U.S. patent application number 11/180044 was filed with the patent office on 2006-02-16 for jnk3 modulators and methods of use.
This patent application is currently assigned to University of Massachusetts, a Massachusetts corporation. Invention is credited to Roger J. Davis, Alan J. Whitmarsh.
Application Number | 20060035303 11/180044 |
Document ID | / |
Family ID | 22033003 |
Filed Date | 2006-02-16 |
United States Patent
Application |
20060035303 |
Kind Code |
A1 |
Davis; Roger J. ; et
al. |
February 16, 2006 |
JNK3 modulators and methods of use
Abstract
The c-Jun NH.sub.2-terminal kinase (JNK) group of MAP kinases
are activated by exposure of cells to environmental stress. The
role of JNK in the brain was examined by targeted disruption of the
gene that encodes the neuronal isoform JNK3. It was found that JNK3
is required for the normal response to seizure activity. Methods of
screening for molecules and compounds that decrease JNK3 expression
or activity are described. Such molecules or compounds are useful
for treating disorders involving excitotoxicity such as seizure
disorders, Alzheimer's disease, Huntington disease, Parkinson's
disease, and ischaemia.
Inventors: |
Davis; Roger J.; (Princeton,
MA) ; Whitmarsh; Alan J.; (Manchester, GB) |
Correspondence
Address: |
J. Peter Fasse;FISH & RICHARDSON P.C.
Suite 3300
60 South Sixth Street
Minneapolis
MN
55402
US
|
Assignee: |
University of Massachusetts, a
Massachusetts corporation
|
Family ID: |
22033003 |
Appl. No.: |
11/180044 |
Filed: |
July 12, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09165522 |
Oct 2, 1998 |
6943000 |
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11180044 |
Jul 12, 2005 |
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60060995 |
Oct 3, 1997 |
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Current U.S.
Class: |
435/15 ; 435/183;
435/194 |
Current CPC
Class: |
A01K 2267/0318 20130101;
C07K 14/82 20130101; A61K 38/00 20130101; A61P 17/02 20180101; A61P
25/00 20180101; A61P 25/14 20180101; A61P 25/16 20180101; A61P
21/00 20180101; A01K 2217/05 20130101; A61P 43/00 20180101; C07K
14/4747 20130101; A01K 2267/03 20130101; A61P 25/08 20180101; A61P
25/28 20180101; A01K 2267/0356 20130101; A61P 9/10 20180101; A01K
67/0276 20130101; A01K 2227/105 20130101; A01K 2217/075 20130101;
A61P 25/02 20180101; C12N 9/1205 20130101; C12N 15/8509
20130101 |
Class at
Publication: |
435/015 ;
435/183; 435/194 |
International
Class: |
C12Q 1/48 20060101
C12Q001/48; C12N 9/12 20060101 C12N009/12 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made, in part, with support from the
National Institutes of Health. The government may have certain
rights in the invention.
Claims
1-17. (canceled)
18. A method for identifying a modulator of c-Jun N-terminal kinase
(JNK3), the method comprising: (a) exposing a cell to a stress
signal in the presence of a test compound under conditions that
allow the cell to phosphorylate c-Jun; and (b) determining the
level of phosphorylated c-Jun in the cell, wherein a difference in
the level of phosphorylated c-Jun determined in (b) compared to the
level of phosphorylated c-Jun in the absence of the test compound
indicates that the test compound is a modulator of JNK3.
19. The method of claim 18, wherein the cell is a neuronal
cell.
20. The method of claim 19, wherein the neuronal cell is a
hippocampal cell.
21. The method of claim 18, wherein the test compound is an
antisense nucleic acid, an oligonucleotide, a ribozyme, a peptide,
a peptidomimetic, an amino acid, a phosphopeptide, a small organic
molecule, or a small inorganic molecule.
22. A method for identifying a modulator of c-Jun N-terminal kinase
(JNK3), the method comprising: (a) exposing a cell to a stress
signal in the presence of a test compound under conditions that
allow the cell to increase AP-1 transcription activity; and (b)
determining the level of AP-1 transcription activity in the cell,
wherein a difference in the level of AP-1 transcription activity
determined in (b) compared to the level of AP-1 transcription
activity in the absence of the test compound indicates that the
test compound is a modulator of JNK3.
23. The method of claim 22, wherein the cell is a neuronal
cell.
24. The method of claim 23, wherein the neuronal cell is a
hippocampal cell.
25. The method of claim 22, wherein AP-1 transcription activity is
measured using a transgene comprising an AP-1 binding site and a
reporter gene.
26. The method of claim 22, wherein the test compound is an
antisense nucleic acid, an oligonucleotide, a ribozyme, a peptide,
a peptidomimetic, an amino acid, a phosphopeptide, a small organic
molecule, or a small inorganic molecule.
27. A method of identifying a compound that modulates
excitotoxicity, the method comprising: (a) identifying a modulator
of c-Jun N-terminal kinase (JNK3) expression; (b) incubating a cell
that can exhibit excitotoxicity with the modulator of JNK3
expression under conditions sufficient for the cell to exhibit
excitotoxicity in the absence of the modulator of JNK3 expression;
and (c) determining excitotoxicity in the cell after (b), wherein a
difference in the level of excitotoxicity determined in (c)
compared to the level of excitotoxicity in the absence of the
modulator of JNK3 expression indicates that the modulator of JNK3
expression modulates excitotoxicity.
28. The method of claim 27, wherein the modulator is an antisense
nucleic acid, an oligonucleotide, a ribozyme, a peptide, a
peptidomimetic, an amino acid, a phosphopeptide, a small organic
molecule, or a small inorganic molecule.
29. A method of identifying a compound that modulates
excitotoxicity, the method comprising: (a) identifying a modulator
of c-Jun N-terminal kinase (JNK3) activity; (b) incubating a cell
that can exhibit excitotoxicity with the modulator of JNK3 activity
under conditions for the cell to exhibit excitotoxicity in the
absence of the modulator of JNK3 activity; and (c) determining
excitotoxicity in the cell after (b), wherein a difference in the
level of excitotoxicity determined in (c) compared to the level of
excitotoxicity in the absence of the modulator of JNK3 activity
indicates that the modulator of JNK3 activity modulates
excitotoxicity.
30. The method of claim 29, wherein the modulator is an
oligonucleotide, a peptide, a peptidomimetic, an amino acid, a
phosphopeptide, a small organic molecule, or a small inorganic
molecule.
31. A method of identifying a compound that modulates
excitotoxicity, the method comprising: (a) identifying a modulator
of c-Jun N-terminal kinase (JNK3) substrate binding; (b) incubating
a cell that can exhibit excitotoxicity with the modulator of JNK3
substrate binding under conditions for the cell to exhibit
excitotoxicity in the absence of the modulator of JNK3 substrate
binding; and (c) determining excitotoxicity in the cell after (b),
wherein a difference in the level of excitotoxicity determined in
(c) compared to the level of excitotoxicity in the absence of the
modulator of JNK3 substrate binding indicates that the modulator of
JNK3 substrate binding modulates excitotoxicity.
32. The method of claim 31, wherein the modulator is an
oligonucleotide, a peptide, a peptidomimetic, an amino acid, a
phosphopeptide, a small organic molecule, or a small inorganic
molecule.
Description
[0001] This application claims priority from provisional
application Ser. No. 60/060,995, filed Oct. 3, 1997.
FIELD OF THE INVENTION
[0003] The invention relates to screening assays for the detection
of inhibitors of protein kinase expression or activity.
BACKGROUND OF THE INVENTION
[0004] Apoptosis, or programmed cell death, is a prominent feature
of the nervous system during normal development and in adult brain
exposed to environmental stress (Kuida et al., Nature, 384:368-372,
1996; Ratan et al., Neurochem., 62:376-379, 1994; Raff et al.,
Science, 262:695-700, 1993). Stress-induced apoptosis has been
implicated in a variety of neurological diseases (Thompson,
Science, 267:1456-1462, 1995) and requires de novo protein and RNA
synthesis (Martin et al., J. Cell. Biol., 106:829-844, 1988;
Oppenheim et al., Dev. Biol., 138:104-113, 1990). Increased
expression of c-Jun protein is associated with neuronal damage
following global ischemia (Neumann-Haefelin et al., Cerebral Flow
Metab, 14:206-216, 1994) or transection of nerve axons in vivo
(Neumann-Haefelin, supra). Increased expression and phosphorylation
of c-Jun have been observed in vitro prior to the apoptotic death
of sympathetic neurons deprived of nerve growth factor (NGF) (Ham
et al., Neuron, 14:927-939, 1995). Moreover, expression of a
dominant negative mutant c-Jun, or treatment with c-Jun antibody
protects NGF-deprived sympathetic neurons from apoptosis (Ham et
al., supra; Estus et al., J. Cell. Biol., 127:1717-1727, 1994).
However, the requirement of c-Jun for stress-induced neuronal
apoptosis has not been tested in vivo since c-Jun deficient mice
die during mid-gestation (Hilberg et al., Nature, 365:179-181,
1993).
[0005] Protein phosphorylation is one important mechanism involved
in the activation of c-Jun in response to environmental stress
signals (Whitmarsh et al., J. Mol. Med., 74:589-607, 1996). c-Jun
N-terminal kinase (JNK, also known as SAPK) is a serine/threonine
protein kinase that phosphorylates two residues (Ser-63 and Ser-73)
on the NH.sub.2-terminal activation domain of c-Jun (Whitmarsh et
al., supra; Derijard et al., Cell, 76:1025-1037, 1994; Kyriakis et
al., Nature, 369:156-160, 1994). Map kinase kinase (MKK) 4 (also
known as SEK1) is a direct activator of JNK in response to
environmental stresses and mitogenic factors (Whitmarsh et al,
supra; Derijard et al, supra; Nishina et al., Nature, 385:350-353,
1997; Yang et al., Proc. Nat. Acad. Sci. USA, 94:3004-3009, 1997;
Sanchez et al., Nature, 372:794-798, 1994). JNK also phosphorylates
ATF2 and other Jun-family proteins which function as components of
the AP-1 transcription factor complex (Whitmarsh et al., supra;
Gupta et al., Science, 267:389-393, 1995; Gupta et al., EMBO J.,
15:2760-2770, 1996). The phosphorylation of these transcription
factors by JNK leads to increased AP-1 transcriptional activity
(Whitmarsh et al., supra). Conversely, the induction of AP-1
transcriptional activity is selectively blocked in cells lacking
MKK4 (Yang et al., supra).
[0006] JNK has been implicated in the apoptosis of
NGF-differentiated PC12 pheochromocytoma cells (Xia et al.,
Science, 270:1326-1311, 1995), one model system of neuronal cell
death in vivo (Batistatou et al., J. Cell. Biol., 122:523-532,
1993). When differentiated PC12 cells are deprived of nerve growth
factor (NGF), JNK activation is observed prior to apoptotic death
(Xia et al., supra). Transfection studies using constitutively
activated and dominant negative mutant components of the JNK
signaling pathway established that JNK is involved in NGF
withdrawal-induced apoptosis of PC12 cells (Xia et al., supra).
[0007] Ten JNK isoforms, resulting from alternative splicing of
three different genes have been identified (Derijard et al., supra;
Kyriakis et al., supra; Gupta et al., supra; Martin et al., Brain
Res. Mol. Brain Res., 35:47-57, 1996). Although the JNK1 and JNK2
isoforms are widely expressed in murine tissues, including the
brain, the JNK3 isoforms are predominantly expressed in the brain
and, to a lesser extent, in the heart and testis.
