U.S. patent application number 12/394988 was filed with the patent office on 2009-08-27 for proteoglycan splice variants as therapeutics and diagnostics for amyloid diseases.
Invention is credited to Judy A. Cam, Joel Cummings, Qubai Hu, Alan D. Snow.
Application Number | 20090214555 12/394988 |
Document ID | / |
Family ID | 40998526 |
Filed Date | 2009-08-27 |
United States Patent
Application |
20090214555 |
Kind Code |
A1 |
Snow; Alan D. ; et
al. |
August 27, 2009 |
Proteoglycan Splice Variants as Therapeutics and Diagnostics for
Amyloid Diseases
Abstract
The identification of novel Syndecan-2 splice variants and their
use in the diagnosis and therapeutic intervention of Alzheimer's
disease and other amyloid diseases. In addition the use of new
animal models expressing or devoid of syndecan-2 splice variants to
effectively screen and identify potential therapeutic compounds for
Alzheimer's disease.
Inventors: |
Snow; Alan D.; (Lynnwood,
WA) ; Hu; Qubai; (Kirkland, WA) ; Cam; Judy
A.; (Bellevue, WA) ; Cummings; Joel; (Seattle,
WA) |
Correspondence
Address: |
PROTEOTECH, INC.
12040 115TH AVE NE
KIRKLAND
WA
98034-6931
US
|
Family ID: |
40998526 |
Appl. No.: |
12/394988 |
Filed: |
February 27, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61031828 |
Feb 27, 2008 |
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Current U.S.
Class: |
424/139.1 ;
435/320.1; 435/6.16; 514/44A; 530/324; 530/350; 530/387.9;
536/23.5 |
Current CPC
Class: |
C12Q 2600/158 20130101;
C07K 14/705 20130101; C12Q 2600/106 20130101; C12Q 2600/136
20130101; C07K 2317/34 20130101; A61P 25/28 20180101; C12Q 2600/156
20130101; C12Q 1/6883 20130101; C07K 16/2896 20130101 |
Class at
Publication: |
424/139.1 ;
536/23.5; 435/320.1; 530/350; 530/324; 435/6; 530/387.9;
514/44.A |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07H 21/04 20060101 C07H021/04; C12N 15/63 20060101
C12N015/63; C07K 14/47 20060101 C07K014/47; A61P 25/28 20060101
A61P025/28; C12Q 1/68 20060101 C12Q001/68; A61K 31/711 20060101
A61K031/711; C07K 16/18 20060101 C07K016/18 |
Claims
1. An isolated polynucleotide consisting of SEQ ID NO: 6.
2. An isolated polynucleotide consisting of SEQ ID NO: 8.
3. A polynucleotide encoding a protein according to SEQ ID
NO:7.
4. A polynucleotide encoding a protein according to SEQ ID
NO:9.
5. A vector comprising SEQ ID NO: 6.
6. A vector comprising at least a portion of nucleotides 124-279 of
SEQ ID NO: 6.
7. A vector comprising SEQ ID NO: 8 or a fragment thereof.
8. A splice variant of syndecan-2 having the sequence of SEQ ID NO:
7.
9. A splice variant of syndecan-2 having the sequence of SEQ ID NO:
9 or a fragment thereof.
10. A polypeptide obtained from the translation of the
polynucleotide of claim 1.
11. A polypeptide comprising at least a portion of amino acids
21-72 of SEQ ID NO: 7.
12. A polypeptide as set forth in SEQ ID NO:9 or a fragment
thereof.
13. A method for detection and/or quantitation of a splice variant
of syndecan-2 in a biological sample, the method comprising;
synthesizing cDNA from mRNA in the sample, amplifying portions of
the cDNA corresponding to the splice variant or fragments thereof
and detecting/quantitating the amplified cDNA.
14. The method of claim 13, wherein the biological sample is
derived from one of tissues, cells or biological fluids.
15. The method of claim 14 wherein said tissues, cells or
biological fluids are derived from humans.
16. The method of claim 13 wherein said biological fluids include
blood, plasma, serum, cerebrospinal fluid, sputum, saliva, urine
and stool.
17. The method of claim 13 wherein said biological fluid is
cerebrospinal fluid.
18. The method of claim 13 wherein the step of amplifying portions
of the cDNA is performed by RT-PCR.
19. The method of claim 18 wherein primers utilized for RT-PCR are
selected from the group consisting of SEQ ID NO:1, SEQ. ID NO:2,
SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
20. The method of claim 13, whereby said method utilizes
quantitative RT-PCR to determine relative levels of the splice
variant in the sample.
21. The method of claim 13 whereby said method is utilized to
diagnose Alzheimer's disease or determine susceptibility or
progression of Alzheimer's disease related to the levels of the
splice variant, wherein elevated or diminished levels of a
particular splice variant are indicative of the presence of,
susceptibility to, or progression of Alzheimer's disease.
22. A method for the treatment of Alzheimer's disease comprising:
administering to a patient a therapeutically effective amount of an
oligonucleotide having a sequence complementary to the
polynucleotide of claim 1 or 2.
23. The method of claim 22 where the oligonucleotide is antisense
DNA or RNA and is complementary to a sequence selected from the
group consisting of SEQ ID NO:6, SEQ ID NO: 8, and fragments
thereof.
24. An antibody that binds to an epitope comprising at least
several of amino acids 21-72 of SEQ ID NO: 7.
25. The antibody of claim 24 where the epitope consisted of amino
acids 50-65 of SEQ ID NO: 7.
26. A method for the treatment of Alzheimer's disease comprising
administering to a patient the antibody of claim 24 or 25.
27. A method for the treatment of Alzheimer's disease comprising
administering to a patient a immunogenic amount of the splice
variant of claim 9.
28. Use of the splice variant of claim 8 or 9 to modulate APP
processing.
29. Use of the splice variant of claim 8 or 9 to modulate activity
of APP secretases.
30. The use according to claim 29 where the APP secretase is
beta-secretase.
Description
TECHNICAL FIELD
[0001] This invention relates to the discovery and identification
of novel proteoglycan splice variants and their utilization for the
diagnosis and therapeutic intervention of Alzheimer's disease and
other amyloid diseases. In addition new animal models to
effectively screen and identify potential therapeutic compounds for
Alzheimer's disease and each of the amyloidoses are described.
BACKGROUND OF THE INVENTION
[0002] Alzheimer's disease (AD) is a degenerative brain disorder
characterized clinically by progressive loss of memory, cognition,
reasoning, judgment and emotional stability that gradually leads to
profound mental deterioration and ultimate death. Alzheimer's
disease is the leading cause of dementia in the elderly, affecting
5-10% of the population over the age of 65 years (Jorm A, A Guide
to the Understanding of Alzheimer's Disease and Related Disorders,
University Press, New York, 1987.). In AD, the parts of the brain
essential for cognitive processes such as memory, attention,
language, and reasoning degenerate. AD is characterized by the
deposition and accumulation of a 39-43 amino acid peptide termed
the beta-amyloid protein, A.beta. (Glenner G G, and C W Wong.
Biochem. Biophys. Res. Comm. 120:885-890, 1984. Husby G, et al.
Bull WHO 71:105-108, 1993. Masters C L, et al. Proc. Natl. Acad.
Sc. USA 82:4245-4249, 1985.). A.beta. is derived from larger
precursor proteins termed beta-amyloid precursor proteins (or APPs)
of which there are several alternatively spliced variants. The most
abundant forms of APPs include proteins consisting of 695, 751 and
770 amino acids (Kitaguchi N, et al. Nature 331:530-532, 1988.
Ponte P, et al. Nature 331:525-527, 1988. Tanzi R E, et al. Nature
331:528-530, 1988.). The small A.beta. peptide is a major
component, which makes up the amyloid deposits of "plaques" in the
brains of patients with AD either as extracellular amyloid plaques
or in blood vessel walls in the parenchyma. In addition, AD is
characterized by the presence of numerous neurofibrillary
"tangles", consisting of paired helical filaments(PHFs) that
abnormally accumulate in the neuronal cytoplasm (Grundke-Iqbal I,
et al. Proc. Natl. Acad. Sci. USA 83:4913-4917, 1986. Kosik K S, et
al. Proc. Natl. Acad. Sci. USA 83:4044-4048, 1986. Lee V M Y, et
al. Science 251:675-678, 1991.). The pathological hallmarks of AD
are therefore the presence of "plaques" and "tangles" with amyloid
being deposited in the central core of plaques. The other major
type of lesion found in AD brain is the accumulation of amyloid in
the walls of blood vessels, both within the brain parenchyma and in
the walls of meningeal vessels that lie outside the brain. A.beta.
amyloid formation, deposition, accumulation and persistence are
believed to play a central role in AD pathogenesis by contributing
to neuronal loss and memory dysfunction. The primary factor(s)
causing amyloid plaque and NFT accumulation leading to the
pathogenesis of AD is not known.
[0003] Previous studies indicate that the accumulation of A.beta.
and amyloid is indeed a causative factor for AD. A.beta. in cell
culture causes degeneration of nerve cells within short periods of
time (Pike C J, et al. Br. Res. 563:311-314, 1991. Pike C J, et al.
J. Neurochem. 64:253-265, 1995.). A.beta. has been found to be
neurotoxic in slice cultures of hippocampus (Harrigan M R, et al.
Neurobiol. Aging 16:779-789, 1995.) and induces nerve cell death in
some forms of transgenic mice (Games D, et al. Nature 373:523-527,
1995. Hsiao K, et al. Science 274:99-102, 1996, Sturchler-Pierrat
C, et al. Proc. Natl. Acad. Sci. 94:13287-13292, 1997.). Previous
studies utilizing amyloid plaque producing transgenic mice also
clearly demonstrate a direct correlation between increased amyloid
plaque burden and behavioral deficits in memory tasks (Choi P Y, et
al. Neuroscience Meeting, Orlando, Fla., November 2002. Janus C, et
al. Nature 408:979-982, 2000. Morgan D, et al. Nature 408:982-985,
2000.). Probably the most convincing evidence that A.beta. amyloid
is directly involved in the pathogenesis of A.beta. comes from
genetic studies in which the production of A.beta. resulted from
mutations in the APP gene (Haas C, et al. Nature Med. 1:1291-1296,
1995. Murrell J, et al. Science 254:97-99, 1991. Van Broeckhoven C,
et al. Science 248:1120-1122, 1990.), and duplication of the APP
locus (Rovelet-Lecrux et al., Nature Genetics, 38:24-26, 2006).
[0004] Important amyloid co-factors that may play a role in the
pathogenesis of AD are specific proteoglycans (PGs) and
glycosaminoglycans (GAGs). Previous studies demonstrated that
particular heparan sulfate proteoglycans (HSPGs) including
perlecan, syndecan-2, glypican, and agrin are specifically
immunolocalized to A.beta.-containing amyloid plaques and/or
cerebrovascular amyloid deposits in AD brain (Perlmutter L S, et
al. Br. Res. 508:13-19, 1990. Snow A D, et al. Am. J. Path.
133:456-463, 1988. Snow A D, and TN Wight, Neurobiol. Aging
10:481-497, 1989. Snow A D, et al. Am. J. Path. 137:1253-1270,
1990. Snow A D, et al. Neuron 12: 219-234, 1994. Su J H, et al.
Neurosc. 51:801-813, 1992. Van Gool D, et al. Dementia 4:308-314,
1993. Van Horssen J, et al. Lancet 2:482-492, 2003, Castillo G M,
et al. J. Neurochem. 69:2452-2465, 1997. Narindrasorasak S, et al.
J. Biol. Chem. 266:12878-12883, 1991. Snow A D, et al. Am. J. Path.
144:337-347, 1994. Snow A D, et al. Arch. Biochem. Biophys.
320:84-95, 1995, Lashley T, et al. Neuropath. Appl. 32:492-504,
2006. Verbeek M M, et al. Am. J. Path. 155:2115-2125, 1999, Lashley
T, et al. Neuropath. Appl. 32:492-504, 2006. Verbeek M M, et al.
Am. J. Path. 155:2115-2125, 1999. Watson D J, et al. J. Biol. Chem.
272:31617-31624, 1997, Cotman S L, et al. Mol. Cell. Neurosc.
15:183-198, 2000. Lashley T, et al. Neuropath. Appl. 32:492-504,
2006. Schultz J G, et al. Europ. J. Neuorsc. 10:2085-2093, 1998.
Verbeek M M, et al. Am. J. Path. 155:2115-2125, 1999. Watanabe N,
et al. FASEB J. published online, Apr. 14, 2004. Watson D J, et al.
