Note: Descriptions are shown in the official language in which they were submitted.
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LRP-mediated Neuronal Calcium Modulators via NMDA
Receptors, and Uses Thereof
Background of the Invention
State~azeht as to Rights to Invesitiotzs Made Under
Fedef~ally-Spohsos~ed Research a~zd Developme~Zt
Part of the work performed during development of this invention utilized
U. S. Government funds. The U. S. Government has certain rights in this
invention.
Field of the havehtion
The present invention relates to methods of treating neurological disorders
with agents that bind to low-density lipoprotein receptor-related protein
(LRP)
receptors. The present invention also relates to methods of modulating calcium
influx and inhibiting cell death in neuronal cells by tr eating the neuronal
cells with
agents that bind to LRP. The present invention also relates to methods of
identifying agents that modulate calcium influx in neuronal cells by binding
to
LRP.
Backgromtd of the Ihve~Ztion
The low-density lipoprotein receptor is one of the best studied examples
of an endocytic receptor, delivering cholesterol containing lipoproteins and
other
ligands to acidic compartments within cells for further metabolism. A family
of
homologous xeceptors plays similar roles in various tissues, including the
very
low-density lipoprotein receptor (VLDL-r), the apolipoprotein E receptor 2
(APOER2), the low-density lipoprotein receptor related protein (LRP), and
megalin (or GP330).
LRP is a widely expressed endocytic receptor which is strongly expressed
in brain on near ons and reactive astrocytes (Rebeclc, G. W., et al., Neuron
11:575-
80 (1993)). LRP is a >600 kDa (4454 amino acid) protein cleaved in the trans-
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Golgi network to form a heterodimer with a single transmembrane spanning
domain, a 515 kDa extracellular region containing 4 ligand binding repeat
regions, multiple EGF and growth factor repeats, and a smaller intracellular
domain containing two NPXY sequences which direct endocytosis of the receptor
to clathrin coated pits (Herz, J., et al., EMBQ J. 7:4119-27 (1988);
Strickland,
D.K., et al., J. Biol. Chem. 265:17401-4 (1990); Winnow, T.E., et al., EMBO J.
15:2632-9 (1996)). LRP has more than 15 identified ligands, which fall into
several broad categories such as proteases, protease inhibitors, such as
activated
alpha-2-macroglobulin (a2M*), protease/protease inhibitor complexes, protein-
lipid complexes, and other proteins and molecules such as lactoferrin. A list
of
the known ligands of LRP can be found in Hussain, M.M., et al. Anhu. Rev.
Nuts.
19: 141-172 (1999). The 39 kDa receptor associated protein (RAP) is an
endoplasmic chaperone protein tightly bound to LRP, which, when used
pharmacologically, specifically blocks and prevents uptake of all known LRP
ligands (Strickland, D.K., et al., J. Biol. Chem. 265:17401-4 (1990);
Williams,
S.E., et al., J. Biol. ChenZ. 267:9035-40 (1992); Medved, L.V., et al., J.
Biol.
Chem. 274:717-27 ( 1999)). Like other members of the LDL receptor family, LRP
binds and imports these ligands into intracellular vesicles, acidified
compartments
where the ligand is released, and the receptor is recycled to the surface. LRP
(Strickland, D.K., et al., J. Biol. Chem. 265:17401-4 (1990)) and APOER2
(Stockinger, W., et al., J. Biol. Chem. 273:32213-21 (1998)) are the only
known
brain receptors for a2M*, mediating clearance of protease/protease inhibitor
complexes.
Summary of the Invention
The present invention is directed to a method of treating a subj ect in need
of treatment of a neurological disorder, the method comprising: administering
to
the subject a pharmaceutically effective amount of an agent that binds to low-
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density lipoprotein receptor-related protein (LRP) receptor of LRP on neuronal
cells, and modulates calcium influx in said neuronal cells.
Additionally, the present invention is directed to a method of inhibiting cell
death in neuronal cells, the method comprising: providing neuronal cells with
an
agent that binds to low-density lipoprotein receptor-related protein (LRP)
receptor
on the neuronal cells, and modulates calcium influx in the neuronal cells.
Similarly, the present invention also is directed to a method of modulating
calcium influx in neuronal cells, the method comprising: treating the neuronal
cells with an agent that binds to low-density lipoprotein receptor-related
protein
(LRP) receptor on the neuronal cells, and modulates calcium influx in the
neuronal cells.
Furthermore, the present invention is directed to a method of identifying
an agent that modulates calcium influx in neuronal cells by binding to low-
density
lipoprotein receptor-related protein (LR.P) receptors on the neuronal cells,
the
method comprising: (a) treating neuronal cells with an agent and assaying for
calcium influx; (b) treating neuronal cells with a known modulator of LRP-
mediated calcium influx and the agent in (a) and assaying for calcium influx;
and
(c) comparing the levels of calcium influx in (a) and (b) to determine if the
agent
in (a) modulates calcium influx by interacting with an LRP receptor.
Details of the present invention will be clear from the description that
follows.
Brief Desca iptioh of the Figures
Figure 1 Shows that a2M* increases [Ca2+)I specifically in neurons. Primary
cultures of mouse cortex were loaded for 30 min with 1 ~,M indo-1/AM and
imaged using a Biorad Multiphoton confocal microscope. The traces represent
a time-course of intracellular calcium concentration in a field of cells in a
single,
representative experiment. Each trace is the average of 6 cells within the
field,
~ std deviation. Not all cells in the mixed cultures responded to a2M*
treatment.
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The cells that did respond resembled neurons morphologically and also
responded
to NMDA application. Non-responders had the generally flat appearance of glia
and/or fibroblasts and did not respond to NMDA addition.
Figure 2 Shows that all cells in the mixed cultures expressed LRP.
Immunohistochemistry using the anti-LRP antibody 8777 revealed that all cells
in the cultures expressed LRP (top left panel). The top right panel is a dual
label
with an antibody to MAP2, which was expressed exclusively in neurons (Izant,
J.G. and McIntosh, J.R., Proc. Nat. Acad. Sci. U.S.A. 77:4741-5 (1980)). The
scale bar is 25 Vim.
Figure 3 Shows that the calcium increase requires extracellular calcium. Cells
were placed in nominally calcium-free buffer (not containing EGTA), and a2M*
was added at approximately t=150 sec. No intracellular calcium increase was
observed. In fact, a small decrease was indicated. The calcium-free buffer was
washed and replaced with calcium containing buffer (2 mM) at t=500 sec. After
replacement of calcium, [Ca2~]I levels increased to about 400 nM, similar to
levels
normally observed after stimulation by a2M* in calcium containing buffers.
This
suggests that a2M* was able to bind to LRP in the absence of calcium and
initiate
a calcium signaling event. The response required extracellular calcium.
Figure 4 Shows that calcium entry occurs through NMDAR channels. In this
experiment, the cells were pretreated with 5 ~,M MIA-801 for 5 minutes, and
the
NMDA antagonist remained in the bath throughout the procedure. At t=350 sec,
a2M* (3 5 nM) was added to the bath, resulting in a small but insignificant
increase
in [Caz~]I in this field of cells. At t=700 sec, NMDA (100 ~M) was added, and
no
change in [Ca2+]I was observed. Glutamate (10 ~,M), however, was capable of
eliciting a calcium response at t=900 sec. This is a representative trace of
n=4
experiments. The trace is the mean ~ std. dev. of n=15 neurons in the field.
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Figure 5 Shows that a2M* increases calcium in neurons in which LRP receptors
are blocked by RAP.
Figure 5A. a2M* (3 5 nM) was added to the bath at around 200 sec, which
elicited a rapid, sustained increase in calcium. At t=650 sec, NMDA (100 ~.M)
was added to the bath, resulting in an additional rise in calcium. The trace
is the
average response from 9 cell bodies in the field ~ std dev.
Figure 5B. RAP (500 nM) was added to the bath at t=200 sec. RAP had
no discernible effect on intracellular calcium. At t=650 sec, a2M* (35 nM) Was
added to the bath, but calcium was unaffected. NMDA was able to elicit a
normal
response in the 7 cells in this field.