SUMMARY OF THE INVENTION
[0008] The invention is based on the discovery that mice lacking
the JNK3 gene (JNK3(-/-)) develop normally and are resistant to
excitotoxic damage, and that JNK3 plays a role in stress-induced
seizure activity, AP-1 transcriptional activation, and
kainate-induced apoptosis of hippocampal neurons. Thus, JNK3 is a
mediator of kainate/glutamate excitotoxicity and a target for
limiting or preventing excitotoxic damage.
[0009] The invention features a method of identifying a candidate
compound that modulates JNK3 expression. The method includes the
steps of incubating a cell that can express a JNK3 protein with a
compound under conditions and for a time sufficient for the cell to
express the JNK3 protein when the candidate compound is not
present. The expression of JNK3 is then measured in the cell in the
presence of the compound. The expression of JNK3 is also measured
in a control cell under the same conditions and for the same time.
The amount of JNK3 expression in the cell incubated in the presence
of the compound and in the control cell is compared. A difference
in JNK3 expression indicates that the compound modulates JNK3
expression. In an embodiment of this method, the compound decreases
JNK3 expression.
[0010] In another embodiment, the invention features a method of
identifying a candidate compound that modulates JNK3 activity. The
method includes the steps of incubating a cell that has JNK3
activity with a compound under conditions and for a time sufficient
for the cell to express JNK3 activity when the candidate compound
is not present. The activity of JNK3 is then measured in the cell
in the presence of the compound. The activity of JNK3 is also
measured in a control cell under the same conditions and for the
same time. The amount of JNK3 activity in the cell incubated in the
presence of the compound and in the control cell is compared. A
difference in JNK3 activity indicates that the compound modulates
JNK3 activity. In an embodiment of this method, the compound
decreases JNK3 activity.
[0011] The invention also includes a method of identifying a
compound that modulates the binding of a JNK3 polypeptide to a
substrate. The method involves comparing the amount of a JNK3
polypeptide bound to a substrate in the presence and absence of a
selected compound. A difference in the amount of binding of a JNK3
polypeptide to the substrate indicates that the selected compound
modulates the binding of a JNK3 polypeptide. In an embodiment of
this method, the binding of a JNK3 polypeptide to a substrate is
decreased.
[0012] Another feature of the invention is a method for generating
a totipotent mouse cell comprising at least one inactivated JNK3
gene. The method includes: a) providing a plurality of totipotent
mouse cells; b) introducing into the cells a DNA construct that
includes a mouse JNK3 gene disrupted by the insertion of a sequence
into the gene, thus the disruption prevents expression of
functional JNK3; c) incubating the cells so that homologous
recombination occurs between the chromosomal sequence encoding JNK3
and the introduced DNA construct; and d) identifying a totipotent
mouse cell that has at least one inactivated JNK gene.
[0013] Also featured in the invention is a method for generating a
mouse homozygous for an inactivated JNK3 gene. The method includes
the steps of: a) providing a totipotent mouse cell that contains at
least one inactivated JNK3 gene; b) inserting the cell into a mouse
embryo and implanting the embryo into a female mouse; c) permitting
the embryo to develop into a neonatal mouse; d) permitting the
neonatal mouse to reach sexual maturity; e) mating two of the
sexually mature mice to obtain a mouse homozygous for the
inactivated JNK3 gene. Such a mouse (homozygous JNK3(-/-)) is
resistant to excitotoxic damage.
[0014] The invention also features methods of treating a patient
having or at risk for a disorder of the nervous system involving
excitotoxicity. The methods include administering to the patient a
therapeutically effective amount of a compound that inhibits JNK3
expression, or a therapeutically effective amount of a compound
that inhibits JNK3 activity. An antisense nucleic acid molecule or
ribozyme can be used as the inhibitory compound. Disorders that can
be treated by these methods include dementias including Alzheimer's
disease, neurodegenerative diseases such as Huntington disease,
cerebrovascular disorders such as ischemia, amyotrophic lateral
sclerosis, trauma including that caused by heat or cold, motor
neuron disease, Parkinson's disease, or seizure disorders including
epilepsy. Neuroendocrine disorders such as those that affect
pituitary glands, adrenal glands, testis, or pancreas (e.g.,
.beta.-cells) can be treated with JNK3 modulators.
[0015] The invention also includes a transgenic non-human mammal
having a transgene disrupting expression of a JNK3 gene, the
transgene being chromosomally integrated into germ cells of the
mammal. In an embodiment of the invention, the mammal is a mouse.
The germ cells of the mammal can be homozygous for the transgene
and the disruption of JNK3 gene expression can be the result of a
null mutation. Another embodiment of the invention includes a cell
line descended from a cell of the mammal having the transgene
disrupting expression of a JNK3 gene.
[0016] A DNA construct comprising a disrupted mouse JNK3 gene is
also featured in the invention. The disruption is by insertion of a
sequence into the gene such that the disruption prevents or
modifies the expression of functional JNK3.
[0017] Unless otherwise specified, "JNK3" can refer both to nucleic
acids and polypeptides, such as the sequences shown in FIGS. 1A-5B
(SEQ ID NOS:1-12; see also, GenBank accession number: U34819 which
corresponds to SEQ ID NO:1 and SEQ ID NO:2; U34820 which
corresponds to SEQ ID NO:4 and SEQ ID NO:5; U07620 which
corresponds to SEQ ID NO:7 and SEQ ID NO:8; L27128 which
corresponds to SEQ ID NO:9 and SEQ ID NO:10; and L35236 which
corresponds to SEQ ID NO:11 and SEQ ID NO:12). SEQ ID NO:3 and SEQ
ID NO:6 represent deduced nucleotide sequences based on the
presumed overlap between the sequences represented by SEQ ID NOS:1
and 4 with the sequence represented by SEQ ID NO:7. JNK3 also
refers to polypeptides that are at least 85% identical to the amino
acid sequences listed above, and to the nucleic acids encoding
those polypeptides. Examples of these sequences and methods of
isolating them are found in Gupta et al., supra, 1996; Kyriakis et
al., supra; Martin et al., Brain Res. Mol. Brain Res., 35:45-57,
1996; and Mohit et al., Neuron, 14:67-78, 1995.
[0018] A "control" cell is a cell that is generally the same, e.g.
genotypically and phenotypically, as the cell to which it is being
compared (e.g., the cells can be sister cells), but which is not
exposed to a test compound.
[0019] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated in their entirety. In case of
conflict, the present specification, including definitions, will
control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
[0020] Other features and advantages of the invention will be
apparent from the following detailed description and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1A is a schematic representation of the nucleic acid
sequence of GenBank Accession No. U34819 (SEQ ID NO:1).
[0022] FIG. 1B is a schematic representation of the amino acid
sequence of GenBank Accession No. U34819 (SEQ ID NO:2).
[0023] FIG. 1C is a schematic representation of the nucleic acid
sequence of SEQ ID NO:3.
[0024] FIGS. 2A-B is a schematic representation of the nucleic acid
sequence of GenBank Accession No. U34820 (SEQ ID NO:4).
[0025] FIG. 2C is a schematic representation of the amino acid
sequence of GenBank Accession No. U34820 (SEQ ID NO:5).
[0026] FIG. 2D is a schematic representation of the nucleic acid
sequence of SEQ ID NO:6.
[0027] FIGS. 3A-B is a schematic representation of the nucleic acid
sequence of GenBank Accession No. U07620 (SEQ ID NO:7).
[0028] FIG. 3C is a schematic representation of the amino acid
sequence of GenBank Accession No. U07620 (SEQ ID NO:8).
[0029] FIG. 4A is schematic representation of the nucleic acid
sequence of GenBank Accession No. L27128 (SEQ ID NO:9).
[0030] FIG. 4B is a schematic representation of the amino acid
sequence of GenBank Accession No. L27128 (SEQ ID NO:10).
[0031] FIG. 5A is a schematic representation of the nucleic acid
sequence of GenBank Accession No. L35236 (SEQ ID NO:11).
[0032] FIG. 5B is a schematic representation of the amino acid
sequence of GenBank Accession No. L35236 (SEQ ID NO:12).
[0033] FIG. 6 is a diagram of the wild type JNK3 gene locus,
targeting vector, and the mutated or disrupted JNK3 gene locus.
[0034] FIG. 7 is a bar graph showing the temporal responses of wild
type and JNK3(-/-) mice to kainic acid (KA) injection.
[0035] FIG. 8 is a bar graph showing the temporal responses of wild
type and JNK3(-/-) mice to pentetrazole (PTZ) injection.
[0036] FIG. 9A is a schematic representation of the nucleic acid
sequence of murine c-Jun (GenBank Accession No. X12740; SEQ ID
NO:13).
[0037] FIG. 9B is a schematic representation of the amino acid
sequence of murine c-Jun (GenBank Accession No. X12740; SEQ ID
NO:14).
[0038] FIGS. 10A-B is a schematic representation of the nucleic
acid sequence of murine c-Fos (GenBank Accession No. V00727; SEQ ID
NO:15).
[0039] FIG. 10C is a schematic representation of the amino acid
sequence of murine c-Fos (GenBank Accession No. V00727; SEQ ID
NO:16)
[0040] FIG. 11 is a bar graph showing the level of KA-induced AP-1
activity at various times after KA induction as reflected by
luciferase activity in JNK3(-/-) mice crossed with transgenic AP-1
luciferase mice.
[0041] FIG. 12 is a bar graph showing the level of KA-induced AP-1
activity as reflected by the relative level of luciferase activity
in hippocampus (HP) and cerebellum (CB) of JNK3(-/-) mice and wild
type(+/+) mice.
[0042] FIG. 13 is a diagram of the proposed chain of molecular
events caused by KA leading to neuronal apoptosis.
[0043] FIG. 14 is a diagram of the trisynaptic connection within
the hippocampal formation.
DETAILED DESCRIPTION
[0044] JNK protein kinase phosphorylates c-Jun and subsequently
increases AP-1 transcriptional activity in response to a specific
group of stress signals (Whitmarsh et al, supra; Yang et al.,
supra). The neural-specific expression of JNK3 may render neurons
particularly susceptible to physiological stress. In the
experiments described herein, a remarkable resistance to kainic
acid (KA)-induced seizures and apoptosis has been observed in
JNK3-deficient mice. The resistance to KA neurotoxicity may be due
to the elimination of a specific stress-response pathway mediated
by the JNK3 isoform of JNK protein kinase. First, the
administration of KA caused the phosphorylation of the
NH.sub.2-terminal activation domain of c-Jun and markedly increased
AP-1 transcriptional activity in wild-type, but not in
JNK3-deficient mice. Second, there was prolonged expression of
phosphorylated c-Jun within the most vulnerable area of the
hippocampus, further indicating that JNK activity may lead to
neuronal apoptosis.
[0045] The findings reported herein are consistent with the
dependence of KA neurotoxicity on excitatory circuitry (Nadler et
al., Brain Res. 195:47-56, 1980). Since JNK3 is widely expressed in
the nervous system and its activity is increased by many different
stress signals (Gupta et al., supra), JNK3 may be involved in
stress-induced apoptosis caused by a wide range of environmental
insults.
[0046] The identification of JNK3 as a critical mediator of
KA-induced excitatory neurotoxicity has clinical implications. The
amino acid sequence of mouse, rat and human JNK3 is highly
conserved (Kyriakis et al., supra; Gupta et al., supra; Martin et
al., supra; Mohit et al., Neuron. 14:67-78, 1995). Moreover, the
expression of the human JNK3 gene is also restricted to the nervous
system and neuroendocrine system, is widely expressed in many brain
subregions (Gupta et al., supra; Mohit et al., supra). It is
therefore likely that the human and rodent JNK3 protein kinases
have related or identical physiological functions. Neurotoxicity of
the excitatory amino acids has been implicated in many neurological
disorders ranging from acute ischemia to chronic neurodegenerative
diseases (Choi, Neuron, 1:623-634, 1988; Lipton et al., N. Engl. J.