J. Biol. Chem. 272:31617-31624, 1997). These HSPGs also accumulate
in transgenic mice overexpressing beta-amyloid precursor protein
(APP) and accumulate in brain concurrent with initial A.beta.
accumulation and deposition (Cummings J A, et al. Annual Meeting of
Neuroscience, Washington, D.C., November 2005, Snow A D, et al. 8th
International Conference on Alzheimer's and Parkinson's disease,
Salzburg, Austria, March 2007). It is believed that HSPGs
facilitate amyloid deposition and/or promote the persistence of
amyloid by inhibiting clearance mechanisms (Snow A D, and T N
Wight, Neurobiol. Aging 10:481-497, 1989.). Consistent with this
hypothesis in vitro studies have revealed that HSPGs such as
perlecan (Narindrasorasak S, et al. J. Biol. Chem. 266:12878-12883,
1991. Snow A D, et al. J. Histochem. Cytochem. 40:105-113, 1992.
Snow A D, et al. Arch. Biochem. Biophys. 320:84-95, 1995.), agrin
(Cotman S L, et al. Mol. Cell. Neurosc. 15:183-198, 2000.) and
glypican (Watson D J, et al. J. Biol. Chem. 272:31617-31624, 1997.)
can bind with high affinity to A.beta. and APPs (Narindrasorasak S,
et al. J. Biol. Chem. 266:12878-12883, 1991.). Additionally, in
vitro and cell culture studies demonstrate that HSPGs protect
A.beta. from protease degradation (Gupta-Bansal R, et al. J. Biol.
Chem. 270:18666-18671, 1995. Nguyen B P, et al. Annual Meeting of
Neuroscience, New Orleans, La. November 2003. Snow A D, et al.
Neuron 12: 219-234, 1994.), supporting a role for HSPGs in
inhibition of A.beta.-degradation and removal in vivo. All of these
studies implicate HSPGs as important co-factors postulated to lead
to the accumulation and persistence of A.beta.. HSPGs are also
specifically co-localized to the PHFs in NFTs in AD brain (Snow A
D, et al. Acta Neuropath. 78:113-123, 1989. Snow A D and G M
Castillo. Amyloid: Int. J. Exp. Clin. Invest. 4:135-141, 1997.). An
alternative hypothesis is that PG's may affect APP processing. Our
results suggest that syndecan-2 splice variants interfere with
.beta.-secretase cleavage of APP which may lead to a reduction in
A.beta. levels. Studies have also demonstrated that highly sulfated
GAGs such as heparan sulfate can induce tau protein to adopt PHF
formation identical to that observed in AD brain (Friedrich M V, et
al. J. Biol. Chem. 294:259-270, 1999. Goedert M, et al. Nature
383:550-553, 1996. Hasegawa M, et al. J. Biol. Chem.
272:33118-33124, 1997. Perez M, et al. J. Neurochem. 67:1183-1190,
1996.). Our results also support that syndecan-2 splice variants
may be relevant to tau NFT formation. Therefore, HSPGs may play an
important role in the pathology of AD.
[0005] Proteoglycans (PGs) usually consist of a protein core to
which are covalently attached one or more glycosaminoglycan (GAG)
chains. GAGs consist of a repeating disaccharide unit containing a
hexuronic acid (either glucuronic acid or iduronic acid) or
hexosamine (glucosamine or galactosamine)(reviewed in Snow A D, and
T N Wight. Neurobiol. Aging 10:481-497, 1989). Different classes of
GAGs include the highly sulfated heparin and heparan sulfate, and
the less sulfated keratan sulfate, dermatan sulfate,
chondroitin-4-sulfate, chondroitin-6-sulfate, and the non-sulfated
hyaluronic acid (reviewed in Snow A D, and TN Wight. Neurobiol.
Aging 10:481-497, 1989). At least 4 different classes of PGs have
been shown to be present in AD brain. These include heparan sulfate
proteoglycans (HSPGs) (Perlmutter L S, et al. Br. Res. 508:13-19,
1990. Snow A D, et al. Am. J. Path. 133:456-463, 1988. Snow A D,
and TN Wight. Neurobiol. Aging 10:481-497, 1989. Snow A D, et al.
Am. J. Path. 137:1253-1270, 1990. Snow A D, et al. Neuron 12:
219-234, 1994. Su J H, et al. Neurosc. 51:801-813, 1992. Van Gool
D, et al. Dementia 4:308-314, 1993. Van Horssen J, P et al. Lancet
2:482-492, 2003.), dermatan sulfate PGs (Snow A D, et al. J.
Histochem. Cytochem. 40:105-113, 1992), chondroitin sulfate PGs
(DeWitt D A, et al. Exp. Neurol. 121:149-152, 1993.) and keratan
sulfate PGs (Snow A D, et al. Exp. Neurol. 138:305-317, 1996.). Of
all these different PGs, evidence indicated that only the HSPGs are
specifically immunolocalized to the A.beta.-containing fibrils both
in the amyloid plaques and in the cerebrovascular amyloid deposits
in AD brain (Perlmutter L S, et al. Br. Res. 508:13-19, 1990, Snow
A D, et al. Am. J. Path. 133:456-463, 1988, Snow A D, and T N
Wight. Neurobiol. Aging 10:481-497, 1989. Snow A D, et al. Am. J.
Path. 137:1253-1270, 1990, Snow A D, et al. Neuron 12: 219-234,
1994. Su J H, et al. Neurosc. 51:801-813, 1992, Van Gool D, et al.
Dementia 4:308-314, 1993. Van Horssen J, et al. Lancet 2:482-492,
2003.). Particular HSPGs that have been immunolocalized or
identified within A.beta.-amyloid deposits in AD brain include
perlecan (Castillo et al. J. Neurochem. 69:2452-2465, 1997,
Narindrasorasak S, et al. J. Biol. Chem. 266:12878-12883, 1991,
Snow A D, et al. Am. J. Path. 144:337-347, 1994, Snow A D, et al.
Arch. Biochem. Biophys. 320:84-95, 1995), syndecan-2 (Lashley T, et
al. Neuropath. Appl. 32:492-504, 2006, Verbeek M M, et al. Am. J.
Path. 155:2115-2125, 1999), agrin (Cotman S L, et al. Mol. Cell.
Neurosc. 15:183-198, 2000, Lashley T, et al. Neuropath. Appl.
32:492-504, 2006, Verbeek M M, et al. Am. J. Path. 155:2115-2125,
1999), and glypican (Lashley T, et al. Neuropath. Appl. 32:492-504,
2006, Schultz J G, et al. Europ. J. Neuorsc. 10:2085-2093, 1998,
Verbeek M M, et al. Am. J. Path. 155:2115-2125, 1999. Watanabe N,
et al. FASEB J. published online, Apr. 14, 2004. Watson D J, et al.
J. Biol. Chem. 272:31617-31624, 1997.). Our own studies indicate
that HSPGs, such as perlecan (which consists of a 400 kDa core
protein with 3 heparan sulfate GAG chains attached) are integral
parts of amyloid deposits in AD brain. Perlecan is present in
isolated amyloid plaque core preparations derived from AD brain as
determined by positive immunostaining and western blotting with
specific perlecan core protein antibodies (Castillo G M, et al.
Soc. Neurosc. Abstr. 22:1172, 1996, Castillo G M, et al. 6th
International Conference on Alzheimer's Disease and Related
Disorders, Amsterdam, July 1998). Perlecan, syndecan-2, glypican
and agrin all not only co-localized to A.beta.-amyloid deposits in
AD brain, but are also present and co-immunolocalized to amyloid
plaques in APP transgenic mice (Cummings J A, et al. Annual Meeting
of Neuroscience, Washington, D.C., November 2005, Snow A D, et al.
8th International Conference on Alzheimer's and Parkinson's
disease, Salzburg, Austria, March 2007). In fact, HS GAGs
accumulate in APP mouse brain concurrent and co-localized with
initial A.beta. accumulation and deposition in brain tissue
(Cummings J A, et al. Annual Meeting of Neuroscience, Washington,
D.C., November 2005, Snow A D, et al. 8th International Conference
on Alzheimer's and Parkinson's disease, Salzburg, Austria, March
2007). HSPG immunoreactivity is localized to diffuse plaques in AD
(Snow A D, et al. Am. J. Path. 133:456-463, 1988, Snow A D, et al.
Am. J. Path. 137:1253-1270, 1990. Snow A D, et al. Am. J. Path.
144:337-347, 1994.) and Down's syndrome brain (Snow A D, et al. Am.
J. Path. 137:1253-1270, 1990.) suggesting that this particular
class of PGs may-in fact represent a primary initiating factor
leading to A.beta. accumulation and persistence. Consistent with
this hypothesis is the observation that in very young Down's
syndrome brain (as early as 1 day after birth), marked HS
accumulation in neuronal cytoplasm occurs prior and much earlier
than the first appearance of A.beta.-deposition (in neurons and
later in the matrix) and fibrillar amyloid (Snow A D, et al. Am. J.
Path. 137:1253-1270, 1990.). In other types of amyloidosis (such as
systemic AA amyloidosis) where the temporal relationship in the
experimental mouse model has been extensively studied, it is clear
that an increase in gene expression of specific HSPGs, such as
perlecan, occurs prior to AA amyloid formation and deposition in
tissues (Ailles L, et al., Lab. Invest. 69:443-448, 1993, Elimova
E, et al. FASEB J. 18:1749-1751, 2004, Snow A D, and R Kisilevsky,
Lab. Invest. 53:37-44, 1985). Furthermore, heparanase
overexpressing transgenic mice that cause a decrease in HS
accumulation renders mice resistant to induction of systemic AA
amyloidosis (Li J P, et al. Proc. Natl. Acad. Sc. 102:6473-6477,
2005) further supporting an important role of HSPGs for the
induction of amyloidosis.
[0006] Perlecan is a large HSPG normally present on all basement
membranes, consisting of 94 exons, coding for a large .about.470
kDa protein core. Perlecan core protein contains a cluster of 3 GAG
attachment sites in domain I (Dolan M, et al., J. Biol. Chem.
272:4316-4322, 1997, Murdoch A D, et al., J. Biol. Chem.
267:8544-8557, 1992). Possible splice variants of perlecan have
been reported for mammalian perlecan (Joseph S J, et al., Develop.
122:3443-3452, 1996.). Syndecan-2 is one of four members of this
single-pass transmembrane family in vertebrates (Kramer K L, and H
J Yost, Ann. Rev. Gen. 37:461-484, 2003). The 22 kDa core protein
is organized into 3 regions: the N-terminal ectodomain containing a
signal sequence, followed by 3 predicted GAG attachment sites, a
transmembrane domain and a highly conserved cytoplasmic domain
(reviewed in Essner J J, et al. Int. J. Biochem. Cell Biol.
38:152-156, 2006).
[0007] The HSPGs, perlecan (Castillo G M, et al. J. Neurochem.
69:2452-2465, 1997. Narindrasorasak S, et al. J. Biol. Chem.
266:12878-12883, 1991, Snow A D, et al. Am. J. Path. 144:337-347,
1994. Snow A D, et al. Arch. Biochem. Biophys. 320:84-95, 1995.),
syndecan-2 (Lashley T, et al. Neuropath. Appl. 32:492-504, 2006.
Verbeek M M, et al. Am. J. Path. 155:2115-2125, 1999), agrin
(Cotman S L, et al. Mol. Cell. Neurosc. 15:183-198, 2000. Lashley
T, et al. Neuropath. Appl. 32:492-504, 2006, Schultz J G, et al.
Europ. J. Neuorsc. 10:2085-2093, 1998, Verbeek M M, et al. Am. J.
Path. 155:2115-2125, 1999, Watanabe N, et al. FASEB J. published
online, Apr. 14, 2004. Watson D J, et al. J. Biol. Chem.
272:31617-31624, 1997.) and glypican (Lashley T, et al. Neuropath.
Appl. 32:492-504, 2006. Schultz J G, et al. Europ. J. Neuorsc.
10:2085-2093, 1998, Verbeek M M, et al. Am. J. Path. 155:2115-2125,
1999. Watanabe N, et al. FASEB J. published online, Apr. 14, 2004.
Watson D J, et al. J. Biol. Chem. 272:31617-31624, 1997.) have been
specifically immunolocalized to amyloid plaques in AD brain. In
addition, our studies have identified these same HSPGs in the
amyloid plaque deposits in APP mouse transgenic brain (FIG.
1)(Cummings J A, et al. Annual Meeting of Neuroscience, Washington,
D.C., November 2005, 102, Snow A D, et al. 8th International
Conference on Alzheimer's and Parkinson's disease, Salzburg,
Austria, March 2007). Sulfated GAGs and polyanions also play a role
in PHF formation such as observed in NFTs in AD brain. In early
studies by Snow et al (Snow A D, et al. Acta Neuropath. 78:113-123,
1989.) cationic dyes retained PGs in tissues and at the electron
microscopic level it was clear that PGs were specifically
co-localized to the PHFs in NFTs, in a specific periodic fashion.
HSPG antibodies also immunolocalized HSPGs to tangles in AD brain
(Goedert M, et al. Nature 383:550-553, 1996, Snow A D, and T N
Wight. Neurobiol. Aging 10:481-497, 1989. Snow A D, et al. Am. J.
Path. 137:1253-1270, 1990. Snow A D and G M Castillo. Amyloid: Int.
J. Exp. Clin. Invest. 4:135-141, 1997.). Evidence by a number of
groups later confirmed that highly sulfated GAGs (i.e. heparan
sulfate and heparin) were potent inducers of tau polymerization
into PHFs (Friedhoff P, et al., Biochem. 37:10223-10230, 1998.
