U.S. patent application number 15/074008 was filed with the patent office on 2016-07-07 for method of regulating neuronal axon elongation.
The applicant listed for this patent is RIKEN. Invention is credited to Hiroyuki KABAYAMA, Katsuhiko MIKOSHIBA.
Application Number | 20160193291 15/074008 |
Document ID | / |
Family ID | 49477818 |
Filed Date | 2016-07-07 |
United States Patent
Application |
20160193291 |
Kind Code |
A1 |
MIKOSHIBA; Katsuhiko ; et
al. |
July 7, 2016 |
METHOD OF REGULATING NEURONAL AXON ELONGATION
Abstract
The present invention includes a treating step of treating
neuronal growth cone with a regulatory factor to regulate
macropinocytosis caused by a repulsive axon guidance molecule. The
present invention can provide a new neuronal axon elongation
regulating method etc.
Inventors: |
MIKOSHIBA; Katsuhiko;
(Saitama, JP) ; KABAYAMA; Hiroyuki; (Saitama,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RIKEN |
Saitama |
|
JP |
|
|
Family ID: |
49477818 |
Appl. No.: |
15/074008 |
Filed: |
March 18, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13869116 |
Apr 24, 2013 |
9314501 |
|
|
15074008 |
|
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61638772 |
Apr 26, 2012 |
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Current U.S.
Class: |
514/17.7 ;
435/375; 435/6.12; 435/6.13; 435/7.21; 514/255.06; 514/44R |
Current CPC
Class: |
A61K 48/00 20130101;
A61K 38/177 20130101; A61K 31/4965 20130101; C07H 21/04 20130101;
A61K 48/005 20130101; G01N 33/5058 20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; G01N 33/50 20060101 G01N033/50; A61K 31/4965 20060101
A61K031/4965 |
Claims
1. A method of increasing neuronal axon elongation, comprising: a
treating step of treating neuronal growth cone with a regulatory
factor to reduce macropinocytosis caused by a repulsive axon
guidance molecule, the treating step inducing promotion of the axon
elongation as a result of reducing the macropinocytosis, wherein
the regulatory factor is an inhibitor of a sodium/proton exchanger
being present in the growth cone.
2. (canceled)
3. The method as set forth in claim 1, wherein the inhibitor is
5-(N-ethyl-N-isopropyl)amiloride or an analog thereof.
4-6. (canceled)
7. The method as set forth in claim 1, wherein the treating step
comprises an administering step of administering an effective
amount of the regulatory factor to a human or non-human animal,
wherein the human or non-human animal is one suffering damage to a
nervous system thereof or is to undergo an operation on its nervous
system thereof.
8-12. (canceled)
13. The method as set forth in claim 3, wherein the inhibitor is
5-(N-ethyl-N-isopropyl)amiloride.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This Continuation application claims benefit to U.S.
Non-provisional application Ser. No. 13/869,116 filed on Apr. 24,
2013, which claims benefit on U.S. Provisional Application Ser. No.
61/638,722 filed on Apr. 26, 2012, the entire contents of which are
hereby incorporated by reference.
TECHNICAL FIELD
[0002] The present invention relates to a new method of regulating
neuronal axon elongation.
BACKGROUND ART
[0003] As is disclosed in Non-patent literature 1 (p. 136-p. 137),
development of neural networks is regulated mainly by action of an
attractive axon guidance factor and a repulsive axon guidance
factor on a growth cone which is located at the tip of a neurite.
The attractive axon guidance factor causes the neurite to grow in a
predetermined direction, and the repulsive axon guidance factor
causes growth cone collapse, thereby reducing neurite
elongation.
[0004] Clarification of a detailed mechanism of regulation of
development of neural networks would lead to induction of
appropriate development of neural networks and induction of
regeneration of neural networks. However, many unclear points still
remain even in a mechanism of induction of growth cone collapse
caused by a repulsive axon guidance factor.
CITATION LIST
Non-Patent Literature
[0005] Non-Patent Literature 1 [0006] Jikken Igaku (Experimental
Medicine) separate volume Bio Science terminology library Cranial
Nerves (third edition) (Published on Sep. 1, 1999)
SUMMARY OF INVENTION
Technical Problem
[0007] A purpose of the present invention is to provide a method of
regulating neuronal axon elongation based on a newly-found
mechanism of induction of growth cone collapse caused by a
repulsive axon guidance factor, and the like.
Solution to Problem
[0008] In order to achieve the above-mentioned purpose, the present
invention includes any one of the following features.
[0009] A method of regulating neuronal axon elongation, comprising:
a treating step of treating neuronal growth cone with a regulatory
factor to regulate macropinocytosis caused by a repulsive axon
guidance molecule.
[0010] A method of screening a regulatory factor for regulating
macropinocytosis dependent on a repulsive axon guidance molecule,
the method comprising: a culturing step of culturing a neuron in
the presence of a candidate of a regulatory factor; a measuring
step of measuring binding activity and/or amount of Syntaxin1B
protein in a growth cone of the neuron; and a sorting step of
sorting the candidate that modulates binding activity and/or amount
of Syntaxin1B protein in the growth cone of the neuron as compared
with a case where the candidate does not exist.
[0011] A neuronal axon elongation regulating agent, comprising: an
inhibitor of a sodium/proton exchanger in a growth cone; and a
regulatory factor that increases binding activity and/or amount of
Syntaxin1B protein in the growth cone.
Advantageous Effects of Invention
[0012] The present invention makes it possible to regulate neuronal
axon elongation.
BRIEF DESCRIPTION OF DRAWINGS
[0013] FIG. 1 is views showing results in Example of the present
invention, which prove that neurotoxin C1-induced vacuoles are
formed by macropinocytosis.
[0014] FIG. 2 is views showing results in Example of the present
invention, which prove that Syx1B (Syntaxin1B) is a negative
regulator of macropinocytosis in growth cones.
[0015] FIG. 3 is views showing results in Example of the present
invention, which prove that macropinocytosis is required for
Sema3A-induced growth cone collapse.
[0016] FIG. 4 is views showing that Sema3A decreases Syx1B protein
in Example of the present invention.
[0017] FIG. 5 is views showing that, in Example of the present
invention, a Syx1B protein level in a growth cone is decreased by
suppression of PI3K and ephrin A2 but not by Jasp.
[0018] FIG. 6 is views showing effects of overexpression of
EGFP-Syx1B on Sema3A- or LY294002 (LY)-induced macropinocytosis and
growth cone collapse in Example of the present invention.
[0019] FIG. 7 is views showing that overexpression of EGFP-Syx1B
suppresses Sema3A- or LY294002 (LY)-induced macropinocytosis and
growth cone collapse in Example of the present invention.
[0020] FIG. 8 is views showing how overexpression of EGFP-tagged
Syntaxin1B protein (EGFP-Syx1B) affected (A) Sema3A-induced
macropinocytosis and (B) prevention of neuronal axon elongation in
Example of the present invention.
DESCRIPTION OF EMBODIMENTS
[0021] The embodiment of the present invention is described in
detail as follow.
[0022] A person skilled in the art appropriately understands that
the term "neuronal axon elongation" used herein has the same
meaning as the term "nerve-cell growth", and those terms can be
replaced with each other.
[0023] As used herein, the expression "a treating step of treating
a neuronal growth cone with a regulatory factor to regulate
macropinocytosis caused by a repulsive axon guidance molecule" has
the same meaning as the expression "an acting step of causing a
regulatory factor to act on a neuronal growth cone, wherein the
regulatory factor regulates repulsive axon guidance
molecule-dependent macropinocytosis", and those expressions can be
replaced with each other.
[0024] [1. Method of Regulating Neuronal Axon Elongation]
[0025] (Outline of Method Regulating Neuronal Axon Elongation)
[0026] A method of regulating neuronal axon elongation in
accordance with the present invention includes a treating step of
treating neuronal growth cone with a regulatory factor to regulate
macropinocytosis caused by a repulsive axon guidance molecule.