Med. 330:613-622, 1994; Rothman et al., Annu. Neurol. 19:105-111,
1986). Previous therapeutic strategies have been focused on the
prevention of calcium influx through cell surface channels, such as
the NMDA-type glutamate receptor. To date, these approaches have
only met with mixed results (Lipton et al., supra). JNK3 is
therefore a target for therapeutic interventions when excitatory
neurotoxicity involves JNK3-mediated apoptosis.
[0047] In the experiments described infra, homologous recombination
was used to generate JNK3-deficient mice, and their responses to
noxious stimuli were examined. KA, a potent excitotoxic chemical,
elicits limbic seizures and neuronal cell death. The neurotoxicity
of KA derives from the direct stimulation of the glutamate receptor
at postsynaptic sites, and an indirect increase in the release of
excitatory amino acids from presynaptic sites. It is
well-documented that systemic application of KA induces the
expression of various cellular immediate early genes (cIEGs),
including c-Jun and c-Fos. Thus, the application of KA triggers a
stress-response pathway in the brain in vivo. The experiments
detailed infra demonstrate that KA induces phosphorylation of c-Jun
and an increase in AP-1 transcriptional activity in the brain of
wild-type mice. However, these effects of KA are markedly
suppressed in the brains of JNK3-deficient mice. Moreover,
JNK3-deficient mice exhibit a remarkable resistance to KA-induced
seizures and apoptosis of hippocampal neurons. These normal mice
treated with KA represent a useful model of human disorders of the
nervous system involving excitotoxicity.
[0048] Based on these experimental results, JNK3 was found to be an
exceptional target for limiting excitotoxic damage. In particular,
JNK3 is a target in screening protocols including protocols to
screen for molecules that regulate JNK3 gene expression, JNK3
binding to its substrates, and JNK3 activity, as described below.
The molecules found in these screens that effectively decrease JNK3
expression or activity are candidate drugs to be used to treat
disorders of the nervous system involving excitotoxicity, including
seizure disorders such as epilepsy, cerebrovascular disorders
including ischemia, metabolic imbalance (e.g., hypoglycemia),
injury due to extreme heat or cold, trauma (e.g. irradiation,
spinal cord injury, pressure, and ionic imbalance), dementias such
as Alzheimer's disease, Parkinson's disease, and neurodegenerative
disorders (e.g., Huntington disease), and motoneuron disease
(including amyotrophic lateral sclerosis) (Thompson, Science,
267:1456-1462, 1995; Coyle et al., Science, 262:689-695, 1993).
Methods of Screening for Molecules that Inhibit JNK3 Activity
[0049] The following assays and screens can be used to identify
compounds that are effective inhibitors of JNK3 activity. The
assays and screens can be done by physical selection of molecules
from libraries, and computer comparisons of digital models of
compounds in molecular libraries and a digital model of the JNK3
active site. The inhibitors identified in the assays and screens
may act by, but are not limited to, binding to JNK3 (e.g., from
mouse or human), binding to intracellular proteins that bind to
JNK3, compounds that interfere with the interaction between JNK3
and its substrates, compounds that modulate the activity of a JNK3
gene, or compounds that modulate the expression of a JNK3 gene or a
JNK3 protein.
[0050] Assays can also be used to identify molecules that bind to
JNK3 regulatory sequences (e.g., promoter sequences), thus
modulating gene expression. See, e.g., Platt, J. Biol. Chem.,
269:28558-28562, 1994.
[0051] The compounds that can be screened by the methods described
herein include, but are not limited to, peptides and other organic
compounds (e.g., peptidomimetics) that bind to a JNK3 protein or
inhibit its activity in any way. Such compounds may include, but
are not limited to, peptides; for example, soluble peptides,
including but not limited to members of random peptide libraries
(see, e.g., Lam et al., Nature 354:82-84, 1991; Houghten et al.,
Nature 354:84-86, 1991), and combinatorial chemistry-derived
molecular libraries made of D- and/or L-amino acids,
phosphopeptides (including, but not limited to, members of random
or partially degenerate, directed phosphopeptide libraries; see,
e.g., Songyang et al., Cell 72:767-778, 1993), and small organic or
inorganic molecules.
[0052] Compounds and molecules are screened to identify those that
affect expression of a JNK3 gene or some other gene involved in
regulating the expression of JNK3 (e.g., by interacting with the
regulatory region or transcription factors of a gene). Compounds
are also screened to identify those that affect the activity of
such proteins (e.g., by inhibiting JNK3 activity) or the activity
of a molecule involved in the regulation of JNK3.
[0053] Computer modeling or searching technologies are used to
identify compounds, or identify modified compounds that modulate or
are candidates to modulate the expression or activity of a JNK3
protein. For example, compounds likely to interact with the active
site of the JWK3 protein are identified. The active site of JNK3
can be identified using methods known in the art including, for
example, analysis of the amino acid sequence of a molecule, and
from a study of complexes formed by JNK3 with a native ligand
(e.g., ATF2 or c-Jun). Chemical or X-ray crystallographic methods
can be used to identify the active site of JNK3 by the location of
a bound ligand such as c-Jun or ATF2.
[0054] The three-dimensional structure of the active site can be
determined. This can be done using known methods, including X-ray
crystallography, which can be used to determine a complete
molecular structure. Solid or liquid phase NMR can be used to
determine certain intra-molecular distances. Other methods of
structural analysis can be used to determine partial or complete
geometrical structures. Geometric structure can be determined with
a JNK3 protein bound to a natural (e.g., c-Jun or ATF2) or
artificial ligand which may provide a more accurate active site
structure determination.
[0055] Computer-based numerical modeling can be used to complete an
incomplete or insufficiently accurate structure. Modeling methods
that can be used are, for example, parameterized models specific to
particular biopolymers such as proteins or nucleic acids, molecular
dynamics models based on computing molecular motions, statistical
mechanics models based on thermal ensembles, or combined models.
For most types of models, standard molecular force fields,
representing the forces between constituent atoms and groups are
necessary, and can be selected from force fields known in physical
chemistry. Information on incomplete or less accurate structures
determined as above can be incorporated as constraints on the
structures computed by these modeling methods.
[0056] Having determined the structure of the active site of a JNK3
protein, either experimentally, by modeling, or by a combination of
methods, candidate modulating compounds can be identified by
searching databases containing compounds along with information on
their molecular structure. The compounds identified in such a
search are those that have structures that match the active site
structure, fit into the active site, or interact with groups
defining the active site. The compounds identified by the search
are potential JNK3 modulating compounds.
[0057] These methods may also be used to identify improved
modulating compounds from an already known modulating compound or
ligand. The structure of the known compound is modified and effects
are determined using experimental and computer modeling methods as
described herein. The altered structure is compared to the active
site structure of a JNK3 protein to determine or predict how a
particular modification to the ligand or modulating compound will
affect its interaction with that protein. Systematic variations in
composition, such as by varying side groups, can be evaluated to
obtain modified modulating compounds or ligands of preferred
specificity or activity.
[0058] Given the teachings herein, additioanl experimental and
computer modeling methods useful to identify modulating compounds
based on identification of the active sites of a JNK3 protein and
related transduction and transcription factors an be developed by
those skilled in the art.
[0059] Examples of molecular modeling systems are the QUANTA
programs, e.g., CHARMm, MCSS/HOOK, and X-LIGAND, (Molecular
Simulations, Inc., San Diego, Calif.). QUANTA provides a modeling
environment for two dimensional and three dimensional modeling,
simulation, and analysis of macromolecules and small organics.
Specifically, CHARMm analyzes energy minimization and molecular
dynamics functions. MCSS/HOOK characterizes the ability of an
active site to bind a ligand using energetics calculated via
CHARMm. X-LIGAND fits ligand molecules to electron density patterns
of protein-ligand complexes. The program also allows interactive
construction, modification, visualization, and analysis of the
behavior of molecules with each other.
[0060] Articles reviewing computer modeling of compounds
interacting with specific proteins can provide additional guidance.
For example, see, Rotivinen et al., Acta Pharmaceutical Fennica
97:159-166, 1988; Ripka, New Scientist 54-57 (Jun. 16, 1988);
McKinaly and Rossmann, Ann. Rev. Pharmaol. Toxicol 29:111-122,
1989; Perry and Davies, OSAR: Quantitative Structure-Activity.
Relationships in Drug Design pp. 189-193 (Alan R. Liss, Inc.,
1989); Lewis and Dean, Proc. R. Soc. Lond. 236:125-140, 141-162,
1989; and, regarding a model receptor for nucleic acid components,
see Askew et al., Am. J. Chem. Soc. 111:1082-1090. Computer
programs designed to screen and depict chemicals are available from
companies such as MSI (supra), Allelix, Inc. (Mississauga, Ontario,
Canada), and Hypercube, Inc. (Gainesville, Fla.). These
applications are largely designed for drugs specific to particular
proteins; however, they may be adapted to the design of drugs
specific to identified regions of DNA or RNA. Commercial sources of
chemical libraries can be used as sources of candidate compounds.
Such chemical libraries can be obtained from, for example, ArQule,
Inc. (Medford, Mass.).
[0061] In addition to designing and generating compounds that alter
binding, as described above, libraries of known compounds,
including natural products, synthetic chemicals, and biologically
active materials including peptides, can be screened for compounds
that are inhibitors or activators.
[0062] Compounds identified by methods described above may be
useful, for example, for elaborating the biological function of
JNK3 gene products and in treatment of disorders in which JNK3
activity is deleterious. Assays for testing the effectiveness of
compounds such as those described herein are further described
below.
In Vitro Screening Assays for Compounds that Bind to JNK3 Proteins
and Genes
[0063] In vitro systems can be used to identify compounds that can
interact (e.g., bind) to JNK3 proteins or genes encoding those
proteins. Such compounds may be useful, for example, for modulating
the activity of JNK3 polypeptides or nucleic acids, elaborating
their biochemistry, or treating disorders caused or exacerbated by
JNK3 expression. These compounds may themselves disrupt normal
function or can be used in screens for compounds that disrupt
normal function.
[0064] Assays to identify compounds that bind to JNK3 proteins
involve preparation of a reaction mixture of the protein and the
test compound under conditions sufficient to allow the two
components to interact and bind, thus forming a complex that can be
detected and/or isolated.
[0065] Screening assays for molecules that can bind to a JNK3
protein or nucleic acid can be performed using a number of methods.
For example, a JNK3 protein, peptide, or fusion protein can be
immobilized onto a solid phase, reacted with the test compound, and
complexes detected by direct or indirect labeling of the test
compound. Alternatively, the test compound can be immobilized,
reacted with JNK3 polypeptide, and any complexes detected.
Microtiter plates can be used as the solid phase and the
immobilized component anchored by covalent or noncovalent
interactions. Non-covalent attachment may be achieved by coating
the solid phase with a solution containing the molecule, and
drying. Alternatively, an antibody specific for JNK3 is used to
anchor the molecule to the solid surface. Such surfaces may be
prepared in advance of use, and stored. JNK3 antibodies anchored by
covalent or noncovalent interactions. Non-covalent attachment may
be achieved by coating the solid phase with a solution containing
the molecule, and drying. Alternatively, an antibody specific for
JNK3 is used to anchor the molecule to the solid surface. Such
surfaces may be prepared in advance of use, and stored. JNK3
antibodies can be produced using conventional methods such as those
described in Coligan et al. (Current Protocols in Immunology, John
Wiley & Sons, Inc., 1994, see Volume 1, chapter 2).
