Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHOD$ OF REDUCING APOLIPOPROTEIN E4-INDUCED INHIBITION
OF NEURON REMODELING
This invention was funded in part with funds from National Institutes of Health
Program Project Grant HL41633. The Government may have certain rights to this
invention.
nescription
This is a continuation-in-part of United States patent application serial no.
60/005,550, filed October 17, 1995, which is hereby incorporated in its entirety herein by
reference.
Techni(~l Field
This invention relates to the treatment of central and peripheral nervous systemdisorders relatiing to neuron remodeling. Specifically, the invention relates to the
reduction of inhibition of neuron remodeling by interfering effectively with theinteraction of apolipoprotein E4 (apoE4) with the neuronal low density lipoprotein
receptor-related protein (LRP) or similar apoE binding receptor. In addition, the
invention provides methods for reducing the apoE4 effects on neurons converting apoE4
to an "apoE3-like" molecule with respect to receptor binding activity, cytoskeletal
4 assembly/stability/activity and neurite extension or remodeling.
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Back~rollnd
ApoE, a 34,000 molecular weight protein is the product of a single gene on
chromosome 19 and exists in three major isoforms de~ign~tetl apoE2, apoE3 and apoE4
5 for review, see Mahley (in press) in: Molecular and Genetic Bases of Neurolo~ical
T)isease 2nd ed.; and Mahley (1988) Science 240:622-630. The different isoforms result
from amino acid substitutions at amino acid residue positions 112 and 158. The common
isoform, apoE3, has a cysteine residue at position 112 and an arginine residue at position
158. The apoE4 isoform differs from apoE3 only at position 112, which is an arginine
10 residue. The apoE2 isoform, associated with type III hyperlipoproteinemia (Mahley
(1988)), differs from apoE3 only at position 158, which is a cysteine residue. ApoE3 and
apoE4 bind normally to the low density lipoprotein (LDL) receptor, whereas apoE2 does
not.
ApoE contains two structural domains: an amino-terminal and a carboxy-terminal
domain. Weisgraber (1994) Adv. Protein Chem. 45:249-302. Each domain is associated
with a specific function. The amino terminal domain contains the lipoprotein receptor
binding region and the carboxy-tennin~l domain contains the major lipid-binding
elements. The two domains appear to interact with each other in an isoform-specific
manner such that amino acid substitutions in one domain influence the function of the
other domain, a phenomenon referred to as domain interaction. Domain interaction is
responsible for the plef~.G,lce of apoE4 for very low density lipoproteins (VLDL)
contrasted with the pl~;;r~ nce of apoE3 for high density lipoproteins (HDL). The
specific amino acid residues in apoE4 that are involved in this interaction have been
identified: arginine-61 in the amino-terminal domain and glutamic acid-255 in the
carboxy-terminal domain. Dong et al. (1994) J. Biol. Chem. 269:22358-22365; and Dong
and Weisgraber (1995) C;rculation 92:I-427-I-428 (abstract).
By redistributing lipids among the cells of different organs, apoE plays a critical
role in lipid metabolism. While apoE exerts this global transport mechanism in
. . .
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chylomicron .md VLDL metabolism, it also functions in the local transport of lipids
among cells within a tissue. Cells with excess cholesterol and other lipids may release
these substances to apoE-lipid complexes or to HDL co~ ini~g apoE, which can
transport the lipids to cells requiring them for proliferation or repair. The apoE on these
lipoprotein particles mediates their interaction and uptake via the LDL receptor or the
LRP.
ApoE plays a neurobiological role. ApoE mRNA is abundant in the brain, where
it is synthesized and secreted primarily by astrocytes. Elshourbagy et al. (1985) Proc.
Natl. Acad. Sci. USA 82:203-207; Boyles et al. (1985) J. Clin. Invest. 76:1501-1513; and
Pitas et al. (1987) Riochem Biophys. Acta 917:148-161. The brain is second only to the
liver in the level of apoE mRNA expression. ApoE-cont~ininE lipoproteins are found in
the cerebrospinal fluid and appear to play a major role in lipid transport in the central
nervous system (CNS). Pitas et al. (1987) J. Biol. Chem 262:14352-14360. In fact, the
major cerebrospinal fluid lipoprotein is an apoE-co"t~ HDL. ApoE plus a source of
lipid promote s marked neurite extension in dorsal root ganglion cells in culture.
Handelmann et al. (1992) J. T ipid Res. 33:1677-1688. ApoE levels drarnatically increase
(about 250-fold) after peripheral nerve injury. Muller et al. (1985) Science 228:4g9-501;
and Ignatius et al. (1986) Proc. Natl. Acad. Sci. USA 83:1125-1129. ApoE appears to
participate bo th in the scavenging of lipids generated after axon degeneration and in the
redistribution of these lipids to sprouting neurites for axon regeneration and later to
Schwann cells for remyelination of the new axons. Boyles et al. (1989) J. Clin. Invest.
83:1015-1031, and Ignatius et al. (1987) Science 236:959-962.
Most recently, apoE has been implicated in Alzheimer's disease and cognitive
performance. Saunders et al. (1993) Neurol. 43:1467-1472; Corder et al. (1993) Science
261 :921 -923; and Reed et al. (1994) Arch. Neurol. 51: 1189-1192. ApoE4 is associated
'~ with the two characteristic neuropathologic lesions of Alzheimer's disease; extracellular
neuritic plaques representing deposits of amyloid beta (AJ) peptide and intracellular
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neurofibrillary tangles representing fil~ment~ of hyperphosphorylated tau, a microtubule-
associated protein. For review, see, McKhann et al. (1984) Neurol. 34:939-944; Selkoe
(1991) Ne-lron 6:487-498; Crowther (1993) C-lrr. Opin. Struct. P~iol. 3:202-206; Roses
(1994) Curr.Neurol. 14:111-141; Weisgraberetal. (1994) Curr. Opin. Lipidol. 5:110- ,.
116; and Weisgraber et al. (1994) Curr. Opin. Struct. Biol. 4:507-515.
Alzheimer's disease is generally divided into three categories: early-onset f~mili~l
disease (occurring before 60 years of age and linked to genes on chromosomes 21 and
14); late-onset f~mili~l disease; and sporadic late-onset disease. Both types of late-onset
disease have recently been linked to chromosome 19 at the apoE locus. Other results
suggest that apoE4 is directly linked to the severity of the disease in late-onset families.
Roses (1994). Recently, cholesterol lowering drugs, the statins, have been suggested for
use in treating Alzheimer's disease by lowering apoE4 levels. WO 95/06470.
The neurofibrillary tangles, which are paired helical filaments of
hyperphosphorylated tau, acclm~ te in the cytoplasm of neurons. Tau is a microtubule-
associated phosphoprotein which normally participates in microtubule assembly and
stabilization; however, hyperphosphorylation impairs its ability to interact with
microtubules. Increased binding of tau by apoE has been suggested as a treatment for
Alzheimer's disease. WO 95/06456.
In vitro tau interacts with apoE3, but not with apoE4. Strittmatter et al. (1994)
Exp. Nellrol. 125:163-171. The interaction of apoE3 with tau may prevent its
hyperphosphorylation, thus allowing it to function normally in stabilizing microtubular
structure and function. In the presence of apoE4, tau could become hyperphosphorylated
and thus inactive, which could promote the formation of neurofibrillary tangles.ApoE4 has recently been associated with decreased learning ability and impaired
memory. Helkala et al. (1995) Nellrosci. Lett~. 191:141-144. ApoE4 has been found to
be a strong predictor of the outcome of patients ~lesign~tc-l as having memory
imp~irment. Note that, apoE4 has been described as a risk factor, rather than a diagnostic.
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Peterson et al. (1995) JAMA 273:1274-1278; and Feskens et al. (1994) E~1 309:1202-
1206.
ApoE interacts with both the LDL receptor and the LRP and undoubtedly with
other apoE-binding receptors on neurons. The LRP has been found to be increased after
brain injury or glial cell conversion to neoplasia. Lopes et al. (1994) FFRS T ett. 338:301-
305. The LRP was previously identified as the-macroglobulin receptor. Strickland et al.
(1991) J. Biol. Chem. 266:13364-13369; and Borth (1992) FASF~ J. 6:3345-3353.
ApoE does not directly bind to the LRP but must first associate with cell surface heparan
sulfate proteoglycans (HSPG). Mahley et al. (1991) Curr. Opin. T ipidol. 2:170-176; and
Ji et al. (1994) J. Biol. Chem. 269:2764-2772. The LRP also binds a nurnber of other
ligands, including t-PA,a2-macroglobulin-protease complex, thrombospondin-1,
Pseudomona(, exotoxin A, the receptor associated protein (RAP) and lactoferrin. The
LRP ligand binding sites have been at least partially described. Orth et al. (1994) J. Biol.
C~hem. 269:21L 117-21122; Godyna et al. (1995) J. Cell. P~iol. 129:1403-1410; Kounnas et
al. (1992) J. Biol. Chem 267:12420-12423, Willnow et al. (1994) J. Cell Sci. 107:719-
726; Meilinger et al. (1995) FFP~S J ett. 360:70-74; Warshawsky et al. (1993) J. Biol.
