U.S. patent application number 10/794425 was filed with the patent office on 2004-12-23 for role of ldl receptor protein-1 (lrp-1) in alzheimer's disease.
Invention is credited to Zlokovic, Berislav V..
Application Number | 20040259159 10/794425 |
Document ID | / |
Family ID | 26901346 |
Filed Date | 2004-12-23 |
United States Patent
Application |
20040259159 |
Kind Code |
A1 |
Zlokovic, Berislav V. |
December 23, 2004 |
Role of LDL receptor protein-1 (LRP-1) in Alzheimer's disease
Abstract
Brain endothelial low-density lipoprotein receptor related
protein-1 (LRP-1) mediates vascular clearance of Alzheimer's
amyloid-.beta. peptide (A.beta.) from the brain. Transport of
A.beta. occurs across the blood-brain barrier (BBB) to the systemic
circulation, but the brain endothelium is compromised in
Alzheimer's disease. The invention is used to diagnose the disease
in symptomatic and asymptomatic individuals, to identify those at
risk for disease or already affected thereby, to determine the
stage of disease or its progression, to intervene earlier in or
alter the disease's natural history, to provide a target for
therapeutic or prophylactic treatments, to screen drugs or compare
medical regimens, to determine the effectiveness of a drug or
medical regimen, or any combination thereof.
Inventors: |
Zlokovic, Berislav V.;
(Rochester, NY) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
1100 N GLEBE ROAD
8TH FLOOR
ARLINGTON
VA
22201-4714
US
|
Family ID: |
26901346 |
Appl. No.: |
10/794425 |
Filed: |
March 8, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10794425 |
Mar 8, 2004 |
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10296168 |
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10296168 |
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PCT/US01/16561 |
May 23, 2001 |
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60206428 |
May 23, 2000 |
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60246268 |
Nov 6, 2000 |
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Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
G01N 2800/52 20130101;
C12Q 2600/158 20130101; C12Q 1/6883 20130101; G01N 33/92 20130101;
G01N 33/6896 20130101; A61P 25/28 20180101; G01N 2800/2821
20130101 |
Class at
Publication: |
435/007.1 |
International
Class: |
G01N 033/53 |
Goverment Interests
[0002] The U.S. government has certain rights in this invention as
provided for by the terms of grants awarded by the National
Institutes of Health.
Claims
What is claimed is:
1. A method of at least diagnosing an individual with Alzheimer's
disease or identifying an individual as being at risk for
developing Alzheimer's disease, said method comprising: (a)
measuring at least abundance of low-density lipoprotein receptor
related protein-1 (LRP-1), abundance of transcripts thereof, or
LRP-1 receptor activity in an individual, (b) comparing the
abundance of LRP-1 protein, abundance of transcripts thereof, or
activity of LRP-1 receptor in said individual to at least one
control, and (c) at least diagnosing said individual with
Alzheimer's disease or identifying said individual as being at risk
for developing Alzheimer's disease when the abundance of LRP-1
protein, abundance of transcripts thereof, or activity of LRP-1
receptor in said individual is decreased relative to said
control.
2. The method of claim 1, wherein the abundance of LRP-1 protein,
abundance of transcripts thereof, or activity of LRP-1 receptor is
measured at least in a brain vascular or systemic endothelial cell
of said individual.
3. The method of claim 1, wherein the abundance of LRP-1 protein,
abundance of transcripts thereof, or activity of LRP-1 receptor is
measured at least in a brain capillary, a temporal artery, a
leptomeningeal artery, or at the blood-brain barrier of said
individual.
4. The method of claim 1, wherein said at least one control is a
value indicative of increased risk for developing Alzheimer's
disease and/or a value indicative of decreased risk for developing
Alzheimer's disease.
5. A method of at least treating an individual with Alzheimer's
disease or at risk for developing Alzheimer's disease, said method
comprising increasing low-density lipoprotein receptor related
protein-1 (LRP-1) mediated removal of amyloid-.beta. peptide
(A.beta.) at the blood-brain barrier in said individual in the
direction from brain to blood.
6. The method of claim 5, wherein removal of A.beta. is increased
by increasing at least LRP-1 expression or receptor activity.
7. The method of claim 5, wherein removal of A.beta. is increased
by increasing at least expression of a ligand of LRP-1 or transport
of said ligand.
8. The method of claim 7, wherein said ligand of LRP-1 is
apolipoprotein E.
9. The method of claim 7, wherein said ligand of LRP-1 is
.alpha..sub.2-macroglobulin.
10. The method of claim 7, wherein LRP-1 mediated removal of
A.beta. is increased by administering at least one proteasome
inhibitor.
11. A method of at least treating an individual with Alzheimer's
disease or at risk for developing Alzheimer's disease, said method
comprising increasing vascular clearance of amyloid-.beta. peptide
(A.beta.) from the brain of said individual.
12. The method of claim 11, wherein vascular permeability to
A.beta. is increased.
13. The method of claim 11, wherein at least angiogenesis or
neovasularization in the brain is increased.
14. A method of determining the effectiveness of at least a drug or
medical regimen for treating Alzheimer's disease, said method
comprising: (a) measuring the abundance of low-density lipoprotein
receptor related protein-1 (LRP-1), abundance of transcripts
thereof, or activity of LRP-1 receptor in the presence of said drug
or medical regimen, and (b) at least determining said drug or
medical regimen is effective when the abundance of LRP-1 protein,
abundance of transcripts thereof, or activity of LRP-1 receptor is
increased in the presence of said drug or medical regimen.
15. The method of claim 14 further comprising decreasing said
abundance of LRP-1, abundance of transcripts thereof, or activity
of LRP-1 receptor prior to treatment.
16. The method of claim 15, wherein said abundance of LRP-1,
abundance of transcripts thereof, or activity of LRP-1 receptor is
decreased by at least one soluble amyloid-.beta. peptide (A.beta.)
and/or aggregate thereof.
17. A novel drug or use of a medical regimen for treating
Alzheimer's disease determined to be effective by the method of
claim 14.
18. The drug or use of a medical regimen of claim 17 which
comprises a proteasome inhibitor.
19. A kit comprised of one or more of a specific binding molecule
for low-density lipoprotein receptor related protein-1 (LRP-1) or a
transcript thereof used to measure abundance of said LRP-1 protein,
abundance of transcripts thereof, or LRP-1 receptor activity,
and/or a non-Alzheimer's disease control containing LRP-1 protein
or a transcript thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation in-part of U.S.
application Ser. No. 10/296,168, filed Jun. 30, 2003, pending;
which was filed under 35 U.S.C. 371 as the national stage of Int'l
Appln. No. PCT/US01/16561, filed May 23, 2001; which claims the
benefit of provisional U.S. Appln. No. 60/206,428 filed May 23,
2000 and U.S. Appln. No. 60/246,268 filed Nov. 6, 2000.
FIELD OF THE INVENTION
[0003] The invention relates to the role of the vascular system in
clearing Alzheimer's amyloid-.beta. peptide (A.beta.) from the
central nervous system (CNS) as mediated by low-density lipoprotein
receptor related protein-1 (LRP-1). Transport of A.beta. occurs
across the blood-brain barrier (BBB) to the systemic circulation,
but the brain endothelium is compromised in Alzheimer's disease.
The invention is used to diagnose symptomatic and asymptomatic
individuals, to identify those at risk for disease or already
affected thereby, to determine the stage of disease or its
progression, to intervene earlier in or alter the disease's natural
history, to provide a target for therapeutic or prophylactic
treatments, to screen drugs or compare medical regimens, to
determine the effectiveness of a drug or medical regimen, or any
combination thereof.
BACKGROUND OF THE INVENTION
[0004] Deposition of amyloid-.beta. peptide (A.beta.) in brain
occurs during normal aging and is accelerated in individuals with
Alzheimer's disease (AD). A.beta. is central to pathology of AD,
and is the main constituent of brain parenchymal and vascular
amyloid (1-6). A.beta. extracted from senile plaques contains
mainly A.beta..sub.1-40 and A.beta..sub.1-42 (7), while vascular
amyloid is predominantly A.beta..sub.1-39 and A.beta..sub.1-40 (8).
Several sequences of A.beta. were found in both lesions (9-11). A
major soluble form of A.beta., which is present in the blood,
cerebrospinal fluid (CSF) (12-14) and brain (15-16) is
A.beta..sub.1-40. In the circulation, CSF and brain interstitial
fluid (ISF), soluble A.beta. may exist as a free peptide and/or
associated with different transport binding proteins such as
apolipoprotein J (apoJ) (17-18), apolipoprotein E (apoE) (19),
transthyretin (20), lipoproteins (21), albumin (22), and alpha-2
macroglobulin (.alpha..sub.2M) (23).
[0005] The neuronal theory argues that soluble brain-derived
A.beta. is a precursor of A.beta. deposits. Neuronal cells secrete
A.beta. in culture (24), which supports this view. An increase in
soluble A.beta. in AD and Down's Syndrome brains precedes amyloid
plaque formation (15, 25, 26), and correlates with the development
of vascular pathology (27). Several cytosolic proteases that may
degrade intracellular A.beta. in vitro cannot degrade extracellular
A.beta. from brain ISF (28) or CSF (29) in vivo. An exception is
enkephalinase that may degrade A.beta..sub.1-42 from brain ISF
(28). However, the physiological importance of this degradation in
vivo remains still unclear since the peptide was studied at
extremely high pharmacological concentrations (30).
[0006] It has been suggested that decreased clearance of A.beta.
from brain and CSF is the main cause of A.beta. accumulation in
sporadic AD (31). Since A.beta. is continuously produced in the
brain, a working hypothesis in this study was that efficient
clearance mechanism(s) must exist at the blood-brain barrier (BBB)
to prevent its accumulation and subsequent aggregation in the
brain. Cell surface receptors such as the receptor for advanced
glycation end products (RAGE) (32-33), scavenger type A receptor
(SR-A) (34), low-density lipoprotein receptor-related protein-1
(LRP-1) (35-38) and LRP-2 (39) bind A.beta. at low nanomolar
concentrations as free peptide (e.g., RAGE, SR-A), and/or in
complex with (.alpha..sub.2M, apoE or apoJ (e.g., LRP-1, LRP-2).