Figure 6 Shows that an antibody to the ligand binding domain of LRP increases
[Ca2+]I , but an antibody to an intracellular domain of LRP does not. This
figure
illustrates two experiments utilizing rabbit polyclonal antibodies directed
against
LRP. In the top trace (circles) 8777, which recognizes the ligand binding
domain
of LRP, was added. Addition of 8777 increased [Ca2+]I in a neuron specific
manner. In the bottom trace (squares), addition of 8704, which recognizes an
intracellular domain of LRP, was unable to elicit an increase in [Caz+]I.
However,
subsequent addition of a2M* was able to generate a calcium response in these
cells.
Detailed Description of the P~efe~~ed Embodiments
The LRP receptor is a widely expressed endocytic receptor which is
strongly expressed on neuronal cells, including neurons and glial cells of the
central and peripheral nervous systems (Rebeck, G. W., et al., Neu~oh 11:575-
80
(1993)). A novel neuron-specific signaling role for LRP is reported herein.
Namely, LRP ligand binding and receptor dimerization led to calcium influx in
neuronal cells via NMDAR channels. A robust, spatially and temporally discrete
calcium signal was observed in neurons treated with ligand competent a2M*,
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which was blocked by RAP. Non-neuronal cells, which also expressed LRP, in
the same cultures did not elicit a calcium response. The calcium signal was
dependent on extracellular calcium and Was blocked by the NMDA receptor
antagonist MK-801 (Tolar, M., et al., J. Neu~osci. 19:7100-10 (1999)). Calcium
influx in neurons also occurred after treatment with 8777, an antibody
directed
against the extracellular domain of LRP, and this response was also blocked
with
MK-801. Calcium entry did not occur after treatment with Fab fragments of
8777, suggesting that receptor dimerization may be critical. These results
demonstrate a novel signaling role for the multifunctional receptor LRP in
neurons.
As used herein, an LRP receptor is a protein that is recognized in the art
as such, and forms a heterodimer with a single tr ansmembr ane spanning
domain,
contains an extracellular region containing 4 ligand binding repeat regions,
contains multiple EGF aild growth factor repeats, and contains a smaller
intracellular domain containing two NPXY sequences (where N symbolizes the
amino acid asparagine, P symbolizes proline, X is any amino acid and Y
symbolizes tyrosine) which direct endocytosis of the receptor to clathrin
coated
pits (Herz, J., et al., EMBO J. 7:4119-27 (1988); Strickland, D.K., et al., J.
Biol.
Chef~z. X65:17401-4 (1990); Winnow, T.E., et al., EMBO J. 15:2632-9 (1996)).
The present invention is directed to a method of treating a subject in need
of treatment of a neurological disorder, the method comprising: administering
to
the subject a pharmaceutically effective amount of an agent that binds to low-
density lipoprotein receptor-related protein (LRP) receptor on neuronal cells,
and
modulates calcium influx in said neuronal cells.
As used herein, the term receptor is meant to include a molecule that binds
to a ligand and causes a cellular or physiological response. The receptor can
be
cytosolic, membrane-bound, membrane-spaaming, or it can be an extracellular
molecule. Additionally, the receptor can be in the form of a monomer or a
multimer (r. e., dimer, trimer, or higher multimer). The term multimer
encompasses
a homomultimer or a heteromultimer. As used herein, the term homomultimer is
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used to mean a multimer molecule where all of the individual proteins or other
molecules that constitute the multimer are identical. A heteromultimer, on the
other hand, is used herein to mean a multimer molecule where any of the
individual proteins or other molecules that constitute the multimer are not
identical. In other words, a monomeric receptor causes the physiological or
cellular response solely, while the multimeric receptor may require two or
more
proteins or other molecules, acting in concert with one another, to cause a
physiological or cellular response. Examples of molecules that can act as
receptors include, but are not limited to, proteins, polysaccharides,
glycoproteins,
proteoglycans, nucleic acids, lipids, and lipoproteins.
Examples of cellular responses that receptors initiate or propagate, which
should be obvious to one skilled in the art, include, but are not limited to
ion influx
or efflux, initiation of second messenger pathways, synthesis of DNA,
translation
of mRNA, entry of the cells into the cell cycle, arrest of the cell in the
cell cycle,
endocytosis, release of molecules from the cell, exocytosis, and apoptosis.
The
cellular response on which the current invention focuses is modulating calcium
influx in the affected neuronal cell.
As used herein, modulation of calcium entry into the cytoplasm of the
affected cell includes such responses as increasing or decreasing the quantity
of
calcium ions that normally enter the cell from the extracellular environment,
in
conjunction with another stimulus. Likewise, modulation of calcium entry into
the
cytoplasm of the affected cell also includes such responses as increasing or
decreasing the quantity of calcium ions that normally enter the cell from the
extracellular environment, in the absence of another stimulus. Additionally,
modulation of calcium influx is meant to encompass increasing or decreasing
the
quantity of release or uptake of calcium ions from or to intracellular stores,
such
as mitochondria, in conjunction with another stimulus. Likewise, modulation of
calcium influx is also meant to encompass increasing or decreasing the
quantity of
release or uptake of calcium ions from or to intracellular stores, such as
mitochondria, in the absence of another stimulus.
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Preferably, upon binding of an agent with LRP, the channel through which
calcium influx in the neuronal cells is mediated is not LRP. More preferably,
the
channel through which calcium influx in the neuronal cells is mediated is
through
a class of receptors that binds to the ligand N-methyl-D-aspartate (NMDA),
dubbed NMDA receptors. NMDA receptors are ligand-gated ion channels that
are a subclass of the larger family of glutamate receptors. A ligand-gated
channel
is a receptor that binds a ligand and subsequently opens to allow the flow of
ions,
such as Nay, I~~ or Ca2+, into or out of the cell. In addition to ligand
binding, the
flow of ions through ligand-gated chamlels is also controlled by the voltage
potential across the plasma membrane separating the cytosol and the
extracellular
space. Specifically, the NMDA receptor is a channel for Ca2+, and it is
thought to
be responsible for the induction of long-term potentiation (LTP) and long-term
depression (LTD). LTP is the phenomenon where a postsynaptic neuron has a
prolonged, increased response to a presynaptic stimulus. LTP is thought to, at
least partially, account for cellular and physiological memory and learning.
Conversely, LTD is the phenomenon where a postsynaptic neuron has a prolonged
decrease response to a presynaptic stimulus.
One embodiment of the current invention is that the agent that binds to
LRP, causes dimerization of the LRP receptors. Subsequently, this dimerization
process regulates calcium influx in the neuronal cells. As used herein,
dimerization
is the process that is well-recognized in the art where two separate proteins
form
an association. The dimer formed may be a heterodimer or a homodimer. The
association forming the dimer can be temporary or permanent. Furthermore, the
association of the two proteins can serve to enhance or diminish the normal
function or signaling capacity of each of the two proteins. Alternatively, the
association of the two proteins can lead to a completely different function,
e.g.,
second messenger propagation, than the normal function of either of the two
proteins.
As used herein, the term agent, ligand or compound is intended to mean
a protein, nucleic acid, carbohydrate, lipid or a small molecule. The types of
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agents or compounds which can be envisioned are limited only by their ability
to
bind to LRP and mediate calcium influx, particularly through the NMDA receptor
channels. LRP has at least four documented binding sites. Each of these four
binding sites has its own ligand binding specificity domains, such that the
various
ligands that LRP binds do not bind to the same binding domain. For example,
although lactoferrin is a ligand of LRP, there are binding sites on LRP that
are
unresponsive to lactoferrin. Thus, in one embodiment, the agent binds to a
site on
LRP that is unresponsive to lactoferrin. In another embodiment, the agent can
bind to a site on the LRP that does not bind to amyloid precursor protein
(APP).
Similarly, the agent that binds to LRP and modulates calcium does not
interfere
with the amount or rate of binding of APP to LRP. Alternatively, the agent
binds
to a site on LRP that is unresponsive to lactoferrin, and the agent also does
not
bind to the APP binding site. Preferably, agents of the present invention
include
agents selected from, but are not limited to, protein-lipid complexes,
proteases,
protease inhibitors, protease-inhibitor complexes, intracellular proteins,
small
molecules, LRP receptor antibodies, and LRP-interacting proteins. Examples of
members of the aforementioned classes, which should be obvious to one skilled
in the art include, but are not limited to, protein-lipid complexes involved
in lipid
and/or cholesterol metabolism such as apolipoprotein, proteases such as
plasminogen, protease inhibitors such as activated alpha-2-macroglobulin,
proteins
such as beta-amyloid precursor protein, small molecules such as
aminoglycosides
(e.g., gentamicin), LRP receptor antibodies. and receptor associated protein
(RAP) or compounds based on the structure of RAP. Accordingly, preferable
agents of the present invention include, but are not limited to, activated
alpha-2-
mace oglobulin, apolipoprotein E, and apolipoprotein E4. A preferred agent of
the
current invention is activated alpha-2-macroglobulin.