Goedert M, et al. Nature 383:550-553, 1996, Hasegawa M, et al. J.
Biol. Chem. 272:33118-33124, 1997, Perez M, et al. J. Neurochem.
67:1183-1190, 1996). Since heparin is only found primarily in mast
cells (not in brain tissue), it is postulated that the heparan
sulfate class of PGs are important in the induction of PHFs as
observed in AD brain.
[0008] Syndecan-2 is widely expressed in many tissues including
brain. In neurons, syndecan-2 is concentrated at synapses in
dimer/multimer clusters playing an essential role in creating
specialized membrane environments for post-synaptic signaling
(Ethell I M, et al., Neuron 31:1001-1013, 2001). The human
syndecan-2 transcript consists of 5 exons, coding for a 22 kDa
protein product that has 201 residues. The first of the GAG
attachment sites in syndecan-2 is encoded by exon 2 and the other 2
GAG attachment sites, representing typical repetitive SGSG amino
acid residues with a flanking cluster of acidic residues encoded by
the combined sequence derived from the boundary of exons 2/3.
[0009] Agrin is also a large PG with the gene encoding a protein
with a predicted MW of 225 kDa. At least 3 HS GAG attachment sites
are present in the amino-terminal half of agrin (Hoch W, et al.,
EMBO J. 13:2814-2821, 1994. Tsen G, et al., J. Biol. Chem.
270:3392-3399, 1995.). The extensive glycosylation in this region
increases the apparent molecular mass of agrin to 600 kDa. The
C-terminal half of agrin is active in acetylcholine receptor
aggregation and contains binding sites for dystroglycan, heparin
and some integrins (Bezakova G, and MA Ruegg, Nat. Rev. Mol. Cell.
Biol. 4:295-308, 2003.). Agrin is expressed as several isoforms in
various tissues.
[0010] Six different glypicans have been identified in mammals
(Esko J D, and S B Selleck, Ann. Rev. Biochem. 71:435-471, 2002.);
they are encoded by 6 independent genes that contain 8-12 exons.
All glypicans are approximately 60-70 kDa in size. The GAG
attachment sites are usually identified as a cluster, which locate
within the last 50 residues at the C-terminus, next to a
glyosylphosphotidy-linositol membrane anchor (Kramer K L, and H J
Yost, Ann. Rev. Gen. 37:461-484, 2003. Veugelers M, et al., J.
Biol. Chem., 274:26969-26977, 1999.).
[0011] It is believed that HSPGs facilitate A.beta. to ultimately
adapt a beta-sheet conformation and into insoluble amyloid fibrils.
Consistent with this hypothesis, HSPGs such as perlecan
(Narindrasorasak S, et al. J. Biol. Chem. 266:12878-12883, 1991.
Snow A D, et al. J. Histochem. Cytochem. 40:105-113, 1992. Snow A
D, et al. Arch. Biochem. Biophys. 320:84-95, 1995.), agrin (Dolan
M, et al. J. Biol. Chem. 272:4316-4322, 1997.) and glypican (Watson
D J, et al. J. Biol. Chem. 272:31617-31624, 1997.) can bind with
high affinity to A.beta. and APPs (Narindrasorasak S, et al. J.
Biol. Chem. 266:12878-12883, 1991). In addition, HSPGs, such as
perlecan, enhance fibrillar A.beta. amyloid deposition and
persistence in brain, when co-infused with A.beta. into rodent
hippocampus (Snow A D, et al. Neuron 12: 219-234, 1994.).
Furthermore, perlecan and HS GAGs can induce A.beta. 1-40 peptides
in vitro to adopt a congophilic Maltese-cross spherical plaque core
appearance identical to that observed in AD brain (Choi P Y, et al.
Neuroscience Meeting, Orlando, Fla., November 2002. Snow A D, et
al. 10.sup.th International Symposium on Amyloid and Amyloidosis,
Tours, France, April 2004.). These studies implicate HSPGs as
important co-factors that may lead to the accumulation and
persistence of A.beta.. Studies indicate that the highly sulfated
GAG chains (and not the core protein) are critical for formation
and acceleration of A.beta. amyloid (as observed in
"plaques")(Castillo G M, et al. J. Neurochem. 72:1681-1687, 1999),
and for tau protein to form PHFs (as observed in
"tangles")(Friedrich M V, et al. J. Biol. Chem. 294:259-270, 1999,
Goedert M, et al. Nature 383:550-553, 1996, Hasegawa M, et al. J.
Biol. Chem. 272:33118-33124, 1997, Perez M, et al. J. Neurochem.
67:1183-1190, 1996). In one study, heparin/HS GAGs in which the
sulfate moieties had been removed, demonstrated a nearly complete
loss of the GAG's ability to accelerate A.beta. amyloid fibril
formation (Castillo G M, et al. J. Neurochem. 72:1681-1687, 1999).
Thus it is postulated that any increase in HS GAG number, leads to
an overall increase in GAG sulfation, which is critical to cause a
formation and acceleration of both A.beta. amyloid fibril and PHF
formation in AD. Studies are therefore needed that characterize the
degree of sulfation in PG GAGs and elucidate the role of sulfation
in A.beta. amyloid fibril and PHF formation in AD.
Amyloid as a Therapeutic Target for Alzheimer's Disease
[0012] 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). A.beta. is derived by protease cleavage from larger
precursor proteins termed .beta.-amyloid precursor proteins (APPs)
of which there are several alternatively spliced variants. The most
abundant forms of the APPs include proteins consisting of 695, 751
and 770 amino acids (Tanzi et al., Nature 31:528-530, 1988).
[0013] The small A.beta. peptide is a major component that makes up
the amyloid deposits of "plaques" 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 hallmark of Alzheimer's disease is
therefore the presence of "plaques" and "tangles", with amyloid
being deposited in the central core of the plaques. The other major
type of lesion found in the Alzheimer's disease brain is the
accumulation of amyloid in the walls of blood vessels, both within
the brain parenchyma and in the walls of meningeal vessels that 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)
[0014] 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 a consequence of the disease. Within the
last few years, studies now indicate that amyloid is indeed a
causative factor for Alzheimer's disease and should not be regarded
as merely an innocent bystander. The Alzheimer's A.beta. protein 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, 1995). Studies suggest that it is
the fibrillar structure (consisting of a predominant .beta.-pleated
sheet secondary structure), characteristic of all amyloids, that is
responsible for the neurotoxic effects. A.beta. has also been found
to be neurotoxic in slice cultures of hippocampus (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., Science 274:99-102, 1996). Injection of the Alzheimer's
A.beta. into rat brain also causes memory impairment and neuronal
dysfunction (Flood et al., Proc. Natl. Acad. Sci. USA 88:3363-3366,
1991; Br. Res. 663:271-276, 1994).
[0015] Probably, the most convincing evidence that A.beta. amyloid
is directly involved in the pathogenesis of Alzheimer's disease
comes from genetic studies. It was discovered that the production
of A.beta. can result from mutations in the gene encoding, its
precursor, .beta.-amyloid precursor protein (Van Broeckhoven et
al., Science 248:1120-1122, 1990; Murrell et al., Science
254:97-99, 1991; Haass et al., Nature Med. 1:1291-1296, 1995). The
identification of mutations in the beta-amyloid precursor protein
gene that cause early onset familial Alzheimer's disease is the
strongest argument that amyloid is central to the pathogenetic
process underlying this disease. Four reported disease-causing
mutations have been discovered which demonstrate the importance of
A.beta. in causing familial Alzheimer's disease (reviewed in Hardy,
Nature Genet. 1:233-234, 1992). All of these studies suggest that
providing a drug to reduce, eliminate or prevent fibrillar A.beta.
formation, deposition, accumulation and/or persistence in the
brains of human patients will serve as an effective
therapeutic.
Modulators of APP Secretases as Therapeutic Targets for Alzheimer's
Disease
[0016] Elucidating APP metabolism and its role in the formation of
A.beta. plaques in AD is becoming increasingly important as
therapeutics for AD and other beta-amyloid protein diseases are
sought. Intracellular trafficking and proteolytic processing of APP
directly influences the amount and type of A.beta. peptide and can
thus have a profound impact on amyloid plaque load.
[0017] Processing of APP in vivo and in cultured cells occurs by
two major pathways (Haass and De Strooper, Science 286(5441):916-9
(1999) and; Selkoe, Physiol Rev. 81(2):741-66, (2001)). Cleavage of
APP at the N-terminus of the A.beta. region by .beta.-secretase and
at the C-terminus by .gamma.-secretases represents the
amyloidogenic pathway for processing of APP. .beta.-secretase
cleaves APP between residues Met.sup.595 and Asp.sup.596 (codon
numbering refers to the APP.sup.695 isoform), and yields A.beta.
peptide plus the .beta.-C-terminal fragment (.beta.CTF or C99).
Following .beta.-secretase cleavage, a second cleavage by
.gamma.-secretase occurs at the C-terminus of A.beta. peptide that
releases A.beta. from CTF. This cleavage occurs in the vicinity of
residue 636 of the C-terminus. .gamma.-secretase can cleave the
C-terminal region at either Val.sup.636 or Ile.sup.638 to produce a
shorter A.beta. peptide (A.beta.1-40) or the longer A.beta. peptide
(A.beta.1-42). The predominant form of A.beta. found in the
cerebrospinal fluid and conditioned media of cultured cells is the
shorter A.beta.40 peptide. Despite its lower abundance, A.beta.42
is the peptide that is initially deposited within the extracellular
plaques of AD patients. In addition, A.beta.42 is shown to
aggregate at a much lower concentration than the A.beta.40 form.
APP can alternatively be processed via a non-amyloidogenic pathway
where .alpha.-secretase cleaves within the A.beta. domain between
Lys.sup.611 and Leu.sup.612, and produces a large soluble
.alpha.-APP domain (sAPP.alpha.) and a .alpha.-C-terminal fragment
(.alpha.CTF or C83). The latter can then be cleaved by
.gamma.-secretase at residue 636 or 638 to release a P3 peptide and
the APP intracellular domain (AICD). The .alpha.-cleavage pathway
is the major pathway used to process APP in vivo; it does not yield
A.beta. peptide (Selkoe, Physiol Rev. 81(2):741-66, (2001). The
characterization of APP cleavage and the related secretases has
provided significant advancement in therapeutic strategies that may
lead to limiting the deposition of A.beta. peptide in the brain,
and eliminate or delay the associated pathological effects in
AD.
BRIEF SUMMARY OF THE INVENTION
[0018] In a first aspect, this invention is the utilization of
novel and specific primer sequences for the detection of
proteoglycan splice variants in human tissues using standard RT-PCR
methodology, known to one skilled in the art. In another aspect,
this invention is the utilization of standard RT-PCR methodology,
utilizing the specific primers described herein, which will aid in
the amplification of each of specific proteoglycan splice variants,
for the ultimate detection of these splice variants in various
human tissues, cells and in biological fluids. In addition,
quantitative competitive RT-PCR techniques can be utilized (Maresh
et al, J. Neurochem. 67:1132-1144, 1996) to determine quantitative
differences in these specific variants in total RNA derived from
human tissues, cells, white blood cells and in biological fluids.
Changes in quantitative levels of these specific proteoglycan
splice variants will aid in the diagnosis and monitoring of
prognosis of patients who demonstrate amyloid and concurrent
specific proteoglycan splice variant and/or specific proteoglycan
accumulation in tissues as part of the pathological process
observed in the amyloid diseases, especially Alzheimer's
disease.
[0019] In another aspect, this invention is the utilization of
syndecan-2 slice variants as specific indicators for the presence
and extent of neurofibrillary tangles in brain by monitoring
biological fluids including, but not limited to, cerebrospinal
fluid, blood, serum, urine, saliva, sputum, and stool.
[0020] In another aspect, this invention is the utilization of the
syndecan-2 slice variants as a specific indicator for the presence
and progression of Alzheimer's disease and/or other amyloid
diseases by monitoring biological fluids including, but not limited
to, cerebrospinal fluid, blood, serum, urine, saliva, sputum, and
stool.
[0021] In another aspect, this invention is the utilization of
purified antibodies to syndecan-2 slice variants as specific
indicators for the presence and progression of Alzheimer's disease
and/or other amyloid diseases by monitoring brain and biological
fluids including, but not limited to, cerebrospinal fluid, blood,
serum, urine, saliva, sputum, and stool.
[0022] In another aspect, this invention is the utilization of the
syndecan-2 slice variants as a specific indicator for the presence
and extent of amyloid plaques in brain by monitoring biological
fluids including, but not limited to, cerebrospinal fluid, blood,
serum, urine, saliva, sputum, and stool.
[0023] In another aspect, this invention is the utilization of the
syndecan-2 slice variants as therapeutics for Alzheimer's disease
due to the effects of syndecan-2 slice variants as modulators of
APP processing and the subsequent reduction of beta secretase
product.