RAGE and SR-A regulate brain endothelial endocytosis and
transcytosis of A.beta. initiated at the luminal side of the BBB
(33), while LRP-2 mediates BBB transport of plasma A.beta.
complexed to apoJ (39). Recent work shows that 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 (73) suggesting that LRP-1 on neurons in vivo does not
mediate A.beta. clearance from the brain. The role of vascular
receptors and BBB transport in the removal of brain-derived A.beta.
was previously unknown.
[0007] In the present study, a technique to measure brain tissue
clearance in mice was developed based on a previous model in the
rabbit (40). This technique was used to determine in vivo the
efflux rates of A.beta..sub.1-40 from the CNS as a function of time
and concentration of peptide, and to characterize vascular
transport and/or receptor-mediated efflux mechanism(s) involved in
elimination of brain-derived A.beta. across the BBB. The study
focused on LRP-1 and its ligands, (.alpha..sub.2M and apoE because
both promote A.beta. clearance in smooth muscle cells (35), neurons
(36, 38), and fibroblasts (37); and the apoE4 genetic locus is
definitely, and the .alpha..sub.2M genetic locus is possibly
associated with increasing the risk for AD (41-42). Reduced
LRP-1-mediated A.beta. transport across the BBB was associated with
AD. At pathological concentrations (greater than 1 .mu.M), A.beta.
promotes LRP-1 degradation in brain endothelium consistent with the
reduced LRP-1 brain capillary levels observed in AD. Thus, loss of
LRP-1 at the BBB mediate brain accumulation of neurotoxic A.beta..
This loss can be prevented.
[0008] This study can be used to improve the understanding of the
pathogenesis of Alzheimer's disease and mechanisms of disease. New
and nonobvious modes of diagnosis and treatment are suggested by
this discovery. Other advantages of the invention are discussed
below or would be apparent to persons in the art from the
disclosure herein.
SUMMARY OF THE INVENTION
[0009] In one embodiment of the invention, reagents are provided in
kit form that can be used for performing the methods such as the
following: 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
databases containing patient information (e.g., genotype, medical
history, symptoms, transcription or translation yields from gene
expression, physiological or pathological findings) are other
products that can be considered aspects of the invention.
[0010] In other embodiments of the invention, these methods for
diagnosis and treatment are provided. For screening of drugs and
clinical trials, the respective drug and medical regimen selected
are also considered embodiments of the invention. The amount and
extent of treatment administered to a cell, tissue, or individual
in need of therapy or prophylaxis is effective in treating the
affected cell, tissue, or individual. One or more
properties/functions of affected endothelium or cells thereof, or
the number/severity of symptoms of affected individuals, may be
improved, reduced, normalized, ameliorated, or otherwise
successfully treated. The invention may be used alone or in
combination with other known methods. Instructions for performing
these methods, reference values and positive/negative controls, and
relational data-bases containing patient information are considered
further aspects of the invention. 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.
[0011] 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.
DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1A shows time-disappearance curves of .sup.14C-inulin
(open circles) and .sup.125I-A.beta..sub.1-40 (60 nM;
TCA-precipitable .sup.125I-radioactivity, filled circles) from the
CNS following simultaneous microinjections of tracers into the
caudate nucleus in mice. Each point is mean.+-.SD, from 3 to 7
animals. FIG. 1B shows two components of .sup.125I-A.beta..sub.1-40
efflux. Vascular transport across the BBB (filled triangles) and
transport by ISF bulk flow (open triangles) were computed with eqs.
3 and 4, using data from FIG. 1A. FIG. 1C shows relative
contributions to A.beta..sub.1-40 efflux by transport across the
BBB (open bar), diffusion by ISF bulk flow (closed bar), and
retention (shaded bar) in the brain were studied at 60 nM
concentrations and calculated from fractional coefficients given in
Table 1.
[0013] FIG. 2 shows time-appearance curves of .sup.14C-inulin (open
circles) and .sup.125I-A.beta..sub.1-40 (60 nM; TCA-precipitable
.sup.125I-radioactivity, filled circles) in the CSF (FIG. 2A) and
plasma (FIG. 2B) following simultaneous microinjections of tracers
into the caudate nucleus in mice. Values are expressed as % of
injected dose (% ID); each point is mean.+-.SD from 3 to 7
animals.
[0014] FIG. 3A shows brain TCA precipitable (open bars) and TCA
non-precipitable .sup.125I-radioactivity (solid bars) following
intracerebral microinjections of .sup.125I-A.beta..sub.1-40 (60 nM)
into the caudate nucleus in mice expressed as a percentage of total
.sup.125I-radioactivity in brain; mean.+-.SD is from 3 to 5
animals. FIG. 3B shows an HPLC elution profile of brain tissue
(right panel) 60 min following intracerebral microinjection of
.sup.125I-A.beta..sub.1-40 (60 nM). Separation was performed for 30
mg of brain tissue on a reverse-phase HPLC column, using a 30-min
linear gradient of 25% to 83% acetonitrile in 0.1% TFA, pH 2.
.sup.125I-A.beta..sub.1-40 eluted at 52% corresponding to the
elution time of A.beta..sub.1-40 standard. The left panel shows
SDS/PAGE analysis of brain tissue supernatant at 30 min (lane a)
and 60 min (lane b) following intracerebral micro-injection of
.sup.125I-A.beta..sub.1-40 (60 nM). The radioactivity in the brain
was eluted as a single peak on HPLC with the same retention time as
the A.beta..sub.1-40 standard. Aliquots of lyophilized sample were
subjected to 10% Tris-tricine SDS/PAGE, transferred to a
nitrocellulose membrane, and exposed to X-ray film. FIG. 3C shows
plasma TCA precipitable (open bars) and TCA nonprecipitable
.sup.125I-radioactivity (solid bars) following intracerebral
microinjections of .sup.125I-A.beta..sub.1-40 (60 nM) into the
caudate nucleus in mice expressed as a percentage of total
.sup.125I-radioactivity in plasma; mean.+-.SD is from 3 to 5
animals.
[0015] FIG. 4A shows concentration-dependent clearance of
A.beta..sub.1-40 from mouse brain. Clearance was determined 30 min
after simultaneous microinjections of .sup.125I-A.beta..sub.1-40 at
increasing concentrations (0.05 nM to 120 nM) along with
.sup.14C-inulin into the caudate nucleus. Clearance by BBB
transport (filled circles) are shown separately from clearance by
ISF bulk flow (open circles). FIG. 4B shows the effects with
(closed bars) or without (open bars) of anti-LRP-1 antibody R777
(60 .mu.g/ml), RAP (0.2 .mu.M and 5 .mu.M), anti-.alpha..sub.2M
antibody (20 .mu.g/ml) and anti-LRP-2 antibody Rb6286 (60 .mu.g/ml)
on brain clearance of .sup.125I-A.beta.1-40 at 12 nM determined 30
min after simultaneous .sup.125I-A.beta..sub.1-40/.sup.14C-- inulin
microinjections. FIG. 4C shows the effects of anti-LRP-1 antibody
R777 (60 .mu.g/ml), anti-RAGE antibody (60 .mu.g/ml), fucoidan (100
.mu.g/ml) and 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH,
10 mM) on brain clearance of .sup.125I-A.beta..sub.1-40 at higher
load of 60 nM determined 30 min after simultaneous
.sup.125I-A.beta..sub.1-40/.sup.14C-- inulin microinjections.
Mean.+-.SD from 3 to 4 animals; *-p<0.05, ns--not
significant.
[0016] FIG. 5 shows the effect of apoE genotype and age on brain
clearance of .sup.125I-A.beta..sub.1-40. Brain clearance of
.sup.125I-A.beta..sub.1- -40 in 2-month old and 9-month old
wild-type mice and apoE KO mice studied at a lower load of
.sup.125I-A.beta..sub.1-40 of 12 nM (FIG. 5A) and a higher load of
60 nM (FIG. 5B). In all studies, .sup.125I-A.beta..sub.1-4- 0
(closed bars) and .sup.14C-inulin (open bars) were injected
simultaneously and clearance determined after 30 min.
[0017] Mean.+-.SD from 3 to 4 animals; *-p<0.05, ns--not
significant in comparison to 2-month old wild-type mice.
[0018] FIG. 6 shows that LRP-1 is down regulated in human brain
endothelium by excess A.beta.. Western blot analyses of LRP-1
.beta.-subunit (LRP-85) expression in brain endothelium exposed for
48 hr to different concentrations of A.beta..sub.1-40,
A.beta..sub.1-42, or mutant A.beta. (Dutch/Iowa40;
DIA.beta..sub.1-40) from 1 nM to 20 .mu.M (FIGS. 6A-6C). RAP (1.25
.mu.M) and anti-LRP-1 .beta.-chain-specific IgG F(ab).sub.2 (25
.mu.g/ml) were incubated for 1 hr prior to the addition of A.beta.
peptides and then for 48 hr simultaneously with different A.beta.
peptides (FIG. 6D). Relative intensity of LRP-1 band in brain
endothelium exposed to different A.beta. peptides was determined by
scanning densitometry and normalized for .beta.-actin. Mean.+-.SEM
(n=3 to 5 independent experiments); *p<0.01; **p<0.05 for the
relative expression of LRP-1 levels in the presence of a given
A.beta. concentration compared to in the absence A.beta..
[0019] FIG. 7 shows that high A.beta. levels reduce LRP-1's
half-life in human brain endothelium. Cells were labeled by
[.sup.35S]-methionine for 1 hr and chased for the indicated times.
FIG. 7A is an autoradiograph of [.sup.35S]-methionine labeled LRP-1
was immunoprecipitated with anti-LRP-1 515 kDa
.alpha.-subunit-specific IgG in the presence or absence of
A.beta..sub.1-42 (20 .mu.M). FIG. 7B is a graph of LRP-1 levels
from 3 independent experiments as in FIG. 7A. *p<0.05;
**p<0.01 in the presence of A.beta. compared to the
corresponding control value. Effects of the proteasomal inhibitor
MG132 (20 .mu.M) on LRP-1 levels in the presence or absence of
A.beta..sub.1-42 (20 .mu.M) was determined at 8 hr (FIG. 7C). FIG.
