U.S. patent application number 12/607049 was filed with the patent office on 2010-05-13 for soluble low-density lipoprotein receptor related protein binds directly to alzheimer's amyloid-beta peptide.
This patent application is currently assigned to The University of Rochester. Invention is credited to Rashid Deane, Berislav V. ZLOKOVIC.
Application Number | 20100119450 12/607049 |
Document ID | / |
Family ID | 35510196 |
Filed Date | 2010-05-13 |
United States Patent
Application |
20100119450 |
Kind Code |
A1 |
ZLOKOVIC; Berislav V. ; et
al. |
May 13, 2010 |
SOLUBLE LOW-DENSITY LIPOPROTEIN RECEPTOR RELATED PROTEIN BINDS
DIRECTLY TO ALZHEIMER'S AMYLOID-BETA PEPTIDE
Abstract
A soluble derivative of low-density lipoprotein receptor related
protein-1 (sLRP-1) binds directly to Alzheimer's amyloid-.beta.
peptide (A.beta.). This binding may be used to detect A.beta. or to
separate A.beta. from the rest of a subject's body. In Alzheimer's
disease, it may be used to provide diagnostic results by detecting
A.beta., treatment by removing A.beta., or both.
Inventors: |
ZLOKOVIC; Berislav V.;
(Rochester, NY) ; Deane; Rashid; (Rochester,
NY) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
The University of Rochester
New York
NY
Socratech L.L.C.
Los Angeles
CA
|
Family ID: |
35510196 |
Appl. No.: |
12/607049 |
Filed: |
October 27, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10558984 |
Dec 2, 2005 |
7608586 |
|
|
PCT/US04/19034 |
Jun 14, 2004 |
|
|
|
12607049 |
|
|
|
|
60477404 |
Jun 11, 2003 |
|
|
|
Current U.S.
Class: |
514/1.1 ;
435/325; 435/7.1 |
Current CPC
Class: |
G01N 33/92 20130101;
G01N 2800/2821 20130101; G01N 33/6896 20130101; C07K 14/4711
20130101; C07K 14/705 20130101; A61K 38/00 20130101 |
Class at
Publication: |
424/9.1 ; 514/2;
435/7.1; 435/325 |
International
Class: |
A61K 49/00 20060101
A61K049/00; A61K 38/00 20060101 A61K038/00; G01N 33/53 20060101
G01N033/53; C12N 5/00 20060101 C12N005/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH
[0002] The U.S. government has certain rights in this invention as
provided for by the terms of grants AG16223 and NS34467 awarded by
the National Institutes of Health.
Claims
1-17. (canceled)
18. A method of binding amyloid-.beta. peptide (A.beta.) in a body
fluid and/or tissue of a subject, said method comprising: (a)
administering a soluble derivative of low-density lipoprotein
receptor related protein-1 (LRP-1), wherein said soluble LRP-1
derivative is comprised of cluster II, cluster IV, or both and (b)
contacting said soluble LRP-1 derivative with at least said body
fluid and/or tissue of said subject such that said A.beta. is
specifically bound by said soluble LRP-1 derivative.
19. The method of claim 18, wherein said soluble LRP-1 derivative
binds said A.beta. inside said subject's body.
20. The method of claim 18, wherein said soluble LRP-1 derivative
binds said A.beta. outside said subject's body.
21. (canceled)
22. The method of claim 18, wherein soluble LRP-1 derivative bound
to AP is inactivated such that amyloid deposits are reduced in said
subject's body.
23-28. (canceled)
29. The method of claim 18, wherein said soluble LRP-1 derivative
is comprised of cluster II.
30. The method of claim 18, wherein said soluble LRP-1 derivative
is comprised of cluster IV.
31. The method of claim 18, wherein said soluble LRP-1 derivative
consists essentially of cluster II, cluster IV, or both.
32. The method of claim 18, wherein said soluble LRP-1 derivative
is comprised of at least one domain which mediates secretion.
33. The method of claim 18, wherein said soluble LRP-1 derivative
is not comprised of a domain which mediates attachment to a lipid
bilayer.
34. The method of claim 18, wherein said soluble LRP-1 derivative
is reversibly attached to a solid substrate.
35. The method of claim 18, wherein said soluble LRP-1 derivative
is irreversibly attached to a solid substrate.
36. The method of claim 18, wherein said soluble LRP-1 derivative
is derived from human.
37. The method of claim 18, wherein said soluble LRP-1 derivative
does not elicit an immune response in human.
38. The method of claim 18, wherein said soluble LRP-1 derivative
further comprises at least one heterologous domain.
39. The method of claim 18, wherein at least one detectable label
is covalently attached to said soluble LRP-1 derivative.
40. The method of claim 38, wherein at least one detectable label
is covalently attached to a heterologous domain of said soluble
LRP-1 derivative.
41. The method of claim 18, wherein said soluble LRP-1 derivative
is comprised of both cluster II and cluster IV.
42. A method of binding amyloid-.beta. peptide (A.beta.) in a body
fluid of a human subject, said method comprising: (a) administering
a soluble fragment of human low-density lipoprotein receptor
related protein-1 (LRP-1) comprising cluster II, cluster IV, or
both and (b) contacting said soluble LRP-1 fragment with at least
said body fluid of said human subject such that said A.beta. is
specifically bound by said soluble fragment of human LRP-1.
43. The method of claim 42, wherein soluble LRP-1 derivative bound
to A.beta. is inactivated such that amyloid deposits are reduced in
said human subject's body.
44. A method of binding amyloid-.beta. peptide (A.beta.) in a human
subject, said method comprising administering an effective amount
of a soluble fragment of human low-density lipoprotein receptor
related protein-1 (LRP-1) comprising cluster II, cluster IV, or
both such that said A.beta. is specifically bound by said soluble
fragment of human LRP-1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
10/558,984, filed Dec. 2, 2005, now U.S. Pat. No. 7,608,586; which
is a U.S. national phase under 35 U.S.C. 371 of Application No.
PCT/U.S. 2004/019034, filed Jun. 14, 2004; which claims the benefit
of provisional Application No. 60/477,404, filed Jun. 11, 2003; the
entire contents of which are incorporated by reference.
FIELD OF THE INVENTION
[0003] The invention relates to a soluble derivative of low-density
lipoprotein receptor related protein-1 (sLRP-1) and its direct
binding to Alzheimer's amyloid-.beta. peptide (A.beta.). This
binding may be used to detect A.beta. or to separate AB from the
rest of a subject's body. In Alzheimer's disease, the invention may
be used to provide diagnostic results by detecting A.beta.,
treatment by removing A.beta., or both.
BACKGROUND OF THE INVENTION
[0004] Amyloid-.beta. peptide (A.beta.) is central to the pathology
of Alzheimer's disease; it is the main constituent of brain
parenchymal and vascular amyloid. A.beta. extracted from senile
plaques contains mainly A.beta..sub.1-40 and A.beta..sub.1-42,
while vascular amyloid is mainly A.beta..sub.1-39 and
A.beta..sub.1-40. The major soluble form of A.beta. which is
present in the blood, cerebrospinal fluid (CSF), and brain is
A.beta..sub.1-40. Soluble A.beta. which is circulating in the
blood, CSF, and brain interstitial fluid (ISF) may exist as free
peptide and/or associated with different transport binding proteins
such as apolipoprotein E (apoE), apolipoprotein J (apoJ),
transthyretin, other lipoproteins, albumin, and
alpha2-macroglobulin (.alpha..sub.2M).
[0005] LRP-1 binds amyloid .beta.-peptide (A.beta.) precursor
protein (APP), apolipo-protein E (apoE) and
.alpha..sub.2-macroglobulin (.alpha..sub.2M), (Herz and Strickland,
2001). But the exact biochemical mechanism(s) by which LRP-1
contributes to the onset of neurotoxic A.beta. accumulations is
unclear. LRP-1 binds secreted APP and influences its degradation
(Kounnas et al., 1995) and processing (Pietrzik et al., 2002)
leading to increased A.beta. production (Ulery et al., 2000). It
also mediates endocytosis of .alpha..sub.2M-A.beta. complexes in
fibroblasts (Narita et al., 1997; Kang et al., 2000) and of
apoE-A.beta. and .alpha..sub.2M-A.beta. complexes in neurons in
vitro (Jordan et al., 1998; Qiu et al., 1999). Overexpression of
functional LRP-1 minireceptors in neurons of Alzheimer's PDAPP mice
results in an age-dependent increase of soluble A.beta. in the
brain (Zerbinatti et al., 2004), which suggests that LRP-1 on
neurons in vivo does not mediate A.beta. clearance from brain.
[0006] Peripheral A.beta. binding agents, e.g., an anti-A.beta.
antibody (DeMattos et al., 2002a), a soluble form of the receptor
for advanced glycation endproducts, sRAGE (Deane et al., 2003)
and/or ganglioside M1 and gelsolin (Matsuoka et al., 2003), rapidly
clear A.beta. from brain in vivo in various transgenic APP
over-expressing mice. The idea that LRP-1 along the brain capillary
membranes is a major clearance mechanism for A.beta. in vivo has
been supported by findings demonstrating that intracerebrally
infused A.beta..sub.1-40 undergoes rapid LRP-1-mediated
transcytosis across the blood-brain barrier (BBB) (Shibata et al.,
2000). Several questions, however, regarding a possible role of
LRP-1 (including A.beta..sub.1-40, A.beta..sub.1-42, and mutant
versions thereof) as a cargo/clearance receptor for brain A.beta.
remained unanswered. Whether A.beta. is a direct ligand for LRP-1
initiating its own efflux from brain through interaction with the
receptor at the BBB is not known. Reduced levels of LRP-1 in the
brain were found in AD (Kang et al., 1997; Kang et al., 2000;
Shibata et al., 2000). Whether high extracellular A.beta.
accumulations affect LRP-1 expression at the A.beta. clearance
site(s) in the brain is not known.
[0007] But it was not previously demonstrated that low-density
lipoprotein receptor related protein-1 (LRP-1) binds directly to
A.beta.. Cell surface receptors such as the receptor for advanced
glycation end products (RAGE), scavenger type A receptor (SR-A),
LRP-1, and low-density lipoprotein receptor related protein-2
(LRP-2) bind A.beta. at low nanomolar concentrations as free
peptide (e.g., RAGE, SR-A), and/or in complex with apoE, apoJ, or
.alpha..sub.2M (e.g., LRP-1,LRP-2). But it was not previously
demonstrated that a soluble derivative of LRP-1 is able to directly
bind A.beta. in a bimolecular interaction.
[0008] WO 01/90758 and U.S. patent application Ser. No. 10/296,168
describe LRP-1's role in mediating vascular clearance of A.beta.
from the brain. It was taught that increasing LRP-1 expression or
its activity can be used to remove A.beta. and thereby treat an
individual with Alzheimer's disease or at risk for developing the
disease. A direct interaction between LRP-1 and A.beta. was not
described, nor was it taught or suggested that the two molecules
are able to bind in solution without another ligand of LRP-1 such
as apoE, apoJ, .alpha..sub.2M, transthyretin, other lipoproteins,
albumin, or RAP.
[0009] Here, it is demonstrated that LRP-1 and A.beta. directly
interact with each other (i.e., the two molecules are sufficient by
themselves to specifically interact with each other) and this
interaction on brain capillary membranes regulates retention of
high .beta.-sheet content neurotoxic A.beta..sub.1-42 and
vasculotropic mutant A.beta. while clearing A.beta..sub.1-40. LRP-1
mediates differential efflux of amyloid .beta.-peptide isoforms
from brain. A.beta..sub.1-40 binds to an immobilized LRP-1 fragment
containing clusters II and IV with high affinity (Kd=0.6 nM to 1.2
nM) compared to A.beta..sub.1-42 and mutant A.beta.. LRP-mediated
A.beta. clearance and transport across the blood-brain barrier in
mice are substantially reduced by high .beta.-sheet content in
A.beta. and deletion of the receptor-associated protein gene.