[0027] The inventors of the present invention showed for the first
time that one part of the mechanism related with the repulsion of
neuronal growth cone is macropinocytosis caused by a repulsive axon
guidance molecule (see also Example). Further, the inventors found
that neuronal axon elongation could be regulated by regulating the
macropinocytosis.
[0028] Note that the repulsive axon guidance molecule is not
limited in kind, and examples of the repulsive axon guidance
molecule encompass Sema3A and ephrin-A2, and preferably Sema3A.
[0029] (Regulatory Factor)
[0030] The regulatory factor regulates macropinocytosis caused by a
repulsive axon guidance molecule in a growth cone. Here, the
expression "regulating macropinocytosis" means either enhancement
or suppression of macropinocytosis as compared with a case where
the regulatory factor is absent.
[0031] The regulatory factor is not particularly limited in kind,
however, preferable examples of the regulatory factor encompass 1)
an inhibitor of a sodium/proton exchanger in a growth cone or 2) a
factor that modulates binding activity and/or amount of Syntaxin1B
protein in a growth cone. Note that "a factor that modulates
binding activity and/or amount of Syntaxin1B protein in a growth
cone" refers to a factor that increases or decreases the binding
activity of Syntaxin1B protein or the amount of Syntaxin1B protein,
as compared with a case where the factor is absent. Note that the
factor may modulate the binding activity and the amount of
Syntaxin1B protein, however, it is preferable that the factor
modulate at least the amount of Syntaxin1B protein. The binding
activity of Syntaxin1B protein can be determined by measuring
binding activity between a biological factor (such as SNAP25,
VAMP2, or Synaptotagmin) and Syntaxin1B protein. Note that, in the
present specification, "A and/or B" is a concept including both `A
and B` and `A or B`.
[0032] The inhibitor of a sodium/proton exchanger in a growth cone
may be any inhibitor that acts as an inhibitor of the sodium/proton
exchanger. The examples of the inhibitor of a sodium/proton
exchanger in a growth cone encompass Amiloride, EIPA, HMA(5-(N,
N-hexamethylene) amiloride), DMA (dimethyl amiloride), HOE-694,
Cariporide, Eniporide, Zoniporide, SM20550, BMS-284640,
T-162559(S), TY-12533, SL-591227, and S-3226 (Reference
Literatures: An overview of inhibitors of Na+/H+ exchanger,
Masereel B, Pochet L, and Laechmann D., European Journal of
Medicinal Chemistry 38 (2003) 547-554). The inhibitor of the
sodium/proton exchanger is preferably
5-(N-ethyl-N-isopropyl)amiloride or an analog thereof such as
Amiloride, EIPA, HMA, or DMA, and more preferably
5-(N-ethyl-N-isopropyl)amiloride. These inhibitors suppress
occurrence of macropinocytosis and growth cone collapse caused by a
repulsive axon guidane molecule, resulting in the cancellation of
growth cone collapse of neuron, more specifically growth cone
collapse of a neurite. As a result of the cancellation of the
growth cone collapse, the neuron can elongate its neurite.
[0033] Among factors that modulate binding activity and/or amount
of Syntaxin1B protein in a growth cone, a factor that increases
binding activity of Syntaxin1B protein and a factor that increases
amount of Syntaxin1B protein can suppress (A) occurrence of
macropinocytosis in a growth cone caused by a repulsive axon
guidance molecule and (B) growth cone collapse. It leads to the
cancellation of growth cone collapse of neuron, more specifically
growth cone collapse of a neurite. As a result of the cancellation
of the growth cone collapse, the neuron can elongate its neurite.
Examples of such factors encompass a nucleic acid molecule which
encodes Syntaxin1B protein, and Syntaxin1B protein itself. Note
that examples of Syntaxin1B protein includes a Syntaxin1B mutant
maintaining a function such as binding activity against a target
molecule which the wild type Syntaxin1B naturally binds. A
Syntaxin1B mutant may have deletion, insertion and/or substitution
of approximately 1-10 amino acids, preferably approximately 1-5
amino acids as long as the mutant maintains a function such as
binding activity.
[0034] Meanwhile, among factors that modulate binding activity
and/or amount of Syntaxin1B protein in a growth cone, a factor that
decreases binding activity of Syntaxin1B protein and a factor that
decreases amount of Syntaxin1B protein induce occurrence of
macropinocytosis in a growth cone and growth cone collapse of a
neuron (neurite). Examples of such factors encompass (i) nucleic
acid molecules such as antisense RNA, shRNA, siRNA, microRNA and
the like, each of which has the function to suppress expression of
a Syntaxin1B gene, and (ii) artificial nucleic acids whose
molecules are equivalent to those of nucleic acids, and each of
which has the function to suppress expression of a Syntaxin1B
gene.
[0035] (Method for Treating Neuron with Regulatory Factor)
[0036] A method for treating neuronal growth cone with the above
regulatory factor can be appropriately selected in accordance with,
for example, a kind of regulatory factor.
[0037] In a case where an individual for administration of the
regulatory factor is a human or an animal, a regulatory factor is
administered to the human or the animal in an amount effective in
regulating neuronal axon elongation. Here, the regulatory factor
may be administered alone, or may be administered as an ingredient
of a pharmasutical composition (described later) suitable for a
purpose of the administration.
[0038] A method for administering a regulatory factor is not
particularly limited. A regulatory factor may be systemically
administered by a method such as oral administration, intravascular
administration into a vein or an artery, or enteral administration.
Alternatively, a regulatory factor may also be locally administered
by a method such as injection (using an injector, or an injection
pump), transdermal administration, or sublingual administration. A
preferable embodiment of administration is such that a regulatory
factor is locally administered, by a method such as injection or
transdermal administration, into a vicinity of a nervous system
(e.g., spinal cord) to be treated.
[0039] A dose (effective amount) of the regulatory factor may be
appropriately set in accordance with age or sex of a human or an
animal to which the regulatory factor is to be administered,
symptoms, administration routes, the number of times the regulatory
factor is administered, or the like. In a case where in vivo assay
with use of the regulatory factor is performed in advance according
to need, it is possible to determine the dose without performing an
excess experiment.
[0040] For example, in a case where the regulatory factor is a
so-called low-molecular compound (e.g.,
5-(N-ethyl-N-isopropyl)amiloride or an analog thereof), a
preferable dose of the regulatory factor falls within a range of,
for example, 0.1 mg or more but 100 mg or less, 1 mg or more but 50
mg or less, and 1 mg or more but 20 mg or less, per kilogram weight
of a human or an animal. The number of times the regulatory factor
is administered is not particularly limited, provided that the
regulatory factor has an effect of regulating neuronal axon
elongation. The number of times the regulatory factor is
administered may be appropriately set in accordance with, for
example, a kind of regulatory factor, the dose, administration
routes, sympoms, or age or sex of a human or an animal.
[0041] A human or a non-human animal as the subject to which the
regulatory factor is to be administered may be any animals,
provided that the human or non-human animal has neurons, however,
it is preferable to select a subject from a group of the mammals
including a human. Mammals, to which the regulatory factor is to be
administered are exemplified by, but not particularly limited in
kind, animals such as a mouse, a rat, a rabbit, a guinea pig, and a
primate other than a human; pet animals such as a dog and a cat;
domestic animals such as a cow and a horse; and a human. Among
these mammals, a human is particularly preferable.
[0042] Examples of a human or a non-human animal to which the
regulatory factor is to be administered encompass an animal having
a dysfunction of a nervous system such as damages to a nervous
system (in particular, spinal cord injury); and erethism such as
atopic deramatitis and pain disorder.