7D is an autoradiograph of [.sup.35S]-methionine labeled LRP-1
immunoprecipitated with anti-LRP-1 515 kDa .alpha.-subunit specific
IgG. FIG. 7E is a graph of LRP-1 levels from 3 independent
experiments as in FIG. 7C. Western blot analysis for immature 600
kDa LRP-1 (LRP-600) in the presence of A.beta..sub.1-40,
A.beta..sub.1-42 or mutant A.beta. (Dutch/Iowa40,
DIA.beta..sub.140) at 20 .mu.M within 48 hr, as demonstrated with
LRP-1-specific IgG directed to the C-terminal region of LRP-1
(5A6). RAP was used at 1.2 .mu.M. Mean.+-.SEM (n=3) in FIGS. 7B and
7D.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS
[0020] Elimination of amyloid-.beta. peptide (A.beta.) from the
brain has been poorly understood. Following intracerebral
microinjections in young mice, .sup.125I-A.beta..sub.14-0 was
rapidly removed from the brain (t.sub.1/2.ltoreq.25 min) mainly by
vascular transport across the blood-brain barrier (BBB)
(.gtoreq.74%). The efflux transport system for A.beta..sub.1-40 at
the BBB was half-saturated (K.sub.0.5) at 15.3 nM and the maximal
transport capacity was reached between 70 and 100 nM.
A.beta..sub.1-40 clearance was inhibited by the receptor associated
protein (44%) and antibodies against low-density lipoprotein
receptor-related protein-1, LRP-1 (58%) and alpha 2-macroglobulin,
.alpha..sub.2M (25%). Clearance was significantly reduced in young
(30%) and old (46%) apolipoprotein E (apoE) knockout mice, and in
old, wild-type mice (55%). There was no evidence that A.beta. was
metabolized in brain interstitial fluid and degraded to smaller
peptide fragments and amino acids prior to its transport across the
BBB into the circulation. LRP-1, although abundant in brain
microvessels in young mice, was down-regulated in older animals
(45%). Down-regulation of vascular LRP-1 correlated with regional
A.beta. accumulation in brains of Alzheimer's disease patients. The
BBB removes A.beta. from the brain largely by an age-dependent,
LRP-1-mediated transport mechanism that is influenced by
.alpha..sub.2M and/or apoE. This mechanism appears to be impaired
in Alzheimer's disease at the level of transcript or protein
abundance or receptor function (e.g., time required for receptor
transcytosis or recycling, efficiency of ligand transport across
the BBB).
[0021] High extracellular accumulation of soluble A.beta. affects
LRP-1 expression at the A.beta. clearance site(s) in the brain. The
interaction of A.beta. and LRP-1 on brain capillary membranes
regulates retention of a high .beta.-sheet content neurotoxic
A.beta..sub.1-42 and vasculotropic mutant A.beta. while clearing
A.beta..sub.1-40. At pathological concentrations, A.beta. down
regulates LRP-1 in brain endothelium by promoting its
proteasome-dependent degradation. This down regulation may be
reversed by one or more inhibitors of the ubiquitin-proteasome
pathway.
[0022] Preparations of endothelial cells, isolated tissues, and in
vitro cell cultures are provided from brain (e.g.,
microvasculature) or other organs (e.g., skin) of individuals at
risk for Alzheimer's disease, affected by the disease, or not. In
particular, tissues like endothelium, smooth muscle, blood vessels
and capillaries of the brain, temporal and leptomeningeal arteries,
or any other tissues that express LRP-1 can be examined. Blood and
bone marrow cells might also be used. They can be obtained as
biopsy or autopsy material; cells of interest may be isolated
therefrom and then cultured. Also provided are extracts of cells;
at least partially purified DNA, RNA, and protein therefrom; and
methods for their isolation. These reagents can be used to
establish detection limits for assays, absolute amounts of gene
expression that are indicative of disease or not, ratios of gene
expression that are indicative of disease or not, and the
significance of differences in such values. These values for
positive and/or negative controls can be measured at the time of
assay, before an assay, after an assay, or any combination thereof.
Values may be recorded on storage medium and manipulated with
computer software; storage in a database allows retrospective or
prospective study. Gene expression (e.g., detected by antibody
staining) and protein activity of LRP-1 (e.g., vascular clearance
of A.beta. from the brain to the systemic circulation) was
decreased in individuals with Alzheimer's disease.
[0023] Polynucleotides representative of genes whose expression is
decreased in Alzheimer's disease may be used to identify, isolate,
or detect complementary polynucleotides by binding assays.
Similarly, polypeptides representative of the gene products that
are decreased in Alzheimer's disease may be used to identify,
isolate, or detect interacting proteins by binding assays.
Optionally, bound complexes including interacting proteins may be
identified, isolated, or detected indirectly though a specific
binding molecule (e.g., antibody) for the gene product that is
decreased in Alzheimer's disease. For the receptor-ligand system
studied here, LRP-1, apoE and .alpha..sub.2M and are interacting
proteins. 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) or for treatment;
conveniently, they are packaged as assay kits or in pharmaceutical
form.
[0024] Assaying Polynucleotides or Polypeptides
[0025] Binding of polynucleotides or polypeptides may take place in
solution or on a substrate. The assay format may or may not require
separation of bound from not bound. 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 polynucleotide or polypeptide,
interacting protein, or specific binding molecule 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.
[0026] Polynucleotide, polypeptide, or specific binding molecule
may 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,
nitro-cellulose, uncharged nylon, or positively-charged nylon;
natural rubber, butyl rubber, silicone rubber, or styrenebutadiene
rubber; agarose or polyacrylamide; crystalline, amorphous, or
impure silica (e.g., quartz) or silicate (e.g., glass);
polyacrylonitrile, polycarbonate, polyethylene, polymethyl
methacrylate, polymethylpentene, polypro-pylene, 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.
[0027] Such reagents would allow capture of a molecule in solution
by specific interaction between the cognate molecules and then
could immobilize the molecule on the substrate. Monitoring gene
expression is facilitated by using an array.
[0028] Polynucleotide, polypeptide, or specific binding molecule
may be synthesized in situ by solid-phase chemistry or
photolithography to directly attach the nucleotides or amino acids
to the substrate. Attachment of the polynucleotide, polypeptide, or
specific binding molecule to the substrate may be 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
polynucleotide, polypeptide, or specific binding molecule 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] Changes in gene expression may be manifested in the cell by
affecting transcripitional initiation, transcript stability,
translation of transcript into protein product, protein stability,
or a combination thereof. The abundance of transcript or
polypeptide can be measured by techniques such as in vitro
transcription, in vitro translation, Northern hybridization,
nucleic acid hybridization, reverse transcription-polymerase chain
reaction (RT-PCR), run-on transcription, Southern hybridization,
cell surface protein labeling, metabolic protein labeling, antibody
binding, immunoprecipitation (IP), enzyme linked immunosorbent
assay (ELISA), radioimmunoassay (RIA), fluorescent or histochemical
staining, microscopy and digital image analysis, and fluorescence
activated cell analysis or sorting (FACS).
[0030] A reporter or selectable marker gene whose protein product
is easily assayed may be used for convenient detection. Reporter
genes 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, respectively), 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). Reporter genes would use cognate substrates
that are preferably assayed by a chromogen, fluorescent, or
luminescent signal. Alternatively, assay product 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.
[0031] A polynucleotide may be ligated to a linker oligonucleotide
or conjugated to one member of a specific binding pair (e.g.,
antibody-digoxygenin/hapten/peptide epitope,
biotin-avidin/streptavidin, glutathione transferase or
GST-glutathione, maltose binding protein-maltose,
polyhistidine-nickel, protein A/G-immunoglobulin).
[0032] The polynucleotide may be conjugated by ligation of a
nucleotide sequence encoding the binding member. A polypeptide may
be joined to one member of the specific binding pair by producing
the fusion encoded such a ligated or conjugated polynucleotide or,
alternatively, by direct chemical linkage to a reactive moiety on
the binding member by chemical cross-linking. Such polynucleotides
and polypeptides may be used as an affinity reagent to identify, to
isolate, and to detect interactions that involve specific binding
of a transcript or protein product of the expression vector.
[0033] Before or after affinity binding of the transcript or
protein product, the member attached to the polynucleotide or
polypeptide may be bound to its cognate binding member. This can
produce a complex in solution or immobilized to a support.
[0034] Construction of Expression Vector
[0035] An expression vector is a recombinant polynucleotide that is
in chemical form either a deoxyribonucleic acid (DNA) and/or a
ribonucleic acid (RNA). The physical form of the expression vector
may also vary in strandedness (e.g., single-stranded or
double-stranded) and topology (e.g., linear or circular). The
expression vector is preferably a double-stranded deoxyribonucleic
acid (dsDNA) or is converted into a dsDNA after introduction into a
cell (e.g., insertion of a retrovirus into a host genome as a
provirus). The expression vector may include one or more regions
from a mammalian gene expressed in the microvasculature, especially
endothelial cells (e.g., ICAM-2, tie), or a virus (e.g.,
adenovirus, adeno-associated virus, cytomegalo-virus, herpes
simplex virus, Moloney leukemia virus, mouse mammary tumor virus,
Rous sarcoma virus, SV40 virus), as well as regions suitable for
gene manipulation (e.g., selectable marker, linker with multiple
recognition sites for restriction endo-nucleases, promoter for in
vitro transcription, primer annealing sites for in vitro
replication). The expression vector may be associated with proteins
and other nucleic acids in a carrier (e.g., packaged in a viral
particle).
[0036] The expression vector further comprises a regulatory region
for gene expression (e.g., promoter, enhancer, silencer, splice
donor and acceptor sites, polyadenylation signal, cellular
localization sequence). Transcription can be regulated by
tetracyline or dimerized macrolides. The expression vector may be
further comprised of one or more splice donor and acceptor sites
within an expressed region; a Kozak consensus sequence upstream of
an expressed region for initiation of translation; downstream of an
expressed region, multiple stop codons in the three forward reading
frames to ensure termination of translation, one or more mRNA
degradation signals, a termination of transcription signal, a
polyadenylation signal, and a 3' cleavage signal. For expressed
regions that do not contain an intron (e.g., a coding region from a
cDNA), a pair of splice donor and acceptor sites may or may not be
preferred. It would be useful, however, to include a mRNA
degradation signal if it is desired to express one or more of the
downstream regions only under the inducing condition. An origin of
replication may be included that allows replication of the
expression vector integrated in the host genome or as an
autonomously replicating episome. Centromere and telomere sequences
can also be included for the purposes of chromosomal segregation
and protecting chromosomal ends from shortening, respectively.
