimer's disease is the strongest argument
that amyloid is central to the pathogenetic process underlying this
disease. Four reported disease-causing mutations have now been
discovered which demonstrate the importance of the beta-amyloid
protein in causing familial Alzheimer's disease (reviewed in Hardy,
Nature Genet. 1:233-234 (1992)). These studies suggest that
providing a drug to reduce, eliminate or prevent fibrillar
beta-amyloid protein formation, deposition, accumulation and/or
persistence in the brains of human patients should be considered an
effective therapeutic.
[0038] Proteoglycans
[0039] Proteoglycans (PGs) are a group of complex macromolecules
which are found in all organs and tissues, intracellularly in a
variety of different cell types, or extracellularly in the matrix
where they are exported for a variety of functions. Proteoglycans
consist of a linear protein core backbone to which one or more
glycosaminoglycan (GAG) chains are covalently linked (Hascall and
Hascall, in Cell Biology of the Extracellular Matrix, Hay editor,
New York, Plenum Press, pp. 39, (1981); Hassell et al., Ann. Rev.
Biochem. 55:539-567 (1986)). The highly anionic GAG chains consist
of repeating disaccharide units, containing 1) hexosamine (either
D-glucosamine or D-galactosamine), and hexuronic acid (either
D-glucuronic acid or L-iduronic acid) (Muir, Am. J. Med. 47:673-690
(1969)). The PGs are traditionally named according to the
identification of the primary GAG present and several major GAGs
have been identified. These are hyaluronic acid, heparan sulfate,
heparin, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan
sulfate and keratan sulfate. Usually the linkage between the GAG
chains and the protein core backbone consists of a
xylose-galactose-galactose attachment region with the xylose
molecule covalently linked to the hydroxyl groups of a serine
residue on the protein core (Roden and Armand, J. Biol. Chem.
241:65-70 (1966)). The exception is hyaluronic acid which has a
backbone consisting of alternating D-glucuronic acid and
D-glucosamine units with no protein component. Keratan sulfate os
the one PG which lacks the typical xylose-serine linkage. It is
linked to protein either via a N-acetylgalactosamine residue linked
to either serine or threonine (in cartilage) or via a
N-acetylglucosamine residue attached directly to an asparagine
residue (in cornea) (Hascall and Hascall, in Cell Biology of the
Extracellular Matrix, Hay editor, New York, Plenum Press, pp. 39,
(1981); Muir, Am. J. Med. 47:673-690 (1969)).
[0040] Heparan Sulfate Proteoglycans: A Common Component of All
Amyloids
[0041] It is clear from the research literature that finding novel
therapeutics for amyloid formation, deposition, accumulation and
persistence is today considered a relevant strategy for Alzheimer's
disease and other amyloid diseases. The major question that
persisted in amyloid research was: why do all amyloids containing
unrelated proteins all form an amyloid fibril with similar
characteristics (i.e. all consist of fibrils of 7-10 nm and contain
a predominant beta-pleated sheet secondary structure)? Is there a
common component that may play a similar role in the pathogenesis
of all amyloids?
[0042] The answer to this central and important question in
understanding the mechanisms involved in amyloid diseases. Early
studies demonstrated that highly sulfated GAGs (later determined to
be specific heparan sulfate PGs) were concurrently deposited with
inflammation-associated amyloid (i.e. AA amyloid) in a well-defined
experimental mouse model (Snow et al., Lab Invest. 56:665-675
(1987)). Later studies demonstrated that heparan sulfate PGs were
temporally and structurally associated with the deposition and
accumulation of AA amyloid in a variety of different tissues (Snow
et al., J. Histochem. Cytochem. 39:1321-1330 (1991)). Specific
staining techniques and immunohistochemical methods then determined
that highly sulfated PGs were a common feature of most, if not all,
amyloids, independent of the specific amyloid protein involved, the
stage of the amyloid disease, and the tissue site of amyloid
deposition (Snow et al., Lab, Invest. 56:120-123 (1987); Am. J.
Path. 133:456-463 (1988); Acta Neuropath. 77:337-342, (1980); Lab.
Invest. 63:601-611 (1990)).
[0043] Studies were then pursued to further try to understand the
potential involvement of specific PGs in Alzheimer's disease
amyloidosis. In initial studies using specific immunohistochemical
probes it was first determined that heparan sulfate PGs were an
important constituent of amyloid in neuritic plaques and
cerebrovascular amyloid deposits (Snow et al., Am. J. Path.
133:456-463 (1988)). It was later revealed that the antibodies
employed for this initial study were in fact those that
specifically recognized the core protein of a large heparan sulfate
PG, known as "perlecan". Heparan sulfate PGs (and specifically
perlecan) were also co-localized to prion protein (PrP) amyloid
plaques in Gerstmann-Straussler syndrome, Creutzfeldt-Jakob
disease, kuru and animal scrapie (Snow et al., Lab. Invest.
63:601-611 (1990)). It was initially postulated that specific
heparan sulfate PGs play important roles in amyloidosis by 1)
influencing amyloidogenic proteins to adapt predominantly
beta-pleated sheet structures (i.e. indicative of amyloid), 2)
determining the anatomical location of amyloid deposition, and 3)
contributing to the stability of amyloid and its inaccessibility to
proteolytic degradation in tissues, thus not allowing the body to
properly degrade and remove unwanted amyloid deposits (Snow and
Wight, Neurobiol. Aging 10:481-497 (1989)).
[0044] Perlecan and/or heparan sulfate PG accumulation in
conjunction with a variety of different amyloid proteins is an
early event, and does not merely represent secondary and
non-specific deposition. In experimental inflammation-associated
amyloidosis, perlecan expression actually precedes AA amyloid
deposition (Ailles et al., Lab. Invest. 69:443-447 (1993))
suggesting that up-regulation of specific PGs may be an initiating
event leading to eventual amyloid formation and/or deposition. In a
previous study (Snow et al., Am. J. Path, 137:1253-1270 (1990)),
the brains of Down's syndrome patients (aged 1 day to 51 years)
were examined to determine the possible sequence of events leading
to beta-amyloid protein (A.beta.) and PG deposition. Down's
syndrome patients, who were completely devoid of any A.beta.
immunoreactivity, demonstrated prominent heparan sulfate
immunoreactivity in neurons as early as 1 day after birth, which
was not observed in similar aged-matched non-Down's syndrome
brains. In older patients, aged 18 and 24 years, diffuse A.beta.
immunoreactivity (which was Congo red negative and therefore
suggestive of non-fibrillar deposits) in the extracellular matrix
was accompanied by co-localized heparan sulfate deposition. In
patients, over the age of 35 years, fibrillar A.beta. deposits in
neuritic plaques and cerebrovascular amyloid accumulation were also
observed with co-localized heparan sulfate immunoreactivity. This
important study suggested that heparan sulfate accumulation within
neurons may be a primary event eventually leading to the
co-accumulation of heparan sulfate and A.beta. in the extracellular
matrix. It is feasible that once the interaction between heparan
sulfate and A.beta. (or its precursor protein) takes place, a
cascade of events occurs which lead to fibril formation, deposition
and eventual persistence.
[0045] The Importance of Heparan Sulfate Proteoglycans in
Alzheimer's Disease
[0046] To date at least four different classes of PGs/GAGs have now
been shown to be present in the A.beta.-containing deposits (i.e.
neuritic plaques, cerebrovascular amyloid) of Alzheimer's disease.
In 1988, it was discovered that heparan sulfate PGs were
specifically present in the amyloid deposits and neurofibrillary
tangles of Alzheimer's disease (Snow et al., Am. J. Path.
133:456-463 (1988)). In later years, the particular type of heparan
sulfate PG found was a large PG with a total molecular weight of
.about.800,000 known as "perlecan". In 1992, a second PG present in
the periphery of neuritic plaques and within neurofibrillary
tangles was discovered, which was a small dermatan sulfate PG known
as decorin (Snow et al., J. Histochem. Cytochem. 40:105-113
(1992)). In 1993, a third class of PGs were discovered, namely
chondroitin sulfate, which were present in the periphery of
neuritic plaques and within neurofibrillary tangles of Alzheimer's
disease brain (DeWitt et al., Exp. Neurol. 121:149-152 (1993)). The
specific type of chondroitin sulfate PG present to this day has yet
to be identified. In 1996, a keratan sulfate PG, known as SV2PG,
was discovered which was localized primarily to synaptic vesicles
in the periphery of neuritic plaques (Snow et al., Exp. Neurol.
138:305-317 (1996)).
[0047] Of all the different specific PGs/GAGs mentioned above, it
has become exceedingly clear that the heparan sulfate PGs may be
the most important class of PG implicated in Alzheimer's disease.
This is due to the fact that heparan sulfate PGs still remain the
only class of PG that has been immunolocalized to 1) all three
characteristic lesions of Alzheimer's disease (i.e. neuritic
plaque, neurofibrillary tangles, and cerebrovascular amyloid
deposits), and 2) specifically to the A.beta.-containing amyloid
fibrils in both neuritic plaques and cerebrovascular amyloid
deposits. The data suggests that perlecan is a major heparan
sulfate PG found within the amyloid core of neuritic plaques and
appears to be present in most, if not all, central nervous system
and systemic amyloids (reviewed in Snow and Wight, Neurobiol, Aging
10:481-497 (1989)). Overproduction of perlecan in a transgenic
animal or transfected cell line has, as yet, not been achieved, and
is needed to develop new models for developing therapeutics for the
amyloid diseases.
[0048] Perlecan Production by Different Cell Types and its
Postulated Roles in the Pathogenesis of Amyloid Diseases
[0049] Perlecan is present on all basement membranes (Dziadek et
al., EMBO J. 4:905-912 (1985); Kato et al., J. Cell Biol.
106:2203-2210 (1988); Murdoch et al., J. Histochem. Cytochem.
42:239-249 (1994)) and was previously cloned from both human
(Murdoch et al., J. Biol. Chem. 267:8544-8557 (1992); Kallunki and
Tryggvason, J. Cell. Biol. 116:559-571 (1992)) and mouse (Noonan et
al., J. Biol. Chem. 266:22939-22947 (1991)). Perlecan is known to
be produced by different cell types including endothelial cells
(Kinsella and Wight, Biochem. 27:2136-2144 (1988); Saku and
Furthmayr, J. Biol. Chem. 264:3514-3523 (1989); Rescan et al., Am.
J. Path. 142:199-208 (1993)), smooth muscle cells (Nikkari et al.,
Am. J. Path. 144:1348-1356 (1994)), fibroblasts (Murdoch et al., J.
Histochem. Cytochem. 42:239-249 (1994); Heremans et al., J. Cell
Biol. 109:3199-3211 (1989)), epithelial cells (Morris et al., In
Vitro Cell Dev. Biol. 30:120-128 (1994); Ohji et al., Invest. Opth.
Vis. Sci. 35:479-485 (1994); Van Det et al., Biochem. J.
307:759-768 (1995)), and synovial cells (Dodge et al., Lab. Invest.
73:649-657 (1995)). Perlecan is also synthesized by bone marrow
derived cells (Grassel et al., Mol. Cell Biochem. 145:61-68 (1995))
and is present in cancerous tissue including metastatic melanomas
(Cohen et al., Cancer Res. 54:5771-5774 (1994)), human breast
tumors (Guelstein et al., Int. J. Cancer 53:269-277 (1993)), and
liver tumors (Kovalsky et al., Acta Biomed. Ateneo Parmense
64:157-163 (1993)). Both F9 embryonal carcinoma cells (which form
parietal endoderm) and P19 embryonal carcinoma cells (which form
cholinergic neurons) also demonstrate marked increased perlecan
expression and synthesis upon differentiation (Chakravarti et al.,
Dev. Dyn. 197:107-114 (1993); Sekiguchi et al., J. Neurosc. Res.
38:670-686 (1994)).
[0050] Perlecan is postulated to play a primary role in the
pathogenesis of Alzheimer's disease (AD) amyloidosis, as well as in
other types of central nervous system and systemic amyloidoses
(reviewed in Snow and Wight, Neurobiol. Aging 10:481-497 (1989)).
Only the heparan sulfate class of PGs have been found to be
immunolocalized to all three major lesions (i.e. neuritic plaques,
neurofibrillary tangles and cerebrovascular amyloid deposits) in
Alzheimer's disease brain and specifically to the beta-amyloid
protein (A.beta.)-containing amyloid fibrils in both amyloid
plaques and congophilic angiopathy (Snow et al., Am. J. Path.
133:456-463 (1988); Snow and Wight, Neurobiol. Aging 10:481-497
(1989); Perlmutter and Chui, Brain Res. Bull. 24:677-686 (1990);
Snow et al., Am. J. Path. 137:1253-1270 (1990); Su et al.,
Neuroscience 51:801-813 (1992); Van Gool et al, Dementia 4:308-314
(1993)). Accumulating evidence suggests that perlecan is a major
heparan sulfate PG present within the A.beta.-containing amyloid
deposits in Alzheimer's disease (Snow et al., Am. J. Path.
133:456-463 (1988); Snow and Wight, Neurobiol. Aging 10:481-497
(1989); Snow et al., Am. J. Path. 137:1253-1270 (1990); Snow et
al., Am. J. Path. 144:337-347 (1994)) and may play a primary role
in A.beta. fibril formation, deposition, accumulation and
persistence. The consistent co-localization of perlecan to A.beta.
deposits which exist in both a fibrillar and non-fibrillar form
(Snow et al., Am. J. Path. 144:337-347 (1994)) is probably due to
perlecan's high affinity interactions with A.beta. (Snow et al., J.
Neuropath. Exp. Neurol. 48:352 (1989) Abstract; Buee et al., Brain
Res. 601:154-163 (1993); Buee et al., Brain Res. 627:199-204
(1993); Snow et al., Arch. Biochem. Biophys. 320:84-95 (1995)) and
with beta-amyloid precursor proteins (Narindrasorasak et al., J.
Biol. Chem. 266:12878-12883 (1991)). Residues 13-16 of A.beta. have
been identified as a perlecan binding site (Snow et al., J.
Neuropath. Exp. Neurol. 48:352 (1989) Abstract; Brunden et al., J.
Neurochem. 61:2147-2154 (1993); Snow et al., Arch. Biochem.
Biophys. 320:84-95 (1995)). This region contains a heparin/heparan
sulfate binding consensus sequence (Cardin and Weintraub,
Arterioscl. 9:21-32 (1989)), and is adjacent to the postulated
alpha-secretase cleavage site on A.beta. (at Lys-16). Once bound,
perlecan is believed to influence the secondary structure and/or
aggregation properties of A.beta. and/or beta-amyloid precursor
proteins (Fraser et al., J. Neurochem. 59:1531-1540 (1992)).
Perlecan also appears to play a role in stabilizing fibrillar
A.beta. amyloid when deposited in vivo (Snow et al., Neuron
12:219-234 (1994); Snow et al., Soc. Neurosc. Abst. 21:1292 (1995)
Abstract), and protects A.beta. from degradation by proteases as
demonstrated in vitro (Gupta-Bansal et al., J. Biol. Chem.
270:18666-18671 (1995)). The combined results described above
suggest that perlecan is an important macromolecule that has now
been implicated at several key steps in the pathogenesis of A.beta.
amyloidosis in Alzheimer's disease. However, due to perlecan's
large size and complex structure (described below), production of
transgenic animals or transfected cells which overproduce perlecan
has, as yet, not been achieved.
[0051] DNA Sequences and the Structure of Perlecan
[0052] The DNA sequence for human perlecan encodes for a protein
core with a molecular weight of approximately 466.564 kDa (Murdoch
et al., J. Biol. Chem. 267:8544-8557 (1992)) whereas the DNA
sequence for mouse perlecan encodes for a protein core with a
molecular weight of approximately 396 kDa (Noonan et al., J. Biol.