The current invention can be useful in treating a subject in need of
treatment of a neurological disorder where aberrant calcium influx in neuronal
cells is either causal or symptomatic. A neurological disorder, as used in the
current context, should be obvious to one skilled in the art, but is meant to
include
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any abnormal physical or mental behavior or experience where neuronal cells
are
involved in the etiology of the disorder, or are affected by the disorder. As
used
herein, neurological disorders encompass disorders affecting the central and
peripheral nervous systems, and include such afflictions as memory loss,
stroke,
dementia, personality disorders, gradual, permanent or episodic loss of muscle
control. Examples of neurological disorders for which the current invention
can
be used include, but are not limited to: Alzheimer's Disease, Parkinson's
Disease,
Huntington's Disease, amyotrophic lateral sclerosis, epilepsy, and stroke.
More
preferably, the current invention can be used to treat Alzheimer's Disease.
As used herein, the term subject can be used to mean an animal, preferably
a mammal including a human or non-human. The term patient is used to indicate
a subject in need of treatment of a neurological disorder.
The treatment envisioned by the current invention can be used for patients
with a pre-existing neurological condition, or for patients pre-disposed to a
neurological disorder. Additionally, the method of the current invention can
be
used to correct cellulax or physiological abnormalities involved with a
neurological
disorder in patients, andJor to alleviate symptoms of a neurological disorder
in
patients, or as a preventative measure in patients.
The present invention is further directed to a method of inhibiting cell
death in neuronal cells, the method comprising: providing neuronal cells with
an
agent that binds to low-density lipoprotein receptor-related protein (LRP)
receptor
and modulates calcium influx in the neuronal cells. The invention ca.n be
practiced
in vitro or iJZ vivo. As used herein, cell death includes a process or event
that
causes the cell to cease or diminish normal metabolism in vivo or in
vita°o. The
various forms and signs of cell death are obvious to those skilled in the art,
but
examples of cell death include, but are not limited to, programmed cell death
(i.e.,
apoptosis), gradual death of the cells as occurs in diseased states (i.e.,
necrosis),
and more immediate cell death such as acute toxicity. The inhibition of cell
death
for which the current invention provides can be a complete or partial
inhibition of
cell death. Likewise, the inhibition of cell death for which the current
invention
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provides can be a complete or partial reversal of the process of cell death.
Preferably, the present invention inhibits cell death by modulating calcium
influx
in neuronal cells. As the current invention contemplates, modulation of
calcium
influx has been previously described herein.
The present invention provides for inhibiting cell death by modulating
calcium influx in neuronal cells. Preferably, the channel through which
calcium
influx in the neuronal cells is mediated is not LRP. More preferably, the
channel
through which calcium influx in the neuronal cells is mediated is through the
NMDA class of receptors.
Agents of the current invention useful for inhibiting neuronal cell death
include any agent that binds to LRP and subsequently modulates calcium influx
in
neuronal cells. Preferably, the agents of the current invention useful for
inhibiting
cell death include, but are not limited to, receptor associated protein (RAP),
small
molecules or peptides that mimic RAP, LRP receptor antibodies, and proteins
that
interact with LRP. Such agents have been previously described herein.
The present invention also is directed to a method of modulating calcium
influx in neuronal cells, the method comprising: treating the neuronal cells
with
an agent that binds to low-density lipoprotein receptor-related protein (LRP)
receptor on neuronal cells and modulates calcium influx in the neuronal cells.
Preferably, the channel through which calcium influx in the neuronal cells is
mediated is not LRP. More preferably, the channel through which calcium influx
in the neuronal cells is mediated is through the NMDA class of receptors. As
the
current invention contemplates, modulation of calcium influx has previously
been
described. The invention can be practiced in vitro or ih vivo.
Preferably, agents that interact with LRP on neuronal cells and modulate
calcium influx include, but are not limited to protein-lipid complexes,
proteases,
protease inhibitors, protease-inhibitor complexes, proteins, small molecules,
LRP
receptor antibodies, and proteins that interact with LRP. Such agents have
been
previously described herein. Furthermore, the types of agents that can be
envisioned include agonists and antagonists of LRP and are limited only by
their
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ability to bind to LRP and modulate calcium influx, particularly through the
NMDA receptor channels. As used herein, an agonist is a protein, nucleic acid,
carbohydrate, lipid or a small molecule that binds to LRP and mimics the
calcium
influx that activated alpha-2-macroglobulin elicits under similar, or
identical,
conditions. The cellular response that the agonist mimics does not have to be
identical in magnitude, duration or character. As used herein, an antagonist
is a
protein, nucleic acid, carbohydrate, lipid or a small molecule that binds to
LRP and
attenuates, or reverses the calcium influx that activated alpha-2-
macroglobulin
elicits Lender similar, or identical, conditions. The cellular response that
the
antagonist prevents does not have to be a total prevention or reversal of the
response that the ligand elicits.
Agents of the present invention that increase calcium influx in neuronal
cells are agonists or stimulators of LRP. As used herein, the term stimulator
is
meant to include any agent that produces an increase in calcium movement into
the cell. Thus, a stimulator of LRP-mediated calcium movement is any agent
that
binds to LRP and causes an increase in calcium movement into or out of the
cell.
A preferred agent of the current invention that is an agonist of~LRP and
modulates
calcium influx in neuronal cells is activated alpha-2-macroglobulin.
Similarly,
preferable agonists of LRP that are used to modulate calcium influx in
neuronal
cells also include antibodies that increase LRP-mediated calcium influx in
neurons
through the NMDA receptor channels, such as 8777. The antibody 8777 is an
antibody that binds to LRP and can be used to block the binding of activated
alpha-2-macroglobulin and other ligands or agents that bind to LRP. The 8777
antibody was obtained from the fusion of spleen cells of mice, which had been
immunized with activated a2M, with the myeloma cell line P3-X63-Ag8.653. The
production and binding specificity of 8777 towards LRP has been previously
described in Strickland, D.K., et al., Biochemistry 27: 1458-1466 (1988), and
Strickland, D.K., et al., J. Biol. Chem. 265:17401-17404 (1990).
Agents of the present invention that decrease calcium influx in neuronal
cells are antagonists or inhibitors of LRP. As used herein, the term inhibitor
is
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meant to include any agent thatproduces a complete or partial blocking of
calcium
movement into the cell. Thus, an inhibitor of LRP-mediated calcium movement
is any agent that binds to LRP and produces a complete or partial blocking of
calcium movement into the cell. Most preferably the agents of the current
invention that are antagonists of LRP modulate calcium influx in neuronal
cells are
receptor associated protein (RAP), antibodies that inhibit LRP-mediated
calcium
influx in neurons through the NMDA receptor chamlels, and small molecules or
peptides that mimic R.AP.
The antibodies used in the invention can be, but are not limited to,
chimeric, humanized, and human and nonhwnan monoclonal and polyclonal
antibodies. Antibodies may be used as an isolated whole antibody, or can be
used
as a source for generating antibody fragments which contain the antigen
binding
site of the antibody. Examples of such antibody fragments include, but are not
limited to the F," the F(ab), the F(ab)Z, fragment and single chain
antibodies.
Various methods known in the art can be used to generate such whole antibodies
or antibody fragments without undue experimentation. For example, apolypeptide
of interest or an antigenic fragment thereof can be administered to an animal
to
induce the production of sera containing polyclonal antibodies. Monoclonal
antibodies can be prepared using a wide of techniques known in the art
including
the use of hybridoma and recombinant technology. See, e.g., Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas
563-681 (Elsevier, N. Y.,1981 ) (said references incorporated by reference in
their
entireties). Recombinant techniques are preferred for generating large
quantities
of antibodies, antibody fragments and single chain antibodies, as described,
for
example, in Pluckthum, BiolTechnology 10:163-167 (1992); Carter et al.,
BiolTechhology 10:167-170 (1992); and Mullinax et al., Biotechhiques 1~:864-
869 (1992). In addition, recombinant techniques may be used to generate
heterobifunctional antibodies.