[0043] Further, a regulatory factor for suppressing occurrence of
macropinocytosis in a growth cone and growth cone collapse is
administered to a human or non-human animal that has a dysfunction
of a nervous system or is to undergo an operation for treating the
dysfunction. In this case, together with the regulatory factor, an
attractive axon guidance molecule may also be administrated to
promote neuronal axon elongation.
[0044] Meanwhile, a regulatory factor for inducing occurrence of
macropinocytosis in a growth cone is administered to a human or a
non-human animal having erethism, so that an undesirable neurite of
a neuron is collapsed. For erethism developed in a vicinity of skin
such as atopic deramatitis, it is preferable that a regulatory
factor be transdermally administered by use of, for example, a
patch. For erethism as pain disorder, it is preferable that a
regulatory factor be locally administered to affected area by
injection (for example, using an injector, or an injection
pump).
[0045] Further, the scope of the present invention encompasses
treating neuronal growth cone with a regulatory factor to regulate
macropinocytosis caused by a repulsive axon guidance molecule in
vitro. Also in case of treating neuronal growth cone with a
regulatory factor to regulate macropinocytosis in vitro, the
regulatory factor may be given into a system in a quantity
effective in regulating neuronal axon elongation. Here, the
regulatory factor may be administered alone, or may be administered
as a component of a pharmasutical composition (described later)
suitable for a purpose of the administration.
[0046] In a case where the regulatory factor is a nucleic acid
molecule, the regulatory factor is introduced into a neuron by use
of, for example, a vector containing an expressible regulatory
factor. Examples of a kind of vector encompass a plasmid vector and
a virus vector (preferably, adenovirus vector, adeno-associated
virus vector, retrovirus vector, and the like). Examples of a
method of introducing a vector into a neuron encompass
electroporation (Nucleic, Acids Res. 15, 1311-1326 (1987)), a
calcium phosphate method (Mol. Cell Biol. 7, 2745-2752 (1987)), and
a lipofection method (Cell 7, 1025-1037 (1994); Lamb, Nature
Genetics 5, 22-30 (1993)).
[0047] Meanwhile, in a case where the regulatory factor is not a
nucleic acid molecule, it may be arranged such that a regulatory
factor is added to a medium in which a neuron is cultured, and then
the medium is caused to contact with the neuron or is introduced
into the neuron.
[0048] In either case, after the regulatory factor is given into a
system in a quantity effective in regulating neuronal axon
elongation, a neuron is cultured in vitro. Note that a neuron which
is treated in an in-vitro system is exemplified by a primary
culture cell and a cell line, but is not particularly limited in
kind.
[0049] (Method of Treating Neuronal Growth Cone with Plurality of
Regulatory Factors in Combination)
[0050] It is possible to use a plurality of kinds of regulatory
factors in combination to treat neuronal growth cone to regulate
macropinocytosis caused by a repulsive axon guidance molecule.
Normally, regulatory factors that suppress macropinocytosis in a
growth cone and growth cone collapse are used in combination, or
regulatory factors that induce macropinocytosis in a growth cone
are used in combination.
[0051] One preferable combination of the regulatory factors is 1)
an inhibitor of a sodium/proton exchanger in a growth cone and 2) a
factor that increases binding activity and/or amount of Syntaxin1B
protein in a growth cone.
[0052] [2. Pharmaceutical Composition]
[0053] The present invention also provides a pharmaceutical
composition containing the regulatory factor as component thereof.
Components other than the regulatory factor which constitute the
pharmaceutical composition are not particularly limited, and can be
mixed with a pharmaceutically acceptable carrier, lubricant,
preservative, stabilizer, humectant, emulsifier, salt for osmotic
pressure adjustment, buffering agent, coloring agent, flavor, a
sweetener, antioxidant, viscosity modifier, etc.
[0054] One preferable example of a pharmaceutical composition is a
combined agent (neuronal axon elongation regulating agent)
containing, as the regulatory factor, both 1) an inhibitor of
sodium/proton exchanger in a growth cone and 2) a factor that
increases binding activity and/or amount of Syntaxin1B protein in a
growth cone.
[0055] The carrier which is pharmaceutically acceptable is not
particularly limited, however, it is preferable that the carrier be
one that has a characteristic of (A) not inhibiting a function
(regulation of neuronal axon elongation) of a regulatory factor in
a case where the carrier is administered together with the
regulatory factor and (B) having no practically adverse effect on a
human or a non-human animal to which a therapeutic agent with the
carrier is to be administered.
[0056] Conventionally known carriers in this field can be widely
used as the carrier. For example, such a carrier is specifically
exemplified by, but not limited to water, various kinds of salt
solutions, alcohols, vegetable oil, polyethylene glycol, gelatin,
lactose, amylose, magnesium stearate, talc, silicic acid, paraffin,
fatty acid monoglyceride, fatty acid diglyceride, hydroxymetyl
cellulose, and polyvinyl pyrrolidone. A kind of carrier may be
appropriately selected in accordance with a dosage form of a
pharmaceutical composition, a method of administering a
pharmaceutical composition, and/or the like.
[0057] The dosage form of the pharmaceutical composition is not
particularly limited, and examples of the dosage form encompass a
tablet, a pill, powder, a liquid medicine, a suspension, an
emulsion, a granule, a capsule, a suppository, a patch, an
ointment/a liniment, and an injection.
[0058] Further, in a case where the regulatory factor is a nucleic
acid molecule, a pharmaceutical composition could be a gene therapy
medicine. The gene therapy medicine may be administered in such a
way that the regulatory factor is directly administered to a human
or a non-human animal by injection, or in such a way that a vector
into which the regulatory factor is incorporated is administered to
a human or a non-human animal by injection. Further, the vector is
not particularly limited, and examples of the vector encompass
vectors applicable to a gene therapy such as an adenovirus vector,
an adeno-related virus vector, an adeno-associated virus vector, a
herpesvirus vector, a vaccinia virus vector, and a retrovirus
vector. Note that the gene therapy medicine may be a liposomal
formulation.
[0059] The nucleic acid molecule of the invention can be applied to
the nucleic acid based drug. In a case of nucleic acid based drug,
the drug derivery system in the public domain including
atelocollagen, aromatic series cation peptide, cationic lipid and
the like, is not particularly limited in kind, can be also used for
the introduction in addition to the above conventional method.
[0060] It is preferable that an expression regulatory sequence to
express the regulatory factor in a neuron is incorporated into the
vector contained in the gene therapy medicine. Here, the expression
regulatory sequence is, for example, a promoter or an enhancer.
[0061] [3. Screening Method and Production Method]
[0062] The present invention further provides a method of screening
the regulatory factor that regulates macropinocytosis which is
dependent on a repulsive axon guidance molecule.
[0063] This screening method includes the following steps in this
order: a culturing step of culturing a neuron in the presence of a
candidate of a regulatory factor; a measuring step of measuring
binding activity against a target molecule which the wild type
Syntaxin1B naturally binds, and/or amount of Syntaxin1B protein in
a growth cone of the neuron; and a sorting step of sorting the
candidate that modulates binding activity against the target
molecule and/or amount of Syntaxin1B protein in the growth cone of
the neuron as compared with a case where the candidate does not
exist.
[0064] The culturing step can be carried out in accordance with a
conventional method for culturing a neuron, except that a candidate
for the regulatory factor is added into culture medium of the
neuron in a system in the culturing step. In view of improving
sensitivity of screening, however, it is more preferable to carry
out the treating step under the colocalization of the candidate
with a repulsive axon guidance molecule. For example, the repulsive
axon guidance molecule is exemplified by Sema3A or ephrin-A2, but
not limited in kind, and preferably Sema3A. Note that for example,
the neuron is exemplified by a primary culture cell and a cell
line, but not particularly limited in kind.
[0065] In the measuring step, binding activity and/or amount of
Syntaxin1B protein in a growth cone of the neuron are/is measured.