Chem. 266:22939-22947 (1991)). A schematic demonstrating the five
structural domains of perlecan is shown in FIG. 1. The genes for
human (Murdoch et al., J. Biol. Chem. 267:8544-8557 (1992);
Kallunki and Tryggvason, Cell Biol. 116:559-571 (1992)) and mouse
(Noonan et al., J. Biol. Chem. 266:22939-22947 (1991)) perlecan
have been cloned and the predicted core protein consists of five
distinct domains (FIG. 1). Domain I contains the proposed heparan
sulfate GAG attachment sites and is unique to perlecan showing no
similarity to other known protein sequences. The location of the
three Ser-Gly consensus heparan sulfate GAG attachment sites at the
N-terminus corresponds with the number and position of known GAG
chains (Kokenyesi and Silbert, Biochem. Biophys. Res. Comm.
211:262-267 (1995)). Domain II is homologous to the LDL binding
domain present in the LDL-receptor, whereas Domain III has homology
to the globule-rod regions of the laminin short arms. Domain IV is
a highly repetitive region with numerous immunoglobulin-like
repeats that show the highest similarity to neural cell adhesion
molecule (N-CAM). Domain V has three globular repeats very similar
to the domain G repeats in the laminin A chain and the equivalent
segment of the A chain homologue, merosin, and two epidermal growth
factor-like regions (Noonan and Hassell, Kidney Int. 43:53-60
(1993)). The perlecan core protein is therefore a unique and large
macromolecule with homology to a number of other well known
proteins.
[0053] Transgenic Models Trying to Mimic the Neuropathology of
Alzheimer's Disease
[0054] A number of transgenic animal models have been produced to
try to mimic some or all of the neuropathology of AD (reviewed in
Greenberg et al., Neurobiol. Aging 17:153-171 (1996)). The
rationale for most of these models was that overproduction of the
beta-amyloid precursor protein (.beta.PP) transgene (containing all
or part of the .beta.PP sequence) could lead to the eventual
development of A.beta. deposits in mouse brain and subsequent
plaque and tangle formation. In some of the first .beta.PP
transgenic animal model studies, (Wirak et al., Science 253:323-325
(1991)) generated transgenic mouse lines containing the A.beta.
sequence under the control of the human .beta.PP promoter. After 1
year, these mice developed A.beta. deposits within hippocampal
neurons and formed aggregates of amyloid-like fibrils. Quon et al
(Nature 352:239-241 (1991)) used a full length .beta.PP-751
sequence linked to a neuron-specific enolase promoter. Transgenic
mice with this construct displayed extracellular A.beta.
immunoreactive deposits, which were infrequently stained with
Thioflavin S, but not by Congo red, suggesting a pre-amyloid like
composition. Kawabata et al., (Nature 354:476-478 (1991)) developed
transgenic mouse lines massively overexpressing a construct
encoding the C-terminal 100 amino acids of .beta.PP under control
of a Thy-1 element. These mice displayed pathology remarkably
similar to that observed in AD including amyloid plaques,
neurofibrillary tangles and neurodegeneration in hippocampus,
neocortex and even cerebellum. While extremely promising, the
report by Kawabata et al., (Nature 354:476-478 (1991)) was
retracted whereas the study by Wirak et al., (Science 253:323-325
(1991)) was questioned (Jucker et al., Science 255:1443-1445
(1992)).
[0055] Recently, two transgenic animal models which probably
produce the most advanced neuropathology observed in animals to
date, have been described. Games et al., (Nature 373:523-527
(1995)) generated transgenic mice using platelet-derived growth
factor (PDGF)-.beta. driving a human .beta.PP minigene encoding the
.beta.PP-717 mutation (valine at residue 717 substituted by
phenylalanine) associated with familial AD. These mice
progressively developed some of the neuropathological hallmarks of
AD including Thioflavin S positive A.beta. deposits, neuritic
plaques, synaptic loss, astrocytosis and microgliosis. This
transgenic animal model was studied more closely by Masliah et al.,
(J. Neurosc. 16:5795-5811 (1996)) who demonstrated the
ultrastructure of the neuritic plaques formed in the brain
parenchyma of these mice. No cerebrovascular amyloid deposition nor
neurofibrillary tangles were observed to date in these mice.
[0056] Another transgenic animal model was recently reported (Hsiao
et al., Science 274:99-102 (1996)) in which the 695-amino acid
isoform of the human .beta.PP containing the double mutation
(Lys.sup.670 to Asn, Met.sup.671 to Leu), which was found in a
large Swedish family with early onset Alzheimer's Disease, was
inserted into a hamster prion protein cosmid vector. These mice
demonstrated a 5-fold increased in A.beta. (1-40) and a 14-fold
increase in A.beta. (1-42/43) accompanied by numerous amyloid
plaques stained with Congo red in cortical and limbic structures.
These transgenic animals had normal learning and memory in spatial
reference and alternating tasks at 3 months of age but showed
impairment by 9-10 months of age. In these mice, no cerebrovascular
amyloid deposition nor neurofibrillary tangles were observed.
[0057] The most recent transgenic models described above may one
day lead to fruitful insights into the pathogenesis of AD and may
ultimately be used to screen and identify new therapeutics to treat
AD. However, these transgenic models do have a major shortcoming.
These models have yet to show convincing cerebrovascular amyloid
deposition and neurofibrillary tangle formation in brain. This
indicates that these transgenic models are missing two of the three
major neuropathological hallmarks of AD (i.e. the other being the
neuritic plaque). Is there a missing factor that these transgenic
models have yet to take into account?
[0058] Heparan Sulfate Containing-Proteoglycans May be the
Additional Factor Necessary for Amyloid Plaque and Neurofibrillary
Tangle Formation in Alzheimer's Disease
[0059] Two very recent studies (Goedert et al., Nature 383:550-553
(1996); Perez et al., J. Neurochem. 67:1183-1190 (1996)) implicate
that heparan sulfate containing-PGs may be necessary additional
factors which may play roles in the formation of neurofibrillary
tangles. In one study, Goedert et al., (Nature 383:550-553 (1996))
found that highly sulfated GAGs such as heparin and heparan sulfate
stimulated different recombinant isoforms of tau protein to form
paired helical filaments in vitro. The paired helical filament
formation was found to be GAG-dependent, and
phosphorylation-independent indicating that the presence of heparan
sulfate GAGs was a key element in neurofibrillary tangle formation.
Highly sulfated GAGs such as heparin and heparan sulfate were
potent inducers of paired helical filament formation, whereas less
sulfated GAGs such as chondroitin sulfate and dermatan sulfate were
much less effective. Non-sulfated GAGs such as hyaluronic acid did
not induce any paired helical filament formation indicating that
sulfation was an important factor. Goedert et al., (Nature
383:550-553 (1996)) also confirmed some earlier work (Snow et al.,
Am. J. Path. 137:1253-1270 (1990)) that heparan sulfate accumulates
in the cytoplasm of neurons prior to the appearance of
neurofibrillary tangles. Heparan sulfate and chondroitin sulfate
have also been detected in tangle-bearing neurons in Alzheimer's
Disease (Perry et al., J. Neurosc. 11:3679-3683 (1991)) and in
neurons bearing tangles in other non-Alzheimer's diseases such as
progressive supranuclear palsy (Dewitt et al., Br. Res. 656:205-209
(1994)). Perez et al., (J. Neurochem. 67:1183-1190 (1996)) also
found heparin to induce different tau protein fragments to assemble
into paired helical filaments in vitro. The induction of paired
helical filaments by heparin/heparan sulfate suggests that PGs
containing this particular class of GAGs may the missing factor
essential for the induction of both amyloid plaques and
neurofibrillary tangles in transgenic animals. Overexpression of
perlecan (a specific heparan sulfate containing-PG) in transgenic
animals may therefore lead to both amyloid plaque and
neurofibrillary tangle formation, and ultimately serve as a new
animal model of Alzheimer's disease.
[0060] Other Co-Components Present in Alzheimer's and in Other
Amyloid Diseases
[0061] Alzheimer's disease is characterized by numerous changes in
the expression levels of various proteins, the biochemical activity
and histopathology of brian tissue, as well as cognitive changes in
affected individuals. Such characteristic changes associated with
Alzheimer's disease have been well documented. The most prominent
change, as discussed herein, is the deposition of A.beta. into
amyloid plaques (Haass and Selkoe, Cell 75:1039-1042 (1993)).
Besides specific PGs, a variety of other molecules have also been
known to be important components of Alzheimer's disease amyloid or
neurofibrillary tangles and include tau protein (Grundke-Iqbal et
al., Proc. Natl. Acad. Sci. USA 83:4913-4917 (1986); Kosik et al.,
Proc. Natl. Acad. Sci. USA 83:4044-4048 (1986); Lee et al., Science
251:675-678 (1991)), apolipoprotein E (Corder et al., Science
261:921-923 (1993); Strittmatter et al., Proc. Natl. Acad. Sci.
90:8098-8102 (1993)), alpha.sub.1-antichymotrypsin (Abraham et al.,
Cell 52:487-501 (1988)), amyloid P component (Coria et al., Lab.
Invest. 58:454-458 (1988)), ubiquitin (Mori et al., Science
235:1641-1644 (1987)), cytokines (McGeer et al., Can. J. Neurol.
Sci. 16:516-527 (1989); reviewed in Rogers, CNS Drugs 4:241-244
(1994)), growth factors (Hefti and Weiner, Ann. Neurol. 20:275-281
(1986); Hefti et al., Neurobiol. Aging 10:515-533 (1989); Kato et
al., Neurosc. 122:33-36 (1991); Tooyama et al., Neurosc. Lett.
121:155-158 (1991)), and complement factors (Eikenbloom et al.,
Virch. Arch. B Cell Pathol. 56:259-262 (1989)). Each of these
components described above may be utilized for the production of
new transgenic animals and/or transfected cells. Overexpression of
perlecan with another amyloid or neurofibrillary tangle component
described above may also lead to new models of amyloidosis (for
Alzheimer's disease and/or other amyloid diseases) and/or models of
tangle neuropathology.
[0062] Alzheimer's Disease Gene Mutations
[0063] A) .beta.PP Mutations
[0064] Certain families are genetically predisposed to Alzheimer's
disease, a condition referred to as familial Alzheimer's disease,
through mutations resulting in an amino acid replacement at
position 717 of the full length protein (Goate et al., supra
(1991); Murrell et al., supra (1991)). Another FAD mutation
contains a change in amino acids at positions 670 and 671 of the
full length protein (Mullan et al., supra (1992)). In one form of
this mutation, the lysine at position 670 is replaced by asparagine
and the methionine at position 671 is replaced by leucine. The
effect of this mutation is to increase the production of A.beta. in
cultured cells approximately 7-fold (Citron et al., Nature
360:672-674 (1992); Lai et al., Science 259:514-516 (1993)).
Additional mutations in .beta.PP at amino acids 669, 670 and 671
have been shown to reduce the amount of A.beta. processed from
.beta.PP (Citron et al., Neuron 14:661-670 (1995)). The .beta.PP
construct with Val at amino acid 690 produces an increased amount
of a truncated form of A.beta..
[0065] .beta.PP expression clones can be constructed to bear a
mutation at amino acids 669, 670, 671, 690, 692, or 717 of the full
length protein. The mutations from Lys to Asn and from Met to Leu
at amino acids 670 and 671, respectively, are sometimes referred to
as the Swedish mutation. Additional mutations can also be
introduced at amino acids 669, 670 or 671 which either increase or
reduce the amount of A.beta. processed from .beta.PP. Some
mutations at amino acid 717 are sometimes referred to as the Hardy
mutation. Such mutations can include conversion of the wild-type
Val717 codon to a codon of Ile, Phe, Gly, Tyr, Leu, Ala, Pro, Trp,
Met, Ser, Thr, Asn, or Gln, A preferred substitution for Val717 is
Phe. These mutations predispose individuals expressing the mutant
proteins to develop Alzheimer's disease. It is believed that the
mutations affect the expression and/or processing of .beta.PP,
shifting the balance towards Alzheimer's pathology. Mutations at
amino acid 669 can include conversion of the wild-type Val669 codon
to a codon for Trp, or deletion of the codon. Mutations at amino
acid 670 can include conversion of the wild-type Lys670 codon to a
codon for Asn or Glu, or deletion of the codon. Mutations at amino
acid 671 can include conversion of the wild-type Met671 codon to a
codon for Leu, Val, Lys, Tyr, Glu or Ile, or deletion of the codon.
A preferred substitution for Lys670 is Asn, and a preferred
substitution for Met671 is Leu. These mutations predispose
individuals expressing the mutant proteins to develop Alzheimer's
disease.
[0066] B) Presenilin 1 and Presenilin 2
[0067] In 1992, evidence for a locus causing early-onset
Alzheimer's disease was reported on the long-arm of chromosome 14
(Schellenberg et al., Science 258:668-671 (1992)). This result was
quickly confirmed by several groups. A positional cloning strategy
was then used to isolate a candidate gene (S182, later re-named
presenilin 1 or PS1) that carried coding region mutations in
families multiply affected by early-onset Alzheimer's disease
(Sherrington et al., Nature 375:754-760 (1995)). Since then more
than 35 different missense mutations have been found in the PS1
gene in over 50 families of different ethnic origins (Van
Broeckhoven, Nat. Genet. 11:230-232 (1995); Clark et al., Nat.
Genet. 11:219-222 (1996)). All PS1 gene mutations reported except
one are missense mutations.
[0068] The mean age of onset of disease in individuals with PS1
mutations is generally earlier than it is in individuals with
.beta.PP mutations, but considerable overlap does exist (the age
range for PS1 is 29 to 62 years, and for .beta.PP is 43 to 62
years). Several of the PS1 gene mutations are found in families of
different ethnic origins suggesting that independent mutational
events have occurred at the same nucleotide.
[0069] The PS1 gene contains 10 protein-coding exons and 2 or 3
additional exons encoding the 5'-untranslated region. The major RNA
transcript of the PS1 gene is about 3 kilobases and is expressed in
various human brian regions. The PS1 protein has 467 amino acids
and is thought to span the cell membrane 8 times, but its function
is unknown.
[0070] Soon after the isolation of the PS1 gene it became clear
from sequence homologies that PS1 was part of a gene family.
Sequences homologous to the PS1 gene were found to map to human
chromosome 1, indicating the existence of a second presenilin gene,
PS2 (also called the STM2 gene). At about the same time, genetic
linkage between Alzheimer's disease and DNA markers on chromosome 1
was detected in a group of Volga Germans with Alzheimer's disease
(Levy-Lahad et al., Science 269:970-973 (1995)). Since the PS2 gene
and the markers linked to the Volga German Alzheimer's disease
locus were very close together, the PS2 gene was sequenced in
affected individuals from several Volga German families, and a
missense mutation causing substitution of asparagine by isoleucine
at codon 141 was identified (Levy-Lahad et al., Science 269:973-977
(1995)). A second mutation, M239V, has been identified in an
Italian pedigree with early-onset familial Alzheimer's disease
(Rogacv et al., Nature 376:775-778 (1996)).
[0071] The PS2 protein contains 448 amino acids and shows 67%
identity with the PS1 protein. The PS2 gene is also widely
expressed in many tissues, but shows more extensive alternative
splicing than PS1 (Prohar et al., Neurorep. 7:1680-1684 (1996)).
However, the overall structures of the PS1 and PS2 proteins are
similar, and all AD-causing mutations are in the nucleotides coding
for amino acids that are conserved between the 2 genes, suggesting
that the 2 proteins serve similar functions and that mutant amino
acids are at positions within the protein critical to function.
[0072] As stated previously, perlecan is a specific heparan sulfate
proteoglycan and a common constituent of all amyloid deposits
regardless of the specific amyloid protein involved. Perlecan is
believed to play a primary role in the pathogenesis of amyloidosis
and contributes to the formation, deposition, accumulation and/or
persistence of amyloid in a variety of tissues and different
clinical settings. Previous animal models or overexpressing a
specific amyloid protein only rarely produce some of the pathology
associated with different amyloid diseases, or produce fibrillar
amyloid in a different location than that observed clinically in
humans, making it extremely difficult to screen in vivo for
potential therapeutics for the various amyloid diseases. In the
present invention, unique restriction sites were used to ligate
together 7 overlapping cDNA clones to produce a single 12 kb cDNA
clone that encodes for mouse perlecan's .sup..about.400 kDa core
protein. In a preferred embodiment, a novel construct, designated
pCA-DI-V, which utilizes a cytomegalovirus enhancer and chick
.beta.-actin promoter, has led to successful overexpression of
mouse perlecan (domains I-V) in transfected cells and in transgenic
mice.