Chem. 268:22046-22054, and Willnow et al. (1994) J. ~iol. Chem. 269:15827-15832.
It has previously been shown that incubation of dorsal root ganglion neurons in
culture with ~-VLDL alters the neurite growth of these cells compared to that of cells
grown in med~ia alone. Handelmann et al. (1992). In the presence of a source of lipid (,~-
VLDL or free cholesterol), neurite outgrowth is greatly enhanced, specifically due to
extensive branching (with little or no increased neurite extension). When the ,~-VLDL
was enriched with exogenous rabbit apoE (equivalent to human apoE3 with respect to the
occurrence of a cysteine residue at position 112) enhanced neurite extension was seen. A
lipid source appears to enhance membrane biosynthesis, whereas the addition of excess
rabbit apoE with a lipid source results in long neuritic extensions and a trimming back of
the branches. It has also been found that the inhibitory effect of apoE4 on neurite
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outgrowth is associated with microtubule polymerization, whereas apoE3 supports
microtubule formation. Nathan et al. (1995) J. Riol. Chem. 270:19791-19799.
Neural plasticity, m~intPn~nce of existing or formation of new synaptic
connections, is critical for normal brain function, including memory. This process can be
compromised by various forms of stress, including, but not limited to, age, deposition of
plaques and neurofibrillary tangles in Alzheimer's disease and oxygen deprivation.
Interference with neuron remodeling can lead to impaired brain function or
neurodegeneration of which dementia and Alzheimer's diseace are extreme examples. In
the case of Alzheimer's disease alone, approximately 4 million individuals are affected in
the United States. With the aging of the population, this number is projected to triple in
the next twenty years. The present health care cost of Alzheimer's disease is estimated at
$90 billion per year in the United States alone. Delaying the average onset of this disease
for even ten years would drastically reduce the financial burdens on society and the
financial and emotional burdens of the families of these patients.
There are currently no effective therapies for arresting (and, more importantly,reversing) the imp~irment of central and peripheral nervous system function once an
irreversible degenerative cascade begins. Likewise, there is no current therapy for
restoration of normal, central and peripheral nervous system function when the induced
stress has a less catastrophic or partially reversible effect compared to the dementias.
All references cited herein, both supra and infra, are hereby incorporated herein by
reference.
1 )isclosure of the Invention
The present invention encomp~c.ses methods of reducing apoE4-induced inhibition
of neuron remodeling. One method comprises ~-lmini~tering to a patient in need thereof,
an effective amount of a therapeutic agent (drug) which interferes with the interaction of
apoE4 and neuronal apoE-binding receptors such as the LRP. As apoE4 interacts with the
LRP by first associating with HSPG on the cell surface and then exerting its effect on the
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LRP, the interaction can be reduced by interfering with either type of interaction both
directly and indirectly.
In another method, apoE4 is "converted" to an "apoE3-like" molecule with respectto receptor binding activity, cytoskeletal assembly/stability/activity, and neurite extension
or remodelin~,.
The invention also includes methods of identifying compounds that are effective
in interfering with the apoE4 domain intçr~ction. These methods are exemplified by the
plasma distribution assay comprising the steps of adding a tracer dose of ~25I-labeled apoE
to plasma, separating the various plasma lipoprotein fractions by gel filtration and
detl?rrninin~ the distribution of 12~I-label among lipoprotein classes. See, e.g. Dong et al.
(1994) J. Biol. Chenl 269:22358-22365.
BriefDescriptior~ ofthe Drawin~
Figure 1 is a sch~m~tic representation of the human apoE cDNA constructs used
to transfect the Neuro-2a cells. NSE promoter (N), exons of apoE have "E" underneath,
the polylinker region has "P" nncl~rne~9th and apoE cDNA has "A" lm(l~rne~th
Figure 2 is two photomicrographs of representati~ve Neuro-2a cells stably
transfected with apoE3 (A) or apoE4 (B) cDNA and grown for 96 hr in N2 medium
20 cont~ininE ~-'VLDL (40 ,ug of cholesterol/ml).
Figure 3 is a series of bar graphs depicting the effect of ,B-VLDL on the number of
neurites per cell (A), neurite branching (B), and neurite extension (C) from control Neuro-
2a cells and from cells stably transfected to express apoE3 or apoE4. In each case, the
solid black bars represent the control, the striped bars represent apoE3 expressing cells
25 and the solid white bars represent apoE4 expressing cells. In all cases the X-axis
represents ,~-~ILDL (,ug cholesterol/ml).
Figure 4 is a graph depicting the effect of ~-VLDL on the percentage of cells
expressing neurites. The cells were incubated as described for the results presented in
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Fig. 3. Four different fielas in each dish were selected, and the percentage of cells
displaying neurites was measured. Data are the means of three different experiments
performed in duplicate (+ S.E.M.). The percentages of cells ~ e~sillg neurites in the
absence of ,B-VLDL were: control cells, 35 + 11 (open squares); apoE3-expressing cells,
32 + 9 (closed circles); apoE4-expressing cells, 25 + 13 (closed squares). *p < 0.025
versus control; **p < 0.005 versus control.
Figure 5 is a bar graph depicting the effect of cerebrospinal fluid (CSF)
lipoproteins on neurite extensions from Neuro-2a cells stably transfected to express
apoE3 or apoE4. Cells were incubated with ~-VLDL or bovine CSF lipoproteins (d c1.21 g/ml) under the conditions described for the results presented in Fig. 3. Each data
point represents the m~;a~ul~lllent of 20-40 neurons. The data are reported as the mean
S.E.M. The calculation of the level of significance of the differences observed was
performed as described for the results obtained in Fig 3. The solid black bars represent
the control. The striped bars represent apoE3 expressing cells. The solid white bars
represent apoE4 expressing cells. *p < 0.025, **p < 0.01, ***p < 0.005.
Figure 6 depicts two photomicrographs of intern~li7~tion of l,l'-dioctadecyl-
3,3,3',3'-tetramethylindocarbocyanine (DiI)-labeled ~-VLDL by Neuro-2a cells stably
transfected with apoE3 (A) or apoE4 (B) cDNA. Cells were grown for 24 hr in N2
medium. Then DiI-labeled ,B-VLDL (5 ~lg of protein/ml medium) were added, and the
incubation was continued for 5 hr at 37~C.
Modes for Carryin~ Out the Invention
In neurons, the cytoskeleton functions in neurite extension and retraction.
Therefore, the studies described herein and by others (E~nflelm~nn (1992); and Nathan et
al. (1994) Science 264:850-852), have focused on the isoform-specific effects of apoE3
and apoE4 on neurite extension and br~nching It has now been found that apoE
modulates the intracellular cytoskeletal apparatus and alters neurite extension and
-
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br~nching Underst~n~ling how the various apoE isoforms alter the cytoskeleton may
shed light on the process of neurofibrillary tangle formation and allow control of apoE-
5 in~ çe~l remodeling of synaptic connections later in life. Stimulation of neurite extension
in vivo is thought to promote nerve regeneration or the ffirmation of synaptic connectionsduring neuronal remodeling in both the cenkal and peripheral nervous system.
A cornparison of the effects of human apoE3 versus human apoE4 showed
pronounced clifferential isoform-specific effects on neurite outgrowth. Nathan et al.
(1994). Compared to a control, hurnan apoE3 plus ,B-VLDL resulted in an increase in
neurite extension, while apoE4 plus ~B-VLDL resulted in a marked decrease in both
neurite branching and extension. Results presented by Nathan et al. (1995) show that
dorsal root gcmglion neurons incubated with apoE4 plus ~-VLDL displayed very short,
stunted neuri tes. This was not a toxic effect of apoE4 since replacement of the apoE4-
15 cont~ininp media with fresh apoE4-lacking media restored the ability of the neurons to
produce neuritic extensions. Furthermore, it has now been found that the apoE3- and
apoE4-specific effects were blocked by addition of an antibody against the receptor
binding domain of apoE or by reductive methylation of critical lysine residues, indicating
that this effect of apoE is receptor-mç~ tc--l
Neuro-2a cells from the cenkal nervous system were used to compare the effects
of apoE on th,e peripheral nervous system neurons described above with the effect on
cortical neuN)ns. Cells of both types respond similarly to apoE. When combined with a
source of lipid, apoE3 stimulated neurite extension, whereas apoE4 inhibited neulite
extension. Nathan et al. (1994) Soc. Neurosci. 20 (Part 2):1033 (Absk.); and Nathan et
al. (1995). Addition of free apoE3 or apoE4 without ,B-VLDL had no effect on neurite
outgrowth. I'hese results further suggest that the effect of apoE on neurons requires the
lipoprotein receptor-mediated uptake of apoE or a combination of apoE and lipid. Free of
lipid, apoE does not bind to either the LDL receptor or the LRP. In contrast, in another
study, using a different neuronal cell line, Holtzman et al. demonstrated that apoE3 with
.
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,B-VLDL stimulated nerve growth factor-induced neurite outgrowth, whereas apoE4 had
no effect. Holtzman et al. (1995) Soc. Nellrosci. 21 (abstr):1009, 400.10.