In order to carry out the measurement easily, it is preferable to
measure the amount of Syntaxin1B protein. Note that the amount of
Syntaxin1B protein may be measured by, for example, a conventional
method in which (i) an amount of transcription (amount of mRNAs) of
Syntaxin1B gene is indirectly grasped by quantifying the amount of
transcription by, for example, realtime PCR or (ii) Syntaxin1B
protein is quantified by an immunologic method.
[0066] The screening step is a step for screening a candidate for a
regulatory factor by comparing, with a control, a candidate that
modulated in binding activity and/or amount of Syntaxin1B protein
in the growth cone of the neuron, which control is a control that
has been subjected to the culturing step through the measuring step
under the same conditions as those of the neuron except that the
candidate was contained in the system. Here, candidates that
decreased binding activity and/or amount of Syntaxin1B protein
could induce macropinocytosis in a growth cone and collapse the
growth cone of a neuron (neurite). Meanwhile, candidates that
increased binding activity and/or amount of Syntaxin1B protein
could reduce macropinocytosis in a growth cone and inhibit growth
cone collapse of a neuron (neurite).
[0067] A Candidate for the regulatory factor which has been
selected by the sorting step is formulated as needed, to prepare
the above pharmaceutical composition (neuronal axon elongation
regulating agent).
SUMMARY
[0068] As described above, each invention of the present
application encompasses any one of the contents below.
1) A method of regulating neuronal axon elongation, comprising: a
treating step of treating neuronal growth cone with a regulatory
factor to regulate macropinocytosis caused by a repulsive axon
guidance molecule. 2) The method as set forth in 1), wherein the
regulatory factor is an inhibitor of a sodium/proton exchanger in
the growth cone. 3) The method as set forth in 2), wherein the
inhibitor is 5-(N-ethyl-N-isopropyl)amiloride or an analog thereof.
4) The method as set forth in 1), the regulatory factor modulates
binding activity and/or amount of Syntaxin1B protein in the growth
cone. 5) The method as set forth in 4), wherein the regulatory
factor increases the binding activity and/or the amount of the
Syntaxin1B protein in the growth cone. 6) The method as set forth
in 5), wherein the regulatory factor contains (A) a nucleic acid
that encodes any one of Syntaxin1B protein and a fragment of
Syntaxin1B protein or (B) any one of Syntaxin1B protein and a
fragment of Syntaxin1B protein. 7) The method as set forth in 2),
further comprising: an administering step of administering an
effective amount of the regulatory factor to (A) a human or a
non-human animal sustaining damage to a nervous system or (B) a
human or a non-human animal to have an operation on a nervous
system, the administering step being carried out before the
treating step. 8) The method as set forth in 5), further
comprising: an administering step of administering an effective
amount of the regulatory factor to (A) a human or a non-human
animal sustaining damage to a nervous system or (B) a human or a
non-human animal to have an operation on a nervous system, the
administering step being carried out before the treating step. 9)
The method as set forth in 2), wherein, as the regulatory factor,
the inhibitor and a regulatory factor for increasing binding
activity and/or amount of Syntaxin1B protein in the growth cone are
used. 10) A method of screening a regulatory factor for regulating
macropinocytosis dependent on a repulsive axon guidance molecule,
the method comprising: a culturing step of culturing a neuron in
the presence of a candidate of a regulatory factor; a measuring
step of measuring binding activity and/or amount of Syntaxin1B
protein in a growth cone of the neuron; and a sorting step of
sorting the candidate that modulates binding activity and/or amount
of Syntaxin1B protein in the growth cone of the neuron as compared
with a case where the candidate does not exist. 11) The screening
method as set forth in 10), wherein the culturing step is carried
out under colocalization with a repulsive axon guidance molecule.
12) A neuronal axon elongation regulating agent, comprising: an
inhibitor of a sodium/proton exchanger in a growth cone;
[0069] and a regulatory factor that increases binding activity
and/or amount of Syntaxin1B protein in the growth cone.
EXAMPLE
[0070] The present invention will be described more specifically
below on the basis of the following Example etc. It should be noted
that the present invention is not limited to the Example etc.
below.
[0071] [Method and Material]
[0072] <Material>
[0073] An F-12 medium (Invitrogen) or an L-15 medium was used as a
culture medium. Alexa Fluor 488-labeled high-molecular-weight (10
kDa) dextran (Alexa Fluor 488-dextran) stock solution (Invitrogen)
was prepared in the F-12 medium with or without neurotoxin C1.
[0074] Anti-Syx1B antibody (purchased from Synaptic Systems) was
generated against synthetic peptide (sequence GKLAIFTDDIKMDSQMT:
SEQ ID NO 1). This sequence of the synthetic peptide corresponds to
amino acid residues 171-187 in mouse Syx1B protein. This sequence
of the synthetic peptide is identical to that present in chick
Syx1B protein cloned in this example. Epoxomicin was purchased from
Peptide Institute, and used at final concentration of 1 .mu.M.
[0075] Neurotoxin C1 was purified from Clostridium botulinum type C
(Reference (Kurazono et al., 1985): Fems Microbiol Lett 30:47-51.)
and used at a final concentration of 200 nM.
[0076] <Cell Culture>
[0077] Primary culcutre DRG neurons were prepared from embryonic
day 7-9 male and female chick (Gallus gallus) embryos in accordance
with a previous report (Reference (Kabayama et al.,
1999):Neuroscience 88:999-1003.). Primary culture DRG neurons were
cultured in an F-12 medium supplemented with 50 ng/ml nerve growth
factor (NGF) (Calbiochem) or in an L-15 medium supplemented with
10% fetal bovine serum, 2 mM glutamine, 5 ng/ml NGF, 0.6% glucose,
and gentamicin (Roche Diagnostics).
[0078] After electroporation, cells were cultured in an F-12-based
medium supplemented with 1.times.MACS Supplement B27 PLUS (Miltenyi
Biotech) or in an L-15-based medium supplemented with 1.times.MACS
Supplement B27 PLUS.
[0079] <Endocytosis of Alexa Fluor 488-Dextran>
[0080] To visualize endocytic activity in growth cones, the cells
were exposed to 0.5 mg/ml (Mr 10,000) fixable Alexa Flour
488-dextran (Invitrogen) for 90 min with 200 nM neurotoxin C1.
[0081] Images of the living growth cones were obtained after
washing the growth cones three times with Alexa Flour
488-dextran-deficient balanced salt saline (BSS) imaging buffer (pH
7.4). The Alexa Flour 488-dextran-deficient BBS imaging buffer
contains the following (in mM): 2 CaCl.sub.2, 5.4 KCl, 115 NaCl, 10
glucose, and 20 HEPES.
[0082] To quantify the percentage of dextran-labeled growth cones,
the cells were cultured in an F-12-based medium and treated with
neurotoxin C1 in the presence of Alexa Fluor 488-dextran for 90
min. Then, the cells were washed three times with the Alexa Fluor
488-dextran-deficient F-12 medium, and immediately after the
washing, the cells were fixed with 4% PFA.
[0083] In the experiments using siRNAs, cells transfected with
siRNAs were cultured in an L-15-based medium as described
previously (Reference (Kabayama et al., 1999):Neuroscience
88:999-1003.) and exposed to 0.5 mg/ml (Mr 10,000) fixable Alexa
Fluor 594-dextran (Invitrogen) for 30 min at 6 h after
electroporation. The cells were fixed after being washed with an
Alexa Fluor 594-dextran-deficient L-15-based medium.
[0084] In the experiments using Sema3A, cells were cultured in an
F-12-based medium and exposed to 0.5 mg/ml Alexa Fluor 594-dextran
for 10 min in the presence of 1 .mu.g/ml Sema3A.
[0085] The effects of macropinocytosis inhibitor
ethylisopropylamiloride (EIPA) on dextran uptake, growth cone
collapse, and neurite elongation were assayed by pretreating DRG
neurons for 15 min with 10 .mu.M or 100 .mu.M EIPA before addition
of dextran.
