U.S. patent application number 12/623776 was filed with the patent office on 2010-10-14 for in vivo modulation of neuronal transport.
This patent application is currently assigned to INSTITUT PASTEUR. Invention is credited to Julien Barbier, Philippe Brulet, Cecile Saint Cloment, Jordi Molgo, Sylvie ROUX.
Application Number | 20100260758 12/623776 |
Document ID | / |
Family ID | 34312741 |
Filed Date | 2010-10-14 |
United States Patent
Application |
20100260758 |
Kind Code |
A1 |
ROUX; Sylvie ; et
al. |
October 14, 2010 |
IN VIVO MODULATION OF NEURONAL TRANSPORT
Abstract
The invention relates to means for in vivo delivery of a
composition into the human or animal central nervous system or
spinal cord, wherein the composition comprises a non-toxic,
proteolytic fragment of tetanus toxoid in association with at least
a molecule having a biological function, and said molecule is
capable of in vivo retrograde axonal transport and transynaptic
transport into the CNS or spinal cord of the human or animal. In a
particular embodiment, the composition comprises a fragment C and a
fragment B of tetanus toxoid or a fraction thereof of at least 11
amino acid residues. The composition can further comprise a
fraction of fragment A of tetanus toxoid.
Inventors: |
ROUX; Sylvie; (Paris,
FR) ; Brulet; Philippe; (Paris, FR) ; Cloment;
Cecile Saint; (Paris, FR) ; Barbier; Julien;
(Paris, FR) ; Molgo; Jordi; (Paris, FR) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
INSTITUT PASTEUR
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE
|
Family ID: |
34312741 |
Appl. No.: |
12/623776 |
Filed: |
November 23, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11375093 |
Mar 15, 2006 |
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12623776 |
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PCT/EP04/10991 |
Sep 15, 2004 |
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11375093 |
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10662808 |
Sep 16, 2003 |
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PCT/EP04/10991 |
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09816467 |
Mar 26, 2001 |
7435792 |
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10662808 |
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09129368 |
Aug 5, 1998 |
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09816467 |
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60065236 |
Nov 13, 1997 |
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60055615 |
Aug 14, 1997 |
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Current U.S.
Class: |
424/134.1 ;
424/94.6; 424/94.65; 435/188; 514/8.3; 514/8.5; 530/350;
530/387.3 |
Current CPC
Class: |
A61P 25/28 20180101;
G01N 33/6896 20130101; C12N 9/52 20130101; C07K 2319/00 20130101;
G01N 33/5035 20130101; A61P 25/00 20180101; G01N 33/5058 20130101;
C07K 14/33 20130101; A61K 38/00 20130101; A61K 47/6415
20170801 |
Class at
Publication: |
424/134.1 ;
530/350; 514/8.3; 514/8.5; 424/94.65; 424/94.6; 530/387.3;
435/188 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 14/33 20060101 C07K014/33; A61K 38/18 20060101
A61K038/18; A61K 38/30 20060101 A61K038/30; A61K 38/48 20060101
A61K038/48; A61K 38/46 20060101 A61K038/46; C07K 19/00 20060101
C07K019/00; A61P 25/00 20060101 A61P025/00; A61P 25/28 20060101
A61P025/28 |
Claims
1. A method for in vivo delivery of a desired composition into
human or animal central nervous system (CNS) or spinal cord,
wherein the method comprises administering to the human or animal a
composition comprising a non-toxic, proteolytic fragment of tetanus
toxin (TT) in association with at least a molecule having a
biological function and said composition is capable of in vivo
retrograde axonal transport and transynaptic transport into the CNS
or the spinal cord of the human or animal and of being delivered at
different areas of the CNS or the spinal cord.
2. The method according to claim 1, wherein the composition is
administered into a muscle.
3. The method according to claim 1, wherein the composition is
administered into a muscle in the vicinity of a neuromuscular
junction.
4. The method according to claim 1, wherein the muscle is selected
in relation with the desired area of the CNS or spinal cord.
5. The method according to claim 1, wherein the composition is
administered into neuronal cells.
6. The method according to claim 1, wherein the composition
comprises a non-toxic, proteolytic fragment of tetanus toxin (TT)
comprising a fragment C and a fragment B or a fraction thereof of
at least 11 amino acid residues in association with at least a
molecule having a biological function selected from the group
consisting of a protein for compensation or modulation of functions
under the control of the CNS or the spinal cord or modulation of
functions in the CNS or the spinal cord or a protein to be
delivered by gene therapy expression system to the CNS or the
spinal cord.
7. The method according to claim 1, wherein the composition
comprises a non-toxic, proteolytic fragment of tetanus toxin (TT)
comprising a fragment C and a fragment B or a fraction thereof of
at least 11 amino acid residues and a fraction of a fragment A
devoid of its toxic activity corresponding to the proteolytic
domain having a zinc-binding motif located in the central part of
the chain between amino acids 225 and 245 in association with at
least a molecule having a biological function selected from the
group consisting of protein for the compensation or the modulation
of functions under the control of the CNS or the spinal cord or
protein to be delivered by gene therapy expression system to the
CNS or the spinal cord.
8. The method according to claim 6 or claim 7, wherein the molecule
is selected from the group consisting of protein SM, BDNF
(Brain-derived neurotrophic factor), NT-3 (Neurotrophin-3), NT-4/5,
GDNF (Gilal cell-line-derived neurotrophic factor), IGF
(Insulin-like growth factor), PNI (protease nexin I), SPI3 (Serine
Protease Inhibitor protein), ICE (Interleukin-1.beta. converting
enzyme), Bcl-2, GFP (green fluorescent protein), endonucleases like
I-SceI or CRE, antibodies, or drugs specifically directed against
neurodegenerative diseases such as latero spinal amyotrophy
(LSA).
9. The method according to claim 8, wherein the composition
comprises a combination of at least two of said molecules.
10. The method according to claim 8, wherein the molecule is
located upstream from the fragment of tetanus toxin.
11. The method according to claim 8, wherein the molecule is
located downstream from the fragment of tetanus toxin.
12. The method according to claim 1, which comprises administering
to the human or animal a vector containing nucleotides encoding the
composition, wherein the vector is capable of in vivo expression in
a muscle and this product is capable of migrating to the CNS or
spinal cord.
13. The method according to claim 12, wherein said vector comprises
a promoter and an enhancer capable of expressing the nucleotides
contained in said vector in the muscle.
14. The method according to claim 13, wherein said vector is the
plasmid pCMV-LacZ-TTC which has been deposited at the C.N.C.M. on
Aug. 12, 1997, under the registration number I-1912.
15. The method according to claim 12 or 13, wherein said vector is
administered into the muscle.
16. The method according to claim 12 or 13, wherein the molecule is
a nucleotide encoding for a protein or a polypeptide linked
chemically to the fragment of tetanus toxin and being transported
and expressed directly in neurons.
17. A hybrid fragment of tetanus toxin comprising a fragment C and
a fragment B or a fraction thereof of at least 11 amino acid
residues capable of transferring in vivo a protein, a peptide, or a
polynucleotide through a neuromuscular junction and at least one
synapse.
18. A hybrid fragment of tetanus toxin comprising a fragment C and
a fragment B or a fraction thereof of at least 11 amino acid
residues and a fraction of a fragment A devoid of its toxic
activity corresponding to the proteolytic domain having a
zinc-binding motif located in the central part of the chain between
amino acids 225 and 245 capable of transferring in vivo a protein,
a peptide or a polynucleotide through a neuromuscular junction and
at least one synapse.
19. An amino acid variant fragment having the same properties as
the hybrid fragment of tetanus toxin according to claim 17 or
18.
20. A polynucleotide variant fragment capable of hybridization
under stringent conditions with the natural tetanus toxin
sequence.
21. A composition containing an active molecule in association with
a hybrid fragment of tetanus toxin according to claim 17 or 18 or
with an amino acid variant fragment according to claim 16.
22. The composition according to claim 21, wherein the active
molecule is selected from the group consisting of protein SMN, BDNF
(Brain-derived neurotrophic factor), NT-3, NT-4/5, GDNF (Glial
cell-line derived neurotrophic factor), IGF (Insulin-like growth
factor), PNI (protease nexin I), SP13 (Serine Protease Inhibitor
protein), ICE, Bcl-2, GFP (green fluorescent protein),
endonucleases like I-SceI or CRE, antibodies or drugs specifically
directed against neorodegenerative diseases such as latero spinal
amyotrophy (LSA).
23. The composition according to claim 21, wherein the active
molecule is a polynucleotide encoding a protein or a polypeptide
with a promoter capable of expression in neurons, and optionally an
enhancer.
24. A vector comprising a promoter capable of expression in muscle
cells and optionally an enhancer, a nucleic acid sequence coding
for the fragment of tetanus toxin according to claim 17 or 18 or
with an amino acid variant fragment according to claim 19
associated with a polynucleotide coding for a protein or a
polypeptide.
25. A method of treatment of a patient or an animal affected with
CNS or spinal cord disease, which comprises delivering a
composition according to claim 21, 22, or 23 to the patient or
animal in an amount effective for treatment of the CNS or spinal
cord disease.
26. A method of treatment of a patient or an animal affected with
CNS or spinal cord disease, which comprises delivering a vector
according to claim 24 to the patient or animal in an amount
effective for treatment of the CNS or spinal cord disease.
27. The method according to claim 1, which comprises administering
to the human or animal a cell or a vector containing nucleotides
encoding the composition, wherein the cell or vector is capable of
in vivo expression in neuronal cells or precursor of neuronal cells
and wherein said cell is reimplanted into the CNS or spinal
cord.
28. The method according to claim 27 wherein said cell or vector
comprises a promoter and an enhancer capable of expressing the
nucleotides contained in said cell in neuronal cells or precursors
of neuronal cells.
29. The method according to claim 27 or 28 wherein the molecule is
a nucleotide encoding for a protein or a polypeptide linked
chemically to the fragment of tetanus toxin and being expressed
directly in neurons.
30. The method according to claim 27 or 28 wherein the molecule is
a nucleotide encoding for a protein or a polypeptide linked
chemically to the fragment of tetanus toxin and being expressed
directly in neurons.
31. A cell or vector comprising a promoter capable of expression in
neuronal cells or precursors of neuronal cells and optionally an
enhancer, a nucleic acid sequence coding for the fragment of
tetanus toxin according to claim 17 or 18 or with an amino acid
variant fragment according to claim 19 associated with a
polynucleotide coding for a protein or a polypeptide.
32. A method of modulating the transport in a neuron of a tetanus
toxin or a fusion protein comprising a fragment C of the tetanus
toxin, wherein the method comprises administering to the neuron a
TrkB receptor agonist or a TrkB receptor antagonist in an amount
sufficient to modulate the neuronal transport of the tetanus toxin
or the fusion protein.
33. The method according to claim 32, wherein the TrkB receptor
agonist is administered, thereby increasing the internalization of
the tetanus toxin or fusion protein at a neuromuscular
junction.
34. The method according to claim 33, wherein the TrkB receptor
agonist is a neurotrophic factor that activates a TrkB
receptor.
35. The method according to claim 34, wherein the neurotrophic
factor is a Brain Derivated Neurotrophic Factor or a Neurotrophin
4.
36. The method according to claim 33, wherein the TrkB receptor
agonist is an antibody that binds to a TrkB receptor, thereby
activating the TrkB receptor.
37. The method according to any one of claim 35 or 36, wherein the
internalization of the fusion protein at the neuromuscular junction
is increased.
38. The method according to claim 32, wherein the TrkB receptor
antagonist is administered, thereby decreasing the internalization
of the tetanus toxin or fusion protein at a neuromuscular
junction.
39. The method according to claim 38, wherein the TrkB receptor
antagonist is an antibody that binds to a TrkB receptor agonist,
thereby reducing activation of a TrkB receptor.
40. The method according to claim 39, wherein the TrkB receptor
agonist is a neurotrophic factor that activates a TrkB
receptor.
41. The method according to claim 40, wherein the neurotrophic
factor is a Brain Derivated Neurotrophic Factor or a Neurotrophin
4.
42. The method according to claim 42, wherein the internalization
of the tetanus toxin at the neuromuscular junction is
decreased.
43. The method according to claim 40, wherein the neurotrophic
factor is administered concurrently with the fusion protein.
44. A method of modulating the transport in a neuron of a tetanus
toxin or a fusion protein comprising a fragment C of the tetanus
toxin, wherein the method comprises administering to the neuron a
GFR.alpha./cRET receptor agonist or a GFR.alpha./cRET receptor
antagonist in an amount sufficient to modulate the neuronal
transport of the tetanus toxin or the fusion protein.
45. The method according to claim 44, wherein the GFR.alpha./cRET
receptor agonist is administered, thereby increasing the
internalization of the tetanus toxin or fusion protein at a
neuromuscular junction.