Despite low A.beta. production in the brain, transgenic mice
expressing low LRP-1-clearance mutant A.beta. develop robust
A.beta. accumulations in the brain earlier than Tg-2576
A.beta.-over-producing mice. At pathological concentrations (>1
.mu.M), A.beta. promotes LRP-1 degradation in brain endothelium
consistent with reduced LRP-1 brain capillary levels observed in
A.beta.-accumulating transgenic mice, A.beta. and patients with
cerebrovascular .beta.-amyloidosis. Thus, low affinity
LRP-1/A.beta. interaction and/or loss of LRP-1 at the BBB mediate
brain accumulation of neurotoxic A.beta..
[0010] Receptor-associated proteins and receptor-mediated cell
signaling are not required. Deletion of the RAP gene (Van Uden et
al., 2002) which is associated with greatly reduced LRP-1
expression in the brain and at the BBB, but not deletion of the
genes for the VLDL receptor or the LDL receptor, almost completely
precluded rapid efflux of A from brain. Consistent with the
findings here, LRP-1 levels were substantially reduced in brain
microvessels in situ in a transgenic A.beta.-accumulating animal
model and patients with AD and cerebro-vascular
.beta.-amyloidosis.
[0011] New and nonobvious pharmaceutical and diagnostic
compositions, and methods of treatment and diagnosis are taught
herein to be applicable to the formation of amyloid and its role in
disease. Other advantages of the invention are discussed below or
would be apparent to a person skilled in the art from that
discussion.
SUMMARY OF THE INVENTION
[0012] A soluble derivative of low-density lipoprotein receptor
related protein-1 (sLRP-1) is provided in one embodiment of the
invention. The soluble LRP-1 derivative may be comprised of one or
more domains derived from LRP-1 and, optionally, one or more
domains not derived from LRP-1 (i.e., heterologous domains which do
not exist in the native protein). It is preferred that at least the
cluster II and/or cluster IV domain(s) is contained therein; it may
consist essentially of only cluster II and/or cluster IV domain(s).
The soluble LRP-1 derivative may or may not contain other optional
domains: a signal domain which directs secretion out of the cell
(e.g., a hydrophobic signal sequence which targets nascent
polypeptide to endoplasmic reticulum, translocates polypeptide
across the membrane, and transports polypeptide with any
modifications through the secretory pathway) and a domain which
attaches a polypeptide to a lipid bilayer (e.g., a transmembrane
domain for docking across or a lipid domain for insertion into the
membrane). The soluble LRP-1 derivative may be reversibly or
irreversibly attached to a solid substrate (e.g., using a covalent
bond which is chemically labile or stable, respectively). It is not
identical to native LRP-1 so one or more domains of the native
amino acid sequence must be mutated (e.g., substitution, addition,
deletion) to make the LRP-1 soluble and to retain its ability to
bind A.beta.. It is also preferred that human or another mammal be
used as the source, and an undetectable immune response be elicited
in the subject in whom the soluble LRP-1 derivative is administered
(e.g., derived from human or a humanized mammalian LRP-1 derivative
infused into a human subject).
[0013] The soluble LRP-1 derivative may be used in treatment as a
medicament (e.g., therapy in a subject having the disease or
prophylaxis in a subject at risk for developing the disease) or
diagnosis as an agent for detection of A.beta.. A therapeutic or
prophylactic composition is comprised of soluble LRP-1 derivative
and at least one pharmaceutically-acceptable carrier (e.g., a
solution of physiological salt and buffer). It may inactivate
A.beta. by removing A.beta. from the subject through the body's
circulatory systems or by machine, or by reducing deposition of
amyloid. A diagnostic composition is comprised of soluble LRP-1
derivative and at least one detectable label (e.g., a moiety for
chromatic, enzymatic, fluorescent, luminescent, magnetic or
paramagnetic, or radioactive detection). The soluble LRP-1
derivative and the detectable label may or may not be covalently
attached. Alternatively, they may be attached though one or more
specific binding pairs. Binding may occur inside or outside the
subject's body, in solution or with one of them immobilized on a
substrate. Soluble LRP-1 derivative bound to A.beta. may be
detected in a specimen prepared from a body fluid or tissue using
laboratory assay (i.e., in vitro diagnostics) or in the body by
fluoroscopic, magnetic resonance, or radiographic imaging (i.e., in
vivo diagnostics).
[0014] Further aspects of the invention will be apparent to a
person skilled in the art from the following detailed description
and claims, and generalizations thereto.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows surface plasmon resonance (SPR) analysis of the
interaction between A.beta. and recombinant LRP-1 fragments. FIG.
1A: A.beta..sub.1-40 (A.beta.40) and A.beta..sub.1-42 (A.beta.42)
binds to LRP-1 cluster II (LRP-1 II) which is immobilized on a CM5
chip at a density of 10 fmol/mm.sup.2 under the conditions
described below. Incubation conditions as described under Materials
and Methods. FIG. 1B: Peptide A.beta..sub.1-40 (A.beta.40),
A.beta..sub.1-42 (A.beta.42), or Dutch/Iowa mutant A.beta..sub.1-40
(DIA.beta.40) binds to immobilized LRP-1 cluster IV (LRP-1 IV)
under the same conditions. FIG. 1C: Kinetic parameters for the
binding of different A.beta. species to LRP-1 were plotted against
the content of .beta.-sheets in A.beta. peptides as determined by
circular dichroism analysis. Apparent affinity constants
K.sub.d(app) were deduced from the ratios of
k.sub.off(app)/k.sub.on(app) as described under Materials and
Methods. Mean.+-.SEM (n=3-5); SEM.ltoreq.5% of the mean in FIGS.
1A-1B. K.sub.d(app) (mean.+-.SD) values were determined from 6 to 9
different concentrations of A.beta. and 3 to 5 independent
measurements at each concentration. RAP, receptor-associated
protein (500 nM); RU, resonance units.
[0016] FIG. 2 shows LRP-1-mediated in vitro clearance of A.beta. by
mouse brain capillaries. FIG. 2A: Rapid saturable A.beta..sub.1-40
uptake on isolated brain capillaries was determined with
.sup.125I-A.beta..sub.1-40 as a ligand (1 nM) in the presence of
increasing concentrations of unlabeled A.beta. (1 nM to 120 nM) at
37.degree. C. within 1 min. A.beta..sub.1-40 brain capillary uptake
at 120 nM was completely inhibited by RAP (1 .mu.M) or
anti-LRP-1-specific N20 polyclonal antibody (.alpha.LRP-1, 60
.mu.g/ml), but not by a non-immune IgG (NI IgG, 60 .mu.g/ml). FIG.
2B: .sup.125I-A.beta..sub.1-40 uptake at 37.degree. C. on isolated
brain capillaries before (total) and after treatment with a cold
stop/strip 0.2 M acetic acid solution (mild acid wash) was blocked
by RAP (500 nM). FIG. 2C: Inhibitory constants K.sub.i for
LRP-mediated brain capillary clearance of A.beta. peptides were
determined using .sup.125I-A.beta..sub.1-40 (2 nM) as a ligand and
unlabeled peptide A.beta..sub.1-40 (A.beta.40), A.beta..sub.1-42
(A.beta.42), Dutch mutant A.beta..sub.1-40 (DA.beta.40), Dutch
mutant A.beta..sub.1-42 (DA.beta.42), or Dutch/Iowa mutant
A.beta..sub.1-40 (DIA.beta.40) at an inhibitory concentration of 40
nM. Ki values are plotted against .beta.-sheet content in A.beta.
determined by circular dichroism. Mean.+-.SEM (n=3-5).
[0017] FIG. 3 shows low LRP-1-mediated A.beta. clearance by brain
microvessels in RAP-null mice. FIG. 3A: LRP-1 levels in brain
capillaries isolated from wild-type and RAP-null (RAP-/-) mice was
determined by Western blot analysis using anti-LRP-1 .beta.-chain
specific IgG (5A6, LRP-85). Scanning densitometry of the intensity
of LRP-1 bands relative to .beta.-actin in wild-type (control, open
bar) and RAP-/- (closed bar) mice. FIG. 3B: LRP-1 and CD31
(endothelial cell marker) were localized in brain tissue sections
(scale bar=50 .mu.m) in wild-type (control) and RAP-null mice using
double immunostaining. FIG. 3C: LRP-1-positive vascular expression
profiles in different brain regions were determined in wild-type
mice (open bars) and RAP-null mice (closed bars). FIG. 3D: Reduced
brain capillary in vitro clearance of .sup.125I-labeled peptide
A.beta..sub.1-40 (A.beta.40), A.beta..sub.1-42 (A.beta.42), or
Dutch/Iowa mutant A.beta..sub.1-40 (DIA.beta.40); in wild-type mice
(open bars) and RAP-null mice (closed bars) was studied at 1 nM
peptide concentration. *P<0.01 RAP-null compared to controls;
anti-LRP-1-specific N20 polyclonal antibody (.alpha.LRP-1, 60
.mu.g/ml). Mean.+-.SEM (n=3-5).
[0018] FIG. 4 shows LRP-1-mediated transport of A.beta. across the
mouse blood-brain barrier (BBB) in vivo. FIG. 4A: LRP-1-mediated
clearance at the BBB of Dutch/Iowa mutant A.beta..sub.1-40
(DIA.beta.40, open points) was compared to wild-type
A.beta..sub.1-40 (A.beta.40, solid points) determined 30 min after
microinjection of radio-iodinated ligands (.sup.125I-A.beta.) in
brain ISF at different A.beta. carrier concentrations (1 nM to 120
nM). RAP (1 .mu.M), anti-LRP-1-specific N20 polyclonal antibody
(.alpha.LRP-1, 60 .mu.g/ml), or non-immune IgG (NI IgG, 60
.mu.g/ml; closed square). **P<0.01 and *P<0.05 for
DIA.beta..sub.1-40 vs. A.beta..sub.1-40. FIG. 4B: Elimination of
.sup.125I-labeled peptide A.beta..sub.1,.sub.40 (A.beta.40),
A.beta..sub.1-42 (A.beta.42), or Dutch/Iowa mutant A.beta..sub.1-40
(DIA.beta.40) from brain ISF via transport across the BBB was
studied at a carrier concentration of 40 nM in the absence or
presence of unlabeled peptide at an inhibittory concentration of
120 nM. Intact .sup.125I-labeled A.beta. monomers was determined by
HPLC analysis of brain homogenates 30 min after microinfusion in
brain ISF (insets above the bars). FIG. 4C: Inhibitory constants
K.sub.i for LRP-1-mediated efflux from brain via transport across
the BBB of peptide A.beta..sub.1-40 (A.beta.40), A.beta..sub.1-42
(A.beta.42), or Dutch/Iowa mutant A.beta..sub.1-40 (DIA.beta.40)
was determined with .sup.125I-A.beta..sub.1-40 at a carrier
concentration of 40 nM and unlabeled peptide at an inhibitory
concentration of 120 nM. K.sub.i values are plotted against
.beta.-sheet content in A.beta. determined by circular dichroism.
FIG. 4D: Peptide A.beta..sub.1-40 (A.beta.40) or A.beta..sub.1-42
(A.beta.42) does not exhibit rapid efflux across the BBB in RAP
null mice (RAP-/-; closed bars) as compared to wild-type mice
(control, open bars). Mean.+-.SEM (n=3-8).
[0019] FIG. 5 shows that A.beta. accumulated in transgenic mice
expressing low LRP-1-clearance mutant A.beta. vs. wild-type A.beta.