[0086] Flourescence images of Alexa Fluor 488 and Alexa Fluor 594
in the growth cone were obtained by GFP optics and Discosoma red
(DsRed) optics, respectively.
[0087] The percentage of growth cones that took up dextran was
calculated from a random selection in the culture. In the
experiments using siRNAs, cells were cotransfected with an
EGFP-expressing vector for visualization of siRNAs. The percentage
of dextarn-labeled growth cones was calculated from all
EGFP-positive growth cones in the culture.
[0088] <Labeling of Growth Cone Vesicles>
[0089] To visualize growth cone vesicles (GCVs), the cells were
exposed to 5 .mu.g/ml Alexa Fluor 594-labeled wheat-germ agglutinin
(WGA) (Invitrogen) for 10 min at 37.degree. C. Subsequently, the
cells were washed three times with a fresh medium prewarmed at
37.degree. C. without Alexa Fluor 594-WGA and were cultured again.
These cells were used for experiments involving treatment with
neurotoxin C1 and Axexa Fluor 488-dextran at 60 min after the
washing.
[0090] <Construction of Expression Vectors>
[0091] Full-length chick Syx1B was obtained from an embryonic day 9
chick DRG cDNA library by PCR. The primers used were
5'-GGAACAGCCATGAAGGACCGC-3' (SEQ ID NO 2) and
5'-CTATAGGCCCAAAGTGCCACAAATG-3' (SEQ ID NO 3), designed on the
basis of previously identified chick Syx1B (NCBI database accession
number NM205074). A nucleotide sequence of cDNA of full-length
chick Syx1B obtained by PCR was registered as Syntaxin1B-2 in the
DNA Data Bank of Japan database (accession number AB244720). This
Syx1B was subcloned into the EcoRI site of the pEGFP-C1 vector
(Clontech).
[0092] <RNAi>
[0093] Two pairs of Syx1B siRNAs (Syx1B-siRNA1 and Syx1B-siRNA2)
and scrambled siRNAs (iGENE Therapeutics) were synthesized by
transcription and purification by use of PAGE. The following siRNA
sequences were used in this experiment.
[0094] (1) Syx1B-siRNA1
TABLE-US-00001 Sense strand (SEQ ID NO 4):
5'-r(GCUCCAAGUUGAAAGCCAUAGAGCAAG)-3' Antisense strand (SEQ ID NO
5): 5'-r(UGCUCUAUGGCUUUCAACUUGGAGCAU)-3'
[0095] (2) Syx1B-siRNA2
TABLE-US-00002 Sense strand (SEQ ID NO 6):
5'-r(CCGAUGAUAUCAAAAUGGACUCGCAAG)-3' Antisense strand (SEQ ID NO
7): 5'-r(UGCGAGUCCAUUUUGAUAUCAUCGGAU)-3'
[0096] (3) Scrambled siRNA
TABLE-US-00003 Sense strand (SEQ ID NO 8):
5'-r(CGAUUCGCUAGACCGGCUUCAUUGCAG)-3' Antisense strand (SEQ ID NO
9): 5'-r(GCAAUGAAGCCGGUCUAGCGAAUCGAU)-3'
[0097] The concentration of those RNAs was 40 nM during
electroporation.
[0098] <Mutagenesis>
[0099] An EGFP-tagged Syntaxin 1B was prepared with a 7 bp silent
mutation at the targeting region of each siRNA. The EGFP-tagged
Syntaxin 1B encoded the wild-type Syx1B protein sequence
(SS-Syx1B).
[0100] A QuikChange Multi Site-Directed Mutagenesis kit
(Stratagene) was used to generate mutations. The following primers
were used (SEQ ID NO 10-11):
TABLE-US-00004 5'-r(GAAGACGGCCAACAAAGTGCGTTCCAAACTGAAGGCGATTGA
ACAGAGCATTGAGCAGGAGGAG)-3'; and
5'-r(CGGGAAGTTGGCCATCTTCACGGACGACATAAAGATGGATTC
TCAGATGACCAAGCAGGCCCTG)-3'.
[0101] <Electroporation>
[0102] In total, 7.5.times.10.sup.5 cells were suspended in an L-15
medium containing 10 .mu.g of a pEGFP vector or a pEGFP-Syx1B
vector and incubated on ice for 10 min.
[0103] The cells were then transferred to chilled cuvettes
(Bio-Rad) and transfected with an electroporator (0.2 V,
950.OMEGA.; Bio-Rad Gene Pulser).
[0104] The cells were immediately suspended in 10 ml of a prewarmed
L-15-based culture medium which is plated onto a Petri dish, were
incubated for 30 min at 37.degree. C. to remove cell debris, and
then were plated on poly-L-lysine- and laminin-coated glass-bottom
culture dishes.
[0105] For quantification of the knockdown efficiency of Syx1B with
siRNAs, the cells were transfected with siRNAs by
electroporation.
[0106] The cells were cultured in an L-15-based medium and
harvested by using a scraper at 6 h after electroporation in order
to be subjected to Western blot analysis.
[0107] For quantifying effects of siRNAs on macropinocytosis,
growth cone collapse, and neurite elongation, the transfected cells
were cultured in an L-15-based medium supplemented with MACS
Supplement B27 PLUS.
[0108] In experiments involving Sema3A, the transfected cells were
cultured in an F-12-based medium supplemented with MACS Supplement
B27 PLUS.
[0109] <Imaging and Quantitative Analysis of Fluorescence
Signals>
[0110] For time-lapse imaging under differential interference
contrast (DIC) and fluorescence microscopy, growth cones that were
not in contact with other cells were selected.
[0111] The growth cones were observed by using an inverted
microscope (IX70; Olympus). The inverted microscope is equipped
with DIC optics, GFP optics (excitation, 490 nm; dichroic mirror,
505 nm; emission, 515 nm), DsRed optics (excitation, 555 nm;
emission, 620 nm), 60.times. objective (numerical aperture 1.25),
and a xenon lamp.
[0112] The images were acquired with a cooled CCD camera (Cool SNAP
HQ; Roper Scientific) driven by MetaMorph (version 7.0) imaging
software (Universal Imaging Corporation). The temperature was
maintained at 37.degree. C. by perfusing the cells with prewarmed
BBS buffer.
[0113] The DIC and fluorescence images were acquired
continuously.
[0114] Measurements were performed by using 2.times.2 binning (1
pixel=0.21.times.0.21 .mu.m).
[0115] The integrated intensity of the growth cone was quantified
by subtracting the nonspecific background fluorescence.
[0116] <Growth Cone Collapse Assay>
[0117] Recombinant human Sema3A/Fc chimera (Sema3A; R & D
Systems) and recombinant mouse ephrin A2/Fc chimera (ephrin A2; R
& D Systems) were used at 1 .mu.g/ml for the growth cone
collapse assay.
[0118] Dissociated DRG neurons from embryonic day 7-9 chicks were
cultured for at least 12 h in an F-12 medium (Invitrogen)
containing 50 ng/ml NGF. To investigate the effect of Sema3A, the
culture medium was changed to an F-12 medium containing 0.1 ng/ml
NGF at 2 h before the growth cone collapse assay. Subsequently, the
culture medium was changed to BSS buffer. The cells were incubated
for 30 min before their use in the growth cone collapse assay. In
experiments using epoxomicin, the cells were treated with 1
.mu.M/ml epoxomicin for 15 min before the addition of 1 .mu.M/ml
Sema3A.
[0119] Growth cone collapse was quantified as the reduction in the
surface area of growth cones. The growth cone area was defined as a
hand-like region extending from a distal part of a neurite to a
growth cone neck whose width is equal to the width of the neurite.