46. The method according to claim 45, wherein the GFR.alpha./cRET
receptor agonist is a neurotrophic factor that activates a
GFR.alpha./cRET receptor.
47. The method according to claim 46, wherein the neurotrophic
factor is a Glial-Derived Neurotrophic Factor.
48. The method according to claim 44, wherein the GFR.alpha./cRET
receptor agonist is an antibody that binds to a GFR.alpha./cRET
receptor, thereby activating the GFR.alpha./cRET receptor.
49. The method according to any one of claim 46 or 47, wherein the
internalization of the fusion protein at the neuromuscular junction
is increased.
50. The method according to claim 44, wherein the GFR.alpha./cRET
receptor antagonist is administered, thereby decreasing the
internalization of the tetanus toxin or fusion protein at a
neuromuscular junction.
51. The method according to claim 50, wherein the GFR.alpha./cRET
receptor antagonist is an antibody that binds to a GFR.alpha./cRET
receptor agonist, thereby reducing activation of a GFR.alpha./cRET
receptor.
52. The method according to claim 51, wherein the GFR.alpha./cRET
receptor agonist is a neurotrophic factor that activates a
GFR.alpha./cRET receptor.
53. The method according to claim 52, wherein the neurotrophic
factor is a Glial-Derived Neurotrophic Factor.
54. The method of claim 53, wherein the internalization of the
tetanus toxin at the neuromuscular junction is decreased.
55. The method according to claim 47, wherein the neurotrophic
factor is administered concurrently with the fusion protein.
56. A composition, comprising a TrkB receptor agonist and a fusion
protein comprising a fragment C of the tetanus toxin fused to a
second protein.
57. The composition according to claim 56, wherein, the TrkB
receptor antagonist is a neurotrophic factor that activates a TrkB
receptor.
58. The composition according to claim 57, wherein the neurotrophic
factor is a Brain Derivated Neurotrophic Factor or a Neurotrophin
4.
59. A composition, comprising a GFR.alpha./cRET receptor agonist
and a fusion protein comprising a fragment C of the tetanus toxin
fused to a second protein.
60. The composition according to claim 59, wherein, the
GFR.alpha./cRET receptor antagonist is a neurotrophic factor that
activates a GFR.alpha./cRET receptor.
61. The composition according to claim 60, wherein the neurotrophic
factor is Glial-Derived Neurotrophic Factor.
62. A method of detecting an effect of a compound on neuronal
transport, comprising administering to a neuron the compound and a
fusion protein comprising a fragment C of the tetanus toxin fused
to a second protein, wherein the second protein is encoded by a
reporter gene, and detecting the second protein to determine the
effect of the compound on neuronal transport.
63. The method according to claim 62, wherein the compound is a
neurotophic factor.
64. A method of screening for a compound that reduces or prevents
transport of a tetanus toxin in a neuron, comprising administering
to the neuron the compound and a fusion protein comprising a
fragment C of the tetanus toxin fused to a second protein, wherein
the second protein is encoded by a reporter gene, detecting the
second protein, and selecting the compound that reduces or prevents
the neuronal transport of the fusion protein.
65. The method according to claim 64, wherein the second protein is
detected at a neuromuscular junction.
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates to the use of part of tetanus toxin
for delivering a composition to the central nervous system of a
human or animal. This invention also relates to a hybrid fragment
of tetanus toxin, a polynucleotide that hybridizes with natural
tetanus toxin, and a composition containing the tetanus toxin
fragment as an active molecule. Further, this invention relates to
a vector comprising a promoter and a nucleic acid sequence encoding
the tetanus toxin fragment.
[0002] Tetanus toxin is produced by Clostridium tetani as an
inactive, single, polypeptide chain of 150 kD composed of three
5DkD domains connected by protease-sensitive loops. The toxin is
activated upon selective proteolytic cleavage, which generates two
disulfide-linked chains: L (light, 50 kD) and H (heavy, 100 kD)
[Montecucco C. and Schiavo G. Q. Rev. Biophys., (1995),
28:423-472].
[0003] Evidence for the retrograde axonal transport of tetanus
toxin to central nervous system (CNS) has been described by Erdmann
et al. [Naunyn Schmiedebergs Arch Phamacol., (1975), 290:357-373],
Price et al. [Science, (1975), 188:945-94], and Stoeckel et al.
[Brain Res., (1975), 99:1-16]. In each of these studies,
radiolabeled toxin was found inside membrane bound vesicles.
Another property was the transynaptic movement of tetanus toxin
that was demonstrated first by autoradiographic localization of
.sup.125I-labeled tetanus toxin in spinal cord interneurons after
injection into a muscle [Schwab and Thoenen, Brain Res., (1976),
105:218-227].
[0004] The structure of this tetanus toxin has been elucidated by
Hefting et al. [J. Biol. Chem., (1977), 252:187-193]. Papain
cleaves the tetanus toxin in two fragments:
[0005] the C terminal part of the heavy chain, 451 amino acids,
also called fragment C; and
[0006] the other part contained the complementary portion called
fragment B linked to the light chain (fragment A) via a disulfide
bond.
[0007] European Patent No. EP 0 030 496 B1 showed the retrograde
transport of a fragment B-II.sub.b to the CNS and was detected
after injection in the median muscle of the eye in primary and
second order neurons. This fragment may consist of "Isofragments"
obtained by clostridial proteolysis. Later, this fragment
B-II.sub.b was demonstrated to be identical to fragment C obtained
by papain digestion by Eisel et al. [EMBO J., 1986,
5:2495-2502].
[0008] This EP patent also demonstrated the retrograde transport of
a conjugate consisting of a I.sub.bc tetanus toxin fragment coupled
by a disulfide bond to B-II.sub.b from axonal endings within the
muscle to the motoneuronal perikarya and pericellular spaces. (The
I.sub.bc fragment corresponds to the other part obtained by papain
digestion as described above by Helting et al.). There is no
evidence that this conjugate was found in second order neurons. The
authors indicated that a conjugate consisting of the fragment
B-II.sub.b coupled by a disulfide bond to a therapeutic agent was
capable of specific fixation to gangliosides and synaptic
membranes. No result showed any retrograde axonal transport or a
transynaptic transport for such conjugate.
[0009] Another European Patent, No EP 0 057 140 B1, showed equally
the retrograde transport of a fragment II.sub.C to the CNS. As in
the European Patent No. EP 0 030 496 B1, the authors indicated that
a conjugate consisting of the fragment II.sub.C and a therapeutic
agent was capable of specific fixation, but no result illustrated
such allegation. This fragment II.sub.C corresponds to the now
called fragment C obtained by papain digestion.
[0010] Francis et al. [J. Biol. Chem., (1995), 270(25):15434-15442]
led an in vitro study showing the internalization by neurons of
hybrid between SOD-1 (Cu Zn superoxide dismutase) and a recombinant
C tetanus toxin fragment by genetic recombination. This recombinant
C tetanus toxin fragment was obtained from Halpern group. (See ref.
11).
[0011] Moreover, Kuypers H. G. J. M and Ugolini G. [TINS, (1990),
13(2):71-75] indicated in their publication concerning viruses as
transneuronal tracers that, despite the fact that tetanus toxin
fragment binds to specific receptors on neuronal membranes,
transneuronal labeling is relatively weak and can be detected only
in some of the synaptically connected neurons.
[0012] Notwithstanding these advances in the art, there still
exists a need for methods for delivering compositions into the
human or animal central nervous system. There also exists a need in
the art for biological agents that can achieve this result.
[0013] Additionally, activity-dependent modification of neuronal
connectivity and synaptic plasticity play an important role in the
development and function, of the nervous system. Recently, much
effort has been dedicated to following such modifications by the
engineering of new optically detectable genetic tools. For example,
fused to a reporter gene such as LacZ or GFP (Green Fluorescent
Protein), the atoxic C-terminal fragment of tetanus toxin (or TTC
fragment) can traffic retrogradely and transsynaptically inside a
restricted neural network either after direct injection of the
hybrid protein (Coen et al., 1997), or when expressed as a
transgene in mice (Maskos et al., 2002). The dynamics of
.beta.gal-TTC clustering at the neuromuscular junction (NMJ) is
strongly dependent on a presynaptic neuronal activity and probably
involves fast endocytic pathways (Miana-Mena at al., 2002).
Neuronal activity may induce this clustering and internalization at
the NMJ by enhancing the secretion and/or action of various
molecules at the synapse.
[0014] Over the past decade, various data indicate that
neurotrophins, a family of structurally and functionally related
proteins, including NGF (Nerve Growth Factor); BDNF (Brain Derived
Neurotrophic Factor); Neurotrophin 3 (NT-3) and Neurotrophin 4
(NT-4), not only promote neuronal survival and morphological
differentiation, but also can acutely modify synaptic transmission
and connectivity in central synapses, thus providing a connection
between neuronal activity and synaptic plasticity (McAllister et
al., 1999; Poo, 2001; Tao and Poo, 2001). The role of these factors
in neurotransmission between motoneurons and skeletal muscle cells
has been studied using Xenopus nerve-muscle co-culture studies,
whereby the treatment of these cultures with exogenous BDNF, NT-3
or NT-4 leads to an increase of synaptic transmission by enhancing
neurotransmitter secretion (Lohof et al., 1993; Stoop and Poo,
1996; Wang and Poo, 1997). Moreover, the muscular expression of
NT-3 and NT-4 (Funakoshi et al., 1995; Xie et al., 1997), as well
as NT-4 secretion (Wang and Poo, 1997) are regulated by electrical
activity. This family of proteins thus provides a molecular link
between electrical neuronal activity and synaptic changes.
[0015] The cellular actions of neurotrophins are mediated by two
types of receptors: the p75.sup.NTR receptor, mainly expressed
during early neuronal development, and a Trk tyrosine kinase
receptor (Bothwell, 1995). The interaction of neurotrophins with
Trk receptors is specific. TrkB and TrkC, are activated by
BDNF/NT-4 and NT-3, respectively, and are expressed by motor
neurons. TrkA, which is expressed by sensory neurons, is activated
by NGF. Recently, evidence for a co-trafficking between TTC and the
neurotrophin receptor p75.sup.NTR has been reported in cultured
motoneurons (Lalli and Schiavo, 2002), as well as the activation by
tetanus toxin and the TTC fragment of intracellular pathways
involving Trk receptors in cultured cortical neurons (Gil et al.,
2003).
[0016] Notwithstanding the knowledge in the art, there still exists
a need for understanding the influences of neurotrophins and other
neurotrophic factors on TIC traffic at the NMJ in vivo and for
developing methods of using these neurotrophins and neurotrophic
factors, and agonists or antagonists thereof, to modulate the
neuronal transport of a tetanus toxin or a fusion protein
comprising a fragment C of the tetanus toxin.
SUMMARY OF THE INVENTION
[0017] This invention aids in fulfilling these needs in the art.
More particularly, this invention provides a method for in vivo
delivery of desired composition into the central nervous system
(CNS) of the mammal, wherein the composition comprises a non-toxic
proteolytic fragment of tetanus toxin (TT) in association with at
least a molecule having a biological function. The composition is
capable of in vivo retrograde transport and transynaptic transport
into the CNS and of being delivered to different areas of the
CNS.
[0018] This invention also provides a hybrid fragment of tetanus
toxin comprising fragment C and fragment B or a fraction thereof of
at least 11 amino acid residues or a hybrid fragment of tetanus
toxin comprising fragment C and fragment B or a fraction thereof of
at least 11 amino acid residues and a fraction of fragment A devoid
of its toxic activity corresponding to the proteolytic domain
having a Zinc-binding motif located in the central part of the
chain between the amino acids 225 and 245, capable of transferring
in vivo a protein, a peptide, or a polynucleotide through a
neuromuscular junction and at least one synapse.
[0019] Further, this invention provides a composition comprising an
active molecule in association with the hybrid fragment of tetanus
toxin (TT) or a variant thereof. The composition is useful for the
treatment of a patient or an animal affected with CNS disease,
which comprises delivering a composition of the invention to the
patient or animal. In addition, the composition of this invention
may be useful to elicit a immune response in the patient or animal
affected with CNS, which comprises delivering a composition of the
invention to the patient or animal.
[0020] Moreover, this invention provides polynucleotide variant
fragments capable of hybridizing under stringent conditions with
the natural tetanus toxin sequence. The stringent conditions are
for example as follows: at 42.0 for 4 to 6 hours in the presence of
6.times.SSC buffer, 1.times.Denhardt's Solution, 1% SDS, and 250
.mu.g/ml of tRNA. (1.times.SSC corresponds to 0.15 M NaCl and 0.05
M sodium citrate; 1.times.Denhardt's solution corresponds to 0.02%
Ficoll, 0.02% polyvinyl pyrrolidone and 0.02% bovine serum
albumin). The two wash steps are performed at room temperature in
the presence of 0.1.times.SCC and 0.1% SDS.
[0021] A polynucleotide variant fragment means a polynucleotide
encoding for a tetanus toxin sequence derived from the native
tetanus toxin sequence and having the same properties of
transport.