The studies described in Nathan et al. (1995) were performed by adding large
quantities of apoE along with ,~-VLDL to the cells in culture. To determine whether
lower levels of endogenously produced apoE would have an effect on neurite outgrowth
from Neuro-2a cells, in the examples provided below, the neuronal cells were transfected
with human apoE cDNA constructs encoding apoE3 or apoE4. Clones of the transfected
cells secreting equal amounts of apoE3 or apoE4 (~50-60 ng of apoE/mg of cell
protein/48 hours) were selected for comparison. The apoE3- and apoE4-secreting cells
grown in serum-free control medium displayed a similar degree of limited neuriteextension. However, when a source of lipid (,B-VLDL) was added to the medium, the
cells had a markedly different growth pattern. The apoE3-secreting cells showed greater
neurite extension than did the apoE4-secreting cells. Thus, even very low levels of
endogenously produced apoE along with a source of lipid revealed the differential effects
of apoE3 versus apoE4. Lipid emulsions of various compositions, as well as
cerebrospinal fluid lipoproteins can be substituted for the ,B-VLDL and appear to serve as
a source of lipid for the cells or as a vehicle for transporting the apoE into a specific
intracellular pathway. The examples presented herein show that the apoE effect on
neurite outgrowth is mediated through the LRP, or a similar apoE-binding receptor, and
that blocking or effectively preventing this interaction inhibits the apoE4 intlucecl
inhibition of neurite outgrowth.
Thus, the invention relates to methods of reducing the apoE4-in~lucec~ inhibition of
neuron remodeling by reducing the interaction of apoE4 and an apoE-binding receptor,
e.g., the LRP.
Further, the invention relates to altering the function of apoE4 by ch~nging thedomain interaction to interfere with the inhibition of apoE4 in neuron remodeling. Any
agent that blocks the interaction of arginine-6 1 with glutamic acid-255 in apoE4 is
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suitable for use in this method. Blocking domain interaction in apoE4 converts apoE4 to
an "apoE3-li]ke" molecule, thereby blunting the undesirable effects of apoE4 on neurite
5 extension. This may also have the effect of switching the apoE4 binding preference from
VLDL to H~IL.
Patients in need of such therapy are selected from those suffering from a wide
range of disorders. For instance, patients particularly suitable for such therapy are those
suffering frorn neurodegeneration or hypoxia. Neurodegeneration may result ~om a10 number of causes, including, but not limited to, Alzheimer's t7i~e~e, trauma, viral
infections, genetic enzyme deficiencies, age-related cognitive decline, and prion diseases.
Viruses which may cause neurodegeneration include, but are not limited to, humanimmunodeficiency virus (HIV) and Epstein-Barr virus. Genetic enzyme deficiencieswhich may cause neurodegeneration include, but are not limited to, deficiency in ,B-N-
15 acetylhexosaminidase which causes Tay-Sachs disease. Age-related cognitive decline is
described, for instance, in Pi~nostic ~nd Statistical Manual of MentalDisorders. Fourth
ed., Washinglton D.C. American Psychiatric Association (1994). Prion diseases include,
but are not limited to, Kuru and Creutzfeldt-Jacob disease. Hypoxia is generally the result
of stroke or is temporary and associated for instance with drowning, airway obstructions
20 or carbon monoxide poisoning.
Neura,n remodeling is also important in otherwise healthy patients. Therefore, the
methods described herein may be suitable for use prophylactically in patients who are
heterozygous or homozygous for apoE4 but do not show overt symptoms of Alzheimer's
disease or other neurodegenerative disorders.
A vari ety of therapeutic agents are suitable for use in the present invention. As
described in the examples below, heparinases, the RAP and lactoferrin all reduce or
abolish apoE4-in(lucecl inhibition of neurite outgrowth. Also, suitable agents include
those that bind specifically to apoE4 and prevent its domain interaction, i.e. small
molecules or antibodies. Agents that disrupt the domain interaction can be selected from
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a wide variety of molecules, including, but not limited to, small molecules, peptides and
antibodies which are designed to bind to arginine-61 or glutamic acid-255 of apoE4. An
assay for screening for agents that disrupt this domain interaction is described in Exarnple
3, below. Essentially, any assay that det~nines whether apoE4 exhibits apoE3 activity is
suitable for use herein.
Hep~rinsl~es or other modifiers of HSPG are effective in vitro in ameliorating the
effects of apoE4 on neuron remodeling. However, their pleiotropic effects render them
unsuitable for human therapy. Nonetheless, effective therapeutic agents include HSPG
analogs which bind to apoE4 to prevent its binding to neurons but do not exert substantial
pleiotropic effects.
The RAP is a glyco~ ehl with an app~t;lll molecular mass of 39-kD in hllm~n~
The RAP specifically associates with gp330 and the LRP, both of which are members of
the LDL receptor gene family. Various RAPs and homologs thereof have been described
and their functional domains have been mapped. For review see, Orlando et al. (1994)
Proc. Natl. Acad. Sci. USA 91 :3161 -3165; and Warshawsky et al. (1995) Biochem.34:3404-3415. The RAP, and portions thereof, are known to block the binding of the
LRP to its ligand t-PA and a2-macroglobulin-protease complexes. Warshawsky et al.
(1994) Ann N.Y. Acad. Sci. pp. 514-517.
Lactoferrin has been shown to bind to the LRP, gp330, and HSPG. Willnow et al.
(1994) J. Riol. Chem. 267:26172-26180;, Mahley et al. (1994) Ann N.Y. ~cad. Sci. USA
737:39-52; and Ji et al. (1994a) ~rterioscler. Thromb. 14:2025-2032. Lactoferrin appears
to be cleared from the bloodstream by binding with LRP. Meilinger et al. (1995).Lactoferrin blocks binding of ligands to both the LRP and HSPG and blocks the HSPG-
LRP pathway. This apparently occurs through the interaction of a region of concentrated
positive charge on the lactoferrin with negatively-charged groups on the HSPG and
negatively-charged amino acids in the ligand binding domain of the LRP.
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As described below, antibodies specific for apoE block the apoE4 induced
inhibition of neuron remodeling. Thus, antibodies to either apoE4 or the LRP can be used
5 therapeutically. In addition, antibodies that inhibit apoE4 domain interaction can be used
therapeuticall y. As described below, methods are known in the art to det~rmine whether
an antibody inhibits the neuron remodeling inhibitory effect of apoE4 whether byinhibiting binding to the LRP or by altering the function of apoE4 to become more
apoE3-like. P~referably, the antibody is monoclonal. More preferably, the antibody is
10 monoclonal and specific for the apoE4 isoform and not apoE3 or apoE2. The term
"antibody" also includes functional portions and equivalents thereof. For instance,
antibodies include any monospecific compound comprised of a sufficient portion of the
light chain valiable region to effect binding to the epitope to which the whole antibody
has binding specificity. The fr~gment~ may include the variable region of at least one
15 heavy or light chain immunoglobulin peptide, and include, but are not limited to, Fab
fragments, Fab2 fragments, and Fv fr~gment~. In addition, the monospecific domains of
antibodies can be produced by recombinant engineering. Such recombinant molecules
include, but are not limited to, fragments produced in bacteria, and murine antibodies in
which the maj ority of the murine constant regions have been replaced with human20 antibody cons~ant regions.
An effi~ctive amount of a therapeutic agent is one which, in in vitro assays,
reduces apoE4 inhibition of neurite outgrowth by at least about 10%, preferably at least
about 50% and most preferably, at least about 90%. The effect on neurite outgrowth can
be measured, Ior instance, by the methods described herein.
The therapeutic agent prevents apoE4 from interacting effectively with neuronal
LRP or other apoE-binding receptors. This prevention can be directed at either the HSPG
and/or the LRl' interactions or by modifying its function to be more apoE3-like and can
directly or indiirectly block binding or otherwise prevent the signal transduction induced
by apoE4 binding. Thus, the therapeutic agents described herein are considered to be
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effective if they prevent mhibition of neurite outgrowth by any of these routes. Thus,
whole proteins, any functional portion thereof, analog or homologue is suitable for use
5 provided it prevents effective interaction of apoE4 and HSPG or LR~, or other apoE-
binding receptors. For instance, changes in the amino acid sequences of the RAP or ~,
lactoferrin and other known ligands of the LRP, or other apoE-binding receptors, that do
not substantially affect their ability to effectively block the interaction of apoE4 and the
LRP are within the scope of the invention. For instance, the invention encomp~ses
10 changes in proteins that result in conservative substitutions of amino acid residues, one or
a few amino acid deletions or additions, and substitution of amino acid residues by amino
acid analogs which do not significantly affect its properties.
Arnino acid residues which can be conservatively substituted for one another
include but are not limited to: glycine/~l~nine; valine/isoleucine/leucine;
15 asparagine/glut~mine; aspartic acid/glutamic acid; serine/threonine; Iysine/arginine; and
phenyl~l~nine/tyrosine. Any conservative amino acid substitution which does not
significantly affect the properties of compositions is encompzl~e~l by the present
invention.
It is within the skill of one in the art to determine whether a particular agent has
20 therapeutic utility, for instance, by ~ltili7ing the methods described herein. It is also
within the skill of one in the art to formulate suitable dosage formats for delivery of the
therapeutic agents. When the site of delivery is the brain, the therapeutic agent must be
capable of being delivered to the brain.