[0066] In the assay, the non-immobilized component is added to the
coated surface containing the immobilized component under
conditions that permit interaction and binding between the two
components. The unreacted components are then removed (e.g., by
washing) under conditions such that any complexes formed will
remain immobilized on the solid phase. The detection of the
complexes can be accomplished by a number of methods known to those
in the art. For example, the nonimmobilized component of the assay
may be prelabeled with a radioactive or enzymatic label and
detected using appropriate means. If the non-immobilized entity was
not prelabeled, an indirect method is used. For example, if the
non-immobilized entity is a JNK3 polypeptide, an antibody against
JNK3 is used to detect the bound molecule, and a secondary, labeled
antibody is used to detect the entire complex.
[0067] Alternatively, a reaction can be conducted in a liquid
phase, the reaction products separated from unreacted components,
and complexes detected (e.g., using an immobilized antibody
specific for a JNK3 protein).
[0068] Cell-based assays can be used to identify compounds that
interact with JNK3 proteins. Cell lines that naturally express such
proteins or have been genetically engineered to not contain the
test compound indicates that the test compound is an inhibitor of
JNK3 activity.
[0069] Inhibitors of JNK3 expression that act on the JNK3 promoter
can be identified using a chimeric gene in which genomic sequences
including the JNK3 promoter are fused to a reporter, for example
firefly luciferase. Cultured cells (including neurons) transformed
with this DNA are screened for the expression of luciferase
activity. Compounds that inhibit luciferase activity in this high
throughput assay can be confirmed by direct measurement of the
endogenous JNK3 protein (by Western blotting) and JNK3 mRNA (by
Northern blotting) using methods known in the art (for example, see
Ausubel et al., Current Protocols in Molecular Biology, John Wiley
& Sons, 1994).
[0070] Candidate inhibitory compounds can be tested further in cell
or tissue cultures as well as animal models. For example, cells
expressing JNK3 are incubated with a test compound. Lysates are
prepared from treated and untreated cells and Western blotted
according to known methods. The blots are probed with antibodies
specific for JNK3. A decrease in the amount of JNK3 expression in
cultures treated with the test compound compared to untreated
controls indicates that the test compound is a candidate for a drug
to treat disorders associated with JNK3 expression.
Assays for Compounds that Interfere with JNK3/JNK3 Substrate
Interactions
[0071] Molecules that disrupt the interaction between JNK3 and its
substrates can be identified using assays that detect
protein-protein interactions. For example, the yeast two-hybrid
method detects protein interactions in vivo. However, an in vitro
assay is preferable because candidate molecules may not be
permeable to the yeast cell wall. An example of an in vitro assay
for such test molecules that disrupt the interaction between JNKC3
and a substrate includes the use of immobilized JNK3 or immobilized
substrate (e.g., c-Jun) and incubation of the immobilized component
with cell lysates or purified proteins in the presence and absence
of a test molecule. In general, the test molecule is tested over a
range of a 100 fold molar excess over the most abundant component
(e.g., the component immobilized or in solution). If the test
molecule is predicted to interact with the immobilized component of
the assay, then it can be pre-incubated with that component before
adding the cell lysate or purified protein. After washing away
unbound material, the bound proteins are detected with antibodies
(e.g., ELISA or Western blot) or through the use of labeled
proteins (e.g. radioactive or fluorescent) using methods known in
the art. Test molecules that decrease the amount of substrate bound
to JNK3 are thus identified as molecules that interfere with
JNK3/JNK3 substrate interactions.
Assays for Compounds that Ameliorate the Effects of JNK3 In
Vivo
[0072] Compounds identified as above, or other candidate compounds
that inhibit JNK3 activity in vitro may be useful for treating
disorders involving JNK3 activity. These compounds can be tested in
in vivo assays, for example, in animal models of disorders
involving JNK3 activity. For example, transgenic mouse models of
ALS have been described (Bruijn and Cleveland, Neuropathol. Appl.
Neurobiol. 22:373-387, 1996; Dal Canto and Gurney, Brain Res. 676:
25-40, 1995; Cleveland et al., Neurology 47: Suppl 2, S54-61) as
have transgenic models of Alzheimer's disease such as the PDAPP
mouse and others (for examples, see Loring et al., Neurobiol. Aging
17:173-182, 1996). MPTP
(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced dopaminergic
neurotoxicity has been used as a model for Parkinson's disease in
rodents and nonhuman primates (for example, Przedborski et al.,
Proc. Nat'l. Acad. Sci. USA 93:4565-4571, 1996).
[0073] Test compounds predicted to inhibit JNK3 activity are
administered to animals, e.g., as described above, that serve as
models for the various disease paradigms. Treated animals are then
assayed for inhibition of JNK3 activity. Such assays may be
indirect or inferential, for example, improved health or survival
of the animal indicates the efficacy of a test compound. Assays can
also be direct, for example, a decrease in JNK3 or c-Jun expression
can be measured by Northern analysis of neural tissue removed from
an animal treated with a test compound. A decrease in the amount of
JNK3 mRNA present in the sample from treated animals compared to
untreated controls indicates that the test compound is inhibiting
JNK3 expression. A decrease in the amount of ca-Jun indicates that
the test compound is inhibiting JNK3 expression or activity.
Antisense Constructs and Therapies
[0074] Treatment regimes based on an "antisense" approach involve
the design of oligonucleotides (either DNA or RNA) that are
complementary to JNK3 mRNAs. These oligonucleotides bind to the
complementary mRNA transcripts and prevent translation. Absolute
complementarily, although preferred, is not required. A sequence
"complementary" to a portion of an RNA, as referred to herein, is a
sequence sufficiently complementary to be able to hybridize with
the RNA, forming a stable duplex; in the case of double-stranded
antisense nucleic acids, a single strand of the duplex DNA may be
tested, or triplex formation may assayed. The ability to hybridize
will depend on both the degree of complementarity and the length of
the antisense nucleic acid. Generally, the longer the hybridizing
nucleic acid, the more base mismatches with an RNA it may contain
and still form a stable duplex (or triplex, as the case may be).
One skilled in the art can ascertain a tolerable degree of mismatch
by use of standard procedures to determine the melting point of the
hybridized complex.
[0075] Oligonucleotides that are complementary to the 5' end of the
message, e.g., the 5' untranslated sequence, up to and including
the AUG initiation codon, are generally most efficient for
inhibiting translation. However, sequences complementary to the 3'
untranslated sequences of mRNAs have also been shown to be
effective for inhibiting translation (Wagner, Nature, 372:333,
1984). Thus, oligonucleotides complementary to either the 5' or 3'
non-translated, non-coding regions of a JNK3 could be used in an
antisense approach to inhibit translation of the endogenous human
homolog of JNK3 mRNA. Oligonucleotides complementary to the 5'
untranslated region of the mRNA should include the complement of
the AUG start codon. Examples of candidate antisense sequences for
the 5' and 3' regions are; 5'-AAG AAA TGG AGG CTC ATA AAT ACC ACA
GCT-3' (SEQ ID NO:17) and 5'-ATT GGA AGA AGA CCA AAG CAA GAG CAA
CTA-3'(SEQ ID NO:18), respectively.
[0076] While antisense nucleotides complementary to the coding
region of a JNK3 gene could be used, those complementary to
transcribed untranslated regions are most preferred. Examples of
this type of candidate sequence are 5'-TAA GTA AGT AGT GCT GTA TGA
ATA CAG ACA-3' (SEQ ID NO:19) and 5'-TAC TGG CAA TAT ATT ACA GAT
GGG TTT ATG-3'(SEQ ID NO:20).
[0077] Antisense oligonucleotides complementary to mRNA coding
regions are less efficient inhibitors of translation, but could be
used in accordance with the invention. Whether designed to
hybridize to the 5', 3', or coding region of a JNK3 mRNA, antisense
nucleic acids should be at least six nucleotides in length, and are
preferably oligonucleotides ranging from 6 to about 50 nucleotides
in length. In specific aspects, the oligonucleotide is at least 10
nucleotides, or at least 50 nucleotides in length.
[0078] Regardless of the choice of target sequence, in vitro
studies are usually performed first to assess the ability of an
antisense oligonucleotide to inhibit gene expression. In general,
these studies utilize controls that distinguish between antisense
gene inhibition and nonspecific biological effects of
oligonucleotides. In these studies levels of the target RNA or
protein are usually compared with that of an internal control RNA
or protein. Additionally, it is envisioned that results obtained
using the antisense oligonucleotide are compared with those
obtained using a control oligonucleotide. It is preferred that the
control oligonucleotide is of approximately the same length as the
test oligonucleotide, and that the nucleotide sequence of the
oligonucleotide differs from the antisense sequence no more than is
necessary to prevent specific hybridization to the target
sequence.
[0079] The oligonucleotides can be DNA or RNA, or chimeric mixtures
or derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the base
moiety, sugar moiety, or phosphate backbone, for example, to
improve stability of the molecule or hybridization. The
oligonucleotide may include other appended groups such as peptides
(e.g., for targeting host cell receptors in vivo), or agents
facilitating transport across the cell membrane (as described,
e.g., in Letsinger et al., Proc. Natl. Acad. Sci. USA 86:6553,
1989; Lemaitre et al., Proc. Natl. Acad. Sci. USA 84:648, 1987; PCT
Publication No. WO 88/09810) or the blood-brain barrier (see, for
example, PCT Publication No. WO 89/10134), or
hybridization-triggered cleavage agents (see, for example, Krol et
al., BioTechniques 6:958, 1988), or intercalating agents (see, for
example, Zon, Pharm. Res. 5:539, 1988). To this end, the
oligonucleotide can be conjugated to another molecule, e.g., a
peptide, hybridization-triggered cross-linking agent, transport
agent, or hybridization-triggered cleavage agent.
[0080] The antisense oligonucleotide may comprise at least one
modified base moiety which is selected from the group including,
but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethyl-aminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-theouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
2-(3-amino-3-N-2-carboxypropl) uracil, (acp3)w, and
2,6-diaminopurine.
[0081] The antisense oligonucleotide can also comprise at least one
modified sugar moiety selected from the group including, but not
limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0082] The antisense oligonucleotide can also include at least one
modified phosphate backbone selected from the group consisting of a
phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a
phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl
phosphotriester, and a formacetal, or an analog of any of these
backbones.
[0083] The antisense oligonucleotide can include an
.alpha.-anomeric oligonucleotide. An .alpha.-anomeric
oligonucleotide forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .beta.-units, the
strands run parallel to each other (Gautier et al., Nucl. Acids.
Res. 15:6625, 1987). The oligonucleotide is a
2'-0-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15:6131,
1987), or a chimeric RNA-DNA analog (Inoue et al., FEBS Lett.
215:327, 1987).
[0084] Antisense oligonucleotides of the invention can be
synthesized by standard methods known in the art, e.g., by use of
an automated DNA synthesizer (such as are commercially available
from Biosearch, Applied Biosystems, etc.). As examples,
phosphorothioate oligonucleotides can be synthesized by the method
of Stein et al. (Nucl. Acids Res. 16:3209, 1988), and
methylphosphonate oligonucleotides can be prepared by use of
controlled pore glass polymer supports (Sarin et al., Proc. Natl.
Acad. Sci. USA 85:7448, 1988).
[0085] The antisense molecules should be delivered to cells that
express JNK3 proteins in vivo. A number of methods have been
developed for delivering antisense DNA or RNA to cells; e.g.,
antisense molecules can be injected directly into the tissue site,
or modified antisense molecules, designed to target the desired
cells (e.g., antisense linked to peptides or antibodies that
specifically bind receptors or antigens expressed on the target
cell surface) can be administered systemically.
