U.S. patent application number 10/788257 was filed with the patent office on 2004-11-18 for model for alzheimer's disease and other neurodegenerative diseases.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Bi, Xiaoning, Lynch, Gary.
Application Number | 20040229209 10/788257 |
Document ID | / |
Family ID | 26916406 |
Filed Date | 2004-11-18 |
United States Patent
Application |
20040229209 |
Kind Code |
A1 |
Lynch, Gary ; et
al. |
November 18, 2004 |
Model for Alzheimer's disease and other neurodegenerative
diseases
Abstract
The present invention provides a model for studying the
development of, and/or pathologies associated with
neurodegenerative diseases, and agents that can alter such
development and/or pathologies. The model of the invention is
especially useful as an Alzheimer's disease model. The model of the
invention provides brain cells and a method for increasing
neurodegenerative disease characteristics in such cells,
especially, induction of neurofibrillary tangles and/or
phosphorylated tau and/or tau fragments and/or the production
and/or release of cytokines and/or microglia reactions and/or
activations and/or inflammation and/or conversion of p35 to p25
and/or the levels and activities of protein kinases by selectively
increasing the concentration of cathepsin D to an effective level,
and/or by lowering the concentration of cholesterol in such cells.
The model also provides a method of reversing such effects, by
inhibiting cysteine protease and mitogen-activated kinase activity,
and especially, by inhibiting calpain, and/or MAP kinase.
Inventors: |
Lynch, Gary; (Irvine,
CA) ; Bi, Xiaoning; (Irvine, CA) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
The Regents of the University of
California
|
Family ID: |
26916406 |
Appl. No.: |
10/788257 |
Filed: |
February 27, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10788257 |
Feb 27, 2004 |
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09917789 |
Jul 31, 2001 |
|
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60283352 |
Apr 13, 2001 |
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60222060 |
Jul 31, 2000 |
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Current U.S.
Class: |
435/4 |
Current CPC
Class: |
G01N 2800/2821 20130101;
G01N 33/5005 20130101; G01N 2333/4709 20130101; G01N 2333/8139
20130101; G01N 33/6896 20130101 |
Class at
Publication: |
435/004 |
International
Class: |
C12Q 001/00; G01N
033/53 |
Goverment Interests
[0002] This invention was made with Government support under Grant
No. 455365-30110, awarded by the National Institute on Aging. The
Government has certain rights in this invention.
Claims
1-12. (Cancelled)
13. A method of determining the effect of a substance on
characteristics of neurodegenerative disease in brain cells, said
method comprising: (A) exposing brain cells to a condition that
decreases an effective concentration of cholesterol in said cells,
(B) maintaining said cells for a time sufficient to induce one or
more characteristics of a neurodegenerative disease in said cells,
(C) adding said substance before, during and/or after said exposing
or said maintaining; and (D) determining whether the presence of
said substance has an effect on said one or more characteristics,
wherein said characteristics are selected from the group consisting
of: (1) the formation of neurofibrillary tangles, (2) an increase
in the phosphorylation of tau, (3) an increase in tau proteolytic
fragments, (4) an increased production and/or release of
brain-produced cytokines TGF-beta, IL-1b or LPS, (5) an increased
microglia reaction or microglial activation, (6) increased
indications of brain inflammatory reactions, (7) decrease in the
levels of p35, (8) decreased activity of cyclin dependent protein
kinase 5 (cdk5), and (9) increased levels of mitogen activated
protein kinase (MAPK).
14. The method of claim 13, wherein said characteristics comprise
an increase in the density of neurofibrillary tangles in said brain
cells.
15. The method of claim 13, wherein said characteristics comprise
an increase in the amount of phosphorylated tau in said brain
cells.
16. The method of claim 13, wherein said characteristics comprise
an increase in the amount of tau proteolytic fragments in said
brain cells.
17. The method of claim 13, wherein said condition comprises
contacting said brain cells with an inhibitor of cholesterol
synthesis.
18. The method of claim 13, wherein said condition comprises
contacting said brain cells with a member of the family of
compounds know as statins.
19. The method of claim 17, wherein said inhibitor is selected from
the group consisting of mevastatin, simvastatin, atorvastatin,
pravastatin, fluvastatin, lovastatin, cerivastatin, and mimetics
thereof.
20. The method of claim 19, wherein said inhibitor is
mevastatin.
21. The method of claim 13, wherein said brain cells are in the
form of a brain slice.
22. The method of claim 21, wherein said brain slice is a
hippocampal slice, an entorhinal cortex slice, an
entorhinohippocampal slice, a neocortex slice, a hypothalamic
slice, or a cortex slice.
23. The method of claim 13, wherein said brain cells are in
vivo.
24. The method of claim 13, wherein said brain cells are
apolipoprotein E-deficient brain cells.
25. The method of claim 13, wherein said brain cells are
apolipoprotein E4-containing brain cells.
26. The method of claim 13, wherein said cells are also contacted
with a cathepsin D-increasing compound.
27-39. (Cancelled)
40. A method of determining the effect of a substance on the
inhibition of characteristics of neurodegenerative disease in brain
cells, said method comprising: (A) exposing brain cells to a
condition that decreases an effective concentration of cholesterol
in said cells, (B) maintaining said cells for a time sufficient to
induce one or more characteristics of a neurodegenerative disease
in said cells, (C) adding a cysteine protease inhibitor before,
during and/or after said exposing or said maintaining; (D) adding
said substance before, during and/or after said exposing or said
maintaining; and (E) determining whether the presence of said
inhibitor has an effect on the inhibition of the development of
said one or more characteristics, wherein said characteristics are
selected from the group consisting of: (1) the formation of
neurofibrillary tangles, (2) an increase in the phosphorylation of
tau, (3) an increase in tau proteolytic fragments, (4) an increased
production and/or release of brain-produced cytokines TGF-beta,
IL-1b, TNF, or LPS, (5) an increased microglia reaction or
microglial activation, (6) increased indications of brain
inflammatory reactions, (7) decrease in the levels of p35, (8)
decreased activity of cyclin dependent protein kinase 5 (cdk5), and
(9) increased levels of mitogen activated protein kinase
(MAPK).
41. The method of claim 40, wherein said characteristics comprise
an increase in the density of neurofibrillary tangles in said brain
cells.
42. The method of claim 40, wherein said characteristics comprise
an increase in the amount of phosphorylated tau in said brain
cells.
43. The method of claim 40, wherein said characteristics comprise
an increase in the amount of tau proteolytic fragments in said
brain cells.
44. The method of claim 40, wherein said condition comprises
contacting said brain cells with an inhibitor of cholesterol
synthesis.
45. The method of claim 40, wherein said condition comprises
contacting said brain cells with a member of the family of
compounds know as statins.
46. The method of claim 44, wherein said inhibitor is selected from
the group consisting of mevastatin, simvastatin, atorvastatin,
pravastatin, fluvastatin, lovastatin, cerivastatin, and mimetics
thereof.
47. The method of claim 46, wherein said inhibitor is
mevastatin.
48. The method of claim 40, wherein said brain cells are in the
form of a brain slice.
49. The method of claim 48, wherein said brain slice is a
hippocampal slice, an entorhinal cortex slice, an
entorhinohippocampal slice, a neocortex slice, a hypothalamic
slice, or a cortex slice.
50. The method of claim 40, wherein said brain cells are in
vivo.
51. The method of claim 40, wherein said brain cells are
apolipoprotein E-deficient brain cells.
52. The method of claim 40, wherein said brain cells are
apolipoprotein E4-containing brain cells.
53. The method of claim 40, wherein said cells are also contacted
with a compound that increases cathepsin D.
54-71. (Cancelled)
72. A method of determining the effect of a substance on the
inhibition of characteristics of neurodegenerative disease in brain
cells, said method comprising: (A) exposing brain cells to a
condition that decreases an effective concentration of cholesterol
in said cells, (B) maintaining said cells for a time sufficient to
induce one or more characteristics of a neurodegenerative disease
in said cells, (C) adding a mitogen activated kinase inhibitor
before, during and/or after said exposing or said maintaining; (D)
adding said substance before, during and/or after said exposing or
said maintaining; and (E) determining whether the presence of said
inhibitor has an effect on the inhibition of the development of
said one or more characteristics, wherein said characteristics are
selected from the group consisting of: (1) the formation of
neurofibrillary tangles, (2) an increase in the phosphorylation of
tau, (3) an increase in tau proteolytic fragments, (4) an increased
production and/or release of brain-produced cytokines TGF-beta,
IL-1b, TNF, or LPS, (5) an increased microglia reaction or
microglial activation, (6) increased indications of brain
inflammatory reactions, (7) decrease in the levels of p35, (8)
decreased activity of cyclin dependent protein kinase 5 (cdk5), and
(9) increased levels of mitogen activated protein kinase
(MAPK).
73. The method of claim 72, wherein said characteristics comprise
an increase in the density of neurofibrillary tangles in said brain
cells.
74. The method of claim 72, wherein said characteristics comprise
an increase in the amount of phosphorylated tau in said brain
cells.
75. The method of claim 72, wherein said characteristics comprise
an increase in the amount of tau proteolytic fragments in said
brain cells.
76. The method of claim 72, wherein said condition comprises
contacting said brain cells with an inhibitor of cholesterol
synthesis.
77. The method of claim 72, wherein said condition comprises
contacting said brain cells with a member of the family of
compounds know as statins.
78. The method of claim 76, wherein said inhibitor of cholesterol
synthesis is selected from the group consisting of mevastatin,
simvastatin, atorvastatin, pravastatin, fluvastatin, lovastatin,
cerivastatin, and mimetics thereof.
79. The method of claim 78, wherein said inhibitor is
mevastatin.
80. The method of claim 72, wherein said brain cells are in the
form of a brain slice.
81. The method of claim 80, wherein said brain slice is a
hippocampal slice, an entorhinal cortex slice, an
entorhinohippocampal slice, a neocortex slice, a hypothalamic
slice, or a cortex slice.
82. The method of claim 72, wherein said brain cells are in
vivo.
83. The method of claim 72, wherein said brain cells are
apolipoprotein E-deficient brain cells.
84. The method of claim 72, wherein said brain cells are
apolipoprotein E4-containing brain cells.
85. The method of claim 72, wherein said cells are also contacted
with a compound that increases cathepsin D.
86. The method of claim 72, wherein said mitogen activated kinase
inhibitor is a MAP kinase inhibitor.
87. The method of claim 72, wherein said mitogen activated kinase
inhibitor is selected from the group consisting of PD98059,
SB203580 and U0126.
88. The method of claim 86, wherein said mitogen activated kinase
inhibitor is PD 98059.
89-94. (Cancelled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/283,352, filed Apr. 13,2001, and the benefit of
U.S. Provisional Application No. 60/222,060, filed Jul. 31, 2000,
the contents of each of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0003] The invention is in the field of models for medical
diseases. Specifically, the invention is in the field of
neurodegenerative disease models, and especially, Alzheimer's
disease models.
BACKGROUND OF THE INVENTION
[0004] As human life span has significantly expanded over the last
century, Alzheimer's disease and other neurodegenerative diseases
will have a growing impact on the quality of life for a large
proportion of the population. For example, Alzheimer's disease is a
leading cause of dementia in the elderly, affecting 5-10% of the
population over the age of 65 years. See A Guide to Understanding
Alzheimer's disease and Related Disorders, edited by Jorm, New York
University Press, New York (1987). Alzheimer's disease often
presents with a subtle onset of memory loss followed by a slow
progressive dementia over several years. The prevalence of
Alzheimer's disease and other dementias doubles every five years
beyond the age of 65. See 1997 Progress Report on Alzheimer's
disease, National Institute on Aging/National Institute of Health.
Alzheimer's disease now affects 12 million people around the world,
and it is projected to increase to 22 million by 2025 and to 45
million by 2050. See Alzheimer's Association Press Release, Jul.
18, 2000.
[0005] The complexity of the brain's architecture and chemistry,
and the complexity of these neurodegenerative brain diseases,
especially Alzheimer's disease, has hampered the development of a
model that mimics many of the changes seen in the human brain. Such
a model is needed in order to identify drugs or other agents that
might be useful in treating, preventing or reversing the effects of
such diseases.
[0006] Alzheimer's disease is histopathologically characterized by
the loss of particular groups of neurons and the appearance of two
principal lesions within the brain, termed senile plaques and
neurofibrillary tangles. See Brion et al., J. Neurochem.
60:1372-1382 (1993). Senile plaques occur in the extracellular
space. A major component of senile plaques is beta-amyloid
(A-beta), a naturally secreted but insoluble peptide formed by
cleavage of amyloid precursor protein (APP). A-beta is a fragment
close to the carboxyterminal domain of APP.
[0007] Neurofibrillary tangles are intraneuronal accumulations of
filamentous material in the form of loops, coils or tangled masses.
They are most abundantly present in parts of the brain associated
with memory functions, such as the hippocampus and adjacent parts
of the temporal lobe. See Robbins Pathologic Basis of Disease,
Cotran et al., 6.sup.th ed. (1999). Neurofibrillary tangles are
commonly found in cortical neurons, especially in the entorhinal
cortex, as well as in other locations such as pyramidal cells of
the hippocampus, the amygdala, the basal forebrain, and the raphe
nuclei.
[0008] Neurofibrillary tangles can also be found during normal
aging of the brain, however, they are found in a significantly
higher density in the brain of Alzheimer's disease patients, and in
the brains of patients with other neurodegenerative diseases, such
as progressive supranuclear palsy, postencephaltic Parkinson
disease, Pick's disease, amylotrophic lateral sclerosis, etc.
Robbins Pathologic Basis of Disease, Cotran et al., 6th ed. (1999),
p.1330. Previous studies suggest that, among other things,
neurofibrillary tangles may significantly contribute to the
cognitive decline associated with the disease and also directly to
neuronal cell death.
[0009] Ultrastructurally, neurofibrillary tangles are composed
predominantly of paired helical filaments ("PHF"). A major
component of PHF is an abnormally phosphorylated form of a protein
called tau and its fragments. Robbins Pathologic Basis of Disease,
Cotran et al., 6th ed., W.B. Saunders Company (1999), p.1300.
[0010] The tau protein (also referred to as "native tau") is a
microtubule-associated phosphoprotein that stabilizes the
cytoskeleton and contributes to determining neuronal shape. See
Kosik & Caceres, Cell Sci. Suppl. 14:69-74 (1991). Tau has an
apparent molecular weight of about 55 kDa. The protease cathepsin D
cleaves tau protein at neutral (cytoplasmic) pH resulting in tau
fragments--one of which has a molecular weight of approximately 29
kDa (referred to by some authors as "tau fragment"). See, e.g.,
Bednarski & Lynch, J. Neurochem. 67:1846-1855 (1996); Bednarski
& Lynch, NeuroReport 9:2089-2094 (1998). Both the tau protein
and 29 kDa tau fragment can be phosphorylated. In a normal brain,
the tau protein and tau fragment typically exist in an
unphosphorylated, or dephosphorylated state. However, in
neurofibrillary tangles, both tau protein and tau fragment can be
found in an abnormally phosphorylated state, a hyperphosphorylated
state. The 29 kDa tau fragment is a major component of
neurofibrillary tangles. Hyperphosphorylation impairs tau protein's
ability to interact with microtubules.
[0011] Bednarski E, and Lynch G, J. Neurochem 67:1846-55 (1996)
cultured hippocampal slices with an inhibitor
[N-CBZ-L-phenylalanyl-L-alanine-diaz- omethyl ketone (ZPAD)] of
cathepsins B and L. The authors reported that this resulted in the
degradation of high molecular weight isoforms of tau protein and
the production of a 29-kDa tau fragment (tau 29).
[0012] Bednarski E, and Lynch G, Neuroreport 9:2089-2094 (1998)
reported that incubating cultured hippocampal slices with
chloroquine or with ZPAD resulted in increases in enzymatically
active cathepsin D and the delayed appearance of a 29 kDa fragment
of the tau protein. The authors proposed that inactivation of
cathepsin L leads to induction of cathepsin D which leads to
aberrant tau proteolysis and that such a pathway is likely to play
an important role in brain aging.
[0013] In addition to the build-up of A-beta and of neurofibrillary
tangles, increasing evidence has pointed to a link between lipid
metabolism and Alzheimer's disease. Epidemiological studies found
that patients with increased plasma cholesterol levels and
cardiovascular diseases have an increased risk of Alzheimer's
disease (Jick, H., et al., Lancet 356:627-631 (2000)). Also,
long-term therapy with the 3-hydroxy-3-methylglutaryl coenzyme A
reductase inhibitors appears to decrease the prevalence of
Alzheimer's disease (Jick, H., et al., Lancet 356:627-631 (2000);
Wolozin, B., et al., Arch. Neurol. 57:1439-1443 (2000)).
[0014] Consistent with a link to lipid metabolism, in vitro
experiments have shown that cholesterol affects the generation and
aggregation of beta amyloid (A-beta) (Bodovitz, S., and Klein, W.
L., J. Biol. Chem. 271:4436-4440 (1996); Xu, H., et al., Proc.
Natl. Acad. Sci. U S A 94:3748-3752 (1997); Howland, D. S., et al.,
J. Biol. Chem. 273:16576-16582 (1998)). Transgenic mice fed a high
cholesterol diet also developed increased amounts of A-beta
deposition (Refolo, L. M., et al., Neurobiol. Dis. 7:321-331
(2000)).
[0015] ApoE-mediated transport of cholesterol into lysosomes is a
critical step for cells to utilize these sterols, which is of
particular importance for mature neurons that mainly rely on
extracellular cholesterol (Brown, M. S., and Goldstein, J. L.,
Annu. Rev. Biochem. 52:223-261 (1983)). Once in the lysosome,
cholesterol and other lipids dissociate from ApoE before being
utilized by the cell (Brown, M. S., and Goldstein, J. L., Annu.
Rev. Biochem. 52:223-261 (1983)).
[0016] Changes in cholesterol levels may be involved in certain
neurodegenerative diseases. For example, accumulation of insoluble
A-beta 1-42 has been found in Niemann-Pick type C (NPC) mutant
cells (Yamazaki, T., et al., J. Biol. Chem. (2000)(epub ahead of
print)). These cells exhibit many pathologic characteristics, one
of which is impaired intracellular transport of cholesterol
(Millard, E. E., et al., J. Biol. Chem. 275:38445-38451 (2000)).
Also, the ApoE4 isoform is a known risk factor for late-onset
Alzheimer's disease.
[0017] Inhibition of cholesterol synthesis enhanced the
phosphorylation of tau in dissociated cell cultures [ref. in
(Sawamura, N., et al., J. Biol. Chem. 57:1439-1443 (2001))].
Likewise, hyperphosphorylation of tau has been demonstrated in cell
cultures prepared from NPC mutant mice (Sawamura, N., et al., J.
Biol. Chem. 57:1439-1443 (2001)). Gradually developing disturbances
in lysosomes, which affect the sorting/trafficking of cholesterol
from lysosomes and late endosomes, may, therefore, be contributors
to the pathologies associated with neurodegenerative diseases and
Alzheimer's disease.
[0018] There has been considerable research into mechanisms
underlying neurodegenerative diseases, including Alzheimer's
disease. Many transgenic animal models of Alzheimer's disease have
been developed and used in an attempt to study the mechanisms of
Alzheimer's disease as well as to screen compounds that may
ameliorate the conditions of Alzheimer's disease. However, many in
vivo or in vitro models lack some of the important features of
Alzheimer's disease, such as neurofibrillary tangles. Thus, there
is an ongoing need to develop a model, especially one useful in
vivo or in vitro, that mimics the pathology of neurodegenerative
diseases including Alzheimer's disease and new ways to investigate
and combat such conditions. The present invention meets these and
other needs.
BRIEF SUMMARY OF THE INVENTION
[0019] The present invention provides a model for Alzheimer's
disease and other neurodegenerative diseases. The model of the
invention provides brain cells, or brain tissue containing the
same, and a method for increasing or decreasing characteristics and
changes indicative of neurodegenerative diseases in such cells,
especially, the amount of neurofibrillary tangles and/or
phosphorylated tau and/or tau fragments and/or the production
and/or release of cytokines and/or microglia reactions and/or
activations and/or inflammation and/or conversion of p35 to p25
and/or the levels and activities of protein kinases and/or any
other characteristic or change indicative of neurodegenerative
diseases in such cells.
[0020] The model of the invention has identified new targets for
therapeutic intervention, and new classes of compounds for the
treatment of neurodegenerative diseases, and especially,
Alzheimer's disease. For example, the model of the invention has
identified the inhibition of tau proteolysis as a new target for
therapeutic intervention. As shown herein, cysteine protease
inhibitors, and specifically, calpain inhibitors, are capable of
inhibiting tau proteolysis and thus the formation of tau fragments.
Such inhibitors prevent the formation of neurofibrillary tangles
(the formation of which have been induced, according to the model
of the invention, by conditions that raise the amount and/or
activity of cathepsin D and/or conditions that lower the amount or
concentration of cholesterol in the brain tissue).
[0021] Accordingly, in one aspect, the invention provides a model
of neurodegenerative disease development, such model being a method
of increasing the amount of neurofibrillary tangles and/or
phosphorylated tau and/or tau fragments and/or the production
and/or release of cytokines and/or microglia reactions and/or
activations and/or inflammation and/or conversion of p35 to p25
and/or the levels and activities of protein kinases, in a suitable
brain cell(s), or brain tissue preparation containing the same, the
method comprising (1) inducing lysosomal dysfunction and
selectively increasing cathepsin D, or, selectively lowering
cholesterol, in the brain cell, to levels sufficient to effect the
desired changes and (2) culturing the brain cell of part (1) for a
period of time sufficient to effect such changes, such changes
including the amount of neurofibrillary tangles and/or
phosphorylated tau and/or tau fragments and/or the production
and/or release of cytokines and/or microglia reactions and/or
activations and/or inflammation and/or conversion of p35 to p25
and/or the levels and activities of protein kinases in such cell
relative to the levels found in control cells. In a further
embodiment, cathepsin D is selectively increased and also
cholesterol is selectively lowered in the brain cells.
[0022] In another aspect, the invention provides a method
comprising: (a) exposing brain cells, or brain tissue preparation
containing the same, to a condition, or contacting brain cells, or
brain tissue containing the same, with a compound that inhibits or
suppresses lysosomal function, increases cathepsin D, or decreases
cholesterol, to a level effective to induce characteristics or
indicia of a brain afflicted with a neurodegenerative disease in
the cells by the continued exposure thereto; and (b) maintaining
the cells for a period of time sufficient to induce such properties
or indicia, wherein such properties or indicia include the amount
of neurofibrillary tangles and/or phosphorylated tau and/or tau
fragments and/or the production and/or release of cytokines and/or
microglia reactions and/or activations and/or inflammation and/or
conversion of p35 to p25 and/or the levels and activities of
protein kinases. In a further embodiment, cathepsin D is
selectively increased and also cholesterol is selectively decreased
in the brain cells, or brain tissue containing the same.
[0023] In yet another aspect, the invention provides brain cells,
or brain tissue containing the same, that have been exposed to
conditions that inhibit or suppress lysosomal function, increase
cathepsin D, or, that selectively decrease cholesterol, to a level
effective to increase the amount of neurofibrillary tangles and/or
phosphorylated tau and/or tau fragments and/or the production
and/or release of cytokines and/or microglia reactions and/or
activations and/or inflammation and/or conversion of p35 to p25
and/or the levels and activities of protein kinases in such brain
cells, or brain tissue preparations containing the same, compared
to such levels in a control. In a further embodiment, the brain
cells, or brain tissue containing the same, have been prepared from
medium in which both cathepsin D is selectively increased and
cholesterol is selectively decreased.
[0024] In yet another aspect, the invention provides brain cells,
or brain tissue containing the same, that contain (in the media or
in the cell), or that have been treated with, a compound that
inhibits or suppresses lysosomal function, increases cathepsin D,
or that lowers cholesterol in such brain cells, or brain tissue
containing the same, to a level effective to increase the amount of
neurofibrillary tangles and/or phosphorylated tau and/or tau
fragments and/or the production and/or release of cytokines and/or
microglia reactions and/or activations and/or inflammation and/or
conversion of p35 to p25 and/or the levels and activities of
protein kinases in such brain cells, or brain tissue containing the
same, compared to such levels in a control. In a further
embodiment, both cathepsin D has been selectively increased and
cholesterol levels have been selectively decreased in the cells as
a result of such compound. In a preferred embodiment, such compound
or its precursor was exogenously administered.
[0025] In yet another aspect, the invention provides a screening
method comprising: (a) contacting brain cells, or brain tissue
containing the same, with a cathepsin D-increasing compound that
increases cathepsin D in the brain cells, or with an agent capable
of decreasing cholesterol, wherein the change in cathepsin D or
cholesterol is sufficient to increase the amount of neurofibrillary
tangles and/or phosphorylated tau and/or tau fragments and/or the
production and/or release of cytokines and/or microglia reactions
and/or activations and/or inflammation and/or conversion of p35 to
p25 and/or the levels and activities of protein kinases in the
brain cells, or brain tissue containing the same; (b) contacting
the brain cells with an agent; and (c) determining whether the
agent modulates the amount of neurofibrillary tangles and/or
phosphorylated tau and/or tau fragments and/or the production
and/or release of cytokines and/or microglia reactions and/or
activations and/or inflammation and/or conversion of p35 to p25
and/or the levels and activities of protein kinases in the brain
cells, as compared to brain cells that are not treated with the
agent. In a further embodiment, both cathepsin D is selectively
increased and cholesterol is selectively decreased in the brain
cells, or brain tissue containing the same, prior to contact with
such agent.
[0026] In yet another aspect, the invention provides a method of
decreasing neurofibrillary tangles, phosphorylated tau and/or tau
fragments, or of preventing the formation of the same, in any
suitable brain cell, or brain tissue containing the same, that
contains, or has been induced to form, such neurofibrillary
tangles, phosphorylated tau and/or tau fragments in such brain
cell, the method comprising (1) selectively inhibiting the activity
of cysteine proteases, and especially of calpain, in the brain cell
and (2) culturing the brain cell containing the selectively
inhibited protease from part (1) for a period of time sufficient to
reduce the amount of neurofibrillary tangles, phosphorylated tau
and/or tau fragments in such cell.
[0027] In yet another aspect, the invention provides a method
comprising (a) exposing the brain cells, or brain tissue containing
the same, to a condition, or contacting the brain cells, or brain
tissue containing the same, with a compound, that inhibits the
activity of cysteine proteases, or at least of a cysteine protease,
and especially calpain, to a level effective to result in a
reduction or lessening in the properties or indicia of a brain
afflicted with a neurodegenerative disease by the continued
exposure to, contact with, or incubation therein, and (b)
maintaining such exposure or contact or incubation for a period of
time sufficient to reduce such properties or indicia, wherein such
properties or indicia include increased amounts of neurofibrillary
tangles, phosphorylated tau and/or tau fragments.
[0028] In yet another aspect, the invention provides brain cells,
or brain tissue containing the same, that have been exposed to a
compound or conditions in which cysteine proteases, and especially
calpain, in such cells are selectively inhibited, and that lack, or
contain a lower amount of neurofibrillary tangles, phosphorylated
tau and/or tau fragments as a result of such inhibition.
[0029] In yet another aspect, the invention provides brain cells,
or brain tissue containing the same, that contain (in the media or
in the cell), or that have been treated with, a compound that
selectively inhibits cysteine proteases, and especially calpain, in
such cells, such brain cells, or brain tissue containing the same,
lacking, or containing, a lower amount of neurofibrillary tangles,
phosphorylated tau and/or tau fragments as a result of such
inhibition.