The blood-brain barrier limits the uptake of many therapeutic agents into the brain
25 and spinal cord from the general circulation. Molecules which cross the blood-brain
barrier use two main mech~ni~m~: free diffusion; and facilitated transport. Because of
the presence of the blood-brain barrier, ~tt~ininp; beneficial concentrations of a given
therapeutic agent in the CNS may require the use of drug delivery strategies. Delivery of
therapeutic agents to the CNS can be achieved by several methods.
14
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W O 97/14437 PCTnJS96/02447
One rnethod relies on neurosurgical techniques. In the case of gravely ill patients
such as accident victims or those suffering from various forms of dementi~) surgical
5 intervention is w~ d despite its ~ n-l~nt risks. For instance, therapeutic agents can
be delivered by direct physical introduction into the CNS, such as intraventricular or
intrathecal injection of drugs. Intraventricular injection may be facilitated by an
intraventricular catheter, for example, attached to a reservoir, such as an Omrnaya
reservoir. Methods of introduction may also be provided by rechargeable or
10 biodegradable devices. Another approach is the disruption of the blood-brain barlier by
substances w]hich increase the permeability of the blood-brain barrier. Examples include
intra-arterial iinfusion of poorly diffusible agents such as mannitol, ph~ ceuticals which
increase cerebrovascular permeability such as etoposide, or vasoactive agents such as
leukotrienes. Neuwelt and Rappoport (1984) Fed. Proc. 43:214-219; Baba et al. (1991) J.
lS Cereb.RloodFlowMetab. 11:638-643;andGennusoetal.(1993)C~ncerInvest. 11:638-
643.
Further, it may be desirable to a~lminict~r the ph~ eutical agents locally to the
area in need of tre~tment; this may be achieved by, for example, local infusion during
surgery, by injection, by means of a catheter, or by means of an implant, said implant
20 being of a porous, non-porous, or gelatinous material, including membranes, such as
silastic membranes, or fibers.
Another method involves ph~rrn:~cological techniques such as modification or
selection of a therapeutic agent to provide an analog which will cross the blood-brain
barrier. Examples include increasing the hydrophobicity of the molecule, decreasing net
25 charge or molecular weight of the molecule, or modifying the molecule, such as to
resemble one normally transported across the blood-brain barrier. Levin (1980) J. Med.
CheIIL 23:682-684; Pardridge (1991) in: Peptide Drug Delivery to the 13r~in and Kostis
- et al. (1994) J. Clin. Ph~rrn~-ol. 34:989-996.
CA 02233848 1998-04-02
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.
Encapsulation of the drug in a hydrophobic environment such as liposomes is alsoeffective in delivering drugs to the CNS. For example WO 91/04014 describes a
5 liposomal delivery system in which the drug is enc~psul~te~l within liposomes to which
molecules have been added that are normally transported across the blood-brain barrier.
Another method of form~ ting the drug to pass through the blood-brain barrier isto encapsulate the drug in a cyclodexkin. Any suitable cyclodexkin which passes through
the blood-brain barrier may be employed, including, but not limited to, J-cyclodextrin, K-
cyclodextrin and derivatives thereof. See generally, U.S. Patent Nos. 5,017,566,5,002,935 and 4,983,586. Such compositions may also include a glycerol derivative as
described by U.S. PatentNo. 5,153,179.
Yet another method takes advantage of physiological techniques such asconjugation of a therapeutic agent to a transportable agent to yield a new chimeric
15 transportable therapeutic agent. For example, vasoactive intestinal peptide analog (VIPa)
exerted its vasoactive effects only after conjugation to a monoclonal antibody (Mab) to
the specific carrier molecule transferrin receptor, which facilitated the uptake of the VIPa-
Mab conjugate through the blood-brain barrier. Pardridge (1991), and Bickel et al. (1993)
Proc. Natl. Acad Sci. USA 90:2618-2622. Several other specific kansport systems have
20 been identified, these include, but are not limited to, those for kansferring insulin, or
insulin-like growth factors I and II. Other suitable, non-specific carriers include, but are
not limited to, pyridinium, fatty acids, inositol, cholesterol, and glucose derivatives.
Certain prodrugs have been described whereby, upon entering the cenkal nervous system,
the drug is cleaved from the carrier to release the active drug. U.S. Patent No. 5,017,566.
The compounds in the instant invention may be used in conjunction with one or
more of these methods to achieve the therapeutically desired result. The choices of
method and dosage scheme thereof are within the skill of one in the art.
The invention also encomp~c~e~ methods for detecting potential therapeutic agents
that reduce the interaction of apoE4 and the LRP. The methods include in vitro ligand
16
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WO 97/14437 PCT~US96/02447
blotting techniques. This can be performed following the separation of cell membrane
proteins (whic,h contain the LRP) or the LRP partially purified from membrane proteins
5 for instance b~y nonreducing sodium dodecylsulfate-polyacrylamide gel electrophoresis
and transfer to a nitrocellulose membrane. Methods of partial purification of the LRP are
described, for instance, by Schneider et al. (1985) Met. Fn7~rnol. 109:405-417. The
membrane is then inc~lb~te~ with apoE and a lipoprotein (e.g. ,~-VLDL) which is labeled,
for instance b!~ biotinylation. Binding of the apoE-~13-VLDL complex to the membrane is
10 then visualized using reagents that detect the label. Agents to be tested for their ability to
block the interaction are added to the nitrocellulose together with apoE and ~-VLDL to
fletennine if the interaction is blocked.
The following examples are provided to illustrate, but not limit, the claimed
invention.
EXAMPLE 1
Tnt~raction of apoF with B RP and Effect on Neurite Olltgrowth
The following materials and methods were used to obtain the results discussed
below.
20 Materials
Dimyri stoylphosphatidylcholine (DMPC), DME/F 12 ( 1 :1 mixture of Dulbecco's
nutrient modified Eagle's medium and Ham's mixture F12), media supplements
(progesterone, putrescine, selenite, and transferrin), sodium chlorate, heparinase,
lactoferrin, triolein, and egg yolk phosphatidylcholine (type XI-E) were purchased from
25 Sigma Chemical Co. (St. Louis, MO), fetal bovine serum (FBS), and insulin from Gibco
(Grand Island, NY), suramin from Miles Inc. (FBA ph~ ceuticals, West Haven, CT),and DiI from Molecular Probes Inc. (Eugene, OR). Neuro-2a was purchased from
- American Type Culture Collection (Rockville, MD). Bovine CSF was obtained from Pel-
Freez, Inc. (Fayetteville, AR).
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Preparation of Lipoproteins and Liposomes
Rabbit ~-VLDL (d < 1.006 g/ml) were isolated from the plasma of New 7.e~1~n~1
white rabbits fed a high-fat, high-cholesterol diet for four days according to the method
described by Kowal (1989) Proc. Natl. Acad. Sci. USA 86:5810-5814. Rabbit VLDL (d
< 1.006 g/ml) were isolated by ultracentrifugation from fasting plasma obtained from
rabbits fed a normal rabbit chow. The VLDL were washed once by ulll~cenllifugation at
d = 1.006 g/ml. Bovine CSF lipoproteins (d < 1.21 g/ml) were isolated by
ultracentrifugation according to the method described by Pitas et al. (1987) J. Biol. Chem
262: 14352- 14360. They were washed once by recentrifugation through a solution of d =
1.21 g/ml. Canine apoE HDLC (d = 1.006-1.02 g/ml) were isolated by ultracentrifugation
and Pevikon electrophoresis from the plasma of foxhounds fed a semisynthetic diet
cont~inin~ hydrogenated coconut oil and cholesterol according to the method described
by Mahley et al. (1977) Am. J. Pathol. 87:205-226. The ,~-VLDL were iodinated
according to the method described by Bilheimer et al. (1972) Biochim. Biophys. Acta
260:212-221, and free iodine was removed by PD10 column chromatography.
The DMPC vesicles were prepared essentially according to the method described
by Innerarity et al. (1979) J. Biol. Chern 254:4186-4190. The DMPC alone (90 mg) or
with the addition of cholesterol (10 mg) was dissolved in benzene and dried by
lyophilization. The lyophilized material was then resuspended in 3 ml of 0.15 M NaCl,
10 mM Tris-Cl, and 1 mM EDTA (pH 7.6) and sonicated for 30 min at 37~C using a
sonifier cell disrupter (Branson 450, Danbury, CT) equipped with a microtip and full
setting at 7 (50 watts). Innerarity (1979). The material was centrifuged for 10 min at
2,000 rpm (37~C), and the supernatant was used for addition to cells. The lipid emulsion
A was prepared according to the methods described Pittman et al. (1987) J. Biol. Chem.
262:2435-2442; and Spooner et al. (1988) J. Riol. Chem. 263:1444-1453. Briefly, the
lipids were mixed together in the following ratio: 100 mg of triolein and 25 mg of egg
yolk phosphatidylcholine and then dried under a stream of nitrogen. The pellet was then
18
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resuspended in S ml of 1~ mM Tris-Cl, 0.1 M KCl, and 1 mM EDTA (pH 8.0) buffer and
sonicated according to the method described by Spooner et al. (1988). The m~ter;~l was
5 then centrifuged for 10 min at 2,000 rpm. The composition of the ~mal emulsion was
2.7:1 for triolein:phosphatidylcholine (wt:wt). The size and morphology of the e~l~ulsion
particles were det~rn in~cl by negative staining electron microscopy.