[0024] In another aspect, this invention is the utilization of a
method which can evaluate a compound or potential therapeutics'
ability to alter (diminish or eliminate) the affinity of a given
amyloid protein (as described herein) or amyloid precursor protein,
to proteoglycan splice variant protein or proteoglycan splice
variant GAGs. By providing a method of identifying compounds which
affect the binding of amyloid proteins, or amyloid precursor
proteins to such proteoglycan splice variant protein or
proteoglycan splice variant derived-GAGs or fragments thereof, the
present invention is also useful in identifying compounds which can
prevent or impair such binding interaction. Thus, compounds can be
identified which specifically affect an event linked with the
amyloid formation, amyloid deposition, and/or amyloid persistence
condition associated with Alzheimer's disease and other amyloid
diseases.
[0025] In another aspect, this invention is the utilization of
peptides or fragments thereof which are specific against new and
unique sequences of any proteoglycan splice variant. These peptides
or fragments thereof can be used as potential blocking therapeutics
for the interaction of the proteoglycan splice variants in a number
of biological processes and diseases (such as in the amyloid
diseases described herein).
[0026] In another aspect, this invention is the utilization and
production of oligonucleotides utilizing the nucleotide sequences
described herein, to be utilized as new molecular biological probes
to detect proteoglycan splice variants in human tissues by standard
in situ hybridization techniques, and Northern blot analysis.
Alternatively oligonucleotides with sequences complementary to
proteoglycan splice variants could be utilized for therapeutic
treatment of amyloid disease, for example antisense RNA using RNA
interference techniques.
[0027] The oligonucleotides of the present invention may be in the
form of RNA or in the form of DNA, which DNA includes cDNA, genomic
DNA, and synthetic DNA. The DNA may be double-stranded or
single-stranded, and if single-stranded may be the coding strand or
non-coding (anti-sense) strand. The coding sequence which encodes
the mature polypeptide may be identical to the coding sequence
shown or may be a different nucleotide sequence as a result of the
redundancy or degeneracy of the genetic code, encodes the same
mature polypeptide as the DNA or the cDNA.
[0028] In another aspect, this invention is the production of new
animal models for the production, deposition, accumulation and/or
persistence of fibrillar A.beta. amyloid in brain as observed in
Alzheimer's disease and Down's syndrome. These new animal models
can also be used to effectively screen and identify new therapeutic
agents that target fibrillar A.beta. amyloid formation, deposition,
accumulation and/or persistence in brain.
[0029] In another aspect, this invention is the utilization of new
animal models for the production, deposition, accumulation and/or
persistence of fibrillar amyloid as observed in each of the other
amyloidoses. These new animal models can also be used for the
evaluation of candidate drugs and therapies for the prevention and
treatment of the amyloidoses as referred to above.
[0030] In another aspect, this invention is the production and
utilization of new transgenic animals that overexpress or knock-out
a particular proteoglycan splice variants in an effort to produce
specific phenotypes associated with a disease and/or pathological
processes, including, but not limited to, Alzheimer's disease
and/or other amyloid diseases.
[0031] In yet another aspect of the invention, syndecan-2 variant
plasmids could be constructed using knowledge and materials known
to one skilled in the art and can be used for Northern blot
analysis of mRNA derived from human tissues, cells, and/or cells in
biological fluids to further determine the size of transcripts. In
addition, Northern blots utilizing the same probes of the invention
can be utilized to quantitate relative levels of syndecan-2 splice
variant mRNA in tissues from normal patients in comparison to those
with specific diseases (such as the amyloid diseases).
[0032] In yet another aspect of the invention, fragments of the
full length gene may be used as a hybridization probe for a cDNA
library to isolate the full length gene and to isolate other genes
which have a high sequence similarity to the gene or similar
biological activity. The probe may also be used to identify a cDNA
clone corresponding to a full length transcript and a genomic clone
or clones that contain the complete syndecan-2 splice variant gene
including regulatory and promoter regions, exons, and introns. An
example of a screen comprises isolating the coding region of the
gene by using the known DNA sequence to synthesize an
oligonucleotide probe. Labeled oligonucleotides having a sequence
complementary to that of the gene of the present invention are used
to screen a library of human cDNA, genomic DNA or mRNA to determine
which members of the library the probe hybridizes to.
[0033] Another aspect of the invention relates to vectors which
includes polynucleotides as described herein, host cells which are
genetically engineered with vectors of the invention and the
production of polypeptides of the invention by recombinant
techniques. Host cells are genetically engineered (transformed or
transduced or transfected) with the vectors of the invention which
may be, for example, a cloning vector or an expression vector. The
vector may be, for example, in the form of a plasmid, a viral
particle, a phage etc. The engineered host cells can be cultured in
conventional nutrient media modified as appropriate for activating
promoters, selecting transformants or amplifying the genes. The
culture conditions, such as temperature, pH and the like, are those
previously utilized with the host cell selected for expression, and
will be apparent to those ordinarily skilled in the art.
[0034] In another aspect of the invention, the polynucleotides of
the present invention may be employed for producing polypeptides by
recombinant techniques. For example, the polynucleotides may be
included in any one of a variety of expression vectors for
expressing a polypeptide. Such vectors included chromosomal,
nonchromosomal and synthetic DNA sequences, e.g., derivatives of
SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids;
vectors derived from combinations of plasmids and phage DNA, viral
DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
However, any other vector may be used as long as it is replicable
and viable in the host.
[0035] In accordance with one aspect of the present invention there
is provided novel peptide sequences encoded within the new
syndecan-2 splice variants described herein, as well as
biologically active and diagnostically or therapeutically useful
fragments, analogs and derivatives thereof. The peptide sequences
described in the present invention are human sequences.
[0036] In accordance with another aspect of the present invention,
there is provided a process for diagnosing Alzheimer's disease or a
susceptibility to Alzheimer's disease related to under-expression
or over-expression of the polypeptide product of a splice variant.
The process comprises determining a mutation in a nucleic acid
sequence encoding the splice variant which is responsible for the
under-expression or over-expression of the polypeptide translated
from the splice variant.
[0037] The present invention accordingly encompasses the expression
of a syndecan-2 splice variant polypeptide, in either prokaryotic
or eukaryotic cells, although eukaryotic expression is preferred.
Preferred hosts are bacterial or eukaryotic hosts including
bacteria, yeast, insects, fungi, bird and mammalian cells either in
vivo, or in situ, or host cells of mammalian, insect, bird or yeast
origin. It is preferred that the mammalian cells or tissue is of
human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse,
goat, dog, or cat origin, but any other mammalian cell may be
used.
[0038] The polynucleotides of the present invention may be utilized
as research reagents and materials for discovery of treatments and
diagnostics to human diseases.
[0039] These and other features and advantages of the present
invention will become more fully apparent when the following
detailed description of the invention is read in conjunction with
the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] The following drawings are illustrative of embodiments of
the invention and are not meant to limit the scope of the
invention. The patent or application file contains at least one
drawing executed in color. Copies of this patent or patent
application publication with color drawing(s) will be provided by
the Office upon request and payment of the necessary fee.
[0041] FIG. 1: are photomicrographs showing that (A) Congo Red, (B)
Perlecan, (C) Syndecan-2 and (D) Agrin immunostain Amyloid Plaques
in Tg2576 Transgenic Mouse Brain.
[0042] FIGS. 2A and C are is a schematic representations of the
Exon Structure of Syndecan-2 (A) and the Newly Identified
Syndecan-2 Splice Variant (C) Containing Consensus Sequence for 4
Additional GAG Chains.
[0043] FIGS. 2B and D are diagrams of the tertiary protein
structure of Syndecan-2 (B) and the Newly Identified Syndecan-2
Splice Variant (D) Containing Consensus Sequence for 4 Additional
GAG Chains.
[0044] FIG. 3A is a schematic representation of the syndecan-2
splice variant, Syn2-vE1a showing the approximate locations of the
primers utilized for PCR.
[0045] FIG. 3B is a photograph of PCR products separated by gel
electrophoresis using primers 398F and v671R.
[0046] FIG. 3C is a photograph of PCR products separated by gel
electrophoresis using primers v533F and 811R
[0047] FIG. 4A is a schematic representation of the syndecan-2
splice variant, Syn2-vE1a showing the approximate locations of the
nested primer sets to enrich PCR products for DNA sequencing.
[0048] FIG. 4B is a photograph of 1.sup.st round PCR products
separated by gel electrophoresis using primers 398F and 1086R.
[0049] FIG. 4C is a photograph of 2.sup.nd round nested PCR
products separated by gel electrophoresis using nested primer sets
398F and v671R.
[0050] FIG. 4D is a photograph of 2.sup.nd round nested PCR
products separated by gel electrophoresis using nested primer sets
v533F and 811R.
[0051] FIG. 5 shows the putative amino acid sequence of Syn2-vE1a
which contains an in-frame 52-residue insertion (grey) that codes
for four extra SG sites (bold and underlined), including a
prominent repeating SGSG sequence flanked by acidic residues.
[0052] FIG. 6A is a photograph of semi-quantitative PCR products
using unpooled single strand cDNA samples reverse transcribed from
RNA isolated from middle temporal cortex of late onset AD patients,
and age-matched non-demented controls.
[0053] FIG. 6B is a photograph of semi-quantitative PCR product
control experiment showing amplification products from
.beta.-actin
[0054] FIG. 6C is a schematic representation of ratios of the DNA
band image from FIGS. 6A and 6B digitally documented, and
quantified with ScionImage software. Relative levels of Syn2-vE1a
were normalized to those of .beta.-actin (FIG. 6B).
[0055] FIG. 7 are photographs of Westerm blots showing the
specificity of rabbit polyclonal anti-Syn2vE1a antibodies as
assessed by Western analysis and epitope-peptide competition
assays.
[0056] FIG. 8A is a photograph of Western blots showing effects of
Syn2vE1a on APP processing in cultured human embryonic kidney 293E
(HEK293E) cells overexpressing APP695 and Syn2vE1a as assessed by
Western analysis. FIG. 8B is a graph plotting the densitometry of
the Western in 8A.
[0057] FIG. 9 are photomicrographs showing that the rabbit
polyclonal anti-Syn2vE1a antibodies specifically stain pyramidal
neurons labeled positive for birefringent tangles in the
hippocampus of an Alzheimer brain as assessed by
immunohistochemical staining. (A), (B) and (D) are pre-adsorbed
with anti-Syn2vE1a (1:1000), (C) is an overlapping image of (B)
under birefringent light showing co-localization of label to
tangles. (D) and (E) are not treated with primary antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0058] Our studies indicate that various PGs are present in AD
lesions, including perlecan, syndecan-2, glypican and agrin. FIG.
1: shows in photomicrographs that Perlecan, Syndecan-2 and Agrin
immunostain the Amyloid Plaques in Tg2576 Transgenic Mouse Brain.A)
Congo red staining of amyloid plaques (arrows) in cortex of
12-month old Tg2576 transgenic mouse X200. B) Perlecan immunostain
of amyloid plaques (arrows) in cortex of a 12-month old Tg2576
transgenic mouse X200. C) Syndecan-2 immunostain (mouse monoclonal
antibody) of amyloid plaques (arrows) in cortex of 12-month old
Tg2576 transgenic mouse (counterstained with Congo red) X200. D)
Agrin immunostain (agrin-33 antibody) in thalamus in a 12-month old
Tg2576 transgenic mouse (counterstained with Congo red). Agrin
deposits are brown, whereas fibrillar amyloid is red X100. PGs may
additionally consist of important splice variants that are unique
and contain increased GAG chain numbers (and increased
sulfation).
[0059] Our studies show that some HSPGs identified are present in
AD lesions, including perlecan, syndecan-2, glypican and agrin
additionally consist of important splice variants that are unique
HSPGs with increased GAG chain numbers (and increased sulfation).
The generation of such HSPG splice variants is hypothesized to be
critical for A.beta. amyloid fibril and PHF formation and
persistence. We have identified a syndecan-2 splice variant that
may contain up to 7 GAG chains in the variant as compared to 3 GAG
chains found normally on the syndecan-2 core protein. The studies
described are believed to have both therapeutic and diagnostic
implications. The surprising discovery of unique proteoglycan
splice variants that may contain additional HS GAG chains (and thus
increased sulfation that drives both A.beta. amyloid and PHF
formation), further implicate their importance in plaque and tangle
development in AD. Identification of novel splice variants that may
be also present in blood and/or CSF and that are indicative of
amyloid plaque or NFT formation in brain will also have exciting
diagnostic implications.
DEFINITIONS
[0060] In this application, the following terms shall have the
following meanings, without regard to whether the terms are used
variantly elsewhere in the literature or otherwise in the known
art.
[0061] A `splice variant` refers to mRNA (or corresponding cDNA)
that arises from an alternative splicing event. Alternative
splicing may arise due to changes at the genomic level or during
RNA processing. Regardless of how it occurs, alternative splicing
results in the insertion or deletion of nucleic acids in the mRNA
relative to the wild type. In general, splice variants can generate
both in-frame and frame-shift amino acid changes. Translation of a
splice variant can result in a polypeptide with an amino acid
sequence distinct from the wild type peptide resulting from
conventional splicing, provided that the addition or deletion of
nucleic acids are in frame. Translation of a splice variant could
also result in a truncated polypeptide where a stop codon is
introduced.