[0022] In addition, the invention provides a vector comprising a
promoter capable of expression in muscle cells and optionally an
enhancer, a nucleic acid sequence coding for the fragment of
tetanus toxin of the invention or an amino acid variant fragment of
the invention associated with a polynucleotide coding for a protein
or a polypeptide of interest. In a preferred embodiment of the
invention the promoter can be the CMV promoter and preferably the
CMV promoter contained in pcDNA 3.1 (In Vitrogen, ref. V790-20), or
the promoter 6 actin as described in Bronson S. V. et al. (PNAS,
1996, 93:9067-9072).
[0023] In addition, the invention provides a vector comprising a
promoter capable of expression in neuronal cells or in precursors
(such NT2(hNT) precursor cells from Stratagene reference #204101)
and optionally an enhancer, a nucleic acid sequence coding for the
fragment of tetanus toxin of the invention or an amino acid variant
fragment of the invention associated with a polynucleotide coding
for a protein or a polypeptide of interest. In a preferred
embodiment of the invention the promoter can be .beta. actin (see
the above reference). These vectors are useful for the treatment of
a patient or an animal infected with CNS disease comprising
delivering the vector of the invention to the patient or animal. In
addition, these vectors are useful for eliciting immune responses
in the patient or animal.
[0024] One advantage of the present invention comprising the
fragment of tetanus toxin (fragment A, B, and C) is to obtain a
better transport of the fragment inside the organism compared with
fragment C. Another advantage of the composition of the invention
is to obtain a well defined amino acid sequence and not a
multimeric composition. Thus, one can easily manipulate this
composition in gene therapy.
[0025] In another embodiment, this invention provides a method of
modulating the transport in a neuron of a neurotoxin, such as the
tetanus toxin, or a fusion protein comprising a fragment C of the
tetanus toxin. These methods comprise administering neurotrophic
factors such as BDNF, NT-4, and GDNF, and agonists and antagonists
thereof, to modulate internalization at a neuromuscular junction of
a neurotoxin or a fusion protein comprising the TTC fragment
according to the invention.
[0026] In one embodiment, these methods further comprise
administering to the neuron a TrkB receptor agonist or a TrkB
receptor antagonist in an amount sufficient to modulate the
neuronal transport of the tetanus toxin or the fusion protein. The
term "modulate" and its cognates refer to the capability of a
compound acting as either an agonist or an antagonist of a certain
reaction or activity. The term modulate, therefore, encompasses the
terms "increase" and "decrease." The term "increase," for example,
refers to an increase in the neuronal transport of a polypeptide in
the presence of a modulatory compound, relative to the transport of
the polypeptide in the absence of the same compound. Analogously,
the term "decrease" refers to a decrease in the neuronal transport
of a polypeptide in the presence of a modulatory compound, relative
to the transport of the polypeptide in the absence of the same
compound. The neuronal transport of polypeptides can be measured as
described herein or by techniques generally known in the art.
[0027] The TrkB receptor agonists include neurotrophic factors that
activate a TrkB receptor, such as a Brain Derivated Neurotrophic
Factor or a Neurotrophin 4. The TrkB receptor agonists can also
include antibodies that bind to TrkB receptors and activate them.
These methods of using TrkB receptor agonists provide useful
methods for enhancing the neuronal transport of a tetanus toxin or
a tetanus toxin fusion protein.
[0028] The TrkB receptor antagonists include antibodies that bind
to a TrkB receptor agonist, such as those described above, and
thereby decrease the activation of a TrkB receptor. For example,
these antibodies can be directed to neurotrophic factors that
activate a TrkB receptor, such as a Brain Derivated Neurotrophic
Factor or a Neurotrophin 4. In addition, TrkB receptor antagonists
include antibodies that bind to TrkB receptors and inactivate them.
These methods of using TrkB receptor agonists provide useful
methods for decreasing or preventing the neuronal transport of a
tetanus toxin or a tetanus toxin fusion protein.
[0029] In another embodiment, these methods further comprise
administering to the neuron a GFR.alpha./cRET receptor agonist or a
GFR.alpha./cRET receptor antagonist in an amount sufficient to
modulate the neuronal transport of the tetanus toxin or the fusion
protein.
[0030] The GFR.alpha./cRET receptor agonists include neurotrophic
factors that activate a GFR.alpha./cRET receptor, such as a
Glial-Derived Neurotrophic Factor. The GFR.alpha./cRET receptor
agonists can also include antibodies that bind to GFR.alpha./cRET
receptors and activate them. These methods of using GFR.alpha./cRET
receptor agonists provide useful methods for enhancing the neuronal
transport of a tetanus toxin or a tetanus toxin fusion protein.
[0031] The GFR.alpha./cRET receptor antagonists include antibodies
that bind to a GFR.alpha./cRET receptor agonist, such as those
described above, and thereby decrease the activation of a
GFR.alpha./cRET receptor. For example, these antibodies can be
directed to neurotrophic factors that activate a GFR.alpha./cRET
receptor, such as a Glial-Derived Neurotrophic Factor. In addition,
GFR.alpha./cRET receptor antagonists include antibodies that bind
to GFR.alpha./cRET receptors and inactivate them. These methods of
using GFR.alpha./cRET receptor agonists provide useful methods for
decreasing or preventing the neuronal transport of a tetanus toxin
or a tetanus toxin fusion protein.
[0032] In these methods, the agonist or antagonist can be
administered to neuronal cells that already contain a tetanus toxin
or a fusion protein. Alternatively, the tetanus toxin or fusion
protein can be administered concurrently with or after the
administration of the agonist or antagonist.
[0033] In one embodiment, the TTC-containing fusion proteins of the
present invention comprises a second protein that is encoded by a
reporter gene, such as the lac Z gene or the Green Fluorescent
Protein gene. Such fusion proteins are useful for visualizing
modulation of the synaptic plasticity in vivo, including in a
human, for example by magnetic resonance imaging. For example, the
fusion proteins can be used to monitor and detect the effects of a
compound, such as a neurotrophic factor, on neuronal transport. In
these methods, the compound and the fusion protein are administered
to a neuron, and the fusion protein is detected to determine the
effect of the compound on the neuronal transport. In addition, the
fusion proteins can be used to detect modifications in trafficking
patterns within a restricted neural network, such as those used in
known animal models for neurodegenerative diseases. The fusion
proteins can also be used in screening methods to detect compounds
that reduce or prevent neuronal transport of a tetanus toxin.
Compounds so identified can be used to prevent or treat tetanus
infections.
[0034] The TTC fragment can also be coupled to a neurotrophic
factor and administered to a patient to treat CNS pathologies
associated with production defects of different factors. The TTC
fragment could also be used as a vector for modulating interactions
with proteins involved in neurodegenerative diseases.
[0035] The present invention also provides compositions comprising
a TrkB receptor agonist or a GFR.alpha./cRET receptor agonist and a
fusion protein comprising a fragment C of the tetanus toxin fused
to a second protein. In one embodiment, the TrkB agonist is a
neurotrophic factor such as a Brain Derivated Neurotrophic Factor
or a Neurotrophin 4. In another embodiment, the GFR.alpha./cRET
receptor agonist is a neurotrophic factor, such as Glial-Derived
Neurotrophic Factor
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] This invention will be more fully described with reference
to the drawings in which:
[0037] FIG. 1 shows the DNA sequence and amino acid sequence of the
TTC fragment cloned in pBS:TTC.
[0038] FIG. 2 shows the details of construct pBS:TTC, which is
further described in Example 1.
[0039] FIG. 3 depicts pGEX:lacZ-TTC construct.
[0040] FIG. 4 shows construct pGEX:TTC-lacZ.
[0041] FIG. 5 depicts the details of the construct
pCMV:lacZ-TTC.
[0042] FIG. 6 shows the confocal immunofluorescence analysis of
GFP-TTC membrane traffic at mature mouse LAL NMJs.
[0043] (A) Two hours after the subcutaneous injection of GFP-TTC in
the vicinity of the LAL muscle, the probe (green) was concentrated
at motor nerve endings of NMJ. Associated intramuscular motor axons
were immunostained (red) with an antibody against NF200. GFP-TTC
labeling was also detected in sensory nerve fibers (arrows) and at
the nodes of Ranvier of myelinated axons (arrowheads). (B) Strong
nodal labeling with GFP-TTC (green) (arrow) in a single living
myelinated axon. Myelin was passively stained with RH414 dye (red).
(C) Two hours after injection as in A, LAL muscle fibers were fixed
and labeled for troponin T by indirect immunofluorescence. (C' and
C'') Inset is a side view image of a NMJ showing that GFP-TTC
staining (green) is located presynaptically. (D-G) LAL was
harvested at various times after GFP-TTC injection and NMJ
identified in red by labeling with TRITC-a-BTX (D'-G'). D-D': 5
min; E-E': 30 min; F-F': 2 h and G-G': 24 h.
[0044] Scale bars: A, 20 .mu.m; B, 8 .mu.m; C: 20 .mu.m; D, 2
.mu.m; E-G, 5 .mu.m.
[0045] FIG. 7 shows that BDNF increases GFP-TTC recruitment at the
NMJ in a dose-dependent manner.
[0046] (A1 A6) The NMJ on LAL muscles was identified by TRITC-a-BTX
labeling 30 min after in vivo co-injection of GFP-TTC with various
amounts of BDNF. The level of GFP fluorescence was quantified over
these areas (see B). An enhancement of axonal labeling (arrows),
more prononced with higher BDNF concentration, was also detected.
Scale bars: 20 .mu.m (B) Confocal sections of the NMJ were
collected for analysis and projections generated. TRIT-a-BTX
labeling determines the area of the NMJ over which the global GFP
fluorescent signal was measured. For each, (n=15-20), the GFP
fluorescence was divided by NMJ area (in .mu.m.sup.2) to obtain the
fluorescence level. Error bars indicate S.D. **P<0.005; Rest, vs
control.
[0047] FIG. 8 depicts the immunofluorescence visualization of TrkB
at the LAL NMJ. Two hours after GFP-TTC injection in LAL, confocal
analysis was performed. (A-B) The fusion protein was identified in
green directly by GFP fluorescence. (C-D) TrkB, identified (in red)
by indirect immunofluorescence (see material and methods), was
located at the NMJ. (E-F) However, when the two projections were
overlaid, no overlap was found between the TrkB and the GFP-TTC
signals.
[0048] Scale bar: Top: 5 .mu.m; Bottom: 2 .mu.m
[0049] FIG. 9 represents the results of experiments elucidating the
mechanisms of GFP-TTC recruitment to the NMJ.
[0050] (A) Quantification of GFP-TTC fluorescence was performed, as
described in FIG. 7 at various time after co-injection with or
without 50 ng BDNF.
[0051] (B) After in vitro loading for 45 min with GFP-TTC, the
excised LAL muscle was faced and SV2 protein detected by indirect
immunofluorescence (red). SV2 labeling was mostly diffuse and
concentrated in a few areas of the NMJ (arrows). Colocalization of
SV2 with GFP-TTC staining was only observed in a very limited
number of areas. Scale bar: 8 .mu.m.
[0052] (C) Treatment with botulinum type-A neurotoxin to block
synaptic vesicles exocytosis and endocytosis. 48 hours after BoTx/A
injection (as described in material and methods), GFP-TTC,
associated or not with 50 ng BDNF, was injected in LAL muscle and
GFP fluorescence quantified as previously. ** P<0.005; t-test,
vs control; * P<0.005; t-test vs BoTx/A treatment
[0053] (D) Comparison of KCl induced depolarization and BDNF
effects on GFP-TTC localization at the NMJ.
[0054] FIG. 10 depicts the localization of GFP-TTC probe in lipid
microdomains.
[0055] (A) 2 hours after intramuscular injection, GFP-TTC was found
in detergent resistant membranes (DRMs) (lanes 4-6) isolated from
gastrocnemius muscle, which also contained the raft marker
caveolin-3. A small amount of GFP-TTC was also detected in the
soluble fraction (lane 12).
[0056] (B) GFP-TTC colocalized with the raft marker GM1 at the NMJ.
NMJ were identified by Alexa 647-a-BTX binding (in blue). Whereas
GFP-TTC was detected in less than 5 min at the NMJ, CT-b requires
3-5 hours. At this time, a diffuse staining which colocalized with
the similar GFP-TTC labeling, was obtained, while a few patches
labeling only for CT-b were also observed.
[0057] (C) Intensity profiles of GFP-TTC (green) and Alexa 594
labeled-CT-b (red) were performed 5 or 24 h after intramuscular
co-injection of both probes in gastrocnemius.
[0058] Scale bar. 5 .mu.m.