Random or targeted integration into the host genome is more likely
to ensure maintenance of the expression vector but episomes could
be maintained by selective pressure or, alternatively, may be
preferred for those applications in which the expression vector is
present only transiently.
[0037] An expressed region may be derived from a gene encoding
LRP-1 or a ligand thereof in operative linkage with a regulatory
region (e.g., constituitive, regulated, or endothelial-specific
promoter and an optional enhancer). The expressed region may encode
a translational fusion. Open reading frames of regions encoding a
polypeptide and at least one heterologous domain may be ligated in
register. If a reporter or selectable marker is used as the
heterologous domain, then expression of the fusion protein may be
readily assayed or localized. The heterologous domain may be an
affinity or epitope tag.
[0038] Screening of Candidate Compounds
[0039] Another aspect of the invention are chemical or genetic
compounds, derivatives thereof, and compositions including same
that are effective in the treatment of Alzheimer's disease and
individuals at risk thereof. The amount that is administered to an
individual in need of therapy or prophylaxis, formulation, and
timing and route of delivery is effective to reduce the number or
severity of symptoms, to slow or limit progression of symptoms, to
inhibit expression of one or more genes that are transcribed at a
higher level in Alzheimer's disease, to activate expression of one
or more genes that are transcribed at a lower level in Alzheimer's
disease, or any combination thereof. Determination of such amounts,
formulations, and timing and route of drug delivery is within the
skill of persons conducting in vitro assays, in vivo studies of
animal models, and human clinical trials.
[0040] A screening method may comprise administering a candidate
compound to an organism or incubating a candidate compound with a
cell, and then determining whether or not gene expression is
increased. The increase in activity may partially or fully
compensate for a change that is associated with or may cause
Alzheimer's disease. Gene expression may be increased at the level
of rate of transcriptional initiation, rate of transcriptional
elongation, stability of the transcript, translation of the
transcript, rate of translational initiation, rate of translational
elongation, stability of protein, rate of protein folding,
proportion of protein in active conformation, functional efficiency
of protein (e.g., activation or repression of transcription), or
combinations thereof. See U.S. Pat. Nos. 5,071,773 and 5,262,300.
High-throughput screening assays are possible.
[0041] The screening method may comprise incubating a candidate
compound with a cell containing a reporter construct, the reporter
construct comprising transcription regulatory region covalently
linked in a cis configuration to a downstream gene encoding an
assayable product; and measuring production of the assayable
product. A candidate compound which increases production of the
assayable product would be identified as an agent which activates
gene expression. See U.S. Pat. Nos. 5,849,493 and 5,863,733.
[0042] The screening method may comprise measuring in vitro
transcription from a reporter construct in the presence or absence
of a candidate compound, the reporter construct comprising a
transcription regulatory region; and determining whether
transcription is altered by the presence of the candidate compound.
In vitro transcription may be assayed using a cell-free extract,
partially purified fractions of the cell-free extract, purified
transcription factors or RNA polymerase, or combinations thereof.
See U.S. Pat. Nos. 5,453,362; 5,534,410; 5,563,036; 5,637,686;
5,708,158; and 5,710,025.
[0043] Techniques for measuring transcriptional or translational
activity in vivo are known in the art. For example, a nuclear
run-on assay may be employed to measure transcription of a reporter
gene. Translation of the reporter gene may be measured by
determining the activity of the translation product. The activity
of a reporter gene can be measured by determining one or more of
the abundance of transcription of polynucleotide product (e.g.,
RT-PCR of GFP transcripts), translation of polypeptide product
(e.g., immunoassay of GFP protein), and enzymatic activity of the
reporter protein per se (e.g., fluorescence of GFP or energy
transfer thereof).
[0044] Inhibition of the ubiquitin-proteasome pathway may be used
to reverse down-regulated expression of LRP-1 (e.g., by aging,
disease, a pathological concentration of A.beta.) or to prevent its
down regulation. Examples of proteasome inhibitors are PS-341 or
derivatives thereof, lactacystin, MG132, MG262, ritonavir, and
TMC-95A.
[0045] Genetic Compounds for Treatment
[0046] Gene activation may be achieved by inducing an expression
vector containing a downstream region related to a gene that is
down regulated (e.g., the full-length coding region or functional
portions of the gene; hypermorphic mutants, homologs, orthologs, or
paralogs thereof) or unrelated to the gene that acts to relieve
suppression of gene activation (e.g., at least partially inhibiting
expression of a negative regulator of the gene). Overexpression of
transcription or translation, as well as over-expressing protein
function, is a more direct approach to gene activation.
Alternatively, the downstream expressed region may direct
homologous recombination into a locus in the genome and thereby
replace an endogenous transcriptional regulatory region of the gene
with an expression cassette. In particular, gene expression of
components of the receptor-ligand system transporting A.beta.
across the blood-brain barrier can be increased by introduction of
an exogenous gene or activating an endogenous gene.
[0047] An expression vector may be introduced into a host mammalian
cell or non-human mammal by a transfection or transgenesis
technique using, for example, chemicals (e.g., calcium phosphate,
DEAE-dextran, lipids, polymers), biolistics, electroporation, naked
DNA technology, microinjection, or viral infection. The introduced
expression vector may integrate into the host genome of the
mammalian cell or non-human mammal. Many neutral and charged
lipids, sterols, and other phospholipids to make lipid carrier
vehicles are known. For example, neutral lipids are dioleoyl
phosphatidylcholine (DOPC) and dioleoyl phosphatidyl ethanolamine
(DOPE); an anionic lipid is dioleoyl phosphatidyl serine (DOPS);
cationic lipids are dioleoyl trimethyl ammonium propane (DOTAP),
dioctadecyldiamidoglycyl spermine (DOGS), dioleoyltrimethyl
ammonium (DOTMA), and
1,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propylamide tetraacetate
(DOSPER). Dipalmitoyl phosphatidylcholine (DPPC) can be
incorporated to improve the efficacy and/or stability of delivery.
FUGENE 6, LIPO-FECTAMINE, LIPOFECTIN, DMRIE-C, TRANSFECTAM,
CELLFECTIN, PFX-1, PFX-2, PFX-3, PFX-4, PFX-5, PFX-6, PFX-7, PFX-8,
TRANSFAST, TFX-10, TFX-20, TFX-50, and LIPOTAXI lipids are
proprietary formulations. The polymer may be polyethylene glycol
(PEG) or polyethylenimine (PEI); alternatively, polymeric materials
can be formed into nanospheres or microspheres. Naked DNA
technology delivers the expression vector in plasmid form to a
cell, where the plasmid may or may not become integrated into the
host genome, without using chemical transfecting agents (e.g.,
lipids, polymers) to condense the expression vector prior to
introduction into the cell.
[0048] Thus, a mammalian cell may be transfected with an expression
vector; also provided are transgenic nonhuman mammals. In the
previously discussed alternative, a homologous region from a gene
can be used to direct integration to a particular genetic locus in
the host genome and thereby regulate expression of the gene at that
locus. Polypeptide may be produced in vitro by culturing
transfected cells; in vivo by transgenesis; or ex vivo by
introducing the expression vector into allogeneic, autologous,
histocompatible, or xenogeneic cells and then transplanting the
transfected cells into a host organism. Special harvesting and
culturing protocols will be needed for transfection and subsequent
transplantation of host stem cells into a host mammal.
Immunosuppression of the host mammal post-transplant or
encapsulation of the host cells may be necessary to prevent
rejection.
[0049] The expression vector may be used to replace the function of
a gene that is down regulated or totally defective or supplement
function of a partially defective gene. Thus, the cognate gene of
the host may be neomorphic, hypomorphic, hypermorphic, or normal.
Replacement or supplementation of function can be accomplished by
the methods discussed above, and transfected mammalian cells or
transgenic nonhuman mammals may be selected for high expression
(e.g., assessing amount of transcribed or translated product, or
physiological function of either product) of the downstream
region.
[0050] Formulation of Compositions
[0051] Compounds of the invention or derivatives thereof may be
used as a medicament or used to formulate a pharmaceutical
composition with one or more of the utilities disclosed herein.
They may be administered in vitro to cells in culture, in vivo to
cells in the body, or ex vivo to cells outside of the individual
that may later be returned to the body of the same individual or
another. Such cells may be diaggregated or provided as solid
tissue.
[0052] 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.
[0053] A reduced fat diet or drug therapy to lower lipids (e.g.,
statins) may be used to alter LRP-1 receptor function in a
beneficial manner. Alternatively, the drug may be used to increase
gene expression of a component of the receptor-ligand system (e.g.,
enhancing transcription or translation). Reducing the systemic
concentration of A.beta. with antibody depletion or filtration of
antibody-A.beta. complexes may favor transport across the
blood-brain barrier. Vasodilation, angiogenesis,
neovascularization, and osmotic shock may be used to increase blood
flow and thereby increase removal of A.beta. from the brain to the
systemic circulation. Another method for increasing removal may be
to increase permeability of the blood vessel with known drugs
(e.g., bradykinin, histamine); tight junctions may be loosened or
the width increased to increase permeability. Other methods for
increasing transcytosis and recycling of LRP-1 may also be used
(e.g., activators of cAMP signalling like theophylline). As
illustrated by the latter, drugs may have one or more of the
aforementioned beneficial affects. 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 A.beta.. Another compound is a
proteasome inhibitor (e.g., MG132).
[0054] Pharmaceutical compositions may be administered as a
formulation adapted for passage through the blood-brain barrier or
direct contact with the endothelium. Alternatively, pharmaceutical
compositions may be added to the culture medium. In addition to the
active compound, such compositions may contain
pharmaceutically-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.
[0055] Pharmaceutical compositions may be administered 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, intramuscular, intravenous,
intra-arterial, intrathecal, and other injection or infusion
techniques, without limitation.
[0056] 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.
[0057] A bolus administered over a short time once a day is a
convenient dosing schedule. Alternatively, the effective daily dose
may be divided into multiple doses for purposes of administration,
for example, two to twelve doses per day. Dosage levels of active
ingredients in a pharmaceutical composition can also be varied so
as to achieve a transient or sustained concentration of the
compound or derivative thereof in an individual, 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.
[0058] The amount of compound administered is dependent upon
factors known to a person skilled in the art such as bioactivity
and bioavailability of the compound (e.g., half-life in the body,
stability, and metabolism); chemical properties of the compound
(e.g., molecular weight, hydrophobicity, and solubility); route and
scheduling of administration; and the like. For systemic
administration, passage of the compound or its metabolite 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.