[0030] In yet another aspect, the invention provides a screening
method comprising: (a) contacting brain cells, or brain tissue
containing the same, with a compound that effectively inhibits the
activity of cysteine proteases, and especially calpain, in the
brain cells, or brain tissue containing the same, wherein the
inhibition of such cysteine proteases, and especially, the
inhibition of calpain, decreases, or prevents an increase in, the
amount of neurofibrillary tangles, phosphorylated tau and/or tau
fragments in the brain cells, or brain tissue containing the same;
(b) contacting the brain cells, or brain tissue containing the
same, with an further agent; and (c) determining whether the agent
of part (b) modulates the amount of neurofibrillary tangles,
phosphorylated tau and/or tau fragments in the brain cells, or
brain tissue containing the same, treated with the agent compared
to the brain cells, or brain tissue containing the same, that are
not treated with the agent.
[0031] In yet another aspect, the invention provides a method of
decreasing the amount of neurofibrillary tangles and/or
phosphorylated tau and/or tau fragments and/or the production
and/or release of cytokines and/or microglia reactions and/or
activations and/or inflammation and/or conversion of p35 to p25
and/or the levels and activities of protein kinases or of
preventing the formation of the same, in any suitable brain cell,
or brain tissue containing the same, that contains, or has been
induced to form such neurofibrillary tangles and/or phosphorylated
tau and/or tau fragments and/or the production and/or release of
cytokines and/or microglia reactions and/or activations and/or
inflammation and/or conversion of p35 to p25 and/or the levels and
activities of protein kinases in such brain cell, the method
comprising (1) selectively inhibiting the activity of a mitogen
activated kinase, and especially of MAP kinase, in the brain cell
and (2) culturing the brain cell containing the selectively
inhibited kinase from part (1) for a period of time sufficient to
reduce the amount of neurofibrillary tangles and/or phosphorylated
tau and/or tau fragments and/or the production and/or release of
cytokines and/or microglia reactions and/or activations and/or
inflammation and/or conversion of p35 to p25 and/or the levels and
activities of protein kinases in such cell.
[0032] In yet another aspect, the invention provides a method
comprising (a) exposing the brain cells, or brain tissue containing
the same, to a condition, or contacting the brain cells, or brain
tissue containing the same, with a compound, that inhibits the
activity of a mitogen activated kinase, and especially MAP kinase,
to a level effective to reduce the properties or indicia of a brain
afflicted with a neurodegenerative disease by the continued
exposure to, contact with, or incubation therein, and (b)
maintaining such exposure or contact or incubation for a period of
time sufficient to reduce such properties or indicia, wherein such
properties or indicia include one or more of neurofibrillary
tangles, phosphorylated tau, and/or tau fragments, the production
of cytokines, the release of cytokines, microglia reactions,
microglia activations, inflammation and/or conversion of p35 to p25
and/or the levels and activities of protein kinases.
[0033] In yet another aspect, the invention provides brain cells,
or brain tissue containing the same, that have been exposed to a
compound or conditions in which a mitogen activated kinase, and
especially MAP kinase, in such cells are selectively inhibited, and
that lack, or contain a lower amount of, neurofibrillary tangles
and/or phosphorylated tau and/or tau fragments and/or the
production and/or release of cytokines and/or microglia reactions
and/or activations and/or inflammation and/or conversion of p35 to
p25 and/or the levels and activities of protein kinases as a result
of such inhibition.
[0034] In yet another aspect, the invention provides brain cells,
or brain tissue containing the same, that contain (in the media or
in the cell), or that have been treated with, a compound that
selectively inhibits a mitogen activated kinase, and especially MAP
kinase, in such cells, such brain cells, or brain tissue containing
the same, lacking, or containing, a lower amount of neurofibrillary
tangles and/or phosphorylated tau and/or tau fragments and/or the
production and/or release of cytokines and/or microglia reactions
and/or activations and/or inflammation and/or conversion of p35 to
p25 and/or the levels and activities of protein kinases as a result
of such inhibition.
[0035] In yet another aspect, the invention provides a screening
method comprising: (a) contacting brain cells, or brain tissue
containing the same, with a compound that effectively inhibits the
activity of a mitogen activated kinase, and especially MAP kinase,
in the brain cells, or brain tissue containing the same, wherein
the inhibition of such a mitogen activated kinase, and especially,
the inhibition of MAP kinases, decreases, or prevents an increase
in, the amount of neurofibrillary tangles and/or phosphorylated tau
and/or tau fragments and/or the production and/or release of
cytokines and/or microglia reactions and/or activations and/or
inflammation and/or conversion of p35 to p25 and/or the levels and
activities of protein kinases in the brain cells, or brain tissue
containing the same; (b) contacting the brain cells, or brain
tissue containing the same, with an further agent; and (c)
determining whether the agent of part (b) modulates the amount of
neurofibrillary tangles and/or phosphorylated tau and/or tau
fragments and/or the production and/or release of cytokines and/or
microglia reactions and/or activations and/or inflammation and/or
conversion of p35 to p25 and/or the levels and activities of
protein kinases in the brain cells, or brain tissue containing the
same, treated with the agent compared to the brain cells, or brain
tissue containing the same, that are not treated with the
agent.
[0036] In preferred embodiments of the above models, methods and
brain cells, or brain tissue containing the same, "wild-type" brain
cells from rats or mice, or brain tissue containing the same,
apoE-deficient brain cells, or brain tissue containing the same, or
apoE4-containing brain cells, or brain tissue containing the same,
are used.
[0037] In yet another aspect, the invention provides a method for
the treatment or prevention of neurodegenerative diseases that are
characterized by tau proteolysis, an accumulation of tau fragments,
or paired helical filaments, or neurofibrillary tangles, such
method comprising the administration of an inhibitor of tau
proteolysis to a patient in need of such treatment or
prevention.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0038] FIGS. 1A-D illustrate morphology of subicular neurons
immunopositive for phosphorylated tau in cultured slices prepared
from apoE-knockout mice. The slices were treated with ZPAD for six
days followed by six-day washout. Panels A and B. Micrographs
showing the variety of routinely encountered structures 1. A
shrunken neuron with a dense, intracellular accumulation of
phosphorylated tau 2. Neurons with immunopositive processes that
appear distended (2a) or fragmented (2b, 2c) at varying distances
from the cell body. 3, Cells with fibril-filled processes that have
separated, or are about to separate, from the soma. 4 & 5,
Neuronal remnants in which the membrane and cytoplasm are lost but
labeled fibrils remain. Panels C and D. Higher magnification images
of cells in panel B. The extended and distorted appearance of the
terminal portion of the labeled process is evident for cell 2b. A
similar effect accompanied by kinking of the neuronal process can
be seen for cell 2a. A remnant neuron marked by heavy stained
fibrils is present in the lower right of the micrograph in panel
D.
[0039] FIGS. 2A and 2B illustrate induction of tangle-like
structures in subfield CA1/subiculum in mouse hippocampal cultures
by ZPAD-treatment. Hippocampal slice cultures incubated with ZPAD
(B) or vehicle (A) for 6 days were stained with monoclonal
antibody. "AT8," that recognizes hyperphosphorylated tau proteins
and neurofibrillary tangles in human tissue. Numerous
immunopositive neurons are present in ZPAD treated slices, while
few if any are found in control tissue (A).
[0040] FIGS. 3A and 3B illustrate ultrastructure of tangle-like
formations using electron microscopic immunogold techniques. FIG.
3A shows a dendritic branch with accumulated organelles resembled
smooth ER (arrows), rough ER (asterisks), or mitochondria (M).
distorted microtubules were found passing through the abnormal
inclusions. Despite these obvious pathologies, plasma membranes and
synaptic apparatus were still distinguishable. Secondary lysosomes
with variable sizes were also frequently encountered in ZPAD
treated tissues (FIG. 3B).
[0041] FIGS. 4A-C-illustrate immunogold analysis and shows that
AT8-ir was found mainly over structures composed of distorted
filaments located throughout dendrites and cell bodies. Enlarged
images showed that filaments were often paired and twisted with
axial periodicity (FIG. 4A, B). Distorted filaments were found
running across each other or waving around, characteristics similar
to early-stage neurofibrillary tangles in Alzheimer's disease (FIG.
4C).
[0042] FIG. 5(A and B). Levels of cathepsin D immunoreactivity in
apoE-deficient and wild-type (WT) mice. Hippocampal slices prepared
from C57BL/6J and C57BL/6J-apoEtm1Unc (apoE-deficient) mice at
postnatal day 10 and cultured for 12-14 days were incubated with
ZPAD or vehicle (Con) for 6 days. Immunoblots probed with
anti-cathepsin D antisera revealed three major bands with apparent
molecular weights of .about.55 kDa, .about.50 kDa, and .about.38
kDa in cultured hippocampal slices, corresponding to the inactive
proenzyme, the active single chain, and the active heavy chain,
respectively (A). ZPAD-treatment increased the first two isoforms
in wild-type tissue, and all three isoforms in the apoE-deficient
slices. Note also that the increase in cathepsin D proteins is
exaggerated in the knockout compared to the wild-type mice:
145+43%, 150+29% and 84+26% vs. 65+29%, 42+22% and 3.0+5.7% (B).
Standard paired t-tests (2-tails) were used for the indicated
statistical comparison.
[0043] FIG. 6. Induction of tangle-like structures in cultured
hippocampal slices prepared from apoE-knockout mice. Slices were
incubated with vehicle (left side) or `ZPAD`, an inhibitor of
cathepsins B and L (right side), for 6 days and then processed for
immunocytochemistry using a monoclonal antibody "AT8" that
recognizes hyperphosphorylated tau proteins, tau fragments, and
neurofibrillary tangles in human tissue. Immunopositive elements
are found in the outgrowth regions of the control slice from an
apoE -/- mouse but not within the hippocampus itself. In contrast,
the ZPAD-treated slice has numerous, densely labeled cells in the
stratum oriens of hippocampal field CA1 and in the subiculum. Note
that the densely packed neurons in the s. pyramidale of field CA3
and in the s. granulosum of the dentate gyrus are not stained
(4.times. objective; scale bar=200 .mu.m).
[0044] FIG. 7. Types and distribution of phosphorylated
tau-immunoreactive neurons in the CA1 region following six days of
ZPAD. Shown is a vertical section that extends across most of the
basal (s. oriens), and the inner third of the apical (s. radiatum),
dendritic fields in field CA1 of a cultured slice that had been
exposed to ZPAD for six days. The majority of the AT8
immunopositive cells were found in the basal dendritic field. The
cell bodies (s. pyramidale) and apical dendrites of the pyramidal
cells, by far the most numerous population of neurons in the
section, were with few exceptions, unlabeled. One of these
immuno-negative neurons is outlined with small circle. The stained
elements were not homogeneous. The cells marked with a "1" appear
to be intact neurons with immunopositive processes and dense
deposits accumulating within the cell body. The labeled neuron
marked as "2" had swollen and distorted dendrites. The elements
marked by a "3" appeared to be remnants of neurons. (25.times.
objective, scale bar=50 .mu.m).
[0045] FIG. 8. Morphology of neurons that are stained by an
antibody that recognizes neurofibrillary tangles. Upper panel.
Immunopositive neurons in cultured slices prepared from apoE -/-
mice. The micrographs are ordered according to a proposed sequence
of pathological steps. [A] Two neurons in the subiculum with
immunopositive cell bodies and primary dendritic branches (white
arrows). Note that other neurons in the field are unlabeled (black
arrows). [B] Neuron with a dense deposit (cap) in one pole of its
cell body. [C] Neuron with pathological swelling (arrow) of a
distal dendrite. [D, E] Cells with pathological dendritic
expansions proximal to the cell body. [F] Exploded process attached
to a dendrite containing fibrous material. Note that the dense
`cap` of immunopositive material covers most of the cell body. [G,
H] Dense caps that do not appear to be associated with somata;
i.e., are likely the remnants of neurons. (100.times. objective,
scale bar=12.5 .mu.m in A, 10 .mu.m in B, 8 .mu.m in C, 15 .mu.m in
D,H; 11 .mu.m in E,G; and 17 .mu.m in F). Lower panel.
Immunopositive neurons in the hippocampus from a human brain
classified as being in the early stages of Alzheimer's disease. The
micrographs are again arranged according to a proposed sequence of
pathologies. [A] Apparently intact pyramidal neuron with a dense
cap and a labeled apical dendrite. [B, C] Neurons with dendritic
swellings. [D, E]. Dendritic expansions proximal to the cell body.
[F, G] Immunopositive caps that do not appear to be attached to
intact neurons. (20 and 40.times. objectives; scale bar=50 .mu.m in
A; scale bar=45 .mu.m in B,D; 30 .mu.m in C, 18 .mu.m in E, 20
.mu.m in F, and 12.5 .mu.m in G).
[0046] FIG. 9. Electron micrographs of CA1 neurons from apoE -/-
slices that were incubated with ZPAD for six days. [A]. Survey
micrograph showing the primary dendrite emerging from the cell
body. Filamentous material (arrows) occupies more than half of the
cross-section of the dendrite. [B]. Higher power image showing the
filaments that occupy the pathological region marked in panel A.
[C]. Micrograph from another dendrite showing that the filaments
form bundles that crisscross each other (arrows). (scale bar=2
.mu.m in A, 0.75 .mu.m in B, 0.4 .mu.m in C).
[0047] FIG. 10. Tangle-like structures are increased in cultured
hippocampal slices by combined lysosomal dysfunction and
disturbance in lipid metabolism. Hippocampal slices were prepared
from 12 day old rat pups, cultured in vitro for 10 days, and
incubated with vehicle only (Cont), and/or a cholesterol metabolism
inhibitor mevastatin (Mev), and/or a cathepsin B and L inhibitor
(ZPAD) plus mevastatin (Mev/ZPAD). Cultured slices were stained
with anti-phosphorylated tau antibody AT8.
[0048] FIG. 11. High magnification micrographs of cultured
hippocampal slices that were treated with vehicle (Cont), ZPAD,
mevastatin (Mev), or mevastatin plus ZPAD (Mev/ZPAD).
[0049] FIG. 12. Generation of phosphorylated tau fragments by
mevastatin and ZPAD treatment. Hippocampal slices were prepared
from 12 day old rat pups, cultured in vitro for 10 days, and
incubated with vehicle only (Cont), and/or a cathepsin B and L
inhibitor (ZPAD), and/or a cholesterol metabolism inhibitor
mevastatin (Mev), and/or mevastatin plus ZPAD (Mev/ZPAD).
[0050] FIG. 13. Level of cdk5 regulatory unit p35 is reduced by
mevastatin treatment. Hippocampal slices cultured in vitro for 12
days were treated with ZPAD, mevastatin (Mev), mevastatin plus ZPAD
(Mev/ZPAD), or vehicle only for 6 days, and Western blots were
stained with anti-p35 antisera. Shown are analytical data from two
separate experiments.
[0051] FIGS. 14A and 14B illustrate the dose response and time
course of p35 following mevastatin(.diamond-solid.) or mevastatin
plus ZPAD (.box-solid.) treatment. For the dose curve experiments,
slices were subjected to mevastatin for 6 days at 0 .mu.M, 1 .mu.M,
5 .mu.M, 10 .mu.M, and 100 .mu.M concentrations. For the time
course experiment, hippocampal cultures were incubated with 10
.mu.M mevastatin for 0, 2, 4, and 6 days. In the mevastatin plus
ZPAD treatment, ZPAD was used at 20 .mu.M.
[0052] FIG. 15. Down regulation of p35 by mevastatin is blocked by
the application of mevalonate. Hippocampal slices were incubated
with vehicle alone/control (lane 1), mevastatin (lane 2),
mevastatin plus ZPAD (lane 3), mevastatin plus EA1 (lane 4),
mevastatin plus cholesterol (lane 5), or mevastatin plus mevalonate
(lane 6).
[0053] FIG. 16. Messenger RNA levels of TGF-beta and IL-10 are
increased by lysosomal dysfunction and interruption of cholesterol
synthesis. Messenger RNAs were extracted from cultured hippocampal
slices that had been incubated with vehicle (Cont), ZPAD (20
.mu.M), mevastatin (Mev, 20 .mu.M), or mevastatin plus ZPAD
respectively (each contained 12 slices) and measured by
RT-PCR/northern blot techniques using a kit from Ambion Inc. Shown
are representatives from three experiments. PD98 and PD98/ZPAD are
groups treated with PD98059 (a mitogen-activated protein kinase
inhibitor) or PD98059 plus ZPAD respectively.
[0054] FIG. 17. Messenger RNA levels of TNF-alpha are increased by
interruption of cholesterol synthesis. Messenger RNAs were
extracted from cultured hippocampal slices that had been incubated
with vehicle (Cont), ZPAD (20 .mu.M), PD98059 (50 .mu.M), PD98059
plus ZPAD, mevastatin (Mev, 20 .mu.M), or mevastatin plus ZPAD
respectively (each contained 12 slices) and measured by
RT-PCR/northern blot techniques using a kit from Ambion Inc.
[0055] FIG. 18. Activation of MAPK is involved in lysosomal
dysfunction induced microglial reaction. Brain tissue was cultured
for 12 days and treated with ZPAD (20 .mu.M) in the presence or
absence of PD98059 (50 .mu.M) for 6 days. Cultured explants were
then sliced and stained by using monoclonal antibody ED-1 which
recognizes reactive microglia, a classical marker of inflammation.
Note that incubation with ZPAD triggered significant reaction of
microglia, and this reaction was completely blocked by
co-application of PD98059. Inhibition of MAPK by itself did not
induce evident change in microglia.
[0056] FIG. 19. Inhibition of cholesterol synthesis causes
activation and transformation of microglia. Rat brain tissues were
cultured for 10 days and incubated with vehicle (Cont), ZPAD (20
.mu.M), mevastatin (Mev, 20 .mu.M), or mevastatin plus ZPAD
(Mev/ZPAD) for 6 days. Cultured brain explants were then sliced and
stained by using monoclonal antibody ED-1.
[0057] FIG. 20. MAPK (ERK1/2) activation by ZPAD and mevastatin
treatment. Hippocampal slices were cultured for 10 days and
incubated with vehicle (lane 1), ZPAD (lane 2), mevastatin (lane
3), PD98059 (lane 4), mevastatin plus ZPAD (lane 5), mevastatin
plus PD98059 (lane 6) and mevastatin plus ZPAD and PD98059 (lane 7)
for 6 days and processed for immunoblot with anti-active MAPK
(Sigma, 1:10,000).
[0058] FIGS. 21A and 21B. Dose response and time course of MAPK
following mevastatin treatment. Cultured hippocampal slices were
treated with mevastatin (.diamond-solid.) or mevastatin plus ZPAD
(.box-solid.). For the dose curve experiments, slices were
subjected to mevastatin for 6 days at 0 .mu.M, 1 .mu.M, 5 .mu.M, 10
.mu.M, and 100 .mu.M concentrations. For the time course
experiment, hippocampal cultures were incubated with 10 .mu.M
mevastatin for 0, 2, 4, and 6 days.
[0059] FIG. 22 illustrates that experimentally-induced lysosomal
dysfunction induced-the conversion of p35 to p25, and that such
conversion was blocked by calpain inhibitors. Hippocampal slices
prepared from rats at postnatal 10 day and cultured for 12-14 days
were incubated with ZPAD and/or vehicle (control) and/or a cysteine
protease inhibitor for 6 days. Immunoblotting carried out using
antisera that recognizes the C-terminal domain of p35 showed that
the CDK5 binding protein p35 was present in cultured hippocampal
slices. Trace amount of p25, the truncated form of p35 that lacks
the N-terminal domain, was also detected. A six day treatment of
the brain cells, or brain tissue containing the same, with ZPAD
resulted in a significant decrease in the amount of p35 polypeptide
and a paralleled increase in the truncated form p25. Such
conversions of p35 to p25 were significantly inhibited in the
presence of calpain inhibitor I.
[0060] FIG. 23 illustrates that tau fragmentation events triggered
by experimentally induced lysosomal dysfunction were blocked by
calpain inhibitors. Immunoblots stained with the
anti-non-phosphorylated antibody (tau 1), revealed that 6-day ZPAD
treatment induced a cleavage of native tau proteins and the
generation of tau fragments that migrated at approximately 40 kDa
and 29 kDa (tau 29). Previous studies have shown that cathepsin D
is a protease whose activation leads to the cleavage of tau and the
generation of tau 29. Incubation of cathepsin D inhibitors
remarkably reduced the production of tau 29 induced by ZPAD
treatment, but the cathepsin D inhibitors failed to block the
increase in the 40 kDa fragments. Such results suggested that
another protease may be activated by the ZPAD treatment. Previous
study had suggested that calpain was able to cleave tau and
generate tau fragments of different length. To test whether calpain
is involved in ZPAD-induced tau cleavage, levels of tau
fragmentation were compared between slices incubated with and
without calpain inhibitors. Results obtained from 16 slices of 2
separated experiments showed that ZPAD-induced tau 29 and tau 40
were almost completely blocked by calpain inhibitor I.
[0061] FIG. 24 illustrates that the induction of tangle-like
structures by ZPAD-treatment was blocked by calpain inhibitors.
Incubation of hippocampal slices with ZPAD for 6-day induced
numerous tangles, in particular, in the border of subiculum and CA1
region. However, when ZPAD was applied in the presence of calpain
inhibitor I, the number of tangles was significantly reduced.
[0062] FIG. 25 illustrates that the induction of tangle-like
structures by ZPAD treatment was blocked by mitogen activate kinase
inhibitors. Incubation of hippocampal slices with ZPAD for 6 days
induced numerous tangles, in particular, in the border of subiculum
and CA1 region. However, when ZPAD was applied in the presence of a
mitogen activate kinase inhibitor, the number of tangles was
significantly reduced.
[0063] FIG. 26. Modulation of biological processing of amyloid
precursor protein by mevastatin treatment is blocked by mevalonate.
Hippocampal slices were incubated with vehicle alone/control (lane
1), mevastatin (lane 2), mevastatin plus ZPAD (lane 3), mevastatin
plus EA1 (lane 4), mevastatin plus cholesterol (lane 5), or
mevastatin plus mevalonate (lane 6).
[0064] FIG. 27. Effects of mevastatin on APP were partially blocked
by MAPKK inhibitor PD98059 but not by inhibitor SB203580 of MAPK
p38. Hippocampal slices were incubated with vehicle alone/control
(lane 1), mevastatin (lane 2), mevastatin plus ZPAD (lane 3),
mevastatin plus PD98059 (lanes 4 and 5), mevastatin plus EA1 (lanes
6 and 7), mevastatin plus cholesterol (lane 8), mevastatin plus
mevalonate (lanes 9 and 12), mevastatin plus SB203580 (lane 10), or
mevastatin plus .gamma.-secretase inhibitor (lane 11).
[0065] FIG. 28 shows the activation of caspase 3 by lysosomal
dysfunction. Hippocampal slices were cultured for 12 days and
incubated with vehicle alone (CONT), ZPAD, or chloroquine (CQN; a
lysosomal inhibitor) for 6 days. Cultures were then homogenized,
and subjected to an ELISA assay to detect the activity of caspase
3, an apoptotic protease. ZPAD treatment caused a marked increase
in the activity of caspase 3.
[0066] FIG. 29. Induction of tangle-like structures by pravastatin
treatment. Shown are images taken form pravastain-treated
hippocampal slices from the subiculum (A), CA1 field (B), and CA3
field (C). Also shown are higher magnification micrographs of
neurons from the CA1 field (D and E).
[0067] FIG. 30. Induction of microglial reactions by mevastatin and
simvastatin treatments. Shown are images of hippocampal areas from
one control animal and an animal treated with simvastatin. CD11b
immunostaining is moderate in control tissue, while it is generally
dense in simvastation treated hippocampus. Higher magnification
images show that the density of microglia is higher in simvasatin
treated tissue than that in the control tissue.
DEFINITIONS
[0068] As used herein, the following terms have the meanings
ascribed to them unless specified otherwise.
[0069] The term "activation" when used to refer to microglial may
refer to a transformation of the microglial, for example, from a
silent/quiet (slim cell body with ramified thin process) state to
an active/macrophage-like (rounded cell body without process)
state. Additionally, the term may refer to an enhanced ability to
express and secrete cytokines.
[0070] "Alzheimer's disease" specifically refers to a condition
associated with: 1) the formation of neuritic plaques comprising
amyloid beta protein and/or neurofibrillary tangles comprising tau
proteins (primarily located in the hippocampus and cerebral cortex)
and, 2) an impairment in both cognitive and non-cognitive
functions, for example, impairment in learning and memory, emotion,
and coordination. "Alzheimer's disease" as used herein includes all
kinds of Alzheimer's disease, including, e.g., early onset family
type Alzheimer's disease and late onset sporadic Alzheimer's
disease.
[0071] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group,
an amino group, and an R group. Examples of amino acid analogs
include homoserine, norleucine, methionine sulfoxide, methionine
methyl sulfonium. Such analogs have modified R groups (e.g.,
norleucine) or modified peptide backbones, but retain the same
basic chemical structure as a naturally occurring amino acid. Amino
acid mimetics refers to chemical compounds that have a structure
that is different from the general chemical structure of an amino
acid, but that functions in a manner similar to a naturally
occurring amino acid. Amino acids may be referred to herein by
either their commonly known three letter symbols or by the
one-letter symbols recommended by the IUPAC-IUB Biochemical
Nomenclature Commission. Nucleotides, likewise, may be referred to
by their commonly accepted single-letter codes (A, T, G, C, U,
etc.).
[0072] "Antibody" refers to a polypeptide substantially encoded by
an immunoglobulin gene or immunoglobulin genes, or fragments
thereof, which specifically binds and recognizes an epitope (e.g.,
an antigen). The recognized immunoglobulin genes include the kappa
and lambda light chain constant region genes, the alpha, gamma,
delta, epsilon and mu heavy chain constant region genes, and the
myriad immunoglobulin variable region genes. Antibodies exist,
e.g., as intact immunoglobulins or as a number of well
characterized fragments produced by digestion with various
peptidases. This includes, e.g., Fab' and F(ab)'2 fragments. The
term "antibody," as used herein, also includes antibody fragments
either produced by the modification of whole antibodies or those
synthesized de novo using recombinant DNA methodologies. It also
includes polyclonal antibodies, monoclonal antibodies, chimeric
antibodies, humanized antibodies, or single chain antibodies. "Fc"
portion of an antibody refers to that portion of an immunoglobulin
heavy chain that comprises one or more heavy chain constant region
domains, CH.sub.1, CH.sub.2 and CH.sub.3, but does not include the
heavy chain variable region. Antibodies that specifically bind to
neurofibrillary tangles, phosphorylated tau and/or tau fragments
can be prepared using any suitable methods known in the art. See,
e.g., Coligan, Current Protocols in Immunology (1991); Harlow &
Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice
(2d ed. 1986); and Kohler & Milstein, Nature 256:495497 (1975).
Such techniques include antibody preparation by selection of
antibodies from libraries of recombinant antibodies in phage or
similar vectors, as well as preparation of polyclonal and
monoclonal antibodies by immunizing rabbits or mice (see, e.g.,
Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature
341:544-546 (1989)). Specific polyclonal antisera and monoclonal
antibodies will usually bind with a K.sub.d of at least about 0.1
mM, more usually at least about 1 .mu.M, preferably at least about
0.1 .mu.M or better, and most preferably, 0.01 .mu.M or better.