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Furthermore, the present invention is directed to a method of identifying
an agent that modulates calcium influx in neuronal cells by binding to low-
density
lipoprotein receptor-related protein (LRP) receptor on the neuronal cells, the
method comprising: (a) treating neuronal cells with an agent and assaying for
calcium influx; (b) treating neuronal cells with a known modulator of LRP-
mediated calcium influx and the agent in (a) and assaying for calcium influx;
and
(c) comparing the levels of calcium influx in (a) and (b) to determine if the
agent
in (a) modulates calcium influx by interacting with an LRP receptor.
Preferably,
the channel through which calcium influx in the neuronal cells is mediated is
not
LRP. More preferably, the channel through which calcium influx in the neuronal
cells is mediated is through the NMDA class of receptors. Accordingly, in one
preferred embodiment of the current invention, the known modulator of LRP-
mediated calcium influx is an NMDA receptor channel antagonist. Examples of
NMDA receptor channel antagonists include, but are not limited to, MK-801,
D(-)-2- Amino- 5- phosphonopentanoic acid, D(-)- 2- Amino- 4-
phosphonobutyric acid, ketamine, ifenprodil or phencyclidine. More preferably,
the NMDA receptor channel antagonist is MK-801. The pharmacological agent
MK-801 is also known as dizocilpine and is described in Woodruff G.N. et al.,
Neuropharmacology 26: 903-9 (1987). As used herein, an NMDA receptor
channel antagonist is a protein, nucleic acid, carbohydrate, lipid or a small
molecule that binds to an NMDA receptor and blocks, attenuates, or reverses
the
calcium influx that N-methyl-D-aspartate elicits Lender similar, or identical,
conditions.
In one embodiment of the current invention, the method of identifying an
agent that modulates calcium influx in neuronal cells is performed on a single
population of cells, and (b) is performed on the identical population after
the agent
in (a) is removed. In another embodiment of the invention, the method of
identifying an agent that modulates calcium influx in cells is performed on
two
nearly identical populations of cells, under the same culture conditions,
where (a)
is performed on one population and (b) is performed on another population, and
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(c) is a comparison of the levels of calcium influx between the two
populations of
cells.
As used herein, assaying for calcium influx can be accomplished by using
any means that can detect differences in intracellular or extracellular
calcium
levels. Such means, which should be obvious to one of ordinary skill on the
art,
include, but are not limited to, the use of fluorescent dyes in conjunction
with
microscopy (calcium imaging) (Grynkiewicz, G., et al., J. Biol. Chem. 260:3440-
50 (1985)), enzyme-linked immunosorbent assays (ELISA), radioactively-labeled
isotopes, and detecting local or systemic changes in membrane potential or
voltage.
The types of agents that can be tested can be proteins, nucleic acids,
carbohydrates, lipids or small molecules. The types of agents or compounds
which can be envisioned are limited only by their ability to bind to L1RP and
modulate LRP-mediated calcium influx in neurons through the NMDA receptor
channels.
The agents of the present invention may be identified andlor prepared
according to any of the methods and techniques known to those skilled in the
art.
These agents, particularly peptide agents and antibody agents, may occur or be
produced as monomer, dimers, trimers, tetrameres or multimers. Such multimers
can be prepared using enzymatic or chemical treatment of the native receptor
molecules or be prepared using recombinant techniques. Preferably, the agents
of
the present invention are selected and screened at random or rationally
selected
or designed using protein modeling techniques.
For random screening, candidate agents are selected at random and
assayed for their ability to bind to LRP and cause calcium influx in neurons.
Any
of the suitable methods and techniques known to those slcilled in the art may
be
employed to assay candidate agents.
For rational selection or design, the agent is selected based on the
configuration of the LRP binding site found on an LRP ligand, e.g. a2M*, or a
ligand binding site found on the L1RP. Any of the suitable methods and
techniques
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known to those skilled in the art may be employed for rational selection or
design.
For example, one skilled in the art can readily adapt currently available
procedures
to generate antibodies, peptides, pharmaceutical agents and the like capable
of
binding to a specific peptide sequence of LRP. Illustrative examples of such
available procedures are described, for example, in Hurby et al. ,
"Application of
Synthetic Peptides: Antisense Peptides," in Synthetic Peptides, A User's
Guide,
W.H. Freeman, NY, pp. 289-307 (1992); Kaspczak et al., Biochemistry 2:9230
(1989); and Harlow, Antibodies, Cold Spring Harbor Press, NY (1990).
The agents of the present invention can alternatively be identified using
modification of methods known in the art. For example, suitable peptide agents
may be identified using the filter binding assay described by Mischak et al.
(Mischalc et al., J. Gefz. Virol. 69:2653-2656 (1988) and Mischak et al.,
Vis°ology
163:19-25 (1988)), wherein the peptide is applied to a suitable membrane, such
as nitrocellulose, and the membrane is saturated with a detergent mixture in
order
to block any non-specific binding. The treated membrane is then incubated with
labeled LRP (labeled with 'ZSI-iodine), to check the specific binding. After
washing and drying of the membrane, specific binding can be visualized by
autoradiography.
Fot~uzulatiozzs azzd Metlzods ofAdzzziuistratiou
As used herein, "a pharmaceutically effective amount" is intended an
amount effective to elicit a cellular response that is clinically significant,
without
excessive levels bf side effects.
A pharmaceutical composition of the invention is thus provided comprising
an agent of the invention useful for treatment of a neurological disorder and
a
pharmaceutically acceptable carrier or excipient.
It will be desirable or necessary to introduce the pharmaceutical
compositions directly or indirectly to the brain. Direct techniques usually
involve
placement of a drug delivery catheter into the host's ventricular system to
bypass
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the blood-brain barrier. Indirect techniques, which are generally preferred,
involve
formulating the compositions to provide for drug latentiation by the
conversion
of hydrophilic drugs into lipid-soluble drugs. Latentiation is generally
achieved
through blocking of the hydroxyl, carboxyl, and primary amine groups present
on
the drug to render the drug more lipid-soluble and amenable to transportation
across the blood-brain barrier. Alternatively, the delivery of hydrophilic
drugs can
be enhanced by intra-arterial infusion ofhypertonic solutions which can
transiently
open the blood-brain barrier.
The blood-brain barrier (BBB) is a single layer of brain capillary
endothelial cells that are bound together by tight junctions. The BBB excludes
entry of many blood-borne molecules. In the invention, the agent can be
modified
for improved penetration of the blood-brain barrier using methods known in the
art. Alternatively, a compound with increase permeability of the BBB can be
administered to the subject. RMP-7, a synthetic peptidergic bradykinin agonist
was reported to increase the permeability of the blood-brain barrier by
opening the
tight junctions betweenthe endothelial cells ofbrain capillaries (Elliott,
P.J. et al.,
Exptl. Neut°ol. 141:214-224 (1996)).
The invention further contemplates the use of prodrugs which are
converted in vivo to the therapeutic compounds ofthe invention (Silverman,
R.B.,
"The Organic Chemistry of Drug Design and Drug Action," Academic Press, Ch.
~ (1992)). Such prodrugs can be used to alter the biodistribution (e.g., to
allow
compounds which would not typically cross the blood-brain barrier to cross the
blood-brain barrier) or the phaxmacokinetics of the therapeutic compound. For
example, an anionic group, e.g., a sulfate or sulfonate, can be esterified,
e.g, with
a methyl group or a phenyl group, to yield a sulfate or sulfonate ester. When
the
sulfate or sulfonate ester is administered to a subject, the ester is cleaved,
enzymatically or non-enzymatically, to reveal the anionic group. Such an ester
can
be cyclic, e.g., a cyclic sulfate or sultone, or two or more anionic moieties
can be
esterified through a linking group. An anionic group can be esterified with
moieties (e.g., acyloxymethyl esters) which are cleaved to reveal an
intermediate
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compound which subsequently decomposes to yield the active compound.
Furthermore, an anionic moiety can be esterified to a group which is actively
transported in vivo, or which is selectively taken up by target organs. The
ester
can be selected to allow specific targeting of the therapeutic moieties to
particular
organs, as described below for carrier moieties.