[0059] 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.
[0060] The amount which is administered to an individual is
preferably an amount that does not induce toxic 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.
[0061] Production of compounds according to present regulations
will be regulated for good laboratory practices (GLP) and good
manufacturing practices (GMP) by governmental agencies (e.g., U.S.
Food and Drug Administration). This requires accurate and complete
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.
[0062] The following examples are merely illustrative of the
invention, and are not intended to restrict or otherwise limit its
practice.
EXAMPLES
[0063] Reagents
[0064] Wild-type and mutant A.beta. (Dutch mutation is E22Q, Iowa
mutation is D23N, and Dutch/Iowa double mutation is E22Q/D23N)
peptides of 40 or 42 amino acids were synthesized, by solid-phase
F-moc (9-fluorenylmethoxycarbonyl) amino acid chemistry, purified
by reverse phase-HPLC and structurally characterized. Aliquots of
the final products were lyophilized and stored at -20.degree. C.
until use. Radioiodination was carried out with Na[.sup.125I] and
lodobeads (Pierce), and the products were resolved by HPLC (39).
Aliquots of radiolabeled A.beta..sub.1-40 were kept at -20.degree.
C. for a maximum of four weeks prior to use. The HPLC analysis
confirmed that more than 99% of radioactivity was present in the
form of non-oxidized monomeric peptide.
[0065] Human recombinant RAP (EMD Biosciences, San Diego, Calif.);
polyclonal 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 the 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
.alpha.g/mi; EMD Biosciences, San Diego, Calif.); and monoclonal
mouse antibodies P2-1 specific for human APP (1:1000, 1 mg/ml),
22C11 which recognizes mouse and human APP (1:100, 0.5 mg/ml;
Chemicon International, Temecula, Calif.), and 66.1 specific for
residues 1-8 of human A.beta. (1:1000, 1 mg/ml) were used.
[0066] Brain Clearance Model in Mice
[0067] Male C57BL/6 wild-type mice, 8-10 weeks old and 9-10 months
old, and male apoE knockout mice (apoE KO) on a C57BL/6 background
(Taconic Farm, German-town, N.Y.), 8-10 weeks old and 9-10 months
old, were studied. CNS clearance of radiolabeled A.beta..sub.1-40
and the inert polar marker, inulin, was determined as described
below (40, 43).
[0068] A stainless steel guide cannula was implanted
stereotaxically into the right caudate-putamen of anesthetized mice
(60 mg/kg i.p. sodium pentobarbital). Coordinates for the tip of
the cannula were 0.9 mm anterior and 1.9 mm lateral to bregma and
2.9 mm below the surface of the brain. The guide cannula and screw
were fixed to the skull with methylmethacrylate (Plastics One,
Roanoke, Va.) and a stylet introduced into the guide cannula.
Animals were observed for one week prior to radiotracer
studies.
[0069] For radioisotope injection, animals were re-anesthetized and
an injector cannula (Plastic One, Roanoke, Va.) attached with
24G-TEFLON tubing (Small Parts, Miami Lake, Fla.) to a 10 .mu.l
gas-tight microsyringe (Hamilton, Reno, Nev.) was used. The amount
of injected tracers was determined accurately using a micrometer to
measure linear displacement of the syringe plunger in the
precalibrated microsyringe. A half microliter of tracer fluid
containing .sup.125I-A.beta..sub.1-40 at varying concentrations
from 0.05 nM to 120 nM was injected over five minutes along with
.sup.14C-inulin. When the effects of different molecular reagents
were tested, those were injected simultaneously with the
radiolabeled peptides.
[0070] Time-response was studied with .sup.125I-A.beta..sub.1-40
from 10 min to 300 min and dose-dependent effects determined at 30
min. The effects of different molecular reagents that may
potentially inhibit .sup.125I-A.beta..sub.1-40 clearance were
studied at 30 min including: rabbit anti-human LRP-1 antibody
designated as R777 that was affinity purified over Sepharose-LRP-1
heavy chain column as described (44) and that immunoprecipitates
mouse LRP-1 as described (44) and blocks LRP-1 -mediated uptake of
APP and thrombospondin in murine fibroblasts (45-46); receptor
associated protein (RAP) (provided by Dr. Bu, Washington
University); rabbit anti-mouse .alpha..sub.2M antibody designated
as YNRMA2M that is specific for mouse .alpha..sub.2M as
demonstrated by radial immunodiffusion and immunoelectrophoresis
(Accurate Scientific Corp, Westbury, N.Y.); rabbit anti-rat gp330
affinity purified IgG designated as Rb6286 that cross reacts with
mouse LRP-2 as reported (47) (provided by Dr. Scott Argraves,
University of S.C.); rabbit anti-human anti-RAGE antibody that
cross-reacts with mouse RAGE (32) (provided by D. Stern, Columbia
University) and fucoidan (Sigma, St. Louis, Mo.).
[0071] Tissue Sampling and Radioactivity Analysis
[0072] Brain, blood and CSF were sampled and prepared for
radioactivity analysis. Degradation of .sup.125I-A.beta..sub.1-40
was initially studied by trichloroacetic acid (TCA) precipitation
assay. Previous studies with .sup.125I-A.beta..sub.1-40o
demonstrated an excellent correlation between TCA and HPLC methods
(33, 48-51). Brain, plasma and CSF samples were mixed with TCA
(final concentration 10%), centrifuged at 14,000 rpm at 4.degree.
C. for 8 min to 10 min, and radioactivity in the precipitate, water
and chloroform fractions determined in gamma counter (Wallac,
Turku, Finland). The intactness of .sup.125I-A.beta..sub.140
injected into the brain was greater than 97% by TCA analysis.
[0073] Degradation of .sup.125I-A.beta..sub.1-40 in brain was
further studied by the HPLC and SDS-PAGE analyses. Following
intracerebral injections of .sup.125I-A.beta..sub.1-40, brain
tissue was homogenized in PBS containing protease inhibitors (0.5
mM phenylmethylsulfonyl fluoride, 1 .mu.g/ml leupeptin, and 1 mM
p-aminobenzamidine) and centrifuged at 100,000 g for 1 hr at
4.degree. C. The supernatant was then lyophilized. The resulting
material was dissolved in 0.005% TFA in water, pH 2, before
injection onto a C4 column (Vydac, The Separation Group, Hesperia,
Calif.). The separation was achieved with a 30-min linear gradient
of 25% to 83% acetonitrile in 0.1 % TFA at a flow rate of 1 ml/min,
as described (57). Under these conditions, the A.beta..sub.1-40
standard eluted at 14.5 min. Column eluants were monitored at 214
nm. The eluted fractions were collected and counted. The intactness
of .sup.125I-A.beta..sub.1-40 injected into the brain by the HPLC
analysis was greater than 97% confirming the results of TCA
analysis.
[0074] 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. Lyophilized HPLC fractions were resuspended in sample
buffer, neutralized, boiled and electrophoresed as previously
reported (39).
[0075] Calculations of Clearance Rates
[0076] The analysis of radioactivity-disappearance curves from the
brain was as reported (40, 43). The percentage of radioactivity
remaining in the brain after microinjection was determined from eq.
1 as % Recovery in brain=100.times.(N.sub.b/N.sub.i) 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.
[0077] In all calculations, the d.p.m. values for .sup.14C-inulin
and the c.p.m. values for TCA-precipitable .sup.125I-radioactivity
were used. Inulin was studied as a metabolically inert polar
reference marker that is neither transported across the BBB nor
retained by the brain (40); its clearance rate, i.e., k.sub.inulin,
provides a measure of the ISF bulk flow and is calculated as
N.sub.b(inulin)/N.sub.i(inulin)=exp (-k.sub.inulin*t) (2).
[0078] For A.beta., there are two possible physiological pathways
of elimination--direct transport across the BBB into the
bloodstream and elimination by ISF bulk flow into the CSF and
cervical lymphatics. It is also possible that A.beta. is retained
within the brain either by binding to its cell surface receptors
directly as a free peptide and/or by binding to different transport
proteins. Thus, according to the model, the fraction of A.beta.
remaining in the brain can be expressed as
N.sub.b(A.beta.)/N.sub.i(A.beta.)=a.sub.1+a.sub.2*e.sup.-k1*t (3),
where a.sub.1=k.sub.2/(k.sub.1+k.sub.2) and
a.sub.2=k.sub.1/(k.sub.1+k.sub.2), and k.sub.1 and k.sub.2 denote
the fractional coefficients of total efflux from the brain and
retention within the brain, respectively.
[0079] The fractional rate constant of A.beta. efflux across the
BBB from brain parenchyma can be calculated by knowing the
fractional rate coefficient of total efflux of A.beta. and inulin
as k.sub.3=k.sub.1-k.sub.(inulin) (4), i.e., as the difference
between the fractional rate constant for total efflux of A.beta.
and the fractional rate constant of inulin. The half-saturation
concentration for the elimination of A.beta. by transport across
the BBB, k.sub.0.5, was calculated from the equation
[1-(N.sub.b/N.sub.i)]*100=Cl.sub.max/(k.sub.- 0.5+N.sub.i) (5),
where Cl.sub.max represents the maximal efflux capacity for the
saturable component of A.beta. clearance across the BBB corrected
for peptide clearance by the ISF flow. Cl.sub.max is expressed as a
percentage of the injected dose, [1-(N.sub.b/N.sub.i)].times.100,
cleared from brain by saturable BBB transport over 30 min.
[0080] The MLAB mathematical modeling system (Civilized Software,
Silver Spring, Md.) was used to fit the compartmental model to the
disappearance curves or percent recovery data with inverse square
weighting.
[0081] Human Brain Endothelial Cells and Metabolic Labeling
[0082] Human brain endothelial cells (BEC) were isolated from rapid
autopsies of neurologically normal young individuals after trauma.
BECs were characterized and cultured, and then incubated with
different A.beta. isoforms at concentrations ranging from 1 nM to
20 .mu.M within 48 hr. Cells were lysed and equal amounts of
proteins (10 .mu.g/mi) were separated by 10% denaturing
polyacrylamide gel electrophoresis (SDS-PAGE), 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.