FIG. 5A: Human APP in the brain of transgenic mice expressing
mutant APP harboring both Dutch and Iowa mutations (Tg-DI mice) was
compared to Tg-2576 mice using immunoblot analysis. FIG. 5B: APP
levels in the brain of Tg-DI mice and Tg-2576 mice at 6 months of
age was determined by quantitative immunoblot analysis. FIG. 5C:
Brain accumulation of low LRP-1-clearance Dutch/Iowa mutant peptide
A.beta..sub.1-40 or A.beta..sub.1-42 (A.beta.40 and A.beta.42,
respectively; black bars) in Tg-DI mice was compared to wild-type
peptide A.beta..sub.1-40 or A.beta..sub.1-42 (A.beta.40 and
A.beta.42, respectively; gray bars) in Tg-2576 mice. FIG. 5D: Early
deposits of Dutch/Iowa mutant A.beta. in the brain of Tg-DI mice at
3 months of age, and abundant deposits at 12 months of age (bar=200
.mu.m) are shown. FIG. 5E: Intracerebral microvascular A.beta.
deposits in Tg-DI mice at 12 months of age was detected by
immunostaining for A.beta. (bar=50 .mu.m). FIG. 5F: LRP-1-positive
brain microvessels in Tg-DI DI mice (black bars) or Tg-2576 mice
(gray bars) were compared to controls (open bars) at 4-6 months of
age. FIG. 5G: LRP-1-positive microvessels in Tg-DI mice (black
bars), Tg-2576 mice (gray bars) and controls (open bars) are
compared at 12 months of age. Mean.+-.SEM (n=4 mice). *P<0.001;
**P<0.01 in FIG. 5B-5C for APP and A.beta. levels in Tg-DI mice
compared to Tg-2576 mice.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0020] Mature low-density lipoprotein receptor related protein-1
(LRP-1) is comprised of five different types of domains: (i)
ligand-binding cysteine-rich repeats, (ii) epidermal growth factor
(EGF) receptor-like cysteine-rich repeats, (iii) YWTD repeats, (iv)
transmembrane domain, and (v) cytoplasmic domain. The signal for
entry into the secretory pathway is cleaved after translocation.
Ligand-binding-type repeats in LRP-1 occur in clusters containing
between two and eleven repeats. Most of the known ligands for LRP-1
that have had their binding sites mapped interact with these
ligand-binding-type domains. They are followed by EGF precursor
homology domains, which are comprised of two EGF repeats, six YWTD
repeats arranged in a propeller-like structure, and another EGF
repeat. Six EGF repeats precede the transmembrane domain. The
cytoplasmic domain is comprised of two NPxY repeats that serve as
docking sites for the endocytosis machinery and for cytoplasmic
adaptor and scaffolding proteins which are involved in cell
signaling. The heavy chain of LRP-1 (515 kDa) contains four
ligand-binding domains (clusters I to IV) and the light chain of
LRP-1 (85 kDa) contains the transmembrane and cytoplasmic domains.
A soluble LRP-1 derivative may be comprised of only the heavy chain
or a fragment thereof.
[0021] LRP-1 recognizes at least 30 different ligands which
represent several families of proteins, which include lipoproteins,
proteinases, proteinase-inhibitor complexes, extracellular matrix
(ECM) proteins, bacterial toxins, viruses, and various other
intracellular proteins. By far the largest group of ligands that
are recognized by LRP-1 are either proteinases or molecules
associated with regulating proteolytic activity. Certain serine
proteinases and metalloproteinases bind directly to LRP-1, while a
number of other proteinases only bind once complexed with their
specific inhibitors. These inhibitors are only recognized by LRP-1
following a conformation change that occurs in them after
proteolytic cleavage or reaction with small amines. In contrast,
LRP-1 recognizes both the native and complexed forms of tissue
factor pathway inhibitor (TFPI). LRP-1 also binds to the multimeric
matrix proteins thrombospondin-1 and thrombospondin-2 and delivers
Pseudomonas exotoxin A and minor-group human rhinovirus into cells.
In addition, LRP-1 recognizes a number of intracellular proteins,
including HSP96, HIV-1 Tat protein, and RAP, an endoplasmic
reticulum resident protein that functions as a molecular chaperone
for LRP-1 and other LDL receptor family members.
[0022] How does LRP-1 specifically recognize this variety of
ligands? Crystallographic and nuclear magnetic resonance studies of
individual ligand-binding domains have revealed that amino acid
sequence variability in short loops of each ligand-binding domain
results in a unique contour surface and charge density for the
repeats. LRP-1 "minireceptors" have been made by fusing different
ligand-binding domains to the LRP-1 light chain and measuring the
ability to mediate the endocytosis of individual ligands following
expression in cells. Alternatively, soluble LRP-1 fragments made by
recombinant technology and representing the different
ligand-binding domains are screened for their ability to bind
different ligands in vitro. For example, the short loops
responsible for A.beta. binding may be grafted onto a heterologous
polypeptide (cf. humanization of rodent antibodies to reduce their
immunogenicity) to make a soluble LRP-1 derivative which may or may
not be attached to a substrate.
[0023] A "fragment" is a particular derivative of LRP-1 with a
molecular weight less than the molecular weight of full-length
LRP-1. The molecular weight of a soluble derivative is preferably
between the molecular weight of a single ligand-binding domain and
the heavy chain of LRP-1 (515 kDa). For example, soluble LRP-1
derivatives may be from about 35 kDa to about 55 kDa, but both
smaller and larger fragment are possible. In particular,
derivatives comprising cluster II (i.e., Arg786 to Leu1165 as
numbered in Herz et al., 1988) and/or cluster IV (i.e., His3313 to
Leu3759 as numbered in Herz et al., 1988) are preferred. The LRP-1
molecule, its amino acid and nucleotide sequence, or its mature
form may be derived from human (e.g., accession CAA32112,
NP.sub.--002323, Q07954 or S02392), other mammals (e.g., cow,
guinea pig, mouse, or rat), or polymorphic and mutant variants
thereof. Although the full-length LRP-1 might be chemically
manipulated (e.g., chemical cleavage or enzymatic proteolysis) to
make polypeptide fragments, genetic manipulation of polynucleotides
to make those fragments by recombinant technology in a bacterium,
mold or yeast, insect, or mammalian cell or organim is preferred. A
genetic chimera may be used to fuse soluble LRP-1 derivative to one
or more heterologous domains; it may be introduced into cells or
organisms (e.g., nuclear transfer, transfection, or transgenesis)
where the polypeptide is translated and processed.
[0024] A preferred method of making a soluble derivative of LRP-1
involves a mutant of the wild-type transmembrane domain (e.g., a
missense or deletion mutation). For example, a stop codon may be
introduced at a site before the transmembrane domain or the
polynucleotide portion encoding the transmembrane and cytoplasmic
domains may be deleted. A minireceptor comprising cluster II and/or
cluster IV may also be synthesized (e.g., by gene splicing or
amplifying with adapter primers) and used. An LRP-1 molecule or
derivative thereof may be attached to the lipid bilayer of a
cellular membrane or another substrate, and then
detached/hydrolyzed to make the soluble LRP-1 derivative. For
example, a proteolytic enzyme may hydrolyze a peptide bond on the
outside of a cell or a lipase may hydrolyze a glycosphingolipid
anchor inserted in the lipid bilayer. Alternatively, soluble LRP-1
derivative may be immobilized on a substrate before, during, or
after binding to A.beta..
[0025] Protein fusions may also be made and used. A heterologous or
the LRP-1 signal domain may be used for translocation across a cell
membrane and transport by the secretory pathway. Soluble LRP-1
derivatives may be glycosylated or otherwise post-translationally
modified. A localization domain (e.g., antibody or another member
of a binding pair) may be used to increase the local concentration
of a soluble LRP-1 derivative in a tissue, organ, or other portion
of a subject's body. For example, biotinylation or a fusion with
streptavidin may localize the LRP-1 derivative to a body part in/or
which the cognate binding member (avidin or biotin, respectively)
is attached. For the receptor-ligand system studied here, LRP-1
ligands (e.g., apoE, apoJ, .alpha..sub.2M) and RAP are not required
to bind A.beta.. Soluble LRP-1 derivative may bind free A.beta. in
solution, or with one of the components in solid phase. After
binding between LRP-1 derivative and A.beta., either or both may be
immobilized on a substrate (e.g., cell, tissue, or artificial solid
substrate) at any time before, during, or after binding. The bound
complex may be isolated or detected. Candidate compounds to treat
Alzheimer's disease may interact with at least one gene,
transcript, or protein which is a component of the receptor-ligand
system to increase receptor activity (i.e., vascular clearance of
A.beta.), and be screened for their ability to provide therapy or
prophylaxis. These products may be used in assays (e.g., diagnostic
methods to detect A.beta. using sLRP-1) or for treatment;
conveniently they are packaged in an assay kit or pharmaceutical
form (e.g., single or multiple dose package).
[0026] Binding of a soluble LRP-1 derivative with A.beta. may take
place in solution or on a substrate. The assay format may or may
not require separation of bound A.beta. from unbound A.beta. (i.e.,
heterogeneous or homogeneous formats). Detectable signals may be
direct or indirect, attached to any part of a bound complex,
measured competitively, amplified, or any combination thereof. A
blocking or washing step may be interposed to improve sensitivity
and/or specificity. Attachment of a soluble LRP-1 derivative to a
substrate before, after, or during binding results in capture of an
unattached species. See U.S. Pat. Nos. 5,143,854 and 5,412,087.
Abundance may be measured at the level of protein and/or
transcripts of a component of the receptor-ligand system.
[0027] A soluble LRP-1 derivative may also be attached to a
substrate. The substrate may be solid or porous and it may be
formed as a sheet, bead, or fiber. The substrate may be made of
cotton, silk, or wool; cellulose, nitrocellulose, nylon, or
positively-charged nylon; natural rubber, butyl rubber, silicone
rubber, or styrenebutadiene rubber; agarose or polyacrylamide;
silicon or silicone; crystalline, amorphous, or impure silica
(e.g., quartz) or silicate (e.g., glass); polyacrylonitrile,
polycarbonate, polyethylene, polymethyl methacrylate,
polymethylpentene, polypropylene, polystyrene, polysulfone,
polytetrafluoroethylene, polyvinylidenefluoride, polyvinyl acetate,
polyvinyl chloride, or polyvinyl pyrrolidone; or combinations
thereof. Optically-transparent materials are preferred so that
binding can be monitored and signal transmitted by light. Such
reagents would allow capture of A.beta. in solution by specific
interaction between the cognate molecules and then could immobilize
A.beta. on the substrate.
[0028] A soluble LRP-1 derivative may be attached to a substrate
through a reactive group as, for example, a carboxy, amino, or
hydroxy radical; attachment may also be accomplished after contact
printing, spotting with a pin, pipetting with a pen, or spraying
with a nozzle directly onto a substrate. Alternatively, the soluble
LRP-1 derivative may be reversibly attached to the substrate by
interaction of a specific binding pair (e.g.,
antibody-digoxygenin/hapten/peptide, biotin-avidin/streptavidin,
glutathione S transferase-glutathione, maltose binding
protein-maltose, polyhistidine-nickel, protein A or
G/immunoglobulin); cross-linking may be used if irreversible
attachment is desired.
[0029] Attaching a reporter, which is easily assayed, to a soluble
LRP-1 derivative may be used for convenient detection. Reporters
include, for example, alkaline phosphatase, .beta.-galactosidase
(LacZ), chloramphenicol acetyltransferase (CAT),
.beta.-glucoronidase (GUS), bacterial/insect/marine invertebrate
luciferases (LUC), green and red fluorescent proteins (GFP and RFP,
respecttively), horseradish peroxidase (HRP), .beta.-lactamase, and
derivatives thereof (e.g., blue EBFP, cyan ECFP, yellow-green EYFP,
destabilized GFP variants, stabilized GFP variants, or fusion
variants sold as LIVING COLORS fluorescent proteins by Clontech).