[0062] With respect to splice variants, `a fragment thereof` refers
to nucleic acid or amino acid sequences which are comprised of at
least a portion of the splice variant sequence or a portion of the
polypeptide sequence translated from the splice variant, that is
novel relative to the wild type. Such a fragment thereof may
additionally include portions of the wild type mRNA or wild type
polypeptide sequence resulting therefrom.
Example 1
Identification of a Syndecan-2 Splice Variant with 4 Additional GAG
Chains
[0063] A novel syndecan-2 splice variant that consists of an exon
insertion coding for 4 extra GAG-chain attachment sites was found
using a comprehensive bioinformatic approach (SEQ ID NO:8).
Initially, we identified the 5' partial sequences of an inserted
exon in Syndecan-2 from the Alternative Splicing and Transcript
Diversity database (ASDT)
(http://www.ebi.ac.uk/asdsrv/Index.cgi?method=PATSEQ&specie=H&product=SIN-
GLE&ensembl_id=ENSG000001 69439&highlight patterns=3). The
database consists of computationally delineated alternative
splicing events as well as literature-based alternative splicing
data; it has been integrated with the Ensemble genome database
(http://www.ensembl.org). Electronic hybridization using the 5'
partial sequences of the inserted exon as an electronic probe was
performed. The analysis led to discovering an EST (Expressed
sequence tag) clone (BG195558) from human EST database at NCBI
(National Center of Biotechnology Information). The EST clone
contains the sequences for the entire inserted exon as well as
downstream exons. Electronic hybridization using the entire
inserted exon as an electronic probe against the sequences of human
syndecan-2 gene (Gene ID: ENSG00000169439) was then conducted. The
results confirmed that the insertion was derived from the middle of
intron 1 of this gene, and that the novel splice sites were in
compliance with the canonical GT/AG. We therefore named the novel
splice variant: syndecan-2 variant E1a (Syn2-vE1a). Syndecan-2
normally consists of a .about.22 kDa core protein encoded by 5
exons. Compared to the syndecan-2 transcript Reference Sequence
(RefSeq; ENSP00000307046), the novel syndecan-2 splice variant has
an exon insertion (E1a) that is in-frame with the downstream codons
(FIG. 2A, SEQ ID NO: 6). Protein sequence analysis indicates that
the inserted exon codes for an extra 52 residues including four
additional putative GAG attachment sites including a repetitive
SGSG sequence flanked by acidic residues (SEQ ID NO: 9). Ser-Gly
consensus sequence for 4 additional GAG attachment sites is shown
by red arrows in FIG. 2C. The identified syndecan-2 (Syn2-vE1a)
splice variant therefore encodes for a putative HSPG containing a
core protein with potentially 7 GAG chains attached (FIG. 2D),
instead of 3 GAG chains found normally on syndecan-2 (FIG. 2B)
Example 2
Detection of Splice Variants in Alzehimer's Disease Brains
[0064] To demonstrate expression of the novel syndecan-2 splice
variant (Syn2-vE1a), characterized by comprehensive bioinformatic
analyses, in cell cultures and brain tissues, we performed RT-PCR
analysis, followed by DNA sequencing. In addition, we also
performed semi-quantitative RT-PCR analysis to examine expression
levels of the variant in Alzheimer disease (AD) and age-matched
control brain samples.
[0065] Two total RNA pools were used for these experiments: (1) the
AD pool, derived from the middle temporal cortex of seven
neuropathologically confirmed late onset AD patients, and (2) the
control pool, derived from the corresponding brain region of six
age-matched non-demented controls. In addition, total RNA isolated
from Hela cells was also analyzed in some experiments. Two
micrograms of total RNA from each of the brain samples was first
reverse transcribed to single strand cDNA with random hexamers. The
single strand cDNA products were then pooled together for
downstream PCR analysis. PCR reactions were initially performed
with two sets of primers 398F/v671R (SEQ ID NO:1/SEQ ID NO:3) and
v533F/811 R (SEQ ID NO:2/SEQ ID NO:4) as shown in FIG. 3A. Primers
v671R (SEQ ID NO:3) and v533F (SEQ ID NO:2) were designed to the
sequences unique to exon E1a. PCR amplification with 35 cycles
revealed DNA bands with the predicted sizes of 274 bp with primer
set 398F/v671R (SEQ ID NO:1/SEQ ID NO:3)(FIG. 3B, indicated by an
arrow) and 453 bp with primer set v533F/811 R (SEQ ID NO:2/SEQ ID
NO:4)(FIG. 3C, indicated by an arrow). These DNA bands were not
observed in negative controls (data not shown). The 274 bp (FIG.
3B) and 453 bp (data not shown) PCR products were also detected in
the RNA sample derived from Hela cells. These results indicate that
Syn2-vE1a is expressed in human brain tissues and Hela cell
cultures. In addition, the amplified Syn2-vE1a DNA bands appeared
to be relatively enriched in the pooled AD sample when compared to
those in the pooled control sample, suggesting a possibility of
up-regulated expression of this variant in AD brain tissues. To
further confirm expression of Syd2-vE1a in human brain tissues, and
to enrich PCR products for DNA sequencing, nested PCR analysis was
also performed (FIG. 4). The 1.sup.st round of PCR was conducted
for 35 cycles with primers 398F (SEQ ID NO:1) and 1086R (SEQ ID
NO:5) (FIG. 4B). The 1.sup.st round PCR products were then diluted
at 1:50, and subjected to two separate 2.sup.nd round nested PCR
analysis. The nested primer set of 398F/v671R (SEQ ID NO:1/SEQ ID
NO:3) produced a 274 bp product shown in FIG. 4C. The nested primer
set of v533F/811R (SEQ ID NO:2/SEQ ID NO:4) produced a 453 bp
product shown in FIG. 4D. As the 1.sup.st-round primers 398F (SEQ
ID NO:1) and 1086R (SEQ ID NO:5) were overlapped with the sequences
that were potentially common to both syndecan-2 reference
transcript (Syn2-wt) and splice variant Syn2-vE1a, they might
amplify both Syn2-wt and Syn2-vE1a, with expected sizes of 689 bp
and 845 bp, respectively (FIG. 4B). Although levels of Syn2-vE1a
appeared to be low or below the detection limit in the 1.sup.st
round PCR (FIG. 4B), the nested PCR specifically enriched the
Syn2-vE1a amplicons to the levels that were sufficient for DNA
sequencing (FIGS. 4C&D). In addition, because the 1.sup.st
round PCR was amplified with primer set that embraces all six exons
including exon E1a, the splice variant Syn2-vE1a may also contain
all those exons present in the Syn2-wt transcript in addition to
the E1a insertion.
Example 3
DNA Sequence Analysis of Splice Variant Syn2-vE1a
[0066] To determine the DNA sequence of Syn2-vE1a, we purified the
274 bp and 453 bp cDNA bands (FIGS. 4C&D) with a gel extraction
kit (QIAGEN, and performed DNA sequencing on the purified samples
with both forward and reverse primers using a commercial DNA
sequencing facility. The DNA sequencing results confirmed that the
sequences of these major PCR products were identical to those
predicted sequences derived from the bioinformatic analysis. The
amino acid sequence of splice variant Syn2-vE1a is shown in FIG. 5
(SEQ ID NO:7). We have confirmed that Syn2-vE1a contains an
in-frame 52 amino acid residue insertion that codes for four extra
SG sites, including a prominent repeating SGSG sequence flanked by
acidic residues. Such motifs are most likely to serve as attachment
sites for glycosaminoglycan side chains on a heparan sulphate
proteoglycan core protein (Zhang L et al. J Biol. Chem. 270:27127,
1995).
Example 4
Comparison of Relative Levels of Splice Variant Syn2-vE1a RNA in
Brain
[0067] To determine relative levels of Syn2-vE1a mRNA in AD vs.
control brain tissues, we performed semi-quantitative PCR analysis
using unpooled single strand cDNA samples reverse transcribed from
RNA isolated from middle temporal cortex of late onset AD patients,
and age-matched non-demented controls (FIG. 6). The 398F/v671R (SEQ
ID NO:1/SEQ ID NO:3) primer set was used for PCR analysis of
Syn2-vE1a with 35-cycle amplification (FIG. 6A). As a control, a
25-cycle PCR amplification of .beta.-actin was also performed in
parallel (FIG. 6B). The PCR products were resolved on 2% agarose
gels. DNA band images were digitally documented, and quantified
with ScionImage software. Relative levels of Syn2-vE1a were
normalized to those of .beta.-actin for potential variations due to
sample loading and PCR amplification efficiency. Consistent with
our previous results, the preliminary quantitative results showed
that expression of Syn2-vE1a appeared to be up-regulated in AD
brain tissues in the majority of samples. The results indicate that
increased expression of Syn2-vE1a coincides with development of
AD.
Example 5
Cloning of Syndecan-2 Variant E1a (Syn2vE1a) into a Mammalian
Expression Vector
[0068] Total RNA was isolated from human adult non-demented frontal
tissues obtained at autopsy from the University of Washington ADRC
Brain Bank and immediately frozen at 80.degree. C. Single stranded
cDNA was synthesized using M-MLV Reverse Transcriptase (Invitrogen;
Carlsbad, Calif., USA) and random priming with hexameric primers
(Invitrogen). All other primers used were also synthesized by
Invitrogen. Mammalian expression constructs, pcDNA3.1-Syn2WT and
pcDNA3.1-Syn2vE1a, were generated as follows. pcDNA3.1-Syn2WT
contains the cDNA sequence coding for the human full-length
syndecan-2 (REFSEQ mRNA: NM.sub.--002998.3). pcDNA3.1-Syn2vE1a
contains the cDNA sequence coding for the human full-length
Syn2vE1a, (SEQ ID NO: 6). Both cDNA inserts were amplified from
human brain single stranded cDNA by PCR with a forward primer,
5'CAGGAGGCTTCGTTTTGC (Synd398-F, SEQ ID NO: 1), and a reverse
primer, 5'TAGAGACACTAAGTTGGAG (Synd1086-R, SEQ ID NO: 5). The PCR
products were then cloned into a pDrive-UA cloning vector (QIAGEN;
Valencia, Calif., USA) as instructed by the manufacturer to
generate pDrive-Syn2WT, and pDrive-Syn2vE1a, respectively. The
Syn2WT and Syn2vE1a inserts were then released by EcoRI digestion
of pDrive-Syn2WT and pDrive-Syn2vE1a, gel-purified with a gel
extraction kit (QIAGEN) as instructed, and subcloned into a
pcDNA3.1 vector at EcoRI sites to generate pcDNA3.1-Syn2WT and
pcDNA3.1-Syn2vE1a, which are driven by a cytomegalovirus
immediate-early promoter. All inserted cDNA sequences were
confirmed by DNA sequencing.
[0069] Expression of these constructs were shown in FIG. 7, and
described in Example 7 below.
Example 6
Production of Polyclonal Antibodies Against Unique Amino Acid
Sequence in Syndecan-2 Variant E1a (Anti-Syn2vE1a)
[0070] The unique amino acid sequence of Syn2vE1a (SEQ ID NO:7) was
analyzed to determine which specific region would be useful for
custom peptide synthesis and the generation of polyclonal
antibodies (Invitrogen). Computer algorithms to determine the
immunogenicity of different peptide regions included the
Kyte/Doolittle model of hydrophilicity, and determinations of
peptide regions for indices of flexibility, protein surface
probability, amphiphilicity, and favorable secondary structure were
used. A segment of 16 amino acids corresponding to
"GIRRAPLYKRHPTGTA" (amino acids 50-65 of SEQ ID NO: 7) was picked
for antibody production due to overall computer index, favorable
secondary structure, peptide location, and posttranslational
modifications (not overlapped with potential GAG-attachment sites).
The region has shown no homology to other proteins using an
advanced-PBAST search on peptide sequences through the NCBI genome
database. Peptide synthesis, purification and site directed KLH
conjugation were performed by Invitrogen. For site directed KLH
conjugation the following peptide was synthesized and used to
immunize rabbits for polyclonal anti-peptide antibody production:
(C)GIRRAPLYKRHPTGTA-amide. The cysteine residue (C was employed for
single point, site-directed conjugation to KLH.
[0071] Two rabbits were immunized with the above peptide for
polyclonal antibody production. Rabbit pre-immune serum, and serum
obtained following peptide immunization were then tested by ELISA,
utilizing the specific peptide sequence described above. The ELISA
data indicated very good, peptide specific antibody titers (not
shown).
[0072] Eight-week post-immunization antisera from each rabbit was
tested by Western analysis for antibody specificity (as described
below). The terminal bleed from both rabbits was combined (70-80 ml
total), as both antisera showed similar specificity and affinity.
The antisera was affinity-purified by Invitrogen using epitope
peptide (described above) affinity chromatography. Purified
antibodies were dialyzed against 1.times.PBS, reconstituted at the
concentration of 1 mg/ml (containing no preservatives), were
aliquoted and stored at -80.degree. C.
Example 7--Specificity of Anti-Syn2Ve1a as Assessed by Western
Analysis
[0073] Specificity of polyclonal anti-Syn2vE1a antibodies were
tested by Western analysis of lysates of human HEK293E cell
cultures that were transiently transfected with pcDNA3.1,
pcDNA3.1-Syn2WT, and pcDNA3.1-Syn2vE1a.