[0073] An "anti-phosphorylated tau protein (or anti-phosphorylated
tau fragment) antibody" is an antibody or antibody fragment that
specifically binds a phosphorylated form of tau protein or its
fragment (and not to the unphosphorylated form). In particular
anti-phosphorylated fragment antibodies recognize tau fragments
that have a molecular weight of about 15-35 kDa, for example, 25-30
kDa. Preferably, antibody that specifically binds to a fragment of
tau, such as, for example, tau 29, and especially preferably to a
tau fragment having a molecular weight of about 33 kDa is used in
embodiments of the invention.
[0074] An "anti-neurofibrillary tangle antibody" is an antibody or
antibody fragment that specifically binds to any component of the
neurofibrillary tangle, e.g., phosphorylated tau and/or tau
fragment.
[0075] The terms "Apolipoprotein E" and "apoE" refer to a protein
that is about 299 amino acids in length and has a molecular weight
of about 34,000 Daltons, and plays a major role in lipid transport
and metabolism. Specifically, apoE functions as a cholesterol
transport protein within the periphery. ApoE is produced in
abundance in brain and apoE-containing lipoproteins are the
principal lipoproteins in the Cerebro-Spinal Fluid (CSF). In the
periphery, apoE expression is dramatically up-regulated in response
to peripheral nerve injury. A similar role for apoE in the central
nervous system (CNS) has been described whereby apoE distributes
cholesterol and phospholipids to neurons after injury. In normal
rodent brain apoE is primarily localized to glial cells, whereas in
normal human brain apoE has been demonstrated in glia and neurons.
After brain injury, intraneuronal apoE is markedly increased in
both rodent and human brain. ApoE acts as a ligand for receptors on
neurons. The terms "apolipoprotein E" and "apoE" are generically
used to refer to either apolipoprotein E protein or gene, and also
the terms can refer to any homologs from rat, mouse, rabbit, guinea
pig, etc., and their variants.
[0076] In humans, three common isoforms of apoE (i.e., apoE2,
apoE3, and apoE4) are encoded by the different alleles 2, 3, and 4.
The three different apoE isoforms differ only by a single amino
acid: apoE2 (cys112, cys158), apoE3 (cys112, arg158) and apoE4
(arg112, arg158). In vitro studies indicate that the three apoE
isoforms have differences. Especially, there is a difference in the
ability of apoE3 and apoE4 to stimulate neurite outgrowth, bind to
amyloid protein, bind to cytoskeletal proteins such as tau and
microtubule associated proteins and protect against oxidative
stress. In general the apoE4 isoform has a detrimental effect when
compared to the apoE3 isoform. For example, in vitro experiments
showed that apoE and apoE3 were able to bind to microtubules and
form stable complexes with the microtubule associated proteins tau
and MAP2c while apoE4 was lacking this ability (Strimmatter et al.,
Exp. Neurol. 125:163-171 (1994)). Current evidence has also
identified the apoE4 allele as a major risk factor for sporadic and
familial late-onset Alzheimer's disease as well as poor clinical
outcome after certain forms of brain injury including that due to
head trauma and spontaneous intracerebral hemorrhage. By contrast,
possession of an apoE2 has been shown to protect against, or delay
the onset of, Alzheimer's disease.
[0077] The terms "apolipoprotein E4" or "apoE4" refer to
apolipoprotein E4 or polymorphic variants, alleles, interspecies
homologs, or conservatively modified variants thereof. The terms
"apolipoprotein E4" and "apoE4" are generically used to refer to
either apolipoprotein E4 protein or gene, as appropriate to the
context. Preferably, apoE4 is from a mammal, e.g., rat, mouse,
human, rabbit, guinea pig, etc., and their variants. The nucleotide
and amino acid sequences of apoE4 is well-known in the art. For
example, the human apoE4 gene is known and has the Genbank
accession number of M10065.
[0078] "Apolipoprotein E4 containing brain cells, or brain tissue
containing the same," or "apoE4-containing brain cells, or brain
tissue containing the same," refer to brain cells, or brain tissue
containing the same, that can express apolipoprotein E4 proteins
and/or contain the apoE4 gene, as will be determined from the
context. Typically, apoE4-containing brain cells, or brain tissue
containing the same, are derived from a transgenic animal that
comprises an exogenous apoE gene, e.g., a human apoE4 gene,
polymorphic variants, alleles, interspecies homologs, or
conservatively modified thereof, which encode an apoE4 protein. The
methods for producing these transgenic animals are well-known in
the art and described in, e.g., U.S. Pat. No. 6,046,381.
[0079] "Brain cells" refers to cells and/or tissue containing the
same. Brain cells can be derived from any brain. For example, for
use in the methods of the invention, brain cells, or brain tissue
containing the same, can be those in or from a normal animal, an
apoE-deficient animal, or an apoE4-containing animal. Preferably,
brain cells, or brain tissue containing the same, are derived from
a mammal, such as a rat, mouse, guinea pig, rabbit, etc. or
transgenic animals with modulated levels of neurofibrillary
tangles, and/or tau proteins, and/or amyloid, and/or amyloid
precursor proteins, and/or Cathepsin D levels, and/or cysteine
protease levels, and/or mitogen activated kinases, and/or lysosomal
enzyme levels, and/or cholesterol levels and/or altered cholesterol
metabolism, synthesis, storage, etc. The pathology modeling and
drug testing brain cell embodiments of the invention can be carried
out in animal models in vivo or in vitro. When provided in an
embodiment in which the cells are cultured in vitro, unless
otherwise indicated, the brain cells, or brain tissue containing
the same, can be provided in any in vitro form capable of culture,
for example, brain tissue that contains cells, or brain sections
such as slices that contain cells, dissociated cells, cells bound
to a solid support or in suspension, etc.
[0080] "BLAST" and "BLAST 2.0" are programs that are used, with the
parameters described herein, to determine percent sequence identity
for the nucleic acids and proteins of the invention. Software for
performing BLAST analyses is publicly available through the
National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always>0) and N
(penalty score for mismatching residues; always<0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) of 10, M=5, N=4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10,
M=5, N=4, and a comparison of both strands. The BLAST algorithm
also performs a statistical analysis of the similarity between two
sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad.
Sci. USA 90:5873-5787 (1993)). One measure of similarity provided
by the BLAST algorithm is the smallest sum probability (P(N)),
which provides an indication of the probability by which a match
between two nucleotide or amino acid sequences would occur by
chance. For example, a nucleic acid is considered similar to a
reference sequence if the smallest sum probability in a comparison
of the test nucleic acid to the reference nucleic acid is less than
about 0.2, more preferably less than about 0.01, and most
preferably less than about 0.001.
[0081] Calpain is a cysteine protease found in brain cells. There
are two major isoforms of calpain in the brain, .mu.-calpain (also
known as calpain I) and m-calpain (also known as calpain II). The
two calpains differ in their calcium requirements but have similar
substrate specificities. Calpain activity can be assayed by
following the cleavage of .alpha.-spectrin as described in WO
00/21550.
[0082] Cathepsin D is a lysosomal protease which is found in the
brain, along with other lysosomal proteases, such as cathepsin B
and cathepsin L. The activities of these proteases change in the
brain with aging. For example, the activity of cathepsin L
decreases by up to 90% during brain aging, while the levels and
activity of cathepsin D increase. See Nakanishi et al., Exp.
Neurol. 126:119-128 (1994). Moreover, the activities of these
cathepsin proteases are inter-related. For example, it was
previously reported that inhibition of cathepsin B and L increases
procathepsin D and its maturation into the active two-chain form
(composed of heavy and light chain) within lysosomes. See Bednarski
& Lynch, Neuroreport 9:2089-2094 (1998); Hoffman et al.,
Neurosci. Lett. 250:75-78 (1998). "Cathepsin D" typically exists in
three forms: the inactive proenzyme having an apparent molecular
weight of about 55 kDa; the active single chain having an apparent
molecular weight of about 50 kDa; and the active double chain form
that consists of a heavy chain having an apparent molecular weight
of about 38 kDa and a light chain of about 14 kDa.
[0083] A "cholesterol-lowering agent" is a compound or other
substance that, at effective levels, depresses the levels of
cholesterol in the brain cells of the invention. The agent may
inhibit the activity or amount of HMG-CoA reductase (an enzyme
involved in cholesterol synthesis in cells) and/or other entities
involved in cholesterol synthesis, degradation, storage, and/or
transport.
[0084] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Specifically,
degenerate codon substitutions may be achieved by generating
sequences in which the third position of one or more selected (or
all) codons is substituted with mixed-base and/or deoxyinosine
residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka
et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol.
Cell. Probes 8:91-98 (1994)). Because of the degeneracy of the
genetic code, a large number of functionally identical nucleic
acids encode any given protein. For instance, the codons GCA, GCC,
GCG and GCU all encode the amino acid alanine. Thus, at every
position where an alanine is specified by a codon in an amino acid
herein, the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide of interest is implicit in each
described sequence. As to amino acid sequences, one of skill will
recognize that individual substitutions, deletions or additions to
a nucleic acid, peptide, polypeptide, or protein sequence which
alters, adds or deletes a single amino acid or a small percentage
of amino acids in the encoded sequence is a "conservatively
modified variant" where the alteration results in the substitution
of an amino acid with a chemically similar amino acid. Conservative
substitution tables providing functionally similar amino acids are
well known in the art. Such conservatively modified variants are in
addition to and do not exclude polymorphic variants and alleles of
the invention.
[0085] The following groups each contain amino acids that are
conservative substitutions for one another:
[0086] 1) Alanine (A), Glycine (G);
[0087] 2) Serine (S), Threonine (T);
[0088] 3) Aspartic acid (D), Glutamic acid (E);
[0089] 4) Asparagine (N), Glutamine (Q);
[0090] 5) Cysteine (C), Methionine (M);
[0091] 6) Arginine (R), Lysine (K), Histidine (H);
[0092] 7) Isoleucine (I), Leucine (L), Valine (V); and
[0093] 8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (see,
e.g., Creighton, Proteins (1984) for a discussion of amino acid
properties).
[0094] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous amino acid or
nucleotide positions selected from the group consisting of from 20
to 600, usually about 50 to about 200, more usually about 100 to
about 150 in which a sequence may be compared to a reference
sequence of the same number of contiguous positions after the two
sequences are optimally aligned. Methods of alignment of sequences
for comparison are well-known in the art. Optimal alignment of
sequences for comparison can be conducted, e.g., by the local
homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482
(1981), by the homology alignment algorithm of Needleman &
Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity
method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444
(1988), by computerized implementations of these algorithms (GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.),
or by manual alignment and visual inspection (see, e.g., Current
Protocols in Molecular Biology (Ausubel et al., eds. 1995
supplement)). A preferred example of algorithm that is suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J.
Mol. Biol. 215:403-410 (1990), respectively.
[0095] The term "control" refers the non-treated condition or
substance. For example, when examining the effect of a compound on
its ability to increase cathepsin D in brain cells, "control" brain
cells could be brain cells that have not been treated with that
compound, or brain cells assayed at the beginning of the experiment
(time=zero) before any compound-induced changes thereto, as will be
clear from the context. In another example, as will be clear from
the context, in some embodiments directed to apoE-deficient brain
cells or apoE4-containing brain cells, the term "control" brain
cells can also refer to normal brain cells (comprising a wild-type
or endogenous apolipoprotein E gene) which have been treated with a
compound that increases an effective concentration of cathepsin D
in the brain cells.
[0096] The term "deficient" refers to a decreased or lower amount
of the indicated substance. For example, apolipoprotein E
"deficient" brain cells, or apoE "deficient" brain cells refer to
brain cells that contain less endogenous apolipoprotein E as
compared to brain cells having wild-type apolipoprotein E genes
(for example, normal brain cells) measured or cultured under
similar conditions. The term deficient may also refer to a variant
that has an altered function, for example, brain cells that are
"deficient" in apoE may contain a variant of apoE that has an
altered function, e.g., in lipid transport, as compared to
wild-type apoE--such altered function not being able to substitute
for the unaltered function.
[0097] By neuronal "degeneration" is meant that one or more
characteristics as described herein as being indicative of a
decline of brain functioning have appeared, are present or
accumulated over time in the brain cells, especially changes in
neuronal tau protein levels or structure (tau phosphorylation, tau
proteolysis, tau fragments, etc) as compared to such
characteristics in normal neurons.
[0098] "Disorder" and "disease" refer to any disorder, disease,
condition, syndrome or combination of manifestations or symptoms
recognized or diagnosed as a disorder. If modified by reference to
a particular disease or by reference to one or more or a set of
manifestations or symptoms, that usage of "disorder" or "disease"
refers to any such disorder, disease, condition, syndrome or
combination of such manifestations or symptoms recognized or
diagnosed as a such disorder.
[0099] The term "effective," as in an "effective concentration of
cathepsin D" or an "effective concentration of cholesterol" refers
to either an amount or an activity of the indicated substance or
condition that is sufficient to achieve the indicated purpose. For
a first example, an effective concentration of cathepsin D to
induce neurofibrillary tangles and/or tau fragmentation, etc.
refers to an amount of cathepsin D or a level or enzymatic activity
of cathepsin D that is sufficient to increase the level of
neurofibrillary tangles, and/or tau fragmentation within a desired
period of time. In a second example, decreasing an effective
concentration of cholesterol to induce neurofibrillary tangles
and/or tau fragments refers to an amount of cholesterol or a level
or activity of agents which synthesize, store, and or transport
cholesterol that is sufficient to increase the level of
neurofibrillary tangles and/or tau fragments within a desired
period of time.
[0100] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., 70% identity, preferably 75%, 80%, 85%, 90%, or 95%
identity or higher over a specified region), when compared and
aligned for maximum correspondence over a comparison window, or
designated region as measured using one of the following sequence
comparison algorithms or by manual alignment and visual inspection.
Such sequences are then said to be "substantially identical." This
definition also refers to the compliment of a test sequence.
Preferably, the identity exists over a region that is at least
about 25 amino acids or nucleotides in length, or more preferably
over a region that is 50-100 amino acids or nucleotides in length.
In most preferred embodiments, the sequences are substantially
identical over the entire length of, e.g., the coding region. For
sequence comparison, typically one sequence acts as a reference
sequence, to which test sequences are compared. When using a
sequence comparison algorithm, test and reference sequences are
entered into a computer, subsequent coordinates are designated, if
necessary, and sequence algorithm program parameters are
designated. Default program parameters can be used, or alternative
parameters can be designated. The sequence comparison algorithm
then calculates the percent sequence identities for the test
sequences relative to the reference sequence, based on the program
parameters.
[0101] The term "immunoassay" is an assay that uses an antibody to
specifically bind an antigen. The immunoassay is characterized by
the use of specific binding properties of a particular antibody to
isolate, target, and/or quantify the antigen."
[0102] By "inducing" a characteristic in a brain cell is meant that
the characteristic appears in the cell, or levels (or the enzymatic
activity) of such characteristic are increased in the cell, after
treatment with the desired agent. By "enhancing" a characteristic
in a brain cell is meant that the levels (or the enzymatic
activity) of the indicated characteristic are increased in the cell
after treatment with the desired agent.
[0103] By "lysosomal function" is meant any activity, enzymatic or
non-enzymatic, that is a property of the lysosomes, including
vesicle trafficking to or from lysosomes, the endocytic pathway,
heterophagy or autophagy, and including the expression and activity
of enzymes that are localized in the lysosomes. By "inhibiting or
suppressing a lysosomal function" is meant lowering or decreasing
one or more such activities from the level or amount of such
activity found in the non-inhibited or non-suppressed state,
including inhibiting or suppressing vesicle trafficking to or from
lysosomes, and including inhibiting or suppressing the expression
or activity of a lysosomal enzyme. Such inhibition or suppression
can be acute or chronic. Examples of lysosomal enzymes that can be
inhibited or suppressed include a lysosomal acid hydrolase,
lysosomal protease, lysosomal nuclease, lysosomal lipase, amylase
and a cathepsin. Cathepsin B, cathepsin H or cathepsin L can be
assayed using methods known in the art, for example, as described
by Barrett, A. J. et al., Meth. Enzymol. 80:535 (1981), Academic
Press, New York, incorporated herein by reference.
[0104] "Lysosomal dysfunction" means an abnormal lysosomal
morphology, chemistry or activity, which is detrimental to
lysosomes or cells. Examples of lysosomal dysfunctions include a
detrimental change, either increased or decreased, in the normal
activity of the endocytic pathway, a detrimental change in
lysosomal morphology, a detrimental change in the intra-lysosomal
pH, and/or the activity(ies) of lysosomal enzyme(s).
[0105] "Neurodegenerative diseases" includes almost all disease in
central nervous system accompanied by neuronal degeneration
including, for example, age-related neurodegenerative diseases,
Alzheimer's disease, frontotemporal dementias, frontotemporal
dementia and Parkinsonism, Huntingon's Disease, ischemia, Pick's
disease, Progressive supranuclear palsy pathology, Parkinson's
Disease, senile dementia, stroke, etc.
[0106] "Neurofibrillary tangles" refer to intraneuronal
accumulations of filamentous material in the form of loops, coils
or tangled masses. Neurofibrillary tangles seen in brain cells are
sometimes referred to herein as "tangle-like structures."
[0107] "Nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form. The term encompasses nucleic acids containing
known nucleotide analogs or modified backbone residues or linkages,
which are synthetic, naturally occurring, and non-naturally
occurring, which have similar binding properties as the reference
nucleic acid, and which are metabolized in a manner similar to the
reference nucleotides. Examples of such analogs include, without
limitation, phosphorothioates, phosphoramidates, methyl
phosphonates, chiral-methyl phosphonates, 2-O-methyl
ribonucleotides, peptide-nucleic acids (PNAs). Unless otherwise
indicated, a particular nucleic acid sequence also implicitly
encompasses conservatively modified variants thereof (e.g.,
degenerate codon substitutions) and complementary sequences, as
well as the sequence explicitly indicated.
[0108] The term "pharmaceutically effective amount" refers to an
amount sufficient to alleviate, in any degree or manner, one or
more of the manifestations or symptoms recognized or diagnosed as
associated with the modifying disorder, the modifying
manifestations, or the modifying symptom.
[0109] The term "phosphorylated tau" includes all forms of tau that
have been phosphorylated, including hyperphosphorylated tau and
"abnormally" phosphorylated tau. Hyperphosphorylated tau is
phosphorylated at both Ser/Thr-Pro and non-Ser/Thr-Pro as compared
to tau in normal tau. In general phosphorylated tau is rare in
mature brain tissues, although there are phosphorylated forms in
developing immature tissues. Thus, phosphorylation of tau in mature
tissue by itself is already abnormal, and such forms of tau are
also referred to as "hyperphosphorylated" tau or as "abnormally"
phosphorylated tau. Moreover, some sites are typically only found
phosphorylated in the phosphorylated form in tau that is in
neurofibrillary tangles, such as Ser 202 (as exemplified herein
below as a marker), Ser 396 and Ser 404. Therefore, tau proteins
phosphorylated at multiple sites, in particular at those sites
found in human neurofibrillary tangles, are also included in the
term hyperphosphorylated tau, or abnormally phosphorylated tau.
[0110] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residues is an analog or mimetic of a corresponding
naturally occurring amino acid, as well as to naturally occurring
amino acid polymers. Polypeptides can be modified, e.g., by the
addition of carbohydrate residues to form glycoproteins. The terms
"polypeptide," "peptide" and "protein" include glycoproteins, as
well as non-glycoprotein.
[0111] The terms "reduces," "reduced," or "reducing," when used to
refer to one or more symptomologies of a disease, refers to any
observable or measurable lessening of that characteristic when the
method or composition of the present invention is compared to prior
art methods or compositions.
[0112] The term "reaction" when used to refer to microglial may
refer to a transformation of the microglial, for example, from a
silent/quiet (slim cell body with ramified thin process) state to
an active/macrophage-like (rounded cell body without process)
state. Additionally, the term may refer to an enhanced ability to
express and secrete cytokines.
[0113] The phrase "selectively (or specifically) hybridizes to"
refers to the binding, duplexing, or hybridizing of a molecule only
to a particular nucleotide sequence under stringent hybridization
conditions when that sequence is present in a complex mixture
(e.g., total cellular or library DNA or RNA).
[0114] The term "selectively" increased or "selectively" decreased
means that the activity or amount of the substance that is being
"selectively" increased or decreased is increased or decreased,
respectively, relative to the activity or amount of such substance
prior to an indicated treatment or relative to that of a control,
or other substance (if named).
[0115] The phrases "specifically binds to" or "specifically
immunoreactive with," when referring to a binding moiety refers to
a binding reaction which is determinative of the presence of a
target antigen in the presence of a heterogeneous population of
proteins and other biologics. Binding moeities include any material
capable of resolving the presence of tau proteins and/or
neurofibrillary tangles, such as antibody, dyes, silver, other
contrast agents etc. Thus, under designated assay conditions, the
specified binding moieties bind preferentially to a particular
target antigen and do not bind in a significant amount to other
components present in a test sample. Specific binding to a target
antigen under such conditions may require a binding moiety that is
selected for its specificity for a particular target antigen. A
variety of immunoassay formats may be used to select antibodies
that are specifically immunoreactive with a particular protein. For
example, solid-phase ELISA immunoassays are routinely used to
select monoclonal antibodies that are specifically immunoreactive
with an antigen. See Harlow and Lane (1988) Antibodies, A
Laboratory Manual, Cold Spring Harbor Publications, New York, for a
description of immunoassay formats and conditions that can be used
to determine specific immunoreactivity. Typically, a specific or
selective reaction will be at least twice background signal or
noise and more typically more than 10 to 100 times background.
Specific binding between an antibody or other binding agent and an
antigen preferably has a binding affinity of at least 10.sup.6
M.sup.-1. Preferred binding agents bind with affinities of at least
about 10.sup.7M.sup.-1, and preferably 10.sup.8 M.sup.-1 to
10.sup.9 M.sup.-1 or 10.sup.10 M.sup.-1.
[0116] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acid, but to
no other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions will be those in which the salt concentration
is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M
sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short probes (e.g.,
10 to 50 nucleotides) and at least about 60.degree. C. for long
probes (e.g., greater than 50 nucleotides). Stringent conditions
may also be achieved with the addition of destabilizing agents such
as formamide. For selective or specific hybridization, a positive
signal is at least two times background, preferably 10 times
background hybridization. Exemplary stringent hybridization
conditions can be as follows: 50% formamide, 5.times.SSC, and 1%
SDS, incubating at 42.degree. C., or, 5.times.SSC, 1% SDS,
incubating at 65.degree. C., with wash in 0.2.times.SSC, and 0.1%
SDS at 65.degree. C. Nucleic acids that do not hybridize to each
other under stringent conditions are still substantially identical
if the polypeptides which they encode are substantially identical.
This occurs, for example, when a copy of a nucleic acid is created
using the maximum codon degeneracy permitted by the genetic code.
In such cases, the nucleic acids typically hybridize under
moderately stringent hybridization conditions. Exemplary
"moderately stringent hybridization conditions" include a
hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at
37.degree. C., and a wash in 1.times.SSC at 45.degree. C. A
positive hybridization is at least twice background. Those of
ordinary skill will readily recognize that alternative
hybridization and wash conditions can be utilized to provide
conditions of similar stringency.
[0117] Two nucleic acid sequences that encode polypeptides are
considered to be "substantially related" if the polypeptide encoded
by the first nucleic acid is immunologically cross reactive with
polyclonal antibodies raised against the polypeptide encoded by the
second nucleic acid. Two nucleic acid sequences that encode
polypeptides are considered to be "substantially identical" if
nucleic acid encoding the first sequence hybridizes to the
complement of nucleic acid that encodes the other molecule under
stringent conditions, as described below. Yet another indication
that two nucleic acid sequences are substantially identical is that
the same primers can be used to amplify the sequences. Generally,
two polypeptides are "substantially identical" if they share an
amino acid sequence identity of at least 85% or differ in sequence
only by conservative substitutions.
[0118] "Transgenic animal" refers to a non-human animal that
comprises an exogenous nucleic acid sequence present as an
extrachromosomal element or stably integrated in all or a portion
of its cells, especially in germ cells.
Detailed Description of the Invention
[0119] I. Characteristics of the Neurodegenerative Disease Brain
Cells of the Invention, or Brain Tissue Containing the Same
[0120] The present invention provides a novel method for triggering
brain cells, or brain tissue containing the same, to induce the
characteristics of a brain cell or tissue from a brain that is
afflicted with a neurodegenerative disease. The present invention
also provides novel methods for inhibiting or preventing the
development of such characteristics of such neurodegenerative
disease in the brain cells.
[0121] The model of the present invention is based on, in part, the
discovery that experimental lysosomal dysfunction, decreases in
cholesterol concentration, and especially a combination of both,
rapidly induce the formation of one or more brain cell
characteristics that are indicative of a decline of neuron
functioning and are associated with, and especially in combination,
definitive for, neurodegenerative diseases and especially
age-related neurodegenerative diseases such as Alzheimer's disease.
According to the invention, exposing a brain cell to conditions
that increase the concentration of cathepsin D ("cathepsin
D-increasing compound"), or exposing a brain cell to conditions
that decrease the concentration of cholesterol ("cholesterol
decreasing compound"), or both, surprisingly trigger the
hyperphosphorylation of the protein tau and tau fragments, the
production of neurofibrillary tangles, the production of tau
fragments, increases in cytokine activity and levels, microglia
activation, and induction of brain inflammatory reactions and other
indicia or characteristics of neurodegenerative diseases. Such
effects are even more surprisingly enhanced when apoE-deficient or
apoE4-containing brain cells, or brain tissue containing the same,
are use in the model. Such exposure can come from altering the
environmental conditions to which the brain cell is exposed, or,
preferably by contacting or treating brain cells, or brain tissue
containing the same, with a compound capable of inducing such
effective levels of cathepsin D and/or of decreasing the
concentration of cholesterol
[0122] Brain cells, or tissue containing the same, maintained under
conditions in which cathepsin D is selectively induced, and/or
cholesterol is selectively decreased, are characterized by the de
novo appearance and/or accumulation of, one or more characteristics
of neurodegenerative diseases in the cells. The accumulation of
such characteristics is relative to the levels present in the cells
at the start of the treatment or exposure to the indicated
condition, and/or relative to the levels present in similar cells
not contained with, or otherwise maintained in the absence of, the
cathepsin D-increasing compound and/or compounds capable of
selectively decreasing cholesterol. Such characteristics
include:
[0123] (1) the formation of neurofibrillary tangles,
[0124] (2) the hyperphosphorylation of tau,
[0125] (3) the fragmentation of tau, that is, tau proteolysis and
especially, increased amounts of the 15-35 kDa forms of tau ("tau
fragments"),
[0126] (4) increased production and/or release of brain-produced
pro-inflammatory cytokines especially TGF-beta (tumor growth factor
beta or TGF1), TGF-alpha, IL1 (interleukin-1), IL1-alpha
(interleukin-1alpha), IL1-beta (interleukin-1beta), IL6
(interleukin-6), IL10 (interleukin-10), TNF (tumor necrosis
factor), TNF-alpha (tumor necrosis factor alpha) and LPS
(lipopolysaccharide), and most especially TGF-beta, IL-1beta and
LPS,
[0127] (5) increased microglia reaction and/or activation,
[0128] (6) increased indications of brain inflammatory reactions,
such as, for example, increased positive staining for HLA-DR (which
detects reactive microglia); increased positive staining for glial
fibrillary acidic protein (GFAP; to detect reactive astrocytes);
the extracellular accumulation of complement proteins, complement
inhibitors, acute phase reactants, growth factors, heat shock
proteins, proteoglycans, lipoproteins, cathepsins, cystatins,
coagulation factors, proteases, protease inhibitors, integrin
adhesion molecules, etc., and
[0129] (7) increased conversion of p35 to p25
[0130] (8) changes in the levels and activities of protein kinases,
for example, cyclin dependent protein kinase 5 (cdk5) and mitogen
activated protein kinase (MAPK).