[0073] For these studies, the constructs are introduced into animal
embryos using standard techniques such as microinjection or
embryonic stem cells. Cell culture based models can also be
prepared by two methods. Cell cultures can be isolated from the
transgenic animals or prepared from established cell cultures using
the same constructs with standard cell transfection techniques.
[0074] The specific constructs that are described preferably employ
a chick .beta.-actin promoter, which causes high expression of
perlecan in all tissues including brain. However, other promoters
may be used which may be selected from the following: the human
.beta.PP gene promoter, mouse .beta.PP gene promoter, rat .beta.PP
gene promoter, metallothionein III gene promoter, metallothionein I
promoter, rat neuron specific enolase gene promoter, mouse neuron
specific enolase promoter, human .beta. actin gene promoter, human
platelet derived growth factor B (PDGF-B) chain gene promoter, rat
sodium channel gene promoter, RNA polymerase I promoter, RNA
polymerase II promoter, polypeptide chain elongation factor 1-alpha
promoter, neurofilament M promoter, neurofilament L promoter, glial
fibrillary acidic protein promoter, prion protein promoter, insulin
promoter, low affinity nerve growth factor receptor (p75) promoter,
mouse myelin basic protein gene promoter, human copper-zinc
superoxide dismutase gene promoter, and mammalian POU-domain
regulatory gene promoter.
[0075] The specific constructs that are described preferably employ
a cytomegalovirus enhancer. However, other enhancers may be used
which may be selected from the following: immunoglobulin kappa
3'-enhancer, lambda enhancer, IgH 3'-enhancer, T cell receptor
alpha enhancer, alpha HS-26 enhancer, alpha HS-40 enhancer, and rat
insulin II gene enhancer.
[0076] The specific constructs that are described led to an
overproduction of perlecan in both COS cells and P19 cells
(embryonic carcinoma cells which differentiate into neuron-like
cells following retinoic acid treatment). Overproduction of
perlecan in P19 cells led to a marked increase in secreted A.beta.
levels and a marked decrease in neuronal survival. As an example
for screening methods for Alzheimer's disease, perlecan transgenic
animals, or perlecan-transfected animal cells, either alone or in
combination with other amyloid disease co-components, are used to
screen for compounds altering the pathological course of
Alzheimer's disease as measured by their effect on .beta.PPs,
A.beta., and numerous other Alzheimer's disease markers in animals,
the neuropathology of the animals, as well as behavioral
alterations in the animals. The production of new transgenic animal
models, and animal cells of amyloid diseases may be used as in vivo
and in vitro screening tools to aid in the identification of lead
therapeutics for the amyloidoses and for the treatment of clinical
manifestations associated with these diseases. The successful
overproduction of perlecan in transfected cells also serves as a
new means to isolate perlecan which will meet the increasing
demands for use of perlecan for a variety of in vitro and in vivo
assays.
[0077] In one embodiment, it is an object of the present invention
to provide a transgenic animal or transfected cell line whose cells
include a recombinant DNA sequence coding for ubiquitous or cell
type specific expression of perlecan or analogs thereof. The mating
of perlecan transgenic mice with transgenic mice overexpressing a
specific amyloid protein or its precursor, or which overproduce
another component implicated in the disease, will produce new
transgenic mice progeny which overexpress both perlecan and a
specific amyloid protein (or its precursor), or both perlecan and a
specific amyloid co-component. These transgenic animals which
overexpress both perlecan and a specific amyloid protein (or its
precursor protein), or both perlecan and a specific amyloid
co-component will lead to the production of new transgenic animals
which display much or all of the pathology associated with a
particular amyloid disease. In addition, perlecan overexpressing
transgenic animals may be mated with transgenic animals which
underexpress a specific amyloid protein or its precursor, or which
underproduce another component implicated in the disease. The
production of these new animals will be effective for the study of
the etiology of various amyloidoses and the efficacy of drugs in
treating each of the amyloid diseases. The amyloid diseases
include, but are not limited to, the amyloid associated with
Alzheimer's disease, Down's syndrome and hereditary cerebral
hemorrhage with amyloidosis of the Dutch type (wherein the specific
amyloid is referred to as beta-amyloid protein or A.beta.), the
amyloid associated with chronic inflammation, various forms of
malignancy and Familial Mediterranean Fever (wherein the specific
amyloid is referred to as AA amyloid or inflammation-associated
amyloidosis), the amyloid associated with multiple myeloma and
other B-cell dyscrasias (wherein the specific amyloid is referred
to as AL amyloid), the amyloid associated with type II diabetes
(wherein the specific amyloid is referred to as amylin or islet
amyloid), the amyloid associated with the prion diseases including
Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru, and
animal scrapie (wherein the specific amyloid is referred to as PrP
amyloid), the amyloid associated with long-term hemodialysis and
carpal tunnel syndrome (wherein the specific amyloid is referred to
as beta.sub.2-microglobulin amyloid), the amyloid associated with
senile cardiac amyloid and Familial Amyloidotic Polyneuropathy
(wherein the specific amyloid is referred to as prealbumin or
transthyretin amyloid), and the amyloid associated with endocrine
tumors such as medullary carcinoma of the thyroid (wherein the
specific amyloid is referred to as variants of procalcitonin).
[0078] One of the major problems in the past in trying to produce
perlecan transgenic mice and perlecan transfected cells was the
large size of the perlecan core protein (.sup..about.400 kDa). A
unique strategy had to be developed which allowed one to produce a
single 12 kb (from mouse) cDNA clone that encoded for the entire
400 kDa perlecan core protein. Another object of the present
invention is to develop a construction strategy using unique
restriction sites in overlapping cDNA clones to produce a single 12
kb cDNA clone that encodes for the entire perlecan core
protein.
[0079] Another object of the present invention is to provide a
method to produce new transgenic animals that overexpress both
perlecan (or portions thereof) and a specific amyloid protein (or
its precursor), or both perlecan and a specific amyloid
co-component. These new transgenic animals can be used for the
studies pertaining to the etiology of various amyloidoses and the
efficacy of drugs in treating each of the amyloid diseases.
[0080] Another object of the present invention is to provide
transgenic animals which have in their cells unique promoter/coding
sequences which can either ubiquitously express perlecan in all
types of tissue, or which can express perlecan in specific types of
tissue. These perlecan transgenic animals can be used to assess the
role that perlecan, and/or portions thereof, play in development
and in a number of relevant biological and/or pathological
processes.
[0081] A further object of the invention relates to the synthesis
and use of promoter/coding constructs which express perlecan alone
or in combination with specific amyloid proteins (or precursor
proteins), or other amyloid co-components, in various tissues of
transgenic animals incorporating such constructs in their genome. A
feature of the transgenic animals which would coexpress both
perlecan and a specific amyloid protein, or both perlecan and a
specific amyloid co-component, will be that these new animals will
provide both prognostic and diagnostic means for the study of
different amyloid diseases and for determining the efficacy of
pharmaceutical drugs in treating specific amyloidoses in a human
subject. Initially, the transgenic animals may be used as in vivo
screening tools to help identify one or more candidate compounds
capable of disrupting the formation, deposition, accumulation
and/or persistence of a given amyloid protein which is associated
with a predisposition to a specific amyloid disease.
[0082] Another object of the present invention relates to providing
transgenic non-human mammals which yield information regarding the
mechanisms (leading to production and deposition) and location of
various amyloids, as well as providing necessary in vivo models for
testing of potential drugs capable of interfering with or
preventing such formation, deposition, accumulation and/or
persistence in specific tissues and organs.
[0083] Yet another object of the present invention relates to the
production of non-human mammals which overexpress, mouse or human
perlecan (and/or any other species from which perlecan cDNA
sequence is known or will become known).
[0084] Yet another object of the present invention is to provide an
animal model for Alzheimer's disease that is constructed using
transgenic technology.
[0085] It is a further object of the present invention to provide
transgenic animals exhibiting one or more histopathologies similar
to those of Alzheimer's disease.
[0086] It is a further object of the present invention to provide
transfected cells expressing one or more A.beta.-containing
proteins at high levels in cell culture media. It is yet a further
object of the present invention to provide transgenic animals
expressing one or more A.beta.-containing proteins at high levels
in brain tissue.
[0087] It is a further object of the present invention to provide a
method of screening potential drugs for the treatment of
Alzheimer's disease using transgenic animal models and transfected
cell lines.
[0088] Yet another object of the present invention relates to the
production of mouse and/or human (or any other species from which
the perlecan cDNA sequence is known or will become known) perlecan
by transfected cells. This invention serves as a new means to
provide for cell culture models for testing of potential drugs
capable of interfering with or preventing A.beta. or .beta.PP (and
constituents thereof) formation, deposition, accumulation and/or
persistence. In addition, these cell cultures can be utilized to
isolate perlecan in sufficient quantities for use in a variety of
different in vitro and in vivo assays.
[0089] This invention can be used either alone or in combination
with other transgenic animals or transfected animal cells which
overproduce or underproduce a given amyloid protein or its
precursor, or which overproduce or underproduce another component
implicated in a given amyloid disease. As an example, a double
transgenic mouse has been made which carry the transgenes for both
perlecan and the C-terminal 99 amino acids of the beta-amyloid
precursor protein of Alzheimer's disease. These model systems
provide new in vivo and in vitro methods for the screening and
evaluation of potential drugs for the treatment of Alzheimer's
disease and other amyloid diseases, and for the production of
perlecan in culture for use in biological systems.
[0090] Before the processes for making and using such transgenic
mice and transfected cells are described, it is to be understood
that these inventions are not limited to particular processes and
materials described as such methods and materials, may vary. It is
also to be understood that the terminology used herein is for the
purpose of describing particular embodiments only, and is not
intended to be limiting.
[0091] A unique feature of the present invention is the strategies
employed to produce a single 12 kb cDNA clone that encodes for
mouse perlecan's 400 kDa core protein. The initial cDNA clones to
murine perlecan were isolated from an expression vector library
prepared from Engelbreth-Holm-Swarm (EHS) tumor mRNA by screening
the library with rabbit antibodies to murine perlecan. The
authenticity of the clones was confirmed by demonstrating an exact
match of amino acid sequences from perlecan peptides with that of
sequence deduced from the cDNA clones. Additional cDNA clones were
obtained by making primer extension libraries and screening them
with existing clones to produce 7 overlapping clones covering
perlecan's 12 kb message. These overlapping clones were then
ligated together using unique restriction sites in the overlapping
regions to produce a single 12 kb cDNA clone that encoded for
perlecan's .sup..about.400 kDa core protein. The single 12-kb
perlecan cDNA was cloned into the NotI/XbaI sites of pBluescript II
SK (Stratagene Cloning System) to generate pBSDI-V.
[0092] Another unique feature of the transgenic mice of the present
invention relates to including general or cell specific promoters
in front of sequences which encode for domains I to V of mouse or
human perlecan. The ability of the transgenic mice to selectively
express perlecan core protein, including any fragments thereof,
distinguishes the present transgenic mice from others.
[0093] The cloned recombinant and/or synthetic DNA sequences used
in connection with the present invention are sequences which encode
a biologically active, refolded proteoglycan, and contains one or
more glycosaminoglycan chains attached to its protein core
backbone.
[0094] Construction of Expression Vectors
[0095] Preferred cDNA clones used in making the transgenic mice and
transfected cells include coding sequences which are initially
inserted in a suitable expression vector for replication and to
confirm production of protein. In a preferred embodiment, a
full-length cDNA of murine perlecan core protein is constructed
from cDNAs for overlapping parts of perlecan isolated from mouse
cDNA libraries (Noonan et al., J. Biol. Chem. 266:22939-22947
(1991)). Such clones are in the pBluescript I vector (a product of
Strategene) and are illustrated in FIGS. 2A-2G. All clones
constructed as described below were verified by sequencing and/or
restriction mapping. Firstly, a plasmid (p Front End) containing
cDNA for the domains I, II and III of perlecan is constructed from
clone 19-J (-XbaI), clone 54 and Clone DIII (FIGS. 2B and 2C). The
1.4-kilobase Not I/Bcl I DNA fragment is isolated from clone 19-J
(XbaI) and cloned into the NotI and Bcl I sites of clone 54 to
produce clone 19-J/54 (FIG. 2B). The 1.8-kilobase Not I/Sal I DNA
fragment is isolated from clone 19-J/54 and ligated onto the Sal I
site of the 3.3-kilobase Sal I/Xba I fragment isolated from clone
DIII. The resulting 5.1-kilobase Not I/Xba I fragment is cloned
into the Not I and Xba I site of pBluescript I to produce clone
pFront End (FIG. 2C). Secondly, a plasmid clone (p Back End)
containing cDNA for most of domain IV of perlecan and all of domain
V, is constructed from clone DV and clone 12 (FIG. 2D). To
introduce a Hind III site into clone DV, the XhoI site in clone DV
is replaced with a Hind III site using Hind III linkers to produce
clone DV wH3. The 3.2-kilobase Xba I/Cla I fragment isolated from
clone 12 is cloned into the XbaI/Cla I sites of clone DV with H3
from which the smaller Xba I/Cla I fragment is previously removed.
The resulting clone is designated p Back End (FIG. 2D). Thirdly,
the Front and Back Ends are connected together to produce clone pF+
B that contains cDNA for the entire core protein of perlecan except
for a small region in the N-terminal part of domain IV (FIG. 2E).
The 5.0-kilobase Not I/Xba I fragment isolated from p Front End is
cloned into the same restriction enzyme sites of p Back End to
produce p F+B (FIG. 2E). Next, a plasmid (p Missing Link)
containing cDNA for the small region of the N-terminal part of
perlecan domain IV is constructed from clone 72 and clone 7 (FIG.
2F). The 1.5-kilobase Not I/Hind III fragment isolated from clone
72 and the 2.2-kb Hind III/Bam HI fragment isolated from clone 7
are cloned into the Not I and Bam HI sites of pBluescript I to
produce clone 72/7 (FIG. 2F). The 190-base pair Ppu MI fragment
isolated from clone 7 is inserted into the same restriction enzyme
site of clone 72/7 from which the smaller Ppu MI fragments are
previously removed. The resulting plasmid is designated as p
Missing Link (FIG. 2F). Finally, cDNA in p Missing Link is inserted
into pF+B to produce pBS DI-V that contains a full-length cDNA for
perlecan (FIG. 2G). The 2.8-kilobase Nru I/Bss HII fragment
isolated from p Missing Link is cloned into the same restriction
sites of pF+B from which the smaller Nru I/Bss HII fragment is
previously removed. The resulting plasmid is designated pBS DI-V
(FIG. 2G).
[0096] The expression vector, pCA-DI-V, is then constructed to
overexpress the entire perlecan core protein under the control of a
cytomegalovirus enhancer and a chick beta-actin promoter (FIGS.
3A-3C). This expression vector contains intron 1 of chick
.beta.-actin gene and intron 3 of rabbit .beta.-globulin gene.
First, a Hind III restriction enzyme site is added next to the Sal
I site in pCAGGSnc (Fukuchi et al., Exp. Exp. Neurol. 127:253-264
(1994)) using a polylinker containing XhoI, Hind III, and Sal I
sites (FIG. 3A). pCAGGSnc is linearized by Sal I digestion and
treated with calf intestinal alkaline phosphatase. The polylinker
containing XhoI, Hind III, and Sal I sites is digested with Xho I
and Sal I and ligated onto the Sal I site of pCAGGSnc to create
pCAGGhnc. In a separate reaction, pBSDI-V which contains full
length perlecan cDNA in pBluescript II SK (Stratagene Cloning
System) is digested with Asn I and a 11.5-kb DNA fragment
containing perlecan cDNA is isolated (FIG. 3B). A polylinker
containing Asn I, Nhe I, and SfuI sites is digested with AsnI and
ligated onto the same restriction enzyme site of the isolated
11.5-kb DNA fragment (FIG. 3B). The isolated fragment is then
digested with Not I and Sfu I and ligated onto the Not I and Cla I
sites of pCAGGShnc which was previously cut with Not I and Cla I
(FIG. 3C). Cla I is compatible with Sfu I. The resulting vector is
designated as pCA-DI-DV; pCA-DI-V is used to establish lines of
transgenic mice and cells.