The therapeutic compounds or agents of the invention can be formulated
to cross the blood-brain-barrier, for example, in liposomes. For methods of
manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and
5,399,331. The liposoxnes may comprise one or more moieties which are
selectively transported into specific cells or organs thus providing targeted
drug
delivery (Ranade, J., Cli~c. Pharmacol. 29:685 (1989)). Exemplary targeting
moieties include folate orbiotin (U.S. Pat. No. 5,416,016), mannosides
(Umezawa
et al., Biochenz. Biophys. Res. Comm. 153:1038 (1988)), antibodies (Bloeman
et al., FEBS Lett. 357:140 (1995); Owais et al., Antimicrob. Agents Chemothe~.
39:180 (1995)), surfactant protein A receptor (Briscoe et al., Am. J. Physiol.
1233:134 (1995)), gp 120 (Schreier et al., J. Biol. Chem. 269:9090 (1994);
Killion and Fidler, Immunomethods 4:273 (1994)).
The pharmaceutical composition can be administered orally, nasally,
parenterally, intrasystemically, intraperitoneally, topically (as by drops or
transdermal patch), bucally, or as an oral or nasal spray. By
"pharmaceutically
acceptable carrier" is intended, but not limited to, a non-toxic solid,
semisolid or
liquid filler, diluent, encapsulating material or formulation auxiliary of any
type.
The term "parenteral" as used herein refers to modes of administration which
include intravenous, intramuscular, intraperitoneal, intrasternal,
subcutaneous and
intraarticular injection and infusion.
A pharmaceutical composition of the present invention for parenteral
inj ection can comprise pharmaceutically acceptable sterile aqueous or
nonaqueous
solutions, dispersions, suspensions or emulsions as well as sterile powders
for
reconstitution into sterile injectable solutions or dispersions just prior to
use.
Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or
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vehicles include water, ethanol, polyols (such as glycerol, propylene glycol,
polyethylene glycol, and the like), carboxymethylcellulose and suitable
mixtures
thereof, vegetable oils (such as olive oil), and injectable organic esters
such as
ethyl oleate. Proper fluidity can be maintained, for example, by the use of
coating
materials such as lecithin, by the maintenance of the required particle size
in the
case of dispersions, and by the use of surfactants.
The compositions of the present invention can also contain adjuvants such
as, but not limited to, preservatives, wetting agents, emulsifying agents, and
dispersing agents. Prevention of the action of microorganisms can be ensured
by
the inclusion of various antibacterial and antifungal agents, for example,
paraben,
chlorobutanol, phenol sorbic acid, and the like. It can also be desirable to
include
isotonic agents such as sugars, sodium chloxide, and the like. Prolonged
absorption of the injectable pharmaceutical form can be brought about by the
inclusion of agents which delay absorption such as aluminum monostearate and
gelatin.
In some cases, in order to prolong the effect of the drugs, it is desirable to
slow the absorption from subcutaneous or intramuscular injection. This can be
accomplished by the use of a liquid suspension of crystalline or amorphous
material with poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution which, in turn, can depend upon crystal
size
and crystalline form. Alternatively, delayed absorption of a paxenterally
administered drug form is accomplished by dissolving or suspending the drug in
an oil vehicle.
Injectable depot forms are made by forming microencapsule matrices of
the drug inbiodegradable polymers such as polylactide-polyglycolide. Depending
upon the ratio of drug to polymer and the nature of the particular polymer
employed, the rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot
injectable formulations are also prepared by entrapping the drug in liposomes
or
microemulsions which are compatible with body tissues.
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The injectable formulations can be sterilized, for example, by filtration
through a bacterial-retaining filter, or by incorporating sterilizing agents
in the
form of sterile solid compositions which can be dissolved or dispersed in
sterile
water or other sterile injectable medium just prior to use.
Solid dosage forms for oral administration include, but are not limited to,
capsules, tablets, pills, powders, and granules. In such solid dosage forms,
the
active compounds are mixed with at least one item pharmaceutically acceptable
excipient or carrier such as sodium citrate or dicalcium phosphate and/or a)
fillers
or extenders such as starches, lactose, sucrose, glucose, mannitol, and
silicic acid,
b) binders such as, for example, carboxymethylcellulose, alginates, gelatin,
polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol,
d) disintegrating agents such as agar-agar, calcium carbonate, potato or
tapioca
starch, alginic acid, certain silicates, and sodium carbonate, e) solution
retarding
agents such as paraffin, f) absorption accelerators such as quaternary
ammonium
compounds, g) wetting agents such as, for example, acetyl alcohol and glycerol
monostearate, h) absorbents such as kaolin and bentonite clay, and I)
lubricants
such as talc, calcium stearate, magnesium stearate, solid polyethylene
glycols,
sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets
and
pills, the dosage form can also comprise buffering agents.
Solid compositions of a similar type can also be employed as fillers in soft
and hardfilled gelatin capsules using such excipients as lactose or milk sugar
as
well as high molecular weight polyethylene glycols and the like.
The solid dosage forms of tablets, dragees, capsules, pills, and granules can
be prepared with coatings and shells such as enteric coatings and other
coatings
well known in the pharmaceutical formulating art. They can optionally contain
opacifying agents and can also be of a composition that they release the
active
ingredients) only, or preferentially, in a certain part of the intestinal
tract,
optionally, in a delayed mariner. Examples of embedding compositions which can
be used include polymeric substances and waxes.
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The active compounds can also be in micro-encapsulated form, if
appropriate, with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration include, but are not limited to,
pharmaceutically acceptable emulsions, solutions, suspensions, syrups and
elixirs.
In addition to the active compounds, the liquid dosage forms can contain inert
diluents commonly used in the art such as, for example, water or other
solvents,
solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol,
ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,
1,3-
butylene glycol, dimethyl formamide, oils (in particular, cottonseed,
groundnut,
corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl
alcohol,
polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include adjuvants
such as wetting agents, emulsifying and suspending agents, sweetening,
flavoring,
and perfuming agents.
Suspensions, in addition to the active compounds, can contain suspending
agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene
sorbitol
and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide,
bentonite, agar-agar, and tragacanth, and mixtures thereof.
Topical administration includes administration to the skin or mucosa,
including surfaces of the lung and eye. Compositions for topical
administration,
including those fox inhalation, can be prepared as a dry powder which can be
pressurized or non-pressurized. In nonpressurized powder compositions, the
active ingredients in finely divided form can be used in admixture with a
larger-
sized pharmaceutically acceptable inert care ier comprising particles having a
size,
for example, of up to 100 ~,m in diameter. Suitable inert carriers include
sugars
such as lactose. Desirably, at least 95% by weight of the particles of the
active
ingredient have an effective particle size in the range of 0.01 to 10 ~,m.
Alternatively, the composition can be pressurized and contain a
compressed gas, such as nitrogen or a liquefied gas propellant. The liquefied
propellant medium and indeed the total composition is preferably such that the
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active ingredients do not dissolve therein to any substantial extent. The
pressurized composition can also contain a surface active agent. The surface
active agent can be a liquid or solid non-ionic surface active agent or can be
a
solid anionic surface active agent. It is preferred to use the solid anionic
surface
active agent in the form of a sodium salt.
The compositions of the present invention can also be administered in the
form of liposomes. As is known in the art, liposomes are generally derived
from
phospholipids or other lipid substances. Liposomes are formed by mono- or
mufti-lamellax hydrated liquid crystals that are dispersed in an aqueous
medium.
Any non-toxic, physiologically acceptable and metabolizable lipid capable of
forming liposomes can be used. The present compositions in liposome form can
contain, in addition to the compounds of the invention, stabilizers,
preservatives,
excipients, and the like. The preferred lipids are the phospholipids and the
phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form
liposomes are known in the art (see, for example, Prescott, Ed., Meth. Cell
Biol.
14:33 et seq (1976)).