[0131] Additional characteristics useful as an indicator of a brain
afflicted neurodegenerative diseases can include, e.g., an
increased amount of lysosomes, the appearance of basophilic
granules in the mossy fiber terminal zone, the presence of
secondary lysosomes with lipofuscin, amyloid deposition, amyloid
plaques, neuritic plaques, synaptic loss, neuritic degeneration,
neuronal death, increased glial elements (astrocytes, microglia),
fragmentation of the amyloid precursor protein, increases in the
levels of Cathepsin D, etc.
[0132] The above characteristics, and others describe herein, are
indicative of a decline of neuron functioning. Such decline may be
the result of a direct effect of the characteristic or an indirect
effect. Characteristics that are the result of a direct effect are
those found in the neurons, for example, an increase in tau
phosphorylation or tau proteolysis and fragmentation.
Characteristics that are the result of an indirect effect are those
that are found in brain cells other than neurons, for example,
induction of glial activation.
[0133] The first, and most preferable characteristic of brain
cells, or brain tissue containing the same, cultured under
conditions that selectively increase cathepsin D, and/or that
selectively decreases levels of cholesterol, according to the
method of the invention is the formation of neurofibrillary tangles
in the cells. The formation of neurofibrillary tangles refers to
the appearance and/or accumulation of intraneuronal deposits that
are composed mainly of paired helical filaments. Generally, such
tangles can be seen with light microscopy. The method of the
invention is especially characterized by its ability to induce the
appearance of "early tangles" in the brain cells, or brain tissue
containing the same. "Early tangles" refer to intraneuronal tangles
typically found at an early stage of neurodegenerative disease,
such as Alzheimer's disease. Under appropriate conditions as
described herein, such "early tangles" are typically formed, or
enhanced levels are detectable, within a few days of culture or
treatment. Such early tangles may appear in the brain cells in any
of day 1, 2, 3, 4, 5 or 6, or even beyond, after initiation of the
appropriate condition in culture or after administration of the
appropriate agent(s) in vivo. Preferably, such early tangles appear
2-6 days in culture embodiments. However, longer periods are
acceptable, especially in the in vivo models, because in vivo
models are not constrained by the same viability considerations as
in vitro models. Morphologically, such tangles mimic early-stage
tangles (i.e., intracellular tangles) found in the brain of
Alzheimer's patients.
[0134] As described above, it was previously shown that
neurofibrillary tangles may contribute to the cognitive decline
associated with neurodegenerative diseases and may also trigger
neuronal cell death in the brain. However, many currently available
in vivo and in vitro models of neurodegenerative diseases lack this
or other key features associated with brain cells from patients
inflicted with these conditions. Thus, the present invention
advantageously provides a model brain cell system, wherein the
brain cells contain, or can be induced to contain, among other
things, neurofibrillary tangles. The appearance and/or
disappearance of such tangles, as a result of the presence or
absence of various therapeutic candidates or culture conditions,
can be monitored and used to assess the value of therapeutic
candidates that might be useful for the treatment of such
conditions or diseases.
[0135] A further characteristic of brain cells maintained according
to the method of the invention so as to induce the formation of
indicia of neurodegenerative disease is the accumulation of levels
(amounts or concentrations) of phosphorylated tau that are greater
than levels found in control cells or in the same cells at the
beginning of the culture. Phosphorylated tau, and especially
abnormally phosphorylated and/or hyperphosphorylated tau, can be
assayed in the cells as a whole, or in subfractions, for example,
in soluble and/or insoluble fractions thereof, including paired
helical filaments.
[0136] A further characteristic of brain cells incubated according
to the method of the invention so as to induce the formation of
neurodegenerative disease indicia is the production or accumulation
of greater amounts of proteolytic fragments of tau, and
specifically, tau fragments having a molecular weight of 15-35 kDa
fragments (tau fragments), when compared to control cells or to the
same cells at the beginning of the culture, especially fragments
having an apparent molecular weight of 33 or 29 kDa. The 29 kDa tau
fragment results from cleavage at amino acids 200-257. Larger
fragments may also be seen. For example, the appearance or
accumulation of a fragment with an apparent molecular weight of 40
kDa is an indicia of neurodegenerative disease. Such proteolytic
products of tau can be unphosphorylated and/or phosphorylated and
can include hyperphosphorylated forms. As above, tau fragments can
be measured in the cells as a whole, or in soluble and/or insoluble
fractions thereof, including paired helical filaments.
[0137] A further characteristic of brain cells incubated according
to the method of the invention so as to induce the formation of
neurodegenerative disease indicia is the increased production
and/or release and/or accumulation of brain-produced cytokines
especially, TGFb (tumor growth factor-beta, or TGF-beta), IL-1b
(interleukin 1 beta) and LPS (lipopolysaccharide), when compared to
control cells or to the same cells at the beginning of the culture.
As above, cytokines and LPS can be measured in the cells as a
whole, or in soluble and/or insoluble fractions thereof, including
the medium.
[0138] A further characteristic of brain cells incubated according
to the method of the invention so as to induce the formation of
neurodegenerative disease indicia is increased microglia reaction
and/or activation. Microglial reaction and/or activation refers to
the fact that when injury or disease affect nerve cells, microglia
in the central nervous system become "active," causing inflammation
in the brain, similar to the manner in which white blood cells act
in the rest of the body. Microglia act like the monocyte phagocytic
system. Microglia can be activated by numerous materials including
complement proteins and beta amyloid protein. Activated microglia
generate large quantities of superoxied anions, with hydroxyl
radicals, singlet oxygen species and hydrogen peroxide being a
downstream product, any of which can be assayed in the preparations
utilized in such methods of the invention. Such microglial
activation may be used with other indicia in the model of the
invention in the embodiments in which brain architecture is
retained to some degree, for example, when a brain slice is
employed, or when brain cells are in vivo.
[0139] Reactive microglia and astrocytes are characterized by their
cell bodies becoming larger, their processes becoming thicker, by
an increase in the GFAP and ED-1 staining, by a proliferation and
clustering of microglia and astrocytes, by infiltration of
peripheral inflammatory cells, for example, white blood cells, and
by formation of gliosis, etc., as compared to that found in the
non-reactive state.
[0140] A further characteristic of brain cells incubated according
to the method of the invention so as to induce the formation of
neurodegenerative disease indicia is the appearance of increased
indications of brain inflammatory reactions. Increased indications
of brain inflammatory reactions can include indications such as,
for example, increased positive staining for HLA-DR (which detects
reactive microglia); increased positive staining for glial
fibrillary acidic protein (GFAP; to detect reactive astrocytes);
the extracellular accumulation of complement proteins, complement
inhibitors, acute phase reactants, growth factors, heat shock
proteins, proteoglycans, lipoproteins, cathepsins, cystatins,
coagulation factors, proteases, protease inhibitors, integrin
adhesion molecules, etc. Such indicia of brain inflammatory
reactions may be used with other indicia in the model of the
invention in the embodiments in which brain architecture is
retained to some degree, for example, when a brain slice is
employed, or when brain cells are in vivo.
[0141] A further characteristic of brain cells incubated according
to the method of the invention so as to induce the formation of
neurodegenerative disease indicia is a change in the levels and
activities of protein kinases, for example, cyclin dependent
protein kinase 5 (cdk5) and mitogen activated protein kinase
(MAPK).
[0142] To be useful in the methods of the invention, it is not
necessary that all of the brain cells in a sample or preparation
exhibit at least one of the above characteristics. Rather, such
preparations are useful even if only some of the brain cells
contained therein exhibit such characteristics. Preparations of
brain cells in accordance with embodiments of the invention
preferably contain at least some cells that contain neurofibrillary
tangles, but not all the cells in the preparation need to exhibit
such tangles. In a preferred embodiment, such changes are found in
the neurons that are in the brain cell preparations. In another
preferred embodiment such changes are found in brain cells in
vivo.
[0143] Not all the characteristics need to be induced by the same
agent or at the same time or to the same degree in preparations
intended to induce brain cells to exhibit the characteristics of
neurodegenerative diseases. Preferably, upon treatment with the
agent that induces lysosomal dysfunction so as to increase
cathepsin D, or with the agent that decreases an effective
concentration of cholesterol, the brain cell's biochemistry,
physiology or morphology is changed to include at least one or more
of:
[0144] (1) the formation of neurofibrillary tangles,
[0145] (2) the hyperphosphorylation of tau, and/or
[0146] (3) the fragmentation of tau, that is, tau proteolysis and
especially, increased amounts of the 15-35 kDa forms of tau.
[0147] The rest of the characteristics:
[0148] (4) increased production and/or release of brain-produced
pro-inflammatory cytokines especially TGF-beta, TGF-alpha, IL1,
IL1-alpha, IL1-beta, IL6, IL10, TNF, TNF-alpha and LPS and most
especially TGF-beta, IL-1beta and LPS,
[0149] (5) increased microglia reaction and/or activation,
[0150] (6) increased indications of brain inflammatory
reactions,
[0151] (7) increased conversion of p35 to p25
[0152] (8) changes in the levels and activities of protein kinases,
for example, cyclin dependent protein kinase 5 (cdk5) or mitogen
activated protein kinase (MAPK), are preferably not relied on
solely but, if assayed, are assayed along with any of
[0153] the formation of neurofibrillary tangles,
[0154] the hyperphosphorylation of tau, and/or
[0155] the fragmentation of tau, that is, tau proteolysis and
especially, increased amounts of the 15-35 kDa forms of tau,
[0156] as indicators of the appearance or disappearance of
characteristics of neurodegenerative disease in the methods and
cultures of the invention.
[0157] Pro-inflammatory cytokines including IL1-alpha, IL1-beta,
IL6, and IL10, TNF, TFN-alpha and TGF (alpha or beta), and
especially TGFbeta1 (also referred to as TGF1), and TNF-alpha, are
useful as indicators of glial activation. Levels of these
cytokines, including levels of their mRNAs, can be quantitated, for
example, by RT-PCR, to assay for glial activation and lysosomal
dysfunction. Assays for such factors are known in the art.
[0158] Activation of the MAP kinase pathways can be monitored as an
indication of glial activation or as an indication of an increased
brain inflammatory condition. The pathways can be summarized as
follows. There are two IL1/TNF-activated kinase cascades, one of
which involves the p38 homologues of MAP kinase and the other of
which involves the p54 homologues of MAP kinase. IL1, TNF,
TGF1beta, etc. activate both pathways. The activation of kinases or
phosphatases of either or of both of these cascades can be assayed
as an indicator of glial activation and/or the induction of a brain
inflammatory reaction.
[0159] The activity of any of a variety of kinases can be assayed
as indicators of glial activation or brain inflammatory reactions.
Such kinases include, for example, GCK1 (Germinal center kinase),
PAK kinase (for example, as identified in Manser, E., Leung, T.,
Salihuddin, H., Zhao, Z. S. and Lim, L. (1994) A brain
serine/threonine protein kinase activated by Cdc42 and Rac1. Nature
367,40-46), MLK3 (mixed lineage kinase-3, also known as SPRK or
PTK1), MLK1, MLK2, MLK4, DLK (also known as Muk), SEK1, SEK2, SAPK
(stress activates protein kinases alpha/beta/gamma), MKK3, MKK6,
p38 MAPK (alpha/beta/gamma/delta), MAPKAPK2, elk1, c-jun, ATF-2 and
hsp27 (heat shock protein-25, also known as hsp25 and hsp28).
Increased activity of the end targets of such cascades, for
example, an increased activity of transcription factors
CHOP/GADD153 and MEF2C, can also be monitored as an indication of
an induced inflammatory state of the brain cells. Conversely,
decreased phosphorylation, or a decreased activity, is indicative
of a lesser inflamed, or non-inflamed state of the brain cells. In
a preferred embodiment, the activation or inactivation of cyclin
dependent protein kinase 5 (cdk5) and mitogen activated protein
kinase (MAPK) are assayed as an indication of an inflammatory or
non-inflammatory state of the brain cells.
[0160] Thus, using the instant invention, agents that enhance or
retard the formation of one or more of the characteristics of
neurodegenerative disorders can be identified, including:
neurofibrillary tangles, tau proteolytic fragments (and especially
the formation of the 15-35 kDa forms of tau), or agents that
enhance or retard the formation of tau hyperphosphorylation, and
increased production and/or release of brain-produced cytokines
especially TGF-beta, IL1-beta, TNF-alpha and LPS, an increased
microglia reaction and/or activation, increased indications of
brain inflammatory reactions, and changes, especially, increases in
the levels and activities of protein kinases, for example, cyclin
dependent protein kinase 5 (cdk5) and mitogen activated protein
kinase (MAPK).
[0161] The above characteristics are also often seen in "brain
aging" and are also useful as models thereof. Age-related
neurodegenerative diseases and neurodegenerative diseases are
characterized by many of the same properties including, e.g., an
increased amount of neurofibrillary tangles and/or lysosomes, the
appearance of basophilic granules in the mossy fiber terminal zone,
the presence of secondary lysosomes with lipofuscin, amyloid
deposition, amyloid plaques, neuritic plaques, synaptic loss,
neuritic degeneration, neuronal death, increased glial elements
(astrocytes, microglia), the fragmentation of the amyloid precursor
protein, increased levels of Cathepsin D, inflammation, etc. Brain
aging refers to a condition in which brain cells mimic the
biochemistry or physiology of brain cells taken from a mature or
elderly animal, especially human. Brain aging is manifested by
significant changes in lysosomal functions and chemistry, e.g., the
proliferation of secondary lysosomes filled with lipofuscin,
decreases in cathepsin L activities and increases in the levels of
cathepsin D. Additional characteristic features of human brain
aging, as is found in neurodegenerative diseases, and especially
Alzheimer's disease, include depletion of synaptic proteins,
meganeurite formation, induction of early-stage tangles, and
accompanying tau proteolysis.
[0162] II. Sources of Brain Cells
[0163] Any suitable source of brain cells can be used in
embodiments of the invention. Typically, brain cells are derived
from a mammal, such as a mouse, rat, guinea pig, rabbit, etc.
Primary cell cultures, including human primary cell cultures, can
also be used in the methods of the invention. Brain cells can be
derived from a normal animal (e.g., comprising a wild-type apoE
gene in the chromosome) or other suitable animals. For example,
apoE-deficient brain cells or apoE4-containing brain cells can be
used in embodiments of the invention. A preferred embodiment
includes in vivo brain cells.
[0164] Among many types of brain cells suitable for embodiments of
the invention, brain cells cultured from apoE-deficient brain cells
or from apoE4-containing brain cells are especially preferred
because they produce neurofibrillary tangles at significantly
enhanced levels when compared to brain cells from control (normal)
brains. The relatively high density with which such tangles form in
apoE-deficient brain cells, or in apoE4-containing brain cells was
not achievable by treatment with only a cathepsin D-increasing
compound in normal brain cells even with a prolonged treatment with
the same cathepsin D-increasing compound. However, a high density
in normal brain cells was achievable by treating the cells under
conditions in which cholesterol synthesis was inhibited or
cholesterol levels were lowered.
[0165] The cultured brain cells, in particular apoE-deficient brain
cells, even without the treatment with a cathepsin D-increasing
compound, have some residual amount of neurofibrillary tangles.
However, the initial density of neurofibrillary tangles in these
untreated brain cells is too low to be regarded as an adequate
model for neurodegenerative diseases, such as Alzheimer's
disease.
[0166] If brain cells with a higher density of neurofibrillary
tangles are desired, apoE-deficient brain cells or apoE4-containing
brain cells can be preferably used in embodiments of the invention.
Alternatively, such brain cells, and even normal brain cells, can
be exposed to the cholesterol limiting embodiments of the invention
as described below.
[0167] Preferably, when apoE-deficient brain cells are used, they
are derived from a transgenic animal. For example, apoE-deficient
brain cells useful in the method of the invention can be derived
from a transgenic animal that carries an altered or ablated and/or
expresses an altered endogenous apolipoprotein E gene (one or both
alleles) that results in undetectable or significantly less amounts
of apolipoprotein E proteins. These transgenic animals are
sometimes referred to as apoE "knockout" animals. "Knock-out"
transgenics can be transgenic animals having a heterozygous
knock-out of the apoE gene or a homozygous knock-out of the apoE
gene. For example, for use in the methods of the invention, the
function and/or expression of the apoE protein in the apoE
"knockout" animal is typically less than about 30%, preferably less
than about 10%, more preferably less than about 5%, still more
preferably less than about 1%, compared to a normal animal with the
wild-type apoE genes. Most preferably, apoE-deficient brain cells
are derived from apoE-knockout animals that have no apoE (i.e.,
null) gene expression.
[0168] Typically, apoE4-containing brain cells can be derived from
a transgenic animal that comprises an exogenous apoE gene, e.g., a
human apoE4 gene, polymorphic variants, interspecies homologs, or
other conservatively modified variants thereof. Preferably, in
these transgenic animals that comprise an exogenous apoE4 gene,
their endogenous apoE gene is completely or partly knocked out.
[0169] Transgenic animals comprising apoE-deficient brain cells can
be produced by recombinant methods known in the art. For example,
the endogenous apoE gene function can be altered or ablated by,
e.g., the deletion of all or part of the coding sequence, or
insertion of a sequence, or substitution of a stop codon. In
another example, the non-coding sequence of the apoE gene in the
chromosome can be modified by, e.g., deleting the promoter region,
the 3' regulatory sequences, enhancers and/or other regulatory
sequences of the apoE gene in the chromosome. In yet another
example, apoE-deficient transgenic animals can be produced by
introducing an anti-sense construct that blocks the expression of
the endogenous apoE gene products. In some cases, it may be
desirable to produce conditional "knock-out" transgenic animals,
wherein the alteration in the apoE gene can be induced by, e.g.,
exposure of the animal to a substance that promotes the apoE gene
alteration postnatally. Preferably, both alleles of the apoE gene
in the chromosome are altered in these transgenic animals.
[0170] The methods for producing transgenic animals are well known
and described in, e.g., U.S. Pat. Nos. 5,464,764, and 5,627,059,
the disclosures of which are incorporated herein by reference. In
particular, the following references describe methods for producing
apoE-deficient homozygous rodents: Plump et al., Cell 71:343-353
(1992); and Gordon et al., Neuroscience Letters 199:1-4 (1995), the
disclosures of which are incorporated herein by reference.
Moreover, some apoE-deficient transgenic animals are commercially
available. For example, apoE-deficient homozygous mice, such as
C57BL/6J-Apoetm1Unc strain, are available from the Jackson
laboratory, Bar Harbor, Me.
[0171] Moreover, apoE4-containing brain cells can be derived from a
transgenic animal that comprises an exogenous apoE gene. For
example, an exogenous apoE gene can be a human apoE4 gene, its
interspecies homologs, polymorphic variants, or conservatively
modified variants thereof. In human, three isoforms (apoE2, apoE3
and apoE4) express variants of apoE. Among these isoforms, apoE4 is
known in the art to encode an apoE protein that is deficient in
various functions. For example, compared to apoE3 that stimulates
neurite extension, apoE4 was shown to inhibit neurite extension.
Nathan et al., Sco. Neurosci. 20(Part 2):1033 (1994). It has also
been suggested that, in vitro, tau interacts with apoE3, but not
with apoE4. Stritmatter et al., Exp. Neurol. 125:163-171 (1994).
Moreover, the human apoE4 isoform has been described as a risk
factor of Alzheimer's disease (see, e.g., Peterson et al., JAMA
273:1274-1278 (1995)). Since brain cells comprising an apoE4 gene
appear to lack many normal functions that other apoE isoforms
possess, like the apoE-deficient brain cells, transgenic animals
that comprise an apoE4 gene or its variants may also be used as a
source of brain cells in embodiments of the invention.
[0172] Such transgenic animals can be produced using various apoE
nucleotide sequences known in the art or conservatively modified
variants thereof. For example, the human apoE4 gene has the Genbank
accession number M10065. The mouse apoE gene has the Genbank
accession number D00466. Other homologs or polymorphic variants of
apoE genes can also be readily identified. For example, homologs or
polymorphic variants of a known apoE gene can be isolated using
nucleic acid probes by screening libraries under stringent
hybridization conditions. Exemplary stringent hybridization
conditions are as follows: a hybridization in a buffer containing
50% formamide, 5.times.SSC, and 1% SDS, at 42.degree. C., or
5.times.SSC, 1% SDS, at 65.degree. C., with wash in 0.2.times.SSC,
and 0.1% SDS at 65.degree. C. In some cases, moderately stringent
conditions may be used to clone homologs or polymorphic variants of
a known apoE gene. An example of a moderately stringent condition
includes a hybridization in a buffer of 40% formamide, 1 M NaCl, 1%
SDS at 37.degree. C., and a wash in 1.times.SSC at 45.degree. C.
The source of homologs can be any species, e.g., rodents, primates,
bovines, canines, human, etc.
[0173] Preferably, the exogenous apoE gene is operably linked with
a mammalian apoE promoter, such as human apoE4 regulatory
sequences. This construct can be introduced into an animal using
methods known in the art. In these transgenic animals comprising an
exogenous apoE gene (e.g., human apoE4 gene), preferably the
endogenous apoE gene is partially or completely knocked out so that
the endogenous apoE expression or function is insubstantial.
Moreover, methods for producing transgenic animals comprising
various human apoE isoforms are described in, e.g., U.S. Pat. No.
6,046,381 and U.S. Pat. No. 5,767,337, the disclosure of which are
herein incorporated by reference.
[0174] Preferably, apoE4-containing brain cells are also derived
from transgenic animals that are genetically modified. As above,
for use in the methods of the invention, the function and/or
expression of the apoE4 protein in the apoE4 animal is typically
less than about 30%, preferably less than about 10%, more
preferably less than about 5%, still more preferably less than
about 1%, compared to a normal animal with the wild-type apoE
genes.
[0175] In some embodiments, it may be desirable to use modified or
mutated versions of apoE genes. For example, a modified version of
a human apoE4 gene, when introduced into a transgenic animal, may
be capable of producing a higher density of neurofibrillary tangles
compared to the unmodified human apoE4 gene. Techniques for in
vitro mutagenesis of cloned genes are well-known in the art and can
be readily applied for making a modified or mutated apoE gene. See,
e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, CSH
Press (1989). The functional effect of a modified or mutated apoE
gene can be further tested in vivo or in vitro. For example, a
transgenic animal comprising a modified or mutated apoE gene can be
produced using the methods known in the art. The change in the
properties in apoE brain cells (e.g., the neurofibrillary tangle or
phosphorylated tau fragment production) can be determined using the
methods described below.
[0176] III. Brain Cell Preparations
[0177] Brain cells and preparations containing the same can be
prepared and processed in any suitable manner. For example, the
brain can be processed in the form of tissue sections, such as
brain slices. Alternatively, the brain tissues can be processed in
the form of dissociated cells. Whether in the form of brain slices,
dissociated cells, or other forms, they will be generically
referred to as "brain cells" herein, unless otherwise
indicated.
[0178] In one embodiment, an in vivo model is used. Such in vivo
models have an advantage in that they retain the native brain
architecture and environment. The effects that are brought about by
the methods of the invention are presented against the background
of a physiological environment that is more likely to mimic such
conditions in humans. In vivo models are also more amenable to long
term analysis than are primary cultures, or brain slice cultures.
Another advantage is that multiple samples can be taken at the same
time from the same animal and from different parts of the
brain.
[0179] Preferably, the brain is processed in the form of brain
slices so that neuronal circuitry or other biological functions are
maintained, but environmental (culture) conditions can be closely
monitored and normalized. A suitable thickness of the brain slice
is readily determinable by those of skill in the art, and may be
varied depending on the culture condition or subsequent analysis
methods. For example, the brain can be sliced in the thickness of
about 200 .mu.m to about 800 .mu.m, preferably about 350 .mu.m to
about 400 .mu.m. The entire brain or portions of the brain can be
processed into slices. For example, suitable brain slices may
include a hippocampal slice, an entorhinal cortex slice, an
entorhinohippocampal slice, a neocortex slice, a hypothalamic
slice, or a cortex slice. Since neurofibrillary tangles tend to
develop more prominently in the hippocampal region, a hippocampal
slice is preferably used.
[0180] Alternatively, the brain can be processed into dissociated
brain cells. The entire brain or selected regions of the brain
(e.g., the hippocampal region) can be dissociated and maintained in
a culture. Generally, the brain tissue is dissected, minced and
digested in an enzyme (e.g., trypsin) for a suitable period of
time. Then cells are centrifuged and plated at a low density in
culture plates, and cultured. The methods for dissociating cells
are well-known in the art. See, e.g., Freshney, Culture of Animal
Cells a Manual of Basic Technique, 3rd ed., Wiley-Liss, New York
(1994), incorporated herein by reference.
[0181] Brain cells in the form of slices or dissociated cells can
be maintained in a culture. Suitable culture conditions for brain
cells are well-known in the art. For example, brain cells can be
placed onto culture plates, preferably on a support, such as a
matrix or membrane, which allows cells to attach. Any suitable
medium can be used in maintaining the culture of brain cells.
Typically, the culture of brain cells is maintained in a medium
that has all the essential nutrients. The culture medium generally
has a neutral pH, e.g., between about pH 7.2 to about 7.8, and is
maintained at a temperature between about 4.degree. C. to about
40.degree. C., typically at about 37.degree. C. The culture of
brain cells is typically maintained in an atmosphere that contains
CO.sub.2, preferably at 5% CO.sub.2. In general, the culture can be
maintained for at least about 60 days with a periodic replacement
of culture medium.
[0182] IV. Assays for Neurofibrillary Tangles, Phosphorylated Tau
and Tau Fragments
[0183] Determination of the neurofibrillary tangles, phosphorylated
tau and/or tau fragments production can be qualitative or
quantitative. In some applications, it may be sufficient to
visually inspect the production of neurofibrillary tangles,
phosphorylated tau and/or tau fragments. For example, it may be
useful to visually observe the timing and the pattern of
neurofibrillary tangle development at different regions of the
brain. In other applications, it may be desirable to quantitate the
neurofibrillary tangles, phosphorylated tau and/or tau fragments
production. Quantitation would be particularly useful in a
screening assay for agents that modulate the production of
neurofibrillary tangles, phosphorylated tau and/or tau
fragments.
[0184] Any suitable methods known in the art can be used to
determine the production of neurofibrillary tangles, phosphorylated
tau and/or tau fragments. For example, brain cells, in the form of
brain slices, brain sections, dissociated cells, or other suitable
forms, can be stained using conventional staining methods. For
example, the brain cells can be fixed and stained with a silver
stain (Bielschowsky) stain or toluidine blue. Then the stained
neurofibrillary tangles can be visualized by microscopy.
[0185] To assess the appearance and density of tangles, light
microscopic evaluation of tangles and tangle formation can be
performed by scanning at 40.times. objective magnification.
Immunoreactive elements can be plotted and images digitized and
stored for any desired area. Especially, the following areas are
preferentially examined: (1) the entorhinal cortical layers II/III;
(2) CA1 str. pyramidale; (3) CA1 str. oriens; (4) subiculum str.
oriens. Typically, five to seven serial sections are collected per
brain slice. The bottom section (i.e., that on the Millipore filter
side of the explant in the examples below) is generally neuron-poor
and therefore not evaluated. Analysis preferably focuses on the
dense paired helical filament (PHF) tau-immunoreactive elements
that are greater than or equal to 2 .mu.m in diameter. Comparisons
from aligned serial sections can be used to identify structures
that are present in more than one section so that individual
elements are not double-counted. With this correction, the densely
stained structures can be quantified and the result expressed per
unit area for each field of analysis. In addition, the types of
paired helical filament tau-positive structures can be catalogued
as shown in FIG. 8. Differences in treatment regimens that affect
the qualities as well as the number of tangles can thus be
determined.