[0086] However, it is often difficult to achieve intracellular
concentrations of the antisense molecule sufficient to suppress
translation of endogenous mRNAs. Therefore, an approach may be used
in which a recombinant DNA construct comprises an antisense
oligonucleotide placed under the control of a strong pol III or pol
II promoter. The use of such a construct to transfect target cells
in a patient will result in the transcription of sufficient amounts
of single stranded RNAs that will form complementary base pairs
with the endogenous JNK3 transcripts and thereby prevent
translation of that mRNA. For example, a vector can be introduced
in vivo such that it is taken up by a cell and directs the
transcription of an antisense RNA. Such a vector can remain
episomal or become chromosomally integrated, as long as it can be
transcribed to produce the desired antisense RNA.
[0087] Such vectors can be constructed by recombinant DNA
technology methods standard in the art. Vectors can be plasmid,
viral, or others known in the art, used for replication and
expression in mammalian cells. Expression of the sequence encoding
the antisense RNA can be by any promoter known in the art to act in
mammalian, preferably human cells. Such promoters can be inducible
or constitutive. Suitable promoters include, but are not limited
to: the SV40 early promoter region (Bernoist et al., Nature
290:304, 1981); the promoter contained in the 3' long terminal
repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797,
1988); the herpes thymidine kinase promoter (Wagner et al., Proc.
Natl. Acad. Sci. USA 78:1441, 1981); and the regulatory sequences
of the metallothionein gene (Brinster et al., Nature 296:39, 1988).
Constructs may also be contained on an artificial chromosome (e.g.,
mammalian artificial chromosome; MAC; Harrington et al., Nature
Genet. 15:345-355, 1997).
[0088] The production of a JNK3 antisense nucleic acid molecule by
any gene therapeutic approach described above results in a cellular
level of JNK3 protein that is less than the amount present in an
untreated individual.
Ribozymes
[0089] Ribozyme molecules designed to catalytically cleave JNK3
mRNAs can also be used to prevent translation of these mRNAs and
expression of JNK3 mRNAs (see, e.g., PCT Publication WO 90/11364;
Saraver et al., Science 247:1222, 1990). While various ribozymes
that cleave mRNA at site-specific recognition sequences can be used
to destroy specific mRNAs, the use of hammerhead ribozymes is
preferred. Hammerhead ribozymes cleave mRNAs at locations dictated
by flanking regions that form complementary base pairs with the
target mRNA. The sole requirement is that the target mRNA have the
following sequence of two bases: 5'-UG-3'. The construction and
production of hammerhead ribozymes is well known in the art
(Haseloff et al.,. Nature 334:585, 1988). Preferably, the ribozyme
is engineered so that the cleavage recognition site is located near
the 5' end of the JNK3 mRNA, i.e., to increase efficiency and
minimize the intracellular accumulation of non-functional mRNA
transcripts.
[0090] Examples of potential ribozyme sites in human JNK3 include
5'-UG-3' sites which correspond to the initiator methionine codon
at, for example, in human JNK3, about nucleotides 224-226, the
codon for a downstream potential initiation site (nucleotides
338-340), and additional codons in the coding region, including
nucleotides 698-670; 740-742; and 935-937.
[0091] The ribozymes of the present invention also include RNA
endoribonucleases (hereinafter "Cech-type ribozymes"), such as the
one that occurs naturally in Tetrahymena Thermophila (known as the
IVS or L-19 IVS RNA), and which has been extensively described by
Cech and his collaborators (Zaug et al., Science 224:574, 1984;
Zaug et al., Science, 231:470, 1986; Zug et al., Nature 324:429,
1986; PCT Application No. WO 88/04300; and Been et al., Cell
47:207, 1986). The Cech-type ribozymes have an eight base-pair
sequence that hybridizes to a target RNA sequence, whereafter
cleavage of the target RNA takes place. The invention encompasses
those Cech-type ribozymes that target eight base-pair active site
sequences present in JNK3 proteins.
[0092] As in the antisense approach, the ribozymes can be composed
of modified oligonucleotides (e.g., for improved stability, or
targeting), and should be delivered to cells which express a JNK3
gene in vivo, e.g., the brain and spinal cord. A preferred method
of delivery involves using a DNA construct "encoding" the ribozyme
under the control of a strong constitutive pol III or pol II
promoter, so that transfected cells will produce sufficient
quantities of the ribozyme to destroy endogenous JNK3 mRNAs and
inhibit translation. Because ribozymes, unlike antisense molecules,
are catalytic, a lower intracellular concentration is required for
efficiency.
[0093] For any of the above approaches, the therapeutic JNK3
antisense or ribozyme nucleic acid molecule construct is preferably
applied directly to the target area (e.g., the focal site of
activity in a seizure disorder, the hippocampus in Alzheimer's
disease, the substantia nigra in patients with Parkinson's
disease), but can also be applied to tissue in the vicinity of the
target area or even to a blood vessel supplying the target
area.
[0094] For gene therapy, antisense, or ribozyme JNK3 expression is
directed by any suitable promoter (e.g., the human cytomegalovirus,
simian virus 40, or metallothionein promoters), and its production
is regulated by any desired mammalian regulatory element. For
example, if desired, enhancers that direct preferential gene
expression in cells under excitotoxic induction can be used to
direct antisense JNK3 expression in a patient with a seizure
disorder.
[0095] JNK3 antisense or ribozyme therapy is also accomplished by
direct administration of the antisense JNK3 or ribozyme RNA to a
target area. This mRNA can be produced and isolated by any standard
technique, but is most readily produced by in vitro transcription
using an antisense JNK3 DNA under the control of a high efficiency
promoter (e.g., the T7 promoter). Administration of antisense JNK3
RNA to target cells is carried out by any of the methods for direct
administration of therapeutic compounds described herein.
Methods of Treating Disorders Involving JNK3 Expression or
Activity
[0096] The invention also encompasses the treatment of disorders,
especially in mammal, such as humans, in which JNK3 plays a
damaging role. A number of disorders or the nervous system
involving excitotoxicity, such as seizure disorders (e.g.,
epilepsy), dementias such as meurodegenerative disorders (e.g.,
Alzheimer's disease, Huntington disease), cerebrovascular disorders
such as ischemia, motor neuron disease (including ALS), injuries
caused by extreme heat or cold, trauma (e.g., irradiation, spinal
cord injury, pressure, and ionic imbalance), metabolic imbalance
(e.g., hypoglycemia) and Parkinson's disease, can be treated by the
methods described herein. Without limiting the invention by
committing to any particular theory, a substantial number of
neurologic disorders are attributable, at least in part, to
excitotoxicity which is mediated by the JNK3 pathway. Thus,
inhibitors of this pathway, identified as described above, are
useful for treatment of disorders involving excitotoxicity.
[0097] Therapy can be designed to reduce the level of endogenous
JNK3 gene expression, e.g., using antisense or ribozyme approaches
to inhibit or prevent translation of a JNK3 mRNA; triple helix
approaches to inhibit transcription of the gene; or targeted
homologous recombination to inactivate or "knock out" a gene or its
endogenous promoter. The antisense, ribozyme, or DNA constructs
described herein can be administered directly to the site
containing the target cells; e.g., specific regions of the brain or
the spinal cord. Antibodies or fragments of antibodies that
recognize JNK3 or a JNK3 substrate, and that have been modified to
be expressed or otherwise enter the cell can also be used
therapeutically.
Effective Dose
[0098] Toxicity and therapeutic efficacy of the compounds of the
invention, e.g., compounds that modulate JNK3 expression or
activity, can be determined by standard pharmaceutical procedures,
using either cells in culture or experimental animals to determine
the LD.sub.50 (the dose lethal to 50% of the population) and the
ED.sub.50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as the ratio
LD.sub.50/ED.sub.50. Polypeptides or other compounds that exhibit
large therapeutic indices are preferred. While compounds that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets such compounds to the site of
affected tissue to minimize potential damage to uninfected cells
and, thereby, reduce side effects.
[0099] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (that is, the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma can
be measured, for example, by high performance liquid
chromatography.
Formulations and Use
[0100] Pharmaceutical compositions for use in accordance with the
present invention can be formulated in conventional manner using
one or more physiologically acceptable carriers or excipients.
[0101] Thus, the compounds and their physiologically acceptable
salts and solvates may be formulated for administration by
inhalation or insufflation (either through the mouth or the nose)
or oral, buccal, parenteral or rectal administration.
[0102] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (for example, pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(for example, lactose, microcrystalline cellulose or calcium
hydrogen phosphate); lubricants (for example, magnesium stearate,
talc or silica); disintegrants (for example, potato starch or
sodium starch glycolate); or wetting agents (for example, sodium
lauryl sulphate). The tablets may be coated by methods well known
in the art. Liquid preparations for oral administration may take
the form of, for example, solutions, syrups or suspensions, or they
may be presented as a dry product for constitution with water or
other suitable vehicle before use. Such liquid preparations may be
prepared by conventional means with pharmaceutically acceptable
additives such as suspending agents (for example, sorbitol syrup,
cellulose derivatives or hydrogenated edible fats); emulsifying
agents (for example, lecithin or acacia); non-aqueous vehicles (for
example, almond oil, oily esters, ethyl alcohol or fractionated
vegetable oils); and preservatives (for example, methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate. Preparations for oral administration may be
suitably formulated to give controlled release of the active
compound.
[0103] For buccal administration the compositions may take the form
of tablets or lozenges formulated in conventional manner.
[0104] The preferred methods of administering the compositions of
the invention are by direct delivery of the compounds to the
central nervous system, preferentially to the brain, especially
near to or directly at the site of the disorder, e.g., the
hippocampus in the case of Alzheimer's disease, the substantia
nigra in the case of Parkinson's disease, and the focal site for
seizure disorders. Accordingly, administration may be into a
ventricle, intrathecal, or intracerebral ventricular. For example,
an Omaya reservoir-shunt with in-line filter can be surgically
placed into the cisternal space. A therapeutic compound in an
appropriate excipient (e.g., phosphate-buffered saline) is
instilled into the shunt by injection on a prescribed basis.
[0105] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, for example,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of, for example, gelatin for use in an inhaler or
insufflator may be formulated containing a powder mix of the
compound and a suitable powder base such as lactose or starch.
[0106] The compounds can be formulated for parenteral
administration by injection, for example, by bolus injection or
continuous infusion. Formulations for injection may be presented in
unit dosage form, for example, in ampoules or in multi-dose
containers, with an added preservative. The compositions may take
such forms as suspensions, solutions or emulsions in oily or
aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents. Alternatively,
the active ingredient may be in powder form for constitution with a
suitable vehicle, for example, sterile pyrogen-free water, before
use.
[0107] The compounds can also be formulated in rectal compositions
such as suppositories or retention enemas, for example, containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0108] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0109] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
[0110] The therapeutic compositions of the invention can also
contain a carrier or excipient, many of which are known to skilled
artisans. Excipients which can be used include buffers (for
example, citrate buffer, phosphate buffer, acetate buffer, and
bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid,
phospholipids, proteins (for example, serum albumin), EDTA, sodium
chloride, liposomes, mannitol, sorbitol, and glycerol. The nucleic
acids, polypeptides, antibodies, or modulatory compounds of the
invention can be administered by any standard route of
administration. For example, administration can be parenteral,
intravenous, subcutaneous, intramuscular, intracranial,
intraorbital, opthalmic, intraventricular, intracapsular,
intraspinal, intracisternal, intraperitoneal, transmucosal, or
oral. The modulatory compound can be formulated in various ways,
according to the corresponding route of administration. For
example, liquid solutions can be made for ingestion or injection;
gels or powders can be made for ingestion, inhalation, or topical
application.
[0111] Methods for making such formulations are well known and can
be found in, for example, "Remington's Pharmaceutical Sciences." It
is expected that particularly useful routes of administration will
be nasal or by direct infusion into the central nervous system.