[0083] Human BEC (4.times.10.sup.5) were pulsed for 1 hr at
37.degree. C. with 400 .mu.Ci of [35S]-methionine (greater than
1000 Ci/mmol; Perkin Elmer, Boston, Mass.) in methionine-free
Dulbecco modified Eagle medium (GIBCO BRL, New York, N.Y.). Cells
were chased at 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 Phosphoimager (Amersham Biosciences,
Piscataway, N.J.).
[0084] Immunocytochemical Analysis in Mice
[0085] Expression of LRP-1 and .alpha..sub.2M in mouse brain was
studied by immunohisto-chemical analysis. Fresh-frozen,
acetone-fixed brain sections of 2-month old and 9-month old
wild-type mice, as well as apoE KO mice were stained using
anti-human LRP R777 antibody that cross reacts with mouse LRP-1
(44-46) (1.5 mg/ml; 1:300 dilution), and anti-mouse .alpha..sub.2M
antibody (as described above 1:250 dilution). R777 was affinity
purified over Sepharose-LRP-1 heavy chain column, as described
(44). The number of positive vessels was counted in ten random
fields by two independent blinded observers and expressed as
percentage per mm.sup.2 of section. The extent and intensity of
staining in cellular elements was quantitated using the Universal
Imaging System and NIH imaging systems. Microvessels were carefully
excluded from the quantitation by suitably varying the magnitudes
of measurement. The relative intensity of cellular staining
(excluding the microvasculature) in brain sections of young mice
was arbitrarily normalized to 1 for purposes of comparison. Routine
control sections included deletion of primary antibody, deletion of
secondary antibody body and the use of an irrelevant primary
antibody.
[0086] Neuropathological Analysis in Humans
[0087] Three AD patients and three neurologically normal,
age-matched controls from the Alzheimer's Disease Research Center
(ADRC) of the University of Southern California were evaluated
clinically and followed to autopsy. Included were three males and
three females, ranging in age from 69 to 99 years.
[0088] Tissue blocks (1 cm.sup.3) were obtained postmortem (range 4
hr to 7 hr; mean 5 hr), fixed in 10% neutral buffered formalin, pH
7.3 (Sigma, St. Louis, Mo.) and embedded in paraffin or snap-frozen
in liquid nitrogen-chilled isopentane. Tissues were sampled from
the superior and middle frontal gyrus (Brodmann's area BA10), and
cerebellar hemisphere.
[0089] Sections were stained with either hematoxylin and eosin or
thioflavin S, a modified Bielschowsky silver impregnation method
(Gallyas stain). Thioflavin S stained sections were viewed through
a Zeiss fluorescence microscope with a narrow band, blue/violet
filter at 400 to 455 nm. Examination was performed by two
independent observers. Diagnosis of AD was according to a modified
CERAD (Consortium to Establish a Registry for Alzheimer's Disease)
protocol (52).
[0090] For immunocytochemical analysis, air-dryed 10 .mu.m cryostat
sections of frontal cortex (Brodmann's area BA10) were used.
Immunocytochemistry was performed using avidin-biotin peroxidase
complexes (ABC method, Vector Laboratories, Burlingame, Calif.).
Antibodies used include: A.beta..sub.1-40 (Chemicon, Temecula,
Calif.), rabbit anti-human, 1:1000 (1 mg/ml); A.beta..sub.1-42,
rabbit anti-human 1:1000 (1 mg/ml); the mouse monoclonal antibody
to the heavy chain of human LRP-1 designated as 8G1 which is
specific for human LRP-1 and recognizes an epitope on the 515 kDa
subunit (53), 1:300 (1.5 mg/ml); and CD105 (clone SMG) (Serotec,
Oxford, England), mouse anti-human 1:100 (0.1 mg/ml). For single
staining with CD105 and LRP, after incubation with primary
antibody, sections were washed three times in PBS, pH 7.4 and
treated with biotinylated anti-mouse IgG for 30 minutes. After
three washes in PBS, slides were incubated with avidin-biotin-HRP
complex for 30 minutes and washed three times in PBS. Binding was
detected with a Vector SG peroxidase detection kit (blue-gray). For
double labeling, after incubation with A.beta. overnight at
4.degree. C., sections were washed three times with PBS and treated
with biotinylated anti-rabbit IgG, washed again, and binding was
detected with Vector NovaRed. Following three washes in PBS, the
second primary antibody (LRP or CD-105) was applied and staining
performed as described for single label. Imaging was accomplished
using a Zeiss Axiophot II microscope equipped with a Spot digital
camera (Spot Diagnostics, Sterling Heights, Mich.).
[0091] Results
[0092] FIG. 1A illustrates brain radioactivity-disappearance curves
of .sup.14C-inulin and .sup.125I-A.beta..sub.1-40 (TCA-precipitable
.sup.125I-radioactivity) studied at a concentration of 60 nM.
Clearance of inulin, a reference ECF maker that is neither
transported across the BBB nor retained by the brain (40, 43),
approximated a single exponential decay, as expected from previous
studies. The clearance curve reflecting total efflux from brain of
.sup.125I-A.beta..sub.1-40 was bi-exponential and much lower than
that for inulin, indicating significant biological transport of
A.beta..sub.1-40 out of the brain. The two components of
A.beta..sub.1-40 efflux, i.e., rapid elimination by vascular
transport across the BBB into the blood and slow elimination
through ISF flow, computed from FIG. 1A with eqs. 3 and 4 are
illustrated in FIG. 1B. FIG. 1B indicates significantly higher
clearance of A.beta. by BBB transport than by ISF bulk flow.
[0093] The half-time, t.sub.1/2, for brain efflux of
A.beta..sub.1-40 and inulin calculated from FIG. 1A and eqs. 2 and
3 was 25.5.+-.2.0 min and 239.0.+-.12.5 min (Table 1),
respectively, a 9.4-fold difference. The half-time of efflux of
A.beta..sub.1-40 across the BBB was 34.6.+-.3.6 min, 6.9-fold
faster than by ISF bulk flow. In addition to efflux, there was also
a slow, time-dependent retention of A.beta..sub.1-40 in brain
parenchyma with a t.sub.1/2 of 164.5 min. As shown in Table 1, the
rate k (min.sup.-1) of clearance of A.beta..sub.1-40 from the brain
was 7.9-fold higher than that for inulin. The relative
contributions of A.beta..sub.1-40 efflux at 60 nM by transport
across the BBB and by ISF bulk flow based on 5 hr measurements were
73.8% and 10.7% respectively, while 15.6% of the dose remained
sequestered within the CNS (FIG. 1C).
1TABLE 1 Clearance rates, k, for .sup.125I-A.beta..sub.1-40 and
.sup.14C-Inulin .sup.125I-A.beta..sub.1-40 .sup.14C-Inulin
Parameter k (min.sup.-1) t.sub.1/2 (min) k (min.sup.-1) t.sub.1/2
(min) Total Efflux 0.0229 .+-. 25.5 .+-. 2.0* 0.0029 .+-. 0.0002
239 .+-. 12.5 0.0023* Transport by 0.0200 .+-. 34.6 .+-. 3.6 None
None BBB 0.0023 Transport by 0.0029 .+-. 239 .+-. 12.5 0.0029 .+-.
0.0002 239 .+-. 12.5 ISF 0.0002 Retention in 0.0042 .+-. 164.5 .+-.
17.6 None None brain 0.0005
[0094] Data are mean.+-.SD from 38 individual experiments;
fractional coefficients, k, were calculated using eqs. 3 and 4;
*-p<0.05 by Students' t-test.
[0095] Following CNS injection, both tracers reached the CSF, and
the CSF time-appearance curves are shown in FIG. 2A. The amount of
.sup.125I-A.beta..sub.1-40 (TCA-precipitable
.sup.125I-radioactivity) determined in the CSF was lower than that
for inulin at each time point, possibly reflecting an active
clearance of A.beta..sub.1-40 from the CSF, as suggested previously
(28). It is noteworthy that at each studied time point the
.sup.125I-labeled material in the CSF was greater than 96%
TCA-precipitable indicating no degradation of the peptide. Both
tracers also appeared in plasma (FIG. 2B), and higher levels of
.sup.125I-A.beta..sub.1-40 TCA-precipitable radioactivity than of
.sup.14C-inulin radioactivity were consistent with active transport
of A.beta..sub.1-40 out of the CNS across the BBB. The absolute
amounts of both tracers in the CSF and plasma were, however, low
due to relatively rapid clearance from the CSF in comparison to
slow ISF bulk flow (29), and significant systemic body clearance
(48), respectively.
[0096] FIGS. 3A and 3B illustrate that .sup.125I-A.beta.1-40 was
not significantly degraded in brain ISF prior to its transport
across the BBB as determined by TCA, HPLC and SDS-PAGE analysis of
.sup.125I-radioactivity in brain. The TCA analysis suggest that
only 4.2% to 9.9% of .sup.125I-radioactivity in brain was not
TCA-precipitable at different time points within 270 min of
intracerebral microinjection of .sup.125I-A.beta..sub.1-40 (FIG.
3A). The HPLC analysis of brain radioactivity confirmed the TCA
results by indicating that 93.7% of the peptide remains intact in
brain ISF at 60 min (FIG. 3B, right). It is noteworthy that
.sup.125I-A.beta..sub.1-40 was greater than 97% intact at the time
of injection as determined both by the HPLC and TCA analyses. The
results were confirmed by SDS/PAGE analysis of lyophilized
ali-quots of HPLC peaks of brain homogenates at different time
points after .sup.125I-A.beta..sub.1-40 injection that indicate
single radioactive band at about 4 kDa (FIG. 3B, left). The
identity of the radio-active components on gels as A.beta..sub.1-40
peptide was confirmed by Western blot analysis using
anti-A.beta.antibody and enhanced chemiluminescence as a detection
system. More than 96% of .sup.125I-radioactivity in the CSF was
TCA-precipitable at studied time points between 15 min and 270 min.
In contrast, degradation products of .sup.125I-A.beta..sub.1-40
were found in plasma (FIG. 3C); the amount of degraded
.sup.125I-A.beta..sub.1- -40 corresponding to TCA nonpreci-pitable
.sup.125I-radioactivity increased from 37.6% to 58.3% from 15 min
to 120 min of intra-cerebral microinjection of intact
.sup.125I-A.beta..sub.1-40 (FIG. 3C). It is noteworthy that the
amount of radioactivity in plasma after 120 min was relatively
small and approached the limits of sensitivity of the TCA
assay.