[0186] An absolute value of the density of tangles in the models of
the invention is not required. Rather, a relative increase in the
density of tangles, as compared to the density found in similar
preparations but from wild-type or in controls, is indicative of
the appearance of neurodegenerative disease changes in the cells of
the method of the invention. In a preferred embodiment, the number
of tangles per unit of space is 20-30% higher in the cells of the
invention that have been exposed to a cathepsin D-increasing
compound and/or a compound that decreases an effective
concentration of cholesterol than it is in normal or control cells.
In a preferred embodiment, such density is greater than 30% and may
be even 100% or more higher than the wild-type or control cells (as
such wild-type or control cells may lack tangles completely).
[0187] Morphologically, cells that contain an increased cathepsin D
generally have lysosomes that are round in shape and that are
distributed homogenously in the cell body. Changes in the shape,
size and numbers of lysosomes and changes in the localization of
enzyme activity from lysosomal localization to cytoplasmic
localization can also be used as indexes by which to assess the
degree of neurodegenerative disease characteristics that have been
induced in the cells according to the invention.
[0188] In another example, the brain cells can be stained by
immunostaining, and the neurofibrillary tangles, phosphorylated tau
and/or tau fragments production can be visualized. In
immunostaining, suitable capture reagents, such as antibodies that
specifically bind to neurofibrillary tangles, phosphorylated tau
and/or tau fragments, can be used. Preferably, antibodies
preferentially bind to neurofibrillary tangles, phosphorylated tau
and/or tau fragments and do not significantly cross-react with
other proteins in the brain cells. For example, the antibodies that
specifically bind to phosphorylated tau proteins and/or tau protein
fragments have less than 50%, preferably less than 30%, more
preferably less than 10% crossreactivity with native tau proteins
that are not phosphorylated.
[0189] Examples of mouse monoclonal antibodies that preferentially
bind phosphorylated tau and/or tau protein fragments over the tau
found in normal adult brain include antibodies 8D8, RT97, 121.5,
BF10 (Miller et al., EMBO J. 5:269-276 (1986)); AT8 (Bierat et al.,
EMBO J. 11:1593-1597 (1992)); SMI31, SMI34, SMI310 (Sternberger et
al., Proc. Natl. Acad. Sci. USA 82:4274-4276 (1985); Sternberger
& Sternberger, Proc. Natl. Acad. Sci. USA 80:6129-6130 (1983));
and ALZ-50 (Wolozin et al., Science 232:648-650 (1986)).
Preferably, AT8 is used in embodiments of the invention to bind
phosphorylated tau protein and/or tau protein fragments.
[0190] In immunostaining, an antibody against neurofibrillary
tangles, phosphorylated tau and/or tau fragments is added to brain
cells, and the brain cells are incubated for a sufficient time to
allow binding between the antibody and neurofibrillary tangles,
phosphorylated tau and/or tau fragments. The antibody may be
labeled with a variety of labels that are detectable. Useful labels
include magnetic beads (e.g., DYNABEADS), fluorescent dyes (e.g.,
fluorescein isothiocyanate, Texas red, rhodamine, and the like),
radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C, or
.sup.32P), enzymes (e.g., horse radish peroxidase, alkaline
phosphatase), and colorimetric labels such as colloidal gold or
colored glass or plastic beads (e.g., polystyrene, polypropylene,
latex, etc.). Alternatively, the antibody may be unlabeled, and a
label may be coupled indirectly. For example, an unlabeled primary
antibody can be added to the culture to bind neurofibrillary
tangles, phosphorylated tau and/or tau fragments, and then a
labeled secondary antibody can be used to amplify the signal for
detection.
[0191] Means of detecting labels are well known to those of skill
in the art. For example, where the label is a radioactive label,
means for detection include a scintillation counter or photographic
film as in autoradiography. Where the label is a fluorescent label,
it may be detected by exciting the fluorochrome with the
appropriate wavelength of light and detecting the resulting
fluorescence. The fluorescence may be detected visually, by means
of photographic film, by the use of electronic detectors such as
charge coupled devices (CCDs) or photomultipliers and the like.
Similarly, enzymatic labels may be detected by providing the
appropriate substrates for the enzyme and detecting the resulting
reaction product. Simple colorimetric labels may be detected simply
by observing the color associated with the label.
[0192] Alternatively, the production of neurofibrillary tangles,
phosphorylated tau and/or tau fragments can be determined using
cell lysate in an immunoassay. An immunoassay can be performed in
any of several formats. These formats include, for example, an
enzyme immune assay (EIA) such as enzyme-linked immunosorbent assay
(ELISA), a radioimmune assay (RIA), a Western blot assay, or a slot
blot assay. For a review of the general immunoassays, see, e.g.,
Methods in Cell Biology: Antibodies in Cell Biology, volume 37
(Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr,
eds., 7th ed. 1991). A general overview of applicable technology
can also be found in Harlow & Lane, Antibodies: A Laboratory
Manual (1988). See, also, U.S. Pat. Nos. 4,366,241; 4,376,110;
4,517,288; and 4,837,168.
[0193] In one embodiment, immunoblotting can be used to quantify
the amount of neurofibrillary tangles, phosphorylated tau and/or
tau fragments produced in brain cells treated with or without a
cathepsin D-increasing compound and/or brain cells treated with or
without (i.e., in the presence or absence of) a cysteine protease
inhibitor. Generally, brain cells are disrupted in an
electrophoresis sample buffer and are treated to obtain a fraction
that contains proteins. The proteins are separated by gel
electrophoresis and transferred to a membrane that binds the
proteins nonspecifically. The location of neurofibrillary tangles,
phosphorylated tau and/or tau fragments on the membrane is
determined using, e.g., a labeled primary antibody or an unlabeled
primary antibody, followed by a labeled secondary antibody. A
detectable label may be, e.g., a radio-label or a fluorescent label
or, an enzyme label. Then the membrane comprising a detectable
label can be scanned, and digitized images can be quantitatively
analyzed by densitometry.
[0194] In another embodiment, a sandwich assay can be performed by
preparing a brain cell lysate sample, and placing the sample in
contact with a solid support on which is immobilized a plurality of
antibodies that bind neurofibrillary tangles, phosphorylated tau
and/or tau fragments. The solid support is then contacted with
detection reagents for neurofibrillary tangles, phosphorylated tau
and/or tau fragments. After incubation of the detection reagents
for a sufficient time to bind a substantial portion of the
immobilized neurofibrillary tangles, phosphorylated tau and/or tau
fragments, any unbound labeled reagents are removed. The detectable
label associated with the detection reagents is then detected. For
example, in the case of an enzyme used as a detectable label, a
substrate for the enzyme that turns a visible color upon action of
the enzyme is placed in contact with the bound detection reagent. A
visible color will then be observed in proportion to the amount of
the neurofibrillary tangles, phosphorylated tau and/or tau
fragments in the sample.
[0195] According to the invention, the production of tau
proteolytic fragments of a size from 15-35 kDa is increased. The
size of tau proteolytic fragments can be determined using
techniques known in the art, for example, gel electrophoresis, and
especially SDS gel electrophoresis, or 2D gel electrophoresis.
[0196] The above described detection methods are merely exemplary,
and other suitable detection methods will be apparent and can be
readily substituted by one of skill in the art.
[0197] V. Treatment of Brain Cells with Cathepsin D-increasing
Compounds to Induce or Enhance the Characteristics of
Neurodegenerative Diseases
[0198] In a preferred embodiment, brain cells (e.g., normal brain
cells, apoE-deficient brain cells, or apoE4-containing brain cells)
are cultured in a medium that provides an effective concentration
of cathepsin D as a result of an agent or compound that selectively
increases the concentration or amount of cathepsin D in the brain
cells. An effective concentration of cathepsin D can be induced, or
increased, in a brain cell by either increasing the amount or
concentration of cathepsin D or by stimulating the catalytic
activity of cathepsin D. The "effective concentration" of cathepsin
D is the concentration that will achieve the indicated result.
[0199] According to the invention, increasing the concentration of
cathepsin D in the brain cells to an effective level results in the
increased production of neurofibrillary tangles, the major
component of which are abnormally phosphorylated tau proteins and
tau protein fragments. Tau protein fragments are generally also
hyperphosphorylated and are composed mainly of fragments containing
the microtubule binding domains and flanks, and are generally of
27-35 kDa in size. In a preferred embodiment, the proteolysis of
tau to fragments of a size of 15-35 kDa is examined. The inventors
have discovered a phosphorylated tau fragment of 33 kDa that is
thought to become a component of the tangles. Therefore, in an
especially preferred embodiment, the amount or levels of the 33 kDa
tau fragment are detected.
[0200] Preferably, such increase in the concentration of cathepsin
D activity or levels is brought about or induced by contacting the
brain cells with a cathepsin D-increasing compound throughout the
entire period of culture during which it is desired to maintain
such selectively increased concentrations or amounts of cathepsin
D. Alternatively, the brain cells can be exposed in intermittent
fashion of desired intervals, or, alternatively, only once at a
desired point in the culture period.
[0201] A selective inhibitor of cathepsin B and L (i.e., ZPAD) can
be used to selectively increase cathepsin D activity or levels
relative to such activity or levels of cathepsin B and L. By
changing the ratio of cathepsin B and/or L to cathepsin D, the
cathepsin D concentration is increased to a concentration effective
to induce the appearance or increase in the desired indicia of
neurodegenerative disease. Selective inhibitors of cathepsin B and
L have been reported to induce abnormally phosphorylated tau
fragments in cultured hippocampal slices of normal rodents.
Abnormally phosphorylated tau fragments assemble into structures
having the appearance, size and epitopes of early-stage
neurofibrillary tangles in human brain. See Bi et al., Exp. Neurol.
158:312-317 (1999). However, the density of neurofibrillary tangles
produced in the normal rodent hippocampal slices was very sparse
compared to the density of neurofibrillary tangles seen in the
brain of Alzheimer's patients.
[0202] Surprisingly, when apoE-deficient or apoE4-containing brain
cells are treated with a cathepsin D-increasing compound, levels of
neurofibrillary tangles and phosphorylated tau proteins and
fragments were elevated and greatly induced. In particular, when
apoE-deficient brain cells were used, a dramatically increased
production of neurofibrillary tangles, phosphorylated tau protein,
and phosphorylated tau fragments was observed. Typically, the
amount of neurofibrillary tangles or phosphorylated tau fragments
seen in these treated apoE-deficient brain cells is at least twice,
sometimes at least ten times greater than the amount of these
materials seen in normal brain cells treated with the same
compound. Also, the amount of neurofibrillary tangles or
phosphorylated tau fragments seen in these treated apoE-deficient
brain cells is at least ten times greater than the amount of these
materials seen in apoE-deficient brain cells that are not treated
with the compound. The density of neurofibrillary tangles in these
apoE-deficient brain cells treated with a cathepsin D-increasing
compound is sufficiently high that it mimics the density of
neurofibrillary tangles typically found in the brain of Alzheimer's
disease patients. Since apoE4-containing brain cells lack many
normal function of apoE, like the apoE-deficient brain cells,
apoE4-containing brain cells can also be used in embodiments of the
invention.
[0203] Preferably, a cathepsin D-increasing compound increases the
effective concentration of cathepsin D in brain cells by at least
about 30%, preferably at least about 50%, more preferably at least
about 80%, most preferably at least about 100%, compared to a
control (e.g., brain cells untreated with the compound). As
described previously, cathepsin D exists in three forms in the
brain--the inactive proenzyme, the active single chain and the
active heavy chain. Any compound that increases one or more of
these cathepsin D forms can be used in embodiments of the
invention.
[0204] Any suitable cathepsin D-increasing compound can be used in
embodiments of the invention. Some of these compounds include
inhibitors of cathepsin B and/or cathepsin L. Examples of these
inhibitors include chloroquine,
N--CBZ-L-phenylalanyl-L-alanine-diazomethylketone,
N--CBZ-L-phenylalanyl-L-phenylalanine-diazomethylketone,
beta-amyloid (amyloid beta protein), and mimetics thereof.
[0205] Other suitable cathepsin D-increasing compounds, and/or
agents which mimic the activity of Cathepsin D (e.g., an inhibitor
of cathepsin B and/or L or a modulator or an agonist of cathepsin
D) are readily determinable by those skilled in the art. For
example, a test compound can be contacted with brain cells. Then
the activity or the amount of cathepsin D in brain cells can be
measured using, e.g., an immunoassay using antibodies against
cathepsin D. For example, antibodies such as Cathepsin D (Ab-2),
Calbiochem can be used.
[0206] The activity or the amount of cathepsin D is then compared
with a control amount (e.g., the amount of cathepsin D in brain
cells that are not treated with the test compound). A test compound
is referred to as a "cathepsin D-increasing compound" if it
increases the activity or the amount of any one or more of
cathepsin D forms (i.e., the inactive proenzyme, the active single
chain or the active heavy chain) by, e.g., at least about 30%,
preferably at least about 50%, more preferably at least about 80%,
most preferably at least about 100%, compared to a control.
[0207] Brain cells can be contacted with a cathepsin D-increasing
compound at any suitable time. For example, brain cells can be
contacted with a cathepsin D-increasing compound when the culture
is first established, or at a later time after maintaining the
culture for a few days. Preferably, brain cells are contacted with
a cathepsin D-increasing compound for a period of 1-18 days,
preferably for a period of 4-8 days. To induce neurofibrillary
tangles and phosphorylated tau fragments, a cathepsin D-increasing
compound is typically added at a concentration of 0.1 .mu.M to
about 500 .mu.M, more typically at a concentration of about 1 .mu.M
to about 100 .mu.M.
[0208] Other modulatory compounds, in addition to a cathepsin
D-increasing compound(s), can be, for example, added in the culture
medium, or at least placed in contact with the brain cells or
tissue containing such cells, to further facilitate the production
of neurofibrillary tangles or other neurodegenerative
characteristics or features in brain cells. Examples of modulatory
compounds include oxidative free radicals (Fe.sup.3+,
H.sub.2O.sub.2, etc.), or inflammatory factors (TGFb, IL-1b,
TNFalpha, LPS, etc.).
[0209] Typically, brain cells in a culture are treated with a
cathepsin D-increasing compound under a condition such that the
amount of neurofibrillary tangles, phosphorylated tau and/or tau
fragments is increased by at least about 10%, or at least about
20%, or at least about 30%, or at least about 40%, or at least
about 50%, or at least about 80%, or at least about 100%, or at
least about 150%, or at least about 200%, compared to a control
(e.g., brain cells that are cultured in substantially the same
condition but without the cathepsin D-increasing compound). Also,
brain cells that are treated with a cathepsin D-increasing compound
generally produce neurofibrillary tangles, phosphorylated tau
and/or tau fragments at a significantly higher level, typically at
least two times, sometimes ten times, more than normal brain cells
treated with the same compound. Preferably, the treatment
conditions (e.g., concentration of a cathepsin D-increasing
compound, a period of incubation, etc.) are selected so that the
density of neurofibrillary tangles, phosphorylated tau and/or tau
fragments produced in apoE-deficient brain cells or in
apoE4-containing brain cells is similar to the density of these
materials in aging brain or the brain of patients with Alzheimer's
disease or other neurodegenerative diseases.
[0210] Brain cells produced in accordance with the present
invention, under conditions in which cathepsin D levels or activity
is increased, have a variety of applications. For example, the
brain cells can be used as an in vitro assay system to screen
libraries or identify agents that modulate the production of
neurofibrillary tangles, phosphorylated tau and/or tau fragments in
the brain, especially agents that decrease or prevent the
accumulation of such characteristics. These agents can be further
tested in other systems and/or in vivo to confirm their efficacy in
modulating the production of neurofibrillary tangles in brain cells
and possibly other conditions and/or pathologies associated with
neurodegenerative diseases, such as the cognitive decline seen in
persons afflicted with such disorders. In another example, the
brain cells can be used to study the morphological pattern of
neurofibrillary tangle formation in the brain. In another example,
the brain cells can be used to study the effect of neurofibrillary
tangle formation in normal aging. Such morphological studies would
provide additional information regarding the pathological process
of neurodegenerative diseases.
[0211] VI. Treatment of Brain Cells with a Cholesterol Decreasing
Compound to Induce or Enhance the Characteristics of
Neurodegenerative Diseases
[0212] According to another embodiment of the invention, decreasing
intracellular cholesterol levels in brain cells, for example, by
inhibiting cholesterol synthesis, can be used to induce the
characteristics of neurodegenerative diseases, and especially
Alzheimer's disease, in that brain cell--even in cells from normal
animals. In a preferred embodiment, the brain cell in which
cholesterol is decreased is a neuron and the characteristics that
are monitored are the formation of tangles and tau fragmentation.
In another embodiment, the brain cells in which cholesterol is
decreased are glia cells and the characteristics that are monitored
are glia activations, glia reactions, and/or cytokine production
and/or release.
[0213] Exposing the brain cell to such cholesterol-lowering agents
or conditions mimics results found when using apoE-deficient brain
cells, or brain cells that contain the apoE4 isotype. The advantage
of the cholesterol-limiting treatment (i.e., inhibition of
cholesterol synthesis and/or lowering of cholesterol levels) is
that relatively high tangle densities can be obtained in normal
cells by such treatment, densities that are otherwise only
obtainable in cells in apoE-deficient brain cells, or in
apoE4-containing brain cells. Accordingly, for the first time, high
densities of neurofibrillary tangles and the appearance of other
characteristics of neurodegenerative diseases can be induced in
brain cells from normal animals in a relatively short period of
time, and thus be useful as a model for studying such diseases and
for identifying agents useful to treat or prevent the same. A
combined inhibition of cholesterol synthesis and lysosomal
dysfunction can be used to further dramatically enhance the
neurodegenerative effects brought about by either manipulation
alone.
[0214] Therefore, in another embodiment, the characteristics of
neurodegenerative diseases, such as, for example
[0215] (1) neurofibrillary tangles,
[0216] (2) the hyperphosphorylation of tau,
[0217] (3) the fragmentation of tau, that is, tau proteolysis and
especially, increased amounts of the 15-35 kDa forms of tau,
[0218] (4) increased production and/or release of brain-produced
pro-inflammatory cytokines especially TGF-beta, TGF-alpha, IL1,
IL1-alpha, IL1-beta, IL6, IL10, TNF, TNF-alpha and LPS and most
especially TGF-beta, IL-1beta and LPS,
[0219] (5) increased microglia reaction and/or activation,
[0220] (6) increased indications of brain inflammatory
reactions
[0221] (7) increased conversion of p35 to p25
[0222] (8) changes in the levels and activities of protein kinases,
for example, cyclin dependent protein kinase 5 (cdk5) and mitogen
activated protein kinase (MAPK),
[0223] are induced by exposing brain cells to a condition that, or
by contacting brain cells with a compound that, inhibits
cholesterol synthesis or otherwise decreases the levels of
cholesterol.
[0224] According to this embodiment, to increase the production of
such characteristics, and especially neurofibrillary tangles and/or
phosphorylated tau and/or tau fragments and/or the production
and/or release of cytokines and/or microglia reactions and/or
activations and/or inflammation and/or conversion of p35 to p25
and/or the levels and activities of protein kinases, brain cells
are contacted with a compound capable of decreasing levels of
cholesterol or inhibiting cholesterol synthesis or otherwise
capable of decreasing the concentration of cholesterol
("cholesterol-lowering compound"). This compound can preferably
decrease either the concentration of cholesterol or the synthesis
of cholesterol in cells and thus decrease the availability of
cholesterol within the cells.
[0225] In one aspect, the invention provides cultured brain cells,
and methods for producing the brain cells, wherein the brain cells
have been treated with a compound that increases cathepsin D to an
effective concentration and with a compound that decreases
cholesterol levels or inhibits cholesterol synthesis to an
sufficient low concentration to result in or to produce increased
amounts of neurofibrillary tangles and/or phosphorylated tau and/or
tau fragments and/or the production and/or release of cytokines
and/or microglia reactions and/or activations and/or inflammation
and/or conversion of p35 to p25 and/or the levels and activities of
protein kinases compared to such indicia in a control (e.g., brain
cells that are untreated with said compound(s)).
[0226] Embodiments of the invention include methods comprising: (a)
culturing brain cells; and (b) contacting the brain cells with a
compound that increases an effective concentration of cathepsin D
and with a compound that decreases an effective concentration of
cholesterol, thereby producing properties of a brain afflicted with
neurodegenerative disease, wherein the properties include increased
neurofibrillary tangles and/or phosphorylated tau and/or tau
fragments and/or the production and/or release of cytokines and/or
microglia reactions and/or activations and/or inflammation and/or
conversion of p35 to p25 and/or the levels and activities of
protein kinases and/or related biochemical changes.
[0227] In some embodiments, a method for increasing neurofibrillary
tangles and/or phosphorylated tau and/or tau fragments and/or the
production and/or release of cytokines and/or microglia reactions
and/or activations and/or inflammation and/or conversion of p35 to
p25 and/or the levels and activities of protein kinases in brain
cells comprises: (a) culturing the brain cells in a medium which
selectively increases an effective concentration of cathepsin D and
that decreases the concentration of cholesterol in the medium and
cells; and (b) optionally, determining the production of and/or
levels of neurofibrillary tangles and/or phosphorylated tau and/or
tau fragments and/or the production and/or release of cytokines
and/or microglia reactions and/or activations and/or inflammation
and/or conversion of p35 to p25 and/or the levels and activities of
protein kinases.
[0228] Preferably, a cholesterol-lowering compound decreases the
effective concentration of cholesterol in brain cells by at least
about 30%, preferably at least about 50%, more preferably at least
about 80%, most preferably at least about 100%, compared to a
control (e.g., brain cells untreated with the compound). Any
compound that lowers cholesterol levels (for example, by inhibiting
cholesterol synthesis or stimulating cholesterol degradation or
lowering the availability of cholesterol) can be used in
embodiments of the invention. Examples which can be used in
embodiments of the invention include compounds which decrease
either the concentration of cholesterol, or the synthesis of
cholesterol, or decreases the availability of cholesterol in
cells.
[0229] Any suitable cholesterol-lowering compound can be used in
embodiments of the invention. Some of these compounds include
inhibitors of hydroxymethylglutaryl coenzyme A (HMG-CoA Reductase)
inhibitors. Examples of these inhibitors include members of the
statin class of compounds, such as, for example, mevastatin,
simvastatin, atorvastatin, pravastatin, fluvastatin, lovastatin,
cerivastatin, and mimetics thereof.
[0230] A further class of compounds includes agents which decrease
the availability of cholesterol within cells. Examples of this
class include agents which bind, immobilize, and/or otherwise
separate cholesterol from other elements found within cells.
[0231] Other suitable cholesterol-lowering compounds, and/or agents
which modulate the activity of cholesterol are readily determinable
by those skilled in the art. For example, a test compound can be
contacted with cells. Then the activity or the amount of
cholesterol in cells can be measured using, e.g., an immunoassay
using antibodies against cholesterol. Alternatively, a test
compound can be contacted with cells and the activity or amount of
HMG-CoA reductase (an enzyme involved in cholesterol synthesis in
cells) and/or other entities involved in cholesterol synthesis,
degradation, storage, and/or transport can be measured using assays
known to one skilled in the art.
[0232] The activity or the amount of cholesterol and/or the
activity or amount of HMG-CoA reductase and/or other entities
involved in cholesterol synthesis, degradation, storage, and/or
transport is then compared with a control amount (e.g., the amount
of cholesterol and/or the activity or amount of HMG-CoA reductase
and/or other entities involved in cholesterol synthesis,
degradation, storage, and/or transport in cells that are not
treated with the test compound). A test compound is referred to as
a "cholesterol-lowering compound" if it decreases the activity or
the amount of cholesterol and/or the activity or amount of HMG-CoA
reductase and/or modulates other entities involved in cholesterol
synthesis, degradation, storage, and/or transport by, e.g., at
least about 30%, preferably at least about 50%, more preferably at
least about 80%, most preferably at least about 100%, compared to a
control.
[0233] Brain cells can be contacted with a cholesterol-lowering
compound at any suitable time. For example, brain cells can be
contacted with a cholesterol-lowering compound when the culture is
first established, or at a later time after maintaining the culture
for a few days. Preferably, brain cells are contacted with a
cholesterol-lowering compound for a period of 1, 2, 3, 4, 5, 10,
20, 30, 40, 50, 60, 90, 120, 150, or 240 days, or preferably, for
in vitro experiments, for a period of 4, 5, 6, 7, 8 or 9 days,
while in vivo experiments preferably have a duration of 30-120
days, or any appropriate period of time to achieve the desired
effect. To induce the formation of neurofibrillary tangles, and/or
phosphorylated tau, and/or tau fragments, and/or microglial
reactions, and/or cytokine reactions, or any other of the indicia
of neurodegenerative brain disease discussed above. For in vitro
experiments a cholesterol-lowering compound is typically added at a
concentration of 0.1 .mu.M to about 500 .mu.M, more typically at a
concentration of about 1 nM, 10 nM or 100 nM to about 100 .mu.M,
and especially 20 .mu.M, or any appropriate amount that achieves
the desired effect. For in vivo experiments a cholesterol-lowering
compound is typically added at a dose of 0.5 to about 50 mgs/kg
body weight of the animal, more typically about 5-40 mgs/kg, and
especially 10-20 mgs/kg, or any appropriate amount that achieves
the desired effect. More than one cholesterol-lowering compound can
be administered at the same time, or sequentially at different
times, to the brain cell preparation or animal.
[0234] Other modulatory compounds, in addition to such
cholesterol-lowering compound(s), can be added in the culture
medium to further facilitate the production of neurofibrillary
tangles or any of the other neurodegenerative features in brain
cells, especially tau fragmentation. Examples of useful modulatory
compounds in this regard include agents capable of modulating those
kinases and/or phosphatases that are involved in cholesterol
metabolism or that interact with cholesterol to affect cell
function, amyloid beta peptide, oxidative free radicals (Fe.sup.3+,
H.sub.2O.sub.2, etc.), or inflammatory factors (TGF-beta, IL-1b,
TNFalpha, LPS, etc.).
[0235] Typically, brain cells in a culture are treated with a
cholesterol-lowering compound under a condition such that the
amount of neurofibrillary tangles and/or phosphorylated tau and/or
tau fragments and/or the production and/or release of cytokines
and/or microglia reactions and/or activations and/or inflammation
and/or conversion of p35 to p25 and/or the levels and activities of
protein kinases is increased by at least about 10%, or at least
about 20%, or at least about 30%, or at least about 40%, or at
least about 50%, or at least about 80%, or at least about 100%, or
at least about 150%, or at least about 200%, compared to a control
(e.g., brain cells that are cultured in substantially the same
condition but without the cholesterol-lowering compound). Also,
brain cells that are treated with a cholesterol-lowering compound
generally produce neurofibrillary tangles and/or phosphorylated tau
and/or tau fragments and/or the production and/or release of
cytokines and/or mnicroglia reactions and/or activations and/or
inflammation and/or conversion of p35 to p25 and/or the levels and
activities of protein kinases at a significantly higher level,
typically at least two times, sometimes ten times more than normal
brain cells treated with the same compound. Preferably, the
treatment conditions (e.g., concentration of a cholesterol-lowering
compound, the period of contact with the brain cells, etc.) are
selected so that the density of neurofibrillary tangles and/or
phosphorylated tau and/or tau fragments and/or the production
and/or release of cytokines and/or microglia reactions and/or
activations and/or inflammation and/or conversion of p35 to p25
and/or the levels and activities of protein kinases produced is
similar to the density of these materials and/or reactions in aging
brain or the brain of patients with Alzheimer's disease or other
neurodegenerative diseases.