EXAMPLES
Example 1
JNK3 Expression
[0112] A 351-bp sequence derived from the 5' region of the mouse
JNK3 cDNA (nucleotides 62-412) was labelled with [.sup.32P] by
random priming and used as a probe to determine the tissue
expression pattern of the JNK3 gene. Northern blot analysis was
performed by standard methods on 2 mg samples of poly(A).sup.+ mRNA
isolated from testis, kidney, skeletal muscle, liver, lung, spleen,
brain, and heart. All Northern blots were probed with
[.sup.32P]-labelled .beta.-actin as a control to ensure loading of
similar amounts of RNA in each lane. A strong signal corresponding
to a 2.7 kb transcript, as well as a weak signal corresponding to a
7.0 kb transcript, were detected in brain. A weak signal
corresponding to a 2.7 kb transcript was also detected in the
heart. A signal corresponding to a 2.4 kb transcript was detected
in the testis. JNK3 expression was not detected in the other
tissues examined.
[0113] In situ hybridization analysis has indicated that JNK3 is
expressed in many regions of the brain (Martin et al., supra).
Total RNA (10 mg) was therefore isolated from different regions of
mouse brain (cerebellum, cerebral cortex, hippocampus, midbrain,
thalamus, and brainstem) using the TRIzol reagent (Gibco-BRL), and
analyzed by Northern blot using the JNK3 probe described above. A
signal corresponding to a 2.7 kb transcript was detected in all
sections of the brain examined, and was most abundant in the
hippocampus.
Example 2
Targeted Disruption of the JNK3 Gene
[0114] To generate JNK3-deficient mice, a targeting vector was
designed to replace an internal 4 kb Mscl-Spel JNK3 genomic
fragment with a PGKneo cassette. A map of the JNK3 gene, the JNK3
targeting vector, and the predicted structure of the mutated JNK3
gene are shown diagrammatically in FIG. 6. Restriction enzyme sites
are indicated (B, BamHI; Hp, HpaI; M, MscI; Nco, NcoI; R, EcoRI;
Spe, SpeI). A 10-kb NotI-EcoRI (the NotI site was vector derived)
JNK3 fragment was cloned from a .lamda. FixII phage library of a
129/Sv mouse strain (Stratagene Inc.). The targeting vector was
constructed by inserting a 4.0 kb MscI fragment from the 5' end of
the JNK3 genomic fragment, a 1.6 kb PGK-neo cassette (Negishi et
al., Nature 376:435-438, 1995) and a 1.8-kb SpeI-NcoI fragment of
the 3' end of the JNK3 fragment into pBluescript KS vector
(Stratagene Inc.) using appropriate linkers. The targeting vector
contains a 2.6-kb PGK tk cassette (Negishi et al., supra) flanking
the 5' end of the JNK3 genomic sequence for negative-selection of
mutant ES cells (Mansour et al., 336:348-352, 1988). The region
replaced in the JNK3 gene by the targeting vector encompasses one
and a half exons encoding amino acids 211 to 267 of JNK3 (as shown
in FIG. 5B). This region includes the tripeptide dual
phosphorylation motif Thr-Pro-Tyr (TPY) that is characteristic of
the JNK group and required for protein kinase activity (Derijard et
al., supra). The two hatched boxes shown in the JNK3 locus
correspond to subdomains VIII and IX (encoding amino acid residues
189-267 in the JNK3 protein shown in FIG. 5B) of JNK3.
[0115] The targeting vector was linearized with NotI and
electroporated into W9.5 embryonic stem (ES) cells. Genomic DNA
from transfectants resistant to G418 (200 mg/ml)(Gibco BRL) and
gancyclovir (2 mM) (Syntex, Pala Alto, Calif.) were isolated and
screened by Southern blot analysis. Southern blot analysis of 104
independent G418- and gancyclovir-resistant clones revealed three
clones containing the desired homologous recombination event
(targeting frequency 2.9%). Chimeric mice were generated by
injecting these ES cells into C57BL/6 (B6) mouse blastocysts.
[0116] Southern blots of EcoRI-restricted DNA derived from the
tails of these chimeric mice were probed with the radiolabeled 351
bp JNK3 probe. EcoRI digestion resulted in a 12 kb band
corresponding to the wild-type (endogenous allele), and a 4.2 kb
band corresponding to the mutant (disrupted allele).
[0117] Two clones mediated germline transmission of the disrupted
JNK3 allele into the next generation of mice. Heterozygotes (+/-)
were intercrossed to generate homozygous mutant mice (-/-) that
were identified by Southern blot analysis of genomic DNA. Total RNA
isolated from mouse brain was examined by Northern blot analysis.
The blot was probed with a random-primed .sup.32P-labeled mouse
JNK3 cDNA probe, then stripped and sequentially reprobed with mouse
JNK1 and .beta.-actin cDNA probes. The major JNK3 transcript in
brain is 2.7 kb, and the JNK1 transcripts in mouse brain are 2.3
and 4.4 kb. Blots hybridized with a JNK3 cDNA probe detected
transcripts in wild-type (+/+), but not in homozygous knockout
(-/-) mice.
[0118] Reverse transcriptase-polymerase chain reaction (RT-PCR)
analysis was used to confirm that JNK3 transcripts were absent in
the homozygous JNK3 (-/-) brain. A JNK1 probe (447 bp) was
amplified from mouse brain RNA by RT-PCR (Yang et al., supra) using
the amplimers 5'-GTGTGCAGCTTATGATGCTATTCTTGAA-3' (SEQ ID NO:21) and
5'CGCGTCACCACATACGGAGTCATC-3' (SEQ ID NO:22). RT-PCR detection
(Yang et al., supra) of JNK3 mRNA in mouse tissues was performed by
RT-PCR using the amplimers: 5'-GTGGAGGAGTTCCAAGATGTCTACT-3' (SEQ ID
NO: 23) and 5'-TGGAAAGAGCTTGGGGAAGGTGAG-3' (SEQ ID NO:24) to yield
specific 537 bp DNA product. RNA isolated from mouse brain was
amplified with primers specific for HPRT as a control. These
experiments confirmed the absence of JNK3 transcripts in the
homozygous JNK3 (-/-) brain.
[0119] Protein kinase assays were performed to show that JNK3 (-/-)
mouse brain was deficient in JNK3 activity. In these experiments,
JNK3 kinase activity in brain lysates was measured after
immunodeletion of JNK1 and JNK2 by in-gel protein kinase assays
using the substrate GST-cJun (Derijard et al., supra) When mouse
hippocampal lysates (30 .mu.g) from wild type (+/+) and homozygous
knockout (-/-) brains were assayed, the 55 kD and 46 kD JNK3
isoforms were detected in wild type but not JNK3 (-/-) mice,
confirming that JNK3 (-/-) mouse brain was deficient in JNK3 kinase
activity. Together, these data demonstrate that the targeted
disruption of the JNK3 gene resulted in a null allele.
[0120] The JNK3(-/-) mice were fertile and of normal size.
Histological surveys of a variety of tissues revealed no apparent
abnormality using hematoxylin and eosin (H & E) staining of
heart, lung, thymus, spleen, lymph nodes, liver, kidney, and
skeletal muscle. JNK3(-/-) and wild-type mouse brains were examined
by immunocytochemical analysis of a pyramidyl neuronal marker
(MAP-2), interneuronal markers calbindin and parvalbumin, an
astrocyte marker (glial fibrillary acidic protein; GFAP; Hsu et
al., J. Histochem. Cytochem. 29:577-580, 1981), and Nissl's stain
(Hsu et al., supra). These studies revealed that JNK3(-/-) mice had
apparently normal development and structural organization of the
brain. A comparable number of motor neurons were found in the
facial nucleus in wild-type and JNK3(-/-) mice (2150-2300 neurons
per nucleus at postnatal day 10, n=4). The neurons were identified
by morphology and were counted by a double-blind assay of serial
sections throughout the facial nuclei of wild-type and JNK3(-/-)
mice. Thus, there is no apparent developmental abnormality,
including cell death, in JNK3 (-/-) mice.
Example 3
JNK3 Deficient Mice are Resistant to KA-Induced Seizures
[0121] JNK3(-/-) mice and their wild-type littermates were injected
intraperitoneally (i.p.) with 30 mg/kg KA to induce seizures
(Ben-Ari, supra). In wild-type mice, the administration of KA first
induced "staring spells" with abnormal body posture, then
progressed to head nodding ("wet-dog shakes"), fore-paw tremor,
rearing, loss of postural control, and eventually, continuous
convulsions. The seizure activities typically subsided one hour
after injection. Wild-type and heterozygous mice developed motor
symptoms of seizures, including rearing, at 30 to 40 minutes
post-injection. The JNK3 (-/-) mice, in contrast, developed much
milder symptoms, mainly consisting of "staring" spells and
occasional myoclonic tremors. At this dose, JNK3 (-/-) mice did not
develop grand mal seizures and recovered much faster than did
wild-type and heterozygous mice. JNK3 (-/-) mice developed seizures
of comparable severity to wild-type mice only at higher dosages of
KA (45 mg/kg, i.p.). However, at this high dose of KA, more than
60% of wild-type mice died from continuous tonic clonic
convulsions, while all of the JNK3(-/-) mice survived. These
results indicate that JNK3(-/-) mice were resistant to the effect
of the excitotoxin KA. Further, JNK3(-/-) mice recovered from the
drug administration more rapidly than wild-type mice (FIG. 7).
Seizure classifications as shown in FIGS. 7 and 8 are: 1, arrest of
motion; 2, myoclonic jerks of the head and neck, with brief
twitching movements; 3, unilateral clonic activity; 4, bilateral
forelimb tonic and clonic activity; and 5, generalized tonic-clonic
activity with loss of postural tone, often resulting in death.
Example 4
Resistance to Pentetrazole (PTZ)-Induced Seizures
[0122] Since the resistance to KA-induced seizures varied between
littermates (+/+ and +/- are less resistant than -/- mice), the
observed differential susceptibility cannot be attributed to a
difference between mouse strains (Schauwecker and Steward, Proc.
Nat. Acad. Sci. USA 94:4103-4108, 1997). However, the resistance of
JNK3 deficient mice to KA-induced seizures could be due to
decreased drug penetration across the blood-brain barrier or an
increased GABA (gamma-aminobutyric acid) inhibitory postsynaptic
potential (IPSP), or the ablation of a specific signal transduction
pathway mediated by the JNK3 protein kinase. To distinguish between
these possibilities, the response of JNK3 (-/-) and wild-type mice
to another epileptogenic agent, pentetrazole (PTZ) (Sigma), was
examined. PTZ was selected due to its ability to induce seizures by
blocking the GABA-IPSPs (Ben-Ari et al., Neurosci. 6:1361-1391,
1981).
[0123] JNK3(-/-) mice and wild-type littermates developed seizures
of comparable severity at all tested dosages of PTZ (30, 40, 50, 60
mg/kg, i.p.; FIG. 8). Moreover, unlike the slow progression of
motor symptoms seen in the KA-induced seizures, PTZ induced abrupt
general tonic-clonic seizures within five minutes after injection,
presumably reflecting that its epileptogenic mechanism works solely
through extracellularly inhibition of the GABA-IPSP. Thus, the
differential susceptibility to KA toxicity in JNK3(-/-) mice can
neither be explained as a consequence of poor drug delivery to the
nervous system nor by potent GABA-IPSPs in the neural circuit.
Furthermore, we examined the expression of the kainate-type
glutamate receptor subunits GluR5-7 (Pharmingen cat. no. 60006E) by
immunocytochemistry using standard methods.