[0097] In another preferred embodiment, a full-length cDNA of human
perlecan core protein can be constructed from cDNAs for overlapping
parts of perlecan isolated from human cDNA libraries. As
demonstrated by Murdoch et al., (J. Biol. Chem. 267:8544-8557
(1992)), commercially available cDNA libraries such as human colon
cDNA library (HL103b, a product of Clontech), or cDNA libraries
prepared from a human fibroblast cell line (CRL 1262, available
from American Tissue Culture Collection) and a human amnion cell
line (WISH also available from American Tissue Culture Collection)
can also be used to isolate overlapping clones covering the entire
coding sequence for human perlecan core protein. These overlapping
clones can be ligated to form a single cDNA using appropriate
restriction sites in the clones in any manner known to those
skilled in the art. The single 14 kb cDNA clone is then cloned into
multiple cloning sites of pCAGGS or pCAGGShnc to create an
expression vector for human perlecan. Expression vectors are not
limited to pCAGGS or pCAGGShnc. Indeed, any other expression
vectors containing the promoters listed below (or any other
promoters that seem appropriate by those skilled in the art) can be
used and the construction of these expression vectors can be
achieved in any manner known to those skilled in the art.
[0098] In another preferred embodiment, a full-length cDNA of
bovine perlecan core protein can be similarly constructed and can
be used to overexpress bovine perlecan in transgenic mice and in
cultured cells. For example, bovine perlecan cDNA can be isolated
by screening a bovine kidney cDNA library (BL3001b, a product of
Clontech) using several parts of mouse cDNA as probes. The isolated
overlapping clones can be ligated into a single cDNA coding for the
entire bovine perlecan and then cloned into any expression vectors,
in any manner known to those skilled in the art.
[0099] Promoter/Perlecan Sequence Fusion Constructs
[0100] A) Promoters That Permit Ubiquitous Expression of
Transgenes
[0101] The following promoters allow transgenes to express at high
levels in virtually all tissues. Therefore such ubiquitous
promoters can be used to make any type of amyloid animal models.
Such useful promoters which can be used to create constructs and
inserted into transgenic animals in connection with the present
invention include, but are not limited to:
[0102] Beta-Actin Promoter
[0103] Beta actin is essential and abundantly expressed in
virtually all cells. pCAGGS (Niwa et al., Gene 108:193-200 (1991))
that contained the beta-actin promoter was used to construct
expression vectors for mouse perlecan and beta-amyloid precursor
protein as in Example 10.
[0104] RNA Polymerase I Promoter
[0105] RNA polymerase I catalyzes synthesis of ribosomal RNA.
Therefore, this promoter permits transgene expression in virtually
all tissues with the exception of erythrocytes. RNA polymerase I
transcripts, however, are poorly translated as mRNA since they
retain a triphosphate at their 5' termini rather than receiving a
trimethyl G cap. In the absence of a trimethyl G cap, ribosomes
appear not to recognize the transcript as message and translation
initiation is impaired. Insertion of an internal ribosome entry
site (IRES) into the 5' leader of the RNA polymerase I transcript
allows high level protein production under control of the RNA
polymerase I promoter (Palmer et al., Nucleic Acid Res.
21:3451-3457 (1993)). Such RNA polymerase promoters with IRES are
available in the plasmid, pMENA (Palmer et al., Nucleic Acid Res.
21:3451-3457 (1993)).
[0106] RNA Polymerase II Promoter
[0107] RNA polymerase catalyzes the synthesis of RNA transcription
from genes and therefore its promoter permits the ubiquitous
expression of transgenes with the exception of erythrocytes. The
RNA polymerase II promoter is cloned in PHBII CATm (Ahearn et al.,
J. Biol. Chem. 262:10695-10705 (1987)).
[0108] Polypeptide Chain Elongation Factor 1-Alpha Promoter
[0109] Polypeptide chain elongation factor 1-alpha (EF-1-alpha)
promotes the GTP-dependent binding of an aminoacyl-tRNA to
ribosomes. EF-1-alpha is one of the most abundant proteins in
eukaryotic cells and expressed in all kinds of mammalian cells. An
expression vector, pEF-BOS, containing EF-1-alpha promoter has been
constructed and demonstrated to be a strong ubiquitous promoter
(Mizushima and Nagata, Nucleic Acids Res. 18:5322 (1990)).
[0110] B) Promoters That Restrict Expression to Specific
Tissues
[0111] Other useful promoters may also be used to produce perlecan
transgenic animals which may prove useful for the ultimate
development of new transgenic animals that mimic the amyloid
diseases. Some useful promoters which can also be used to create
constructs and inserted into transgenic animals in connection with
the present invention include, but are not limited to:
[0112] Platelet-Derived Growth Factor (PDGF) Promoter
[0113] This promoter has recently been used to produce transgenic
mice that overexpress the beta-amyloid precursor protein and that
deposit fibrillar beta-amyloid protein deposits in an extracellular
location (Games et al., Nature 373:523-527 (1995)). It is possible
that when perlecan expression is driven by a PDGF promoter, these
resulting transgenic mice may prove more useful for the ultimate
development of a transgenic mouse model for Alzheimer's disease and
Down's syndrome (i.e. beta-amyloid protein) amyloidosis. A plasmid,
psisCAT6a, that contains the PDGF-.beta. chain promoter is
available (Sasahara et al., Cell 64:217 (1991)).
[0114] Neurofilament M or L Promoters
[0115] These promoters demonstrate a high level of expression and
are found in connection with the most abundant neural protein. They
are characterized by central nervous system/peripheral nervous
system neuronal-specific expression. The mouse gene for this
promoter is a published sequence and the isolation of the promoter
region is necessary in order to use the promoter in connection with
the present invention. The neurofilament L promoters (Begemann et
al., Proc. Natl. Acad. Sci. USA 87:9042-9046 (1990)) and M
promoters (Begemann et al., Mol. Brain Res. 15:99-107 (1992)) have
been characterized and are available.
[0116] Glial Fibrillary Acidic Protein (GFAP) Promoters
[0117] Such promoters are characterized by murine specificity and
CNS/PNS glial-specific expression. The promoter has been
characterized and is available (Balcarek et al., Nucleic Acids Res.
13:5527-5543 (1985)).
[0118] Methallothionein III (MT-III) Promoters
[0119] MT-III is a third member of the methallothionein gene family
and its expression appears to be restricted to brain in mice.
MT-III is abundant in human brain and is immunohistochemically
localized to astrocytes in the grey matter (Uchida et al., Neuron
7:337-347 (1991)). In the Alzheimer's disease cortex, the number of
MT-III positive astrocytes are drastically reduced and this
reduction is highly correlated with the abundance of
neurofibrillary tangles and curly fibers (Uchida et al., Neuron
7:337-347 (1991)). The involvement of astrocytes in beta-amyloid
protein amyloidosis as observed in Alzheimer's disease has been
previously suggested (Potter et al., Prog. Brain Res. 94:447-458
(1992)). The MT-III promoter has been cloned and characterized
(Palmiter et al., Proc. Natl. Acad. Sci. USA 89:6333-6337
(1992)).
[0120] Prion Protein Promoter.
[0121] This promoter has been shown to drive position-independent
copy number-dependent transgene product expression in the mouse
brain (Scott et al., Cell 59:847-857 (1989); Prusiner et al., Cell
63:673-686 (1990)). This promoter was also used to create
transgenic mice that overexpress the beta-amyloid precursor protein
(Hsiao et al., Neuron 15:1203-1218 (1995)).
[0122] Insulin Promoter.
[0123] The insulin promoter can be used for the development of a
transgenic mouse model to mimic islet amyloidosis as observed in
patients with type II diabetes (Verchere et al., Proc. Natl. Acad.
Sci. USA (in Press) (1996)). The insulin promoter can be used to
achieve the specific expression of transgene in beta-cells
(Sarvetnick et al., Cell 52:773-782 (1988)). The human insulin
promoter has been characterized and is available.
[0124] Neuron-Specific Enolase Promoter
[0125] This promoter directs the neural-specific expression of a
reporter gene in transgenic mice (Fross-Petter et al., Neuron
5:187-197 (1990)) and has been used to make transgenic mice which
overexpress the beta-amyloid precursor protein (Quon et al., Nature
352:239-241 (1991)).
[0126] Low-Affinity Nerve Growth Factor Receptor (p75) Promoter
[0127] p75 is mainly expressed in central and peripheral
cholinergic neurons. The activity of p75 promoter has been tested
in cultured neurons (Taiji et al., Mol. Cell. Biol. 12:2193-2202
(1992)). Since loss of cholinergic neurons in brain represents an
important feature of Alzheimer's disease, this promoter can be
useful to create such models.
[0128] Methallothionein I Promoter
[0129] This promoter was used to create transgenic mice that
produce a mutant form of human transthyretin as a potential model
for type I familial amyloidotic polyneuropathy (Yi et al., Am. J.
Path. 138:403-412 (1991)). These mice develop amyloid deposits in
the gastrointestinal tract, cardiovascular system, and kidney, but
not in the peripheral nervous system where amyloid deposition
occurs in humans. Therefore, this promoter may be effective for
producing perlecan transgenic mice and/or transgenic mice which
overexpress both perlecan and transthyretin, for a new animal model
of prealbumin/transthyretin amyloidosis.
[0130] Other known promoters that are tissue specific may also be
used to produce animal models of amyloidoses affecting specific
organ systems. Such promoters can be used by those skilled in the
art. In addition, new promoters may become available which may be
also used by those skilled in the art to drive the expression
within desired tissues or organs for the ultimate production of
amyloid transgenic mice.
[0131] It is understood that modifications which do not
substantially affect the activity of various embodiments of this
invention are also included within the definition of the invention
provided herein. Accordingly, the following examples are intended
to illustrate but not limit the present invention.
EXAMPLE 1
Expression of Domains I-V of Murine Perlecan in COS and P19
Cells
[0132] To facilitate the expression of murine perlecan in mammalian
cells, the expression vector, designated pCA-DI-V was transfected
into an African green monkey kidney cell line (COS), and into
embryonic carcinoma cells (P19 cells) which upon stimulation with
the appropriate amount of retinoic acid differentiate into
neuron-like cells. This transfection was done by liposome-mediated
gene transfer (Lipofectamine, a product of Life technologies)
according to the manufacturer's protocol. The neomycin-resistance
gene that confers G418-resistance was also transfected together
with the pCA-DI-V vector. By screening the transfected cells with
G418, 20 clones for each cell line (COS and P19 cells) were
isolated and subjected to Western blot analysis to determine the
levels of perlecan expression. More precisely, COS-7 cells (African
green monkey kidney cell line; available from American Tissue
Culture Collection, ATCC# CRL-1651) and P19 cells (Rudnicki and
McBurny in Teratocarcinoma and Embryonic Stem Cells: A Practical
Approach, IRL Press, Washington, D.C., pp. 19-50,(1987)) were
cultured in Dulbecco's modified Eagle's medium (DMEM) containing
10% fetal bovine serum, 100 .mu.g/ml streptomycin and 100 units/ml
penicillin (regular medium). 1-2.times.10.sup.5 cells/60 mm dish
were inoculated one day before transfection of DNA. The cells were
washed with Opti-MEM I Reduced-Serum Medium (a product of Life
Technologies) prior to transfection to remove serum from the
culture. Three .mu.g of pCA-DI-V, 0.1 .mu.g of pMAMneo (a product
of Clontech Laboratories Inc.) and 20 .mu.l of Lipofectamine were
mixed, incubated for 15 minutes at room temperature and added to
the cells cultured in Optim-MEM I Reduced Serum Medium. The COS-7
and P19 cells were incubated at 37.degree. C. in a humidified
atmosphere of 5% CO.sub.2 for 18 and 6 hours, respectively, and
Optim-MEM I Reduced-Serum medium was replaced with regular medium.
Two days after transfection, the cells were treated with 0.05%
trypsin and 530 .mu.M EDTA and replaced at a concentration of
10,000 cells/100 mm dish with selection medium (regular medium with
800 .mu.g/ml G418). Two to three weeks after transfection, emerging
resistant colonies were isolated using clonal rings and propagated
for storage and Western blot analysis. For each cell line,
approximately 20 clones were isolated.
EXAMPLE 2
Up-Regulation of Perlecan Levels in Transfected COS and P19 Cells
as Revealed by Western Blot Analyses
[0133] To determine the potential differences in perlecan levels in
transfected versus non-transfected COS and P19 cells, the media and
cell layers were analyzed by Western blotting with a specific
monoclonal antibody (HK-102) against perlecan core protein. From
each cell culture dish, the media was collected for analysis as
described below, making sure that equal volumes of media were
analyzed from either transfected or non-transfected cells. The cell
layers were harvested by scraping into urea buffer (urea buffer
contained 7 M urea, 0.2 M NaCl, 0.1% (w/v) CHAPS, 50 mM Tris-HCl,
pH 8.0, containing a protease inhibitor cocktail including 10 mM
EDTA, 10 mM NEM, 10 mM 6-aminohexanoic acid, 5.0 mM
benzamidine-HCl, and 1 mM PMSF).
[0134] For COS cells, PGs from cell layer and media extracts were
then purified by DEAE-Sephacel ion-exchange chromatography.
Briefly, cell layer and media extracts were pooled separately, and
supplemented with 0.5% Triton X-100 (v/v) and applied to a 1 ml
DEAE-Sephacel column packed in a plastic syringe equilibrated with
urea buffer (urea buffer contained 7 M urea, 0.2 M NaCl, 0.1% (w/v)
CHAPS, 50 mM Tris-HCl, pH 8.0, containing a protease cocktail
including 10 mM EDTA, 10 mM NEM, 10 mM 6-aminohexanoic acid, 5.0 mM
benzamidine-HCl, and 1 mM PMSF). Proteins and non-PGs were removed
by first washing the column with 5 column volumes of urea buffer
(described above) containing 1% Triton X-100 and 0.25M NaCl. Bound
PGs were then eluted with 5 column volumes of urea buffer
containing 3M NaCl. Equal proportions of each sample were then
precipitated by adding 3.5 volumes of 95% ethanol, 1.5% potassium
acetate (w/v), cooled on dry ice for 1 hour and centrifuged (using
an Eppendorf 5415C desktop centrifuge) at 14,000.times.g for 20
minutes. Samples were then left either undigested, or digested with
heparinase/heparitinase and/or chondroitinase ABC. For
heparinase/heparitinase digestion, samples were suspended in 50
.mu.l of 0.2M Tris-HCl, 5 mM calcium acetate (pH 7.0) and digested
with a mixture of heparinase I, II, and III (i.e. heparitinase)
(Sigma Chemical Co.) used at 1U each (approximately 10 mU/.mu.g
protein) in 30 .mu.l of glycerol:distilled water (1:1). Following
addition of 10 .mu.l of a 10.times. protease inhibitor cocktail (as
described above), heparinase/heparitinase containing samples were
incubated overnight at 41.degree. C. and ethanol precipitated (as
described above), prior to separation by SDS-PAGE. Samples treated
with chondroitinase ABC were digested with 100 mU of chondroitin
ABC lyase (ICN Biochemicals) in Tris-buffered solution (50 mM Tris,
50 mM sodium acetate, 10 mM EDTA, and protease inhibitor cocktail
as described above, pH 7.5) at 37.degree. C. for 2 hours. Each
sample was then heated for 5 minutes in a boiling water bath,
loaded onto SDS-PAGE and electrophoresed at 100V for 45 minutes
along with pre-stained molecular weight protein standards (a
product of Bio-Rad). SDS-PAGE were performed according to the
procedure of Laemmli (Nature 227:680-685 (1970)) using a
Mini-Protean II electrophoresis system with precast 4-15%
polyacrylamide gels. Samples were run under non-reducing conditions
for use with a monoclonal antibody (HK-102) against perlecan core
protein. Following SDS-PAGE, separated proteins were transferred to
nitrocellulose using a Mini transblot electrophoresis transfer
cell. Electrotransfer was performed at 100V for 2 hours. Following
transfer, membranes were rinsed in water and blocked overnight with
0.15% (w/v) bovine serum albumin, 1% (v/v) normal goat serum, 100
mM Tris-HCl, and 3 mM NaN.sub.3 (pH 7.4). Blots were probed with a
monoclonal antibody (HK-102) which recognizes the perlecan core
protein, diluted 1:3000 in TBS containing 100 mM Tris-HCl, 50 mM
NaCl, 0.05% Tween-20, and 3 mM NaN.sub.3 (pH 7.4) (TTBS). Blots
were incubated with primary antibody for 3 hours, washed with TTBS
three times (10 minutes each), followed by a 1 hour incubation in
biotinylated goat-anti-rat Ig's (IgG and IgM) (Jackson
Immunoresearch, West Groven, Pa.) diluted 1:1000 with TTBS. The
membranes were then rinsed three times (10 minutes each) with TTBS,
probed for 30 minutes with avidin alkaline phosphatase conjugate
(Vectastain), rinsed again (as described above), followed by the
addition of an alkaline phosphatase substrate solution
(Vectastain). Following color development, the reaction was stopped
by flushing the membranes with double-distilled water.