Reporters would use cognate substrates that are preferably assayed
by a chromogen, fluorescent, or luminescent signal. Alternatively,
the soluble LRP-1 derivative may be tagged with a heterologous
epitope (e.g., FLAG, MYC, \ SV40 T antigen, glutathione
transferase, hexahistidine, maltose binding protein) for which
cognate antibodies or affinity resins are available.
[0030] A soluble LRP-1 derivative may be joined to one member of
the specific binding pair by genetically ligating appropriate
coding regions in an expression vector or, alternatively, by direct
chemical linkage to a reactive moiety on the binding member by
chemical cross-linking. They may be used as an affinity reagent to
identify, to isolate, and to detect interactions that involve
specific binding with A.beta.. This can produce a complex in
solution or immobilized to a support.
[0031] A soluble LRP-1 derivative may be used as a medicament,
diagnostic agent, or used to formulate therapeutic or diagnostic
compositions with one or more of the utilities disclosed herein.
They may be administered in vitro to a body fluid or tissue in
culture, in vivo to a subject's body, or ex vivo to cells outside
of the subject that may later be returned to the body of the same
subject or another. Fluids and tissues may be further processed
after a specimen is taken from the subject's body and before
laboratory assay. For example, cells may be diaggregated or lysed,
or provided as solid tissue. The specimen may be stored in dry or
frozen form prior to assay.
[0032] Compounds or derivatives thereof may be used to produce a
medicament or other pharmaceutical compositions. Use of
compositions which further comprise a pharmaceutically acceptable
carrier and compositions which further comprise components useful
for delivering the composition to an individual are known in the
art. Addition of such carriers and other components to the
composition of the invention is well within the level of skill in
this art.
[0033] The concentration of free A.beta. may be decreased by
binding to a soluble LRP-1 derivative or removing A.beta. bound to
a soluble LRP-1 derivative through the body's circulation (e.g.,
reticuloendothelial system) or by machine (e.g., affinity
chromatography, electrophoresis, filtration, precipitation).
Efficacy of treatment may be assessed by removal of A.beta. from a
subject's body or reducing deposition of amyloid in the subject's
body. This may be accomplished in an animal model or in a human
where the amount and/or the location of may be detected with
soluble LRP-1 derivative. It should be noted that the modes of
treatment described herein differ significantly from the mechanism
described in U.S. Pat. No. 6,156,311 which identifies a role for
low-density lipoprotein receptor related protein in endocytosis and
degradation of amyloid-.beta. precursor protein (APP).
[0034] Labels or other detectable moieties may be attached to
soluble LRP-1 derivatives or contrast agents may be included for
structural imaging: e.g., X-ray computerized tomography (CT),
magnetic resonance imaging (MRI), or optical imaging. Functional
imaging such as Single Photon Emission Computed Tomography (SPECT)
may also be used. A soluble LRP-1 derivative may be labeled (e.g.,
gadolinium) for MRI evaluation of amyloid load in the brain or
vascular system. A soluble LRP-1 derivative may be labeled (e.g.,
.sup.76Br, .sup.123I) for SPECT evaluation of amyloid load in the
brain with a blood-brain barrier (BBB) permeabilizing agent, or for
evaluating cerebral amyloid angiopathy with or with the BBB
permeabilizing agent.
[0035] Reagents may also be provided in a kit for use in performing
methods such as, for example: diagnosis, identification of those at
risk for disease or already affected, or determination of the stage
of disease or its progression. In addition, the reagents may be
used in methods related to the treatment of disease such as the
following: evaluation whether or not it is desirable to intervene
in the disease's natural history, alteration of the course of
disease, early intervention to halt or slow progression, promotion
of recovery or maintenance of function, provision of targets for
beneficial therapy or prophylaxis, comparison of candidate drugs or
medical regimens, or determination of the effectiveness of a drug
or medical regimen. Instructions for performing these methods,
reference values and positive/negative controls, and relational
data-bases containing patient information (e.g., genotype, medical
history, disease symptoms, transcription or translation yields from
gene expression, physiological or pathological findings) are other
products that can be considered aspects of the invention.
[0036] The amount and extent of treatment administered to a subject
in need of therapy or prophylaxis is effective in treating the
affected subject. The invention may be used alone or in combination
with other known methods. The individual may be any animal or
human. Mammals, especially humans and rodent or primate models of
disease, may be treated. Thus, both veterinary and medical methods
are contemplated.
[0037] A pharmaceutical or diagnostic composition containing one or
more soluble LRP-1 derivatives may be administered as a formulation
adapted for passage through the blood-brain barrier or direct
contact with the endothelium. Alternatively, compositions may be
added to the culture medium. In addition to the soluble LRP-1
derivative(s), such compositions may contain
physiologically-acceptable carriers and other ingredients known to
facilitate administration and/or enhance uptake (e.g., saline,
dimethyl sulfoxide, lipid, polymer, affinity-based cell
specific-targeting systems). The composition may be incorporated in
a gel, sponge, or other permeable matrix (e.g., formed as pellets
or a disk) and placed in proximity to the endothelium for
sustained, local release. The composition may be administered in a
single dose or in multiple doses which are administered at
different times.
[0038] A pharmaceutical or diagnostic composition containing one or
more soluble LRP-1 derivatives may be administered into the body by
any known route. By way of example, the composition may be
administered by a mucosal, pulmonary, topical, or other localized
or systemic route (e.g., enteral and parenteral). The term
"parenteral" includes subcutaneous, intradermal, subdermal,
intramuscular, intrathecal, intra-arterial, intravenous, and other
injection or infusion techniques, without limitation.
[0039] Suitable choices in amounts and timing of doses,
formulation, and routes of administration can be made with the
goals of achieving a favorable response in the individual with
Alzheimer's disease or at risk thereof (i.e., efficacy), and
avoiding undue toxicity or other harm thereto (i.e., safety).
Therefore, "effective" refers to such choices that involve routine
manipulation of conditions to achieve a desired effect.
[0040] A bolus of one or more soluble LRP-1 derivatives
administered into the body over a short time once a day is a
convenient dosing schedule. Alternatively, the effective daily dose
of soluble LRP-1 derivative(s) may be divided into multiple doses
for purposes of administration, for example, two to twelve doses
per day. Dosage levels of soluble LRP-1 derivative(s) in a
pharmaceutical composition can also be varied so as to achieve a
transient or sustained concentration in an individual's body,
especially in and around vascular endothelium of the brain, and to
result in the desired therapeutic response or protection. But it is
also within the skill of the art to start doses at levels lower
than required to achieve the desired therapeutic effect and to
gradually increase the dosage until the desired effect is achieved.
Similarly, dosage levels of soluble LRP-1 derivative(s) in a
diagnostic composition may be varied to achieve the desired
sensitivity and specificity of detection of A.beta. in an
individual's body.
[0041] The amount of soluble LRP-1 derivative(s) administered is
dependent upon factors known to skilled artisans such as its
bioactivity and bioavailability (e.g., half-life in the body,
stability, and metabolism); chemical properties (e.g., molecular
weight, hydrophobicity, and solubility); route and scheduling of
administration; and the like. For systemic administration, passage
of soluble LRP-1 derivative(s) or metabolite(s) thereof through the
blood-brain barrier is important. It will also be understood that
the specific dose level to be achieved for any particular
individual may depend on a variety of factors, including age,
gender, health, medical history, weight, combination with one or
more other drugs, and severity of disease.
[0042] The term "treatment" of Alzheimer's disease refers to, inter
alia, reducing or alleviating one or more symptoms in an
individual, preventing one or more symptoms from worsening or
progressing, promoting recovery or improving prognosis, and/or
preventing disease in an individual who is free therefrom as well
as slowing or reducing progression of existing disease. For a given
individual, improvement in a symptom, its worsening, regression, or
progression may be determined by an objective or subjective
measure. Efficacy of treatment may be measured as an improvement in
morbidity or mortality (e.g., lengthening of survival curve for a
selected population). Prophylactic methods (e.g., preventing or
reducing the incidence of relapse) are also considered treatment.
Treatment may also involve combination with other existing modes of
treatment (e.g., ARICEPT or donepezil, EXELON or rivastigmine,
anti-amyloid vaccine, mental exercise or stimulation). Thus,
combination treatment with one or more other drugs and one or more
other medical procedures may be practiced.
[0043] The amount of soluble LRP-1 derivative(s) which is
administered to an individual is preferably an amount that does not
induce toxic or other deleterious effects which outweigh the
advantages which result from its administration. Further objectives
are to reduce in number, diminish in severity, and/or otherwise
relieve suffering from the symptoms of the disease as compared to
recognized standards of care. The invention may also be effective
against neurodegenerative disorders in general: for example,
dementia, depression, confusion, Creutzfeldt-Jakob disease,
Huntington's disease, Parkinson's disease, loss of motor
coordination, multiple sclerosis, stroke, and syncope.
[0044] Production of a soluble LRP-1 derivative will be regulated
for good laboratory practices (GLP) and good manufacturing
practices (GMP) by appropriate governmental regulatory agencies.
This requires accurate and comprehensive recordkeeping, as well as
monitoring of QA/QC. Oversight of patient protocols by agencies and
institutional panels is also envisioned to ensure that informed
consent is obtained; safety, bioactivity, appropriate dosage, and
efficacy of products are studied in phases; results are
statistically significant; and ethical guidelines are followed.
Similar oversight of protocols using animal models, as well as the
use of toxic chemicals, and compliance with regulations is
required.
[0045] For therapeutic uses, an appropriate regulatory agency would
specify acceptable levels of purity (e.g., lack of extraneous
protein and nucleic acids); sterility (e.g., lack of microbes);
lack of host cell contamination (e.g., less than 0.5 Endotoxin
Units/nil); and potency (e.g., efficiency of gene transfer and
expression) for biologics. Another objective may be to ensure
consistent and reproducible production of a soluble LRP-1
derivative, which may improve the potency of the biologic while
being compatible with the good manufacturing practices used to
ensure a pure, sterile, and pyrogen-free product.
[0046] The following examples are merely illustrative of the
invention, and are not intended to restrict or otherwise limit its
practice.
EXAMPLES
[0047] It was recently shown that LRP-1 functions as a clearance
receptor for A.beta. at the blood-brain barrier. LRP-1-mediated
A.beta. transcytosis is initiated at the abluminal (brain) site of
the endothelium and is therefore directly responsible for
eliminating A.beta. from brain interstitial fluid into blood.
A.beta. clearance can be influenced by apoE and .alpha..sub.2M,
known ligands for LRP-1, but formation of A.beta. complexes with
either of those ligands have not been shown in the central nervous
system (CNS) in vivo during relatively rapid clearance studies
(Shibata et al., 2000). Therefore, whether LRP-1 binds directly to
free A.beta. is determined herein.