Dosaging
One of ordinary skill will appreciate that effective amounts of the agents
of the invention can be determined empirically and can be employed in pure
form
or, where such forms exist, in pharmaceutically acceptable salt, ester or
prodrug
form. The agents can be administered to a subject, in need of treatment of a
neurological disorder, as pharmaceutical compositions in combination with one
or
more pharmaceutically acceptable excipients. It will be understood that, when
administered to a human patient, the total daily usage of the agents or
composition
of the present invention will be decided by the attending physician within the
scope
of sound medical judgement. The specific therapeutically effective dose level
for
any particular patient will depend upon a variety of factors: the type and
degree
of the cellular response to be achieved; activity of the specific agent or
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composition employed; the specific agents or composition employed; the age,
body weight, general health, sex and diet of the patient; the time of
administration,
route of administration, and rate of excretion of the agent; the duration of
the
treatment; drugs used in combination or coincidental with the specific agent;
and
like factors well lcnown in the medical arts. For example, it is well within
the skill
of the art to start doses of the agents at levels lower than those required to
achieve
the desired therapeutic effect and to gradually increase the dosages until the
desired effect is achieved.
For example, satisfactory results are obtained by oral administration of the
compounds at dosages on the order of from 0.05 to 10 mg/kg/day, preferably 0.1
to 7.5 mg/kg/day, more preferably 0.1 to 2 mglkg/day, administered once or, in
divided doses, 2 to 4 times per day. On administration parenterally, for
example
by i.v. drip or infusion, dosages on the order of from 0.01 to 5 mg/kg/day,
preferably 0.05 to 1.0 mg/kg/day and more preferably 0.1 to 1.0 mg/kg/day can
be used. Suitable daily dosages for patients are thus on the order of from 2.5
to
500 mg p.o., preferably 5 to 250 mg p.o., more preferably 5 to 100 mg p.o., or
on
the order of from 0.5 to 250 mg i.v., preferably 2.5 to 125 mg i.v. and more
preferably 2.5 to 50 mg i.v.
Dosaging can also be arranged in a patient specific manner to provide a
predetermined concentration of the agents in the blood, as determined by
techniques accepted and routine in the art (HPLC is preferred). Thus patient
dosaging can be adjusted to achieve regular on-going blood levels, as measured
by HPLC, on the order of from 50 to 1000 ng/ml, preferably 150 to 500 ng/ml.
It will be readily apparent to one of ordinary skill in the relevant arts that
othex suitable modifications and adaptations to the methods and applications
described herein can be made without departing from the scope of the invention
or any embodiment thereof.
The following Example serves only to illustrate the invention, and is not
to be construed as in any way to limit the invention.
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Example
Materials ahd Methods
Primary cultures of mouse cortex were prepared from embryonic day
15-17 CD1 mice. The cortices were isolated and triturated in Ca2~ free PBS and
plated onto 35 mm poly-lysine coated culture, dishes at a density of
2x106 cells/ml in neurobasal medium containing 10% fetal bovine serum
(Intergen,
NY), 2 mM glutamine, 100 U/ml penicillin and 100 ~,g/ml streptamycin. After
45-60 min at 37°C, supernatants containing unattached cells were
removed, and
attached cells were incubated in neurobasal medium supplemented with 1X B27
(Gibco, Gaithersburg, MD). After 48 hrs, 5 ~g/ml cytosine-(3-D-
arabinofuranoside (Sigma, St. Louis) was added for 48 hrs in serum containing
media, and then the media was replaced with neurobasal plus B27. Cultures were
used between 7-14 days after plating. Although highly enriched for neurons,
the
cultures contained some non-neuronal cells.
Alpha-2-macroglobulin (Sigma) was activated (a2M*) by incubating with
100 mM methylamine at pH=7.6 in PBS for 1 hr at room temperature, followed
by dialysis in PBS for 24 hours at 4 ° C, with at least 3 buffer
changes. Native a2M
was treated identically except for the addition of methylamine.
For calcium imaging experiments, indo-1/AM (Calbiochem, La Jolla, CA)
was mixed with 20% pluronic F- 127 (Molecular Probes, Eugene, Oregon) in
DMSO and then added to the culture dishes at a final concentration of 1 ~,M
indo-
1/AM and 0.02% pluronic F-127 for 30 min (Grynkiewicz, G., et al., J. Biol.
Cheyn. X60:3440-50 (1985)). The cells were then washed and maintained in
Hanks Balanced Salt Solution (HBSS), supplementedwith 1 g/L glucose, pH=7.4.
The dishes were placed on the stage of an upright microscope (Olympus
BXSOVJI), and imaged with a mufti-photon confocal microscope (Biorad,
Hercules, CA). A femtosecond pulsed Ti:Saphire laser (Spectra Physics,
Mountain View, CA) tuned to 725 nm, provided approximately 300 mW of
excitation power. External PMTS (Hamamatsu, Hamamatsu City, Japan) which
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did not require de-scanning the emission signal were used to capture the two
wavelength channels which were discriminated with interference filters
corresponding to 390 nm, 65 nm band pass, and 495 nm, 20 nm band pass
(Chroma Technology Corp., Brattleboro, Vermont). A 60x water immersion
objective, NA=0.9 (Olympus, Japan), was used to view the cells.
Time course experiments were performed by acquiring an image pair
(512x512 pixels, 8 bits per pixel) at a relatively slow rate (generally, 0.2
Hz) and
saving the images to disk. Regions of interest (ROI) within an image were
selected, corresponding to the cell bodies of single cells. The average
intensity
from within each ROI was obtained for each emission wavelength, the
appropriate
background level was subtracted, and the ratio was calculated. The ratio
reflects
changes in intracellular calcium ([Ca2+]I), independently of excitation
strength,
concentration of indo-1, volume of the cell, or the optical path. The ratios
were
converted to calcium concentration after calibrating the dye in vitro with a
series
of calcium buffers (Molecular Probes), and plotted as a function of time.
Fab fragments were generated from the polyclonal antibody as follows.
8777 (Strickland, D.K., et al., Biochemistry 27: 1458-1466 (1988), and
Strickland, D.K., et al., J. Biol. Chem. 265:17401-17404 (1990)) was dialyzed
against 20 mM sodium phosphate,10 mM EDTA, pH 7.0, and mixed with 0.5 mL
Pierce immobilized papain in 20 mM sodium phosphate, 10 mM EDTA, pH 7.0
containing 20 mM cysteine. Digestion was carried out at 37°C for 12 h
with
gentle mixing. Following digestion, the digest was applied to Protein A
Sepharose, and the nonbinding Fab fragments were collected. The Fab fragments
were analyzed by immunoblotting cell extracts (using 5 ~glml), which revealed
positive reactivity against LRP and no other proteins.
Statistics were performed using a paired Student's t-test. Data from each
cell within an experiment was averaged, and statistics were performed based on
the number of experiments. Data are expressed as mean ~ standard deviation.
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Results
Primacy cultures of mouse cortex were loaded with the fluorescent calcium
indicator indo-1/AM. Addition of methylamine-activated a2M (a2M*, 35 nM)
elicited an increase in [Ca2+~I in a subset of cells within the mixed cultures
(Figure 1). a2M* increased intracellular calcium in responsive cells from
88 ~ 29 nM to 396 ~ 22 nM (n=24 expts, 211 cells, p<0.001 ). Unresponsive
cells
did not exhibit a significant increase in [Ca2+~I, (106 ~ 22 nM vs 107 ~ 18
nM,
n = 8 expts, 26 cells, NS, p>0.05).
Morphologically, the responding cells resembled neurons, and the non-
responding cells had the appearance of glia or fibroblasts. To help
distinguish the
identity of cells after an experiment, NMDA (100 ~,M) was added to the bath.
Non-neuronal cells generally do not respond to NMDA (Beaman-Hall, C.M.,
et al., J. Neu~ochem. 71:1993-2005 (1998)), whereas neurons that do express
NMDA receptors allow calcium entry in the presence of NMDA (Grant, E.R.,
et al., J. Biol. Chem. 272:647-56 (1997)). Using this criterion, the
responding
cells were all identified as neurons. Greater than 95% of NMDA responsive
cells
responded to a2M* (203 out of 208 cells), whereas greater than 90% of all non
NMDA responsive cells failed to show a calcium response to a2M* (67 of 72).
This functional marker was confirmed using immunocytochemistry for MAP-2 to
identify neurons (Figure 2).
The time course and magnitude of the response to bath application of
a2M* varied to some extent even among neurons within a field. The calcium
response occurred within several tens of seconds after ligand addition, and,
in
most cases, the response was sustained for several tens of minutes until the
end
of the experiment. However, occasionally the calcium response was transient,
returning to baseline within several minutes. No consistent difference in
these
subpopulations in terms of response to NMDA or in morphology was noted.