Preparation c~Expression Vectors
The e~pression vectors were assembled in the pBSSK plasmid (Stratagene, La
10 Jolla, CA). The constructs contained the rat neuron-specific enolase (NSE) promoter
(kindly provicied by Dr. J. G. Sutcliffe, Scripps Clinic and Research Foundation, La Jolla,
CA), which has been previously used to direct neuron-specific e~les~ion of the human
amyloid precursor protein and ,~-galactosidase in transgenic mice. Quon et al. (1991)
~ature 352:239-241; and Forss-Petter (1990) Neuron 5:187-197. In addition, the
15 construct contained the first exon (noncoding), the first intron, and the first six bases of
the second exon (prior to the initiation methionine) of the human apoE gene, followed by
the apoE cDNA.
The apoE4 construct was identical except that it also contained the third intron(Fig. 1). The noncoding region of the fourth exon was downstream from the cDNA,
20 followed by 1 12 bp of the 3'-fl~nking sequence of the human apoE gene that contains the
polyadenylation signal. The apoE constructs for insertion in these expression vectors
were kindly provided by Drs. S. Lauer and J. Taylor of the J. David Gladstone Institutes.
The orientation of the cDNAs was confirmed by sequencing, using an Applied
Biosystems automated sequencer. The final constructs were referred to as NSE-E3 (for
25 apoE3 cDNA) and NSE-E4 (for apoE4 cDNA) (Fig. 1). Plasmid DNA was purified bytwo rounds of cesium chloride gradient ultracentrifugation according to the method
described by Sambrook et al. (1989) Molecular Clonin~: A T ~horatory Manual. 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. To test the constl~cts,
Chinese h~m~ter ovary cells and human embryonic kidney 293 cells were transiently
19
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kansfected (lipofectin-mediated), and the concentration of apoE in the medium was
measured as described below. Similar levels of explc~s~ion of apoE3 and apoE4 were
5 achieved.
Production of Stably Transfected Ne~ro-2a Cell Lines
Cells at 20-30% confluence were cokansfected with pSV2neo and either NSE-E3
or NSE-E4 using a calcium phosphate pleci~ilalion protocol es~nti~lly as described by
Chen et al. (1988) RioTechniques 6:632-638. Control cells were transfected with
10 pSV2neo alone, following the same protocol. Stably transfected cells were selected by
growth in DME/F 12 media cont~ining 10% FBS and 400 ,uglml of G418 (Geneticin,
Gibco). Individual G418-resistant colonies were selected and expanded. Secretion of
human apoE3 or apoE4 by the transfected cells was verified by Western blotting of the
conditioned media.
15 ApoE Quantitation
Intracellular, cell-surface-bound, and secreted apoE were qll~ntit~tç-1 in cellsm~int~ined for 96 hr in N2 medium, a serum- and lipid-free medium (DME/F12
cont~ining growth supplements as described in Bottenstein et al. (1980) F.xp. Cell Res.
129:361-366), with or without added ~-VLDL (40 ,ug cholesterol per ml). The medium
20 was changed once at 48 hr. The secreted apoE reported is that present in the medium
following the second 48 hr incubation. The media were collected and, after the addition
of protease inhibitors, centrifuged to elimin~tc suspended cells. The cell monolayers were
washed with PBS and incubated for 1 hr at 4~C with 2 ml of DMEM/F12 cont~ining 25
mM Hepes and 10 mM suramin, a polyanion that is able to release apoE bound to the cell
25 surface. Ji et al. (1994). The apoE was precipitated from the medium and the suramin
exkact by addition of 50 ,ug/ml of fumed silica (Sigma, St. Louis, MO) and centrifugation
at 13,000 x g for 10 min.
Each pellet was washed three times with sterile water and dissolved in gel-loading
buffer. Cellular apoE was extracted from the cells, following suramin removal of surface-
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bound apoE, using STEN buffer (50 mM Tris-Cl, pH 7.6, cont~inin~ 150 mM NaCl, 2
mM EDTA, l C~o NP-40, 20 mM PMSF, and 5 ~Lg/ml l~u~c~lhl). Samples were
electrophoresed on 5-20% polyacrylamide gradient gels ccnt~ining sodium dodecyl
sulfate, according to the method described by Ji et al. (1994) J. Biol. Cheln 269:13429-
13436. The proteins were transferred to nitrocellulose paper by blotting and treated with
an anti-human apoE polyclonal antiserum (1: 1,000 dilution) raised in rabbit (generously
provided by Dr. K. H. Weisgraber, Gladstone Institutes). The nitrocellulose immml~blot
was then incublated with donkey anti-rabbit secondary antibody conjugated to horseradish
peroxidase (1 :';,000 dilution) (Amer.~h~m, Arlington Heights, IL). After washing to
remove unbowld antibody, the immunocomplex was detected using an ECL kit
(Amersham), according to the m~nllf~cturer's instructions. Qll~ntit~tion of the level of
apoE bound, intern~li7~-1 and secreted by the cells was accomplished by densitometric
sc~nninp~ (Ambis Scanner, San Diego, CA) and based on a standard curve of purified
human plasma apoE3 and apoE4.
Neurite Outgrowth
Cells were grown in DME/F12 co~ i.,g 10% FBS and G418 (400 ~lg/ml). On
the day the exp~eriment was initi~t~-l the cells were subcultured into 35 mm plates in
DME/F12 with 10% FBS. The cells were allowed to adhere to the plastic plates for 2 hr at
37~C, and then the culture medium was changed to N2 medium with or without
increasing concentrations of lipoproteins. After 48 hr at 37~C, the media were replaced
with the same medium (with or without lipoproteins), and the incubation was continued
for an additional 48 hr. (The CSF lipoproteins were dialyzed against N2 medium prior to
addition to the cells.) The cells then were washed with DME/F12 cont~ining 0.2% BSA,
nonspecifically stained for 1 hr at 37~C with DiI added in DMSO according to the method
described by Nathan et al. (1994) Science 264:850-852, and fixed with 2.5%
- glutaraldehyde in PBS (v/v). Neurons were imaged in fluorescence mode with a confocal
laser sc~nning system (MRC-600, BioRad, Hercules, CA), and the images were digitized
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with an Image-1/AT image analysis system (Universal Images, West Chester, PA). The
neuronal images were coded before characterization, and the following variables were
5 measured: 1) number of neurites (defined as cell surface projections at least one-half the
cell diameter) on each neuron; 2) neurite branching (the nurnber of branch points on each
neurite); and 3) neurite extension (the length of the longest neurite, measured from the
cell body). Typically, in each experiment the neurites of 20 to 40 neurons per plate were
measured and the results preserved as the mean ~t S.E.M.
In studies on the effect of the inhibitors of lipoprotein binding to the LRP, cells
were incubated for 1 hr at 37~C in N2 medium cont~inin~ the indicated concentrations of
either lactoferrin, chlorate, or heparinase or with the receptor-associated protein (RAP).
Then the ,~-VLDL were added, and the incubation was con~inued for a total of 96 hr. The
reagents, except for ~-VLDL, were re-added every 24 hr. The media and ~-VLDL were
15 replaced after 48 hr.
Cell Association and Degradation of 1251-~-VLDL
The cells were grown for 24 hr in 35 mm dishes in N2 medium alone. Then '2sI-
,B-VLDL (3 ,ug of protein per ml of medium) were added, and the incubation was
continn~d for 16 hr at 37~C. The medium was analyzed for TCA-soluble lipoprotein20 degradation products according to the method described by Goldstein et al. (1983) Met.
F.n7~lnol. 98:241-260. The cells were placed on ice, washed with PBS con~inin~ 0.2%
BSA, and dissolved in 0.1 N NaOH. Lipoprotein cell association was determined bymeasuring cellular radioactivity using a gamma counter (Beckman ~'J~mm~ 8000,
Beckman Instrllm~nt.~, Fullerton, CA) and according to the method described by
25 Goldstein et al. (1983).
Cell Association of DiI-labeled ~- VLDL
The cells were grown for 24 hr in 35 mm dishes in N2 medium. Then DiI-labeled
,~-VLDL (4 ~Lg of protein per ml of medium), was prepared according to the methods
described by Pitas et al. (1983) Arteriosslerosis 3:2-12; and Pitas et al. (1981)
22
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W O 97/14437 PCTnUS96~2~47
Arteriosclerosis 1: 177- 185, were added, and the incubation was continued for 5 hr at
37~C. The ce]ls were then washed with PBS and fixed with 4% paraformaldehyde in PBS
(v/v). Uptake of DiI-labeled ,B-VLDL was visualized by fluorescence microscopy. To
quantitate the amount of DiI-labeled lipoprotein in the cells at the end of the incubation,
the cells were scraped, using two 0.5 ml aliquots of PBS, and lyophili7~cl The DiI was
extracted from the dried cell pellet with methanol and analyzed using a
spectrofluorometer (excitation 520 nm, emission 570 nm). Pitas et al. (1983). Standards
of DiI in methanol were used for ~ ion.