[0048] The major binding sites of LRP-1 are contained in clusters
II and IV of .alpha.-subunit which bind most of the currently
mapped known ligands of LRP-1, e.g., apoE, .alpha..sub.2M, tissue
plasminogen activator, plasminogen activator inhibitor-1, APP,
factor VIII, and lactoferrin. FIGS. 1A-1B show high affinity
binding of soluble monomeric A.beta..sub.1-40 to immobilized LRP-1
clusters II and IV with K.sub.d values of 0.57.+-.0.12 nM and
1.24.+-.0.01 nM, respectively, determined by the surface plasmon
resonance (SPR) analysis. In contrast, A.beta..sub.1-42 and
vasculotropic mutant A.beta. (double mutant Dutch/Iowa40 model
peptide; Van Nostrand et al., 2001) exhibit greatly reduced binding
affinity for LRP-1 clusters II and IV by 6- and 9-fold and 28- and
12-fold, respectively, compared to A.beta..sub.1-40. The K.sub.d
values for A.beta..sub.1-42 binding to LRP-1 II and IV clusters
were 3.00.+-.0.11 nM and 10.10+0.03 nM, respectively, and for
mutant A.beta. (Dutch/Iowa40) 15.10.+-.0.10 nM and 15.30.+-.0.07
nM, respectively. These data suggest that in vitro LRP-1
preferentially interacts with A.beta..sub.1-40 compared to
A.beta..sub.1-42 and mutant A.beta..
[0049] Binding of all A.beta. peptides to LRP-1 clusters II and IV
was abolished by RAP, an LRP-1 antagonist (FIGS. 1A-1B). In
contrast, the present findings show the affinity of A.beta. species
to bind to immobilized LRP-1 fragments was greatly reduced by high
content of .beta.-sheets in A.beta. (FIG. 1C), as determined by the
circular dichroism analysis (Zlokovic et al., 1996; Golabek et al.,
1996). These results raise a possibility that if LRP-1 is a major
clearance receptor for A.beta. in the brain, then direct
interaction with LRP-1 will mediate preferential clearance of
A.beta..sub.1-40 from brain interstitial fluid (ISF) while favoring
the retention of A.beta..sub.1-42 and mutant A.beta..
[0050] According to the amyloid hypothesis, neurotoxic
A.beta..sub.1-42 accumulation in the brain is a major event
initiating AD pathogenesis (Hardy and Selkoe, 2002). Increased
A.beta..sub.1-42 accumulation could be associated with increased
A.beta. production as in familial forms of AD and/or impaired
A.beta. clearance as in a late-onset AD (Selkoe, 2001; Zlokovic and
Frangione, 2003). Increased levels of A.beta. in the brain lead to
formation of neurotoxic A.beta. oligomers and progressive synaptic,
neuritic and neuronal dysfunction (Walsh et al., 2002; Dahlgren et
al., 2002; Kayed et al., 2003; Gong et al., 2003). Missense
mutations within A.beta. associate mainly with vascular deposits,
as in patients with Dutch mutation (G to C at codon 693, Glu to Gln
at position 22) and Iowa mutation (G to A at codon 694, Asp to Asn
at position 23). Vasculotropic Dutch (E22Q) or Iowa (D23N) mutant
A.beta. exhibit enhanced fibrillogenesis and toxicity to cerebral
vascular cells, while Dutch/Iowa double mutant A.beta. (E22Q,D23N),
a model peptide used in the present study, has accelerated
pathogenic properties compared to both Dutch and Iowa vasculotropic
mutants (Van Nostrand et al., 2001).
[0051] To confirm that direct interaction with LRP-1 predisposes to
accumulation of A.beta..sub.1-42 (a mutant A.beta.) and clearance
of A.beta..sub.1-40, in vitro clearance of the A.beta. species was
studied by isolated mouse brain capillaries. FIG. 2A shows rapid
saturable uptake of A.beta..sub.1-40 at the abluminal side of brain
capillaries that follows Michaelis-Menten kinetics (Kd=10.+-.2 nM).
Uptake of A.beta..sub.1-40 was abolished by RAP and an anti-LRP-1
antibody suggesting that LRP-1 is involved. Interaction of
A.beta..sub.1-40 with LRP-1 on capillary membranes initiated almost
instantaneous internalization of the ligand. Mild acid wash
treatment indicated that, after stripping membrane-bound
.sup.125I-A.beta..sub.1-40, about 80% of A.beta..sub.1-40 still
remains associated with brain capillaries. This suggests a rapid
internalization of the ligand within 1 min (FIG. 2B), which is
consistent with rapid endocytotic function of the receptor (Li et
al., 2001a; 2001b). These data also indicate that binding of
reduced A.beta. to its carrier proteins apoE and .alpha..sub.2M is
not required for its brain capillary clearance. However, these
A.beta. chaperones may still influence A.beta. clearance by
enhancing its uptake by neurons (Jordan et al., 1998; Qiu et al.,
1999) and/or accelerating its extracellular deposition (Golabek et
al., 1996).
[0052] A series of cross-inhibition experiments using
.sup.125I-labeled A.beta..sub.1-40 as a test-ligand and different
unlabeled A.beta. peptides as inhibitors was performed to determine
the relative affinity of A.beta. species for LRP-dependent brain
capillary clearance. The kinetic inhibitory constants K.sub.i
determined from the velocity ratios (Zlokovic et al., 1996)
revealed that Dutch A.beta..sub.1-40, wild-type A.beta..sub.1-42,
Dutch A.beta..sub.1-42 and Dutch/Iowa A.beta..sub.1-40 exhibit 6,
14, 18 and 22-fold lower affinities for LRP-mediated clearance on
brain capillaries than A.beta..sub.1-40, respectively (FIG. 2C). As
for the in vitro binding (FIG. 1C), the affinity of A.beta. species
for brain capillary clearance was inversely related to the content
of .beta.-sheets in A.beta. and/or the loss of negative charges
caused by mutations within the A.beta., i.e., one for Dutch mutant
and two for double Dutch and Iowa mutant (Van Nostrand et al.,
2001).
[0053] RAP is involved in maintaining proper folding of LRP-1 and
preventing its premature interaction with cognate ligands in the
endoplasmic reticulum (ER). Deletion of the RAP gene results in
substantial reduction in LRP-1 levels in the brain (Van Uden et
al., 2002). Here, Western blot analysis (FIG. 3A) and
immunostaining of brain tissue in situ (FIG. 3B) showed that the
amount of LRP-1 in brain capillaries of RAP null mice was decreased
by greater than 75% compared to controls. LRP-positive brain
vascular profiles in a RAP null mouse model was reduced in several
brain regions, (e.g., cortex, hippocampus and thalamus) from 60% to
70% in controls to 14% to 16% in RAP null mice as indicated by
double staining for LRP-1 and endothelial cell marker CD31 (FIG.
3C). To validate LRP-1 as a critical clearance receptor for
A.beta., clearance of .sup.125I-labeled A.beta..sub.1-40,
A.beta..sub.1-42 and mutant A.beta. (Dutch/Iowa40) by brain
microvessels isolated from RAP null mice and control mice was
compared. These results demonstrate that deletion of the RAP gene
results in about 80% reduction in vascular clearance of all studied
A.beta. isoforms in vitro (FIG. 3D). In contrast, deletion of the
genes for the LDL receptor or the VLDL receptor did not result in a
change in A.beta. clearance at the abluminal side of brain
capillaries.
[0054] Next, it was determined whether LRP-1 in vivo mediates
differential efflux of A.beta. peptides across the BBB in mice as
it mediates differential A.beta. brain capillary clearance in
vitro. Transport out of the brain of [.sup.125I]-labeled
A.beta..sub.1-40, A.beta..sub.1-42, and mutant A.beta.
(Dutch/Iowa40) microinfused simultaneously with .sup.14C-inulin
(reference marker) into the mouse brain ISF space was measured (as
described Shibata et al., 2000). Clearance of .sup.125I-labeled
A.beta. peptides across the BBB was calculated after correction for
the passive diffusion of tracers via the ISF bulk flow using the
elimination rate of .sup.14C-inulin. At concentrations comparable
to physiological levels of soluble A.beta. in brain ISF (i.e., less
than or equal to 1 nM) (Cirrito et al., 2003), A.beta..sub.1-40
wild-type was cleared rapidly from brain across the BBB within few
seconds. In contrast, clearance of mutant A.beta. (Dutch/Iowa40)
was slow and only 40% of the infused peptide was cleared across the
BBB within 30 min (FIG. 4A). At higher concentrations, mutant
A.beta. was almost devoid of clearance at the BBB, while wild-type
A.beta..sub.1-40 exhibited still a substantial clearance. These
data are consistent with reduced clearance of mutant
A.beta..sub.1-40 (Dutch) from the cerebrospinal fluid in guinea
pigs (Monro et al., 2002) and decreased clearance of mutant A.beta.
(Dutch/Iowa40) on brain capillaries in vitro observed in this study
(FIGS. 2C and 3D). RAP and an anti-LRP-1 antibody, but not
non-immune immunoglobulin G (NI IgG in FIG. 4A), almost completely
abolished A.beta. elimination from brain confirming a critical role
of LRP-1 for A.beta. clearance from brain in vivo.
[0055] A significant (.rho.<0.05) cross-inhibition of
[.sup.125I]-A.beta..sub.1-40 clearance at the BBB by unlabeled
A.beta..sub.1-42 and mutant A.beta. (Dutch/Iowa40), and a
pronounced greater than 95% inhibition of [.sup.125I]-labeled
A.beta..sub.1-42 and mutant A.beta. clearance by unlabeled
wild-type A.beta..sub.1-40 (FIG. 4B), indicated that all A.beta.
peptides share the same LRP-1-mediated efflux mechanism to exit the
brain, and that A.beta..sub.1-40 exerts a significant retention
effect on A.beta..sub.1-42 and mutant A.beta. in vivo. The K.sub.i
values determined with .sup.125I-A.beta..sub.1-40 as a test-ligand
and unlabeled A.beta. peptides as inhibitors indicated that the
affinity of A.beta. for LRP-1-mediated clearance in vivo is
remarkably reduced by the high .beta.-sheet content (FIG. 4C).
A.beta..sub.1-42 and mutant A.beta. exhibited 8- and 15-fold lower
affinity for LRP-1-mediated efflux at the BBB in vivo. All A.beta.
test-ligands microinfused in brain ISF remained greater than 97% in
their monomeric forms as intact peptides during short-term
clearance studies within 30 min and over the range of A.beta.
concentrations less than 100 nM, as reported (Shibata et al., 2000;
Zlokovic et al., 2000), and demonstrated by the HPLC analysis (FIG.
4B, insets) and SDS-PAGE analysis of brain homogenates.
[0056] To further confirm the role of LRP-1 in rapid efflux of
A.beta. from the brain in vivo, clearance of .sup.125I-labeled
A.beta..sub.1-40 and A.beta..sub.1-42 in RAP null mice was compared
to control mice. As expected based on in vitro brain capillary
clearance data (FIG. 3D), there was 75% to 85% inhibition of
A.beta..sub.1-40 and A.beta..sub.1-42 rapid efflux across the BBB
in RAP null/severely depleted LRP-1 mice (FIG. 4C). Crossing RAP
null mice with APP overexpressing mice doubles the amount of
amyloid deposits (Van Uden et al., 2002) which is consistent with
the present findings demonstrating that deletion of the RAP gene
almost completely eliminates rapid A.beta. clearance at the
BBB.
[0057] To validate the clearance hypothesis for endogenous A.beta.,
accumulation of A.beta. in transgenic Dutch/Iowa (Tg-DI) mice
expressing low levels of human APP under the control of a Thy 1.2
neuronal promoter harboring the Dutch and Iowa vasculotropic
mutations were compared to Tg-2576 APP overexpressing mice (Hsiao
et al., 1996). Tg-DI mice produce mutant A.beta. (Dutch/Iowa) that
compared to the wild-type A.beta..sub.1-40 binds to LRP-1 with
significantly lower affinity (FIGS. 2B-2C) and exhibits low
LRP-1-clearance on brain capillaries (FIGS. 2B and FIG. 3D) and
across the BBB (FIGS. 4A and 4D). At 3, 6 or 12 months of age, the
level of APP in the brain of Tg-DI mice was considerably lower than
in Tg-2576 mice, as determined by the quantitative immunoblot
analysis of brain homogenates (FIGS. 5A-5B). Despite about 24-fold
lower levels of human APP (FIG. 5B), the Tg-DI mice still exhibited
robust brain accumulations of mutant A.beta. earlier than Tg-2576
mice overproducing wild-type A.beta. (FIG. 5C), i.e., by 15- and
5-fold higher for the A.beta..sub.1-40 and A.beta..sub.1-42
isoforms, respectively, at 6 months of age.