[0059] FIG. 11 shows a comparison of GFP-TTC and CT-b localization
in motoneuron cell bodies. Twenty four hours after .beta.-gal-TTC
(A) or GFP-TTC and CT-b (B-E) intramuscular injection in
gastrocnemius muscle, mice were perfused intracardially and their
spinal cords removed. (A) X-gal reaction on spinal cord transerve
sections showed labeling in motoneuron cell bodies but also in
neurites (inset). (B) GFP-TTC and CT-b were detected on
longitudinal section of spinal cord in a significant number of
motoneurons. (C) Probes were detected in vesicles highly
concentrated in cell bodies but also in neurites. (D) In neuronal
extensions, GFP-TTC and CT-b were detected in different
vesicular-like structures. Note that only few of them were positive
for both probes. (E) Note that neither GFP-TTC nor CT-b were
detected in the nucleus as shown in one optical section.
[0060] Scale bars: A, 0.2 mm; inset, 50 .mu.m; B, 20 .mu.m; C, 10
.mu.m; D, 5 .mu.m; E, 2 .mu.m.
DETAILED DESCRIPTION
[0061] Tetanus toxin is a potent neurotoxin of 1315 amino acids
that is produced by Clostridium tetani (1, 2). It prevents the
inhibitory neurotransmitter release from spinal cord interneurons
by a specific mechanism of cell intoxication (for review see ref
3). This pathological mechanism has been demonstrated to involve
retrograde axonal and transynaptic transport of the tetanus toxin.
The toxin is taken up by nerve endings at the neuromuscular
junction, but does not act at this site; rather, the toxin is
transported into a vesicular compartment and travels along motor
axons for a considerable distance until it reaches its targets. The
transynaptic movement of tetanus toxin was first demonstrated by
autoradiographic localization in spinal cord interneurons after
injection into a muscle (4). However, previous studies of
transynaptic passage of tetanus toxin from motoneurons were limited
by the rapid development of clinical tetanus and death of the
experimental animal (4, 5, 6).
[0062] A fragment of tetanus toxin obtained by protease digestion,
the C fragment, has been shown to be transported by neurons in a
similar manner to that of the native toxin without causing clinical
symptoms (7, 8, 9, 10). A recombinant C fragment was reported to
possess the same properties as the fragment obtained by protease
digestion (11). The fact that an atoxic fragment of the toxin
molecule was able to migrate retrogradely within the axons and to
accumulate into the CNS led to speculation that such a fragment
could be used as a neurotrophic carrier (12). A C fragment
chemically conjugated to various large proteins was taken up by
neurons in tissue culture (13) and by motor neurons in animal
models (ref. 12, 14, and 15). According to the invention the
fragment of tetanus toxin consists of a non-toxic proteolytic
fragment of tetanus toxin (TT) comprising a fragment C and a
fragment B or a fraction thereof of at least 11 amino acid residues
or a non-toxic proteolytic fragment of tetanus toxin (TT)
comprising a fragment C and a fragment B or a fraction thereof of
at least 11 amino acids residues and a fraction of a fragment A
devoid of its toxic activity corresponding to the proteolytic
domain having a zinc-binding motif located in the central part of
the chain between the amino acids 225 and 245 (cf. Montecucco C.
and Schiavo G. Q. Rev. Biophys., (1995), 28:423-472). Thus the
fraction of the fragment A comprises, for example, the amino acid
sequence 1 to 225 or the amino acid sequence 245 to 457, or the
amino acid sequence 1 to 225 associated with amino acid sequence
245 to 457.
[0063] The molecule having a biological function is selected from
the group consisting of protein of interest, for example, for the
compensation or the modulation of the functions under the control
of the CNS or the spinal cord or the modulation of the functions in
the CNS or the spinal cord, or protein of interest to be delivered
by gene therapy expression system to the CNS or the spinal cord.
The proteins of interest are, for example, the protein SMN
implicated in spinal muscular atrophy (Lefebvre et al., Cell,
(1995), 80:155-165 and Roy et al., Cell, (1955), 80:167-178);
neurotrophic factors, such as BDNF (Brain-derived neurotrophic
factor); NT-3 (Neurotrophin-3); NT-4/5; GDNF (Glial
cell-line-derived neurotrophic factor); IGF (Insulin-like growth
factor) (Oppenheim, Neuron, (1996), 17:195-197; Thoenen et al.,
Exp. Neurol., (1933), 124:47-55 and Henderson et al., Adv. Neurol.,
(1995), 68:235-240); or PNI (protease nexin I) promoting neurite
outgrowth (this factor can be used for the treatment of Alzheimer
disease (Houenou et al., PNAS, (1995), 92:895-899)); or SPI3 a
serine protease inhibitor protein (Safaei, Dev. Brain Res., (1997),
100: 5-12); or ICE (Interleukin-1.beta. converting Enzyme) a factor
implicated in apoptosis, to avoid apoptosis (Nagata, Cell, (1997),
88:355-365); or Bd-2, a key intracellular regulator of programmed
cell death (Jacobson, M.D. (1997), Current Biology, 7:R277-R281);
or green fluorescent protein (Lang et al., Neuron, (1997),
18:857-863) as a marker, enzyme (ex: .beta.-Gal); endonuclease like
I-SceI (Choulika A., et al. (1995), Molecular and Cellular biology,
15, (4):1968-1973 or CRE (Gu H., et al. (1994), Science,
265:103-106); specific antibodies; drugs specifically directed
against neurodegenerative diseases such as latero spinal
amyotrophy. Several molecules can be associated with a TT
fragment.
[0064] In association means an association obtained by genetic
recombination. This association can be realized upstream as well as
downstream to the TT fragment. The preferred mode of realization of
the invention is upstream and is described in detail; a downstream
realization is also contemplated. (Despite Halpem at al., J. Biol.
Chem., (1993), 268(15):11188-11192, who indicated that the
carboxyl-terminal amino acids contain the domain required for
binding to purified gangliosides and neuronal cells.)
[0065] The desired CNS area means, for example, the tongue which is
chosen to direct the transport to hypoglossal motoneuron; the arm
muscle which is chosen to direct the transport to the spinal cord
motoneurons.
[0066] For this realization of transplantation of a neuron to the
CNS or the spinal cord see Gage, F. H. et al. (1987), Neuroscience,
23:725-807, "Grafting genetically modified cells to the brain:
possibilities for the future."
[0067] The techniques for introducing the polynucleotides to cells
are described in U.S. Pat. Nos. 5,580,859 and 5,589,466, which is
relied upon and incorporated by reference herein. For example, the
nucleotides may be introduced by transfection in vitro before
reimplantation in area of the CNS or the spinal cord.
[0068] A chemical linkage is considered for a particular embodiment
of the invention and comprises the association between the TT
fragment and a polynucleotide encoding the molecule of interest
with its regulatory elements, such as promoter and enhancer capable
of expressing said polynucleotide. Then the TT fragment allows the
retrograde axonal transport and/or the transynaptic transport, and
the product of the polynucleotide is expressed directly in the
neurons. This chemical linkage can be covalent or not, but
preferably covalent performed by thiolation reaction or by any
other binding reaction as described in "Bioconjugate Techniques"
from Gret T. Hermanson (Academic press, 1996).
[0069] The axonal retrograde transport begins at the muscle level,
where the composition of interest is taken up at the neuromuscular
junction, and migrates to the neuronal body of the motoneurons
(which are also called the first order neurons) in the CNS or
spinal cord. First order neurons mean neurons that have
internalized the composition of interest, and thus in this case,
correspond to motoneurons.
[0070] The transynaptic retrograde transport corresponds to
interneuron communications via the synapses from the motoneurons,
and comprises second order neurons and higher order neurons (fourth
order corresponding to neurons in the cerebral cortex).
[0071] The different stages of the neuronal transport are through
the neuromuscular junction, the motoneuron, also called first order
neuron, the synapse at any stage between the neurons of different
order, neuron of order second to fourth order, which corresponds to
the cerebral cortex.
[0072] In one embodiment of this invention, it is shown that a
.beta.-gal-TTC (TT-fragment C) hybrid protein retains the
biological activities of both proteins in vivo. Therefore, the
hybrid protein can undergo retrograde and transneuronal transport
through a chain of interconnected neurons, as traced by its
enzymatic activity. These results are consistent with those of
others who used chemically conjugated TTC, or TTC fused to other
proteins (12, 13, 14, 15). In these in vitro analyses, the activity
of the conjugated or hybrid proteins was likewise retained or only
weakly diminished. Depending on the nature of the TTC fusion
partner, different types of potential applications can be
envisioned. For example, this application can be used to deliver a
biologically active protein into the CNS for therapeutic purposes.
Such hybrid genes can also be used to analyze and map synaptically
connected neurons if reporters; such as lacZ or the green
fluorescent protein (GFP; 29) gene, were fused to TTC.
[0073] The retrograde transport of the hybrid protein may be
demonstrated as follows. When injected into a muscle, .beta.-gal
activity rapidly localized to the somata of motoneurons that
innervate the muscle. The arborization of the whole nerve, axon,
somata and dendrites can easily be visualized. However, in
comparison to the neurotropic viruses, the extent of retrograde
transneuronal transport of the hybrid protein from the hypoglossal
neurons indicates that only a subset of interconnected neurons is
detected, although most areas containing second-order interneurons
have been identified by the .beta.-gal-TTC marker. Transneuronal
uptake is mostly restricted to second order neurons. In such
experiments, when a limited amount of a neuronal tracer is injected
into a muscle or cell, only a fraction will be transported through
a synapse, thereby imposing an experimental constraint on its
detection. Presently, the most efficient method, in terms of the
extent of transport, relies on neurotropic viruses. Examples
include: alpha-herpes viruses, such as herpes simplex type 1
(HSV-1), pseudorabies virus (PrV), and rhabdoviruses (24, 25).
Viral methods are very sensitive because each time a virus infects
a new cell, it replicates, thereby amplifying the signal and
permitting visualization of higher order neurons in a chain.
Ultimately, however, one wants to map a neuronal network in an in
vivo situation such as a transgenic animal. Here, the disadvantage
of viral labeling is its potential toxicity. Most viruses are not
innocuous for the neural cell, and their replication induces a
cellular response and sometimes cell degeneration (24).
Furthermore, depending on experimental conditions, budding of the
virus can occur leading to its spread into adjoining cells and
tissues.
[0074] Differences in mechanisms of transneuronal migration could
also account for the restricted number of neurons labeled by
.beta.-gal-TTC. Matteoli et al have provided strong evidence that
the intact tetanus toxin crosses the synapses by parasitizing the
physiological process of synaptic vesicle recycling at the nerve
terminal (22). The toxin probably binds to the inner surface of a
synaptic vesicle during the time the lumen is exposed to the
external medium. Vesicle endocytosis would then presumably provide
the mechanism for internalization of the toxin. Because the TTC
fragment is known to mimic the migration of the toxin in vivo, it
could therefore direct the fusion protein along a similar
transynaptic pathway. If this hypothesis is confirmed, it would
strongly suggest that synaptic activity is required for the
transneuronal transport of .beta.-gal-TTC. Therefore, only active
neuronal circuits would be detected by the hybrid protein. The
possible dependence of .beta.-gal-TTC on synaptic vesicle
exocytosis and endocytosis could be further investigated, since
techniques are now available to record synaptic activity in neural
networks in vitro (30). In contrast, the transneuronal pathway of
neurotropic viruses has not yet been elucidated and could be
fundamentally different, involving virus budding in the vicinity of
a synapse. Finally, the transneuronal transport of the hybrid
protein might depend on a synaptic specificity, although the
tetanus toxin is not known to display any (7, 23). It is therefore
likely that a virus would cross different or inactive synapses. In
summary, the restricted spectrum of interneuronal transport, in
addition to its non-toxicity, make the .beta.-gal-TTC hybrid
protein a novel and powerful tool for analysis of neural
pathways.
[0075] One advantage of the fusion gene of the invention for
neuronal mapping is that it derives from a single genetic entity
that is amenable to genetic manipulation and engineering. Several
years ago, a technique based on homologous recombination in
embryonic stem cells was developed to specifically replace genes in
the mouse (31, 32). This method generates a null mutation in the
substituted gene, although in a slightly modified strategy, a
dicistronic messenger RNA can also be produced (33, 34). When a
reporter gene, such as E. coli lacZ, is used as the substituting
gene, this technique provides a means of marking the mutated cells
so that they can be followed during embryogenesis. Thus, this
technique greatly simplifies the analysis of both the heterozygote
expression of the targeted gene as well as the phenotype of null
(homozygous) mutant animals.
[0076] Another advantage of this invention is that the composition
comprising the fusion gene may encode an antigen or antigens. Thus,
the composition may be used to elicit an immune response in its
host and subsequently confer protection of the host against the
antigen or antigens expressed. These immunization methods are
described in Robinson et al., U.S. Pat. No. 5,43,578, which is
herein incorporated by reference. In particular, the method of
immunizing a patient or animal host comprises introducing a DNA
transcription unit encoding comprising the fusion gene of this
invention, which encodes a desired antigen or antigens. The uptake
of the DNA transcription unit by the host results in the expression
of the desired antigen or antigens and the subsequent elicitation
of humoral and/or cell-mediated immune responses.