Immunodetection of perlecan core protein using the monoclonal
(HK-102) antibody was employed in at least three different cell
culture experiments in order to determine reproducibility. For
comparisons perlecan was isolated from the Engelbreth-Holm-Swarm
tumor as previously described (Castillo et al., J. Biochem.
120:433-444 (1996)) and digested with heparinase I, II and III (as
described above) prior to Western blotting.
[0135] As shown in FIG. 4, Western blot analyses of PGs isolated
from the cell layer of non-transfected COS cells demonstrated no
immunoreactivity on Western blots probed with a monoclonal antibody
(HK-102) which recognizes perlecan core protein (FIG. 4, lanes 1
and 3). On the other hand, Western blot analyses of PGs isolated
from the cell layer of transfected COS cells overexpressing the
entire perlecan core protein, revealed a .about.400 kDa band using
the monoclonal antibody (HK-102) specific for perlecan core protein
(FIG. 4, lane 2 and lane 4). Chondroitinase ABC digestion did not
appear to cause a shift in the .about.400 kDa band in the cell
layer of transfected cells when compared to undigested sample (not
shown), suggesting that little to no chondroitin sulfate chains
were present on the perlecan core protein produced. On the other
hand, a slight shift and/or tighter .about.400 kDa band was
observed in the cell layer of transfected cells following
heparinase/heparitinase digestion (FIG. 4, lane 4) when compared to
undigested sample (FIG. 4, lane 2), suggesting that the perlecan
core protein produced in the cell layer of transfected cells
contained heparan sulfate chains that were either shorter or less
in number than normal (i.e. perlecan is usually thought to contain
3 glycosaminoglycan chains attached to its core protein). The
perlecan core protein band on immunoblots from cell layer of
transfected COS cells (FIG. 4, lanes 2 and 4) was similar in
position to that of pure perlecan isolated from the
Engelbreth-Holm-Swarm tumor (FIG. 4, lane 5). However, whereas
isolated perlecan gave a characteristic doublet at .about.400 kDa
(FIG. 4, lane 5), the cell layer from transfected COS cells
demonstrated a single major band at .about.400 kDa (FIG. 4, lane 2
and lane 4).
[0136] Similar results were obtained by Western blot analyses of
PGs isolated from the media (FIG. 5) of non-transfected (FIG. 5,
lanes 1 and 3) and transfected (FIG. 5, lanes 2 and 4) COS cells,
when probed with the monoclonal antibody (HK-102) against perlecan
core protein. A marked accumulation of perlecan core protein
immunoreactivity was observed in transfected COS cells (FIG. 5,
lanes 2 and 4) in comparison to non-transfected cells (FIG. 5,
lanes 1 and 3). Chondroitinase ABC digestion did not cause a shift
in the .about.400 kDa band isolated from the media of transfected
cells (FIG. 5, lane 4) when compared to undigested sample (FIG. 5,
lane 2), suggesting that little to no chondroitin sulfate chains
were present on the perlecan core protein secreted. On the other
hand, a slight shift and/or tighter .about.400 kDa band was
observed in the media of transfected cells following
heparinase/heparitinase digestion when compared to undigested
sample from transfected media (not shown), suggesting that the
perlecan core protein produced in the media of transfected cells
contained heparan sulfate chains that were either shorter in length
or less in number than normal. Perlecan core protein band on
immunoblots from media of transfected COS cells (FIG. 5, lanes 2
and 4) was similar in position to that obtained from pure perlecan
(FIG. 5, lane 5), and a similar characteristic double band at
.about.400 kDa was observed in media from transfected COS cells
(FIG. 5, lanes 2 and 4).
[0137] The fact that the perlecan bound to DEAE-Sephacel for
isolation indicated that the perlecan most likely contained
glycosaminoglycan chains. Since heparinase/heparitinase digestion,
and not chondroitinase ABC caused a slight shift in the position of
the .about.400 kDa core protein on Western blots of both media and
cell layer from transfected COS cells, it suggested that heparan
sulfate GAG chains (although shorter in length or less in number
than usual) were present on the perlecan core protein of
transfected cells. Nonetheless, this study represents the first
time that successful transfection of the entire perlecan core
protein has been achieved, and represented the first important
critical step for the development of perlecan transgenic mice over
expressing the entire .about.400 kDa core protein.
[0138] For P19 cells, following isolation of PGs from cell layer
and media, and SDS-PAGE, as described above, the proteins were
transferred to PVDF membrane (a product of Millipore) at 260 mA for
18 hours using the Bio-Rad Transblot System. The membranes were
blocked with PBS containing 5% nonfat dried milk (w/v), 0.02%
sodium azide, and 0.02% Tween 20, then incubated at 4.degree. for
18 hours with primary antibodies (polyclonal or monoclonal
antibodies recognizing perlecan core protein) and immunostained
with an enhanced chemiluminescence system (a product of Amersham).
As found with the overexpression of perlecan core protein in
transfected COS cells, the western blot analyses of P19 cell layer
and media revealed high levels of perlecan production in
transfected cells, whereas non-transfected cells showed little to
no immunoreactivity (not shown). These studies confirmed that
overexpression of the perlecan core protein could be achieved in
transfected COS and P19 cells by the construction strategy used in
the present invention.
EXAMPLE 3
Up-Regulation of Secreted A.beta. in Media of P19 Cells
Overexpressing Both Perlecan and .beta.PP-695
[0139] A P19 derived cell clone had previously been established and
designated P19 695B2, which overexpresses the 695 amino acid
isoform of the human beta-amyloid precursor protein (.beta.PP-695)
(Fukuchi et al., J. Neurochem. 66:2201-2204 (1996)). In this
example, the P19 695B2 cells were transfected with the pCA-DI-V
vector, and stable transformants designated P19 695B2 PCB6 and P19
695B2 PCD3, which overexpress both perlecan and .beta.PP-695 were
established using the methods described in Example 1.
Overexpression of .beta.PP-695 and perlecan was confirmed by
Western blotting as in Example 2.
[0140] To demonstrate that overexpression of perlecan in P19 695B2
D3 led to increased perlecan protein production, perlecan was
isolated and analyzed by Western blotting in the presence or
absence of heparitinase and/or chondroitinase ABC pretreatment (to
degrade heparan sulfate and/or chondroitin/dermatan sulfate GAGs,
respectively) as described in Example 2. As shown in FIG. 6, P19
cells only overexpressing .beta.PP-695 (i.e. P19 695 B2) did not
demonstrate detectable perlecan in cell lysates or media. On the
other hand, P19 cells overexpressing both perlecan and .beta.PP-695
(i.e. P19 695B2 PC D3) demonstrated a marked increase in perlecan
protein in both cell lysates and media as demonstrated by Western
blots using a specific perlecan polyclonal antibody (FIG. 6).
Cellular perlecan was identified as a .about.400 kDa band or smear
using the specific perlecan antibody (FIG. 6). Heparitinase
digestion (to remove heparan sulfate GAG chains) only slightly
increased the immunodetection of cellular perlecan, suggesting that
cellular perlecan may be lacking full length heparan sulfate GAG
chains. On the other hand, the .about.400 kDa band of perlecan
isolated from media was visualized only after heparitinase and
heparitinase/Chondroitinase ABC treatments (FIG. 6, arrow),
indicating that secreted perlecan produced in these cells had
heparan sulfate GAG chains. Thus, overexpression of perlecan in P19
cells leads to overproduction of perlecan in media which may be
useful for the isolation of perlecan in relatively large
quantities.
[0141] To determine the potential effects of perlecan
overexpression on .beta.PP and A.beta. levels in P19 cells, the
media and cell layers of P19 cells overexpressing both perlecan and
.beta.PP-695 (i.e. P19 695B2 PC B6 and P19 695B2 PC D3) were
compared to cells overexpressing .beta.PP-695 only (i.e. P19 695 B2
PC), by immunoprecipitation followed by Western blotting with
specific antibodies (6E10 which recognizes human A.beta., and 6561
which recognizes the C-terminus of .beta.PP). All cells (P19 695B2,
P19 695B2 PC B6, and P19 695B2 PC D3) were induced to differentiate
into neurons by treatment with retinoic acid as previously
described (Fukuchi et al., J. Neurochem. 58:1863-1873 (1996)). On
the 8th day of differentiation, the media was collected and
centrifuged at 12,000.times.g for 1 hour and the supernatant was
made to 10 mM Tris, 1% Nonidet P-40, 0.5% cholic acid, 0.1% sodium
dodecyl sulfate (SDS), and 5 mM EDTA, 2 .mu.g/ml leupeptin and 0.1
.mu.g pepstain (pH 8.0). After preabsorption with 50 .mu.l of
protein A-agarose (previously coupled to rabbit antibody against
mouse IgG), the media was incubated with primary antibodies 6E10
and 40 .mu.l of protein A-agarose (pretreated as described above)
for 16 hours. Precipitates were washed three times with TBS
containing 1% Nonidet P-40, 5 mM EDTA, 2 .mu.g/ml leupeptin, 2 mM
phenylmethyl sulfonylfluoride and was boiled in 20 l of 2X laemmli
buffer for 5 minutes before loading onto 7.5% (to detect higher
molecular weight bands) or 16.5% (to detect low molecular weight
bands) SDS polyacrylamide gels. The cells were lysed by adding 2X
Laemmli buffer (1X=62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol,
5% 2-mercaptoethanol, 0.001% bromophenol blue), boiled for 5
minutes, and sheared with a 26-gauge needle. Protein concentrations
were determined by Bio-Rad Protein Assay (a product of Bio-Rad
laboratories). Aliquots corresponding to 30 .mu.g of protein were
applied to 7.5% SDS-polyacrylamide gel electrophoresis (PAGE) and
Tris-Tricine 16.5% SDS-PAGE. Following electrophoresis, proteins
were electrotransferred to a polyvinylidine difluoride (PVDF)
membrane (Immobilon-P, a product of Millipore). The membrane was
blocked with phosphate-buffered saline (PBS) containing 5% nonfat
dried milk (w/v), 0.02% sodium azide, and 0.02% Tween 20, then
incubated at 4.degree. C. for 16 hours with the 6561 antibody
(which recognizes amino acids 681-695 of .beta.PP-695) or 6E10
antibody (which recognizes human A.beta.) and immunostained with an
enhanced chemiluminescence system (a product of Amersham Co.). The
relative concentration of the protein was determined by
densitometric scanning (Molecular Analyst/PC, a product of
Bio-Rad).
[0142] As shown in FIG. 7, .beta.PP was evident in both the cell
lysates and media of transfected P19 cells overexpressing
.beta.PP-695 (i.e. P19 695B2) as discrete or smeared bands at
.about.100 and 130 kilodaltons (arrow) following Western blots
using the 6561 or 6E10 antibodies. On the other hand, a
.about.4-fold increase in .beta.PP levels in the media was apparent
in transfected P19 cells overexpressing both perlecan and
.beta.PP-695 (i.e. P19 695B2 PC B6 and P19 695B2 PC D3) (FIG. 7).
The P19 695B2 PC D3 cell clone also demonstrated a .about.4-fold
increase in .beta.PP levels in the cell lysates. These studies
indicate that overexpression of both perlecan and .beta.PP-695
leads to a marked increase in .beta.PP levels in the media and cell
layer of P19 cells, as compared to P19 cells which overexpress only
.beta.PP-695. Overexpression of perlecan may therefore contribute
to changes in .beta.PP metabolism and levels.
[0143] Besides an effect on increasing levels of .beta.PP in both
media and cell, perlecan overexpression also led to a marked
increase in the secretion of A.beta. into the media. As shown in
FIG. 8, Western blot analysis utilizing the 6E10 antibody barely
detected A.beta. (FIG. 8) in media from cells overexpressing
.beta.PP-695 only (i.e. P19 695B2). On the other hand,
overexpression of both perlecan and .beta.PP-695 (i.e. P19 695B2 PC
B6 and P19 695B2 PC D3) led to a marked increase in A.beta. levels
in the media (FIG. 8, arrow). When secreted A.beta. levels were
normalized to levels of cellular and secreted .beta.PP, an
8-10-fold increase was demonstrated in P19 cells which overexpress
both perlecan and .beta.PP-695 (FIG. 9). These results indicate
that overexpression of perlecan leads to an increase in secreted
A.beta. levels.
EXAMPLE 4
Overexpression of Perlecan Leads to Degeneration of P19-Derived
Neurons
[0144] To study the effects of perlecan overexpression on neuronal
survival, P19 cells overexpressing perlecan only (designated P19 PC
C2 and P19 PC D2) were also induced to differentiate into neurons.
These additional P19 cell lines were isolated by stable
transfection of pCA-DI-V and perlecan overexpression in these two
clones was confirmed by Western blot analysis as described in
Example 1 and Example 2. Transformed cell clones overexpressing
perlecan only (P19 PC C2, P19 PC D2), perlecan and .beta.PP-695
(P19 695B2 PC B6 and P19 695B2 PC D3), .beta.PP-695 only (P19
695B2) and their parental cells (P19) were induced to differentiate
into neuronal cells by treatment with retinoic acid. Proliferating
non-neuronal cells were eliminated from the cultures by treatment
with 10 .mu.M of cytosine .beta.-D-arabinofuranoside (Ara C). Eight
days following initial treatment with retinoic acid, surviving
neurons were quantified by measuring produced formazan from MTS
according to the manufacturer's protocol (Cell Titer 96 Aqueous
Assay, a product of Promega). For each cell clone 5 independent
cultures were prepared and subjected to statistical analysis. The
results are shown in FIG. 10. Cell survival of the clones
overexpressing perlecan only (i.e. P19 PC C2, P19 PC D2) were
significantly lower than control P19 cells (i.e. P19), and in
comparison to cells overexpressing .beta.PP-695 only (i.e. P19
695B2). The P19 695B2 PC D3 line which overexpresses both perlecan
and .beta.PP-695 also showed a decrease in neuronal survival,
especially when compared to cells which only overexpress
.beta.PP-695 (i.e. P19 695B2). All of the cells of the P19 695B2
PCB6, which overexpress both perlecan and .beta.PP-695 died out.
These studies therefore demonstrated that overexpression of
perlecan also has an effect on neuronal cell survival. Therefore,
cells which overexpress perlecan only, and perlecan and .beta.PP
may be utilized a screening tools for the development of new
therapeutic and preventive agents for Alzheimer's disease. In a
preferred embodiment, anti-Alzheimer's compounds can be tested and
will be deemed effective if they cause a lowering of secreted
A.beta. levels in the media, and/or increase neuronal survival.
EXAMPLE 5
Construction of Perlecan Transgenic Expression Plasmids
[0145] The expression vector, pCA-DI-V (FIG. 3C), was used to
prepare DNA for microinjection into mouse fertilized eggs. pCA-DI-V
was digested with Hind III enzyme and electrophoresed on agarose to
separate the bacterial DNA sequences from the DNA construct to be
used for microinjection. The DNA construct that contained the
cytomegalovirus enhancer/beta-actin promoter, cDNA for the entire
perlecan core protein, and a polyadenylation signal were
electrophoresed onto a DEAE-cellulose membrane. The DNA on the
membrane was eluted by a high-salt buffer (50 mM Tris-HCl, pH 8.0,
1M NaCl, and 10 mM EDTA, pH 8.0) and extracted with phenol and
chloroform. After precipitating the DNA by ethanol, the DNA was
resuspended in Tris-EDTA (10 mM Tris-HCl, pH 7.5, 0.25 mM EDTA) and
used for microinjection into fertilized eggs.