[0236] VII. Treatment of Brain Cells with a Cysteine Protease
Inhibitor to Prevent or Reverse the Characteristics of
Neurodegenerative Diseases
[0237] According to a further model of the invention, the above
indicia of neurodegenerative disease can be prevented or reversed
by exposing brain cells to a cysteine protease inhibitor, and
preferable a calpain inhibitor. Specifically such protease
inhibitor, and especially calpain inhibitor, reverses the effects
of the lysosomal dysfunction and/or cholesterol-lowering, and
decreases, or prevents the formation of:
[0238] (1) neurofibrillary tangles,
[0239] (2) the hyperphosphorylation of tau,
[0240] (3) the fragmentation of tau, that is, tau proteolysis and
especially, increased amounts of the 15-35 kDa forms of tau,
[0241] (4) increased production and/or release of brain-produced
pro-inflammatory cytokines especially TGF-beta, TGF-alpha, IL1,
IL1-alpha, IL1-beta, IL6, IL10, TNF, TNF-alpha and LPS and most
especially TGF-beta, IL-1beta and LPS,
[0242] (5) increased microglia reaction and/or activation,
[0243] (6) increased indications of brain inflammatory
reactions
[0244] (7) increased conversion of p35 to p25
[0245] (8) changes in the levels and activities of protein kinases,
for example, cyclin dependent protein kinase 5 (cdk5) and mitogen
activated protein kinase (MAPK).
[0246] Cysteine protease inhibitors, and specifically calpain
inhibitors, are therefore useful to identify agents or compounds
that might modulate the effects of the cysteine protease inhibitor,
for example, induce or enhance the effects, or interfere with the
same.
[0247] The term "calpain inhibitor" refers to a compound that
inhibits the proteolytic action of calpain-I or calpain-II, or
both, but preferably calpain-I. The term calpain inhibitors as used
herein include those compounds having calpain inhibitory activity
in addition to or independent of their other biological activities.
A wide variety of compounds have been demonstrated to have activity
in inhibiting the proteolytic action of calpains. Examples of
calpain inhibitors that are useful in the practice of the invention
include N-acetyl-leucyl-leucyl-me- thional (ALLM or calpain
inhibitor II), N-acetyl-leucyl-leucyl-norleucinal (ALLN or calpain
inhibitor 1), calpain inhibitor III
(carbobenzoxy-valyl-phenylalanal; Z-Val-Phe-CHO), calpain inhibitor
IV (Z-LLY-FMK; Z-LLY--CH.sub.2F where Z=benzyloxycarbonyl), calpain
inhibitor V (Mu-Val-HPh-FMK where Mu is morphlinoureidyl and Hph is
homophenylalanyl), calpeptin (benzyloxycarbonyldipeptidyl aldehyde;
Z-Leu-Nle-CHO), calpain inhibitor peptide (Sigma No. C9181),
calpastatin, acetyl-calpastatin (acetyl calpain inhibitor fragment,
184-210), leupeptin, mimetics thereof and combinations there,
AK275, MDL28170 and E64. Additional calpain inhibitors are
described in the following U.S. patents, incorporated herein by
reference, U.S. Pat. No. 5,716,980; U.S. 5,714,471; U.S. Pat. No.
5,693,617; U.S. Pat. No. 5,691,368; U.S. Pat. No. 5,679,680; U.S.
Pat. No. 5,663,294, U.S. Pat. No. 5,661,150; U.S. Pat. No.
5,658,906; U.S. Pat. No. 5,654,146; U.S. Pat. No. 5,639,783; U.S.
Pat. No. 5,635,178; U.S. Pat. No. 5,629,165; U.S. Pat. No.
5,622,981; U.S. Pat. No. 5,622,967; U.S. Pat. No. 5,621,101; U.S.
Pat. No. 5,554,767; U.S. Pat. No. 5,550,108; U.S. Pat. No.
5,541,290; U.S. Pat. No. 5,506,243; U.S. Pat. No. 5,498,728; U.S.
Pat. No. 5,498,616; U.S. Pat. No. 5,461,146; U.S. Pat. No.
5,444,042; U.S. Pat. No. 5,424,325; U.S. Pat. No. 5,422,359; U.S.
Pat. No. 5,416,117; U.S. Pat. No. 5,395,958; U.S. Pat. No.
5,340,922; U.S. Pat. No. 5,336,783; U.S. Pat. No. 5,328,909; U.S.
Pat. No. 5,135,916.
[0248] Preferably the concentration of such inhibitor in the fluid,
culture medium, milieu, or other environment contacting the brain
cells of the invention is a concentration of 1 nM to 1 mM, and
preferably 10 nM, 100 nM, 1 .mu.M, 10 .mu.M, 100 .mu.M and
especially 20 .mu.M, or any appropriate amount that achieves the
desired effect.
[0249] The cysteine protease inhibitor, and especially, the calpain
inhibitor, can be added at the beginning of the culture of the
brain cells, or intermittently during the culture, as desired. The
inhibitor can be one that is active metabolically intracellularly,
or that acts by binding to the outer membrane and inducing a
cascade that ultimately results in an inhibition and/or reversal of
the desired characteristic of neurodegenerative disease that is
being measured in the culture. Two or more inhibitors can be
simultaneously added, or sequentially added.
[0250] Accordingly, a target class of compounds for a screening
method can be identified according to the invention by a method
comprising: (a) contacting brain cells with a cathepsin
D-increasing compound that increases cathepsin D to an effective
concentration in the brain cells, and/or contacting brain cells
with a cholesterol-lowering compound wherein the increased
concentration of cathepsin D and/or the decreased concentration of
cholesterol is effective to increase the amount of neurofibrillary
tangles, phosphorylated tau and/or tau fragments in the brain
cells; (b) contacting the brain cells with a cysteine protease
inhibitor; and (c) determining whether the cysteine protease
inhibitor modulates the amount of neurofibrillary tangles,
phosphorylated tau and/or tau fragments in the brain cells treated
with the cysteine protease inhibitor compared to the brain cells
that are not treated with the cysteine protease inhibitor.
[0251] The present invention thus provides a novel
target--inhibition of tau proteolysis by a cysteine protease
inhibitor, and especially by a calpain inhibitor--for intervention
and treatment of Alzheimer's disease, neurodegenerative diseases,
and related disorders, such as senile dementias, progressive
supranuclear palsy, corticobasal degeneration, frontotemporal
dementias, Parkinsonism, Pick's disease, etc., and for diminishing
the occurrence of neurofibrillary tangles and/or tau fragmentation
events capable of resulting in the formation of neurofibrillary
tangles and/or tau-related pathologies.
[0252] That cysteine protease inhibition, and especially calpain
inhibition can reverse the characteristics of neurodegenerative
diseases, and especially tangle formation, is especially surprising
because the art has taught that hyperphosphorylated tau in the
paired helical filaments is resistant to degradation by calpain. As
much as five times the levels of calpain are needed to completely
degrade paired helical filament tau as compared to "normal" tau
(Mercken, M. et al., FEBS Lett 38:10-14 (1995); Yang, L.-S. and
Ksiezak-Reding, H., Eur. J. Biochem. 233:9-17 (1995))
[0253] In a preferred embodiment of this aspect of the invention,
the present invention provides a cysteine protease inhibitor, and
particularly an inhibitor of the class of cysteine proteases known
as calpains, that affects the central nervous system in a manner
that alleviates the symptomologies of Alzheimer's disease, senile
dementia, and related disorders, such as Pick's disease. Further,
the present invention provides the advantage of alleviating the
symptomologies of Alzheimer's disease by inhibiting the formation
of neurofibrillary tangles and related tau fragmentation events
characteristic of such diseases, of which there is a need in the
art.
[0254] In a preferred embodiment, the present invention provides
novel methods for ameliorating certain conditions associated with
neurodegenerative disease and/or neurodegenerative diseases, such
as Alzheimer's disease, Pick's disease, senile dementia, etc. In
accordance with embodiments of the invention, a host afflicted with
a neurodegenerative disease, such as Alzheimer's disease, Pick's
disease, etc., is treated with a cysteine protease inhibitor, e.g.,
by administering a pharmaceutically effective amount of an agent
capable of inhibiting the activity of a member of the calpain class
of proteases.
[0255] In another embodiment of the invention brain cells (e.g.,
normal brain cells, apoE-deficient brain cells, apoE4-containing
brain cells, and/or other transgenically altered brain cells) are
treated with a cysteine protease inhibitor, e.g., by contacting the
brain cells with an agent capable of inhibiting the activity of a
member of the calpain class of proteases. The administration of the
agent to the host, and/or the contacting of the agent with the
brain cells, then results in a decreased amount of tau
fragmentation events which can lead to the formation of
neurofibrillary tangles, and the decreased formation of
neurofibrillary tangles, or the degradation of tangles that have
already been formed.
[0256] While some features of neurodegenerative disease or
neurodegenerative diseases have been partially remedied by other
classes of therapeutics, a key feature such as the reduction of tau
fragmentation and/or a reduction in the density of neurofibrillary
tangles in the brain was missing in these treatment modalities. The
present invention advantageously provides a therapeutic treatment
for a host and/or a treatment for brain cells, wherein the host
and/or the brain cells comprise, among other things, reduced levels
of tau fragmentation and reduced levels of neurofibrillary
tangles.
[0257] In the present invention, any suitable host or brain cells
can be treated. Preferably, hosts are human, and are believed to be
afflicted with a neurodegenerative disease, such as Alzheimer's
disease, Pick's disease, or a related disorder such as senile
dementia, etc. Preferably, brain cells are from a mammal, such as
rat, mouse, guinea pig, rabbit, etc. In some embodiments,
apoE-deficient brain cells or apoE4-containing brain cells, or
other brain cells from a transgenic animal, can be treated.
[0258] In one aspect, the invention provides compounds, and methods
for using such compounds to decrease the formation of
neurofibrillary tangles and/or tau fragments compared to a control
(e.g., a host not given said compound(s) and/or brain cells that
are untreated with said compound(s)). Embodiments of the invention
include methods comprising:
[0259] (a) identifying a host thought to be afflicted with a
disorder or disease believed to comprise abnormal tau fragmentation
events and/or increased levels of neurofibrillary tangles; and
[0260] (b) administering to such host a compound that inhibits a
member of the calpain class of cysteine proteases, wherein, as a
result of the administration of the compound, the characteristics
of neurodegenerative disease are lessened or decreased, and
preferably, there are decreased levels of neurofibrillary tangles,
and/or there are decreased levels of tau fragments and/or decreases
in related tau-mediated pathologies.
[0261] In other embodiments, a method is provided for decreasing
neurofibrillary tangles and/or tau fragmentation in brain cells,
the method comprising contacting brain cells with a medium under
conditions which, or in the presence of sufficient amounts of a
compound that, inhibit one or more members of the calpain class of
cysteine proteases, and preferably calpain I.
[0262] In another embodiment, the invention provides a target class
of compounds for a screening method comprising:
[0263] (a) contacting brain cells with a cathepsin D-increasing
compound and/or a cholesterol-decreasing compound that increases
cathepsin D and/or decreases cholesterol in the brain cells to
levels effective to increase the amount of neurofibrillary tangles,
phosphorylated tau and/or tau fragments in the brain cells;
[0264] (b) contacting the brain cells with a cysteine protease
inhibitor, and preferably an inhibitor of calpain; and
[0265] (c) determining whether the cysteine protease inhibitor
modulates the amount of neurofibrillary tangles, phosphorylated tau
and/or tau fragments in the brain cells treated with the cysteine
protease inhibitor compared to the brain cells that are not treated
with the cysteine protease inhibitor.
[0266] VIII. Screening Assays
[0267] Screening assays can be performed in vitro or in vivo. To
produce brain cells comprising neurofibrillary tangles,
phosphorylated tau and/or tau fragments, etc., the methods
described above can be used.
[0268] The advantage of using brain cells in the form of slices or
in vivo animal testing for the screening assays is that since the
neuronal circuitry and other biological functions are more intact
in brain slices and in vivo, compared to dissociated brain cells,
the experimental conditions better mimic the physiological
condition of the brain.
[0269] Preferably, the concentration of cathepsin D, and/or the
synthesis (and/or levels) of cholesterol, and other culture
conditions are adjusted so that the density of neurofibrillary
tangles, phosphorylated tau and/or tau fragments in the brain cells
(prior to contacting with an agent) is similar to the density of
these materials found in neurodegenerative diseases, such as
Alzheimer's disease. ApoE-deficient brain cells or apoE4-containing
brain cells can be used.
[0270] To screen agents that modulate the production of
neurofibrillary tangles, phosphorylated tau and/or tau fragments,
brain cells are contacted with a test agent. An "agent" refers to
any molecule, including, e.g., a chemical compound (organic or
inorganic), or a biological entity, such as a protein, sugar,
nucleic acid or lipid, that modulates the amount of neurofibrillary
tangles, phosphorylated tau and/or tau fragments in brain cells.
Generally, a test agent is added to the culture medium in the range
from 0.1 nM to 10 mM, and/or an animal is administered a dose of
0.5 to 50 mgs/kg.
[0271] Agents can be obtained from a wide variety of sources,
including libraries of synthetic or natural compounds. For example,
libraries of natural compounds in the form of bacterial, fungal,
plant and animal extracts can be tested. Known pharmacological
agents may be subjected to directed or random chemical
modifications, e.g., alkylation, esterification, amidification,
etc. to produce a library of structural analogs. Alternatively, a
library of randomly or directed synthesized organic compounds or
biomolecules (e.g., oligonucleotides and oligopeptides) can be used
as a source of agents. Preparation and screening of combinatorial
libraries are well known to those of skill in the art. See, e.g.,
U.S. Pat. No. 5,010,175, PCT Publication No. WO 93/20242, PCT
Publication No. WO 92/00091, Chen et al., J. Amer. Chem. Soc.
116:2661 (1994), U.S. Pat. No. 5,539,083.
[0272] Since the production of neurofibrillary tangles,
phosphorylated tau and/or tau fragments is correlated with the
increased concentration of cathepsin D in brain cells, an inhibitor
of cathepsin D may be effective in reducing the production of
neurofibrillary tangles or other neuropathological lesions.
Accordingly, a library of putative cathepsin D inhibitors can be
used as a source of agents in a screening assay. Methods for
producing a library of potential cathepsin D inhibitors are known.
For example, a combinatorial library of agents against the active
site of cathepsin D was previously synthesized by others based on
the crystal structure of cathepsin D. See Kick et al., Chem. Biol.
4:297-307 (1997). The library of these agents can be screened by
methods in accordance with embodiments of the invention.
[0273] An agent can be contacted with brain cells at any suitable
time. For example, an agent can be contacted with brain cells prior
to contacting the brain cells with a cathepsin D-increasing
compound, and/or a compound that lowers the cholesterol to an
effective concentration in the cells. In another example, the brain
cells can be contacted with the agent after the brain cells are
contacted with a cathepsin D-increasing compound and/or a compound
that lowers cholesterol to an effective concentration in the cells.
Preferably, the brain cells can be contacted simultaneously with
the agent and the cathepsin D-increasing compound and/or a compound
that lowers cholesterol to an effective concentration in the cells.
Generally, brain cells are contacted with an agent for a period of
time sufficient to allow the agent to penetrate the cells and to
take an effect. Typically, the brain cells and an agent are
contacted for a period of between about 1 minute to about 30 days,
preferably between about 30 minutes to about 6 days. Typically,
during this time, the culture of brain cells is maintained at a
temperature between about 4.degree. C. to about 40.degree. C.,
preferably at 37.degree. C., at atmosphere containing about 0 to
10% CO.sub.2. Other suitable experimental conditions are readily
determinable by those skilled in the art.
[0274] A number of assays known in the art can be used to determine
the effect of candidate agents on the production of neurofibrillary
tangles, phosphorylated tau and/or tau fragments in brain cells.
For example, various staining or immunoassays described above can
be used, and the details of these assay techniques will not be
repeated in this section. Other suitable assays will be readily
determinable by those of skill in the art, and can be applied in
detecting the production of neurofibrillary tangles, phosphorylated
tau and/or tau fragments.
[0275] In determining whether an agent modulates the cathepsin D
and/or cholesterol-induced production of neurofibrillary tangles,
phosphorylated tau and/or tau fragments in brain cells, experiments
are typically carried out with a control. A control can be, e.g.,
adding no agent or adding a different amount or type of agent and
extrapolating and determining the zero amount. A statistically
significant difference in a test amount (e.g., brain cells treated
with a test agent) and a control amount (e.g., brain cells
untreated with a test agent) of neurofibrillary tangles,
phosphorylated tau and/or tau fragments indicates that the test
agent modulates the production of neurofibrillary tangles of
phosphorylated tau fragments. For example, inhibition of
neurofibrillary tangles, phosphorylated tau and/or tau fragment
production is achieved when the test amount of neurofibrillary
tangles or phosphorylated tau or tau fragments relative to the
control amount is about 90% (e.g., 10% less than the control),
optionally 80% or less, 70% or less, 60% or less, 50% or less, 40%
or less, or 25-0%.
[0276] Brain cells in accordance with embodiments of the invention
provide a model for the development of the biochemical
characteristics of neurodegenerative diseases, such as Alzheimer's
disease. In regular rats, mevastatin produces similar types and
amounts of pathologies as observed in ApoE-knockout mice, and
mevastatin plus ZPAD in regular rats produces results similar to
those found in apoE-knockout mice treated with ZPAD. However, just
as useful are normal rats treated with an agent that can lower the
concentration of cholesterol since neurofibrillary tangles and
phosphorylated tau protein/tau fragments are induced at a higher
density, mimicking early-stage tangles found in Alzheimer's disease
and other neurodegenerative diseases.
[0277] ApoE-deficient brain cells and apoE4-containing brain cells
provide a cost and time efficient in vitro model to study such
diseases. For example, apoE-deficient brain cells or
apoE4-containing brain cells produced in accordance with
embodiments of the invention can be used to screen agents that may
modulate the production of neurofibrillary tangles, phosphorylated
tau and/or tau fragments in the brain cells. Efficacious agents
that are identified by in vitro screening methods described herein
can be further tested to determine their efficacy in vivo. Some of
these agents can potentially be useful as therapeutic compounds for
neurodegenerative diseases, including Alzheimer's disease.
[0278] In another aspect, the invention provides screening assays
to identify cysteine protease inhibitors that modulate the amount
of tau fragments. Additionally, such inhibitors may be assayed for
their ability to inhibit the formation of tau fragments in the
aforementioned assay system. For example, such screening methods
would comprise:
[0279] (a) contacting brain cells with an agent capable of
modulating the activity or levels of a cysteine protease;
[0280] (b) determining whether the agent modulates the amount of
neurofibrillary tangles, tau fragmentation and/or the production of
phosphorylated tau in the brain cells treated with the agent
compared to the brain cells that are not treated with the
agent.
[0281] Thus, the inhibition of tau proteolysis can be used as an
assay for a new calpain inhibitor, especially a calpain inhibitor
that has therapeutic utility in the treatment or prevention of
neurological disorders, and, especially, Alzheimer's disease and/or
Pick's disease.
[0282] In another aspect, the invention provides screening assays
that identify MAP kinase inhibitors that modulate the amount of
neurofibrillary tangles and/or phosphorylated tau and/or tau
fragments and/or the production and/or release of cytokines and/or
microglia reactions and/or activations and/or inflammation and/or
conversion of p35 to p25 and/or the levels and activities of
protein kinases. Additionally, such inhibitors may be assayed for
their ability to inhibit the amount of neurofibrillary tangles
and/or phosphorylated tau and/or tau fragments and/or the
production and/or release of cytokines and/or microglia reactions
and/or activations and/or inflammation and/or conversion of p35 to
p25 and/or the levels and activities of protein kinases in the
aforementioned assay system. For example, such screening methods
can include:
[0283] (A) contacting brain cells with an agent that modulates the
activity or levels of a MAP kinase; and
[0284] (B) determining whether the agent modulates the amount of
neurofibrillary tangles and/or phosphorylated tau and/or tau
fragments and/or the production and/or release of cytokines and/or
microglia reactions and/or activations and/or inflammation and/or
conversion of p35 to p25 and/or the levels and activities of
protein kinases in the brain cells treated with the agent as
compared to the brain cells that are not treated with the agent,
and
[0285] (C) identifying those agents that decrease or that increase
one or more of neurofibrillary tangles and/or phosphorylated tau
and/or tau fragments and/or the production and/or release of
cytokines and/or microglia reactions and/or activations and/or
inflammation and/or conversion of p35 to p25 and/or the levels and
activities of protein kinases in the brain cells treated with the
agent as compared to the brain cells that are not treated with the
agent.
[0286] In a further embodiment, such agents are used in brain cells
treated with such agent to increase or decrease, respectively, one
or more of such neurofibrillary tangles and/or phosphorylated tau
and/or tau fragments and/or the production and/or release of
cytokines and/or microglia reactions and/or activations and/or
inflammation and/or conversion of p35 to p25 and/or the levels and
activities of protein kinases that such agent increased or
decreased in the screening assay of the invention.
[0287] MAP kinase inhibitors that would be useful in this regard
are known in the art. PD98059
(2-2(Amino-3-methoxyphenyl)4H-1-benzopyran-4-one) is a specific
inhibitor of mitogen-activated protein kinase kinase (MAPKK).
SB209580
(4-[5-(4-Fluorophenyl)-2-[4-(methylsulphonyl)phenyl]-1H-imidzaol-
-4yl]pyridine) is a highly selective inhibitor of p38
mitogen-activated protein kinase (p38 MAPK) and also inhibits
cycoloxygenase-1 and -2, and thromboxane synthase. PD98059 and
SB203580 are especially useful at concentrations of 5-100 .mu.M
range. U0126 (1,4-Diamino-2,3-dicyano-1,4-b-
is[2-aminophenylthio]butadiene; Promega) is a selective inhibitor
of MAP kinase kinase. U0126 is more potent and inhibits MEK-1 and
MEK-2 with an IC.sub.50 value of 0.07 and 0.06 .mu.M, respectively.
Preferred concentrations of U0126 are 5-20 .mu.M.
[0288] The compounds can be employed in a free base form or in a
salt form (e.g., as pharmaceutically acceptable salts). Examples of
suitable pharmaceutically acceptable salts include inorganic acid
addition salts such as hydrochloride, hydrobronide, sulfate,
phosphate, and nitrate; organic acid addition salts such as
acetate, galactarate, propionate, succinate, lactate, glycolate,
malate, tartrate, citrate, maleate, fumarate, methanesulfonate,
salicylate, p-toluenesulfonate, and ascorbate; salts with acidic
amino acids such as aspartate and glutamate; alkali metal salts
such as sodium salt and potassium salt, alkaline earth metal salts
such as magnesium salt and calcium salt; ammonium salt; organic
basic salts such as trimethylamine salt, triethylamine salt,
pyridine salt, picoline salt, dicyclohexylamine salt, and
N,N-dibenzylethylenediamine salt; and salts with basic amino acids
such as the lysine salt and arginine salts. The salts may be in
some cases be hydrates or ethanol solvates.
[0289] The manner in which the compounds are administered in vivo
can vary. The compounds can be administered by inhalation (e.g., in
the form of an aerosol either nasally or using delivery articles of
the type set forth in U.S. Pat. No. 4,922,901 to Brooks et al., the
disclosure of which is incorporated herein by reference in its
entirety); topically (e.g., in lotion form); orally (e.g., in
liquid form within a solvent such as an aqueous or non-aqueous
liquid, or within a solid carrier); intravenously (e.g., within a
dextrose or saline solution); as an infusion or injection (e.g., as
a suspension or as an emulsion in a pharmaceutically acceptable
liquid or mixture of liquids); intrathecally; intracerebro
ventricularly; or transdermally (e.g., using a transdermal patch).
Although it is possible to administer the compounds in the form of
a bulk active chemical, it is preferred to present each compound in
the form of a pharmaceutical composition or formulation for
efficient and effective administration. Exemplary methods for
administering such compounds will be apparent to the skilled
artisan. For example, the compounds can be administered in the form
of a tablet, a hard gelatin capsule or as a time release capsule.
As another example, the compounds can be delivered transdermally
using the types of patch technologies available from Novartis and
Alza Corporation. The administration of the pharmaceutical
compositions of the present invention can be intermittent, or at a
gradual, continuous, constant or controlled rate to a warm-blooded
animal, (e.g., a mammal such as a mouse, rat, cat, rabbit, dog,
pig, cow, monkey or human). In addition, the time of day and the
number of times per day that the pharmaceutical formulation is
administered can vary. Administration preferably is such that the
active ingredients of the pharmaceutical formulation interact with
receptor sites within the body of the subject that effect the
functioning of the central nervous system. More specifically, in
treating a neurodegenerative disease, administration preferably is
such so as to optimize the effect upon those relevant protease
and/or kinase subtypes (e.g., those which have an effect upon the
functioning of the central nervous system), while minimizing the
effects upon protease and/or kinase subtypes in muscle and ganglia.
Other suitable methods for administering the compounds of the
present invention are described in U.S. Pat. No. 5,604,231 to Smith
et al., the disclosure of which is incorporated herein by reference
in its entirety
[0290] The following examples are offered by way of illustration,
not by way of limitation.
EXAMPLES
Example 1
[0291] I. Materials and Methods
[0292] a. Preparation of Mouse Hippocampal Slice Cultures
[0293] Hippocampal slices were prepared from 10 to 13 day old
C57BL/6J (wild-type) and C57BL/6J-ApoEtm1Unc (ApoE-knockout) mice
obtained from the Jackson laboratory, Bar Harbor, Me. Pups were
placed under light bromo-chloro-trifluoroethane anesthesia (Sigma,
St. Louis, Mo.), and killed by decapitation. After removing the
brains, the hippocampus was dissected and subsequently placed on a
McIIwain tissue chopper where slices (400 .mu.m thick) were
obtained and placed in a solution of cutting medium consisting of
Minimum Essential Medium (MEM) with Earle's salts (Gibco, Grand
Island, N.Y.), 25 mM HEPES buffer, 10 mM Tris base, 10 mM glucose,
and 3 mM MgCl.sub.2, pH 7.20. Hippocampal slices were then placed
onto the membranes of Millicell-CM culture inserts (Millipore
Corp., Bedford, Mass.) in 6 well culture cluster plates and 1 ml of
media per well using the methods described by Stoppini et al., J.
Neurosci. Methods 37(2):173-82 (1991). The culture medium was
described previously by Bednarski et al., J. Neurosci.
17(11):4006-21 (1997). The cultures were incubated in a 37.degree.
C. atmosphere containing 5% CO.sub.2 and the culture medium was
replaced every other day until the initiation of experiments. Each
culture cluster plate contained hippocampal slices from either two
wild-type or two apoE-knockout mice and individual wells were used
for matched control and experimental treatment groups.
[0294] After maintaining the slices with normal culture medium
(Bednarski et al., J. Neurosci. 17(11):4006-21 (1997)) in vitro for
12-14 days, slices were incubated with culture medium containing
either 20 .mu.M N-CBZ-L-phenylalanyl-L-alanine-diazomethyl-ketone
(ZPAD; BACHEM Bioscience, Inc., Torrance, Calif.), an inhibitor of
cathepsins B and L (Shaw & Dean, Biochem. J. 186:385-390
(1980); Green & Shaw, J Biol Chem. 256:1923-1928 (1981);
Richardson et al., J. Cell Biol. 107: 2097-2107 (1988)) or vehicle
(dimethylsulfoxide; DMSO, 0.01%-0.04%) for six days. This treatment
media was exchanged every other day.