[0074] Human Embryonic Kidney (HEK) 293E cells (CRL-10852; ATCC)
were cultured in a regular growth media (RGM) that contained
Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen) supplemented
with 10% fetal bovine serum at 37.degree. C. in a cell culture
incubator supplemented with 5% CO.sub.2. A 0.25% trypsin/0.03% EDTA
solution was used to release cells from culture dishes. For
transient transfection, HEK293E cells were grown to 80-90%
confluence in 6-well plates, and transfected with pcDNA3.1,
pcDNA3.1-Syn2WT, or pcDNA3.1-Syn2vE1a. Transfection was mediated by
polyethyleneimines (PEI)(Polysciences, Inc.) as described by Hu et
al. (J Biol. Chem. 2005, 280:12548). Three micrograms of plasmid
DNA and 15 .mu.l of PEI (1 mg/ml in H.sub.2O) were used. Eighteen
hours after transfection, cells were fed with fresh RGM.
Forty-eight hours after transfection cell lysates were collected
for Western analysis. Briefly, the cell monolayer was washed once
with PBS, and directly lysed in 200 .mu.l of 2.times. Laemmli
sample buffer (75 mM Tris-HCl, pH 8.4, 4% SDS, 20% glycerol, 50 mM
DTT, 0.004% bromphenol blue) and iced for 15 min. Lysates were
collected into a tube, boiled at 100.degree. C. for 10 min without
centrifugation, and stored at -80.degree. C. for Western
analysis.
[0075] For Western blotting, proteins in lysates were separated in
4-12% Bis/Tris Criterion XT gels (Bio-Rad; Hercules, Calif., USA),
with buffer systems recommended by the manufacturer. After
electrophoresis, proteins bands were transferred onto Immobilon-PSQ
membranes using Bio-Rad Criterion.TM. Blotters, and a corresponding
transfer buffer system (Bio-Rad). Transfer was conducted at 0.4 A
(constant) for 90-120 min. All transferred membranes were blocked
with 5% milk in PBS+0.05% Tween-20 for 30-60 min at room
temperature, and incubated with pre-immune serum (1:20,000), 8-week
anti-Syn2vE1a antisera (1:20,000), affinity purified anti-Syn2vE1a
antibodies (1:20,000), or a goat polyclonal antibody against
syndecan-2 (1:2,000) (sc-9492; Santa Cruz Biotechnology, Santa
Cruz, Calif., USA) overnight at 4.degree. C., and then with
HRP-conjugated secondary antibody (Vector) at 1:4000 at room
temperature for 2 h. Protein bands were visualized with an ECL
system (GE Healthcare) by exposing to autoradiography films. For
re-probing membranes with a different antibody, membranes were
stripped with Restore.TM. PLUS Western blot stripping buffer
(Thermo Scientific; Rockford, Ill., USA), and reprobed with the
next primary antibody. PhotoShop was used for image scanning and
processing. Quantitation of relative intensities of protein bands
on autoradiographic films was performed by image quantification
with the ScionImage software downloaded from
http://www.scioncorp.com.
[0076] For peptide pre-absorption, 20 .mu.l of crude antisera were
incubated with the same volume of 3 mg/ml of synthetic epitope
peptide provided by Invitrogen at 37.degree. C. for 2 hours,
followed by centrifugation at 12,000.times.g at 4.degree. C. for 15
min. The supernatant was used as peptide pre-absorbed antibodies.
For pre-absorbing purified antibodies, 100:1 (peptide:antibody)
molar ratio was used.
[0077] FIG. 7 shows the specificity of rabbit polyclonal
anti-Syn2vE1a antibodies assessed by Western analysis and epitope
peptide competition assays. Lysates (10 .mu.l per lane) of HEK293E
cell cultures without transfection (NT) or with transient
transfection of pcDNA3.1 (3.1), pcDNA3.1-Syn2WT (WT) or
pcDNA3.1-Syn2vE1a (E1a) were analyzed by Western analysis, and
probed with pre-immune serum (FIG. 7A; lanes 1-4), peptide
pre-absorbed 8-week anti-Syn2vE1a antisera (FIG. 7A; lanes 5-8),
and unabsorbed 8-week anti-Syn2vE1a antisera (FIG. 7A; lanes 9-14).
The results showed that the anti-Syn2vE1a antibody specifically
recognized proteins bands ranging from 37-250 kDa (Lanes 12-14;
FIG. 7A; lanes 13 and 14 were loaded with reduced amounts of
lysates, 3 and 1 .mu.l, respectively). Relatively discrete bands
were observed at 35 kDa and 70 kDa, which may represent monomers
and dimers of Syn2vE1a, respectively. It has been shown that the
syndecan family proteins, especially syndecan-2, form strong,
detergent-resistant dimers mediated by transmembrane domains (Dews
and Mackenzie, Proc Natl Acad Sci USA. 2007, 104:20782). The high
molecular-weight smear between 75-250 kDa likely represent
GAG-modified Syn2vE1a, as treatment with heparinases
I-III/chondroitinase ABC could partially remove the smear (not
shown). Importantly, the anti-Syn2vE1a antibody does not
cross-react with Syn2WT (lane 1, FIG. 7A). Expressions of the
Syn2WT and Syn2vE1a in the lysates were confirmed by Western
analysis with a goat anti-syndecan-2 antibody that recognizes both
Syn2WT and Syn2vE1a (FIG. 7B). Pre-immune serum did not detect any
specific protein bands (FIG. 7A; lanes 1-4). In addition, peptide
pre-absorption blocked more that 98% of anti-Syn2vE1a signals (Lane
8, FIG. 7A). Similar results were also seen with affinity-purified
anti-Syn2vE1a antibodies (not shown). Together, these results
indicate that anti-Syn2vE1a antibody specifically recognizes
Syn2vE1a, and does not react with Syn2WT.
Example 8
Syn2vE1a Selectively Reduces Secretions of Beta Cleavage Products
of APP in HEK293E Cell Cultures as Assessed by Western Analysis
[0078] Mammalian expression constructs, pcDNA3.1-APP695-myc, were
obtained from previous studies described by Yang et al. (J Biol.
Chem. 2006, 281:4207). pcDNA3.1-APP695-myc contains the cDNA
sequence coding for the human full-length APP695 that was inserted
at BamHI and EcoRI sites of a pcDNA3.1-myc/His vector (Invitrogen).
The vector is driven by a cytomegalovirus immediate-early
promoter.
[0079] HEK293E cell cultures grown in 6-well plates were
transiently co-transfected with pcDNA3.1-APP695-myc and pcDNA3.1,
or pcDNA-Syn2vE1a (1.5 .mu.g of each plasmid DNA). The transfection
was performed as described above. 48-hr post transfection,
conditioned media was collected, and centrifuged at 8000.times.g
for 10 min at 4.degree. C. to remove cell debris. Cell lysates were
also collected in 200 .mu.l of 2.times. Laemmli sample buffer.
Proteins in both lysates or conditioned media were separated in
4-12% Bis/Tris Criterion XT gels (Bio-Rad), and incubated with
primary antibodies for overnight at 4.degree. C., and with
HRP-conjugated secondary antibody (Vector) at 1:4000 at room
temperature for 2 h. Membranes were probed for APP with a rabbit
polyclonal antibody specifically recognizing the C-terminus of
APP695 (amino acids 676-695; Sigma) at 1:50,000, sAPP.alpha. with
mAb 6E10 at 1:20,000 (Covance), sAPP.beta. with a polyclonal
antibody specific for secreted APP.beta. at 1:5000 (Covance),
.beta.-actin with mAb C4 at 1:200,000 (Sigma), and Syn2vE1a with
anti-Syn2vE1a antibody at 1:20,000.
[0080] FIG. 8A shows that co-expression of APP695 with Syn2vE1a
reduces levels of secreted APP.beta. (sAPP.beta.), a
.beta.-cleavage product of APP, in conditioned media of HEK293E
cell cultures when compared to co-transfection with pcDNA3.1
(Vector), as assessed by Western analysis. Quantitative
densitometry analysis of Western blots revealed a 62% reduction in
sAPP.beta. levels (p<0.001) (FIG. 8B). In contrast,
co-transfaction with Syn2vE1a did not significantly alter levels of
secreted APP.alpha. (sAPP.alpha.), an .alpha.-cleavage product of
APP, and cellular APP (APP) (FIG. 8A-B). The results indicate that
Syn2vE1a may selectively affect the .beta.-cleavage pathway of
APP.
Example 9
Immunolocalization of the Syn2vE1a to the Neurofibrillary Tangles
of Alzheimer's Disease
[0081] Polyclonal anti-Syn2vE1a was then used to immunolocalize the
syndecan-2 variant in brains of patients with Alzheimer's disease.
Hippocampal sections from an autopsy-confirmed Alzheimer's disease
brain obtained from the University of Washington ADRC were
utilized. From paraffin embedded material, 6-8 .mu.M serial
sections were cut and placed on gelatin coated slides. Amyloid
containing plaques and neurofibrillary tangles were identified
following Congo red staining (Puchtler et al, Appl Pathol. 3:5-17,
1985) when viewed under polarized light. Detection of Syn2vE1a was
achieved using purified anti-Syn2vE1a antibody at a dilution of
1:1000 (FIGS. 9A, 10.times. magnification; 9B-C, 20.times.
magnification). Controls consisted of staining an adjacent serial
section with no primary antibody (FIG. 9E, 20.times.
magnification), or with the purified anti-Syn2vE1a antibody
pre-reabsorbed with excess (100.times. molar ratios) epitope
peptide (FIG. 9D, 20.times. magnification) as described above.
Immunostaining of tissue sections was accomplished using the avidin
biotin complex (Hsu et al, J. Histochem. Cytochem. 29:577 580,
1981). For immunocytochemical staining the primary antibody was
tested at different dilutions to obtain the best specificity with
the least background staining. Only the optimal dilutions of
primary antibody are reported.
[0082] Congo red staining in these tissue sections had previously
revealed numerous amyloid plaques and neurofibrillary tangles when
stained with Congo red and viewed under polarized light (not
shown). The Syn2vE1a antibody revealed staining of pyramidal
neurons (FIGS. 9A-B), positive for ghost tangles and intraneuronal
tangles which were identified under birefrigent light (FIG. 9C;
this is an overlapping image of FIG. 9B viewed under birefrigent
lights) or by positive Congo red staining on adjacent serial
sections (not shown). Preabsorption experiments completely
eliminated any positive immunostaining, indicating the specificity
of the antibody used (FIG. 9D). In addition, sections from
Alzheimer's disease brains immunostained with preimmune serum did
not show any positive immunostaining of neurofibrillary tangles
(not shown). This study therefore demonstrated that in Alzheimer's
disease brain the syndecan-2 variant E1a (Syn2vE1a) was localized
specifically with the neurofibrillary tangles present in brain.
Peptides, Amino Acids and GAGs
[0083] The polypeptides referred to in the present invention may be
a natural polypeptide, a synthetic polypeptide or a recombinant
polypeptide. The fragments, derivatives or analogs of the
polypeptides to any of the syndecan-2 splice variants referred to
herein may be a) one in which one or more of the amino acid
residues are substituted with a conserved or non-conserved amino
acid residue and such substituted amino acid residue may or may not
be encoded by the genetic code, or b) one in which one or more of
the amino acid residues includes a substituent group, or c) one in
which the mature polypeptide is fused with another compound, such
as a compound used to increase the half-life of the polypeptide
(for example, polyethylene glycol), or d) one in which the
additional amino acids are fused to the mature polypeptide, such as
a leader or secretory sequence or a sequence which is employed for
purification of the mature polypeptide or a proprotein sequence.
Such fragments, derivatives and analogs are deemed to be within the
scope of the invention.
[0084] The polypeptides of the present invention include the
polypeptides or fragments therein contained within the deduced
amino acid sequences of syndecan-2 splice variant as shown in the
sequence listing, as well as polypeptides which have at least 70%
similarity (preferably 70% identity) and more preferably a 90%
similarity (more preferably a 90% identity) to the polypeptides
described above.
[0085] Fragments or portions of the polypeptides of the present
invention may be employed for producing the corresponding
full-length polypeptides by peptide synthesis; therefore, the
fragments may be employed as intermediates for producing the full
length polypeptides. Fragments of portions of the polynucleotides
of the present invention may be used to synthesize full-length
polynucleotides of the present invention.
[0086] The polypeptides of the present invention may be a naturally
purified product, or a product of chemical synthetic procedures, or
produced by recombinant techniques from a prokaryotic or eukaryotic
host (for example, by bacterial, yeast, higher plant, insect and
mammalian cells in culture). Depending upon the host employed in a
recombinant procedure, the polypeptides of the present invention
may be glycosylated or may be non-glycosylated. Polypeptides of the
invention may also include an initial methionine amino acid
residue.
[0087] Syndecan-2 polypeptides of the present invention of can be
synthesized according to known method steps, including portions of
disclosed new syndecan-2 polypeptides, conservative substitution
derivatives thereof or functional derivatives thereof.