[0058] Consistent with early accumulation of A.beta., Tg-DI mice
developed early A.beta. plaque-like deposits in the cortex and
hippocampus at 3 months of age (FIG. 5D, left), while Tg-2576 mice
initially presented A.beta. deposits at about 9 months of age, as
reported (Hsiao et al., 1996; Kawarabayashi et al., 2001). The
A.beta. plaque-like deposits in Tg-DI mice were abundant at 12
months (FIG. 5D, right), but the majority presented as diffuse
plaques similar as in patients with the Dutch and Iowa A.beta.
mutations. Significant intracerebral vascular association of
A.beta. in Tg-DI mice (FIG. 5E) was suggestive of a clearance
problem at the level of brain's blood vessels, consistent with
prominent cerebrovascular pathology in Dutch and Iowa patients
(Vinters and Farag, 2003). Plasma levels of mutant A.beta. in Tg-DI
mice were extremely low, i.e., less than 25 pM, corroborating low
efflux of mutant A.beta. from brain (FIGS. 4A and 4D). In Tg-2576
mice, the ratio of A.beta..sub.1-42:A.beta..sub.1-40 in plasma was
1:10, while the ratio in brain varied between 1:3 to 1:2 at 6 and
12 months of age, respectively (FIG. 5C). These results suggest
lower clearance of endogenous A.beta..sub.1-42 relative to
A.beta..sub.1-40, as would be expected from substantially lower
LRP-1-mediated clearance of exogenous A.beta..sub.1-42 by brain
capillaries (FIGS. 2B and 3D) and across the BBB (FIGS. 4B-4D).
[0059] Increased fibrillogenic properties of mutant A.beta.
observed in vitro (Van Nostrand et al., 2001) may contribute to its
decreased clearance from brain in vivo. However, the presence of
mainly non-fibrillar A.beta. parenchymal deposits in Tg-DI mice
within the first 12 months of age would argue against the
possibility that enhanced fibrillogenicity is the primary mechanism
for reduced efflux of endogenous mutant A.beta. from brain. To
better understand the relationship between brain capillary LRP-1
and accumulation of A.beta., the expression of LRP-1 in brain
microvessels in situ was next studied in Tg-DI and Tg-2576 mice.
Surprisingly double immunostaining Tor brain endothelial LRP-1 and
endothelial cell marker CD31 indicated substantial reduction of
LRP-1-positive vascular profiles in several brain regions in Tg-DI
and Tg-2576 mice, i.e., only 5% to 20% and 25% to 30% of
microvessels in 4- to 6-month old Tg-DI and Tg-2576 mice were
positive for LRP-1, respectively, compared to 65% to 75% in
age-matched littermate controls (FIG. 5F). The decrease in LRP-1
vascular profiles at 12 months of age was also more pronounced in
A.beta.-accumulating transgenic mice than in controls (FIG. 5G). A
significant age-dependent decrease of LRP-1 at the BBB was
consistent with reported down-regulation of LRP-1 in the brain
during normal aging. The most obvious early reductions observed in
LRP-1 expression at the BBB in Tg-DI mice at 4 months to 6 months
of age correlated well with significantly higher A.beta.
accumulation in these mice relative to Tg-2576 mice (FIG. 5C).
[0060] The present study reveals that direct interaction between
LRP-1 and A.beta. in brain endothelium may critically influence
neurotoxic and vasculotropic A.beta. accumulations by promoting
retention of A.beta. species with high .beta.-sheet content and
genetic mutations within A.beta. while clearing soluble
A.beta..sub.1-40. Mutations within AR do not significantly affect
the affinity of mutant A.beta. to bind to sLRP-1 cluster II or
cluster IV. In contrast to LRP-1, the receptor for advanced
glycation end-products (RAGE) mediates continuous influx of
circulating A.beta. into the brain and is overexpressed in brain
vasculature in transgenic APP models and in AD (Deane et al.,
2003). There is a possibility that increased activity of LRP-1
receptor at the blood-brain barrier or in the vascular system will
reduce levels of A.beta. in the CNS by acting directly to free
A.beta.. Applications include subjects with familial forms of
Alzheimer's disease (FAD) with cerebral amyloid angiopathy (CAA),
such as patients with Dutch or Iowa mutations (FAD/CAA). Because
the LRP-1 cluster II or IV domain binds efficiently to wild-type
and mutant A.beta., they can be used for diagnostic purposes in
Alzheimer's disease, FAD/CAA, and Down syndrome as imaging agents
in the brain to visualize changes associated with vascular
pathology.
[0061] Since soluble LRP-1 derivatives bind A.beta., they can be
used to promote egress of A.beta. from brain into blood. The levels
of A.beta. free and bound to soluble LRP-1 derivative can be used
to develop a double sandwich ELISA diagnostic blood test in
Alzheimer's disease, FAD/CAA, and Down syndrome. The mechanism of
action may be sequestration of circulating wild-type or mutant
A.beta. over hours or days, similar to other peripheral
A.beta.-binding agents such as anti-A.beta. antibody, gelsolin,
GM1, and sRAGE. Using a brain perfusion model (Deane et al., 2003),
it was shown that either sLRP-1 cluster II or cluster IV sequesters
A.beta. in the systemic circulation, and prevents A.beta. transport
across the blood-brain barrier into the brain. In contrast to other
A.beta.-binding agents, use of one or more soluble LRP-1
derivatives provides the advantages that (1) they should be
well-tolerated by a subject and avoids an immune or
neuroinflammatory response in the brain and cerebral blood vessels,
which can be a serious complication of anti-A.beta. antibody
therapy, and (2) their binding affinities for A.beta. is much
higher than gelsolin, GM1, or sRAGE.
[0062] These properties of soluble LRP-1 derivatives can also be
used to lower the level of A.beta. in the brain of transgenic
Alzheimer's disease mice, transgenic FAD/CAA mice, or Alzheimer's
disease and FAD/CAA patients by sequestering A.beta. over long
periods of time (e.g., months, years) and possibly in the CNS
itself. For this purpose, one or more soluble LRP-1 derivatives can
be used alone, or in combination with agents that permeabilize the
blood-brain barrier (e.g., insulin-like growth factor-1, RMP-7),
neuroprotective agents (e.g., activated protein C as described in
Guo et al., 2004), or other therapies to lower AP in an individual:
immunization or vaccination against A.beta.; administration of
ganglioside, gelsolin, or sRAGE; inhibiting beta/gamma
secretase-mediated processing of amyloid precursor protein; osmotic
opening of the blood-brain barrier (Neuwelt et al., 1985);
normalization of cerebrospinal fluid production (Silverberg et al.,
2003); or combinations thereof.
MATERIALS AND METHODS
Reagents
[0063] Wild-type and mutant A.beta. (Dutch40, Dutch42,
Dutch/Iowa40) peptides were synthesized, by solid-phase F-moc
(9-fluorenylmethoxycarbonyl) amino acid synthesis, purified by
reverse phase-HPLC and structurally characterized (as described in
Burdick et al., 1992; Van Nostrand et al., 2001). Recombinant LRP-1
fragments encompassing clusters II and IV were produced using
stable transfected baby hamster kidney cell lines (as described in
Westein et al., 2002). Human recombinant RAP (EMD Biosciences, San
Diego, Calif.), poly-clonal goat anti-human LRP-1 N20 antibody
which cross reacts with mouse LRP-1 (1:200, Santa Cruz Biotech,
Santa Cruz, Calif.), monoclonal mouse antibody against C-terminal
domain of human LRP-1 .beta.-chain which cross reacts with mouse
LRP-1 (5A6, 1:350, 5 .mu.g/ml; EMD Biosciences, San Diego, Calif.),
monoclonal mouse antibody against human LRP-1 .alpha.-chain
(8G1,1:240, 5 .mu.g/ml; EMD Biosciences, San Diego, Calif.),
monoclonal mouse antibody (mA.beta.) P2-1 specific for human APP
(1:1000, 1 mg/ml), mA.beta. 22C11 which recognizes mouse and human
APP (1:100, 0.5 mg/ml; Chemicon International, Temecula, Calif.),
mA.beta. 66.1 to residues 1-8 of human A.beta. (1:1000, 1 mg/ml)
(Deane et al., 2003), rat anti-mouse CD.beta. antibody (1:200, BD
Pharmigen, Lexington, Ky.) and polyclonal rabbit antibody to human
von Willebrand Factor, vWF (1:200, DAKO, Carpinteria, Calif.) were
used.
Surface Plasmon Resonance Analysis
[0064] LRP-1 clusters II and IV were immobilized at CM5 chips at a
density of 10-20 fmol/mm.sup.2 and incubated with A.beta..sub.1-40,
A.beta..sub.1-42, and mutant A.beta. (Dutch/Iowa40) (0 nM to 50 nM)
in 150 mM NaCl, 0.005% (v/v) TWEEN surfactant and 25 mM HEPES
buffer (pH 7.4) at a flow rate of 5 .mu.l/min for 2 min at
25.degree. C. (as described in Westein et al., 2002). RAP was used
at 500 nM. Ligand solution was replaced with buffer to initiate
dissociation. The data were analyzed to calculate apparent
association rate constants k.sub.on(app) and apparent dissociation
rate constants k.sub.off(app) using a single-site binding model (as
described in Westein et al., 2002). Apparent affinity constants
K.sub.d(app) were inferred from the ratio
k.sub.off(app)/k.sub.on(app). Data are based on three to five
measurements using six to nine different concentrations for each
measurement. Data are presented as the mean.+-.SEM. Analysis was
performed using BIACORE X biosensor system (Uppsala, Sweden) and
BIA evaluation 3.0 software (Biocore, Sweden).
Secondary Structure Analysis
[0065] Secondary structure of peptides was analyzed by circular
dichroism (as described in Zlokovic et al., 1996 and Golabek et
al., 1996). Briefly 20 .mu.g to 25 .mu.g of hexafluoroisopropanol
treated seedless peptide was initially dissolved in 980 .mu.l of 10
mM phosphate buffer, pH 7.4, and centrifuged to remove any
precipitated or undissolved material. The CD spectrum was recorded
within 24 hr (corresponding to the time of peptides use for in
vitro and in vivo assays) using a 1 mm path length cell in an Aviv
202 CD spectrometer (Proterion, Piscataway, N.J.). Results are
expressed as molar ellipticity and the percentage of .alpha.-helix,
(3-sheet, (3-turn and random coil determined for each peptide.
Under the present conditions, there was neither formation of high
molecular weight oligomers, as confirmed by gel exclusion
chromatography and dot blots with oligomer-specific antibodies (as
described in Kayed et al., 2003), nor fibrillar and aggregated
forms, as confirmed by atomic force microscopy.
Radioiodination of A.beta.
[0066] Radioiodination of A.beta. peptides was carried out using a
mild lactoperoxidase method. Typically 10 .mu.g of A.beta. was
labeled for 18 min at room temperature with 2 mCi of Na[.sup.125I].
After radiolabeling, preparations were processed by reverse-phase
HPLC using a Vydac C4 column and a 30 min linear gradient of 25% to
40% acetonitrile in 0.059% trifluoroacetic acid to separate the
monoiodinated non-oxidized form of A.beta. (which is the tracer
being used) from diiodinated A.beta., non-labeled non-oxidized
A.beta., and oxidized A.beta. species. The material in the peaks
eluted from HPLC was determined by MALDI-TOF mass-spectrometry to
ensure the purity of the radiolabeled species. For MALDI-TOF mass
spectrometry, A.beta. peptides were labeled under identical
conditions using Na[.sup.127I] instead of the radioactive nuclide.