Association oJ~ApoE with Lipid Particles
ApoE3, and apoE4 were iodinated using Bolton-Hunter reagent (DuPont NEN,
Boston, MA) according to the method described by Innerarity et al. (1983) J. l~iol. Chem.
258:12341-12347, and then incub~tecl with the lipid particles for 1 hr at 37~C. The
samples were then fractionated by chromatography on a Superose 6 column (10/50 HR,
Pharmacia Fine Chemicals, Uppsala, Sweden) and eluted with 1 mM EDTA in PBS at aconstant flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected and analyzed for
cholesterol and triglyceride, and the l25I-apoE content was measured in a Beckman 8000
counter (Beckman Instruments) and according to the method described by Dong et al.
(1994) J. Biol Chem 269:22358-22365.
~tatistical Analysis
Data were analyzed using a paired t-test.
~FSUT TS
The levels of apoE secreted into the medium, bound to the cell surface, and
accumulated intracellularly by the stably transfected Neuro-2a cells expressing human
apoE3 or apoE4 were assessed by Western blot analysis and qll~ntit~ted by densitometry.
The results obtained are presented in Table. 1.
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Table 1
ApoE3 or apoE4 secreted, releasable by suramin, or present inside cells stably transfected
5 with apoE3 or apoE4 cDNA
Cells SecretedReleasable Intracellular
ApoE3-expressing ng of apoE/mg of
cell protein
Clone #1 54 6.2 140
+~-VLDL 56 7.2 119
Clone #3 44 4.9 259
+,~-VLDL 45 4.3 251
ApoE4-expressing
Clone #4 60 6.7 215
+,~-VLDL 63 5.3 231
Clone #5 69 8.0 135
+,B-VLDL 62 6.5 128
Clone #6 89 5.2 111
+,B-VLDL 87 5.6 105
To obtain the results depicted in Table 1, transfected cells were incubated for 96 hr
in medium with or without ~-VLDL (40 ,ug cholesterol/ml). The medium was changed at
48 hr. ApoE secreted in the last 48 hr, intracellular, and suramin-releasable (surface-
bound) apoE were quantitated at the end of the 96 hr of incubation as described in Nathan
et al. (1995). The data are the mean of two separate determinations. The duplicates did
not differ by more than 12%.
The results depicted in Table 1 indicate that the cells secreted 44-54 ng of apoE3
and 60-89 ng of apoE4 per mg of cell protein in 48 hr. The apoE3- and apoE4-secreting
cells had similar amounts of apoE bound to the cell surface (releasable by suramin
treatment), ranging from 4.9 to 8.0 ng of apoE per mg of cell protein. The intracellular
content of apoE in the two apoE3-expressing cell lines was 140 and 259 ng of apoE per
24
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W O 97/14437 PCT~US96/02447
mg of cell protein. Similar amounts of intracellular apoE (1 1 1-215 ng/mg) were seen in
the apoE4-expressing cell lines. The addition of ~-VLDL to the cells did not have a
significant effèct on the amount of apoE secreted, surface-bound, or present within the
apoE3- or apoE4-secreting cells (Table 1).
In initial experiments, two Neuro-2a cell lines that secreted similar amounts ofapoE3 (clone 1, 54 ng/mg of cell protein) and apoE4 (clone 4, 60 ng/mg of cell protein)
(Table 1) were used to e~nnine neurite growth. When these cells were grown in N2medium in the absence of ,B-VLDL, there were no a~ l differences in neurite
outgrowth between the apoE3- and apoE4-secreting cells. However, incubation of the
cells in N2 medium co~ -VLDL resulted in a markedly different pattern in the
neurite outgrc,wth from these cells. ApoE3-secreting cells incubated with ,~-VLDL
developed long neurites (Fig. 2A), whereas in apoE4-secreting cells neurite outgrowth
was suppressed (Fig. 2B).
Differ,ences in neurite outgrowth in the absence and presence of increasing
concentrations of ,B-VLDL were ~ led by measuring the number of neurites per cell,
neurite br~nching, and neurite extension (Figs. 3A, B, and C, respectively). The values
for the non-apoE kansfected control cells incubated for 96 hr in N2 medium in the
absence of ,~- VLDL are set at 100%. The expression of either apoE3 or apoE4 by the
transfected Neuro-2a cells did not influence neurite number, branching, or extension when
the cells were grown in N2 medium in the absence of added lipoprotein (Figs. 3A, B, and
C). To obtain the results depicted in Fig. 3, cells (clone #1 for apoE3-expressing and
clone #4 for apoE4 t;~lessillg) were incubated for 96 hr in N2 medium alone or in
medium cont ~ining increasing concentrations of ,B-VLDL. The media were changed at 48
hr. The cells were stained with DiI and fixed, and the indicated parameters weremeasured. Each data point was obtained by the measurement of 20-50 cells expressing
neurites in four separate experiments. The data are presented as the percentage of the
value obtained with control cells with N2 medium alone. The data are the mean + the
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S.E.M. As depicted in Fig. 3, the average values obtained with control cells incubated
with N2 mediurn alone were: A: neurites per cell = 3; B: branch points per neurite = 2; C:
5 average neurite length = 155 ~Lm.
For calculation of the level of significance for the effect of added ,13-VLDL,the
results in the presence of ,~-VLDL are, compared to the data obtained with the same cells
in the absence of ,13-VLDL (i.e., grown in N2 medium alone). *p < 0.025; **p < 0.010;
***p < 0.005.
However, as shown in Fig. 3A, the addition of ~-VLDL resulted in an increase in
the number of neurons in the control cells and in the cells secreting apoE3 (significantly
increased at 40 ,ug of 13-VLDL cholesterol/ml compared with apoE3-secreting cells in N2
medium). On the other hand, in the presence of high concentrations of ,B-VLDL, the
Neuro-2a cells secreting apoE4 showed a significant reduction in the number of neurites
15 per cell as compared with the apoE4-secreting cells in the N2 medium.
As previously described for DRG cells (Handelmann et al. (1992) J. T ipids ~es.
33:1677-1688; and Nathan et al. (1994)), the addition of 13-VLDL alone resulted in
increased branching of neurites. As shown in Fig. 3B, addition of ,~-VLDL to the non-
apoE-transfected cells resulted in a significant increase in neurite br~nchin~ In addition,
20 at the highest concentration of ,~-VLDL cholesterol, the apoE3-secreting cells displayed
enhanced br~nching by comparison with the apoE3-secreting cells grown in N2 medium
alone. In contrast, the apoE4-secreting cells tended to show decreased branching when
incubated with ,13-VLDL; however, this decrease did not reach statistical significance.
Neurite extension was increased in the Neuro-2a cells secreting apoE3 when they
25 were incubated with the highest concentrations of ,B-VLDL. In contrast, in the apoE4-
secreting cells neurite extension was very significantly suppressed even at the lowest
concentration of ,B-VLDL used (Fig. 3C).
The results described in Fig. 3 were based on a comparison of cells having neuritic
outgrowths and did not take into account those Neuro-2a cells without neuritic extensions.
26
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Approximately 25-30% of the Neuro-2a cells in N2 medium possessed neurite extensions
(defined as a cell-surface projection of at least one-half the cell diameter). However, as
5 shown in Fig. 4, it was apparent that in the presence of ,3-VLDL, the number of apoE3-
secreting cells cleveloping neurites increased markedly to 60-70% of the total. On the other
hand, the numbler of apoE4-secreting cells developing neuritic extensions was significantly
reduced, compared with the control or apoE3-secreting cells. Thus, the apoE3-secreting cells
incubated with l3-VLDL not only had longer neuritic extensions but also showed an increase
10 in the number of cells with neurites. The apoE4-secreting cells grown in the presence of ~-
VLDL showed :fewer neurites, and those that were produced were much shorter.
To ensure that the differential effect of ~B-VLDL on neurite outgrowth in the apoE3-
and apoE4-secreting cells was not due to clonal variation or to differences in the secretion or
intracellular content of apoE in the various cell lines, additional experiments were performed
15 with the other sl ably transfected cell lines secreting apoE3 or apoE4. Incubation of these
cells with ,B-VLDL also resulted in dirr~ Lial effects of apoE3 and apoE4 on neurite
outgrowth. The results obtained are ~lese.lled in Table 2.
Table 2
Effect of ~- YLDL ~40 llg cholesterol/ml medium) on the number of neurites per cell, neurite
branching, and neurite extension from cells stably transfected with apoE3 or apoE4
Number of Neurites Branching Extension
Cell type (% of values obtained with control cells in N2 medium alone)
ApoE3-w.,~l~,;,sillg
Clone #1 165 + 30 186 ~t 39 186 + 13
Clone #2 150 ~t 25 180 + 15 190 + 23
Clone #3 170 + 39 175 + 20 180 ~ 25
ApoE4-expre:ssing
Clone #4 43 ~ 25 65 + 2641 + 9
Clone #S 49 ~ 15 70 ~ 3150 ~t 15
Clone #6 53 ~ 19 60 + 2545 + 19
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In Table 2, the level of secretion of apoE by clones #1, #3, #4, #5, and #6 is as
described for Table 1. Clone #2 secreted 36 ng of apoE3/mg of cell protein/48 hr.