The width of each neurite was calculated by measuring the width of
the neurite region at a location that was 30 .mu.m apart from the
distal part of each neurite. This calculation was adopted because
the mean length of the control growth cone was less than 30 .mu.m.
If a growth cone collapsed completely or the width of the growth
cone was less than that of the each neurite, the neurite region at
the location that was 30 .mu.m apart from the distal part of the
neurite was considered as the growth cone neck.
[0120] <Macropinocytosis Assay in NIH3T3 Cells>
[0121] NIH3T3 cells transfected with pEGFP or pEGFP-Syx1B were
exposed to a medium containing 2.5 mg/ml of Alexa Fluor 594-dextran
in the presence of serum for 30 min. Macropinocytosis in the NIH3T3
cells was quantified by measuring the integrated fluorescence
intensity of internalized Alexa Fluor 594-dextran in the cells.
[0122] <Western Blot Analysis>
[0123] The proteins were extracted in a passive lysis buffer
(Promega). Then, 15 .mu.g of protein extract was subjected to 12.5%
SDS-PAGE and transferred to a PVDF membrane (Millipore). The blots
were blocked with 2% skimmed milk in PBS containing 0.1% Tween 20,
incubated in the presence of affinity-purified rabbit anti-Syx1B (1
.mu.g/ml; Synaptic Systems) or rabbit polyclonal anti-actin
antibody (1:4000 dilution; Sigma), and incubated in the presence of
HRP-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch).
Immunoreactive bands were visualized with an Enhanced
Chemiluminescence Plus detection system (GE Healthcare).
[0124] For visualization of polyubiquitination of endogenous
proteins, cells were cultured for 6 h with an L-15-based medium and
pretreated with 1 .mu.M epoxomicin 15 min before the 15 min Sema3A
treatment. Total proteins were extracted with passive lysis buffer
[10 nM ubiquitin aldehyde (Peptide Institute) and complete protease
inhibitor cocktail (Roche)]. Polyubiquitinated proteins were
analyzed by Western blot analysis using anti-ubiquitin antibody
(Roche).
[0125] <Immunocytochemistry>
[0126] After Sema3A or vehicle treatment, the cells were fixed with
4% PFA for 1 h at 37.degree. C., followed by permeabilization with
0.3% Triton X-100 in PBS for 2 min at a room temperature.
[0127] The cells were then immediately washed three times with a
blocking solution (1% BSA and 0.1% Triton X-100 PBS), followed by
incubation with the blocking solution for 1 h at a room
temperature, and were then incubated in the presence of anti-Syx1B
(0.5 .mu.g/ml) and anti-synaptosomal-associated protein 25 (SNAP25)
antibody (1:500 dilution, clone SM-81; Sternberger Monpclonals) for
2 h at a room temperature. Immunofluorescence signals were
visualized by incubation with Alexa Fluor 488-labeled donkey
anti-rabbit antibody (Invitrogen) and Alexa Fluor 594-labeled
donkey anti-mouse antibody (Invitrogen). The integrated
fluorescence intensity of Syx1B in the growth cones was normalized
to that of the SNAP 25 protein.
[0128] <Statistical Analysis>
[0129] All the data are representative of at least three
independent experiments. The results are expressed as the
mean.+-.SEM. The data were analyzed with the GraphPad Prism 4.0
program (Graph-Pad Software). p<0.05 was considered
statistically significant.
[0130] [Results]
[0131] <Syx11B is a Negative Regulator of
Macropinocytosis>
[0132] During 90 min neurotoxin C1 treatment, primary culture DRG
neurons were exposed to an Alexa Fluor 488-dextran-containing
medium. The growth cones were imaged after washing cells with Alexa
Fluor 488-dextran-deficient BBS imaging buffer. Unlike the
controls, neurotoxin C1-treated cells exhibited marked accumulation
of Alexa Fluor 488-dextran in the DIG-identifiable vacuoles of
living growth cones (FIGS. 1A-F).
[0133] The fusion of WGA-labeled small GCVs has been postulated to
form neurotoxin C1-induced vacuoles (Reference: Igarashi et al.,
1996_J Cell Biol 134:205-215.). However, WGA-fluorescent puncta
were not colocalized with Alexa Fluor 488-dextran-positive large
vacuoles in the neurotoxin C1-treated growth cones (FIG. 1G).
[0134] Although neurotoxin C1-induced large vacuoles are known to
move in a retrograde manner (Reference: Igarashi et al., 1996_J
Cell Biol 134:205-215.), the WGA-labeled vesicles in the neurotoxin
C1-treated growth cones were not transported in this manner,
indicating that the neurotoxin C1-induced vacuoles were not formed
by the fusion of WGA-labeled GCVs.
[0135] Next, whether neurotoxin C1-induced endocytosis is
macropinocytosis was investigated using EIPA, a
macropinocytosis-specific inhibitor. Neurotoxin C1-induced uptake
of Alexa Fluor 594-dextran (FIGS. 1H, I, L, M, P) and reduction in
growth cone surface area (FIGS. 1H, I, Q) were blocked by 15 min
pretreatment with 100 .mu.M EIPA (FIGS. 1J, K, N-Q). These results
indicate that neurotoxin C1-induced large vacuoles are formed by
macropinocytosis and not the fusion of WGA-labeled vesicles.
[0136] Note that FIG. 1 is views showing that neurotoxin C1-induced
vacuoles are formed by macropinocytosis. FIGS. 1A-F show DIC,
fluorescence, and merged images of a living growth cone in the
presence of Alexa Fluor 488-dextran, A-C show DIC, fluorescence,
and merged images treated with neurotoxin C1, and D-F show DIC,
fluorescence, and merged images of a control buffer. The arrows in
FIG. 1 indicate large vacuoles which were colocalized with Alexa
Fluor 488-dextran.
[0137] FIG. 1G shows that WGA-labeled GCVs were not colocalized
with neurotoxin C1-induced large vacuoles. More specifically, a DIC
image of a living growth cone treated with neurotoxin C1,
fluorescence images of Alexa Fluor 488-dextran (green) and Alexa
Fluor 594-WGA (red), and merged images of the DIC image and the
fluorescence images. The white arrowheads indicate Alexa Fluor
488-dextran localized to large vacuoles and the black arrows
indicate Alexa Fluor 594-WGA fluorescent puncta.
[0138] FIGS. 1H-O show DIC images and fluorescence images of a
fixed growth cone treated with a control buffer (H, L, J, N) or
neurotoxin C1 (I, M, K, O). FIGS. 1H, I, L, and M each show an
experiment in the presence of DMSO, FIGS. 1J, N, K, and O each show
an experiment in the presence of 100 .mu.M EIPA.
[0139] FIG. 1P shows the percentage of growth cones labeled with
Alexa Fluor 594-dextran during 90 min neurotoxin C1 or control
buffer treatment in the presence of DMSO or 100 .mu.M EIPA
(**p<0.01, ***p<0.001, two-tailed non-repeated-measures ANOVA
followed by Newman-Keuls post hoc multiple comparison test.).
[0140] FIG. 1Q shows a growth cone area treated with neurotoxin C1
or a control buffer in the presence of DMSO or 100 .mu.M EIPA
(***p<0.01, two-tailed non-repeated-measures ANOVA followed by
Newman-Keuls post hoc multiple comparison test.). The error bars
(in P and Q) indicate SEM. The scale bar indicates 5 .mu.m.
[0141] siRNAs for Syx1B (Syx1-siRNAs) was used to investigate
whether knockdown of this molecule caused macropinocytosis, growth
cone collapse, and reduced neurite elongation. The endogenous Syx1B
level normalized to actin protein in Syx1-siRNA-transfected DRG
neurons was significantly reduced to 53.4.+-.3.1% (p<0.0001,
two-tailed unpaired t test) as compared with control
siRNA-transfected DRG neurons (FIG. 2A).