EXAMPLE 6
Collection and Injecting the Eggs with the DNA Construct
[0146] The transgenic organisms of the invention all include within
a plurality of their cells a cloned recombinant or synthetic DNA
sequence which is believed to relate to the pathogenesis of the
amyloid diseases. More specifically, the transgenic organisms
contain specific sequences of exogenous genetic material which are
comprised of a specific promoter sequence which allows for
production of perlecan core protein. Since it is possible to
produce transgenic organisms of the invention, a general
description will be given of the production of transgenic organisms
by referring generally to exogenous genetic material. This general
description can be adapted by those skilled in the art in order to
incorporate the above-described specific DNA sequences into
organisms and obtain expression of those sequences utilizing the
materials and methods described below. For more information
regarding the production of transgenic mice refer to U.S. Pat. No.
4,873,191 issued Oct. 10, 1989 (incorporated herein by reference to
disclose methods producing transgenic mice), and to the scientific
publications referred to and cited therein.
[0147] One-cell stage embryos (F2 of C57BL/6J.times.SJL) were
collected from the oviducts of Fl female mice of
C57BL/6J.times.SJL/J that had been already mated with Fl male mice
of C57BL/6J.times.SJL/J. These embryos should be early enough to
distinguish male pronuclei from female pronuclei. Cumulus cells
surrounding oocytes were removed by hyaluronidase treatment, washed
properly and incubated at 37.degree. C. in an atmosphere of 5%
CO.sub.2 for a certain time prior to DNA injection. Any method
which allows for the addition of the exogenous genetic material can
be utilized as long as it is not destructive to the cell, nuclear
membrane or other existing cellular or genetic structures. The
exogenous genetic material is preferably inserted into the nucleic
genetic material by microinjection. Microinjection of cells and
cellular structures is known and is used in the art. About 1-2
picoliters (p1) of the DNA construct prepared in Example 5 (about
100-200 copies of the transgene per pl) was microinjected into a
male pronucleus. Approximately 100 embryos were injected with the
DNA construct described in Example 5. The injected embryos were
incubated from several hours to one day and then transplanted to
the oviducts of pseudopregnant ICR foster mothers of Day 1 of
pregnancy. The day when a vaginal plug was recognized was recorded
as Day 1. The transplanted foster mothers were fed until a delivery
of the fetus. After delivery, neonates (25 pups) were nursed by the
foster mothers for 4 weeks until weaning. At the time of weaning,
the tails of the neonates were cut for Southern blot analysis to
determine the integration of transgenes into mouse chromosomes. The
founder mice, judged as transgenic by Southern blot analysis, were
back-crossed with C57BL/6J mice to establish lines of transgenic
mice with C57BL/6J genetic background.
EXAMPLE 7
Determination of Transgene Copy Numbers
[0148] Pups produced from microinjection of the DNA construct
(pCA-DI-V) were grown to 4 to 6 weeks old and then the tails of the
pups were cut. The DNA was isolated from the tails using QIAamp
tissue kit (a product of Qiagen Inc.). 10 .mu.g of DNA isolated
from mouse tails were digested with Sca I restriction enzyme and
separated by electrophoresis on a 0.9% agarose gel. Additionally,
pCA-DI-V was digested with Sca I restriction enzyme and 0.045 ng (1
copy), 0.225 ng (5 copies) and 0.45 ng (10 copies) of the digested
PCA-DI-V were also separated on the same agarose gel as a reference
positive control. The separated DNA was then blotted onto a nylon
membrane. The membrane was prehybridized for 6 hours at 42.degree.
C. in 50% formamide, 5.times. saline sodium citrate (SSC;
1.times.SSC=0.15M NaCl and 0.015 M sodium citrate, pH 7.0),
5.times. Denhardt's solution (1.times. Denhardt's solution=0.02%
each of Ficoll, polyvinylpyrrolidone, and bovine serum albumin),
0.1% sodium dodecyl sulfate (SDS), and 100 mg/ml of denatured
herring sperm DNA and then hybridized in the above solution
containing 10% dextran sulfate and .about.10.sup.7 cpm of a
radiolabeled probe at 42.degree. C. for 18 hours. The radiolabeled
probe was the 1.7-kb Sca I fragment of mouse perlecan cDNA (bp
5523-7215 of perlecan cDNA) (Noonan et al., J. Biol. Chem.
266:22939-22947 (1991)). The membrane was then washed at 65.degree.
C. in 0.2.times.SSC and 0.5% SDS. The membrane was exposed to Kodak
XAR film with an enhancer screen, at -80.degree. C. for 4 days. As
shown in FIG. 11, out of approximately 25 pups, the integrated
transgene in the mouse genome was identified as a 1.7-kb fragment
on Southern blot in four founder mice (designated #3595, 3697, 3763
and 3694 in FIG. 11). Founder mouse #3595 was found to have 5-10
copies of pCA-DI-V whereas founder mouse #3697, 3763 and 3694 had
approximately one copy of pCA-DI-V.
EXAMPLE 8
Confirmation of Perlecan Overexpression in Perlecan Transgenic
Mice
[0149] The founder mice (#3595, 3697, 3763 and 3694) were mated
with C57BL/6J mice. Each of the male founders (#3694 and 3595) were
housed in one cage together with two C57BL/6J female mice, and each
of the female founders (#3697 and 3763) were housed in one cage
together with one C57BL/6J male mouse. Every female mated gave
birth to 8-12 mice indicating that the founders were fertile. The
mouse tails from the progeny are cut to perform Southern blot
analysis as described above to demonstrate the transgene
segregation. Mice determined as transgenic by Southern blot
analysis are mated with C57BL/6J mice for propagation.
[0150] Levels of transgene expression in the transgenic mice were
determined by Northern blotting for mRNA, Western blotting and
immunostaining for protein. Most of the techniques which are used
to perform Northern and Western blot analysis are widely practiced
in the art and most practitioners are familiar with the standard
resource materials as well as specific conditions used in the
procedures.
[0151] Northern Blotting: Three mice were sacrificed by cervical
dislocation under anesthesia; T 213, a 3-month old progeny from
perlecan transgenic founder 3595 (5 copy founder); T 156, a 3-month
old progeny from perlecan transgenic founder 3694 (1 copy founder),
and C 196, a 3-month non-transgenic control litter-mate of T 213.
Tissues including brain, heart, liver, kidney, spleen and intestine
were quickly removed and homogenized in a denaturing solution
consisting of 4M guanidinium thiocyanate, 25 mM sodium citrate, pH
7.0; 0.5% sarcosyl, 0.1M 2-mercaptoethanol) and passed through a
syringe fitted with a 27 gauge needle. After adding sodium acetate,
the RNA was extracted with phenol/chloroform and precipitated with
isopropanol. The precipitated RNA was resuspended again with the
above denaturing solution and precipitated with isopropanol. After
rinsing the RNA with 75% ethanol, the RNA was resuspended with
sterile double distilled water. 20 .mu.g of total RNA from each
tissue was electrophoresed through a 1% agarose-formaldehyde gel,
followed by capillary transfer to a nylon membrane. A perlecan
probe (mouse perlecan cDNA, bp 5523-7215 of perlecan cDNA as
described in Noonan et al., J. Biol. Chem. 266:22939-22947 (1991))
was radiolabeled with .sup.32P dCTP to a specific activity of
4.times.10.sup.8 cpm/.mu.g using a random primed labeling kit (a
product of Boehringer Mannheim). The membrane was hybridized for 20
hours at 42.degree. C. in 50% formamide 5.times.SSC
(1.times.SSC=0.15M NaCl, 0.015M sodium citrate, pH 7.0), 0.1% SDS
and 100 .mu.g/ml of denatured herring sperm DNA. Following
hybridization, the membrane was washed twice at 65.degree. C. in
0.2.times.SSC and 0.2% SDS for 30 minutes each. The membrane was
then exposed to Kodak XAR film with enhancer screens at -70.degree.
C. for 6 hours. The results are shown in FIG. 12. High levels of
expression of the transgene mRNA with the expected 11.6 kb size,
were observed in brain, heart, kidney, spleen and intestine of T
213 (progeny of founder with 5 copies of transgene). Levels of
perlecan mRNA in T 156 (progeny of founder with 1 copy of
transgene) were much lower than those in T 213 (FIG. 12). No
endogenous mRNA for perlecan was detected in any of the C 196
tissues. This study demonstrated that perlecan overexpression was
particularly evident in brain and in other tissues of transgenic
mice carrying .about.5 copies of the transgene.
[0152] Western Blotting: Levels of the protein products from the
transgene in the transgenic mice were determined by Western blot
analysis. The tissues (i.e. brain, heart, liver, kidney, spleen,
intestine and pancreas) isolated from transgenic T 213 and control
C 196 were homogenized in 2X Laemmli buffer (1X=62.5 mM Tris-HCl,
pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.001%
bromophenol blue), boiled for 5 min, and sheared with a 26-gauge
needle. Protein concentration was determined by Bio-Rad Protein
Assay (a product of Bio-Rad). Aliquots corresponding to 30 .mu.g of
protein were applied to 4-20% gradient gels for SDS-PAGE. Following
electrophoresis, proteins were electrotransfered to a
polyvinylidine difluoride (PVDF) membrane (Immobilon-P, Millipore).
The membrane was blocked with phosphate-buffered saline containing
5% nonfat dried milk (w/v), 0.02% sodium azide, and 0.02% Tween 20,
then incubated at 4.degree. C. for 16 hours with perlecan
monoclonal or polyclonal antibodies which recognize perlecan core
protein, and immunostained with an enhanced chemiluminescence
system (a product of Amersham). The results are shown in FIG. 13.
Substantial increased amounts of transgene protein product (i.e.
perlecan detected as a .about.400 kDa band or smear) were observed
in brain, heart, liver, kidney, and pancreas of T 213 in comparison
to control (i.e. C196). This study demonstrated that perlecan
overproduction successfully occurred in brain and in other tissues,
and was apparent in a 3-month old perlecan transgenic mouse.
[0153] Immunostaining: For immunostaining studies, animals were
first sacrificed by deep anesthesia. The tissues of two 5-month old
transgenic progenies from the 3595 (5 copy founder) and two 5-month
old non-transgenic litter-mates were subjected to immunostaining.
The tissues including brain, heart, intestine, kidney and pancreas
were removed, fixed by 10% formalin and embedded in paraffin by
procedures known to those skilled in the art. 10 .mu.m sections
were then cut and subjected to the avidin-biotin immunoperoxidase
method to detect perlecan using perlecan specific antibodies and
the Vectastain ABC kit (a product of Vector Laboratories) as
described below. Endogenous peroxidase was eliminated by treatment
with 3% hydrogen peroxide for 30 minutes after deparaffinization of
the sections. After rinsing with distilled water, the sections were
blocked with 15% goat serum in Tris-buffered saline (TBS) for 60
minutes at room temperature and incubated with a perlecan primary
polyclonal antibody (which specifically recognizes the core protein
of perlecan; antibody of Dr. J. Hassell) in 0.1M TBS containing 15%
goat serum for 16 hours at 4.degree. C. The sections were then
rinsed in 0.1 M TBS containing 1% goat serum and incubated with
biotinylated goat anti-rabbit IgG secondary antibody for 1 hour at
room temperature. After washing, the sections were incubated with
Vectastain reagent for 60 minutes at room temperature. Peroxidase
activity was then detected by treatment with 3,3'-diaminobenzidine.
Strong immunoreactivity for perlecan was evident in transgenic mice
but not in control liter-mates, especially in the epithelium of the
choroid plexus, the myofibrils of the heart, the acidophil cells of
the gastric gland, the glomeruli of the kidney, and the cells in
the islets of Langerhans in pancreas. In addition, transgenic mice,
but not control litter-mates demonstrated moderate perlecan
immunostaining in the C3A neuronal layer of the hippocampus, the
Purkinje cell layer of the cerebellum and the large neurons of the
brain stem. FIG. 14 demonstrates strong perlecan immunostaining in
kidney glomeruli (FIG. 14A, arrows), and in the C3A neuronal layer
of the hippocampus (FIG. 14C, arrows) of a 5-month old perlecan
transgenic mouse. In comparison, control litter-mates demonstrate
weak to no staining for perlecan in kidney glomeruli (FIG. 14B) and
hippocampal neurons (FIG. 14D). These results confirm that perlecan
overexpression leads to perlecan protein accumulation in specific
sites in brain and in systemic organs.
EXAMPLE 9
Production of Beta-Amyloid Precursor Protein Mice as a First Step
in Producing a New Animal Model of Alzheimer's Disease
Amyloidosis
[0154] Four founder lines of transgenic mice that constitutively
overproduce the signal sequence and the 99 amino acid C-terminal
region of the human beta-amyloid precursor protein were
established. The cDNA for the signal sequence and the 99 amino acid
C-terminal region (the construct referred to as S.beta.C) of the
human beta-amyloid precursor protein (hereinafter called .beta.PP)
was placed under the control of a cytomegalovirus enhancer and a
chick beta-actin promoter in pCAGGS (FIG. 15A) (Niwa et al., Gene
108:193-200 (1991)). To overexpress S.beta.C under control of a
cytomegalovirus enhancer and a chick .beta. actin promoter, an
expression vector, pCA-S.beta.C was constructed. Using the LMG2
human brain stem lambda gt11 cDNA library (a product of ATCC,
catalog #ATCC37432), a partial cDNA of the human .beta.PP,
extending from base pairs 901-2851 (Kang et al., Nature 325:733-736
(1987)) was isolated. The EcoRI/CIaI fragment (bp 1796-2473; Kang
et al., Nature 325:733-736 (1987)) of the cDNA was subcloned into
pBluescript KS (a product of Stratagene) at the same restriction
enzyme sites to create pKS-.beta.C (FIG. 15A). In a separate
reaction the signal peptide coding sequence for .beta.PP and the
sequence for the first 3 amino acids of the beta-amyloid protein
(BamHI site+bp-4-51+bp 1789-1795+EcoRI site; bps are in Kang et
al., Nature 325:733-736 (1987)) was chemically synthesized (FIG.
15A). This synthetic fragment was also ligated to the EcoRI/ClaI
fragment (bp 1796-2473) of .beta.PP in pKS-.beta.C using BamHI and
EcoRI sites to create the pKS-S.beta.C plasmid. This plasmid was
digested with BamHI and SaII and the 0.8-kb fragment coding for
S.beta.C of .beta.PP was isolated. The SaII site of the 0.8-kb
fragment was ligated onto one of the XhoI sites of the expression
vector, PCAGGS (Niwa et al., Gene 108:193-200 (1991)), which was
previously digested with XhoI (FIG. 15B). The other ends were
ligated after blunt end formation with the Klenow fill-in reaction
to create pCA-S.beta.C (FIG. 15B). Fertilized eggs were injected
into pCA-S.beta.C after removing bacterial sequence from
pCA-S.beta.C. Four independent lines of transgenic mice were
established. To determine the levels of mRNA expression from the
transgene, the total RNA from transgenic mice was isolated and
analyzed by Northern blot using a radiolabeled human cDNA probe (bp
1795-2851; Kang et al., Nature 325:733-736 (1987)). The high levels
of expression of mRNA with the expected size, 1.06-kb from the
transgene were observed in virtually all tissues examined including
the brain (Fukuchi et al., Am J Path 149:219-227 (1996)). Native
mRNA with a size of 3.2 kb was also observed in the brain and
kidney. On Western blots, .about.11-17 kDa protein products from
the transgene were observed in lung, muscle, intestine, liver and
kidney in transgenic mice, whereas no corresponding fragments were
observed in Western blots from the same tissues of non-transgenic
mice. A 3-5 fold increase in the C-terminal fragments of .beta.PP
was found in the brains of transgenic mice compared to controls.