[0077] Neural cells establish specific and complex networks of
interconnected cells. If a gene were mutated in a given neural
cell, we would expect this mutation to have an impact on the
functions of other, interconnected neural cells. With these
considerations in mind, a genetic marker that can diffuse through
active synapses would be very useful in analyzing the effect of the
mutation. In heterozygous mutant animals, the cells in which the
targeted gene is normally transcribed could be identified, as could
the synaptically connected cells of a neural network. In a
homozygous animal, the impact of the mutation on the establishment
or activity of the neural network could be determined. The
feasibility of such an in vivo approach depends critically on the
efficiency of synaptic transfer of the fusion protein, as well as
its stability and cellular localization.
[0078] Another extension of the invention is to gene therapy
applied to the CNS. This invention provides for uptake of a
non-toxic, enzyme-vector conjugate by axon terminals and conveyance
retrogradely to brainstem motoneurons. A selective retrograde
transynaptic mechanism subsequently transports the hybrid protein
into second-order connected neurons. Such a pathway, which
by-passes the blood-brain barrier, can deliver macromolecules to
the CNS. In fact, pathogenic agents, such as tetanus toxin and
neurotropic viruses, are similarly taken up by nerve endings,
internalized, and retrogradely transported to the nerve cell
somata. In such a scenario, the lacZ reporter would be replaced by
a gene encoding a protein that provides a necessary or interesting
activity and/or function. For example, the human CuZn superoxide
dismutase, SOD-1, and the human enzyme
.beta.-N-acetylhexosaminidase A, HexA, have been fused or
chemically coupled to the TTC fragment (13; 16), and their uptake
by neurons in vitro was considerably increased and their enzymatic
functions partially conserved. Combined with the in vivo
experiments described here using .beta.-gal-TTC, a gene therapy
approach based on TTC hybrid proteins appears to be a feasible
method of delivering a biological function to the CNS. However,
ways have to be found to target the TTC hybrid proteins, which are
likely to be sequestrated into vesicles, to the appropriate
subcellular compartment. Such a therapeutic strategy could be
particularly useful for treating neurodegenerative and motoneuron
diseases, such as amyotrophy lateral sclerosis (ALS, 35), spinal
muscular atrophies (SMA, 36, 37), or neurodegenerative lysosomal
storage diseases (38, 39). Injection into selected muscles, even in
utero, could help to specifically target the appropriate neurons.
In addition, such an approach would avoid the secondary and
potentially toxic effects associated with the use of defective
viruses to deliver a gene (40, 41).
Example 1
Plasmid Constructions
[0079] (A) TTC Cloning:
[0080] Full length TTC DNA was generated from the genomic DNA from
the Clostridium Tetani strain (a gift from Dr. M. Popoff, Institut
Pasteur) using PCR. Three overlapping fragments were synthesized:
PCR1 of 465 by (primer 1: 5'-CCC CCC GGG CCA CCA TGG TTT TTT CAA
CAC CAA TTC CAT TTT CTT ATT C-3' (SEQ ID NO:4) and primer 2: 5'-CTA
AAC CAG TAA ITT CTG-3' (SEQ ID NO:5)), PCR2 of 648 by (primer 3:
5'-AAT TAT GGA CTT TAA AAG ATT CCG C-3' (SEQ ID NO:6) and primer 4:
5'-GGC ATT ATA ACC TAC TCT TAG AAT-3' (SEQ ID NO:7)) and PCR3 of
338 by (primer 5: 5'-AAT GCC TTT AAT AAT CTT GAT AGA AAT-3' (SEQ ID
NO:8) and primer 6: 5'-CCC CCC GGG CAT ATG TCA TGA ACA TAT CAA TCT
GTT TAA TC-3' (SEQ ID NO:9)). The three fragments were sequentially
introduced into pBluescript KS+ (Stratagene) to give pBS:TTC
plasmid. The upstream primer 1 also contains an optimized
eukaryotic Ribosome Binding Site (RBS) and translational initiation
signals. Our TTC fragment (462 amino acids) represents the amino
acids 854-1315 of tetanus holotoxin, i.e. the carboxy-terminal 451
amino acids of the heavy chain, which constitute the fragment C
plus 11 amino acids of the heavy chain that immediately precede the
amino terminus of the fragment C. The DNA sequence and amino acid
sequence of the TTC fragment cloned in pBS:TTC is shown in FIG. 1.
The construct pBS:TTC is shown in FIG. 2.
[0081] (B) pGEX:lacZ-TTC:
[0082] pGEXJacZ was obtained by cloning a SmaI/XhoI lacZ fragment
from the pGNA vector (a gift from Dr. H. Le Mouellic) into pGEX
4T-2 (Pharmacia). PCR was used to convert the lacZ stop codon into
an NcoI restriction site. Two primers (upstream: 5'-CTG AAT ATC GAC
GGT TTC CAT ATG-3' (SEQ ID NO:10) and downstream: 5'-GGC AGT CTC
GAG TCT AGA CCA TGG CTT TTT GAC ACC AGA C-3' (SEQ ID NO:11)) were
used to amplify the sequence between NdeI and XhoI, generating
pGEX:lacZ (NcoI) from pGEX:lacZ. pGEX:lacZ-TTC was obtained by
insertion of the TTC NcoI/XhoI fragment into pGEX:lacZ (NcoI),
fusing TTC immediately downstream of the lacZ coding region and in
the same reading frame. FIG. 3 shows the details of the
pGEX:lacZ-TTC construct.
[0083] (C) pGEX:TTC-lacZ:
[0084] pBS:TTC was modified to change NcoI into a BamHI restriction
site (linker 5'-CAT GAC TGG GGA TCC CCA GT-3' (SEQ ID NO:12)) at
the start of the TTC DNA, to give pBS:TTC (BamH/) plasmid. pGEX:TTC
was obtained by Boning The TTC BamHI/SmaI fragment from pBS:TTC
(BamH/) into pGEX 4T-2 (Pharmacia). PCR was used to convert the TTC
stop codon into an NheI restriction site. Two primers (upstream:
5'-TAT GAT AAA AAT GCA TCT TTA GGA-3' (SEQ ID NO:13) and
downstream: 5'-TGG AGT CGA CGC TAG CAG GAT CAT TTG TCC ATC CTT C-3'
(SEQ ID NO:14)) were used to amplify the sequence between NsiI and
SmaI, generating pGEX:TTC (NheI) from pGEX:TTC. The lacZ cDNA from
plasmid pGNA was modified in its 5' extremity to change Sac into an
NheI restriction site (linker 5'-GCT AGC GC-3' (SEQ ID NO:15)).
pGEX:TTC-lacZ was obtained by insertion of the lacZ NheI/XhoI
fragment into pGEX:TTC (NheI), fusing lacZ immediately downstream
of the TTC coding region and in the same reading frame. The details
of the construct of pGEX:TTC-lacZ are shown in FIG. 4.
[0085] (D) pCMV:lacZ-TTC:
[0086] pCMV vector was obtained from pGFP-C1 (Clontech
laboratories) after some modifications: GFP sequence was deleted by
a BglII/NheI digestion and relegation, and SacII in the polylinker
was converted into an AscI restriction site (linkers 5'-GAT ATC GGC
GCG CCA GC-3' (SEQ ID NO:16) and 5'-TGG CGC GCC GAT ATC GC-3' (SEQ
ID NO:17)).
[0087] pBluescript KS+ (Stratagene) was modified to change XhoI
into an AscI restriction site (linker 5'-TCG ATG GCG CGC CA-3' (SEQ
ID NO:18)), giving pBS (AscI) plasmid. pBS:lacZ-TTC was obtained by
cloning a XmaI lacZ-TTC fragment from pGEX lacZ-TTC into pBS
(AscI). pCMV:lacZ-TTC was obtained by insertion of the lacZ-TTC
XmM/AscI fragment into pCMV vector at the XhoI and AscI sites (XhoI
and XmnI was eliminated with the clonage), putting the fusion
downstream of the CMV premotor. FIG. 8 shows the details of the
construct pCMV:lacZ-TTC. Plasmid pCMV:lacZ-TTC was deposited on
Aug. 12, 1997, at the Collection Nationale de Cultures de
Microorganisms (CNCM), Institut Pasteur, 25, Rue de Docteur Roux,
F-75724, Paris Cedex 15, France, under Accession No. 1-1912.
Example 2
Purification of the Hybrid Protein
[0088] The E. coli strain SR3315 (a gift from Dr. A. Pugsley,
Institut Pasteur) transfected with pGEX:lacz-TTC was used for
protein production. An overnight bacterial culture was diluted
1:100 in LB medium containing 100 .mu.g/ml ampicillin, and grown
for several hours at 32.degree. C. until an OD of 0.5 was reached.
Induction from the Ptac promoter was achieved by the addition of 1
mM IPTG and 1 mM MgCl.sub.2 and a further 2 hrs incubation. The
induced bacteria were pelleted by centrifugation for 20 min at 3000
rpm, washed with PBS and resuspended in lysis buffer containing 0.1
M Tris pH 7.8, 0.1M NaCl, 20% glycerol, 10 mM EDTA, 0.1%
Triton-X100, 4 mM DTT, 1 mg/ml lysosyme, and a mixture or
anti-proteases (100 .mu.g/ml Pefablok, 1 .mu.g/ml leupeptin, 1
.mu.g/ml pepstatin, 1 mM benzamidine). After cell disruption in a
French Press, total bacterial lysate was centrifuged for 10 min at
30000 rpm. The resulting, supernatant was incubated overnight at
4.degree. C. with the affinity matrix Glutathione Sepharose 4B
(Stratagene) with slow agitation. After centrifugation for 5 min at
3000 rpm, the matrix was washed three times with the same lysis
buffer but without lysosyme and glycerol, and then three times with
PBS. The resin was incubated overnight at 4.degree. C. with
Thrombin (10 U/ml; Sigma) in PBS in order to cleave the
.beta.-gal-TTC fusion protein from the Glutatione-S-transferase
(GST) sequence and thereby elute it from the affinity column.
Concentration of the eluted fusion protein was achieved by
centrifugation in centricon X-100 tubes (Amicon; 100,000 MW cutoff
membrane).
[0089] Purified hybrid protein was analyzed by Western blotting
after electrophoretic separation in 8% acrylamide SDS/PAGE under
reducing conditions followed by electrophoretic transfer onto
nitrocellulose membranes (0.2 mm porosity, BioRad). Immunodetection
of blotted proteins was performed with a Vectastain ABC-alkaline
phosphatase kit (Vector Laboratories) and DAB color development.
Antibodies were used as follows: rabbit anti-.beta.-gal antisera
(Capel), dilution 1:1000; rabbit anti-TTC antisera (Calbiochem),
dilution 1:20000. A major band with a relative molecular mass of
180 kDa corresponding to the .beta.-Gal-TTC hybrid protein was
detected with both anti-.beta.-Gal anti-TTC antibodies.
Example 3
Binding and Internalization of Recombinant Protein in
Differentiated 1009 Cells
[0090] The 1009 cell line was derived from a spontaneous testicular
teratocarcinoma arising in a recombinant inbred mouse strain
(129.times.B6) (17). The 1009 cells were grown in Dulbecco's
modified Eagle's medium (DMEM) containing 10% fetal calf serum and
passaged at subconfluence. In vitro differentiation with retinoic
acid and cAMP was performed as described (18). Eight days after
retinoic acid treatment cells were used for the internalization
experiments with either the hybrid protein or .beta.-gal.
[0091] Binding and internalization of the p-Gal-TTC fusion were
assessed using a modified protocol (16). Differentiated 1009 cells
were incubated for 2 hrs at 37.degree. C. with 5 .mu.g/ml of
.beta.-Gal-TTC or O-Gal protein diluted in binding buffer (0.25%
sucrose, 20 mM Tris acetate 1 mM CaCl2, 1 mM MgCl.sub.2, 0.25%
bovine serum albumin, in PBS). The cells were then incubated with 1
.mu.g/ml Pronase E (Sigma) in PBS for 10 min at 37.degree. C.,
followed by washing with proteases inhibitors diluted in PBS (100
.mu.g/ml Pefablok, 1 mM benzamidine).
[0092] The cells were faced with 4% formalin in PBS for 10 min at
room temperature (RT) and then washed extensively with PBS.
.beta.-gal activity was detected on fixed cells by an overnight
staining at 37.degree. C. in X-Gal solution (0.8 mg/ml X-Gal, 4 mM
potassium ferricyanide, 4 mM potassium ferrocyanide, 4 mM
MgCl.sub.2 in PBS). For electron microscopy, the cells were further
fixed in 2.5% glutaraldehyde for 18 hrs, and then processed as
described (19).