Next the specificity of the response for the activated form of a2M, a2M*
Was examined. Treatment of neurons with native a2M (70 nM) had no effect on
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intracellular calcium (139 ~ 80 nM vs 144 ~ 80 nM, n= 4 expts, 54 cells, NS,
p>0.05). Likewise, to test the possibility that residual methylamine was
initiating
the increase in calcium, methylamine was added at concentrations up to 100 ~.M
directly to the cultures with no effect on intracellular calcium. Thus,
activated
a2M appears to be critical for the calcium response.
To test the possibility that the a2M* induced increase in intracellular
calcimn is an indirect effect of synaptic activity in the cultures, a2M* was
added
in the presence of 2-5 ~.M tetr odotoxin (TTX). At this concentr anon, the
cultur ed
neurons are unable to generate action potentials. However, a2M* was capable of
eliciting a calcium response even in the presence of TTX. Tlus result
indicates that
the observed calcium response elicited by a2M* is not an indirect result of
synaptic glutamate release.
Although the time-course of the calcium response in neurons was not
suggestive of calcium release from intracellular stores, this hypothesis was
tested
by adding a2M* in the absence of extracellular calcium (Figure 3). Under this
condition, a2M* (35 nM) was unable to increase intracellular calcium (82 ~ 20
nM
vs 64 ~ 14 nM, n=3 expts, 31 cells, NS, p>0.05). This indicates that the
observed
calcium entry is not from the release of calcimn from intracellular stores.
When
the calcium-free buffer was replaced with a calcium-containing buffer,
intracellular
calcium increased to typical stimulated levels (2b6 ~ 70 nM, p<0.05). This
suggests that 1) the a2M* was able to bind to its receptor in the absence of
calcium; 2) receptor-mediated processes allowing calcium entry were activated
in
the absence of calcium; and 3) the source of the calcium entry is from the
extracellular environment through plasma membrane calcium channels. Consistent
with this idea, pretreating the cultures with the non-specific calcium channel
blockers NiCl2, (2-5 mM, 89 ~ 8 nM vs 107 ~ 23 nM, n=5 expts, 25 cells, NS,
p>0.05) or CoCl2 (86 ~ ??nM vs 107 ~ 23 nM, 5 mM, n=4 expts, 44 cells, NS,
p>0.05)) abolished the calcium response to a2M*.
To test whether NMDAR channels might be involved in the response,
cultures were pretreated with 5 ~M MIA-801, a potent NMDA receptor
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antagonist. This treatment abolished the [Ca2+], response mediated by a2M*
(Figure 4) (84 ~ 12 nM vs 113 ~ 26 nM, n=4 expts, 60 cells, NS, p>0.05). This
result demonstrates that the calcium signal observed by activation of LRP with
a2M* is mediated by calcium entry through NMDA receptors. This also explains
the observation that the response is specific for neurons in the mixed
cultures.
A variety of other channel antagonists were tested, however none were
able to prevent the a2M*-stimulated calcium increase when used alone,
including
nimodipine, c~-conotoxin, and w-agatoxin IVA, as shown below (Table 1).
Table 1
Channel AntagonistType of channel Result
affected
Ca2+ -removal All Ca2+ channels Blocked Ca2k response
NiClz(2-5 mM) All Ca2+ chamiels Blocked Ca2~ response
CoCl2 (5 mM) All Caz+ channels Blocked Ca2~ response
MK801 NMDAR channels Blocked Caz~ response
ee-agatoxin IVA P/Q-type Ca2+ channelsNo effect
(2 pM)
nimodipine (5 L-type CaZ~ channelsNo effect
pM)
co-conotoxin N-type Ca2+ channelsNo effect
(1 pM)
tetrodotoxin Na2+ channels No effect
(5 pM)
Table 1 The effect of channel blockers on the a2M*-mediated calcium response.
The table lists
the channel blockers used (at the indicated concentrations), as well as the
target of the Mockers,
and the experimental result. Each experimental test was performed in at least
three cultures.
a2M*-induced calcium influx was examined to test if this phenomenon was
mediated by LRP. Pre-incubation with a specific physiologic inhibitor of LRP,
receptor associated protein (RAP, 500 ~M), blocked the response to a2M*
(114 ~ 17 nM vs 125 ~ 9 nM, n=4 expts, 27 cells, NS, p>0.05, Figure 5). RAP
blocks ligand-receptor interactions with all members of the LDL receptor
family
proteins. Next, an anti-LRP antibody was used which specifically interacts
with
LRP but not other members of the LDL receptor family. The rabbit polyclonal
antibody 8777, directed against the ligand binding repeat region of LRP, was
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added to neuronal cultures at a concentration of 10 p,g/ml (Figure 6). This
resulted in an immediate, marked increase in neuronal calcium (84 ~ 13 nM vs
728 ~ 426 nM, n=8 expts, 92 cells, p< 0.01 ) but no response in non-neuronal
cells.
The response to 8777 was blocked completely when the cultures were pretreated
with 5 ~M MK-801 (102 ~ 12 nM vs 120 ~ 4 nM, n=3 expts, 61 cells, NS,
p<0.05). The rabbit polyclonal antibody 8704 (Strickland, D.K., et al., J:
Bial.
Chem. 265:17401-17404 (1990)), which is directed against a C-terminal portion
of LRP, did not elevate intracellular calcium (865 nM vs 1205 nM, n=3 expts,
29 cells, NS, p>0.05). Thus, the ability of antibody R77? to recognize the
ligand
binding region of LRP activated the calcium response.
Both a2M'~, which is tetrameric, and the bivalent 8777 antibody
potentially lead to dimerization of the ligand binding domains of LRP. To test
the
possibility that dimerization of the receptor or cross-linking of the ligand
binding
sites is important for the calcium response in neurons, Fab fragments were
derived
from 8777. Western blot analysis showed that the Fab fragments specifically
recognized LRP. Addition of up to 275 ~,g/ml of Fab showed no response
(79 ~ 9 nM vs 80 ~ 13 nM, n=5 expts, 59 cells, NS, p>0.05). To further examine
this issue, and to test the hypothesis that endocytosis is sufficient to
evolve a
calcium signal, neuronal responses to another LRP ligand, which is bound and
readily endocytosed by LRP, but is monomeric, was examined. Lactoferrin is
readily taken up by neuxons via LRP (Qiu, Z., et al., J. Neu~ochem. 73:1393-8
(1999)), but lactoferrin (up to 5 ~,M) did not evoke a calcium response
(79 ~ 9 nM vs 89 ~ 21 nM, n=4 expts, 32 cells, NS, p>0.05). These results
demonstrate that although the Fab fragments and lactoferrin bound to the
receptor, they were unable to activate calcium entry, thus supporting the
hypothesis that dimerization of LRP may be important for the calcium signaling
event.
It is possible that a2M* concentrations might provide information about
the local microenvironment to neurons, and therefore might alter local
dendritic
calciwn levels in a spatially restricted fashion. As a means of examining the
spatial
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characteristics of the response, several picoliters of a a2M* (5 ~,M) solution
was
applied via micropipette to various regions of individual neurons using a
pressure
pulse. The delivery of a2M* was restricted to a circular area with a radius of
about 25 Vim, and this was the only area that responded with a calcium
increase.
The response was localized, and decays within several tens of seconds. Indeed,
a second pressure pulse was able to stimulate the same area again, without
affecting the calcium concentration in the cell body. In a similar experiment,
positioning the pipette near the cell body was capable of eliciting a calcium
transient that was restricted to the cell body, and did not spread to the
dendrites.
Thus, the response can be spatially restricted, and may not lead to a global
increase in calcium. Furthermore, the calcium response does not lead to a
large
cellular depolarization, which, if above threshold, would be expected to
increase
calcium everywhere in the cell.
Discussion
Members of the LDL receptor family, including LRP, have been studied
extensively as multiligand endocytic receptors (Herz, J., et al., EMBO J.
7:4119-
27 (1988); Herz, J., J. Biol. Chem. 265:21355-62 (1990); Bu, G., et al., J.
Biol.
Chem. 269:29874-82 (1994)). The data presented herein support several novel
conclusions suggesting that LRP serves an unexpected role as a signaling
receptor
as well. Stimulation of neurons by the LRP ligand a2M* elicits a robust
calcium
response, which is local to the area of stimulation, and which is temporally
linked
to the stimulus. The response appears to require LRP receptor dimerization.