[0142] Syx1B-siRNA-transfected cells exhibited strong accumulation
of Alexa Fluor 594-dextran in growth cones (FIGS. 2B, F, J), growth
cone collapse (FIGS. 2B, K), and reduced neurite elongation (FIG.
2L) under physiological conditions, whereas control
siRNA-transfected growth cones exhibited no such effect (FIGS. 2C,
G, J-L). These Syx1B knockdown-mediated phenotypes were reversed by
15 min pretreatment with 10 .mu.M EIPA (FIGS. 2D, H, J-L).
[0143] Because siRNA-induced RNA interference in mammalian systems
may have off-target effects (Reference: Jackson et al., 2003_Nat
Biotechnol 21:635-637.), whether Syx1B-siRNA-induced phenotypes can
be suppressed by reintroducing Syx1B into DRG neurons was
investigated.
[0144] An EGFP-tagged syntaxin 1B with a 7 bp silent mutation at
the siRNA-targeting region, which encoded the wild-type Syx1B
protein sequence (SS-Syx1B), was prepared. As predicted, SS-Syx1B
was expressed in growth cones cotransfected with Syx1B-siRNA (FIG.
2E). SS-Syx1B expression suppressed Syx1B-siRNA-induced uptake of
Alexa Fluor 594-dextran (FIGS. 2I, J), growth cone collapse (FIGS.
2E, K), and reduced neurite elongation (FIG. 2L). Furthermore,
Syx1B overexpression inhibited macropinocytosis in NIH3T3 cells
(not shown).
[0145] Note that FIG. 2 is views showing that Syx1B is a negative
regulator of macropinocytosis in growth cones. FIG. 2A shows a
result of Western blot analysis of endogenous Syx1B expression
level in DRG neurons transfected with Syx1B-siRNA or control
siRNAs.
[0146] FIGS. 2B-I show fluorescence EGFP (top, white) and Alexa
Fluor 594-dextran (bottom, white) fluorescence images of a fixed
growth cone cotransfected with the indicated siRNAs and EGFP or
EGFP-labeled SS-Syx1B in the presence of DMSO or 10 .mu.M EIPA. The
blue arrowheads indicate incorporated Alexa Fluor 594-dextran. The
insert in FIG. 2F is an enlargement of a merged image of EGFP (B,
green) and dextran (F, magenta).
[0147] FIG. 2J shows the percentage of siRNA-transfected growth
cones labeled with Alexa Fluor 594-dextran under the various
conditions shown in FIGS. 2B-I. FIG. 2K shows a growth cone area of
siRNA-transfected growth cones shown in FIGS. 2B-I. FIG. 2L shows a
neurite length of siRNA-transfected cells shown in FIGS. 2B, C, and
E. *p<0.05, **p<0.01, ***p<0.001 (two-tailed
non-repeated-measures ANOVA followed by Newman-Keuls post hoc
multiple comparison test.). The error bars (FIGS. 2J-L) show SEM.
The scale bar indicates 10 .mu.m.
[0148] <Sema3A Signaling Decreases Syx1B Protein Level>
[0149] Sema3A-induced uptake of Alexa Flour 594-dextran (FIGS. 3A,
B, E, F, I) and growth cone collapse (FIGS. 3B, J) were suppressed
by 15 min pretreatment of cells with 10 .mu.M EIPA (FIGS. 3C, D, G,
H-J).
[0150] These results indicated that macropinocytosis was required
for Sema3A-induced growth cone collapse and raised the possibility
that Sema3A-induced macropinocytosis was mediated by the
suppression of Syx1B, because Syx1B-reduction-triggered
macropinocytosis was required for growth cone collapse (see FIG.
2).
[0151] To examine the possibility, the effect of Sema3A on Syx1B
expression in growth cones was investigated. Immunocytochemistry
revealed that the protein level of Syx1B in growth cones was
reduced by 10 min Sema3A treatment (FIGS. 4A, C, I). The protein
level of Syx1B normalized to that of SNAP25 (FIGS. 4B, D) in the
growth cones was significantly reduced by 10 min Sema3A treatment
(FIG. 4I).
[0152] This rapid suppression of Syx1B suggested that the protein
is degraded by the proteasome. To test this, cells were exposed to
a medium containing 1 .mu.M epoxomicin, a specific proteasome
inhibitor. As a result of the above epoxomicin treatment,
Sema3A-induced suppression was suppressed (FIGS. 4E-H, I).
[0153] When Western blot analysis with anti-ubiquitin antibody was
performed using cell lysate prepared from cells treated with Sema3A
in the presence of 1 .mu.M epoxomicin, a broad band with high
molecular weight was increased in response to Sema3A (FIG. 4J).
These results indicate that Sema3A activates the
ubiquitin-proteasome pathway.
[0154] The cells were treated for 10 min with the PI3K inhibitor
LY294002 [2-(4-morpholinyl)-8-phenyl-1 (4H)-benzopyran-4-one] and
the effect of the PI3K inhibitor on syx1B protein expression was
then evaluated. The protein level of Syx1B normalized to that of
SNAP25 in the growth cones was significantly reduced by 10 min
LY294002 treatment (FIG. 5A).
[0155] Expression of Syx1B normalized to SNAP25 was decreased by 10
min treatment with ephrin A2 (FIG. 5B). In contrast, expression of
Syx1B normalized to SNAP25 was not decreased in growth cones
treated with jasplakinolide (Jasp), an inhibitor of F-actin
depolymerization (FIG. 5C). Note that jasplakinolide (Jasp) induces
growth cone collapse-like morphological changes.
[0156] These results indicate that reduction of Syx1B protein
levels is not secondary to the morphological changes in growth
cones during growth cone collapse. Together, these results suggest
that Sema3A-PI3K signaling decreases Syx1B protein in growth cones,
thereby inducing macropinocytosis.
[0157] Note that FIG. 3 is views showing that macropinocytosis is
required for Sema3A-induced growth cone collapse. FIGS. 3A-H show
DIC images and fluorescence images of growth cones subjected to 10
min treatment with BSA (A, C, E, G) or Sema3A (B, D, F, H) in the
presence of DMSO (A, B, E, F) or 10 .mu.M EIPA (C, D, G, H). The
blue arrowheads indicate incorporated Alexa Fluor 594-dextran. The
insert in FIG. 3F is an enlargement of a merged image of DIC (FIG.
3B) and dextran (FIG. 3F, magenta). FIG. 3I shows the percentage of
growth cones labeled with Alexa Fluor 594-dextran under the
indicated conditions shown in FIGS. 3A-H. FIG. 3J shows a growth
cone area of growth cones shown in FIGS. 3A-H. *p<0.05,
**p<0.01 (two-tailed non-repeated-measures ANOVA followed by
Newman-Keuls post hoc multiple comparison test). The error bars
indicate SEM. The scale bar indicates 10 .mu.m.
[0158] FIG. 4 is views showing that Sema3A decreases Syx1B protein.
FIGS. 4A-H show immunofluorescence images of endogenous Syx1B (A,
C, E, G) and SNAP25 (B, D, F, H) in growth cones treated with BSA
(A, B, E, F) or Sema3A (C, D, G, H) for 10 min in the presence of
DMSO (A-D) or epoxomicin (E-H). FIG. 4I shows a relative amount of
endogenous Syx1B protein normalized to SNAP25 in the growth cones
(***p<0.001, two-tailed non-repeated-measures ANOVA followed by
Newman-Keuls post hoc multiple comparison test.). FIG. 4J shows
Poly-ubiquitination of endogenous proteins by Sema3A treatment in
the presence of epoxomicin. Cell lysate was prepared from cells
treated for min with Sema3A or BSA in the presence of 1 .mu.M
epoxomicin, immunoblotted with anti-ubiquitin antibody to detect
ubiquitinated proteins as smear bands. Asterisk indicates
Poly-ubiquitinated proteins. The error bars indicate SEM. The scale
bar indicates 10 .mu.m.