The amount of the C-terminal fragments in the intestine was at
least 50 times greater than that observed in the brain of
transgenic mice, and at least 3 times greater than that observed in
the heart of transgenic animals (Fukuchi et al., Am. J. Path.
149:219-227 (1996)).
[0155] Six-month, 9-month, 12-month, 14-month and 16-month-old
transgenic mice bearing CA-S.beta.C were sacrificed for
immunohistochemical and histopathological analysis. Nontransgenic
siblings, ranging from 12-16 months were used as controls. By
immunohistochemical analysis, using antibodies 1282 (a rabbit
polyclonal antiserum raised against beta-amyloid protein; Tamaoka
et al., Proc. Natl. Acad. Sci. USA 89:1345-1349 (1992)) and 10D5
(directed against amino acids 1-16 of beta-amyloid protein; Hyman
et al., J. Neuropath. Exp. Neurol. 51:76-83 (1992)), large
immunoreactive deposits were found in the lamina propria of the
mucosa of the small intestine from transgenic mice >12 months of
age, but not in younger mice. These deposits were positive for
Congo red (indicative of amyloid) demonstrating a red/green
birefringence as viewed under polarized light and consisted of
fibrils 7-10 nm in diameter as observed by electron microscopy.
Surprisingly, these beta-amyloid deposits were also found to be
positive for perlecan using perlecan specific core protein
antibodies. The accumulation of both beta-amyloid and perlecan in
these deposits suggests that both components are necessary for
amyloid deposition and serves as further evidence for the basis of
the present invention. Although fibrillar beta-amyloid deposits
were observed in the small intestine in these transgenic mice, no
pathological changes were observed in the brains of transgenic mice
indicating that other co-factors, such as perlecan, are probably
necessary for the ultimate formation, deposition and persistence of
fibrillar beta-amyloid protein in brain.
EXAMPLE 10
Production of Perlecan-Amyloid Protein Double Transgenic
Animals
[0156] Perlecan-amyloid protein transgenic animals are produced by
the mating of perlecan transgenic mice with, for example,
beta-amyloid precursor protein transgenic animals. Double
transgenic mice carrying both transgenes (CA-S.beta.C and perlecan)
were created by mating the two lines of transgenic mice. Generally,
25% of emerging pups from mating heterozygous parents for each
transgene should carry both transgenes, and 50% of emerging pups
from mating heterozygous mice for one transgene with homozygous
mice for the other transgene should carry both transgenes.
Homozygous mice for CA-S.beta.C transgenes were established by
mating heterozygous mice for CA-S.beta.C. The homozygotes for
CA-S.beta.C was confirmed by crossing the homozygous mice for
CA-S.beta.C to C57BL/6J non-transgenic mice. A homozygous male for
CA-S.beta.C was crossed to a heterozygous female (a progeny from
founder 3595) for the perlecan transgene. Eight pups were produced
by the mating and the tails from the pups were cut to extract the
DNA for Southern blot analysis. The DNA was hybridized with
radiolabeled perlecan (bp 5523-7215 of perlecan cDNA in Noonan et
al., J. Biol. Chem. 266:22939-22947 (1991)) and beta-amyloid (bp
1796-2473 in Kang et al., Nature 325:733-736 (1987)) probes. The
results are shown in FIG. 16. Pup #552 was found to positive for
both perlecan and beta-amyloid precursor protein (C-terminal
portion which contains the A.beta. region) (shown in FIG. 16) and
verified the successful production of a double transgenic mouse
which carried transgenes for both perlecan and beta-amyloid.
EXAMPLE 11
Screening of Compounds for Treatment of Alzheimer's and Other
Amyloid Diseases
[0157] The transgenic animals, transfected cells, or animal cells
derived from transgenic animals, can be used to screen compounds
for a potential effect in the treatment of Alzheimer's disease and
other amyloidoses using standard methodology. In such screening
assays, the compound is administered to the animals, or introduced
into the culture media of transfected cells, or cells derived from
these animals, over a period of time and in various dosages. Then
the animals or animal cells are examined for alteration in amyloid
protein or amyloid precursor protein processing, expression levels,
or localization of other Alzheimer's disease or other amyloid
disease markers, and/or histopathology. In the case of animals
utilized for Alzheimer's disease, behavior tests are implemented
utilizing standard known memory tests known to those skilled in the
art. In general, any improvement in behavioral tests, alteration in
Alzheimer's disease-associated markers, reduction in the severity
of Alzheimer's disease-related histopathology, reduction in the
expression of A.beta. or .beta.PP cleavage products, and/or changes
in the presence, absence or levels or other compounds that are
correlated with Alzheimer's disease which are observed in treated
animals, relative to untreated animals, is indicative of a compound
useful for treating Alzheimer's disease. The specific proteins,
and/or histopathology of those proteins, that are associated with
and characteristic of Alzheimer's disease are referred to herein as
markers. Expression or localization of these markers characteristic
of Alzheimer's disease has either been detected, or is expected to
be present, in the disclosed transgenic animals. These markers can
be measured or detected, and those measurements compared between
treated and untreated transgenic animals to determine the effect of
a tested compound.
[0158] Markers useful for Alzheimer's disease screening assays are
selected based on detectable changes in these markers that are
associated with Alzheimer's disease. Many such markers have been
identified in Alzheimer's disease and are expected to be present in
the disclosed animals. These markers fall into several categories
based on their nature, location or function. Preferred examples of
markers useful in Alzheimer's disease screening assays are
described below, grouped as A.beta.-related markers, plaque-related
markers, cytoskeletal and neuritic markers, inflammatory markers,
and neuronal and neurotransmitter-related markers.
[0159] A. A.beta.-Related Markers
[0160] Expression of the various forms of .beta.PP and .beta.B can
be directly measured in treated and untreated transgenic animals
both by immunocytochemistry and by quantitative ELISA measurements.
Currently, it is known that two forms of .beta.PP products are
found, .beta.PP and A.beta. (Haass and Selkoe, Cell 75:1039-1042
(1993)). They have been shown to be intrinsically associated with
the pathology of Alzheimer's disease in a time dependent manner.
Therefore, preferred assays compare age-related changes in .beta.PP
and A.beta. expression in the transgenic mice. Generally, specific
forms of A.beta. can be assayed, either quantitatively or
qualitatively using specific antibodies. When referring to amino
acid positions in forms of A.beta., the positions correspond to the
A.beta. region of .beta.PP. Amino acid 1 of A.beta. corresponds to
amino acid 672 of .beta.PP, and amino acid 42 of A.beta.
corresponds to amino acid 714 of .beta.PP.
[0161] Also preferred as targets for assay measurement are .beta.PP
markers. For example, different forms of secreted .beta.PP (termed
.beta.PP-alpha and .beta.PP-.beta.) can also be measured (Seubert
et al., Nature 361:260-263 (1993)). .beta.PP forms are also
preferred targets for assays to assess the potential for compounds
to affect Alzheimer's disease. The absolute level of .beta.PP and
.beta.PP transcripts, the relative levels of the different .beta.PP
forms and their cleavage products, and localization of .beta.PP
expression or processing are all markers associated with
Alzheimer's disease that can be used to measure the effect of
treatment with potential therapeutic compounds. The localization of
.beta.PP to plaques and neuritic tissue is an especially preferred
target for these assays.
[0162] Quantitative measurement can be accomplished using many
standard assays. For example, transcript levels can be measured
using RT-PCR and hybridization methods including RNase protection
assays, Northern blot analysis, and R-dot analysis. .beta.PP and
A.beta. levels can be assayed by ELISA, Western blot analysis, and
be comparison of immunohistochemically stained tissue sections.
Immunohistochemical staining can also be used to assay localization
of .beta.PP and A.beta. to particular tissues and cell types.
[0163] B. Plaque-Related Markers
[0164] A variety of other molecules are also present in plaques of
individuals with Alzheimer's disease, and their presence in plaques
and neuritic tissue can be detected. Preferred plaque markers are
tau protein (Grundke-Iqbal et al., Proc. Natl. Acad. Sci. USA
83:4913-4917 (1986); Kosik et al., Proc. Natl. Acad. Sci. USA
83:4044-4048 (1986); Lee et al., Science 251:675-678 (1991)),
apolipoprotein E (Corder et al., Science 261:921-923 (1993);
Strittmatter et al., Proc. Natl. Acad. Sci. USA 90:8098-8102
(1993)), alpha.sub.1-antichymotrypsin (Abraham et al., Cell
52:487-501 (1988)), amyloid P component (Coria et al., Lab. Invest.
58:454-458 (1988)), ubiquitin (Mori et al., Science 235:1641-1644
(1987)), cytokines (McGeer et al., Can. J. Neurol. Sci. 16:516-527
(1989); reviewed in Rogers, CNS Drugs 4:241-244 (1994)), growth
factors (Hefti and Weiner, Ann. Neurol. 20:275-281 (1986); Hefti et
al., Neurobiol. Aging 10:515-533 (1989); Kato et al., Neurosc.
122:33-36 (1991); Tooyama et al., Neurosc. Lett. 121:155-158
(1991))), complement factors (Eikenbloom et al., Virch. Arch. B
Cell Pathol. 56:259-262 (1989)), advanced glycosylation end
products (Smith et al., Proc. Natl. Acad, Sci. USA 91:5710 (1994)),
receptor for advanced glycosylation products, growth inhibitory
factor, as well as heparan sulfate PGs (Snow et al., Am. J. Path.
133:456-463 (1988)), chondroitin sulfate PGs (DeWitt et al., Exp.
Neurol. 121:149-152 (1993)), dermatan sulfate PGs (Snow et al., J.
Histochem. Cytochem. 40:105-113 (1992)) and keratan sulfate PGs
(Snow et al., Exp. Neurol. 138:305-317 (1996)). While the above
markers can be used to detect specific components of plaques and
neuritic tissue, the location and extent of plaques can also be
determined by using well known histochemical stains, such as Congo
red and Thioflavin S.
[0165] C. Cytoskeletal and Neuritic Markers
[0166] Changes in cytoskeletal markers can also be used in
Alzheimer's disease screening assays to determine the effect of
compounds on Alzheimer's disease. Many of the changes in
cytoskeletal markers occur in the neurofibrillary tangles or
dystrophic neurites associated with plaques. The following are
preferred cytoskeletal and neuritic markers that exhibit changes in
and/or an association with Alzheimer's disease. These markers can
be detected, and changes can be determined, to measure the effect
of compounds on the disclosed transgenic animals, or transgenic
animals formed by mating with the disclosed animals. Spectrin
exhibits increased breakdown in Alzheimer's disease. Tau and
neurofilaments display an increase in hyperphosphorylation in
Alzheimer's disease, and levels of ubiquitin increase in
Alzheimer's. Tau protein, ubiquitin, MAP-2, neurofilaments, heparan
sulfate PGs, dermatan sulfate PGs, chondroitin sulfate GAGs and
keratan sulfate PGs are localized to plaques and neuritic tissues
in Alzheimer's disease and in general change from their normal
localization. GAP43 levels are decreased in the hippocampus in
Alzheimer's disease.
[0167] D. Inflammatory Markers
[0168] Alzheimer's disease is also known to stimulate an
inflammatory response, with a corresponding increase in
inflammatory markers (McGeer et al., Can. J. Neurol. Sci.
16:516-527 (1989); Rogers, CNS Drugs 4:241-244 (1994)). The
following are preferred inflammatory markers that exhibit changes
in and/or an association with Alzheimer's disease. Detection of
changes in these markers are useful in Alzheimer's disease
screening assays. Acute phase proteins and glial markers such as
alpha1-antitrypsin, C-reactive protein, alpha2-macroglobulin
(Tooyama et al., Molecular and Chem. Neuropath. 18:153-160 (1993)),
glial fibrillary acidic protein, Mac-1, F4/80, and cytokines, such
as interleukin-1alpha and .beta., TNF-alpha, interleukin-8,
MIP-1alpha (Kim et al., J. Neuroimmunology 56:127-134 (1995)),
MCP-1 (Kim et al., J. Neurol. Sci. 128:28-35 (1995); J.
Neuroimmunol. 56:127-134 (1995)) and interleukin-6, all increase in
Alzheimer's disease and are expected to increase in the disclosed
animals. Complement markers such as C3d, C1q, C5, C4d, C4 bp, and
C5a-C9 are localized in plaques and neuritic tissue. Major
histocompatibility complex (MHC) glycoproteins, such as HLA-DR and
HLA-A, HLA-D, HLA-C increase in Alzheimer's disease. Microglial
markers, such as CR3 receptor, MHCI, MHCII, CD31, CD11a, CD11b,
CD11c, CD68, CD45RO, CD45RO, CD45RD, CD18, CD59, CR4, CD45, CD64,
and CD44 (Akiyama et al., Br. Res. 632:249-259 (1993)) increase in
Alzheimer's disease. Additional inflammatory markers useful in
Alzheimer's disease screening assays include alpha2-macroglobulin
receptor, fibroblast growth factor (Tooyama et al., Neurosc. Letts.
121:155-158 (1991)), ICAM-1 (Akiyama et al., Acta Neuropath.
85:628-634 (1993)), lactotransferrin (Kawamata et al., Am. J. Path.
142:1574-1585 (1993)), C1q, C3d, C4d, C5b-9, Fc gamma RII, CD8
(McGeer et al., Can. J. Neurol. Sc. 16:516-527 (1989)), LCA (CD45)
(Akiyama et al., J. Neuroimmunol. 50:195-201 (1994)), CD18 (beta-2
integrin) (Akiyama and McGeer, J. Neuroimmunology 30:81-93 (1990)),
CD59 (McGeer et al., Br. Res. 544:315-319 (1991)), vitronectic
(McGeer et al., Can. J. Neurol. Sci. 18:376-379 (1991)),
vitronectin receptor, beta-3 integrin, Apo J, clusterin (McGeer et
al., Br. Res. 579:337-341 (1992)), type 2 plasminogen activator
inhibitor (Akiyama et al., Neurosc. Lett. 164:233-235 (1993)), CD44
(Akiyama et al., Br. Res. 633:249-259 (1993)), midkine (Yasuhara et
al., Biochem. Biophys. Res. Comm. 192:246-251 (1993)), macrophage
colony stimulating factor receptor (Akiyama et al., Br. Res.
639:171-174 (1994)), MRP14, 27E10, and interferon-alpha (Akiyama et
al., J. Neuroimmunol. 50:195-201 (1994)). Additional markers which
are associated with inflammation or oxidative stress include
4-hydroxynonenal-protein conjugates (Uchida and Stadtman, Meth.
Enz. 233:371-380 (1994)), IkappaB, NFkappaB (Kaltschmidt et al.,
Mol. Aspects. Med. 14:171-190 (1993)), cPLA.sub.2 (Stephenson et
al., Neurobiol. Dis. 3:51-63 (1996)), COX-2 (Chen et al.,
Neuroreport 6:245-248 (1995)), matrix metalloproteinases (Backstrom
et al., J. Neurochem. 58:983-992 (1992)), membrane lipid
peroxidation, protein oxidation (Smith et al., Proc. Natl. Acad.
Sci. USA 88:10540-10543 (1991)) and diminished ATPase activity
(Mark et al., J. Neurosc. 15:6239 (1995)). These markers can be
detected, and changes can be determined to measure an effect of
compounds on the disclosed transgenic animals.
[0169] E. Neuronal and Neurotransmitter-Related Markers
[0170] Changes in neuronal and neurotransmitter biochemistry have
been associated with Alzheimer's disease. In Alzheimer's, there is
profound reduction in cortical and hippocampal cholinergic
innervation. This is evidenced by the dramatic loss of the
synthetic enzyme choline acetyltransferase and decreased
acetylcholinesterase, synaptosomal choline uptake and synthesis and
release of acetylcholine (Sims et al., J. Neurochem. 40:503-509
(1983)), all of which are useful markers. These markers can be used
in Alzheimer's disease screening assays to determine the effect of
compounds on Alzheimer's disease. There is also a loss of basal
forebrain neurons and the galanin system becomes hypertrophic in
Alzheimer's disease.