[0093] For immunohistochemical labeling, cells were fixed with 4%
paraformaldehyde in PBS for 10 min at RT then washed extensively
with PBS, followed by a 1 hr incubation at RT with 2% BSA/0.02%
Triton X-100 in PBS. Cells were co-incubated in primary antibodies
diluted in 2% BSA/0.02% Triton X-100 in PBS for 2 hrs at RT.
Antibodies used were a mouse anti-neurofilament antibody (NF 200
Kd, dilution 1:50; Sigma) or the rabbit anti-TTC antibody
(dilution, 1:1000). The labeling was visualized using fluorescent
secondary antibodies: Cy3, goat anti-rabbit IgG (dilution 1:500;
Amersham) or anti-mouse IgG with extravidin-FITC (dilution 1:200;
Sigma). Cells were mounted in moviol and visualized with
epifluorescence.
Example 4
In Vivo Recombinant Protein Injection
[0094] 14-week old B6D2F1 mice were obtained from IFFA-CREDO. The
animal's tongue muscle was injected using an Hamilton syringe (20
.mu.l per animal) while under general anesthesia with 3% Avertin
(15 .mu.l/g of animal). The protein concentration was 0.5 to 5
.mu.g/.mu.l in PBS; therefore, mice received approximately 10 to
100 .mu.g per injection. Animals were kept alive for 12 hrs to 48
hrs post-injection to permit migration of the injected protein, and
in no case were any tetanus symptoms detected. The mice were
sacrificed by intracardiac perfusion with 4% paraformaldehyde in
PBS while under deep anesthesia. Brains were harvested, rinsed in
PBS and incubated in 15% sucrose overnight at 4.degree. C., then
mounted in tissue-tek before sectioning, 15 .mu.m thick slices
using a cryostat.
Example 5
Histology, Immunohistology, and X-Gal Staining
[0095] For in toto X-Gal staining of the dissected brain and
tongue, mice (10 animals) were sacrificed and fixed as described
above. The brain was further cut with a scalpel along a median
plane and directly incubated for 12 hrs in X-Gal solution.
[0096] For immunohistology, sections were incubated in a 1:5000
dilution of anti-TTC antibody in 2% BSA/0.02% Triton X-100 in PBS
overnight at 4.degree. C. after nonspecific antibody binding sites
were blocked by a 1 hr incubation in the same buffer. Antibody
detection was carried out using the Vectastain ABC-alkaline
phosphatase kit with DAB color development. For X-Gal staining,
sections were incubated in X-Gal solution and counterstained for 30
sec with hematoxylin 115 (v/v) in PBS. Histology on adjacent
sections was done after X-Gal staining, using a 30 sec incubation
in hematoxylin/thionin solution. All sections were mounted in
moviol before eight microscopy analysis.
Example 6A
Internalization of the .beta.-Gal-TTC Fusion Protein by Neurons In
Vitro
[0097] Differentiation of 1009 cells with retinoic acid and cAMP in
vitro yields neuronal and glial cells (18, 20). X-Gal staining or
immunolabeling were performed after incubation with the
.beta.-gal-TTC fusion protein or with either the .beta.-gal or TTC
proteins alone. Only when the hybrid protein was incubated with
differentiated 1009 cells was a strong X-Gal staining detected in
cells having a neuronal phenotype. No signal was detected when
.beta.-gal alone was incubated under the same conditions. A similar
X-Gal staining pattern was obtained after pronase treatment of the
cells to remove surface bound proteins, indicating that the hybrid
protein had been internalized. The intracellular localization of
the hybrid protein was further. confirmed by electron microscopic
analysis of X-Gal-stained cells. Furthermore, the enzymatic
activity observed in axons seemed to be localized in vesicles
associated with filaments, which is in agreement with previous work
on TTC fragment or native tetanus toxin (14, 21, 22). Co-labeling
with anti-TTC and anti-neurofilament antibodies revealed that
.beta.-gal activity co-localized with TTC fragment in neuronal
cells. No glial cells were labeled with either antibody.
Example 6B
Internalization of the TTC-.beta.-Gal Fusion Protein by Neurons In
Vitro
[0098] The method used for the internalization was identical to
that described in Example 6 above. The results show efficiently
internalization of the hybrid as in Example 6 above.
Example 7
Retrograde Transport of the Hybrid Protein In Vivo
[0099] To study the behavior of the .beta.-gal-TTC protein in vivo,
the hybrid protein was tested in a well characterized neuronal
network, the hypoglossal system. After intramuscular injection of
.beta.-gal-TTC protein into the mouse tongue, the distribution of
the hybrid protein in the CNS was analyzed by X-Gal staining.
Various dilutions of the protein were injected and sequential time
points were analyzed to permit protein transport into hypoglossal
motoneurons (XII), and its further transneuronal migration into
connected second order neurons.
[0100] A well-defined profile of large, apparently retrogradely
labeled neurons was clearly evident in the hypoglossal structure,
analyzed in toto at 12 hrs post-injection. A strong labeling was
also apparent in the hypoglossal nerve (XIIn) of the tongue of the
injected mice. At the level of muscle fibers, button structures
were observed that might reflect labeling of neuromuscular
junctions where the hybrid protein was internalized into nerve
axons. These data demonstrate that the .beta.-gal-TTC hybrid
protein can migrate rapidly by retrograde axonal transport as far
as motoneuron cell bodies, after prior uptake by nerve terminals in
the tongue. This specific uptake and the intraaxonal transport are
similar to the properties that have been described for the native
toxin (6, 21, 23).
[0101] Transport of the hybrid protein was examined in greater
detail by analyzing X-Gal-stained brain sections. Motoneurons of
the hypoglosial nucleus became labeled rapidly, with 12 hrs being
the earliest time point examined. Most of the label was confined to
neuronal somata, the cell nuclei being unlabeled. The intensity of
the labeling depends upon the concentration of the .beta.-gal-TTC
protein injected: when 10 .mu.g of protein was injected, only the
hypoglossal somata were detected, whereas with 25 to 50 .mu.g a
fuzzy network of dendrites was visualized; transynaptic transfer
was detected with 100 .mu.g of hybrid protein. An identical
distribution of label was observed then brain sections were
immunostained with an anti-TTC antibody, demonstrating that
.beta.-gal and TTC fragment co-localize within cells. Finally,
injection of .beta.-gal alone did not result in labeling of the
hypoglossal nuclei and therefore confirms that transport of the
hybrid protein is TTC-dependent. Labeling with an anti-TTC antibody
was less informative than detection of .beta.-gal activity; for
instance, the nerve pathway to the brain could not be visualized by
anti-TTC immunostaining. At 18 hrs post-injection, labeling was
observed in the hypoglossal nuclei: all motoneuron cell bodies and
the most proximal part of their dendrites were very densely
stained. In contrast, no labeling was ever detected in glial cells
adjoining XII motoneurons or their axons. Our results are in
accordance with others who reported an identical pattern of
immunolabeling after injection of the TTC fragment alone (9).
Transneuronal transfer is detectable after 24 hrs. An additional 24
hrs and beyond did not yield a different staining.
Example 8
Transneuronal Transport of the Hybrid Protein
[0102] Second order interneurons, as well as higher order neurons
that synapse with the hypoglossal motoneurons, have been
extensively analyzed using conventional markers, such as the wheat
germ agglutinin-horseradish peroxidase complex (WGA-HRP) or
neurotropic viruses such as alpha-herpes (24) and rhabdoviruses
(25). An exhaustive compilation of regions in the brain that
synaptically connect to the hypoglossal nucleus has also been
described recently (25). In this invention, the distribution of the
.beta.-gal-TTC fusion depended on the initial concentration of
protein injected into the muscle and the time allowed for transport
after injection. Up to 24 hrs post-injection, labeling was
restricted to the hypoglossal nuclei. After 24 hrs, the
distribution of second order transneuronally labeled cells in
various regions of the brain was consistent and reproducible. Even
at longer time points (e.g. 48 hrs), labeling of the hypoglossal
nucleus remained constant. At higher magnification, a discrete and
localized staining of second-order neurons was observed, suggesting
that the hybrid protein had been targeted to vesicles within cell
somata, synapses and axons. A similar patchy distribution was
previously described for tetanus toxin and TTC fragment alone (14,
21, 22).
[0103] Intense transneuronal labeling was detected in the lateral
reticular formation (LRF), where medullary reticular neurons have
been reported to form numerous projections onto the hypoglossal
nucleus (26, 27). .beta.-gal activity was detected bilaterally in
these sections. Label led LRF projections formed a continuous
column along the rostrocaudal axis, beginning lateral to the
hypoglossal nucleus, with a few neurons being preferentially
stained in the medullary reticular dorsal (MdD) and the medullary
reticular ventral (MdV) nuclei. This column extends rostrally
through the medulla, with neurons more intensely labeled in the
parvicellular reticular nucleus (PCRt, caudal and rostral). After
48 hrs, cells in MdD and PCRt were more intensely stained. A second
bilateral distribution of medullary neurons projecting to the
hypoglossal nucleus was detected in the solitary nucleus (Sol) but
the labeling was less intense than in the reticular formation,
presumably because relatively few cells of the solitary nucleus
project onto the hypoglossal nucleus (26). However, no labeling was
found in the spinal trigeminal nucleus (Sp5), which has also been
shown to project onto the hypoglossal nucleus (26). Transynaptic
transport of the .beta.-gal-TTC protein was also detected in the
pontine reticular nucleus caudal (PnC), the locus coeruleus (LC),
the medial vestibular nucleus (MVe) and in a few cells of the
inferior vestibular nucleus (IV). These cell groups are known to
project onto the hypoglossal nucleus (25), but their labeling was
weak, probably because of the greater length of their axons. A few
labeled cells were observed in the dorsal paragigantocellular
nucleus. (DPGI), the magnocellular nucleus caudal (RMc), and the
caudal raphe nucleus (R); their connections to the hypoglossal
nucleus have also been reported (25). Finally, labeled neurons were
detected bilaterally in midbrain projections, such as those of the
mesencephalic trigeminal nucleus (Me5), and a few neurons were
stained in the mesencephalic central gray region (CG). These latter
nuclei have been typed as putative third order cell groups related
to the hypoglossal nucleus (25).
[0104] Neurons in the motor trigeminal nucleus (Mo5) and the
accessory trigeminal tract (Acs5) were also labeled, along with a
population of neurons in the facial nucleus (N7). However,
interpretation of this labeling is more ambiguous, since it is
known that motoneurons in these nuclei also innervate other parts
of the muscular tissue, and diffusion of the hybrid protein might
have occurred at the point of injection. Conversely, these nuclei
may have also projected to the tongue musculature via nerve XII,
since neurons in N7 have been reported to receive direct
hypoglossal nerve input (28). This latter explanation is consistent
with the fact that labeling in these nuclei was detected only after
24 hrs; however, this point was not further investigated.
[0105] Together, the data summarized in Table 1 clearly establish
transneuronal transport of the .beta.-gal-TTC fusion protein from
the hypoglossal neurons into several connected regions of the
brainstem.
TABLE-US-00001 TABLE 1 Transneuronal transport of the lacZ-TTC
fusion from the XII nerve: labeling of different cells types in the
central nervous system. Cell groups 12-18 hrs 24-48 hrs First order
neurons First category: XII, hypoglossal motoneurons ++ +++ Second
category: N7, facial nu - ++ Mo5, motor trigeminal nu - ++ Acs5,
accessory trigeminal nu - + Second order cell groups MdD, medullary
reticular nu, dorsal - ++ MdV, medullary reticular nu, ventral -
+/- PCRt, parvicellular reticular nu, caudal - ++ PCRt,
parvicellular reticular nu, rostral - ++ Sol, solitary tract nu - +
DPGi, dorsal paragigantocellular nu - +/- PnC, pontine reticular
nu, caudal - + RMc, magnocellular reticular nu - +/- R, caudal
raphe nu - +/- MVe, medial vestibular nu - + IV, inferior
vestibular nu - +/- LC, locus coeruleus - + Me5, mesencephalic
trigeminal nu (*) - + CG, mesenphalic central gray (*) - +/- (*)
Represents second order cell groups that also contain putative
third order neurons (see text). -, no labeling; + to +++, increased
density of label; +/- weak labeling. 16 animals were analysed for
the 12-18 hrs p.i. data; 6 animals were analysed for the 24-48 hrs
p.i. data.
[0106] In another embodiment of the invention, we have constructed
a fusion protein (GFP-TTC) comprising the C-terminal fragment of
tetanus toxin and the GFP reporter gene, and have demonstrated its
effectiveness to map a simple neural network retrogradely and
transsynaptically in transgenic mice. (Maskos et al., 2002). The
GFP-TTC fusion protein permits the visualization of membrane
traffic at the presynaptic level of the neuromuscular junction and
can be detected optically without immunological or enzymatic
reactions. The GFP-TTC fusion protein, therefore, permits
observation of active neurons with minimal disturbance of their
physiological activities.