[0295] Cysteine protease inhibitors (calpain inhibitor I, III,
calpeptin; Calbiochem, San Diego, Calif.) were added at 10-100 mM
alone or in combination with ZPAD.
[0296] b. Histology
[0297] To prepare semithin sections, control and treated slices
from both wild-type and apoE-deficient mice were fixed in a
solution of 0.1M phosphate buffer ("PB"; pH 7.2), containing 1.5%
paraformaldehyde and 1.5% glutaraldehyde. After a period of two to
three hours, the solution was removed and the slices were rinsed
three times in phosphate buffered saline ("PBS"; 50 mM phosphate
buffer, 0.9% NaCl, pH 7.3). At this time, slices were postfixed in
2% osmium tetroxide in PB for one hour, dehydrated in a series of
alcohols and embedded in Polybed-812. Semithin (1 .mu.m thick)
sections were cut on a Sorvall Porter-Blum ultramicrotome and
stained with a solution of 0.1% toluidine blue. Digitized images
were imported using a Sony DKC-5000 camera attached to a Zeiss
microscope and processed using Adobe Photoshop.RTM..
[0298] c. Immunoblotting
[0299] Control and ZPAD-treated slices were collected in ice-cold
10 mM Tris-HCl harvest buffer consisting of 0.32 M sucrose, 2 mM
EDTA, 2 mM EGTA, and 0.1 mM leupeptin, pH 7.4, and centrifuged at
12,000.times.g for 5 minutes at 4.degree. C. At this point, the
pellets were resuspended in lysis buffer (8 mM HEPES, 1 mM EDTA,
0.3 mM EGTA, pH 8.0) and sonicated. The Bradford analysis was
performed (Bradford, M, Anal. Biochem 72:248-254 (1976)) and
100-120 .mu.g of protein from each sample was denatured by boiling
for 5 min with 2.5% (wt/vol) sodium dodecyl sulfate (SDS) and 3%
2-mercaptoethanol and then subjected to SDS-PAGE on 10% linear
gradient gels. See Laemmli et al., J. Mol. Biol. 47:69-85 (1970).
Resolved proteins were then transferred to nitrocellulose membranes
as described by Towbin et al., Biotech. 24:145-9 (1992), incubated
in 3% gelatin in Tris-buffered saline ("TBS"; NaCl 8 g, KCl 0.2 g,
Tris base 3 g in 1 liter distilled water, pH 7.4) for 1 hour at RT
followed by incubation with 1% gelatin in TBS with 0.5% Tween 20
("TTBS") containing an antibody that recognized tau-1 (1:100;
Boehringer Mannheim, Indianapolis, Ind.) at RT overnight.
Antibodies were localized by using the anti-IgG-alkaline
phosphatase conjugates and the 5-bromo-4-chloro-3-indolyl-phosphate
and nitro blue tetrazolium substrate system. Relative optical
densities and areas of immunobands were quantified using the NIH
image analysis system.
[0300] d. Immunohistochemical Procedures
[0301] For immunocytochemical staining, control and ZPAD-treated
slices from both wild-type and apoE-deficient mice were fixed in 4%
paraformaldehyde in 0.1 M phosphate buffer ("PB") at 4.degree. C.
overnight, rinsed once in a solution of phosphate buffered saline
PBS (PB: phosphate buffer: 0.1M Na.sub.2HPO.sub.4 and
NaH.sub.2PO.sub.4; PBS: phosphate buffered saline: NaCl 8 g, KCl
0.2 g, Na.sub.2HPO.sub.4 1.44 g, KH.sub.2PO.sub.4 0.24 g, dissolved
in 1 liter distilled water, pH 7.4) cryoprotected in 20% sucrose in
0.1 M PB, sectioned on a freezing microtome at 25 .mu.m parallel to
the broad upper surface of the explant and mounted onto sterile
Fisher Superfrost/Plus slides. After slides were preincubated with
10% normal goat serum (NGS) with 0.3% Triton X-100 for 1 hour at
room temperature ("RT"), sections were incubated with monoclonal
anti-PHF, AT8 (1:1000; Innogenetics, Belgium), in 5% NGS, at
4.degree. C. overnight. The following day, the sections were rinsed
in PBS and then incubated in biotinylated anti-mouse IgG (1:200,
Vector) for 2-3 hours at RT, followed by avidin-biotin conjugate
("ABC") (1:100, Vector) diluted in PBS for 1 hour. The binding of
the antibody was localized by using the avidin-biotin system
(1:100, Vector) with kit reagents and diaminobenzidine as
chromagen. As a control, tissue was processed through all
incubations as described above except the primary antisera was
omitted from the initial incubation.
[0302] e. Postembedding Immunocytochemistry
[0303] Lowicryl resin-embedded ultrathin sections (of 70-80 nm
thickness) were picked up on either pioloform-coated nickel slot
grids or pioloform-coated 400 mesh nickel grids. The grids were
incubated on drops of blocking solution, followed by incubation on
drops of primary antibodies (AT8 or PHF-1). After the incubation
with primary antibodies, the sections were washed in TBS (three
times for 10 min each) and in 50 mM Tris-HCl, pH 7.4, containing
0.9% NaCl ("TBS*"; once for 10 min; TBS* has 50 mM Tris-HCl and
0.9% NaCl without KCl, while TBS has 25 mM Tris-HCl and KCl) ) and
incubated on drops of goat anti-mouse IgG coupled to 10 nm gold
particles (British BioCell Int.). The secondary antibodies were
diluted 1:100 in TBS* containing 0.05% polyethylene glycol 20000
(BDH; Merck) and 1% gelatin for 2 hr at 28.degree. C. After
additional washing in TBS* (three times for 10 min each) and PBS
(once for 10 min), the sections were post-fixed in 2%
glutaraldehyde in PBS for 2 min at room temperature and then washed
in bidistilled water (three times for 10 min each). Finally, the
sections were contrasted with saturated aqueous uranyl acetate
followed by staining with lead citrate.
[0304] f. Electron Microscopic Analysis
[0305] For electron microscopic immunogold labeling, cultures were
subjected to freeze substitution techniques as previously described
(Schwarz et al., Scann. Microsc. 3 (Suppl.):57-63 (1989); Van
Lookem et al., J. Histochem. Cytochem. 39:1267-1279 (1991)). In
brief, the specimens were cryoprotected by immersion in graded
concentrations of glycerol (10, 20, and 30%) in phosphate buffer
and plunged into liquid propane (-170.degree. C.) in a cryofixation
unit (KF 80; Reichert, Wien, Austria). The samples were then
immersed in 0.5% uranyl acetate dissolved in anhydrous methanol
(-90.degree. C.) in a cryosubstitution unit (AFS; Reichert). The
temperature was raised in steps of 4.degree. C./h to -45.degree. C.
Samples were washed with anhydrous methanol and infiltrated with
Lowicryl HM20 resin at 45.degree. C. with a progressive increase in
the ratio of resin to methanol. Polymerization was carried out with
UV light (360 nm) for 48 h.
[0306] Ultrathin sections were cut with a Reichert ultramicrotome,
mounted on nickel grids and processed for immunogold cytochemistry
(Ottersen, Anat. Embryol. 180:1-15 (1989)). In brief, the sections
were treated with a saturated solution of NaOH in absolute ethanol
(2-3 s), rinsed in phosphate buffer and incubated sequentially in
the following solutions (at room temperature): (i) 0.1% sodium
borohydride and 50 mM glycine in Tris buffer (5 mM) containing
0.01% Triton X-100 and 0.3% NaCl ("TBNT"; 10 min); (ii) 0.5%
powdered milk in TBNT (10 min); (iii) primary antibody (AT8, 1:100;
2h); (iv) same solution as in (ii) (10 min); and (v)
gold-conjugated secondary antibodies (10 or 20 nm particles)
diluted 1:20 in TBNT containing powdered milk and polyethylene
glycol (5 mg/ml, 2 h). Finally, the sections were counterstained
and electron micrographs obtained by a Philips CM10 transmission
electron microscope.
[0307] II. Results
[0308] Morphological Studies
[0309] 1. Morphology of Cultured Hippocampal Slices
[0310] Both wild-type and apoE-knockout mice hippocampal slices
that were maintained in vitro for 12-14 days had morphologies
similar to the morphologies of the hippocampus in vivo. The
lamination of the hippocampus was clearly distinguishable even
though the pyramidal neurons were slightly less compacted, in
particular those of CA1 subfield. Neurons showed large centrally
located nuclei and well differentiated prominent apical and basal
dendrites. Within the cytoplasm, few basophilic organelles were
found. No obvious morphological differences were observed between
cultures from wild-type and knockout mice at the light microscopy
level, although lack of efficient sprouting following culturing has
been reported for apoE-deficient hippocampal cultures (Teter et
al., Neuros. 91:1009-6 (1999)).
[0311] 2. Morphological Changes Induced by Suppression of
Cathepsins B and L
[0312] Incubation with 20 .mu.M ZPAD for six days resulted in an
increase in the number of basophilic granules in both wild-type and
apoE-deficient hippocampal slices. Based on their size and
distribution and their similar appearance to those found in earlier
studies of ZPAD-treated rat cortical, hippocampal, hypothalamic,
and entorhino-hippocampal slices (Bednarski et al., J. Neurosci.
17:4006-21 (1997); Bi et al., Exp. Neurol. 158:312-327 (1999); Yong
et al., (1999); Exp. Neurol. 157:150-160 (1999)), these granules
represent lysosomes. While densely stained organelles were evident
in all subfields of hippocampus, a clear accumulation was found in
CA3 subfield along fiber like structures that laminated the cell
bodies on their apical dendrite side. From their location, these
lysosomes appeared to be mostly contained in mossy fibers that
project from granule cells to CA3 pyramidal neurons. The increase
in the number of lysosomes and the appearance of clusters of
basophilic granules in the mossy fiber terminal zone were observed
in cultures from both wild-type and apoE-deficient mice. However,
quantitative analyses of digitized images revealed that both
phenomena were substantially enhanced in apoE-deficient hippocampal
slice cultures. Additional pathologies were also found in the
knockout cultures but were rare in wild-type ones. First, numerous
large dark granules were found in the regions where apical
dendrites end, in both CA1 and CA3 subfields. These granules are
probably debris from degenerated cells. Second, neuropil in the
molecular layers surrounding the hippocampal fissure thinned out
and became more transparent, which also indicates neuritic
degeneration. Finally, large neurons contained inclusions of
different sizes and eccentrically localized nuclei were also
frequently observed.
[0313] Enhanced Upregulation of Cathepsin D in apoE-deficient
Hippocampal Slices
[0314] Immunoblotting was used to compare the concentrations of
cathepsin D in slices from wild-type versus knockout mice. Cultured
hippocampal slices had three major bands with apparent molecular
weights of .about.55 kDa, .about.50 kDa, and .about.38 kDa,
corresponding to the inactive proenzyme, the active single chain,
and the active heavy chain of cathepsin D, respectively (FIG. 5A).
ZPAD treatment for six days reliably increased the first two
isoforms in cultured wild-type slices. The proenzyme increased by
65.+-.29% (mean.+-.s.e.m.) relative to that in yoked controls that
were not infused with ZPAD (p<0.0001, paired t-test, n=9, FIG.
5A). A smaller increase was obtained for the single chain form
42.+-.22% (p<0.0001) but there were no evident effects on the
heavy chain (3.0.+-.5.7%, p>0.5). The differential effect of
ZPAD across the isoforms was highly significant (p<0.0001,
F=37.3, ANOVA), as were the differences in the increases between
subunits (p<0.01).
[0315] ZPAD produced more striking increases in the concentration
of all isoforms of cathepsin D in apoE-knockout slices (FIG. 5A).
Relative to yoked knockout controls, the inhibitor increased the
proenzyme by 145.+-.43% and the single chain by 150.+-.29%. In
contrast to the results obtained for the wild-type slices, ZPAD
also caused a marked increase in the cathepsin D heavy chain
relative to control values (84.+-.26%, p=0.0006). It is noteworthy
that the differential increase in the pro-enzyme versus single
chain found for the wild-type slices did not occur in the slices
from apoE-knockouts. The differences between wild-type and
apoE-knockout slices with regard to ZPAD induced increases in
cathepsin D isoforms were statistically significant (FIG. 5B).
[0316] In all, upregulation of cathepsin D in response to lysosomal
dysfunction was substantially greater in apoE-deficient mice than
in wild-type controls.
[0317] Enhanced Induction of Tangle-like Structures in Cultured
Slices Prepared from apoE-knockout Rodents
[0318] 1. Immunocytochemical Studies
[0319] Immunocytochemical staining was carried out using monoclonal
antibody "AT8" that recognizes full sized tau protein and tau
protein fragments phosphorylated at residues Ser-202 and
Thr-205.
[0320] Hippocampal slices from wild-type were treated with ZPAD.
After six days of treatment with ZPAD, thick filaments that were
densely stained by antibodies against hyperphosphorylated tau were
occasionally found within neurons in superficial layers of
entorhinal cortex. The intracellular location and appearance of
these structures corresponded to published descriptions of
early-stage tangles. More mature tangle-like profiles were found at
a number of sites after 12-day incubations. Immunoblots indicated
that essentially all phosphorylated tau labeling in the slices
involved proteins approximately 15-35-kDa in size, confirming that
the immunostained filamentous structures were composed of tau
fragments. While these results established that early-stage tangles
follow from lysosomal dysfunction in cultured slices, the number of
such profiles per unit area was far below that found in Alzheimer's
disease brains.
[0321] Hippocampal slices from the apoE-knockout mice were treated
with ZPAD as described above. Typical results are summarized in
FIG. 2. Shown are sections through the CA1/subicular transition
zone from a control slice (FIG. 2A) and from a slice incubated with
ZPAD for six days, followed by six days washout (FIG. 2B). The
material has been immunostained with antibodies developed against
human tangles and that recognize phosphorylated tau and its 15-35
kDa fragment. These survey micrographs demonstrate that
immunopositive structures are present in large numbers in the
experimental slice, something that was not achieved with wild-type
slices even after prolonged incubations. The dense structures were
also absent from the control slices. The results shown in FIG. 2
are typical of effects obtained with different litters tested over
a period of several months.
[0322] Closer examination revealed a number of types of "AT8"
immunopositive structures. These different structures possibly
represent cells at various stages of tangle formation, depicting a
progression from early-stage tangles to cell death. Higher power
micrographs of typical immunopositive structures are shown in FIG.
1. The cell marked as #1 has a dense structure at one pole of its
soma and a thin, lightly labeled process. Other cells (e.g., #2c)
have stained twisted processes emerging from the soma and ending in
fragments. Another version of this can be seen in cell #3. In this
instance the cell body is connected to a bulbous structure by a
filament. The cell adjacent to #3 has a similar profile as well as
a nearby sphere filled with well-stained filaments. It should be
noted that the somata in most of these cases are anatomically
distorted. The panel on the right side shows examples in which the
cell appears to have ruptured and the immunostained fibrils have
extruded into the extracellular space (#4 and #5). The selected
cells (1-5) may represent stages of a progression from the
intracellular buildup of tau fragments, to the development of
intracellular tangles and abnormal processes, to cell death with
persistence of the tangles.
[0323] Slices from wild-type mice without ZPAD treatment showed
slight neuropil AT8-immunoreactivity and without evident cell body
staining. In contrast, some of the untreated slices from
apoE-deficient mice had low to moderate numbers of densely
AT8-immunoreactive (AT8-ir) structures that were limited to the
subiculum and hippocampal field CA1a (Sub/CA1a) and mainly located
in stratum oriens. The number of these structures was significantly
increased by application of ZPAD for six days and the affected
areas expanded from Sub/CA1a to Sub/CA1a-c (FIG. 2B). Quite often
AT8-ir neurons with larger cell bodies were also encountered in
stratum lacunosum-molecular of field CA1. Occasionally AT8-ir cells
were found in stratum oriens of field CA3. In contrast, hippocampal
cultures from wild-type mice treated with ZPAD exhibited far fewer
AT8-ir structures and the territory containing these structures was
also much smaller. Unlike the case of ZPAD-treated apoE tissues
where large numbers of AT8-ir structures were found in almost every
single section, the incidence in ZPAD-treated wild-type sections
was also lower.
[0324] To study the ultrastructure of tangle-like formations,
electron microscopic immunogold technique was used. Numerous
intracellular inclusions were found in cells that had been treated
with ZPAD for 6 days. Shown in FIG. 3A is a dendritic branch with
accumulated organelles resembling smooth ER (arrowheads), rough ER
(asterisks), or mitochondria (M). Distorted microtubules were found
passing through the abnormal inclusions. Despite these obvious
pathologies, plasma membranes and synaptic apparatus were still
distinguishable. Secondary lysosomes with variable sizes were also
frequently encountered in ZPAD-treated tissues (FIG. 3B).
Immunogold analysis showed that AT8-ir was found mainly over
structures composed of distorted microtubules located throughout
dendrites and cell bodies. Enlarged images showed that microtubules
were often paired and twisted with axial periodicity (FIG. 4A and
B). Distorted microtubules were found running across each other or
waving around, characteristics similar to early-stage
neurofibrillary tangles in Alzheimer's disease (FIG. 4C).
[0325] 2. Immunoblotting Studies
[0326] Immunoblots carried out using the anti-nonphosphorylated tau
protein antibody, tau-1, detected a few moderately to densely
immunopositive bands at .about.50-55 kDa that corresponded to the
different isoforms of native tau proteins in untreated hippocampal
slice cultures from both apoE-knockout and wild-type mice.
Occasionally, other stained bands were observed migrating at
apparent molecular weights of 15-35 kDa, and were assumed to be
breakdown products of tau. Tau isoforms and the breakdown product
did not differ significantly between the two groups. Six days of
ZPAD treatment resulted in a reduction in the native tau proteins
in hippocampal slices from both apoE-knockout mice and wild-type
mice. A statistic analysis showed that ZPAD-induced reduction was
significantly greater in the knockout group than in wild-type
group: 35.+-.1.1% versus 22.+-.2.6% (n=6, p<0.001). In parallel
to the reduction of native tau, ZPAD treatment also increased the
levels of the 15-35 kDa fragments in both groups. While the
increase appeared to be larger in apoE-knockout mice than in
wild-type mice, 277.+-.20% vs 240.+-.19%, the difference did not
reach statistic significance.
Discussion
[0327] The above results provide, among other things, the
following. 1) Tangle-like structures can be induced in culture
slices in a medium which triggers lysosomal dysfunction and/or
selectively increases cathepsin D. 2) Incubating cultured
hippocampal slices from apoE-deficient mice with an inhibitor of
cathepsins B and L for 6 days resulted in the formation of
tangle-like structures in the subiculum and hippocampal field CA1
that was far more numerous than in wild-type mice. 3) Electron
microscopic immunogold analysis revealed that the tangle-like
structures were composed of distorted microtubules that had
paired-helical like features. 4) Degradation of tau proteins was
significantly greater in apoE-knockout than in wild-type mice.
[0328] Thus, the present invention provides a first instance in
which tangle-like profiles have been induced in culture slices.
Moreover, the present invention provides clear evidence, for the
first time, for the relationship between a predisposing condition
of Alzheimer's disease, apoE, and the formation of neurofibrillary
tangles, one of the major pathologies in Alzheimer's disease. The
location of tangle-like structures corresponds to that in tissues
from Alzheimer's disease patient. The tangle-like structures are
composed mainly of tau fragments that are similar in size as
discovered in neurofibrillary tangles in Alzheimer's disease.
[0329] Neurofibrillary tangles have long been recognized as the
hallmarks of Alzheimer's disease and the existence of a close
correlation between the presence and distribution of
neurofibrillary tangles and the degree of cognitive impairment in
Alzheimer's disease further emphasizes the critical role of tau
pathology in the development of the disease. Hyperphosphorylated
tau proteins tend to dissociate from microtubule and assemble into
paired helical filaments. Other factors proposed to facilitate the
aggregation of tau include oxidation, polyanions, and nucleation.
In vitro tests have demonstrated that all tau isoforms are able to
aggregate, however, tau fragments containing the repeat domain
exhibit faster kinetics in in vitro assembly tests. Thus, not
wishing to be bound by a theory, fragmentation of tau could be a
significant factor that enhances the aggregation of tau and causes
the generation of tangle like structures.
[0330] Incubation of hippocampal slice cultures from mice with an
inhibitor of cathepsins B and L resulted in 15-35 kDa tau fragments
and AT8-ir structures that resembled early-stage tangles. However,
generation of large numbers of tangle-like structures was only
observed in apoE-knockout mice. Hyperphosphorylated tau
immunopositive neurons were also found in field subiculum and CA1
areas in some untreated apoE-knockout slices, even though the
numbers were much smaller than in ZPAD-treated apoE slices. Further
statistic analysis showed that the incidence of the spontaneous
AT8-ir neurons found in apoE-knockout mice was also lower than that
in ZPAD-treated wild-type tissue. Not wishing to be bound by a
theory, these results suggest that while apoE deficiency and
lysosomal dysfunction are both facilitating factors for the
formation of tangle-like structures, lysosomal dysfunction seems
more potent. The effects of these two factors are not simply
additive because the number of tangle-like structures in
ZPAD-treated apoE tissues was more than double that in
apoE-untreated or ZPAD-treated wild-type slices. Thus, lack of apoE
gene makes the tissue extremely susceptible to pathologies
associated with lysosomal dysfunction.
[0331] ApoE is a major risk factor for late onset sporadic
Alzheimer's disease: apoE is co-localized with the neurofibrillary
tangles and senile plaques, and the burden of both
A-beta-containing plaques and neurofibrillary tangles is increased
in a dose-dependent manner in Alzheimer's disease patients with
apoE4. In vitro experiments showed that apoE2 and apoE3 were able
to bind to microtubules and form stable complexes with the
microtubule-associated proteins tau and MAP2c while apoE4 lacks
this ability (Strittmatter et al. (1994), supra). Not wishing to be
bound by a theory, apoE3, by binding to tau, protects tau from
being hyperphosphorylated and thus prevents the generation of
intracellular neurofibrillary tangles. On the other hand, the
formation of apoE-tau complex has been shown to be dependent on the
phosphorylation state of tau; phosphorylation of Ser262 within the
microtubule binding domain of tau has been shown to prevent binding
of apoE (Huang et al., Neurosci Lett. 192:209-12 (1995)).
Therefore, the phosphorylation state of tau proteins, altered by
missing the stabilization effect from apoE (that is more like
apoE3), could be one of the reasons that more tangle-like
structures were formed in knockout mice.
[0332] Among other things, the present invention provides that
tangle-like structures can be induced in brain cells by contacting
the brain cells with a medium that triggers lysosomal dysfunction
and/or increases cathepsin D. Moreover, the present results
demonstrated that the absence of apoE significantly enhanced the
susceptibility of the tissue to insults that caused lysosomal
dysfunction, and the induction of neurofibrillary tangles.
Example 2
Increases in Cathepsin D Associated with Lysosomal Dysfunction are
Enhanced in Apolipoprotein E-Knockout Mice
[0333] As apoE is currently the only confirmed risk factor for
late-onset Alzheimer's disease, tests were undertaken to determine
whether upregulation of cathepsin D, a sign of Alzheimer's disease
pathology, is more pronounced in slices from apoE-deficient than in
wild-type mice. Immunoblotting stained with anti-cathepsin D showed
that homogenates of cultured hippocampal slices exhibited three
major bands with apparent molecular weights of .about.55 kDa,
.about.50 kDa, and .about.38 kDa, corresponding to the inactive
proenzyme, the active single chain, and the active heavy chain of
cathepsin D, respectively (FIG. 5A). ZPAD treatment for six days
reliably increased the first two isoforms in cultured slices from
wild-type mice. The proenzyme increased by 65.+-.29%
(mean.+-.s.e.m.) relative to that in controls that were not infused
with ZPAD (p<0.0001, paired t-test, n=9, FIG. 5B). A smaller
increase was obtained for the single chain form (42.+-.22%;
p<0.0001), but there were no evident effects on the heavy chain
(3.0.+-.5.7%, p>0.5). The differential effect of ZPAD across the
isoforms was highly significant (p<0.0001, F=37.3, ANOVA) as
were the differences in increases between subunits (p<0.01).
[0334] ZPAD produced more striking increases in the concentration
of all isoforms of cathepsin D in apoE-knockout slices (FIG. 5A).
The proenzyme was increased by 145.+-.43% and the single chain by
150.+-.29%. In evident contrast to the results obtained for the
slices from wild-type mice, ZPAD also caused a marked increase in
the cathepsin D heavy chain (84.+-.26%, p<0.01). It is
noteworthy that the differential increase in pro-enzyme versus
single chain found in slices from wild-type mice did not occur in
slices from apoE-knockouts. The differences between wild-type and
apoE-knockout with regard to ZPAD-induced increases in cathepsin D
isoforms were statistically significant (FIG. 5B).
[0335] In all, the results demonstrate that upregulation of
cathepsin D in response to lysosomal dysfunction is substantially
greater in apoE-deficient mice than in wild-type controls.
Example 3
Regional Induction of Intraneuronal Neurofibrillary Tangles in
Cultured Slices Prepared from apoE-knockout Mice
[0336] Cultured hippocampal slices prepared from apoE-deficient
mice were exposed to an inhibitor of cathepsins B and L and then
processed for immunocytochemistry using antibodies against human
paired helical filaments. Dense, immunopositive deposits were found
in the subiculum, stratum oriens of field CA1, and the hilus of the
dentate gyrus. This distribution agrees with that described for
tangles in AD. The appearance of the labeled structures fell into
categories that correspond to previously proposed stages in the
progression of intraneuronal neurofibrillary tangles in human
hippocampus. Electron microscopic analyses confirmed that
microtubule disruption and twisted filaments were present in
neurons in the affected areas. These results support the hypothesis
that partial lysosomal dysfunction is a contributor to Alzheimer's
disease and suggest a simple model for studying an important
component of the disease.
[0337] Cultured hippocampal slices were prepared from 10-12 day old
C57BL/6J-apoE.sup.tmIUnc (apoE-knockout or apoE -/-) or C57BL/6J
(wild-type) mice and kept in vitro for 12-14 days before being
exposed to medium containing ZPAD, a selective inhibitor of
cathepsins B and L (Bahr, B. A., et al., Exp. Neurol. 129:1-14
(1994); Heinonen, O., et al., Neuroscience 64:375-384 (1995);
Heffernan, J. M., et al., Exp. Neurol. 150:235-239 (1998)) or
vehicle (DMSO, 0.04%) for 6 days. Immunocytochemical staining was
carried out using monoclonal antibody "AT8" which recognizes the
full-length human tau protein (and tau fragments) phosphorylated at
residues Ser-202 and Thr-205. The results described here involved
detailed analysis of multiple sections from 30 apoE -/- and 30
wild-type slices treated with ZPAD. A smaller number of vehicle
alone slices in each group were also examined. Double labeling to
confirm the identification of cells as neurons was carried out in
four apoE -/- slices using an antibody against neuronal nuclear
protein.
[0338] Slices from knockout mice were comparable in size and
appearance to their wild-type counterparts. Some, but not all,
untreated apoE -/- slices had labeled cells in the outgrowth zone
that develops in the first week after explantation. However,
immunopositive neurons were only rarely found within the dendritic
and cell body layers of hippocampus itself (FIG. 6, right
panel).