The
response is neuron specific, and is mediated through NMDAR channels. Non
neuronal cells containing LRP within the same culture wells did not respond to
a2M* with calcium influx.
LRP is implicated as the mediator of this response because it is an a2M*
receptor on neurons (Rebeck, G.W., et al., Neuron 11:575-80 (1993); Bu, G.,
et al., J. Biol. Chem. 269:29874-82 (1994)), and the effect is blocked by RAP
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which is a specific physiologic inhibitor of the LDL family of receptors. That
LRP
is specifically involved is demonstrated by the observation that an antibody
directed against the extracellular domain of LRP can also induce calcium
influx in
neurons. Of note, both a2M*, which is tetrameric, and the bivalent 8777
antibody
could potentially lead to dimerization of the receptor. The Fab fragments of
the
same antibody and a monomeric ligand, lactoferrin, did not evoke a calcium
response, supporting the conclusion that dimerization may play a role in
calcium
signaling. LRP is present on both neurons and astrocytes in culture and in
adult
brain (Rebeck, G.W., et al., Neuron 11:575-80 (1993); Bu, G., et al., J. Biol.
Chem. 269:29874-82 (1994); Bu, G., et al., J. Biol. Chem. 269:18521-8 (1994)),
although only neurons have an LRP mediated calcium response. It is possible
that
there may be a neuron-specific intracellular adapter protein that mediates
opening
of NMDAR channels after LRP dimerization, or that LRP may interact directly
with NMDA receptors.
Links between a2M* and calcium influx have been examined in several
systems. Previous studies in macrophages (Misra, U.K., J. Biol. Chem.
269:18303-6 (1994)) and trabecular meshworlc cells (Howard, G.C., et al.,
Arch.
Biochenz. Biophys. 333:19-26 (1996)) suggest 2 classes of a2M methylamine
receptors: LRP and a separate "signaling receptor." Stimulation of the latter
leads to a rapid rise in intracellular calcium in macrophages, which is not
blocked
by RAP or altered by LRP antibodies. By contrast, a2M* binding to LRP does
not appear to induce a calcium influx in macrophages, consistent with the lack
of
response that was observed in non-neuronal cells.
In neurons, the role of LDL receptor family members in calcium influx may
2S be complex. A rise in intracellular calcium in hippocampal neurons has also
been
observed after treatment with apolipoprotein E (Hartmann, H., et al., Biochem.
Biophys. Res. Commuvc. 200:185-92 (1994)); these apolipoprotein E-evoked Caz+
increases are dependent on extracellular calcium and blocked by the Caz+-
channel
antagonists nickel and en-Agatoxin-IVa, implicating activation of P/Q type
Caz+-
channels (Muller, W., et al., Brain Pathol. 8:641-53 (1998)). More similar to
the
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current findings, proteolytic fragments of apolipoprotein E or a tandem dimer
repeat peptide derived from apolipoprotein E, elicited calcium responses in
both
hippocampal cultures and chick sympathetic neurons, with the calcium increases
being blocked by RAP and by the NMDA receptor antagonist MIA-801 (Tolar, M.,
et al., J. Neurosci. 19:7100-10 (1999)). The relationship among these various
experimental systems must be more closely examined, but the accumulating
evidence strongly suggests that, in neurons, stimulation of LRP by ligands
such as
a2M* or apolipoprotein E leads to a calcium signaling event. It is possible
that
this acts as a neuronal sensor for proteolytic activity or lipid breakdown
within a
dendrite's microenvironment.
The influx of calcium into spatially segregated dendritic elements due to
LRP-mediated activation of NMDAR channels is likely to locally impact a wide
variety of downstream signaling cascades, including IP3, protein kinase C, and
calcium/calmodulin dependent kinase (Alkon, D.L., et al., Tends Neunosci.
21:529-37 (1998)). This cascade implies that a2M* interaction with LRP may
provide a novel mechanism of altering local dendritic excitability, and thus
synaptic efficacy. a2M* has previously been implicated in inhibiting long term
potentiation (Cavus, L, et al., J. Neu~osci. Res. 43:282-8 (1996)). Another
LRP
ligand, tissue plasminogen activator (tPA), contributes to activity-dependent
synaptic plasticity in the hippocampus via LRP (Zhuo, M., et al., J. Neurosci.
20:542-549 (2000)). It is possible that the LRP-mediated NMDAR channel
activation and calcium influx that is seen might contribute to these
phenomena.
In addition to its well established role as a multiligand endocytic receptor,
there is some precedence for LRP having a role in neuronal signaling pathways.
a2M*, apolipoprotein E, and other LRP ligands have been shown to promote
neurite outgrowth via LRP (Holtzman, D.M., et al., P~oc. Natl. Acad. Sci. U.
S.A.
92:9480-4 (1995); Ishii, M., et al., B~aih Res. 737:269-74 (1996); Mori, T.,
et al.,
Brain Res. 567:355-7 (1991); Postuma, R.B., et al., FEBSLett. 428:13-6
(1998)).
Moreover, the intracellular domain of LRP can bind both disabled and FE65,
adapter proteins implicated in signal transduction (Trommsdorff, M., et al.,
J.
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Biol. Chem. 273:33556-60 (1998)). LRP has also been reported to interact with
aheterotrimeric G protein (Goretzki, L. and Mueller, B. M., Biochem. J.
336:381-
6 (1998)). Treatment of LRP-expressing cell lines with the LRP ligands
lactoferrin or urokinase-type plasminogen activator caused a significant
elevation
in cAMP and stimulated PKA activity in a dose-dependent manner (Goretzki, L.
and Mueller, B. M., Biochem. J. 336:381-6 (1998)).
The idea that members of the LDL receptor family could act as signaling
receptors (Cooper, J.A. & Howell, B.W., Cell 97:671-4 (1999)) in the central
nervous system recently received dramatic support from the observation that
APOER2/VLDL-r double null animals develop areelerphenotype (Trommsdorff,
M., et al., Cell 97:689-701 (1999)), due to inactivation of the reelin-
disabled
signaling pathway. Reelin is a ligand for APOER2 and VLDL-r, supporting the
idea that these receptors directly mediate reelin signal transduction
(Trommsdorff,
M., et al., Cell 97:689-701 (1999); D'Arcangelo, G., et al., Neuron 24:471-9
(1999)). It is interesting to note that both VLDL-r and APOER2 are also
strongly
expressed on mature neurons (Christie, R.H., et al., J. Neuropathol.,. Exp.
Neurol.
55:491-8 (1996); Clatworthy, A.E., et al., Neurosci. 90:903-11 (1999)), and
APOER2 has been reported to be an a2M~ receptor (Stockinger, W., et al, J.
Biol. Chem. 273:32213-21 (1998)). The data regarding a specific anti-LRP
antibody clearly implicate LRP itself, but do not rule out a role for APOER2
in
calcium signaling. These data, taken together with the current demonstration
that
LRP is also a potent signaling receptor in neurons, suggest a major role for
the
LDL receptor family in brain development and function.
Several LRP ligands have been strongly implicated in the pathophysiology
of Alzheimer's disease. A(3, the maj or constituent of senile plaques, is a
peptide
fragment of the amyloid precursor protein, itself a protease inhibitor and
ligand for
LRP (Kounnas, M.Z., et al., Cel182:331-40 (1995)). ApolipoproteinE and a2M*
bind A(3 and the complexes can be cleared by LRP (Qiu, Z., et al., J.
Neu~ochem.
73:1393-8 (1999); Jordan, J., et al., J. Neu~osci. 18:195-204 (1998); Narita,
M.,
et al., J. Neu~ochem. 69:1904-11 (1997)). Genetic studies strongly implicate
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polymorphisms in the apolipoprotein E gene, and also in the a2M and the LRP
genes, in affecting risk for late onset Alzheimer's disease (see Hyman, B.T.,
et al.,
Aoch. Neurol. 57:646-650 (2000) for review). Finally, it should be noted that
APP contains a NPXY domain in its carboxyl terminus, and APP can bind DAB 1
and Fe65 adaptor proteins which also interact with LRP (Trommsdorff, M., et
al.,
J. Baol. Cheni. 273:33556-60 (1998)). The current observations that LRP may be
both an endocytic and a signaling receptor may thus be of relevance to the
role of
LRP and its ligands in Alzheimer's disease.