[0097] Clearance of A.beta. in young mice was
concentration-dependent (FIG. 4A). The efflux transport system was
half-saturated (K.sub.0.5) at 15.3 nM of A.beta..sub.1-40. The
plateau or maximal clearance capacity was reached between 70 and
100 nM, and further increases in A.beta. concentration resulted in
progressively greater retention of the peptide in the brain. In
contrast, clearance of .sup.14C-inulin did not change with
increasing concentrations of A.beta. suggesting a physiologically
intact BBB (FIG. 4A).
[0098] The next set of experiments was designed to characterize the
BBB transport system responsible for the transcytosis of A.beta..
Brains were loaded with .sup.125I-A.beta..sub.1-40 either at 12 nM
(FIG. 4B) or 60 nM (FIG. 4C) and clearance determined at 30 min in
the absence (open bars) or the presence (solid bars) of several
drugs that may act as potential inhibitors and/or competitors of
export. FIG. 4B indicates that both LRP-1 antibody (60 .mu.g/ml)
and RAP (200 nM) produced significant, 58% and 30% respectively,
reductions in A.beta. clearance from the brain in comparison to
vehicle treated controls; a further decrease in A.beta. clearance
to 44% was obtained by increasing the concentration of RAP to 5
.mu.M. A significant 25% inhibition in A.beta. clearance was also
obtained in the presence of anti-.alpha..sub.2M antibody (20
.mu.g/ml). In contrast, anti-LRP-2 antibody (FIG. 4B) and anti-RAGE
antibody (FIG. 4C) did not affect A.beta. clearance. Fucoidan, a
specific ligand for SR-A, produced a modest increase in the
clearance, possibly by blocking the binding of A.beta. to
parenchymal SR-A receptors, thereby allowing more peptide to be
available for clearance. At higher A.beta. loads (FIG. 4C),
anti-LRP-1 antibody produced a 53% decrease in clearance similar to
that observed at a lower load (FIG. 4B), but A.beta. recovery
approached that of .sup.14C-inulin, suggesting drainage of the
peptide almost exclusively through ISF bulk flow. Clearance of
.sup.14C-inulin was not affected by any of the studied molecular
reagents. BCH, a substrate that specifically blocks L-system for
amino acids, does not affect clearance of A.beta. across the BBB
which excludes the possibility that .sup.125I-A.beta..sub.1-40 is
degraded to .sup.125I-tyrosine that is transported out of the CNS
instead of .sup.125I-A.beta..sub.1-40.
[0099] Next, the effect of apoE and aging was studied by
determining A.beta. clearance in 2-month old and 9-month old apoE
KO mice and wild-type mice, using 12 nM (FIG. 5A) or 60 nM (FIG.
5B) of .sup.125I-A.beta..sub.1-40. FIG. 5A shows that the clearance
of A.beta. was reduced by 30% in young apoE KO mice, and by about
55% and 40% in 9-month old wild-type and apoE KO mice,
respectively. These results were confirmed at a higher load of
A.beta., and observed decrease in clearance was 46% in 9-month old
apoE KO mice (FIG. 5B).
[0100] The effect of different concentrations of A.beta. on LRP-1
levels in primary human brain endothelial cells (BEC), a clearance
site for A.beta. in the brain in vivo, was studied.
A.beta..sub.1-40, A.beta..sub.1-42, and/or mutant A.beta.
(Dutch/Iowa4o) at low concentrations (less than 10 nM) do not alter
significantly LRP-1 expression within 48 hr. But at concentrations
which were substantially higher than the levels of A.beta. that can
be transported via LRP-1 across the BBB (i.e., greater than or
equal to 1 .mu.M), all of the A.beta. species progressively
decreased LRP-1 levels in BEC, as demonstrated for LRP-1 85 kDa
.beta.-subunit (FIGS. 6A-6C) and 515 kDa .alpha.-subunit. RAP and
an anti-LRP-1 specific antibody, but not a control non-immune IgG,
block down-regulation of LRP-1 by a high level of A.beta.
suggesting that different A.beta. species at pathological
concentrations hypersaturate LRP-1 resulting in the receptor's
down-regulation.
[0101] To examine whether ligand-induced down-regulation of LRP-1
by excess A.beta. peptide is due to enhanced degradation of the
receptor, pulse chase experiments were performed with human BEC and
the half-life (t.sub.1/2) of LRP-1 in the absence or presence of
excess A.beta. was determined. A.beta..sub.1-42 (20 .mu.M)
decreases t.sub.1/2 for LRP-1 from 9.6 hr to 6.4 hr (FIGS. 7A-7B).
A similar reduction in LRP-1 turnover rate was shown with
A.beta..sub.1-40 and mutant A.beta.. MG132, an inhibitor of
proteasome-dependent LRP-1 degradation, increased the t.sub.1/2 for
LRP-1 in both control and A.beta.-treated cells (FIGS. 7C-7D). This
confirmed that LRP-1 delivery into the degradative pathway in brain
endothelium is regulated by the proteasome pathway and that
saturation of LRP-1 by A.beta. accelerates its proteasome-dependent
degradation. Control immunoblot analysis with an anti-LRP-1
C-terminal antibody that recognizes immature 600 kDa form of LRP-1
confirmed that the levels of immature LRP-1 are not affected by
A.beta. treatment (FIG. 7E). Thus, binding of internalized
extracellular A.beta. to immature LRP-1 in the ER compartment is
not implicated in LRP-1 degradation by extracellular A.beta., as
expected, in contrast to receptor's down-regulation by simultaneous
overexpression of secreted ligands such as apoE, resulting in
retention and degradation of LRP-1 in the ER.
[0102] Immunocytochemical studies with anti-LRP-1 R777 antibody
staining (5 .mu.g/ml) confirmed abundant expression of LRP-1 in
brain microvessels (including capillaries, small venules and
arterioles) and significant parenchymal cellular staining
(including neurons) in 2-month old mice. There was a significant
reduction in LRP-1 positive vessels in 9-month old mice in
comparison to 2-month old mice; the number of LRP-1 positive
vessels dropped from 94% in 2-month old to 52% in 9-month old mice.
Quantitative analysis of LRP-1 positive parenchymal cells
(excluding blood vessels) showed a trend towards reduced staining
in older animals, though the difference was not statistically
significant. Similarly, there was no significant difference in the
number of .alpha..sub.2M-positive microvessels or parenchymal cells
in brain between young and old mice. It is noteworthy that present
staining for .alpha..sub.2M was not able to distinguish between
circulating .alpha..sub.2M and .alpha.2M expressed on
microvessels.
[0103] Frontal cortex of AD patients (Brodmann's area BA10)
revealed moderate to marked neuritic plaques and A.beta. deposits
in all AD patients and parenchymal and vascular amyloid in two of
the three AD patients. Controls revealed no neuritic plaques or
A.beta. in parenchyma and only meningeal vascular A.beta. in one of
the three patients. Staining with anti-LRP-1 monoclonal 8G1 (5
.mu.g/ml) in the frontal cortex of control patients revealed
moderate vascular staining in capillaries and arterioles, as well
as neuronal staining. There was reduced LRP-1 staining in AD
tissues, including regions with A.beta..sub.1-40 or
A.beta..sub.1-42 positive plaques and vessels. However, the
immediate subcortical white matter showed more robust vascular
staining for LRP-1, and absence of staining for A.beta., both in AD
and controls. Anti-CD105, which identifies vascular endothelium,
revealed ample staining of capillaries and arterioles of frontal
cortex in controls and moderately reduced numbers of stained
vessels in AD tissues. Cerebellum revealed equivalent vascular
staining with anti-LRP-1 and CD105 in AD and control sections and
no anti-A.beta..sub.1-40 or A.beta..sub.1-42 positive staining was
seen in either AD or control tissues.
[0104] The number of LRP-1-positive staining cerebral vessels
dropped from about 45% in age-matched controls to about 12% in AD.
They were barely detectable in patients with cerebrovascular
Dutch-type .beta.-amyloidosis. Based on in vitro results (see FIGS.
6 and 7), accumulation of A.beta. adjacent to brain vasculature in
patients with AD and with cerebrovascular .beta.-amyloidosis may
down regulate LRP-1 at the BBB in vivo.
[0105] Discussion
[0106] This study demonstrates the importance of vascular transport
across the BBB in clearing A.beta. from the brain into circulation.
Moreover, this transport mechanism is shown to be mediated mainly
by LRP-1 in brain microvascular endothelium, and that transport of
brain-derived A.beta. out of the CNS may be influenced by LRP-1
ligands, .alpha..sub.2M and apoE. This vascular clearance mechanism
for A.beta. is age-dependent, and lower clearance rates in older
animals correlate with decreased vascular abundance of LRP-1.
[0107] The capability of BBB to remove A.beta. was significant in
younger animals. The elimination time, t.sub.1/2, for
A.beta..sub.1-40 at 60 nM was 25 min or 9.4-fold faster than for
inulin, an ECF marker used to determine the ISF bulk flow rate
(40). The major component of the CNS efflux of A.beta. was
transport across the BBB into the vascular system. The clearance of
A.beta. across the BBB was time- and concentration-dependent. At
very low concentra-tions, i.e. less than 2 nM as found normally in
mouse brain (54-55), A.beta..sub.1-40 was elimi-nated from brain at
a rate, on an average 3.5-fold faster than at a load of 60 nM that
may compare to concentrations of A.beta..sub.1-40 found in the
brains of transgenic APP animals at 3 to 4 months (54-55). The
efflux transport system was half-saturated at 15.3 nM of
A.beta..sub.1-40 and appears to be fully saturated at
concentrations between 70 nM and 100 nM. Thus, this efflux
transporter may be completely saturated by higher levels of A.beta.
as found in the brains of older transgenic APP animals (54-55),
which in turn may lead to vascular accumulation of A.beta. and
development of prominent deposits of cerebrovascular amyloid, as
recently described (56-57).
[0108] In this study, significant metabolism or degradation of
A.beta..sub.1-40 within 5 hr was not observed, in contrast to a
recent report suggesting that A.beta..sub.1-42 is degraded by
enkephalinase (neprilysin) in brain within minutes (28). There may
be a possibility that A.beta..sub.1-40 and A.beta..sub.1-42 are
differentially processed in the brain. The physiological relevance
of the proposed degradation mechanism for A.beta..sub.1-42 (28),
however, remains unclear since the peptide was studied at extremely
high concentrations of about 240 .mu.M that are not found even in
the brains with severe .beta.-amyloidosis (30). As shown by
pharmacological studies, these high concentrations of A.beta. may
impair local BBB integrity (58-59), which in turn may contaminate
brain ISF with blood and/or plasma that possesses A.beta.-degrading
activity (48), as confirmed in this study.