[0339] Numerous, densely stained neurons were present within
hippocampus and retrohippocampal cortex in nearly all of 30 apoE
-/- slices treated with ZPAD for 6 days. These were numerous in the
subiculum and field CA1 and relatively uncommon in dentate gyrus
and field CA3. Within CA1 the profiles were usually much more
frequent in the stratum oriens than in the pyramidal cell layer or
stratum radiatum. This can be seen clearly in the higher power
micrograph presented in FIG. 8. This figure also illustrates the
extent to which most neurons and their processes were unstained by
the PHF antibody, even in apoE -/- slices treated with ZPAD. Note,
for example, that the densely packed cell bodies in stratum
pyramidale, as well as the profusion of apical dendritic branches
in stratum radiatum, are barely detectable in the micrograph. The
pattern seen in FIGS. 6 and 8 held for most slices; a common
variation involved the presence of significant numbers of labeled
cells in the s. pyramidale and s. radiatum of field CA1 and in the
hilus of the dentate gyrus.
[0340] Effects of the type just described were not seen after much
longer treatments in prior studies using cultured slices from rat
hippocampus (Jicha, G. A., et al., J Neurosci 19:7486-7494 (1999)).
Wild-type mice were intermediate between rats and apoE -/- mice.
Slices with clear anatomical landmarks in the CA1/subiculum
boundary region were selected for estimating the magnitude of the
difference between the two mouse groups. Counting was done in a 0.3
mm.sup.2 box centered over the stratum oriens/pyramidal layer at
the CA1/subicular border. The number of AT8 immunopositive profiles
in the knockouts was 193.8.+-.15 (mean.+-.s.e.m., n=5) and
112.6.+-.13 (n=5) in the wild-types, a difference that was highly
significant (p=0.005, two-tail t-test). These results confirm that
the apoE mutation contributes to the formation of intraneuronal
neurofibrillary tangles.
[0341] While ZPAD had robust and reliable effects across apoE -/-
slices, the immunopositive cells within a given slice were not
homogeneous in appearance. Most of the labeled neurons were
shrunken and had `polar caps`; i.e. dense deposits located
eccentrically within the somata. Examples of these are marked as
`1` in FIG. 7. The degree of cell shrinkage can be appreciated by
comparing the immunopositive elements to the unlabeled neuron
outlined within the stratum pyramidale. In many cases the cells had
immunopositive processes extending away from the somata for
considerable distances. Neurons with labeled, pathological
dendrites (`2` in FIG. 7) as well as `caps` of labeled material
unconnected to cell bodies (`3` in FIG. 7) were also commonplace
throughout the stratum oriens and subiculum. The isolated `caps`
may be remnants of neurons. Double labeling experiments (not shown)
confirmed that the shrunken profiles were neurons.
[0342] The variety of densely stained elements found in the
ZPAD-treated apoE -/- slices is not unlike the diversity of
intraneuronal NFTs found in hippocampus during early-stage
Alzheimer's disease and/or other related disorders. This point is
illustrated in FIG. 8. The upper panels, from an apoE slice, are
higher power micrographs organized according to a progression along
the lines proposed for NFT development in Alzheimer's disease
(Chin, J. Y., et al., J Neuropathol Exp Neurol 59:966-971 (2000);
Auer, I. A., et al., Acta Neuropathol (Berl) 90:547-551 (1995)).
The steps are as follows: Panel A. Essentially intact neurons with
immunopositive cell bodies and dendrites; Panels B & C. Dense,
localized deposits within the cell body accompanied by evident
dendritic abnormalities such as clubbing (arrow); Panels D & E.
Expansion of initial dendritic segments; Panel F. Loss of dendritic
organization, sometimes accompanied by the growth of large filament
filled structures resembling growth cones (arrows); Panels G &
H. Disappearance of the neuron leaving a cap of labeled
material.
[0343] The bottom panels of FIG. 8 are from field CA1 of an
early-stage human Alzheimer's disease brain. Immunostaining was
carried out with the same procedures and antibody used for the
sections from the apoE slices. Panel A shows a typical neuron with
labeled dendrite and cell body `cap` (arrow). Panels B, C, & D
illustrate the dendritic abnormalities that are commonplace at this
stage of the disease. Note the apparent clubbing and fragmentation
(arrows) at sites removed from the cell body. Panel E shows a
swelling proximal to the soma; examples of neuronal remnants are
found in panels F & G.
[0344] Electron microscopic analyses of zones with labeled neurons
confirmed that the pathological changes detected with PHF
antibodies were accompanied by the development of aberrant
filaments. The proximal apical dendrite was often nearly filled
with a dense plexus of filamentous material, as shown in the
micrograph in FIG. 9A. Closer examination of the filaments from
this (FIG. 9B) and other (e.g., FIG. 9C) neurons shows them to be
long twisting structures that frequently cross each other (arrows).
Figures of this type were unique to ZPAD-treated slices.
[0345] Several lines of evidence indicate that NFTs assemble from
mixtures of tau and tau fragments (von Bergen, et al., Proc Natl
Acad Sci USA 97:5129-5134 (2000); Pei, J.-J., et al., J Neuropathol
Exp Neuro 58:1010-1019 (1999)), and it is likely that tau
proteolysis is an essential step in tangle formation. Accordingly,
immunoblots were used to test if accelerated breakdown of tau might
account for the enhanced build-up of intraneuronal NFTs in the apoE
-/- slices. The antibody `tau-1` detected a set of tau isoforms
(50-55 kDa) in untreated hippocampal slice cultures from both apoE
-/- and wild-type mice. Six days of ZPAD treatment caused a
22.+-.2.6% (mean+s.e.m.) reduction in native tau in slices from
wild-type animals and a 35.+-.1.1% reduction in apoE-knockout
slices (n=6, p<0.001, t-test).
Example 4
Induction of Early-Stage Neurofibrillary Tangles is Triggered by
Cholesterol-Lowering Agents; these Effects are Enhanced by
Lysosomal Dysfunction and Include Glial Reactions and the
Upregulation of Cytokines
[0346] The inhibition of cholesterol synthesis induced tangle-like
structures in cultured rat hippocampal slices and this effect was
markedly enhanced in the presence of ZPAD.
[0347] To test the effect of manipulating intracellular lipid
levels on lysosomal dysfunction-induced tangle formation, cultured
rat hippocampal slices were incubated with ZPAD in the presence of
the lipid metabolism inhibitor, mevastatin. Incubation of rat
hippocampal slices with ZPAD results in only a small number of
anti-phosphorylated tau (AT8) immunoreactive (ir) structures.
However, in the presence of a cholesterol-lowering agent
(mevastatin), ZPAD treatment caused robust induction of tangle-like
structures. As shown in FIG. 10, and FIG. 11, these AT8-ir cells
exhibited similar morphological characteristics as those found in
cultured slices from apoE-deficient mice. Moreover, the regional
distribution of AT8-ir in the ZPAD plus mevastatin treated slices
were mainly observed in the subiculum and field CA1, areas showing
AT8-ir neurons in apoE-deficient cultures. AT8 immunostaining is
moderately increased in ZPAD or mevastatin-treated tissue, and a
further increase is present in the mevastatin plus ZPAD treatment
groups. Similar results were observed in six different
experiments.
[0348] These results suggest that cholesterol-lowering agents, and
specifically inhibitors of lipid metabolism, and more specifically
inhibitors of cholesterol synthesis, may produce significant
neurodegenerative-like pathologies. It is noteworthy that
incubation of cultured slices with mevastatin alone resulted in
tangle-like structures and that knockout animals were not used.
[0349] Immunoblotting results showed that the combined application
of mevastatin and ZPAD induced a novel tau breakdown product with
an apparent molecular weight of .about.33 kDa when probed with
tau-1 antibody that reacts with the non-phosphorylated protein.
Western blots probed with AT8 antibody that recognized the
phosphorylated forms, showed that the 33 kDa breakdown products
were markedly enhanced in samples from mevastatin/ZPAD AND
mevastatin only (FIG. 12). These results suggest that the 33 kDa
breakdown products exist in both phosphorylated and
non-phosphorylated form, and the ZPAD plus mevastatin treatment
increases the former more than the latter.
[0350] Immunoblotting was carried out by using anti-non
phosphorylated tau antibody, tau-1 or anti-phosphorylated tau
antibody, AT8. Densitometric analysis of blots stained with tau-1
antibody showed that while ZPAD treatment induced decreases in the
native tau proteins and increases in fragments at apparent
molecular weights of 40, 33, and 29 kDa, combined application of
ZPAD and mevastatin enhanced the increase in levels of p33
fragments (FIG. 12, upper panel). The lower panel of FIG. 12 shows
levels of p33 tau phosphorylated at residues 199 and 202 (detected
with AT8 antibody.) *, p<0.05, ** p<0.01.
[0351] Several protein kinases have been shown to be involved in
the phosphorylation of tau proteins. Among these are cyclin
dependent protein kinase 5 (cdk5) and mitogen activated protein
kinase (MAPK). FIG. 13 shows that treatment of cultured hippocampal
slices with mevastatin induced significant decreases in the levels
of p35, the regulatory component of cdk5.
[0352] FIGS. 14A and 14B illustrate the dose response and time
course of p35 following mevastatin or mevastatin plus ZPAD
treatment. Hippocampal slices were cultured from 12 day-old rat
pups and kept in vitro for 12 days before exposure to mevastatin.
For the dose curve experiments. slices were subjected to mevastatin
for 6 days at 0 .mu.M, 1 .mu.M, 5 .mu.M, 10 .mu.M, and 100 .mu.M
concentrations. For the time course experiment, hippocampal
cultures were incubated with 10 .mu.M mevastatin for 0, 2, 4, and 6
days. In the mevastatin plus ZPAD treatment, ZPAD was used at 20
.mu.M. Hippocampal slices were collected, homogenized, and
subjected to SDS-PAGE electrophoresis. Immunoblots were then probed
with anti-p35 sera that were raised against the C-terminal domain
of p35.
[0353] Down regulation of p35 by mevastatin is blocked by the
application of mevalonate (FIG. 15). Hippocampal slices were
prepared from apoE-knockout mice at postnatal day 13, cultured in
vitro for 12 days, and then incubated with vehicle alone (control),
mevastatin, mevastatin plus ZPAD, EA1, cholesterol, or mevalonate,
a product of HMG-CoA reductase. Down regulation of p35 induced by
mevastatin is completely blocked by mevolonate.
[0354] Increasing evidence has indicated that inflammation is an
important component of AD-related pathology (Akiyama, H., et al.,
Neurobiol. Aging 21:383421 (2000); Rogers, J., et al., Neurobiol.
Aging 17:681-686 (1996); Eikelenboom, P., et al., Exp. Neurol.
154:89-98 (1998)). For instance, classic features of immune
reactions have been found in Alzheimer's disease brains, including
increases in proinflammatory cytokines, activation of microglia and
astrocytes, and the existence of complement proteins in neuritic
plaques. Epidemiological studies have shown that the use of
non-steroidal anti-inflammatory drugs reduces the risk and slows
the progression of the disease.
[0355] To determine whether experimentally-induced tangle-like
structures were also associated with inflammatory reactions, mRNA
levels for several cytokines were analyzed by the RT-PCR technique.
Additionally, the activation of mitogen-activated protein kinase
(MAPK) has been implicated in the activation of microglia, thus
tests were undertaken to determine the effects of an inhibitor of
MAPK kinase (PD98059), and to assess if MAPK is involved in
neurofibrillary tangle formation. mRNA levels for certain cytokines
were decreased by PD98059 treatment (FIG. 16). Treatment of
cultured hippocampal slices with mevastatin or ZPAD-triggered
increases in cytokines TGF and IL-10. The combination of mevastatin
and ZPAD (Mev/ZPAD) markedly increased the levels of both TGF and
IL-10 mRNA. PD98 and PD98/ZPAD are groups treated with PD98059 (a
mitogen-activated protein kinase inhibitor) or PD98059 plus ZPAD
respectively. Upregulation of cytokine mRNAs is specific to the
disruption of lipid metabolism.
[0356] RT-PCR and Northern Blot Analyses of Cytokines.
[0357] Increases in pro-inflammatory cytokines including IL1-alpha,
IL1, IL6, and IL10, TNF-alpha, and TGF have been reported in
Alzheimer's disease brains. To characterize glial reaction
following ZPAD and mevastatin treatment, the levels of mRNA for
these cytokines were analyzed by RT-PCR.
[0358] Levels of mRNA for the cytokines were quantified by RT-PCR
and northern blot analysis, following protocols outlined in the
RT-PCR kit (Ambion Inc.). The results demonstrated that
experimentally-induced lysosomal dysfunction and/or application of
mevastatin (20 .mu.M) increased mRNA levels of TGF-beta and
IL-10.
[0359] Treatment of cultured hippocampal slices with mevastatin
triggered increases cytokine TNF-alpha. (FIG. 17). Note, only the
inhibitor of cholesterol metabolism, mevastatin, appeared to
markedly increase the level of TNF-alpha.
[0360] Immunocytochemical Studies of Microglial Activation
[0361] Immunocytochemical studies using the monoclonal antibody
ED-1 that recognizes reactive microglia were used at 1:1000
dilution following standard immunohistochemical procedures.
Microglial activation was determined by measuring cell numbers,
optical density, and size of cell bodies using the program NIH
Image.
[0362] Brain tissue was cultured for 12 days and treated with ZPAD
(20 .mu.M) in the presence or absence of PD98059 (50 .mu.M) for 6
days (FIG. 18). Cultured explants were then sliced and stained by
using monoclonal antibody ED-1 which recognizes reactive microglia,
a classical marker of inflammation. Note that incubation with ZPAD
triggered significant reaction of microglia, and this reaction was
completely blocked by co-application of PD98059. Inhibition of MAPK
by itself did not induce evident changes in microglia.
[0363] Rat brain tissues were cultured for 10 days and incubated
with vehicle (Cont), ZPAD (20 .mu.M), mevastatin (Mev, 20 .mu.M),
or mevastatin plus ZPAD (Mev/ZPAD) for 6 days (FIG. 19). Cultured
brain explants were then sliced and stained by using monoclonal
antibody ED-1, a classical marker for reactive microglia and
macrophages. Note that treatment with ZPAD triggered significant
reaction of microglia that became larger and their cell bodies were
filled with ED1 immunopositive granules. Treatment with mevastatin
induced dramatic morphologic changes of microglia; these cells
became round and lost their characteristic thin processes. However,
ED1-stained granules that resemble phagosomes were evident in most
cells, suggesting the transformed cells maintained their phagocytic
character.
[0364] FIG. 20 is an immunoblot using anti-active MAPK (Sigma,
1:10,000). FIG. 20 demonstrates that MAPK (ERK1/2) was activated by
ZPAD and mevastatin treatment in the hippocampal slices. Not only
did application of mevastatin and ZPAD activate MAPK, but also this
effect was blocked by MAPKK inhibitor PD98059.
[0365] FIGS. 21A and 21B show the response and time course of MAPK
following mevastatin treatment. Cultured hippocampal slices were
treated with mevastatin or mevastatin plus ZPAD. For the dose curve
experiments, slices were subjected to mevastatin for 6 days at 0
.mu.M, 1 .mu.M, 5 .mu.M, 10 .mu.M, and 100 .mu.M concentrations.
For the time course experiment, hippocampal cultures were incubated
with 10 .mu.M mevastatin for 0, 2, 4, and 6 days. In the mevastatin
plus ZPAD treatment, ZPAD was used at 20 .mu.M. Immunoblots were
probed with the monoclonal anti-MAPK/ERK antibody that recognizes
the diphosphorylated (activated) isoforms of MAPK.
[0366] Summary
[0367] These results demonstrate that: a) compounds that inhibit
cathepsin D block the formation of tau fragments; b) upregulation
of cathepsin D in response to lysosomal impairment is greater in
brain tissue from apoE-knockout mice than in brain tissue from
wild-type controls or from rats; c) neurofibrillary tangles are far
more frequent and develop more quickly after the onset of lysosomal
dysfunction in brain tissue from apoE-knockout mice than in brain
tissue from wild-type controls or from rats; d) disturbance of
cholesterol synthesis and/or availability and/or levels of
cholesterol induces neurofibrillary tangles and/or phosphorylated
tau and/or tau fragments and/or the production and/or release of
cytokines and/or microglia reactions and/or activations and/or
inflammation and/or conversion of p35 to p25 and/or the levels and
activities of protein kinases, and these effects are further
enhanced by lysosomal dysfunction; e) the increases in
neurofibrillary tangles and/or phosphorylated tau and/or tau
fragments and/or the production and/or release of cytokines and/or
microglia reactions and/or activations and/or inflammation and/or
conversion of p35 to p25 and/or the levels and activities of
protein kinases triggered by lysosomal dysfunctions and/or
increases in Cathepsin D and/or decreases in cholesterol levels are
blocked by inhibitors of mitogen-activated kinases, f) inflammation
co-exists with early-stage neurofibrillary tangles. The instant
invention reproduces cardinal features of neuropathology including:
hyperphosphorylation of tau, fragmentation of tau, formation of
neurofibrillary tangles, increased production and/or release of
cytokines, increased microglia reaction and/or activation,
increased inflammation, and/or increased conversion of p35 to p25
changes in the levels and activities of protein kinases, and/or
other characteristics of neurodegeneration, including Alzheimer's
disease.
Example 5
Tau Fragmentation and the Formation of Neurofibrillary Tangles is
Blocked by Inhibition of the Cysteine Protease Calpain
[0368] Cultures of hippocampal slices were prepared from 10-12 days
old rats. Slices were kept in vitro for 12-14 days before being
exposed to a medium containing ZPAD (a selective inhibitor of
cathepsins B and L) and/or vehicle (DMSO, 0.04%) for 6 days and/or
a cysteine protease inhibitor (calpain inhibitor I).
A. Lysosomal Dysfunction Induced Conversion of p35 to p25 was
Blocked by Calpain Inhibitors
[0369] Immunoblotting carried out using antisera that recognizes
the C-terminal domain of p35 showed that the CDK5 binding protein
p35 was present in cultured hippocampal slices. Trace amount of
p25, the truncated form of p35 that lacks the N-terminal domain,
was also detected. A six day treatment of the brain cells with ZPAD
(a selective inhibitor of lysosomal hydrolases cathepsin B and L)
resulted in a significant decrease in the amount of p35 polypeptide
and a paralleled increase in the truncated form p25. Such
conversions of p35 to p25 were significantly inhibited in the
presence of calpain inhibitor I (see FIG. 22).
B. Tau Fragmentation Events Triggered by Experimentally Induced
Lysosomal Dysfunction were Blocked by Calpain Inhibitors
[0370] Imnunoblots stained with the anti-non-phosphorylated
antibody (tau 1), revealed that 6-day ZPAD treatment induced a
truncation of native tau proteins and the generation of tau
fragments that migrated at about 40 kDa, 29 kDa (tau 29), and 15-35
kDa. Previous studies have shown that cathepsin D is a protease
whose activation leads to the cleavage of tau. Incubation with
cathepsin D inhibitors remarkably reduced the production of tau
15-35 induced by ZPAD treatment, but the cathepsin D inhibitors
failed to block the increase in the 40 kDa fragments. Such results
suggested that another protease may be activated by the ZPAD
treatment. A previous study had suggested that calpain was able to
cleave tau and generate tau fragments of different lengths. See
Mercken et al., FEBS letters, 368 (1995). To test whether calpain
is involved in ZPAD-induced tau cleavage, levels of tau
fragmentation were compared between slices incubated with and
without calpain inhibitors. Results obtained from 2 separate
experiments showed that ZPAD-induced tau 15-35 and tau 40 were
almost completely blocked by calpain inhibitor I (See FIG. 23).
C. ZPAD-Induced Tangles were Blocked by Calpain Inhibitors
[0371] Incubation of hippocampal slices with ZPAD for 6 days
induced numerous tangles, in particular, in the border of subiculum
and CA1 region. However, when ZPAD was applied in the presence of
calpain inhibitor I, the number of tangles was significantly
reduced (See FIG. 24).
[0372] The above results provide, among other things, the
following. 1) -The formation of tangle-like structures can be
inhibited by contacting brain cells with a cysteine protease
inhibitor. 2) The formation of tangle-like structures, induced in
brain cells by contacting such cells with a medium which
selectively increases cathepsin D, can be inhibited by contacting
the cells with a cysteine protease inhibitor. 3) Degradation of tau
proteins was significantly inhibited by contacting the brain cells
with a cysteine protease inhibitor.
[0373] Thus, the present invention provides a first instance in
which tau proteolysis capable of triggering the formation of
neurofibrillary tangles has been inhibited by a cysteine protease
inhibitor. Moreover, the present invention provides clear evidence,
for the first time, for the relationship between cysteine
proteases, tau proteolysis, and the formation of neurofibrillary
tangles--one of the major pathologies in Alzheimer's disease. The
location of tau proteolysis and tangle-like structures inhibited in
brain cells by such protease inhibitors corresponds to that in
tissues from Alzheimer's disease patient. Such tangle-like
structures are composed mainly of tau fragments that are similar in
size as discovered in neurofibrillary tangles in Alzheimer's
disease.
[0374] Neurofibrillary tangles have long been recognized as the
hallmarks of Alzheimer's disease and the existence of a close
correlation between the presence and distributions of
neurofibrillary tangles and the degree of cognitive impairment in
Alzheimer's disease further emphasizes the critical role of tau
pathology in the development of the disease.
[0375] Hyperphosphorylation and fragmentation of tau have both been
previously proposed to be key steps involved in the aggregation of
tau into paired-helical filaments and thus key steps in the
production of neurofibrillary tangles. Therefore, one way that
calpain could facilitate tangle formation is through indirectly
increasing the phosphorylation of tau. Calpain could trigger such
phosphorylation by cleaving p35 to p25 (p25 is known to be more
active than p35 with regard to the phosphorylation of tau).
[0376] In vitro tests have demonstrated that all tau isoforms are
able to aggregate, however, tau fragments containing the repeat
domain exhibit faster kinetics in in vitro assembly tests. Thus,
not wishing to be bound by a theory, fragmentation of tau could be
the key factor that enhances the aggregation of tau and causes the
generation of neurofibrillary tangles, and therefore the inhibition
of such fragmentation of tau by the application of a cysteine
protease inhibitor may be a viable therapeutic option for diseases
and disorders comprising pathologies related to tau fragmentation.
These results significantly extend the range of neurodegenerative
disease features that can be induced and/or inhibited in brain
cells.
Example 6
Induction of Tangle-Like Structures by ZPAD Treatment was Blocked
by Mitogen-Activated Kinase Inhibitors
[0377] Incubation of hippocampal slices with ZPAD for 6 days
induced numerous tangles, in particular, in the border of subiculum
and CA1 region. However, when ZPAD was applied in the presence of a
mitogen-activated kinase inhibitor, the number of tangles was
significantly reduced (FIG. 25).
Example 7
Modulation of Biological Processing of Amyloid Precursor Protein by
Mevastatin Treatment is Blocked by Mevalonate
[0378] Hippocampal slices were prepared from apoE-knockout mice at
postnatal day 13, cultured in vitro for 12 days, and then incubated
with vehicle alone (control), mevastatin, mevastatin plus ZPAD,
EA1, cholesterol, or mevalonate, a product of HMG-CoA reductase
(FIG. 26). Tissues were processed for immunoblotting and assessed
by monoclonal antibody 22C11, which recognizes the N-terminal
domain of amyloid precursor protein (APP). Mevastatin treatment
markedly increased the levels of full length APP and induced a
novel band with molecular weight slightly lower than the native
APP. Whether this new product is due to proteolysis or changes in
protein maturation is under investigation. When mevastatin was
applied in the presence of mevalonate, its effects on APP were
completely blocked.
Example 8
Effects of Mevastatin on APP were Partially Blocked by MAPKK
Inhibitor PD98059, but not by Inhibitor SB203580 of MAPK p38
[0379] Hippocampal slices were incubated with vehicle
alone/control, mevastatin, mevastatin plus ZPAD, mevastatin plus
PD98059, mevastatin plus EA1, mevastatin plus cholesterol,
mevastatin plus mevalonate, mevastatin plus SB203580, or mevastatin
plus .gamma.-secretase inhibitor (FIG. 27). These results showed
that treatment of hippocampal slices with mevastatin rapidly and
markedly decreased levels of p35, increased activated forms of
MAPK, and increased levels and proteolytically processed APP. The
observation that both decrease in p35 and increase in APP were
completely blocked in the presence of mevalonate, the product of
HMG-CoA reductase, strongly indicates that the effects of
mevastatin are due to disruptions in cholesterol biosynthesis. The
effects of mevastatin on APP bioprocesses were partially blocked by
MAPKK inhibitor PD98059 but not inhibitor of MAP kinase p38
SB203580, suggesting MAPK/Erk1/2 is involved in mevastatin induced
changes in APP metabolisms.
Example 9
Lysosomal Dysfunction Induces Increased Activity of Caspase 3
[0380] Hippocampal slices were cultured for 12 days and incubated
with vehicle alone, ZPAD, or chloroquine (CQN; a lysosomal
inhibitor) for 6 days (FIG. 28). Cultures were then homogenized,
and subjected to an ELISA assay to detect the activity of caspase
3, an apoptotic protease. ZPAD treatment caused a marked increase
in the activity of caspase3.
Example 10
Pravastatin, Simvastatin, and Mevastatin Produce
Neurodegeneration--In Vitro and In Vivo
[0381] Pravastatin treatment induces the formation of tangle-like
structures (FIG. 29). Cultured rat hippocampal slices of 12 days in
vitro were treated with 20 .mu.M pravastatin for 6 days and
processed from immunohistological studies with the monoclonal
antibody AT8. The subiculum, CA1 field, and CA3 field of the
hippocampus were examined by photomicroscopy.
[0382] Microglial reactions are induced by mevastatin and
simvastatin treatments (FIG. 30). Male Sprague-Dawley rats age 2
1/2 months were injected daily (i.p.) with vehicle (n=3),
mevastatin (10 mg/kg, n=3), or simvasatin (10 mg/kg, n=4) for 39
days, and killed by with overdose of sodium pentobarbital (200
mg/kg, i.p.) and perfused intracardially with phosphate buffered
saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 M
phosphate buffer (PB, pH 7.4). Brains were then removed, postfixed
in perfusate for 5-6 hors, and cryoprotected in 15% sucrose/PB
followed by 30% sucrose/PB (12-24 hours each) at 4.degree. C.
Coronal sections were cut at 20-30 .mu.m by using a freezing
microtome and collected into PBS. Immunostaining was performed as
described for the in vitro experiments using monoclonal antibody
CD11b that reacts with both active and non-active microglia.
[0383] Shown are images of hippocampal areas from one control
animal and an animal treated with simvastatin. CD11b immunostaining
is moderate to dense in control tissue, while it is generally dense
in simvastatin treated hippocampus. Higher magnification images
show that the density of microglia is higher in simvasatin treated
tissue than that in the control tissue.
Discussion
[0384] The present invention provides novel materials, such as
brain cells (e.g., normal, apoE-deficient, apoE4-containing) as
models of neurodegenerative diseases, and methods for inducing
and/or preventing the induction of characteristics of such diseases
in brain cells so that such cells can be used as a model of
neurodegenerative diseases, including Alzheimer's disease.
[0385] While specific examples have been provided, the above
description is illustrative and not restrictive. Any one or more of
the features of the previously described embodiments can be
combined in any manner with one or more features of any other
embodiments in the present invention. Furthermore, many variations
of the invention will become apparent to those skilled in the art
upon review of the specification. The scope of the invention
should, therefore, be determined not with reference to the above
description, but instead should be determined with reference to the
appended claims along with their full scope of equivalents.
[0386] All publications and patent documents cited in this
application are incorporated by reference in their entirety for all
purposes to the same extent as if each individual publication or
patent document were so individually denoted. By their citation of
various references in this document, applicants do not admit any
particular reference is "prior art" to their invention.
* * * * *
References