[0088] Chemical polypeptide synthesis is a rapidly evolving area in
the art, and methods of solid phase polypeptide synthesis are
well-described in the following references, hereby entirely
incorporated by reference (Merrifield, J. Amer. Chem. Soc.
85:2149-2154, 1963; Merrifield, Science 232:341-347, 1986; Fields,
Int. J. Polypeptide Prot. Res. 35, 161, 1990).
[0089] Recombinant production of Syndecan-2 polypeptide can be
accomplished according to known method steps. Standard reference
works setting forth the general principles of recombinant DNA
technology include Watson, Molecular Biology of the Gene, Volumes I
and II, The Benjamin/Cummings Publishing Company Inc., publisher,
Menlo Park, Calif. 1987; Ausubel et al, eds., Current Protocols in
Molecular Biology, Wiley Interscience, publisher, New York, N.Y.
1987; 1992; and Sambrook et al, Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor Laboratory, publisher,
Cold Spring Harbor, N.Y. 1989, the entire contents of which
references are herein incorporated by reference.
[0090] The polypeptides of the present invention may be utilized as
research reagents and materials for discovery of treatments and
diagnostics for human diseases.
Diagnostic Applications--Use of Primers and/or Nucleic Acids
[0091] The invention provides in one aspect methods of diagnosis of
amyloidosis, which method comprises analyzing the expression of the
Syndecan-2 splice variants in a sample. In a particular embodiment,
the invention provides methods of assaying a sample for splice
variants of Syndecan-2 which method comprises, making cDNA from
messenger RNA (mRNA) in the sample, amplifying portions of the
complementary DNA (cDNA) corresponding to the Syndecan-2 splice
variant and detecting the amplified cDNA, characterized in that the
amplified cDNA is used in the diagnosis and to monitor the
prognosis of the amyloidoses. The sample on which the assay is
performed is preferably of body tissue or body fluid. The sample
may be a piece of tissue obtained by biopsy, or a fine needle
aspirate of cells. Alternatively, it may be a sample of blood or
urine or another body fluid, such as a cervical scraping or a
non-invasively obtained sample such as sputum, urine or stool.
[0092] The primers described can be utilized for the specific
detection of Syndecan-2 splice variants in RNA derived from
tissues, cells, and/or cells in biological fluids in human tissues
using standard RT-PCR methodology, knowledgeable to one skilled in
the art.
[0093] In addition, the primers can be used for quantitative
competitive RT-PCR to determine the quantitative differences in
these specific Syndecan-2 variants in total RNA derived from human
tissues, cells, white blood cells and/or cells in biological
fluids. Changes in quantitative levels of these Syndecan-2 splice
variants will aid in the diagnosis and prognosis of patients who
demonstrate amyloid and concurrent Syndecan-2 splice variant
accumulation in tissues as part of the pathological process in the
amyloid diseases. In a preferred embodiment, specific primers are
utilized (as described above) for quantitative RT-PCR to determine
levels of specific Syndecan-2 splice variants in patients with an
amyloid disease in comparison to age-matched controls. The specific
syndecan-2 splice variants which are determined to be significantly
elevated or diminished in tissues, cells and/or cells in biological
fluids in a type of amyloidosis will aid in the diagnosis and
monitoring of the prognosis of a given patient afflicted with a
particular amyloid disease. Elevated or diminished levels of a
particular Syndecan-2 splice variant will be indicative of
Syndecan-2 splice variant deposition, accumulation and/or
persistence which will correlate with amyloid deposition,
accumulation and/or persistence in a given patient. Increasing
elevations of a particular Syndecan-2 splice variant in a biopsy or
biological fluid sample obtained from a patient at regular
intervals (ie. every 6 months) may suggest continued deposition and
accumulation of this syndecan-2 splice variant in conjunction with
amyloid, implicating a worsening of the disease. Such diagnostic
assays as described above may be produced in a kit form.
[0094] This invention is also related to the use of the Syndecan-2
splice variant gene as a diagnostic. Detection of a mutated form of
Syndecan-2 splice variants will allow diagnosis of a disease or a
susceptibility to a disease which results from overexpression or
underexpression of Syndecan-2 splice variants. Individuals carrying
mutations in the human Syndecan-2 splice variant gene may be
detected at the DNA level by a variety of techniques. Nucleic acids
for diagnosis may be obtained from a patient's cells, from blood,
urine, saliva, tissue biopsy, stool and autopsy material. The
genomic DNA may be used directly for detection or may be amplified
enzymatically by using PCR prior to analysis. RNA or cDNA may also
be used for the same purpose. As an example, PCR primers
complementary to the nucleic acids encoding the Syndecan-2 splice
variants can be used to identify and analyze mutations. For
example, deletions and insertions can be detected by a change in
size of the amplified product in comparison to the normal genotype.
Point mutations can be identified by hybridizing amplified DNA to
radiolabeled Syndecan-2 splice variant RNA or alternatively,
radiolabeled Syndecan-2 splice variant antisense DNA sequences.
Perfectly matched sequences can be distinguished from mismatched
duplexes by RNase A digestion or by differences in melting
temperatures.
[0095] Sequencing differences between the reference gene and genes
having mutations may be revealed by the direct DNA sequencing
method. In addition, cloned DNA segments may be employed as probes
to detect specific DNA segments. The sensitivity of this method is
greatly enhanced when combined with PCR. For example, a sequencing
primer is used with double-stranded PCR product or a
single-stranded template molecule generated by a modified PCR. The
sequence determination is performed by conventional procedures with
radiolabeled nucleotide or by automatic sequencing procedures with
fluorescent-tags. Genetic testing based on DNA sequence differences
may be achieved by detection of alteration in electrophoretic
mobility of DNA fragments in gels with or without denaturing
agents. Small sequence deletions and insertions can be visualized
by high resolution gel electrophoresis. DNA fragments of different
sequences may be distinguished on denaturing formamide gradient
gels in which the mobilities of different DNA fragments are
retarded in the gel at different positions according to their
specific melting or partial melting temperatures (Myers et al,
Science 230:1242, 1985). Sequence changes at specific locations may
also be revealed by nuclease protection assays, such as RNase and
S1 protection or the chemical cleavage method (e.g., Cotton et al,
Proc. Natl. Acad. Sci. U.S.A, 85:4397-4401, 1985). Therefore, the
detection of a specific DNA sequence may be achieved by methods
such as hybridization, RNase protection, chemical cleavage, direct
DNA sequencing or the use of restriction enzymes (e.g., Restriction
Fragment Length Polymorphisms (RFLP)) and Southern blotting of
genomic DNA. In addition to more conventional gel-electrophoresis
and DNA sequencing, mutations can also be detected by in situ
analysis.
[0096] Yet another aspect of the invention is to make
oligonucleotides utilizing the nucleotide sequences described
herein, to be utilized as new molecular biological probes to detect
Syndecan-2 splice variants in human tissues by standard in situ
hybridization techniques, knowledgeable by one skilled in the art.
In a preferred embodiment, this includes the utilization of the
nucleic acid sequences described
[0097] Yet another aspect of the present invention is to provide a
method which can evaluate a compound's ability to alter (diminish
or eliminate) the affinity of a given amyloid protein (as described
herein) or amyloid precursor protein, to syndecan-2 splice variant
protein or syndecan-2 splice variant-derived GAGs. By providing a
method of identifying compounds which affect the binding of amyloid
proteins, or amyloid precursor proteins to such syndecan-2 splice
variant protein or syndecan-2 splice variant derived-GAGs or
fragments thereof, the present invention is also useful in
identifying compounds which can prevent or impair such binding
interaction. Thus, compounds can be identified which specifically
affect an event linked with the amyloid formation, amyloid
deposition, and/or amyloid persistence condition associated with
Alzheimer's disease and other amyloid diseases as described
herein.
[0098] In the case in which the amyloid is immobilized, it is
contacted with fee syndecan-2 splice variant polypeptides,
syndecan-2 splice variant derived-GAGs or fragments thereof, in the
presence of a series of concentrations of test compound. As a
control, immobilized amyloid is contacted with free syndecan-2
splice variant polypeptides, syndecan-2 splice variant
derived-GAGs, or fragments thereof in the absence of the test
compound. Using a series of concentrations of syndecan-2 splice
variant polypeptides, syndecan-2 spice variant derived-GAGs or
fragments thereof, the dissociation constant (K.sub.d) or other
indication of binding affinity of amyloid-syndecan-2 splice variant
binding can be determined. In the assay, after the syndecan-2
splice variant polypeptides, syndecan-2 splice variant
derived-GAGs, or fragments thereof is placed in contact with the
immobilized amyloid for a sufficient time to allow binding, the
unbound syndecan-2 splice variant is removed. Subsequently, the
level of syndecan-2 splice variant-amyloid binding can be observed.
This information is used to determine first qualitatively whether
or not the test compound can prevent or reduce binding between
syndecan-2 splice variant and amyloid. Secondly, the data collected
from assays performed using a series of test compound at various
concentrations, can be used to measure quantitatively the binding
affinity of the syndecan-2 splice variant-amyloid complex and
thereby determine the effect of the test compound on the affinity
between syndecan-2 splice variant an amyloid. Using this
information, compounds can be identified which modulate the binding
of syndecan-2 splice variant to amyloid and thereby prevent or
reduce the amyloid formation, deposition, accumulation and/or
persistence, and the subsequent development and persistence of
amyloidosis.
Therapeutic Applications--Use of Primers and/or Nucleic Acids
[0099] Another aspect of the present invention is to provide a
potential therapeutic using antisense technology. Antisense
technology can be used to control gene expression through
triple-helix formation or antisense DNA or RNA, both of which
methods are based on the binding of a polynucleotide to DNA or RNA.
For example, the 5' coding portion of the polynucleotide sequence,
which encodes for the mature polypeptides of the present invention
is used to design an antisense RNA oligonucleotide of from about 10
to 40 base pairs in length. A DNA oligonucleotide is designed to be
complementary to a region of the gene involved in transcription
(Lee et al, Nucleic Acids Res. 6:3073, 1979; Cooney et al, Science
241:456, 1988; Dervan et al, Science 251:1360, 1991), thereby
preventing transcription by steric blocking and hence the
production of syndecan-2 splice variants. The antisense RNA
oligonucleotide hybridizes to the mRNA in vivo and blocks
translation of the mRNA molecule into syndecan-2 splice variants
(Okano, J. Neurochem. 56:560, 1991). The oligonucleotides described
above can also be delivered to cells such that the antisense RNA or
DNA may be expressed in vivo to inhibit production of the
syndecan-2 splice variants.
[0100] Alternatively, RNA interference (RNAi) may be utilized to
inhibit gene expression via the micro RNA (miRNA) or small
interfering RNA (siRNA) pathways. (Song, E et al., Nature
Med:347-351, 2003; de Fougerolles, A., et al., Nature Reviews Drug
Discovery 6:443-453, 2007; Iorns, E., Nature Reviews Drug Discovery
6:556-568, 2007; and Hammond, S. M., et al., Nature Reviews
Genetics 2:110-119, 2001).
[0101] The syndecan-2 splice variant polypeptides of the present
invention and antagonists which are polypeptides may also be
employed in accordance with the present invention by expression of
such polypeptides in vivo which is often referred to as "gene
therapy". For example, cells from a patient may be engineered with
a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with
the engineered cells then being provided to the patient to be
treated with the polypeptide. Such methods are well known in the
art. For example, cells may be engineered by procedures known in
the art by use of a retroviral particle containing RNA encoding a
polypeptide of the present invention.
[0102] Preparations of syndecan-2 splice variant polypeptides for
parenteral administration include sterile aqueous or non-aqueous
solutions, suspensions, and emulsions, which may contain auxillary
agents or excipients which are known in the art. Pharmaceutical
compositions such as tablets, pills, tablets, caplets, soft and
hard gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid
drops, elixers, suspensions, emulsions, solutions, syrups, tea
bags, aerosols (as a solid or in a liquid medium), suppositories,
sterile injectable solutions, sterile packaged powders, can be
prepared according to routine methods and are known in the art.
[0103] For example, administration of such a composition may be by
various parenteral routes such as subcutaneous, intravenous,
intradermal, intramusclular, intraperitoneal, intranasal,
transdermal or buccal routes. Alternatively, or concurrently,
administration may be by the oral route. Parenteral administration
can be by bolus injection or by gradual perfusion over time.
[0104] A preferred mode of using a syndecan-2 splice variant
polypeptides, or pharmaceutical composition of the present
invention is by oral administration or intravenous application.
[0105] A typical regimen for preventing, surpressing or treating
syndecan-2 splice variant-related pathologies, such as comprises
administration of an effective amount of a syndecan-2 splice
variant polypeptide, administered over a period of one or several
days, up to and including between one week and about 24 months.
[0106] It is understood that the dosage of the syndecan-2 splice
variant polypeptide of the present invention adminstered in vivo or
in vitro will be dependent upon the age, sex, health, and weight of
the recipient, kind of concurrent treatment, if any, frequency of
treatment, and the nature of the effect desired. The most preferred
dosage will be tailored to the individual subject, as is understood
and determinable by one of skill in the art, without undue
experimentation.