[0171] In addition to changes in the markers described above in
Alzheimer's disease, there is also atrophy and loss of basal
forebrain cholinergic neurons that project to the cortex and
hippocampus (Whitehouse et al., Science 215:1237-1239 (1982)), as
well as alterations of entorhinal cortex neurons (Van Hoesan et
al., Hippocampus 1:1-8 (1991)). Based upon these observations
measurement of these enzyme activities, neuronal size, and neuronal
count numbers are expected to decrease in the disclosed transgenic
animals and are therefore useful targets for detection in
Alzheimer's disease screening assays. Basal forebrain neurons are
dependent on nerve growth factor. Brain-derived neurotrophic factor
(BDNF) may also decrease in the hippocampus and is therefore a
useful target for detection in Alzheimer's disease screening
assays. In addition, screening assays that measure the effect of
compounds on neurotransmitter receptors can possibly be used to
identifying compounds useful in treating Alzheimer's disease.
[0172] In addition to the well-documented changes in the
cholinergic system, dysfunction in other receptor systems such as
the serotinergic, adrenergic, adenosine and nicotine receptor
systems, has also been documented. Markers characteristic of these
changes, as well as other neuronal markers that exhibit both
metabolic and structural changes in Alzheimer's disease are listed
below. Changes in the level and/or localization of these markers
can be measured using similar techniques as those described for
measuring and detecting the earlier markers.
[0173] The following are preferred cytoskeletal and neuritic
markers that exhibit changes in and/or an association with
Alzheimer's disease. Levels of cathepsin D, cathepsin B, cathepsin
G, and neuronal thread protein, and phosphorylation of elongation
factor-2, increase in Alzheimer's. Cathepsin D, cathepsin B,
protein kinase C and NADPH are localized in plaque and neuritic
tissue in Alzheimer's disease. Activity and/or levels of nicotine
receptors, 5-HT.sub.2 receptor, NMDA receptor, alpha2-adrenergic
receptor, synaptophysin, synaptoglycan (SV2PG), p65, glutamine
synthetase, glucose transporter, PPI kinase, drebrin, GAP43,
cytochrome oxidase, heme oxygenase, calbindin, adenosine A1
receptors, mono amine metabolites, choline acetyltransferase,
acetylcholinesterase, and symptosomal choline are all reduced in
Alzheimer's disease.
[0174] Additional markers that are associated with Alzheimer's
disease or after treatment of cells with A.beta. include a)
cPLA.sub.2 which is upregulated in Alzheimer's disease (Stephenson
et al., Neurobiol. Dis. 3:51-63 (1996)), b) heme oxygenase-1 (Smith
et al., Am. J. Path. 145:42-47 (1994)), c-jun (Anderson et al.,
Exp. Neurol. 125:286-295 (1994)), c-fos (Zhang et al., Neurosci.
46:9-21 (1992)), HSP27 (Renkawek et al., Acta NeuroDath. 87:511-519
(1994)), HSP70 (Cisse et al., Acta Neuropath. 85:233-240 (1993))
and MAP5 (Geddes et al., J. Neurosc. Res. 30:183-191 (1991)), which
are induced in Alzheimer's disease and in cortical cells after
A.beta. treatment and c) junB, junD, fosB, fra1 (Estus et al., J.
Cell Biol. 127:1717-1727 (1994)), cyclin D1 (Freeman et al., Neuron
12:343-355 (1994)), NGFI-A (Vaccarino et al., Mol. Br. Res.
12:233-241 (1992)), and NGFI-B, which are induced in cortical cells
after A.beta. treatment.
[0175] F. Measuring the Amounts and Localization of Alzheimer's
Disease Markers
[0176] Quantitative measurements can be accomplished using many
standard assays. For example, transcript levels can be measured
using RT-PCR and hybridization methods including RNase protection,
Northern analysis, and R-dot analysis. Protein marker levels can be
assayed by ELISA, Western analysis, and by comparison of
immunohistochemically stained tissue sections. Immunohistochemical
staining can also be used to assay localization of protein markers
to particular tissues and cell types. The localization and the
histopathological association of Alzheimer's disease markers can be
determined by histochemical detection methods such as antibody
staining, laser scanning confocal imaging, and electron and
immunoelectron microscopy.
[0177] In the case of receptors and enzymatic markers, activity of
the receptors or enzymes can be measured. For example, the activity
of neurotransmitter metabolizing enzymes such as choline
acetyltransferase and acetylcholine esterase can be measured using
standard radiometric enzyme activity assays.
[0178] G. Screening Assays Using Cultured Cells
[0179] Screening assays for determining the therapeutic potential
of compounds can also be performed using cells derived from animals
transgenic for the disclosed perlecan constructs or cell cultures
stably transfected with the disclosed constructs, or from cells
derived from progeny derived from the mating of perlecan transgenic
mice with other transgenics implicated in Alzheimer's disease. For
example, P19 cells which overproduce perlecan also demonstrate a
marked increase in A.beta. levels in the media. In a preferred
embodiment, a potential therapeutic compound for Alzheimer's
disease is tested on perlecan transfected P19 cells, or other cells
types that also demonstrate increased A.beta. production due to
perlecan transfection. For such testing, derived transgenic cells
or transfected cell cultures, following an overnight incubation at
37.degree. C. in an incubator equilibrated with 5% carbon dioxide,
will have the media removed and replaced with media containing a
compound to be tested for a 2 hour pretreatment period with the
cells then incubated as above. At the end of the pretreatment
period, the media are again removed and replaced with fresh media
containing the compound to be tested (as described above) and cells
are incubated for an additional 2 to 16 hours. After treatment,
plates are centrifuged in a Beckman GPR at 1200 rpm for 5 minutes
at room temperature to pellet cellular debris from the conditioned
media. From each well, 100 .mu.l of conditioned media or
appropriate dilutions thereof are transferred into an ELISA plate
precoated with antibody 266 (an antibody directed against amino
acids 13 to 28 of AS) as described in International patent
Application No. 94/10569 and stored at 4.degree. C. overnight. An
ELISA assay employing any labelled antibody against A.beta. (such
as 4G8 from Senetek) can be run to measure the amount of A.beta.
produced. Different capture and detection antibodies can also be
used. In addition, various cytotoxicity assays, such as the MTS
assay described in Example 6 of the disclosed invention, can be
utilized to determine whether the compound protect cells from
neurodegeneration or neuronal death.
[0180] H. Screening for Compounds Effective in Other Amyloid
Diseases
[0181] Use of the perlecan transgenic mice, transfected cells, or
cells derived from transgenic mice as disclosed in the present
invention, can be used either alone or in combination with other
transgenic mice, transfected cells, or cells derived from other
transgenic mice implicated in a given amyloid disease, to produce
new animal and cell culture models of amyloid diseases (other than
the Alzheimer's-type of amyloid disease). For example, the mating
of perlecan mice with transgenic animals which overproduce islet
amyloid (amylin) may produce progeny which exhibit a new animal
model completely mimicking islet amyloid deposition in pancreas. In
such transgenic animals amyloid formation, deposition, accumulation
and/or persistence of an amyloid protein (other than the
Alzheimer's disease A.beta.) is occurring in peripheral tissues,
making these animals useful for the screening of anti-amyloid
compounds to determine if said compounds affect or diminish
accumulation of a specific amyloid protein in the tissues. Initial
analysis may include, but is not limited to, detecting levels and
immunolocalization of a given amyloid protein using specific
anti-amyloid antibodies which are commercially available including
those against AA amyloid. AL amyloid, islet amyloid (i.e. amylin),
beta.sub.2-microglobulin, transthyretin/prealbumin, procalcitonin,
and PrP amyloid (brain amyloidosis), and comparison of
compound-treated versus placebo-treated animals. In addition,
qualitative and quantitative assessment of the extent of amyloid
can be made following detection by specific amyloid stains such as
Congo red (and viewed under polarized light) or Thioflavin S
(viewed under fluorescent light). In such assessments, a given
compound is administered to an animal at various dosages, for
various treatment periods, and at varying stages of amyloid
formation, deposition accumulation and persistence in tissues. A
given compound is said to be effective by reducing or eliminating
the amyloid by a correlative decrease or elimination of amyloid
protein immunostaining, Congo red staining (as viewed under
polarized light) or Thioflavin T fluorescence. In addition,
electron microscopic analysis can be used for the detection and
localization of amyloid fibrils in a given tissue, and qualitative
and quantitative assessments can be made prior-to and following
compound treatments in groups of animals. In all of the screening
assays described above, the effective compounds will be the ones
which diminish or eliminate amyloid in a particular tissue or
animal.
[0182] New Amyloid Disease Models
[0183] The strategy of crossing perlecan transgenic mice with
transgenic mice that overexpress any other amyloid protein, or
another component implicated in a given amyloid disease, will also
produce new animal models of different amyloidoses which can be
utilized as screening tools to identify new therapeutics for the
treatment of amyloidoses.
[0184] In one preferred embodiment, perlecan transgenic mice mated
with transgenic mice overexpressing any isoform of beta-amyloid
precursor protein (and/or its portions thereof) produce transgenic
progeny that overexpress both perlecan and .beta.PP. This new
transgenic animal model can be used as a screening tool to identify
potential therapeutics targeting A.beta. amyloidosis as observed in
Alzheimer's disease, Down's syndrome and cerebral hemorrhage with
amyloidosis of the Dutch type.
[0185] In another preferred embodiment, perlecan transgenic mice
are mated with transgenic mice wherein the A.beta.-containing
protein consists of all or a contiguous portion of a protein
selected from a group consisting of .beta.PP-770, .beta.PP-770
bearing a mutation in one or more of the amino acids selected from
the group consisting of amino acids 669, 670, 671, 690, 692 and
717, .beta.PP-751, .beta.PP-751 bearing a mutation in one or more
of the amino acids selected from the group consisting of amino acid
669, 670, 671, 690, 692 and 717, .beta.PP-695, and .beta.PP-695
bearing a mutation in one or more of the amino acids selected from
the group consisting of amino acid 669, 670, 671, 690, 692 and 717,
wherein the A.beta.-containing protein includes amino acids 672 to
714 of human .beta.PP.
[0186] In another preferred embodiment, perlecan transgenic mice
are mated with transgenic mice which overexpress presenilin 1,
presenilin 2, or transgenics which contain mutations to either
presenilin 1 or presenilin 2.
[0187] In another preferred embodiment, perlecan transgenic mice
are mated with transgenic mice which overexpress a protein selected
from the group consisting of laminin, type IV collagen, heparan
sulfate PGs, chondroitin sulfate PGs, dermatan sulfate PGs, keratan
sulfate PGs, glypican, syndecan, syndecan-3, neurocan, phosphacan,
aggrecan, decorin, biglycan, hyaluronan, amyloid P component,
alpha.sub.1-antichymotrypsin, cathepsin D, cathepsin G, cathepsin
B, neuronal thread protein, nicotine receptors, 5-HT.sub.2
receptor, NMDA receptor, alpha2-adrenergic receptor, synaptophysin,
p65, glutamine synthetase, glucose transporter, PPI kinase, GAP43,
cytochrome kinase, heme oxygenase, calbindin, adenosine A1
receptors, choline acetyltransferase, acetylcholinesterase, glial
fibrillary acidic protein, alpha1-antitrypsin, C-reactive protein,
alpha2-macroglobulin, interleukin-1alpha, interleukin-1.beta.,
TNF-alpha, interleukin-6, HLA-DR, HLA-A, HLA-D, HLA-C, CR3
receptor, MHC I, MHC II, CD 31, CR4, CD45, CD64, CD4, spectrin, tau
protein, ubiquitin, MAP-2, apolipoprotein E, apolipoprotein E4,
apolipoprotein E2, apolipoprotein E3, nerve growth factor,
brain-derived neurotrophic factor, advanced glycosylation end
products, receptor for advanced glycosylation end products, COX-2,
CD18, C3, fibroblast growth factor, CD44, ICAM-1, lactotransferrin,
C1q, C3d, C4d, C5b-9, gamma R1, Fc gamma RII, CD8, CD59,
vitronectin, vitronectin receptor, beta-3 integrin, Apo J,
clusterin, type 2 plasminogen activator inhibitor, midline,
macrophage colony stimulating factor receptor, MRP14, 27E10,
interferon-alpha, S100.beta., cPLA.sub.2, c-jun, c-fos, HSP27,
HSP70, MAP5, membrane lipid peroxidase, protein carbonyl formation,
junB, junD, fosB, fra1, cyclin D1, p53, NGFI-A, NGFI-B, I-kappa-B,
NF-kappa-B, interleukin-8, MCP-1, MIP-1alpha, matrix
metaloproteinases, and 4-hydroxynonenal-protein conjugates.
[0188] In another preferred embodiment, perlecan transgenic mice
mated with transgenic mice overexpressing AL amyloid (and/or its
portions thereof) produce transgenic progeny that overexpress both
perlecan and AL amyloid. This new transgenic animal model can be
used as a screening tool to identify potential therapeutics
targeting AL amyloidosis as observed in multiple myeloma and other
.beta.-cell dyscrasias.
[0189] In another preferred embodiment, perlecan transgenic mice
mated with transgenic mice overexpressing islet amyloid (amylin)
produce transgenic progeny that overexpress both perlecan and
amylin. This new transgenic animal model can be used as a screening
tool to identify potential therapeutics targeting islet amyloidosis
as observed in type II diabetes.
[0190] In another preferred embodiment, perlecan transgenic mice
mated with transgenic mice overexpressing PrP amyloid produce
transgenic progeny that overexpress both perlecan and PrP protein.
This new transgenic animal model can be used as a screening tool to
identify potential therapeutics targeting PrP amyloidosis as
observed in Creutzfeldt-Jakob disease, Gerstmann-Straussler
syndrome, kuru and animal scrapie.
[0191] In yet another preferred embodiment, perlecan transgenic
mice mated with transgenic mice overexpressing
beta.sub.2-microglobulin produce transgenic progeny that
overexpress both perlecan and beta.sub.2-microglobulin. This new
transgenic animal model can be used as a screening tool to identify
potential therapeutics targeting beta.sub.2-microglobulin
amyloidosis as observed in long-term hemodialysis and carpal tunnel
syndrome.
[0192] In another preferred embodiment, perlecan transgenic mice
mated with transgenic mice overexpressing prealbumin/transthyretin
produce transgenic progeny that overexpress both perlecan and
prealbumin/transthyretin. This new transgenic animal model can be
used as a screening tool to identify potential therapeutics
targeting prealbumin/transthyretin amyloidosis as observed in
senile cardiac amyloid and Familial Amyloidotic Polyneuropathy.
[0193] In yet another preferred embodiment, perlecan transgenic
mice mated with transgenic mice overexpressing variants of
procalcitonin produce transgenic progeny that overexpress both
perlecan and variants of procalcitonin. This new transgenic animal
model can be used as a screening tool to identify potential
therapeutics targeting the amyloid associated with endocrine tumors
such as in medullary carcinoma of the thyroid.
[0194] In yet another preferred embodiment, perlecan transgenic
mice are mated with "knock-out" transgenic mice which do not
express, or express at much reduced levels than normal, a given
amyloid protein or protein implicated to be important in amyloid
disease as listed above. These new transgenic animal models can be
used as a screening tool to identify potential therapeutics
targeting various amyloid diseases.
[0195] In yet another preferred embodiment, transgenic mice that
overexpress both perlecan and any amyloid protein, (amyloid
precursor protein and/or a fragment thereof), is produced by
injection of both perlecan and amyloid protein DNA constructs into
fertilized mouse eggs (one cell stage embryos). The embryos bearing
both the DNA constructs are transferred to the oviducts of
pseudopregnant foster mothers. The tails from the emerging pups are
cut for Southern blot analysis or PCR to determine the chromosomal
integration of both the transgenes. Pups determined positive for
both the transgenes are crossed with other mice (example: C57BL/6J)
to establish lines of transgenic mice bearing both the DNA
constructs.
[0196] Throughout this application various publications have been
referenced within parentheses. The disclosures of these
publications in their entireties are hereby incorporated by
reference in this application in order to more fully describe the
state of the art to which this invention pertains.
[0197] Although the invention has been described with reference to
the disclosed embodiments, those skilled in the art will readily
appreciate that the specific experiments detailed are only
illustrative of the invention. It should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
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