[0107] We have also previously shown that, without neural activity,
localization of a TTC fusion protein at the NMJ is impaired
(Miana-Mena et al., 2002). In this aspect of the invention,
therefore, we investigated in vivo, the influence of neurotrophic
factors on neuronal localization and internalization of GFP-TTC and
the mechanisms by which certain neurotrophic factors influence
neuronal trafficking in vivo. We found that localization of GFP-TTC
at the NMJ is rapidly induced by neurotrophic factors such as Brain
Derivated Neurotrophic Factor (BDNF), Neurotrophin 4 (NT-4), and
Glial-Derived Neurotrophic Factor (GDNF) but not by Nerve Growth
Factor (NGF), Neurotrophin 3 (NT-3), and Ciliary Neurotrophic
Factor (CNTF).
[0108] Co-injection of various amounts of BDNF with the GFP-TTC
probe induces an increase of the fluorescence measured at the
neuromuscular junction (NMJ). This effect, which is detectable as
early as 5 min after injection and reaches a maximum level at about
30 min after injection, indicates that BDNF treatment enhances
neuronal endocytosis. Among other functions, BDNF stimulates the
secretion of neurotransmitter from Xenopus nerve muscle co-cultures
and from hippocampal neurons (Lohof et al., 1993; Tyler and
Poco-Miller, 2001). Since tetanus toxin is known to enter neurons
by means of synaptic vesicle endocytosis (Matteoli et al., 1996),
BDNF might increase GFP-TTC internalization through enhancement of
synaptic vesicle turnover. In our study, BDNF effects persisted
after BoTx/A treatment, which blocks exocytosis and endocytosis of
synaptic vesicles, showing that BDNF increases the kinetics and
localization of a TTC-containing fusion protein at the NMJ through
another endocytic pathway. Therefore, intramuscular injection of
GFP-TTC and visualization of transport mechanisms revealed at least
two different endocytic pathways: a dathrin-dependent and a
clathrin-independent pathways. We found that after intramuscular
injection of GFP-TTC, it displayed characteristics consistent with
localization in lipid rafts, including biochemical colocalization
with caveolin 3 and colocalization with GM1, a raft marker
identified by CT-b binding (Orlandi and Fishman, 1998; Wolf et al.,
1998). Accordingly, the clathrin-independent pathway used by
GFP-TTC, appears to involve lipid microdomains. Analysis by
confocal microscopy revealed morphologically two different
labelings. Firstly, a GFP-TTC diffuse staining, which partially
overlaps with the synaptic vesicle SV2 but also with the raft
marker CT-b, indicating a mixing of synaptic vesicles and lipid
rafts. Secondly, highly fluorescent domains, which are detected
before and persist after the more diffuse pattern and that appear
to be invaginations or infoldings of the synaptic membrane. These
GFP-TTC patches contained only CT-b labeling. Indeed, lipid
microdomains which play a role in cellular functions such as
vesicular trafficking and signal transduction (Simons and Toomre,
2000), can move laterally and cluster into larger patches (Harder
et al., 1998). They might also be specific zones of exocytosis in
the presynaptic compartment, undergoing a rapid form of internal
traffic in response to retrograde signaling from target cells.
Similar infolding and cistemae structures have been described in
frog motor nerve terminals which replenish the pool of synaptic
vesicles in a manner dependent upon neuronal activity (Richards et
al., 2000). In CHO cells, tubular caveolae have also been described
(Mundy et al., 2002).
[0109] Based on the kinetics of probes for NMJ localization, we
observed different trafficking behaviours for GFP-TTC and CT-b. It
has been postulated that targeting of toxin into the cell depends
on the structure and function of endogenous ganglioside receptors,
which could couple toxins to specific lipid raft microdomains (Wolf
et al., 1998). Thus, in vivo, endogenous or injected BDNF might
increase the amount of lipid microdomains containing TTC receptors.
Tetanus toxin and cholera toxin bind to different gangliosides,
known as GD1b/GT1b and GM1, respectively. Hence, the difference we
observed in the dynamics of recruitment at the presynaptic motor
nerve terminal may be relevant to different lipid microdomains
having specific glycosphingolipids and protein composition.
Neuronal membranes are rich in gangliosides and different
microdomains are likely to co-exist on the cell surface. Indeed,
Thy-1 and PrP prion protein, two functionally different GPI
proteins, are found in adjacent microdomains (Madore et al., 1999).
Similarly, syntaxins are concentrated in cholesterol-dependent
microdomains, which are distinct from rafts containing GPI-linked
proteins (Lang et al., 2001).
[0110] Like BDNF, NT-4 was also found to increase the concentration
of GFP-TTC at the NMJ, whereas NGF and NT-3 had no effect. Since
the TrkB receptor is specifically activated by BDNF and NT-4, TrkB
activation might be involved in this neoronal trafficking.
Interestingly, high-frequency neuronal activity and synaptic
transmission have been shown to elevate the number of TrkB
receptors on the surface of cultured hippocampal neurons (Du et
al., 2000), apparently by recruiting extra TrkB receptors to the
plasma membrane (Meyer-Franke et al., 1998). Moreover, TrkB is
highly enriched in lipid microdomains from neuronal plasma membrane
(Wu et al., 1997). However, no specific colocalization between
GFP-TTC and TrkB or p-Trk receptors were detected at the NMJ. Thus,
TrkB may act indirectly on the detected traffic at the presynaptic
motor nerve membrane.
[0111] It is worth noting that the TTC fragment has been detected
in cultured motoneurons in the same vesicles as p75.sup.NTR (Lalli
and Schiavo, 2002). This colocalization may be explained by the
tight association of p75, which is expressed mainly during
development and in pathological conditions, with GT1b ganglioside
(Yamashita et al., 2002). Binding of neurotrophins to their Trk
receptors leads to phosphorylation of tyrosine residues that are
recognized by several intracellular signaling proteins. Such
interactions lead to the activation, by means of a kinase cascade,
of the MAP kinase, PI 3-kinase and phospholipase-C-.gamma. pathways
(for review see (Huang and Reichardt, 2003)). Many of the
intermediates in these signaling cascades are also present in lipid
rafts (Simons and Toomre, 2000; Tsui-Pierchala et al., 2002).
Activation of PKA is required for translocation of activated
p75.sup.NTR to lipid rafts (Higuchi et al., 2003). Similarly, the
coreceptor GFR.alpha.1, which binds GDNF and thus allows activation
of the c-RET tyrosine kinase receptor; localize to lipid rafts.
GFR.alpha.1 recruits RET to lipid rafts after GDNF stimulation and
results in strong and continuous signal transduction (Paratcha et
al., 2001; Tansey et al., 2000).
[0112] Another neurotrophic factor, GDNF, also induced GFP-TTC
localization at the NMJ. GDNF, however, activates a different
receptor (i.e., a GFR.alpha./cRET receptor) than BDNF and NT-4.
Because BDNF/NT-4 and GDNF activate different receptors, we
postulated that component(s) of their activation pathways may
activate the trafficking of GFP-TTC receptors in specific lipid
microdomains. Indeed, various stimuli can lead to internalization
of caveolae, a specialized form of lipid rafts. Thus, simian virus
40 stimulates its internalization in caveolae and transport via
caveosomes (Pelkmans et al., 2001). Similarly, the albumin-docking
protein pg60 activates its transendothelial transport by
interaction with caveolin-1 and subsequent activation of Src kinase
signaling (Minshall et al., 2000). Recently, it has been reported
that tetanus toxin can activate, through the TTC fragment,
intracellular pathways involving Trk receptors, extracellular
signal-regulated kinases (ERK) and protein kinase C isoforms (Gil
et al., 2001; Gil et al., 2000; Gil et al., 2003). In this way,
tetanus toxin could therefore autoactivate its neuronal endocytosis
via an uncoated pathway rather than by clathrin-dependent pathway
to avoid the lysosomal degradation.
[0113] Finally, we have demonstrated that GFP-TTC trafficking is
regulated by neurotrophic factors. By visualization of GFP-TTC
trafficking, our data show that BDNF can stimulate both
clathrin-coated and uncoated endocytic pathways, presumably via
TrkB activation. Since tetanus toxin, as other pathogens or toxins,
uses constitutive mechanisms for its internalization and traffic in
cells we have been able to visualize with GFP-TTC, a physiological
response to neurotrophic factors.
[0114] This aspect of the invention is further discussed in the
following examples.
Example 9
GFP-TTC Localization at the NMJ
[0115] To determine the characteristics of the GFP-TTC distribution
at the NMJ, a single injection of the GFP-TTC fusion protein was
performed in the immediate vicinity of the Levator auris longus
(LAL) muscle and at various times after the injection, the LAL was
removed and examined as a whole mount. As LAL is a thin and flat
muscle consisting of only a few layers of fibers, the entirety of
the neuromuscular preparation with associated nerves could be
examined by confocal analysis (FIG. 6A). As shown in FIG. 6,
GFP-TTC rapidly concentrates at the NMJ, as identified by the
staining of muscle nicotinic acetylcholine receptors with
TRITC-conjugated .alpha.-bungarotoxine (.alpha.-BTX). A patchy
clustering of GFP-TTC was observed after approximately 5 min
following the deposit of the fusion protein onto the surface of the
LAL muscle (FIGS. 6D and D'). After 30 min, a more diffuse staining
was observed that was distributed over the entire surface of the
NMJ (FIGS. 6E and E'), and which persisted for about 2 h (FIGS. 6F
and F'). Immunostaining experiments, performed with an antibody
that recognizes troponin T confirmed that GFP-TTC is concentrated
mainly in presynaptic motor nerve terminals of the NMJ (FIGS. 6C
and C'). We could also detect a strong GFP-TTC labeling at the
nodes of Ranvier of intramuscular myelinated axons and in sensory
nerve fibers (FIGS. 6A and B; arrows and arrowheads respectively).
It is likely that most of the GFP-TTC probe was internalized within
24 h, since only a few fluorescent patches persisted at the NMJ 24
h after its injection (FIGS. 6G and G').
Example 10
Influence of BDNF on GFP-TTC Trafficking in Motor Nerve
Terminals
[0116] To assess whether exogenously applied neurotrophins affected
GFP-TTC recruitment in motor nerve terminals, increasing
concentrations of BDNF (2.5-250 ng) were co-injected with GFP-TTC
in the vicinity of LAL muscles, while control mice were injected
with GFP-TTC alone. Mice were sacrificed and LAL muscles harvested
30 min after injection. GFP fluorescence was quantified by confocal
microscopy analysis at NMJs, after identification by
TRITC-.alpha.-BTX labeling. BDNF injection produced a statistically
significant concentration-dependent enhancement of GFP-TTC
fluorescence at the NMJ, with the highest effect obtained with 50
ng BDNF (FIG. 7B and Table 2) while higher doses (100 and 250 ng)
resulted in weaker elevations in the level of GFP-TTC concentration
at the NMJ (1.72.+-.0.12 and 1.15.+-.0.22 fold respectively). The
higher GFP-TTC axonal labeling observed at these higher doses (FIG.
7A, arrows), probably correlates to an enhanced internalization of
the probe.
[0117] In TrkB mutant mice, a physiological phenotype in the facial
nerve nucleus, which innervates LAL muscle has been reported (Klein
et al., 1993; Silas-Santiago et al., 1997). To exclude the
possibility that the BDNF effect observed could be LAL specific, a
different muscle, the gastrocnemius, was also analyzed. Thirty
minutes after injecting GFP-TTC (.+-.BDNF 50 ng) in gastrocnemius,
muscles were fixed, removed and serially sectioned. For each
muscle, different serial sections were quantified for GFP-TTC
fluorescence at the motor, nerve terminals as described in material
and methods. We found that the BDNF-dependent increase of GFP-TTC
concentration at the NMJ, closely resembled that observed in LAL
(1.51.+-.0.12 fold increase vs 2.12.+-.0.19 respectively).
Example 11
Influence of Other Neurotrophic Factors on GFP-TTC Localization at
Motor Nerve Terminals
[0118] We also examined the effect of five additional trophic
factors on GFP-TTC localization at the NMJ, including the
neurotrophins NT-3; NT-4 and NGF as well as the neurocytokine CNTF
(Ciliary Neurotrophic Factor), a member of the LIF cytokine family,
and GDNF (Glial-Derivated Neurotrophic Factor), a member of the
TGF-.beta. superfamily (Table 2). Many BDNF actions in neurons are
mediated via the high affinity receptor tyrosine kinase TrkB, which
is also the receptor for NT-4. Like BDNF, NT-4 also induced GFP-TTC
localization at the NMJ (a 1.54.+-.023 fold increase). A level of
induction similar to NT-4 was also observed for GDNF (Table 2). On
the other hand, even at high concentrations, neither NGF, NT-3, nor
CNTF exhibited a significant effect on GFP-TTC localization.