[0109] Consistent with the hypothesis that cytosolic peptidases
have little access to A.beta. peptides secreted or injected into
brain ISF (28) or CSF (29), it has been recently reported that
insulin-degrading enzyme (IDE) cannot not degrade A.beta. in brain
in vivo following intracerebral injection of radiolabeled peptide
(28). This is in contrast to in vitro degradation of
.sup.125I-A.beta..sub.1-40 by IDE from brain and liver cytosol
fractions (60). Since IDE is an intracellular protease, it is not
surprising that IDE may not be able to process A.beta. from brain
ISF, in particular if peptide clearance is faster than its cellular
uptake, as suggested here and in a previous study (28). It is
noteworthy that brain endothelial cells in vitro (33) and
astrocytes (61) do not catabolize A.beta., in contrast to activated
microglial cells that secrete a specific metalloproteinase which
degrades A.beta. in vitro (61). Neuronal cells metabolize A.beta.
in vitro by an LRP-1-dependent mechanism that may require apoE or
.alpha..sub.2M (38). The rate of this degradation, however, is
about 50- to 100-fold slower than by transport across the BBB in
vivo.
[0110] Transport of A.beta. out of the CSF was not associated with
significant degradation of peptide in the CSF (29). Lower CSF
levels of A.beta..sub.1-40 in comparison to inulin may suggest an
active transport of A.beta. from the CSF to the blood, possibly
across the choroid plexus or leptomeningeal vessels, as shown
previously (29). Higher levels of radiolabeled A.beta. in the
plasma relative to inulin confirm vascular transport of the peptide
out of the CNS. Although, present results indicate that
brain-derived A.beta. could contribute to the pool of circulating
peptide, the degradation in plasma, systemic metabolism and body
clearance tend to reduce the levels of circulating peptide, as
shown previously (48). Under these experimental conditions, the
levels of radiolabeled A.beta. in the circulation were two to three
orders of magnitude lower than the brain levels, thus making
re-entry of radiolabeled A.beta. into the brain very unlikely since
the blood-to-brain transport of A.beta. normally operates down the
concentration gradient (39, 62-65). In addition, the apoJ system
that transports blood-borne A.beta. into the brain is saturated
under physiological conditions (64) that may facilitate the efflux
of A.beta. from brain. Previous studies have shown that circulating
free A.beta. is also metabolized during its transport across the
BBB (48-49, 65-66), possibly by pericytes, which represent a major
enzymatic barrier for the transport of several peptides and
proteins across the BBB (67).
[0111] The affinity of neprilysin to its physiological substrates
(e.g., enkephalins, tachykinins, atrial natriuretic peptide) and/or
different synthetic peptides is in the low millimolar range (68).
In contrast, the levels of A.beta. in the brain are normally in low
nanomolar range, and in transgenic models of brain amyloidosis they
vary from 40 to 250 mmol/kg from 3 to 12 months (54). Thus, under
physiological and/or pathological conditions, A.beta. will likely
bind to its high affinity cell surface receptors such as RAGE
and/or SR-A and/or high affinity transport binding proteins, e.g.,
.alpha..sub.2M, apoE and apoJ, that all react with low nanomolar
level of peptide corresponding to their K.sub.D values.
[0112] In the present study, anti-LRP-1 antibodies inhibited
A.beta..sub.1-40 clearance by about 55%, both at lower (12 nM) and
higher loads (60 nM) of the peptide, suggesting the involvement of
LRP-1 in vascular elimination of A.beta. from the brain. RAP, a
chaperone protein that facilitates proper folding and subsequent
trafficking of LRP-1 and LRP-2 (69), also inhibited A.beta.
clearance. RAP binds to multiple sites on LRP and antagonizes
binding of all known LRP ligands to both LRP-1 and LRP-2 in vitro
(69), as well as to LRP-2 in vivo at the blood side of the BBB
(39). In the present study, RAP at higher concentrations produced
comparable inhibition of A.beta. clearance as an anti-LRP-1
antibody. It is interesting that anti-LRP-1 antibody almost
completely inhibited vascular transport of A.beta. at higher
concentrations of peptide, which may indicate that LRP-1 could be
of primary importance in eliminating the peptide from the brain. At
a lower load of the peptide (i.e., 12 nM) though, neither of the
molecular reagents was able to abolish clearance of A.beta., which
suggests that in addition to LRP-1, there may be an alternative,
highly sensitive BBB transport mechanism(s) that eliminates the
peptide from the brain at very low concentrations. The molecular
nature of this putative "second" transport system is not presently
known, although present data suggest that RAGE and LRP-2 are
unlikely to be involved in rapid elimination of A.beta. from brain.
The fact that fucoidan, an SR-A ligand moderately increased
clearance of A.beta. suggests that inhibition of SR-A receptors in
brain may decrease CNS sequestration of the peptide, thus allowing
more peptide to be available for enhanced clearance across the
BBB.
[0113] The role of LRP-1 in promoting A.beta. clearance in vitro in
smooth muscle cells, neurons and fibroblasts by .alpha..sub.2M and
apoE has been suggested (35-38), although at significantly slower
rates than across the BBB, as demonstrated in the present study.
High affinity in vitro binding of A.beta. to .alpha..sub.2M and
lipidated apoE3 and apoE4 and a lower affinity binding to
delipidated apoE isoforms has been well documented (23, 70).
Binding/uptake studies in mouse embryonic fibroblasts, wild type
and deficient in LRP-1, confirmed that free A.beta. is not a ligand
for LRP-1 (37, 45). The possible role of the two LRP-1 ligands in
elimination of A.beta. by vascular transport is suggested by
inhibition of A.beta. clearance with anti-.alpha..sub.2M
antibodies, and significantly reduced clearance in apoE KO animals
by 30% and 46% at 2 months and 9 months of age, respectively, in
comparison to wild-type young controls. In relation to these
findings, it is interesting to note that recent studies indicated
that lack of endogenous mouse apoE in both the APP.sup.V717F and
APPsw mouse models of AD results in less A.beta. deposition and no
fibrillar A.beta. deposits in the brain (57, 71). This suggests
that mouse apoE strongly facilitates A.beta. fibrillogenesis. It is
possible that mouse apoE also plays a role in clearance of soluble
A.beta. across the BBB as suggested by the current studies but that
its ability to influence A.beta. aggregation in APP transgenic mice
is dominant. In 10 contrast to the effects of mouse apoE, a recent
study demonstrates that human apoE isoforms suppresses early
A.beta. deposition in APP.sup.V717F mice (72). Further studies in
this model will be useful to determine whether this suppressive
effect of human apoE isoforms on early A.beta. deposition is
secondary to effects on facilitating of A.beta. transport across
the BBB. Although the present study does not rule out the
possibility that A.beta. clearance by neurons, vascular smooth
muscle cells and fibroblasts shown in vitro (35-38) may also occur
in vivo, vascular transport across the BBB seems to be of primary
importance for rapid elimination of A.beta. from brain in vivo.
[0114] Since normal aging is associated with A.beta. accumulation
in brain (1), and there is a significant, time-dependent and
progressive accumulation of A.beta. with age in transgenic APP
animals (54-55), it was hypothesized that the clearance mechanism
of A.beta. is impaired in older animals, and is also possibly
impaired in elderly humans.
[0115] The present findings of about 55-65% inhibition of A.beta.
clearance in 9-month old wild-type animals in comparison to young
(2-month old) animals confirmed this hypothesis. Immunocytochemical
studies indicated a significant reduction in the number of LRP-1
positive cerebral blood vessels from 94% in 2-month old to 52% in
9-month old mice, which correlated well with the observed
reductions in the clearance capacities between the two age groups.
Interestingly, downregulation of vascular LRP-1 correlated well
with regional parenchymal and vascular accumulation of A.beta. in
brains of Alzheimer's patients compared to age-matched controls. In
brain areas where LRP-1 vascular expression remains prominent, as
in the white matter, no accumulation of A.beta. was found in
Alzheimer's brains.
[0116] Moreover, excess A.beta. amplifies its own accumulation in
brain ISF by accelerating LRP-1's degradation at the BBB clearance
site. LRP-1 on endothelium can be reduced by a high level of
A.beta.. This concentration-dependent effect can be prevented by
RAP or anti-LRP-1 specific antibody. Cells affected or unaffected
by disease, aged or young cells, cells incubated with wild-type or
mutant A.beta. (or aggregates thereof), normal or reduced levels of
LRP-1 on cells, or combinations thereof may be used in screening
assays. The effect of candidate drugs and/or medical regimens for
treating Alzheimer's disease may be evaluated using such cells. For
example, LRP-1 degradation, transport, and/or synthesis by be
manipulated to prevent or reverse the down regulation seen in
Alzheimer's disease.
[0117] These results are illustrative of the discovery that the
vascular system plays an important role in regulating the levels of
A.beta. in the brain. The findings further suggest that if the
levels of A.beta. in brain extracellular space exceed the transport
capacity of the clearance mechanism across the BBB, or if the
vascular transport of the peptide were impaired, as for example, by
down-regulation of LRP-1, this would result in accumulation of
A.beta. in the brain and possibly formation of amyloid plaques.
Previous studies have demonstrated a major role of the BBB in
determining the concentrations of A.beta. in the CNS by regulating
transport of circulating A.beta. (33, 39, 49-51, 62-66). The
present study extends this hypothesis by showing that vascular
transport across the BBB out of the brain may represent a major
physiological mechanism that prevents accumulation of A.beta. and
amyloid deposition in brain.
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[0190] All references (e.g., articles, books, patents, and patent
applications) cited above are indicative of the level of skill in
the art and are incorporated by reference.
[0191] 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. More-over, "comprising" allows
the inclusion of other elements in the claim, "comprising
essentially of" allows the inclusion of other elements in the claim
that do not materially affect operation of the invention, and no
particular relationship between or among elements of a claim is
meant unless such limitation is explicitly recited (e.g.,
arrangement of components in a product claim, order of steps in a
method claim).
[0192] 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.
* * * * *