5 Surface-bound and intern~li7~cl apoE was not ~uallLi~ted for clone #2. The conditions for
incubation with ,~-VLDL are as described for Fig. 3. Each data point was obtained by the
measurement of 25-40 cells. The data are the mean ~ S.E.M.
As sllmm~ri7~d in Table 2, in the presence of ~-VLDL, all of the apoE4-secretingcells showed a significant reduction in the number of neurites expressed, branching, and
10 neurite extension, whereas the apoE3-secreting cells displayed an increased number of
neurites, increased branching, and increased extension as compared to cells grown in N2
medium lacking a source of lipoprotein.
To determine whether apoE4 blocks neurite extension in the presence of ,B-VLDL
or whether it incl~lces neurite retraction, the cells were incubated for 48 hr in N2 medium
15 alone to stimulate neurite outgrowth. The medium was changed, and the cells incubated
for an additional 48 or 96 hr in media with ,B-VLDL (40 ~lg of cholesterol per ml). The
addition of ~-VLDL did not decrease the extension of neurites of apoE4-expressing cells
compared with cells incubated in N2 medium alone. Therefore, apoE4 in the presence of
,B-VLDL, inhibits neurite extension directly and does not cause a retraction of neurites
20 that have already extended.
Other lipoproteins were used to detPrrnine if any lipid vehicle carrying apoE
would substitute for 13-VLDL. Incubation of the apoE3- or apoE4-~ ssi~lg cells with
rabbit VLDL, a lipoprotein rich in triglyceride, resulted in similar effects on neurite
extension as obtained with ,3-VLDL. The results are presented in Table 3.
28
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Table 3
Ef~ect of ~-VL.DL, VLDL or lipid emulsions on neurite extension from cells stably
transfected wilih apo~3 or apoE4 cDNA
ApoE3- apoE4-expressing
Tre~tment Control ex~l~ssillg
Lipid Mean Size
composition (nm + % of value obtained with control cells
(wtlwtlwt) S.D.) in N2 m~ m alone
N2 alone , , 100 + 10 110 + 15 115 ~ 11
,B-VLDL CHOL:~lg:PL43.7 ~ 25.6 120 l 15 160 ~ 18a 60 + 13a
(5-6:0.4:1)
VLDL CHOL:~lg:PL39.5 + 18.7 110 + 11 155 + 21a 61 ~t 19a
(1 :7.4: 1)
Emul A ,ug:PL (2.7:1)35.8 + 14.9 95 ~ 14 150 ~ 12a 75 ~ 12a
To obtain the results depicted in Table 3, cells (clone #1 for apoE3-expressing and
clone #4 for apoE4-e2~lcssillg) were incubated for 96 hr in N2 medium alone or
cont~ining the indicated concentrations of particles: ,B-VLDL, 40 ~lg cholesterol/ml
medium (this c orresponds to 5 llg triglyceride/ml medium); VLDL, 5 ~Lg triglyceride/ml
medium; emulsion A, 5 ,ug triglyceride/ml medium. CHOL = cholesterol; ,ug =
triglyceride; PL = phospholipid. Each data point was obtained by the measurement of
30,40 cells expressing neurites in three separate experiments. The data are the mean +
S.E.M. ap < 0.010 versus control***.
As shown in Table 3, when the Neuro-2a cells secreting apoE3 were incubated
with VLDL, thiey showed an increase in neurite extension, whereas the apoE4-secreting
cells in the presence of VLDL showed an inhibition of neurite extension. In other
experiments, human LDL and canine apoE HDLC, an apoE-enriched plasma high density
lipoprotein (H~DL) inclu(~ed by cholesterol feeding and resembling apoE-cont~ining
lipoproteins in the CSF (Pitas et al. (1987)), also were used. The apoE3- and apoE4-
secreting Neuro-2a cells did not respond to LDL (40 llg cholesterol/ml) (i.e., there was no
difference in neurite extension as compared with control cells grown in N2 medium
29
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alone). On the other han~, incubation of apoE HDLC (40)1g cholesterol/ml) with the
apoE4-secreting or apoE3-secreting cells resulted in only a small reduction or increase in
5 neurite extension, respectively (control cells in N2 medium, l 00%; apoE4-secreting cells
plus HDLC, 85,90% of the value obtained with N2 medium; apoE3-secreting cells plus
HDLC,110% of the value obtained with N2 medium).
Liposomes and lipid emulsions also were used in an attempt to define the type oflipid vehicle required for the delivery of the apoE. The DMPC emulsion alone or DMPC
10 complexed with cholesterol were incubated with the apoE3- and apoE4-secreting cells for
96 hr at increasing phospholipid concentrations of up to 45,ug phospholipid and 5 ~Lg
cholesterol/ml medium (higher concentrations were toxic to the cells).
In these studies, there was no effect on neurite outgrowth with either of the apoE-
transfected Neuro-2a cells. Previously, it was shown that apoE complexes with DMPC
15 and mediates high-affinity binding to the LDL receptor. Pitas et al. (1980) J. ~iol. Chem
255:5454-5460. On the other hand, a lipid emulsion particle (emulsion A in Table 3),
which was a triglyceride- and phospholipid-cont~ining spherical particle (approximately
35.8 nm), caused a significant enhancement of neurite extension in the apoE3-secreting
cells and was associated with an inhibition of outgrowth in the apoE4-secreting cells.
20 Thus, specific combinations of lipids and/or a unique particle size may be required to
elicit the apoE isoform, specific effects on neurite outgrowth. It is interesting to note that
the delivery of cholesterol to the cells does not appear to be required for the differential
effect.
Additional studies using the lipoproteins from bovine CSF suggest that natural
25 lipoproteins in the CNS may mediate the isoform-specific effects of apoE3 and apoE4.
As shown in Fig.5, addition of lipoproteins isolated from CSF (d < 1.21 g/ml) to the cells
caused an inhibition of neurite outgrowth from the apoE4-expressing cells and an increase
in outgrowth from the apoE3-expressing cells. When CSF lipoproteins were used at a
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concentration of 40 ,ug lipoprotein cholesterol/ml, the effect was sirnilar to that obtained
using ,~-VLDL, at the sarne concentration.
CSF lipoproteins (d < 1.21 g/ml) were analyzed for protein and cholesterol content
and apolipoprotein composition. The ratio of cholesterol to protein was approximately
1: 1, similar to data reported for canine CSF. Pitas et al. (1987). The bovine CSF
lipoproteins (d < 1.21 g/ml) contained only apoE and apoA-I when separated by sodiurn
dodecyl sulfate polyacrylamide gel eleckophoresis and vi~u:~1i7t~1 by Coomassie Brilliant
10 Blue st~ining These results are similar to those reported previously for human and
canine CSF lipoproteins. Pitas et al. (1987); and Roheim et al. (1979) Proc. Natl. ~cad.
Sci. USA 76:4646-4649.
The ability of the neuroblastoma cells to bind, illt~rnZ~li7f':7 and degrade ,B-VLDL
was e~mined to cletPrrnine whether the differences in neurite outgrowth in the apoE3-
15 and apoE4-expressing cells was due to a different ability of the secreted apoE3 and apoE4
to stim~ te the delivery of apoE and/or lipoprotein lipids to the cells. In these studies,
l2sI-,B-VLDL were used to quantitate the binding, uptake, and degradation of thelipoproteins in the Neuro-2a cells. The results are presented in Table 4.
Table 4
Cell association and degradation of '25I-,~-VLDL by stably transfected and control cells
l2sI-~-VLDL
Cell association Degradation
~Cell type (ng of lipol,loteill protein/mg of cell protein)
Control cells 750 + 16 2,467 ~ 331
Apo]_3-t;~le~i,lg cells 671 + 40a 1,945 ~ 219
Apo]_4-expressingcells 662+50a 1,788~t 188b
To obtain the results depicted in Table 4, cells were incubated for 24 hr in N2
medium alone. The l2sI-,~-VLDL (3 ~Lg protein/ml medium) were then added, and after
16 hr at 37~C lhe lipoprotein cell association (bound and illte~ li7.~?d) and degradation by
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Neuro-2a cells were measured. The data reported are the mean of two ~cpdldl~
experiments performed in duplicate (+ S.D.). Control = cells transfected with pSV2neo
alone. In Table 4, a represents <0.05 versus control and b le~ senl~ <0.01 versus control.
The results presented in Table 4 indicate that the total amount of cell-associated
(bound and intPrn~li7~ 25I-,B-VLDL was very similar in the apoE3- and apoE4-
secreting cells (both were slightly lower than that seen in the non-apoE-transfected
control cells). The degradation of l2sI-~-VLDL by the apoE3- and apoE4-secreting cells
10 was similar. There was a small (but statistically significant) decrease in the degradation
of 125I-,B-VLDL by the apoE4-secreting cells when compared with the non-apoE-
transfected control Neuro-2a cells.