Typically the specific activities obtained with this protocol were
in the range of 45 to 65 .mu.Ci/.mu.g of peptide. Rapid radiolysis
of A.beta. is possible and, therefore, the quality of each
preparation was rigorously monitored. For brain capillary uptake
studies and animal clearance studies, in most experiments the
preparations were used within 24 hr of labeling such that greater
than 99% was TCA precipitable. If used within 72 hr of labeling,
the radiolabeled peptides were stabilized in ethanol as a quenching
agent. Prior to in vitro study or infusion into animals, HPLC
purification of the tracer was performed. HPLC/SDS-PAGE analysis
was used to confirm the monomeric state of infused radiolabeled
A.beta.. The secondary structure of A.beta. remained unchanged by
iodination as confirmed by CD analysis.
Brain Capillary Uptake
[0067] To study uptake at the abluminal brain side of capillaries,
capillaries from wild-type mice and RAP null mice were isolated
from brain using a modified procedure (as described in Wu et al.,
2003). Brain capillaries were incubated with .sup.125I-labeled
A.beta..sub.1-40, A.beta..sub.1-42 and mutant A.beta.
(Dutch/Iowa40) at concentration of 1 nM in mock CSF at 37.degree.
C. for 1 min. Incubation medium contained 1 mM sodium perchlorate
to prevent free iodide uptake. Self- and cross-inhibition studies
were performed with unlabeled A.beta..sub.1-40 from 1 nM to 120 nM,
unlabeled A.beta..sub.1-42 or mutant A.beta. (Dutch40, Dutch42,
Dutch/Iowa40) at 40 nM, RAP at 500 nM, and LRP-1-specific
polyclonal N20 antibody at 60 .mu.g/ml. Ice-cold stop/strip
solution (0.2 M acetic acid, pH 2.6, 0.1 M NaCl), was added to one
set of experiments as a mild acid wash to strip membrane-bound
A.beta. and estimate the amount of A.beta. that was internalized
(as described in Melman et al., 2002).
Brain Clearance Studies
[0068] All studies were performed according to National Institutes
of Health guidelines using an approved institutional protocol. CNS
clearance of .sup.125I-A.beta..sub.1-40, .sup.125I-mutant A.beta.
(Dutch/Iowa40) and .sup.125I-A.beta..sub.1-42 was determined
simultaneously with .sup.14C-inulin (reference marker) in male
C57BL/6 mice, RAP null mice and littermate controls 8-10 weeks old
(as described in Shibata et al., 2000). Briefly a stainless steel
guide cannula was implanted stereotaxically into the right
caudate-putamen of anesthetized mice (0.5 mg/kg ketamine and 5
mg/kg xylazine I.P.). Coordinates for tip of the cannula were 0.9
mm anterior and 1.9 mm lateral to the bregma and 2.9 mm below the
surface of the brain. Animals were allowed to recover after surgery
prior to radiotracer studies. Clearance experiments were performed
before substantial chronic processes have occurred, as assessed by
histological analysis of negative tissue staining for astrocytes
(glial fibrillar acidic protein) and activated microglia
(antiphosphotyrosine), but allowing time for repair of the BBB to
large molecules, that was typically 4 hr to 6 hr after the cannula
insertion (Cirrito et al., 2003). Tracer fluid (0.5 .mu.l)
containing [.sup.125I]-A.beta. and .sup.14C-inulin (reference
molecule) was injected over 5 min via an ultramicropump with a
MICRO4 controller (World Precision Instruments, Sarasota, Fla.)
into brain ISF. When the effects of the different unlabeled
molecular reagents were tested, they were injected simultaneously
with radiolabled ligands. For self-inhibition studies, the uptake
of .sup.1251-A.beta..sub.1-40 and .sup.125I-mutant A.beta.
(Dutch/Iowa40) was studied over a range of carrier concentrations
from 0.5 nM to 120 nM. For cross-inhibition studies, efflux of
.sup.125I-test-peptides was studied at a carrier concentration of
40 nM and the inhibitory concentration of unlabeled A.beta.
peptides at 120 nM. Brain and blood were sampled 30 min after
tracers injection and prepared for radioactivity analysis by TCA,
HPLC and SDS-PAGE/immunoprecipitation analysis to determine the
molecular forms of test-tracers. Gamma counting for
.sup.125I-radioactivity was performed using WALLAC VIZARD gamma
counter (Perkin Elmer, Meriden, Conn.) and beta-counting for
.sup.14C-inulin using a TRI-CARB 2100 liquid scintillation counter
(Perkin Elmer), Meriden, Conn. Previous studies with
.sup.125I-labeled A.beta. peptides demonstrated an excellent
correlation between TCA and HPLC methods. .sup.125I-labeled
A.beta..sub.1-40, A.beta..sub.1-42 or mutant A.beta. (Dutch/Iowa40)
injected into the brain ISF was greater than 99% intact by TCA/HPLC
analysis. The A.beta. standards eluted between 29.1 and 31.2 min
for different A.beta. peptides. For SDS-PAGE analysis, TCA
precipitated samples were resuspended in 1% SDS, vortexed and
incubated at 55.degree. C. for 5 min, then neutralized, boiled for
3 min, homogenized, and analyzed by electrophoresis in 10%
Tris-Tricine gels followed by fluorography. These methods have been
previously described (Zlokovic et al., 1996; Shibata et al., 2000;
Deane et al., 2003).
Calculations
[0069] .sup.125I-A.beta. brain capillary uptake was corrected for
the distribution of .sup.14C-inulin (extracellular space marker)
and determined as the tissue to medium ratio as: c.p.m. for
TCA-precipitable .sup.125I-radioactivity (mg capillary
protein)/c.p.m. for TCA-precipitable .sup.125I-radioactivity (ml
medium) (1) (as described in Shibata et al., 2000). Briefly the
percentage of radioactivity remaining in the brain after
microinjection was determined as % recovery in
brain=100.times.(N.sub.b/N.sub.i) (2), where, N.sub.b is the
radioactivity remaining in the brain at the end of the experiment
and N.sub.i is the radioactivity injected into the brain ISF, i.e.,
the d.p.m. for .sup.14C-inulin and the c.p.m. for TCA-precipitable
.sup.125I-radioactivity (intact A.beta.). The percentage of A.beta.
cleared through the BBB was calculated as
[(1-N.sub.b(A.beta.)/N.sub.i(A.beta.))-(1-N.sub.b(inulin)/N.sub.i(inulin)-
)].times.100, using a standard time of 30 min (3). Efflux of
A.beta. from brain ISF via transport across the BBB at different
concentrations of peptides, J.sub.out, was calculated as
[(1-N.sub.b(A.beta.))-(1-N.sub.b(inulin)/N.sub.i(inulin))]/T.times.C.sub.-
A.beta. (4) where C.sub.A.beta.is A.beta. concentration in the
infusate. The half-saturation concentration for A.beta. elimination
via BBB transport, K.sub.m, was calculated from
J.sub.out=Cl.sub.max/(K.sub.m+C.sub.A.beta.) (4), where Cl.sub.max
(pmol/s/L ISF) represents the maximal efflux capacity for the
saturable A.beta. efflux across the BBB corrected for the rate of
ISF flow. The K.sub.m value for A.beta..sub.1-40 uptake by isolated
brain capillaries was calculated using Michaelis-Menten analysis.
The inhibitory constants, K.sub.i, were calculated from the
velocity ratios (Zlokovic et al., 1996) as
K.sub.i=(J.sub.i.times.K.sub.m.times.C.sub.i)/(J.sub.out-J.sub.i)(K.su-
b.m+C.sub.A.beta.40), where C.sub.i and C.sub.A.beta.40 were the
inhibitory concentrations of test-A.beta. peptide and
A.beta..sub.1-40 in the infusate in vivo or incubation medium in
vitro. Kinetic constants were obtained by a non-linear regression
curve fitting (PRISM 3.0 software).
Transgenic Mice
[0070] Tg-2576 mice in a C57BL6/SJL background (Hsiao et al., 1996)
and Tg-DI (Dutch/Iowa) mice in C57BL/6 background (Davis et al.,
unpublished observations) were used. Human APP (770 isoform) cDNA
harboring the Swedish (KM670/671NL), Dutch (E693Q), and Iowa
(D694N) mutations was subcloned between exons II and IV of a
Thy-1.2 expression cassette (a gift from Dr. F. LaFerla, University
of California, Irvine). The 9 kb transgene was liberated by
Notl/Pvul digestion, purified, and microinjected into pronuclei of
C57BL/6 single-cell embryos at the Stony Brook Transgenic Mouse
Facility. Founder transgenic mice were identified by Southern blot
analysis of tail DNA. Transgenic offspring were determined by PCR
analysis of tail DNA using the following primers for human APP to
generate a 500 base pair product.
Quantification of A.beta.
[0071] Soluble and insoluble pools of A.beta. peptides were
determined by ELISA of carbonate extracted forebrain tissue and of
guanidine lysates of the insoluble pellets resulting from the
carbonate extracted brain tissue, respectively (DeMattos et al.,
2002b). Levels of total A.beta. were compared between Tg-2576 and
Tg-DI mice.
Histological Analysis
[0072] For neuropathological analysis on mouse brain tissue in
Tg-2576 and Tg-DI mice, tissue sections were cut from mouse brain
hemispheres in the sagittal plane either at 5 .mu.m (paraffin
embedded fixed tissue) or 14 .mu.m (fresh frozen tissue). A.beta.
immunoreactive deposits were identified with human specific
mono-clonal mouse antibody 66.1 to A.beta. (Deane et al., 2003).
For LRP-1 staining on brain microvessels in RAP null, Tg-2576,
Tg-DI, and wild-type mice, 14 .mu.m frozen acetone-fixed tissue
sections were double immunostained for LRP-1 and CD31 (endothelial
marker). LRP-1-specific IgG (5A6) was used as a primary antibody.
Biotinylated anti-mouse IgG was used as a secondary antibody and
was detected with fluoresceinated streptavidin (1:1000, Vector
Laboratories, Burlingame, Calif.). M.O.M kit (Vector Laboratories,
Burlingame, Calif.) was used to block endogenous IgG (as described
in Sata et al., 2002). For CD31 staining, mouse CD31-specific IgG
was used as a primary antibody, and Alexa Fluor 594 donkey anti-rat
IgG (1:500, Molecular Probes, Eugene, Oreg.) as a second-dary
antibody.
Human Brain Endothelial Cells
[0073] Human brain endothelial cells (BEC) were isolated from rapid
autopsies of neurologically normal young individuals after trauma.
BECs were characterized and cultured (as described in Cheng et al.,
2003) and incubated with different AQ isoforms at concentrations
ranging from 1 nM to 20 .mu.M within 48 hr. Cells were lysed and
equal amounts of proteins electrophoresed (10 .mu.g/ml) on 10%
SDS-polyacrylamide gel, transferred onto nitrocellulose membrane
and probed with 5A6 (.beta.-chain) or 8G1 (.alpha.-chain) human
anti-LRP-1-specific IgGs. The relative density of each protein was
determined by scanning densitometry using .beta.-actin as an
internal control.
Metabolic Labeling
[0074] Human BEC (4.times.10.sup.5) were pulsed for 1 hr at
37.degree. C. with 400 pCi of [.sup.35S]-methionine (greater than
1000 Ci/mmol; Perkin Elmer Life Science, Boston, Mass.) in
methionine-free Dulbecco modified Eagle medium (GIBCO BRL, New
York, N.Y.) (as described in Guenette et al., 2002). Cells were
chased at the indicated times within 48 hr. Cell lysates were
immunoprecipitated with anti-LRP-1 515 kDa .alpha.-chain specific
IgG (8G1) on SDS-PAGE. The intensity of signal was quantified in
pixels using Storm 860 Phosphorlmager (Amersham Biosciences,
Piscataway, N.J.).