[0124] Pyramidal neurons in the hippocampal CA1 subfield were most
prominently labeled by the Glu5-7 antibody. Both wild-type and
JNK3(-/-) mice showed prominently labeled apical dendrites arising
from lightly labeled somata in the CA1 subfield of the hippocampus,
a pattern similar to the primate hippocampus (Good et al., Brain
Research 624:347-353, 1993). In addition to kainate-type subunit
GluR5-7, the expression pattern of the GluR1 subunit that is
essential to various glutamate receptor subtypes and the
intracellular calcium-binding proteins parvalbumin and calbindin
that may buffer the influx of extracellular calcium were also
indistinguishable between JNK3(-/-) and wild-type mice. Together,
these results indicate no apparent structural abnormality that
might be responsible for the resistance of JNK3(-/-) mice to
KA-induced excitotoxicity.
Example 5
Attenuation of KA-Induced Phosphorylation of c-Jun
[0125] The systemic administration of KA in wild-type mice may
induce a stress-response pathway mediated by the JNK3 protein
kinase. To explore this possibility, the expression of the
immediate-early genes c-fos and c-jun (Morgan et al., Annu. Rev.
Neurosci. 14:421-451, 1991; Smeyne et al., Nature 363:166-169,
1993; Kasof et al., J. Neurosci. 15:4238-4249, 1995) was examined
to determine whether KA imposed an equivalent level of noxious
stimulation on wild-type and JNK3(-/-) mice. Total RNA was
extracted from the hippocampi of mice sacrificed before and at 0.5,
2, 4, or 8 hours after KA injection (30 mg/kg, i.p.), and Northern
blots were probed with murine c-fos and c-jun probes. The c-jun
probe was a 207 bp fragment corresponding to nucleotides 888-1094
(FIG. 9) of the murine c-Jun cDNA. The c-Fos probe was a 347 bp
fragment of the murine c-Fos gene (exon 4; base pairs 2593-2939)
(FIG. 10). Both JNK3(-/-) and wild type mice exhibited a comparable
level of rapid induction of c-fos and c-jun transcripts, which
gradually declined four hours after injection.
[0126] To further define this phenomenon, the distribution of
KA-induced c-Fos and c-jun immunoreactivity along the synaptic
circuit of the hippocampus was examined. In these experiments,
homozygous mutant and control wild-type mice were killed and fixed
by transcardial perfusion of 4% paraformaldehyde at 2 or 6 hours
after the injection of KA (30 mg/kg, i.p.). Brains from both groups
were removed, post-fixed for one hour, and sectioned on a Vibratome
(40 mm thick). Tissue sections were processed by
immunocytochemistry to detect the expression of c-Jun (Santa Cruz,
cat# sc-45), c-Fos (Santa Cruz, cat# sc-52), and phospho-specific
c-Jun (Ser-73) (New England Biolabs, #9164S). Sections were floated
in a solution of the primary antibody (diluted 200.times. in PBS)
and incubated overnight at room temperature. Secondary antibody
incubation, avidin-biotin conjugated peroxidase (Vectastain Elite
ABC kit, Vector Lab.), and DAB (3,3'-diaminobenzidine, Sigma)
reactions were performed using standard procedures (Hsu et al.,
supra). In the absence of KA, there was no detectable c-Fos
expression and only a few c-Jun-positive cells within the dentate
gyrus. Two hours after KA injection (30 mg/kg, i.p.), there was a
large increase in c-Fos immunoreactivity throughout the hippocampal
region that was the same in both wild-type and JNK3(-/-) mice.
Simultaneously, there was an increase in the number of
c-Jun-positive cells in the dentate gyrus and the CA3 region of the
hippocampus in both wild type and JNK3(-/-) mice. By six hours
after KA injection, the expression of c-Jun extended to the CA1
region in both wild-type and JNK3(-/-) mice. The induction of c-Fos
and C-Jun is generally accepted as an indicator of neuronal
activity following noxious stimulation (Morgan et al., supra). The
comparable induction level, time-course, and distribution of c-Jun
and c-Fos-labeled cells suggests that JNK3(-/-) and wild-type mice
were subject to an equivalent level of noxious stress by systemic
administration of KA.
[0127] C-Jun is activated by phosphorylation of the
NH.sub.2-terminal activation domain by JNK. The expression of
phosphorylated c-jun provides another measure of whether JNK-like
activity was present in JNK3(-/-) mice. The expression of
phosphorylated c-Jun was examined using an antibody raised against
c-jun phosphorylated at Ser-73, one of the sites phosphorylated by
JNK (Whitmarsh et al., supra; Derijard et al., supra; Kyriakis et
al., supra). Prior to challenge with KA, no cells were labeled by
the antibody in either wild type or JNK3(-/-) mice. By two hours
after KA injection, there was a high level of phosphorylated c-Jun
in the dentate gyrus and the CA3/CA4 region of the hippocampus in
wild-type mice. In contrast, only a trace amount of phosphorylated
c-Jun was detected in the JNK3(-/-) mice. Thus, there was either a
decreased level or less sustained phosphorylation of c-Jun in the
JNK3(-/-) mice.
[0128] In addition, there was a dynamic change of the distribution
of phosphorylated c-Jun in the wild type mouse hippocampus. By six
hours after KA injection, the expression of phosphorylated c-Jun
subsided in the dentate gyrus and progressed to a restricted area
in the hippocampal CA3 region. Under higher magnification, it was
apparent that the expression of phosphorylated c-Jun surrounded a
focus of cell destruction. In contrast, no labeling of
phosphorylated c-Jun was detected in the JNK3(-/-) mice at the same
time point. The hippocampal CA3 region is well documented as the
most vulnerable structure to the KA excitotoxicity, presumably due
to both a high KA binding affinity (Berger et al., supra) and a
potent excitatory synaptic connection between CA3 pyramidal neurons
(Westbrook et al., Brain Research 273:97-109, 1983). These results
indicate that JNK3 is required for the phosphorylation of c-Jun
induced by KA.
Example 6
Attenuation of KA-Induced AP-1 Transcriptional Activity
[0129] Since the phosphorylation of c-Jun is an important initial
event during the induction of AP-1 transcriptional activity
(Whitemarsh et al., supra; Yang et al., supra), whether the
observed attenuation of c-Jun phosphorylation would lead to
decreased induction of AP-1 transcriptional activity in JNK3 (-/-)
mice was examined. JNK(-1-) mice were crossed with transgenic AP-1
luciferase. (AP1-luc) mice (Rincon et al., Embo. J. 13:4370-4381,
1994) and progeny back crossed. The JNK3(-/-)/API-Luc(-/+) mice
were used in experiments with JNK3(+/+) mice to compare the level
of KA-induced AP-1 transcriptional activity in the presence or
absence of JNK3. The AP1-luc mice contain the firefly luciferase
gene under the control of four copies of a consensus AP-1 binding
site in the context of the minimal rat prolactin promoter. It has
been established that the expression of luciferase in these mice is
due to the presence of the AP-1 regulatory element.
[0130] In the luciferase assay, mice were sacrificed at intervals
after injection of KA (30 mg/kg, i.p.), and relative luciferase
activity compared with that detected in hippocampal lysates
obtained from mice injected with vehicle (saline). Mice were
decapitated, brains were dissected, and brain tissues were
immediately lysed in buffer containing 25 mM Hepes pH 7.4, 1%
TRITON.RTM.X-100, 1 mM EDTA, 1 mM phenylmethyl sulfonyl fluoride,
and 10 .mu.g/ml leupeptin (Promega, Madison, Wis.). Luciferase
activity was measured as described in Rincon and Flavell (Embo. J.
13:4370-4381, 1994). The injection of KA (30 mg/kg, i.p.) caused a
large induction of AP-1 transcriptional activity in the hippocampus
of wild-type mice, as evidenced by the induction of luciferase
activity. Luciferase activity in wild type mice was detectable by
six hours, gradually increased to the peak at three days, and
persisted for at least seven days (FIG. 11). Control experiments
demonstrated that the injection of vehicle (saline) did not cause
induction of luciferase activity in the AP1-luc mice.
[0131] The relative luciferase activity in the hippocampus and
cerebellum prepared from wild-type (+/+) and JNK3 (-/-) mice was
measured following KA injection. The results are shown in FIG. 12.
Each time point represents the mean of three to five (+SEM)
individual animals. The induction of luciferase activity was most
prominent in the hippocampus, where a markedly greater induction of
phosphorylation of c-Jun was observed, as compared to the
cerebellum and the cerebral cortex. The induction of AP-1 activity
was significantly reduced in the JNK3 (-/-) mice with the AP1-luc
transgene as compared to the wild type mice. At 15 hours after
injection of KA, there was approximately four-fold greater AP-1
activity in the hippocampus of wild-type mice compared with
JNK3(-/-) mice. At three days after injection, the AP-1 activity
was more than six times higher in the hippocampus of wild-type
compared with JNK3(-/-) mice. Together, these data demonstrate that
the disruption of the JNK3 gene suppressed KA-induced
phosphorylation of c-Jun and AP-1 transcription activity in the
hippocampus in vivo.
Example 7
Resistance to KA-Induced Apoptosis
[0132] One unique feature of KA among other epileptogenic agents is
its potency in inducing neuronal cell death (Ben-Ari, supra; Schwob
et al., supra). Since this property of cell destruction is
paralleled by a sustained level of AP-1 transcriptional activity,
it has been suggested that AP-1 mediates KA-induced-neuronal death
(Kasof et al., supra; Schwarzschild et al., J. of Neurosci.
17:3455-3466, 1997). Wild-type and JNK3(-/-) mouse brains were
therefore examined after treatment with KA to determine whether the
attenuation of AP-1 transcriptional activity in JNK3 (-/-) mice
altered the extent of neuronal damage (Ben-Ari, supra; Ben-Ari et
al., supra; Schwob et al., Neurosci. 5:991-1014, 1980).
[0133] These experiments were performed as follows. Wild-type and
JNK3 (-/-) mice were killed and fixed by transcardial perfusion of
4% paraformaldehyde and 1.5% glutaraldehyde three days after the
injection of KA (30 mg/kg, i.p.). Semithin and thin sections of
brain were prepared using a Vibratome and embedded in Epon. Tissue
blocks were prepared using a microtome with a diamond tube for 1
.mu.m-thick semithin sections examined by toluidine blue staining,
and for ultrathin sections examined by electron microscopy. Nissl's
stain was used for initial examination of damage to the hippocampus
(Kluver et al., J. Neuropath. Exp. Neuro. 12:400-403, 1953). GFAP
immunocytochemistry was also used to assess cell destruction in the
hippocampus (Hsu et al., supra). Nissl's staining was also
performed as described above. TUNEL assays, used to evaluate
apoptosis, were performed using cryostat sections (50 .mu.m) of
cerebral hemispheres that were cryoprotected with sucrose. The
TUNEL assay was modified from the terminal deoxynucleotidyl
transferase (TdT)-mediated dUTP nick end labeling assay (Gavrieli
et al., J. Cell. Biol. 119:493-501, 1992). Briefly, tissue
sections, directly mounted on a salinated slide, were permeablized
with 2% TRITON.RTM. X-100 (20 minutes at room temperature) and then
incubated for nick end-labeling for 2 hours at 37.degree. C. using
0.32 U/.mu.l TdT (Boehringer Mannheim, cat# 220582) and 2 .mu.M
digoxigenin-11-dUTP (Boehringer Mannheim, cat# 1573152) in a final
volume of 40 .mu.l. The tissues were incubated with
anti-digoxigenin antibody (Boehringer Mannheim, cat# 1333062)
diluted 500-fold, and processed for immunocytochemistry using
standard procedures (Hsu et al., supra).