[0159] FIG. 5 is views showing that the Syx1B protein level in a
growth cone is decreased by the suppression of PI3K and ephrin A2
but by Jasp. FIGS. 5A-C show a relative amount of endogenous Syx1B
protein normalized to SNAP25, in the growth cones treated with
LY294002 (LY, FIG. 5A), ephrin A2 (FIG. 5B), and Jasp (FIG. 5C).
Note that the treatment of the growth cones with each drug is
carried out for 10 min. ***p<0.001 (unpaired t test). The error
bars indicate SEM. The scale bar indicates 10 .mu.m.
[0160] <Syx1B Couples Macropinocytosis to Sema3A-Induced Growth
Cone Collapse>
[0161] A gain-of-function analysis was next performed by
maintaining Syx1B expression at high levels after Sema3A treatment
in the overexpression experiment. The overexpression of EGFP-Syx1B
reduced Sema3A- or LY294002-induced macropinocytosis in the growth
cones (FIGS. 6A-D, I-L and FIGS. 7A, C), and reversed the Sema3A-
or LY294002-induced reduction in growth cone area (FIGS. 6A-D, I-L
and FIGS. 7B, D), whereas the overexpression of EGFP alone had no
such effect (FIGS. 6E-H, M-P and FIGS. 7A-D).
[0162] As illustrated in FIGS. 8A-C, the overexpression of
EGFP-Syx1B reduced Sema3A-induced macropinocytosis in growth cones,
however, the overexpression of EGFP alone had no such effect.
Further, the overexpression of EGFP-Syx1B suppressed both reduction
in areas of Sema3A-induced growth cones (FIGS. 8A, B, D) and
retraction of Sema3A-induced neurite (FIGS. 8A, B, E). This
indicates that syntaxin1B-reduction-triggered macropinocytosis is
one process of causing inhibition of Sema3A-dependent axon
elongation including growth cone collapse and axon retraction.
[0163] Note that the experiment, whose results are shown in FIG. 8,
was carried out as in the case of the above <Growth cone
collapse assay> using Sema3A. pEGFP or pEGFP-syntaxin1B was
introduced into cells by electroporation, and the cells were
cultured in an F-12 medium and were subjected to a Sema3A
treatment. Then, a vacuole obtained after the Sema3A treatment, an
area of a growth cone, and a neurite elongation rate were measured
by time-lapse imaging. An area of the vacuole was obtained by
quantifying a maximum value obtained during 10 min after Sema3A was
added to the vacuole. An area of a growth cone was quantified at 0
(zero) min and 10 min after Sema3A was added. A neurite elongation
rate was obtained by quantifying a length obtained before the
Sema3A treatment and a length obtained at 10 min after the Sema3A
treatment, and then comparing the lengths. All data were obtained
from GFP-positive cells.
[0164] Note that FIG. 6 is views showing effects of overexpression
of EGFP-Syx1B on Sema3A- or LY294002 (LY)-induced macropinocytosis
and growth cone collapse. FIGS. 6A-P show fluorescence images of
pEGFP- or pEGFP-Syx1B-transfected growth cones subjected to 10 min
treatment with BSA (FIGS. 6A, C, E, G), Sema3A (FIGS. 6B, D, F, H),
DMSO (FIGS. 6I, K, M, O), or LY (FIGS. 6J, L, N, P) in the presence
of Alexa Fluor 594-dextran. Top panels show green fluorescence of
EGFP or EGFP-Syx1B-expressing growth cones. Bottom panels show
Alexa Fluor 594-dextran incorporated into growth cones. The inserts
in H and P show a merged image of EGFP (FIG. 6F, green) and dextran
(FIG. 6H, magenta) and a merged image of EGFP (FIG. 6N, green) and
dextran (P, magenta), respectively. The scale bar indicates 10
.mu.m.
[0165] FIG. 7 is views showing that overexpression of EGFP-Syx1B
suppresses Sema3A- or LY294002 (LY)-induced macropinocytosis and
growth cone collapse. FIG. 7 shows the percentage of growth cones
labeled with Alexa Fluor 594-dextran (FIGS. 7A, C) and growth cone
area (FIGS. 7B, D) of EGFP- or EGFP-Syx1B-transfected growth cones
subjected to 10 min treatment with BSA, Sema3A, DMSO, or LY294002
in the presence of Alexa Fluor 594-dextran. *p<0.05,
**p<0.01, ***p<0.001 (two-tailed non-repeated-measures ANOVA
followed by Newman-Keuls post hoc multiple comparison test.). The
error bars indicate SEM.
[0166] FIG. 8 is views showing, in Example of the present
invention, effects of overexpression of EGFP-tagged Syntaxin1B
protein (EGFP-Syx1B) on (A) Sema3A-induced macropinocytosis and (B)
inhibition of neuronal axon elongation. FIG. 8A shows a series of
time-lapse images of a pEGFP-transfected growth cone (control), and
FIG. 8B shows a series of time-lapse images of a
pEGFP-Syx1B-transfected growth cone. Sema3A was added into a medium
immediately after the experiment was started (at 0 min). The arrow
in FIG. 8A indicates a vacuole obtained at 2 min after the
experiment was started. The broken line in FIG. 8A indicates a
leading edge of a growth cone. FIG. 8C indicates a maximum area of
a vacuole in a growth cone of a pEGFP- or pEGFP-Syx1B-transfected
DRG neuron subjected to the Sema3A treatment for 10 min.
***P<0.001, two-tailed unpaired t-test. FIGS. 8D and 8E show (i)
a change in area of a growth cone and (ii) a change in neurite
elongation rate, respectively, the changes (i) and (ii) each being
induced by Sema3A. ***P<0.001, two-tailed paired t-test. The
error bars in FIGS. 8C, D, and E indicate SEM. The scale bar
indicates 10 .mu.m.
[0167] The present invention is not limited to the description of
the embodiments above, and can be modified in numerous ways by a
skilled person as long as such modification falls within the scope
of the claims. An embodiment derived from a proper combination of
technical means disclosed in different embodiments is also
encompassed in the technical scope of the present invention.
[0168] All cited references cited in this specification are
incorporated into the specification by referring the cited
references as if disclosed matters of such cited references were
described in the specification.
INDUSTRIAL APPLICABILITY
[0169] The present invention can provide a novel method of
regulating neuronal axon elongation etc.
Sequence CWU 1
1
11117PRTArtificial SequenceSynthetic peptide 1Gly Lys Leu Ala Ile
Phe Thr Asp Asp Ile Lys Met Asp Ser Gln Met 1 5 10 15 Thr
221DNAArtificial Sequenceprimer sequence 2ggaacagcca tgaaggaccg c
21325DNAArtificial Sequenceprimer sequence 3ctataggccc aaagtgccac
aaatg 25427RNAArtificial SequencesiRNA sequence 4gcuccaaguu
gaaagccaua gagcaag 27527RNAArtificial SequencesiRNA sequence
5ugcucuaugg cuuucaacuu ggagcau 27627RNAArtificial SequencesiRNA
sequence 6ccgaugauau caaaauggac ucgcaag 27727RNAArtificial
SequencesiRNA sequence 7ugcgagucca uuuugauauc aucggau
27827RNAArtificial SequencesiRNA sequence 8cgauucgcua gaccggcuuc
auugcag 27927RNAArtificial SequencesiRNA sequence 9gcaaugaagc
cggucuagcg aaucgau 271064DNAArtificial Sequenceprimer sequence
10gaagacggcc aacaaagtgc gttccaaact gaaggcgatt gaacagagca ttgagcagga
60ggag 641164DNAArtificial Sequenceprimer sequence 11cgggaagttg
gccatcttca cggacgacat aaagatggat tctcagatga ccaagcaggc 60cctg
64
* * * * *