[0107] The total dose required for each treatment may be
administered by multiple doses or in a single dose. A syndecan-2
splice variant polypeptide may be adminstered alone or in
conjunction with other therapeutics directed to syndecan-2 splice
variant-related pathologies, such as Alzheimer's disease or amyloid
diseases.
[0108] Effective amounts of a syndecan-2 splice variant polypeptide
or composition are about 0.01 g to about 100 mg/kg body weight, and
preferably from about 10 .mu.g to about 50 mg/kg body weight, such
as 0.05, 0.07, 0.09, 0.1, 0.5, 0.7, 0.9, 1, 2, 5, 10, 20, 25, 30,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg/kg.
[0109] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions, which
may contain auxillary agents or excipients which are known in the
art. Pharmaceutical compositions comprising at least one syndecan-2
splice variant polypeptide, such as 1-10 or 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10 syndecan-2 splice variant polypeptides, of the present
invention may include all compositions wherein the syndecan-2
splice variant polypeptide is contained in an amount effective to
acheive its intended purpose. In addition to at least one
syndecan-2 splice variant polypeptide, a pharmaceutical composition
may contain suitable pharmaceutically acceptable carriers, such as
excipients, carriers and/or auxiliaries which facilitate processing
of the active compounds into preparations which can be used
pharmaceutically.
[0110] Pharmaceutical compositions comprising at least one
syndecan-2 splice variant polypeptide may also include suitable
solutions for administration intravenously, subcutaneously,
dermally, orally, mucosally, rectally or may by injection or
orally, and contain from about 0.01 to 99 percent, preferably about
20 to 75 percent of active component (i.e. polypeptide) together
with the excipient. Pharmaceutical compositions for oral
administration include pills, tablets, caplets, soft and hard
gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid
drops, elixers, suspensions, emulsions, solutions, and syrups.
Use of Syndecan-2 Splice Variants for Production of New Animal
Models--Infusion Models for Alzheimer's Disease and Down's Syndrome
Amyloidosis
[0111] The production of each of the syndecan-2 splice variants in
sufficient quantities can also be utilized to produce new animal
models of the amyloidoses. For purposes of this application,
syndecan-2 splice variants can refer to a) syndecan-2 splice
variants which contain both core protein and attached GAG chains,
b) syndecan-2 core protein only, or c) syndecan-2 GAG chains
derived from syndecan-2 splice variants, or any fragments or
combinations of any of the above. For example, as a new model of
Alzheimer's disease amyloidosis, syndecan-2 splice variants can be
continuously infused in combination with beta-amyloid protein
(A.beta.) into the hippocampus of groups of rats or mice. In a
preferred embodiment syndecan-2 splice variant (25 .mu.g) is
dissolved in water in a microcentrifuge tube containing 50 .mu.g of
A.beta. (1-40) or (1-42). Using the described methods of Snow et al
(Neuron 12:219-234, 1994) herewith incorporated by reference, the
syndecan-2 splice variant+A.beta. is continuously infused for 1
week into hippocampus (via stereotaxic surgeries knowledgeable by
one skilled in the art) of groups (usually 10) of 3 month old
Sprague-Dawley rats. Following the 1 week infusion the animals are
sacrificed and the brains are removed as described in Snow et al
(Neuron 12:219-234, 1994), and 6-8 .mu.m serial sections spanning
through the entire infusion site are cut from paraffin embedded
blocks or from frozen sections. The extent of amyloid deposition
per animal is then detected by Congo red staining (as viewed under
polarized light) or Thioflavin S fluorescence and quantitated in a
blind study using an arbitrary scoring method as described by Snow
et al (Neuron 12:219-234, 1994). The use of the syndecan-2 splice
variant peptides and/or proteins in this model can be used as a
rapid model of fibrillar A.beta. amyloid deposition, accumulation
and persistence in vivo. In addition, this model may be used to
rapidly screen potential therapeutics targeting fibrillar A.beta.
amyloid formation, deposition, accumulation and/or persistence. In
a preferred embodiment, syndecan-2 splice
variant+A.beta.+therapeutic compound is directly infused into the
hippocampus (as described above) of a group of animals and
comparisons are made to a group of animals infused with only
syndecan-2 splice variant+A.beta.. Compounds or drugs found to
reduce amyloid formation, deposition, accumulation and/or
persistence (as determined by Congo red or Thioflavin S scoring) in
vivo are then identified as having potential therapeutic value.
[0112] In another preferred embodiment, the potentially therapeutic
compound can be tested to reduce amyloid persistence over prolonged
periods of time. In this model, groups of animals (usually 10
animals per group) are infused with syndecan-2 splice
variant+A.beta.+therapeutic compound and directly compared to
groups of animals (usually 10 animals per group) infused with
syndecan-2 splice variant+A.beta.3. Following a 1 week infusion (as
described above), the cannulae are removed with the animals under
anesthesia, and the animals are then allowed to recover until
sacrifice 1, 3, 6 or 12 months later. Serial sections are cut and
amyloid is scored as described above. It is expected that
persistent amyloid deposits can be observed in animals infused with
the syndecan-2 splice variant+A.beta.. Potent therapeutic compounds
will be those that effectively reduce the amount of amyloid
observed in comparison to those animals not given the therapeutic
compound. These compounds can therefore be referred to as compounds
which effectively reduce amyloid persistence in vivo.
[0113] In yet another preferred embodiment, potentially therapeutic
compounds can be tested for reducing or eliminating pre-formed
amyloid deposits. In this model, two groups of animals (usually 10
animals per group) are infused with syndecan-2 splice
variant+A.beta.. Following a 1 week infusion (as described above),
the cannulae and osmotic pumps are changed (with the animals under
anesthesia), and a new cannulae connected by vinyl tubing to a new
osmotic pump, contains either vehicle only (ie. double distilled
water) or the potential therapeutic compound. Following a 1 week
continuous infusion of either the vehicle or the potential
therapeutic compound of interest, the animals are sacrificed.
Serial sections are then cut through the entire infusion site and
the extent of amyloid is measured by arbitrary blind scoring as
described above. Potent therapeutic compounds will be those that
are able to effectively remove pre-formed amyloid deposits. It is
anticipated that little to no reduction in the amount of amyloid
will be observed in the group of animals infused with vehicle only.
These compounds can therefore be referred to as therapeutic
compounds which effectively reduce pre-formed amyloid deposits in
vivo.
Syndecan-2 Splice Variant Transgenic Animals
[0114] In accordance with the disclosure of means and methods of
making transgenic animals, in particular transgenic mice, which
disclosure is found in U.S. patent application Ser. No. 08/870,987
by K. Fukuchi, A. Snow and J. Hassell, filed Jun. 6, 1997, and
which is hereby incorporated by this reference as if fully set
forth, another aspect of the invention is to produce new transgenic
animals that overexpress or knock-out a particular syndecan-2
splice variant in an effort to produce specific phenotypes
associated with a number of diseases and/or pathological processes.
For the production of these new syndecan-2 splice variant
transgenic animals, this would generally involve ligating the
splice variant cDNA sequence from the plasmid clones (described
herein) into the correct region of normal human syndecan-2 cDNA
(available in an expression vector with correct promoter and
enhancer regions as described in the incorporated reference above).
The syndecan-2 splice variant expression vector would then be
inserted into mouse eggs or embryonic stem cells and transgenic
mice would be produced through known, routine methods as described
in the incorporated reference above. Production of these transgenic
mice, and the mating of these mice with transgenic animals which
overexpress a given amyloid protein or its precursor protein, will
produce progeny that develop much or all of the phenotypic
pathology of a given amyloid disease. The production of new
transgenic animal models of amyloid diseases may be used as in vivo
screening tools to aid in the identification of lead therapeutics
for the amyloidoses and for the treatment of clinical
manifestations associated with these diseases (as described in the
incorporated reference above). The successful overproduction of
syndecan-2 splice variants in transfected cells also serves as a
new means to isolate these syndecan-2 splice variants which will
meet the increasing demands for use of syndecan-2 splice variants
for a variety of in vitro and in vivo assays.
[0115] All references cited are herein incorporated by reference.
Sequence CWU 1
1
9118DNAHomo sapiens 1caggaggctt cgttttgc 18218DNAHomo sapiens
2tcaagggaga ggacgcag 18319DNAHomo sapiens 3aaggtgccac tgatgttgg
19419DNAHomo sapiens 4ctgagtcaga gaggtgaac 19519DNAHomo sapiens
5tagagacact aagttggag 196891DNAHomo sapiens 6gcaggaggct tcgttttgcc
ctggttgcaa gcagcggctg ggagcagccg 50gtccctgggg aatatgcggc gcgcgtggat
cctgctcacc ttgggcttgg 100tggcctgcgt gtcggcggag tcgaacacaa
cggcggtcaa gggagaggac 150gcagaaagat caggctcagg cctgggtctt
gggcatcttc aaggaactcg 200ctttctcagt ggtataagaa gagccccact
ctataagagg catccgacag 250gaacagccaa catcagtggc accttccaaa
gagcagagct gacatctgat 300aaagacatgt accttgacaa cagctccatt
gaagaagctt caggagtgta 350tcctattgat gacgatgact acgcttctgc
gtctggctcg ggagctgatg 400aggatgtaga gagtccagag ctgacaacat
ctcgaccact tccaaagata 450ctgttgacta gtgctgctcc aaaagtggaa
accacgacgc tgaatataca 500gaacaagata cctgctcaga caaagtcacc
tgaagaaact gataaagaga 550aagttcacct ctctgactca gaaaggaaaa
tggacccagc cgaagaggat 600acaaatgtgt atactgagaa acactcagac
agtctgttta aacggacaga 650agtcctagca gctgtcattg ctggtggagt
tattggcttt ctctttgcaa 700tttttcttat cctgctgttg gtgtatcgca
tgagaaagaa ggatgaagga 750agctatgacc ttggagaacg caaaccatcc
agtgctgctt atcagaaggc 800acctactaag gagttttatg cgtaaaactc
caacttagtg tctctattta 850tgagatcact gaacttttca aaataaagct
tttgcataga a 8917253PRTHomo sapiens 7Met Arg Arg Ala Trp Ile Leu
Leu Thr Leu Gly Leu Val Ala Cys1 5 10 15Val Ser Ala Glu Ser Asn Thr
Thr Ala Val Lys Gly Glu Asp Ala 20 25 30Glu Arg Ser Gly Ser Gly Leu
Gly Leu Gly His Leu Gln Gly Thr 35 40 45Arg Phe Leu Ser Gly Ile Arg
Arg Ala Pro Leu Tyr Lys Arg His 50 55 60Pro Thr Gly Thr Ala Asn Ile
Ser Gly Thr Phe Gln Arg Ala Glu 65 70 75Leu Thr Ser Asp Lys Asp Met
Tyr Leu Asp Asn Ser Ser Ile Glu 80 85 90Glu Ala Ser Gly Val Tyr Pro
Ile Asp Asp Asp Asp Tyr Ala Ser 95 100 105Ala Ser Gly Ser Gly Ala
Asp Glu Asp Val Glu Ser Pro Glu Leu 110 115 120Thr Thr Ser Arg Pro
Leu Pro Lys Ile Leu Leu Thr Ser Ala Ala 125 130 135Pro Lys Val Glu
Thr Thr Thr Leu Asn Ile Gln Asn Lys Ile Pro 140 145 150Ala Gln Thr
Lys Ser Pro Glu Glu Thr Asp Lys Glu Lys Val His 155 160 165Leu Ser
Asp Ser Glu Arg Lys Met Asp Pro Ala Glu Glu Asp Thr 170 175 180Asn
Val Tyr Thr Glu Lys His Ser Asp Ser Leu Phe Lys Arg Thr 185 190
195Glu Val Leu Ala Ala Val Ile Ala Gly Gly Val Ile Gly Phe Leu 200
205 210Phe Ala Ile Phe Leu Ile Leu Leu Leu Val Tyr Arg Met Arg Lys
215 220 225Lys Asp Glu Gly Ser Tyr Asp Leu Gly Glu Arg Lys Pro Ser
Ser 230 235 240Ala Ala Tyr Gln Lys Ala Pro Thr Lys Glu Phe Tyr Ala
245 2508156DNAHomo sapiens 8aacacaacgg cggtcaaggg agaggacgca
gaaagatcag gctcaggcct 50gggtcttggg catcttcaag gaactcgctt tctcagtggt
ataagaagag 100ccccactcta taagaggcat ccgacaggaa cagccaacat
cagtggcacc 150ttccaa 156952PRTHomo sapiens 9Asn Thr Thr Ala Val Lys
Gly Glu Asp Ala Glu Arg Ser Gly Ser1 5 10 15Gly Leu Gly Leu Gly His
Leu Gln Gly Thr Arg Phe Leu Ser Gly 20 25 30Ile Arg Arg Ala Pro Leu
Tyr Lys Arg His Pro Thr Gly Thr Ala 35 40 45Asn Ile Ser Gly Thr Phe
Gln 50
* * * * *
References