[0134] The damage to the hippocampus caused by KA was initially
examined by Nissl's stain. The KA-induced cell loss caused either a
breach of staining of the pyramidal neurons in the CA3 region or a
diffuse sparse staining throughout the CA1 subfield. To corroborate
the cell destruction revealed by Nissl's stain, the TUNEL method
was applied to detect apoptosis. Groups of small pyknotic nuclei
and positively TUNEL-labeled cells were found in the hippocampal
CA3 subfield devoid of Nissl's staining. Similarly, a high
percentage of pyramidal neurons showing pyknotic nuclei and
shattered apical dendrites and numerous strongly TUNEL-labeled
cells were located in the hippocampal CA1 subfield which exhibited
decreased Nissl's staining. Since the TUNEL method and the pyknosis
morphology only indicated the extent of cell damage at one time
point of a dynamic process, immunostaining of damage-induced GFAP
was also used as an independent assessment of the extent of cell
destruction in the hippocampus. Consistent with the patterns of
Nissl's, toluidine, and TUNEL staining, an increased number of
strongly GFAP-labeled astrocytes was found either in the
hippocampal CA3 or CA1 regions. Thus, a combination of Nissl's
stain, GFAP immunocytochemistry, TUNEL method, and toluidine stain
of semithin sections was used to classify the KA-induced damage in
the mouse hippocampus.
[0135] A total of 17 wild-type and 18 JNK3(-/-) mice were examined.
Results are shown in Table 1 (below). The table was compiled from
two sets of data. First, wild-type (n=11) and JNK3 (-/-) (n=10)
mice were sacrificed on the fifth day after a single injection of
KA (30 mg/kg, i.p.). Second, wild-type (n=6) and JNK3 (-/-) (n=8)
mice received an injection of KA (30 mg/kg, i.p.) for five
consecutive days and examined two days following the final
injection. The severity of the hippocampal damage in wild-type mice
was comparable in experiments using both protocols. The ratio of no
cell loss/CA3 lesion/CA3+CA1 lesion was 2/7/2 in the
single-injection experiments, and 2/2/2 in the multiple injection
experiments. TABLE-US-00001 TABLE 1 Kainate-indUced neuronal damage
(number of animals) JNK3 genotype +/+ -/- No perceivable cell loss
4 18 Selective CA3 cell loss 9 0 Including CAl cell loss 4 0
[0136] The hippocampal CA3 region was the most susceptible to
KA-induced damage in the wild-type mice (9/17; 53%). Cell loss was
indicated by decreased crystal violet staining in the CA3 region.
Using the TUNEL method (Gavrieli et al., supra), which identifies
DNA fragmentation in the dying cells, groups of labeled cells were
found in the damaged region. A cluster of pyknotic nuclei was found
in the CA3 region in toluidine-stained semithin sections. As a
result of KA-induced damage, there was selective glial
proliferation confined to the CA3 region, as indicated by the
strong immunostaining of GFAP. In some wild-type animals, massive
cell loss was observed throughout the entire hippocampal CA1 region
(4/17; 24%). Similarly, damage to the CA1 region was revealed by
decreased crystal violet staining, positively TUNEL-labeled cells,
pyknotic nuclei, shattered apical dendrites of pyramidal neurons,
and both hypertrophy and proliferation of GFAP-positive
astrocytes.
[0137] In contrast, there was no apparent hippocampal damage in any
of the JNK3(-/-) mice examined (n=18). The pattern of the Nissl's
stain, TUNEL assay, toluidine blue staining of semithin sections,
and GFAP immunostaining of the hippocampal region in the JNK3(-/-)
mice was indistinguishable from that of untreated wild-type mice.
Moreover, although JNK2(-/-) mice developed seizures of comparable
severity at the sublethal dose of 45 mg/kg KA (a dose that is
lethal for more than 60% of wild-type mice due to continuous
convulsions), cell damage was nevertheless found in a much smaller
percentage of animals (2/15, 13%; p<0.005 by chi-square
analysis, d.f.=1).
[0138] Methods of assessing apoptosis (e.g., TUNEL assay) can be
used to evaluate whether JNK3 modulator is affecting apoptosis.
Example 8
Electron Microscopic Analysis of Ultrastructural Changes Associated
with KA-Induced Neuronal Damage
[0139] Cortical neurons in vitro undergo either apoptosis or
necrosis depending on the extracellular concentration of the
glutamate analog N-methyl-D-aspartate-(NMDA) (Bonfoco et al., Proc.
Natl. Acad. Sci. USA 92:7162-7166, 1995). The distinction between
apoptosis versus necrosis in KA-induced neuronal damage is critical
since necrosis is generally thought to represent a consequence of
acute mechanical insult that is incompatible with an active cell
death program involving de novo protein synthesis. The TUNEL
results (supra) indicate the involvement of apoptosis. To further
examine whether the neuronal death in vivo due to KA induction was
apoptotic or necrotic, electron microscopy was employed to
investigate the ultrastructural changes in the degenerated
hippocampal neurons. The microscopic analysis suggested a series of
morphological changes indicating neuronal damage in the wild-type
mouse as a consequence of apoptosis. The initial event after KA
injection (30 mg/kg i.p.) appeared to be compaction and segregation
of chromatin in pyramidal neurons into electron-dense masses that
abutted on the inner surface of the nuclear envelope. In contrast,
the nuclei of the hippocampal neurons in the JNK3(-/-) mice
following KA injection contained homogeneous electron-lucent
euchromatin. At later stages, in wild type mice, there was
convolution of the nuclear outline and condensation of the
cytoplasm. The double-layered structure of the nuclear envelope in
wild type mice remained largely intact in all of these
morphological stages. Eventually the degenerated neurons
disintegrated resulting in numerous membrane-bounded apoptotic
bodies. These morphological features are all consistent with the
hallmarks of apoptosis (Kerr et al., Br. J. Cancer 26:239-257,
1972). Thus, it appeared that KA triggered a genetic program within
the damaged neuron leading to apoptosis, which was abrogated in
JNK3-deficient neurons.
[0140] These results suggest that KA-induced phosphorylation of the
NH.sub.2-terminal activation domain of c-Jun leads to increased
AP-1 transcriptional activity and neuronal apoptosis. Without
limitation to a particular theory, a proposed chain of molecular
events caused by KA that lead to neuronal apoptosis is shown in
FIG. 13.
[0141] Although systemic administration of KA causes cell damage
predominantly localized in the hippocampal CA3 area, the
significance of JNK3 in stress-induced neuronal apoptosis is not
only restricted to this region. Several lines of evidence indicate
that the particular vulnerability of the CA3 hippocampal neurons to
KA is due to their unique cellular and synaptic properties. First,
the hippocampal CA3 and CA4 regions have the highest density of
KA-receptors (Berger et al., Neurosci. Lett. 39:237-242, 1983).
Second, the recurrent synaptic excitation is particularly potent in
the hippocampal CA3 region (Miles et al., J. Physiol. (London)
373:397-418, 1986). The recurrent excitation of the CA3 pyramidal
neurons may sustain JNK3 signaling and therefore rapidly induce KA
excitotoxicity. The observed progression of c-Jun phosphorylation
from the dentate gyrus to the CA3 region is reminiscent of the
synaptic circuitry of the hippocampus. A diagram of the trisynaptic
connection within the hippocampal formation is shown in FIG. 14.
The first synaptic relay (1) is from the afferent perforant path
(pp) onto the granule cell of the dentate gyrus (DG). The second
relay (2) follows the mossy fiber (mf) from the dentate gyrus to
the CA3 hippocampal neurons. The third relay (3) is from the
hippocampal CA3 to the CA1 region along the Schaffer collaterals
(Sch). There are recurrent synaptic interactions of pyramidal
neurons in the CA3 region.
Example 9
Assays for Detection of Inhibitors of JNK3 Protein Kinase
Activity
[0142] Inhibitors of JNK3 can be identified in protein kinase
assays. These assays can be performed using JNK3 purified from
tissue (e.g., brain) or with recombinant enzyme. The recombinant
JNK3 can be isolated from bacteria, yeast, insect, or mammalian
cells using standard procedures. Assays of endogenous (natural)
JNK3 are known in the art and assays of recombinant JNK3 have been
described previously (Gupta et al., EMBO J. 15:2760-2770,
1996).
[0143] The protein kinase activity of JNK3 can be measured using
ATP and protein substrates for JNK3 in an in vitro assay. These
substrates include, but are not limited to, the transcription
factors ATF2 and Elk-1 (Gupta et al., 1996, supra). The
incorporation of phosphate into the substrate can be measured by
several methods. One example is to measure the incorporation of
radioactive phosphate (e.g., .sup.32P) into the substrate. The
incorporation into the substrate can be measured following removal
of unincorporated radioactivity by precipitation with
trichloroacetic acid and recovery on phosphocellulose paper or by
polyacrylamide gel electrophoresis. The radioactivity can be
monitored by scintillation counting, phosphorimager analysis, or by
autoradiography. In general, methods for automated high throughput
screens would not use radioactive materials. For this purpose a
method is used to detect the phosphorylated substrate without a
radioactive probe. In one approach the electrophoretic mobility of
the substrate is examined. For example, ATF2 demonstrates a marked
reduction in electrophoretic mobility following phosphorylation by
JNK on Thr-69 and Thr-71 (Gupta et al., Science 267:389-393,
1995).
[0144] A second approach is to detect the phosphorylation of the
substrate using immunochemical methods (e.g. ELISA). Antibodies
that bind specifically to the phosphorylated substrates are
prepared (monoclonal and polyclonal) and are commercially available
(e.g., New England Biolabs, Promega Corp., and Upstate
Biotechnology Inc.). The extent of substrate phosphorylation is
then measured by standard ELISA assay using secondary antibodies
coupled molecules suitable for to spectrophotometric or
fluorometric detection using methods known in the art.
[0145] Molecules that inhibit JNK3 can be identified in a high
throughput screen. A molecule that is a preferred candidate to
treat excitotoxic disorders inhibits JNK3, but not other protein
kinases, including related MAP kinases. Candidate molecules once
identified can be optimized using combinatorial chemical methods or
by the synthesis of related molecules. These molecules represent
candidate drugs that can be tested for JNK3 therapy.
Example 10
Assays for Detection of Inhibitors of JNK3 Activation
[0146] The JNK protein kinases are activated by dual
phosphorylation on Thr and Tyr within protein kinase sub-domain
VIII (Davis, Trends Biochem. Sci. 19:470-473, 1994). These sites of
activating phosphorylation are conserved in JNK3 (Gupta et al.,
1996, supra). Molecules that inhibit the activation of JNK3 by
interfering with the phosphorylation of JNK3 can be identified by
measurement of JNK3 activation in the presence and absence of
candidate molecules. Cells expressing JNK3, e.g., neuronal cells,
neuroendocrine cells, or cells that are engineered to express
recombinant JNK3 (Gupta et al., 1996 supra), are exposed to
environmental stress (e.g., depolarization, excitotoxic agents, UV
radiation, heat, and anoxia) to activate JNK3. The state of JNK3
activation can be assessed by several methods. For example, JNK3
can be isolated, washed free of the candidate inhibitor, and the
activation state of JNK3 monitored by protein kinase assay (supra).
Alternatively, the activation of JNK3 can be probed using
immunological methods using antibodies that bind to the Thr and Tyr
phosphorylated (activated) form of JNK3. Antibodies that bind
specifically to the Thr and Tyr phosphorylated enzyme can be
prepared (monoclonal and polyclonal) and are commercially available
(e.g., from New England Biolabs and Promega Corp.). The extent of
substrate phosphorylation can then be measured by a standard ELISA
assay using secondary antibodies coupled to spectrophotometric or
fluorometric detection.
Other Embodiments
[0147] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
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