Statistical Analysis
[0075] Data were analyzed by multifactorial analysis of variance,
Student's t-test, and Dunnett's t test.
REFERENCES
[0076] Burdick et al. (1992) Assembly and aggregation properties of
synthetic Alzheimer's A4/.beta. amyloid peptide analogs. J. Biol.
Chem. 267:546-554. [0077] Cheng et al. (2003) Activated protein C
blocks p53-mediated apoptosis in ischemic human brain endothelium
and is neuroprotective. Nature Med. 9:338-342. [0078] Cirrito et
al. (2003) In vivo assessment of brain interstitial fluid with
microdialysis reveals plaque-associated changes in amyloid-.beta.
metabolism and half-life. J. Neurosci. 23:8844-53. [0079] Dahlgren
et al. (2002) Oligomeric and fibrillar species of amyloid-.beta.
peptides differentially affect neuronal viability. J. Biol. Chem.
277:32046-32053. [0080] Deane et al. (2003) RAGE mediates
amyloid-.beta. transport across the blood-brain barrier and
accumulation in brain. Nature Med. 9:907-913. [0081] DeMattos et
al. (2002a) Brain to plasma amyloid-A.beta. efflux: A measure of
brain amyloid burden in a mouse model of Alzheimer's disease.
Science. 295:2264-226. [0082] DeMattos et al. (2002b) Clusterin
promotes amyloid plaque formation and is critical for neuritic
toxicity in a mouse model of Alzheimer's disease. Proc. Natl. Acad.
Sci. USA 99:10843-10849. [0083] Golabek et al. (1996) The
interaction between apolipoprotein E and Alzheimer's amyloid
beta-peptide is dependent on beta-peptide conformation. J Biol.
Chem. 271:10602-10606. [0084] Gong et al. (2003) Alzheimer's
disease-affected brain: Presence of oligomeric A.beta. ligands
(ADDLs) suggests a molecular basis for reversible memory loss.
Proc. Natl. Acad. Sci. USA 100:10417-10422. [0085] Guenette et al.
(2002) Low-density lipoprotein receptor-related protein levels and
endocytic function are reduced by overexpression of the FE65
adaptor protein, FE65L1. J. Neurochem. 82:755-762. [0086] Guo et
al. (2004) Activated protein C prevents neuronal apoptosis via
protease activated receptors 1 and 3. Neuron 41:563-572. [0087]
Hardy and Selkoe (2002) The amyloid hypothesis of Alzheimer's
disease: Progress and problems on the road to therapeutics.
Science. 297:353-356. [0088] Herz and Strickland (2001) LRP: A
multifunctional scavenger and signaling receptor. J. Clin. Invest.
108:779-784. [0089] Herz et al. (1988) Surface location and high
affinity for calcium of a 500-kd liver membrane protein closely
related to the LDL-receptor suggest a physiological role as
lipoprotein receptor. EMBO J. 7:4119-4127 [0090] Hsiao at al.
(1996) Correlative memory deficits, A.beta. elevation, and amyloid
plaques in transgenic mice. Science 274:99-102. [0091] Jordan et
al. (1998) Isoform-specific effect of apolipoprotein E on cell
survival and .beta.-amyloid-induced toxicity in rat hippocampal
pyramidal neuronal cultures. J. Neurosci. 18:195-204. [0092] Kang
et al. (1997) Genetic association of the low-density lipoprotein
receptor-related protein gene (LRP), an apolipoprotein E receptor,
with late-onset Alzheimer's disease. Neurology 49:56-61. [0093]
Kang et al. (2000) Modulation of amyloid .beta.-protein clearance
and Alzheimer's disease susceptibility by the LDL receptor-related
protein pathway. J. Clin. Invest. 106:1159-1166. [0094]
Kawarabayashi et al. (2001) Age-dependent changes in brain, CSF,
and plasma amyloid A.beta. protein in the Tg2576 transgenic mouse
model of Alzheimer's Disease. J. Neuroscience. 21:372-381. [0095]
Kayed et al. (2003) Common structure of soluble amyloid oligomers
implies common mechanism of pathogenesis. Science 300:486-489.
[0096] Kounnas et al. (1995) -LDL receptor-related protein, a
multifunctional ApoE receptor, binds secreted beta-amyloid
precursor protein and mediates its degradation. Cell 82:331-340.
[0097] Li et al. (2001a) Differential functions of members of the
low density lipoprotein receptor family suggested by their distinct
endoytosis rates. J. Biol. Chem. 276:18000-18006. [0098] Li et al.
(2001b) Identification of a major cyclic AMP-dependent protein
kinase A phosphorylation site within the cytoplasmic tail of the
low-density lipoprotein receptor-related protein: Implication for
receptor-mediated endocytosis. Mol. Cell. Biol. 21:1185-1195.
[0099] Matsuoka at al. (2003) Novel therapeutic approach for the
treatment of Alzheimer's disease by peripheral administration of
agents with an affinity to A.beta.-amyloid. J Neurosci. 23:29-33.
[0100] Melman et al. (2002) Proteasome regulates the delivery of
LDL receptor-related protein into the degradation pathway. Mol.
Biol. Cell 13:3325-3335. [0101] Monro et al. (2002) Substitution at
codon 22 reduces clearance of Alzheimer's amyloid-beta peptide from
the cerebrospinal fluid and prevents its transport from the central
nervous system into blood. Neurobiol. Aging 23:405-412. [0102]
Narita et al. (1997) .alpha..sub.2-Macroglobulin complexes with and
mediates the endocytosis of .beta.-amyloid peptide via cell surface
low-density lipoprotein receptor-related protein. J. Neurochem.
69:1904-1911. [0103] Neuwelt et al. (1985) Osmotic blood-brain
barrier modification: Monoclonal antibody, albumin, and
methotrexate delivery to cerebrospinal fluid and brain.
Neurosurgery 17:419-423. [0104] Pietrzik et al. (2002) The
cytoplasmic domain of the LDL receptor-related protein regulates
multiple steps in APP processing. EMBO. J. 21:5691-5700. [0105] Qiu
et al. (1999) .alpha..sub.2-Macroglobulin enhances the clearance of
endogenous soluble .beta.-amyloid peptide via low-density
lipoprotein receptor-related protein in cortical neurons. J.
Neurochem. 73:1393-1398. [0106] Sata et al. (2002) Hematopoietic
stem cells differentiate into vascular cells that participate in
the pathogenesis of atherosclerosis. Nature Med. 8:403-409. [0107]
Selkoe (2001) Clearing the brain's amyloid cobwebs. Neuron
32:177-180. [0108] Shibata et al. (2000) Clearance of Alzheimer's
amyloid-A.beta..sub.1-40 peptide from brain by low-density
lipoprotein receptor-related protein-1 at the blood-brain barrier
J. Clin. Invest. 106:1489-1499. [0109] Silverberg et al. (2003)
Alzheimer's disease, normal-pressure hydrocephalus, and senescent
changes in CSF circulatory physiology: A hypothesis. Lancet Neurol.
2:506-511. [0110] Ulery et al. (2000) Modulation of .beta.-amyloid
precursor protein processing by the low density lipoprotein
receptor-related protein (LRP). Evidence that LRP-1 contributes to
the pathogenesis of Alzheimer's Disease. J. Biol. Chem.
275:7410-7415. [0111] Van Nostrand et al. (2001) Pathogenic effects
of D23N Iowa amyloid .beta.-protein. J. Biol. Chem.
276:32860-32866. [0112] Van Uden et al. (2002) Increased
extracellular amyloid deposition and neurodegeneration in human
amyloid precursor protein transgenic mice deficient in
receptor-associated protein. J. Neurosci. 22:9298-9304. [0113]
Vinters and Farag (2003) Amyloidosis of cerebral arteries. Adv.
Neurol. 92:105-112. [0114] Walsh et al. (2002) Naturally secreted
oligomers of amyloid .beta. protein potently inhibit hippocampal
long-term potentiation in vivo. Nature 416:535-539. [0115] Westein
et al. (2002) The .alpha.-chains of C4b-binding protein mediate
complex formation with low density liporpoprotein receptor-related
protein. J. Biol. Chem. 277:2511-2516. [0116] Wu et al. (2003) A
simple method for isolation and characterization of mouse brain
microvascular endothelial cells. J. Neurosci. Meth. 130:53-63.
[0117] Zerbinatti et al. (2004) Increased soluble amyloid .beta.
peptide and memory deficits in amyloid model mice overexpressing
the LDL receptor-related protein. Proc. Natl. Acad. Sci. USA
101:1075-1080. [0118] Zlokovic and Frangione (2003)
Transport-clearance hypothesis for Alzheimer's disease and
potential therapeutic implications. A.beta. Metabolism in
Alzheimer's Disease. Ed. T. Saido (Landes Bioscience) 114-122.
[0119] Zlokovic et al. (1996) Glycoprotein 330/megalin: Probable
role in receptor-mediated transport of apolipoprotein J alone and
in a complex with Alzheimer's disease amyloid-A.beta. at the
blood-brain and blood-cerebrospinal fluid barriers. Proc. Natl.
Acad. Sci. USA 93:4229-4236. [0120] Zlokovic et al. (2000)
Clearance of amyloid-A.beta.-peptide from brain: Transport or
metabolism? Nature Med. 6:718-719.
[0121] Patents, patent applications, books, and other publications
cited herein are incorporated by reference in their entirety.
[0122] All modifications and substitutions that come within the
meaning of the claims and the range of their legal equivalents are
to be embraced within their scope. A claim using the transition
"comprising" allows the inclusion of other elements to be within
the scope of the claim; the invention is also described by such
claims using the transition "consisting essentially of" (i.e.,
allowing the inclusion of other elements to be within the scope of
the claim if they do not materially affect operation of the
invention) and the transition "consisting" (i.e., allowing only the
elements listed in the claim other than impurities or
inconse-quential activities which are ordinarily associated with
the invention) instead of the "comprising" term. For example,
"consisting essentially of cluster II and/or cluster IV" would
allow the inclusion of other functional domains if the latter did
not affect binding of A.beta. while "consisting of cluster II
and/or cluster IV" would prohibit the inclusion of other functional
domains. Any of these three transitions can be used to claim the
invention.
[0123] It should be understood that an element described in this
specification should not be construed as a limitation of the
claimed invention unless it is explicitly recited in the claims.
For example, variants of LRP-1 are known as homologs, mutations,
and polymorphisms in the known nucleotide and amino acid sequences.
Thus, the granted claims are the basis for determining the scope of
legal protection instead of a limitation from the specification
which is read into the claims. In contradistinction, the prior art
is explicitly excluded from the invention to the extent of specific
embodiments that would anticipate the claimed invention or destroy
novelty.
[0124] Moreover, no particular relationship between or among
limitations of a claim is intended unless such relationship is
explicitly recited in the claim (e.g., the arrangement of
components in a product claim or order of steps in a method claim
is not a limitation of the claim unless explicitly stated to be
so). All possible combinations and permutations of individual
elements disclosed herein are considered to be aspects of the
invention. Similarly, generalizations of the invention's
description are considered to be part of the invention.
[0125] From the foregoing, it would be apparent to a person of
skill in this art that the invention can be embodied in other
specific forms without departing from its spirit or essential
characteristics. The described embodiments should be considered
only as illustrative, not restrictive, because the scope of the
legal protection provided for the invention will be indicated by
the appended claims rather than by this specification.
* * * * *