In a parallel experiment, the cells were incubated with DiI-labeled ,~-VLDL to
visualize the intern~li7~ion of the lipoproteins in the apoE3- and apoE4-secreting cells by
15 fluorescence microscopy. Following intern~ tion, DiI is trapped in the Iysosomes, and
the fluorescent intensity of the cells, therefore, is proportional to the total amount of
lipoprotein internalized and degraded. Pitas et al. (1983). In these studies, no dirr~-t;nce
in the uptake of DiI-labeled ,~-VLDL was observed in the apoE3- and apoE4-secreting
cells (Fig. 6). Extraction and quantitation of the DiI from cells incubated with DiI-labeled
,~-VLDL (40 ,ug of cholesterol per ml) for 16 hr at 37~C confirmed the visual impression
that the uptake of DiI-labeled ~-VLDL was similar in the apoE3- and apoE4-secreting
cells. The control cells incorporated 8.9 ~ 0.4 ng of DiI per mg of cell protein, while the
apoE3- and apoE4-expressing cells incorporated 10.2 + 1.0 and 10.8 + 0.3 ng of DiI per
mg of cell protein, respectively.
To demonstrate that apoE binds to the lipid particles when it is present at the
concenkations secreted by the cells, radiolabeled apoE3 or apoE4 was incubated with the
,~-VLDL, VLDL, or emulsion A for 1 hr at 37~C (100 ng of apoE with 40 ~Lg of ,~-VLDL
cholesterol or 100 ng of apoE with either S ~Lg of VLDL or emulsion A kiglyceride) and
fractionated by FPLC. Approximately 70% of the apoE was associated with the ,B-VLDL
-
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and 50% with the VLDL and emulsion A. There was no difference in the amount of
apoE3 or apoE4 associated with the lipid particles.
EXAMPLE 2
Specific Inhibition of apoF l~in(1ir~ to apoF, R;n~lin~ Receptor
To determine which receptor was involved in me~ tin~ the dir~lellLial effects ofapoE3 and apo]_4 on neurite outgrowth, inhibitors that block the binding and
10 int~rn~li7~tion of apoE-enriched lipoproteins by the HSPG-LRP pathway, but not by the
LDL receptor pathway, were used. The effect on neurite outgrowth was then determined.
Priior to the addition of ,B-VLDL, the cells were preincubated for 1 hr with either
heparinase (20 units/ml) and chlorate (20 mM), with the RAP (5 ~Lg/ml), or with
lactoferrin (10 llg/ml). The binding of apoE-enriched lipoproteins to the LRP requires
15 their initial binding to cell-surface HSPG. Heparinase and chlorate cleave and reduce the
sulfation of cel]-surface HSPG, respectively. Ji et al. (1993) J. Biol. Chem 268: 10160-
10167; and Humphries et al. (1989) Met. Fn7~mol. 179:428-434. Lactoferrin blocksbinding of lipoproteins to both HSPG and LRP, whereas the RAP primarily blocks the
binding of apoE-enriched lipoproteins to the LRP. All of these reagents previously have
20 been shown to iinhibit the uptake of apoE-enriched ,B-VLDL by the LRP. Mahley et al.
(1994) Ann N.Y. Acad. Sci. 737:39-52; Ji et al. (1993); Ji et al (1994a); and Willnow et
al. (1992) J. Liol. Chem. 267:2G172-26180. As previously shown in Fig. 3, ,B-VLDL
alone stimnl~te,d the outgrowth of neurites. The ~tim~ tion of neurite outgrowth by ,B-
VLDL was further enh~nced in the apoE3-expressing cells and markedly inhibited in the
25 apoE4-secreting cells (Table 5).
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Table 5
E~ect of chlorate, heparinase, the RAP, and lactoferrin in the presence of ~-VLDL on
neurite extension from cells stably transfected with apoE3 or apoE4 cDN~
Tre~tmen~ ControlApoE3-~ ;ssing ApoE4-expressing
% of value obtained with control
cells in N2 medium alone
N2 alone 100 + 8 105 + 10 103 + 9
~-VLDL (40,ug cholesterol/ml) 160 + 13 209 + 13a 70 + 4b
,B-VLDL + chlorate (20 mM) and 159 + 14 163 + 20C 138 ~t 12
10 heparinase (20 units/ml)
~-VLDL + RAP (5,ug/ml)d 176 ~t 11 179 + 15 160 + 16
,B-VLDL + lactoferrin (10 ~lg/ml) 128 ~ 16 154 + lgc 130 + 12
To obtain the results depicted in Table 5, cells were incubated for 1 hr in N2
15 medium alone or conf~inin~ the indicated concenkations of chlorate, heparinase, RAP, or
lactoferrin. Then the ~-VLDL were added, and the incubation was continued for a total of
96 hr. The reagents, except for ~-VLDL, were re-added every 24 hr. The media and ,B-
VLDL were changed at 48 hr. Each data point was obtained by measuring 30,40 neurons
expressing neurites in two separate experiments. Data are the mean ~ S.E.M. ap < 0.05,
20 bp < 0.01 versus value obtained with control cells (non-apoE-expressing cells incubated
with ~-VLDL). cp < 0.05 versus apoE3-~ s~ g cells with ,B-VLDL alone. dIn a
parallel set of experiments,5 Jlg/ml of RAP did not block the binding of DiI-labeled LDL
to the Neuro-2a cells.
The results depicted in Table 5 indicate that the addition of chlorate and
25 heparinase or the RAP did not block the stimulatory effect of ,B-VLDL on neurite
outgrowth in the control cells (Neuro-2a cells not expressing apoE), suggesting that the
effect of ,13-VLDL alone is mediated by the LDL receptor; however, these reagents
blocked the isoform-specific effects in the cells secreting apoE (Table 5). Chlorate and
heparinase treatment of the cells or the addition of the RAP prevented the stimulation of
34
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W O 97114437 PCTnUS96/0~447
neurite extension in the apoE3-expressing cells incubated with ,B-VLDL (that is,significantly decreased the ~-VLDL, induced neurite extension in the Neuro-2a cells
5 secreting apoE,3). Moreover, chlorate and heparinase or the RAP blocked the inhibition
of neurite extension seen in the apoE4-~ essing cells (that is, the apoE4-expressing
cells in the presence of ,B-VLDL did not demonstrate inhibition of neurite extension but,
in fact, showe~ increased extension) (Table S). In the presence of hep~rin~ce and chlorate
or the RAP, in. the apoE-secreting cells, neurite outgrowth was similar to that observed
10 when ,B-VLDI, were added to the control cells in the absence of apoE (Table 5).
Therefore, in the presence of these reagents, the LDL receptor, mediated effect of ~-
VLDL was not blocked. Lactoferrin also blocked the effects of apoE3 and apoE4 onneurite outgrowth, however, it also slightly suppressed the effect of ~-VLDL on neurite
extension in the control cells. These data show that inhibition of the interaction between
15 ,~-VLDL and the HSPG-LRP pathway prevents the differential effects of apoE3 and
apoE4 on neurite outgrowth (Table 5).
In dorsal root ganglion or neuroblastoma cells, apoE3 plus a source of lipid
supports and facilitates neurite extension. ApoE3 appears to accllm~ te widely in cell
bodies and neurites, stabilize the cytoskeleton and support neurite elongation, and directly
20 or indirectly mlodulate microtubule assembly. ApoE4, on the other hand, does not appear
to accumulate within neurons or support neurite extension, and may even destabilize the
microtubule a~)paldlus. The apoE4 effect appears to be mediated via the LRP pathway.
Individuals with apoE4 clearly have normal neuronal development early in life.
However, apo]_4 may exert its detrimental effects later in life, by not allowing or
25 supporting remodeling of synaptic connections. This effect may be important in the
pathogenesis of Alzheimer's disease. Alternatively, apoE4 may contribute to Alzheimer's
disease by aiding the formation of dense, complicated, possibly toxic plaques of A~
~ peptide. At present, the ~dlhway whereby apoE affects the development of Alzheimer's
disease remains speculative.
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- EXAMPLE 3
Methods of detection of a~ents that interfere with the apoF.4 dorn~in interaction
ApoE4 is iodinated using the Bolton-Hunter reagent (New Fngl:~nd Nuclear Corp.,
Boston, MA) as previously described by Innerarity et al. (1979) J. Riol. Chern 254:4186-
4190, with specific activities ranging from 200 to 1100 dpm/ng. The iodinated apoE4
(0.5-2 mg in 50-10 ml 0.1 M NH4HCO3) is incubated with the test reagent or compound
and the mixture is added to 250 ml of plasma from normal subjects at 37~C for 2 h.
l O Plasma is then fractionated into the various lipoprotein classes by chromatography on a
Superose 6 column (10/50 HR, Pharmacia Fine Chemicals, Uppsala, Sweden) eluted with
20 mM sodium phosphate (pH 7.4), contslininp; 0.15 M NaCl. The column flow rate is 0.5
ml/min, 0.5 ml fractions are collected, and the l25I content is determined in a Beckman
8000 gamma counter (Beckman Instruments, Fullerton, CA). Reagents that i~ ;.r~l~ with
apoE4 domain interaction will shift the preference of the "modified" apoE4 from VLDL
to HDLs, resulting in a distribution that resembles that of apoE3 (run in parallel as a
control).
Although the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of underst~n~ling, it will be ~ to
those skilled in the art that certain changes and modifications may be practiced.
Therefore, the description and examples should not be construed as limiting the scope of
the invention, which is deline~ted by the appended claims.
36
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