TABLE-US-00002 TABLE 2 Effect of various neurotrophic factors on
nerve terminal's GFP-fluorescence level 30 min after in vivo
GFP-TTC injection. Relative increase in Receptor fluorescence level
BDNF TrkB 2.12 .+-. 0.19** NT-4 TrkB 1.49 .+-. 0.23** NT-3 TrkC
0.94 .+-. 0.05 NGF TrkA 1.06 .+-. 0.06 CNTF CNTFR.alpha. 0.95 .+-.
0.05 GDNF GFR.alpha./cRET 1.51 .+-. 0.02* GFP-TTC was co-injected
with increasing concentrations of neurotrophic factors and GFP
fluorescence quantified 30 min after as previously described. Mean
of relative increase of GFP fluorescence of 2 or 3 independent
experiments are indicated. Maximum fold induction was obtained for
50 ng of neurotrophic factor injected except for NT-3 (2.5 ng). **P
< 0.005; *P < 0.05 t-test vs control.
Example 12
Comparison of Trk Receptors Distribution and GFP-TTC Localization
at Motor Nerve Endings
[0119] Detection of either TrkB mRNA or protein in adult skeletal
muscle and motoneurons has been reported in several studies
(Funakoshi et al., 1993; Gonzalez et al., 1999; Griesbeck et al.,
1995; Yan et al., 1997). Since our results indicated that the BDNF
effect on GFP-TTC localization is dependent on TrkB receptor
activation, it was of interest to determine whether GFP-TTC
colocalized with TrkB at the NMJ of LAL muscles. Consistent with
previous studies (Gonzalez et al., 1999; Sakuma et al., 2001), TrkB
immunostaining was confined to the NMJ (FIG. 8). In the presynaptic
side, TrkB staining was adjacent to, but not colocalized to the
dusters of GFP-TTC labeling. Similar results were also obtained
with an antibody that recognizes the activated Trk receptors
(p-Trk, data not shown). This observation suggests that the
mechanism whereby BDNF has an influence on the concentration of
GFP-TTC at the nerve terminals, does not involve a direct
interaction between TrkB and GFP-TTC or its receptors.
Example 13
Mechanisms Involved in BDNF effect on GFP-TTC Concentration at the
NMJ
[0120] Possible explanations for the BDNF-induced enrichment of
GFP-TTC at the NMJ could involve an elevated rate of localization
of the probe at the NMJ, and/or an increased neuronal endocytosis
of the probe. Confocal analysis performed 5, 15, 30, 60 and 120 min
after GFP-TTC injection (.+-.BDNF 50 ng) showed maximal labeling
intensity at 30 min with BDNF injection, whereas in controls, the
maximal staining occurred at 1 h and reached a level lower than
that obtained with BDNF co-injection. After the first hour, similar
levels of GFP-TTC were recorded at the NMJ in both conditions (FIG.
9A). These results are in accordance with previous results in
Xenopus nerve-muscle co-culture indicating a time-limiting effect
of BDNF (Lohof et al., 1993).
[0121] In vitro, tetanus neurotoxin internalization in neurons
appears to involve both coated and uncoated-vesicular pathways
(Herreros at al., 2001; Matteoli et al., 1996). Experiments
performed either in vitro on excised LAL muscles with the endocytic
fluid marker RH414 (data not shown), or immunostained against the
SV2 synaptic vesicle proteins (FIG. 9B) and synaptophysin (data not
shown) showed some overlapping with GFP-TTC labeling, indicating
that the endocytosis of GFP-TTC was in part via recycling of
neuronal synaptic vesicles. To differentiate between
clathrin-dependent and clathrin-independent endocytic pathways, we
used treatment with botulinum neurotoxin serotype A (BoTx/A), which
blocks neurotransmitter release and endocytosis in motor nerve
terminals (de Paiva at al., 1999). When BoTx/A was applied 48 hours
before GFP-TTC injection, the probe level at the NMJ was markedly
decreased by 50% (FIG. 9C), indicating that both dathrin-dependent
and independent pathways are used to a comparable degree.
[0122] Enhanced synaptic transmission produced by application of
exogenous BDNF; NT-3 or NT-4 involves a potentiation of
neurotransmitter release (Lohof at al., 1993; Stoop and Poo, 1996;
Wang and Poo, 1997). The increasing amount of GFP-TTC at the NMJ
induced by BDNF injection could therefore be due in part to an
elevated recycling of synaptic vesicles. To explore this
hypothesis, increased exocytosis and endocytosis of synaptic
vesicles were induced by GFP-TTC injection in a high-potassium
medium. Five minutes after injection, exposure to high K.sup.+
medium or BDNF induced a similar increase of GFP-TTC level at the
NMJ. However, after 30 min, the effect of high K.sup.+ was no
longer detectable, whereas maximal induction was reached with BNDF
at this time (FIG. 9D). Finally, even after neurotransmitter
release and synaptic vesicle recycling were blocked by BoTx/A, an
increased GFP-TTC signal was induced by BDNF treatment (FIG. 9C)
with an amplitude comparable to that recorded in the non-paralyzed
control NMJ (2.05 fold increase vs 2.12 respectively). Taken
together, these results indicate that BDNF enhances an alternative
endocytic pathway that appears to involve uncoated vesicles.
Example 14
Evidence for Association of GFP-TTC in Detergent-Insoluble Membrane
at the Neuromuscular Junction
[0123] Binding of TTC to plasma membranes involves association to
polysialogangliosides GD1b and GT1b, as well as a N-glycosylated 15
kDa protein. These three components partition preferentially in
membrane microdomains called rafts. In vitro, TTC has been shown to
associate with such microdomains in NGF-differentiated PC12 cells
and in cultured spinal cord neurons (Herreros et al., 2001; Vyas,
2001). To test in vivo whether GFP-TTC associated to lipid rafts,
gastrocnemius muscles were submitted to detergent extraction to
isolate lipid microdomains after GFP-TTC intramuscular injection.
Twelve fractions from the discontinuous sucrose gradient were
collected and analyzed for distribution of GFP-TTC.
[0124] Neurons do not contain caveolin or morphologically distinct
caveolae (Anderson, 1998), but significant fractions of cholesterol
and glycosphingolipids are found in detergent-insoluble complexes,
which are indistinguishable using the criteria of detergent
insolubility from those associated with caveolae (Schnitzer et al.,
1995). Thus, caveolin 3, a specific muscular caveolar marker (Tang
et al., 1996), was used to identify the detergent-resistant
fractions. Immunoblot analysis revealed that GFP-TTC co-migrated
with raft microdomains, which contain caveolin 3 (FIG. 10A).
[0125] To investigate whether the GFP-TTC patches observed in vivo
in motor nerve terminals correspond to lipid microdomains, we
performed co-staining with Alexa 594-conjugated cholera toxin-B
fragment (CT-b). CT-b specifically binds to ganglioside GM1, which
is enriched in cholesterol-rich membrane microdomains, and is
commonly used as a marker for membrane rafts and caveolae (Orlandi
and Fishman, 1998; Schnitzer et al., 1995; Wolf et al., 1998).
GFP-TTC and Alexa 594-conjugated CT-b fragment were co-injected
into the gastrocnemius and confocal analysis was performed 1; 3; 5;
9 and 24 h later, with the NMJ being identified by AlexaFluor
647-conjugated .alpha.-BTX (FIG. 10B). Although the GFP-TTC
labeling of motor nerve terminals was easily visualized in less
than the 5 min necessary to process the tissue (FIGS. 6D and D'),
CT-b was detectable at the NMJ only several hours after injection
(3-5 h). Thus, the dynamics of trafficking of CT-b and TTC
receptors to active synapse are clearly different. However, after 5
h, the distribution obtained for CT-b was similar to GFP-TTC
staining, as characterized by diffuse staining and patches having
an extensive overlap of the staining patterns obtained with
GFP-TTC, indicating a localization of the TTC probe in lipid
microdomains in motor nerve endings (FIG. 10B). Twenty four hours
after gastrocnemius injection, both toxins had been internalized
since only few patches, most of them positive for both toxins,
persisted at the NMJ (FIGS. 10B and C). At this time, GFP-TTC and
CT-b staining were detected in the same motoneuron cell bodies in
the ventral horn of the spinal cord, but in different vesicular
compartments (FIG. 11). Taken together, these results indicate that
GFP-TTC used different lipid microdomains for neuronal binding
and/or internalization pathways than CT-b.
MATERIALS AND METHODS
Antibodies and Reagents
[0126] Rabbit ant-GFP polyclonal antibodies was obtained from
Invitrogen (1:5000 dilution). Mouse monoclonal antibody against
caveolin 3 was from Transduction Laboratories (1:500). The
monoclonal anti-neurofilament 200 (clone NE14) and the rabbit
polyclonal antitroponin T were obtained from Sigma. AlexaFluor
594-conjugated Cholera toxin subunit B (CT-b); AlexaFluor
488-conjugated goat-antirabbit IgG, AlexaFluor 647-conjugated
.alpha.-bungarotoxin (.alpha.-BTX) and RH414 were obtained from
Molecular Probes. Cy3-conjugated goat anti-rabbit IgG and
Cy3-conjugated rat anti-mouse IgG were from Jackson Laboratories.
TRITC-conjugated .alpha.-bungarotoxin was obtained from Calbiochem.
The rabbit anti-TrkB (794) and the anti-p-Trk polyclonal antibody
were obtained from SantaCruz. The monoclonal antibody against
synaptic vesicle protein SV2, developed by K. Buckley, was obtained
from the Developmental Studies Hybridoma Bank developed under the
auspices of the NICHD and maintained by The University of Iowa,
Department of Biological Sciences, Iowa City. Monoclonal antibody
against synaptic vesicle synaptophysin protein was obtained from
Chemicon. The goat anti-rabbit and anti-mouse IgG antibodies
conjugated to horseradish peroxydase were obtained from Pierce as
well as the SuperSignal detection reagent. Recombinant neurotrophic
factors rat CNTF; human NT3; human NT-4, human BDNF, human GDNF and
purified mouse NGF 7S were purchased from Alomone labs.
Neurotrophic factors were prepared as stock solutions (10 .mu.g/ml)
and kept in aliquots at -80.degree. C.
[0127] In Vivo Intramuscular Injection.
[0128] Experiments were performed in accordance with French and
European Community guidelines for laboratory animal handling.
Six-week-old Swiss female mice were obtained from Charles River
Breeding Laboratories. Intramuscular injections of .beta.-gal-TTC,
GFP-TTC fusion proteins, produced as previously described (Coen et
al., 1997), or AlexaFluor 594-conjugated CT-b were intramuscular
injected into the gastrocnemius muscle or subcutaneously in the
immediate vicinity of the Levator auris longus (LAL) muscle on
anesthetized mice. For fluorescence quantification, 25 .mu.g of
GFP-TTC fusion protein were injected in PBS in 50 .mu.l final
volume. For immunodetection or biochemical extraction, 50 .mu.g of
GFP-TTC probe were used. When co-injections with neurotrophic
factors were performed, the volume injected was kept constant (50
.mu.l). For injection in high K.sup.+, a physiological solution
containing 60 mM KCl was co-injected with the probe.
[0129] Botulinum Type-A Toxin Injection
[0130] Clostridium botulinum type-A toxin (BoTx/A) was injected
subcutaneously as a single dose of 0.05 ml containing about 0.5
.mu.g of the purified neurotoxin in the vicinity of the LAL muscle
of female Swiss mice (body weight 24-27 g). 48 h after BoTx/A
treatment, a time sufficient for inducing muscle paralysis in the
LAL due to blockade of neurotransmitter release (de Paiva et al.,
1999), GFP-TTC (25 .mu.g) was injected associated or not with BDNF
(50 ng) in the vicinity of the LAL muscle. Mice were killed by
intracardial injection of PFA 4% 30 min after injection and LAL
muscle harvested and processed for confocal analysis.
[0131] In Vitro Analysis of GFP-TTC Localization and Confocal
Acquisition
[0132] LAL muscles with their associated nerves were isolated from
female Swiss-Webster mice (20-25 g), killed by dislocation of the
cervical vertebrae. LAL muscles were mounted in
Rhodorsii.sup.R-lined organ baths (2 ml volume) superfused with a
standard oxygenated physiological solution of the following
composition (mM): NaCl 154; KCl 5; CaCl.sub.2 2; MgCl.sub.2 1;
HEPES buffer 5 (pH=7.4) and glucose 11. Muscles were loaded for 45
min in the dark and at room temperature with both 25 .mu.g GFP-TTC
and 30 .mu.M of RH414, dissolved in standard solution or, for
synaptic vesicle recycling, in high K.sup.+ isotonic solution (with
60 mM KCl replacing 60 mM NaCl). Preparations were washed out of
the GFP-TTC and RH414 dye, and rinsed several times with dye-free
standard medium before being imaged with a Leica TCS SP2 confocal
laser scanning microscope system (Leica Microsystems, Germany)
mounted on a Leica DM-RXA2 upright microscope equipped with a
.times.40 water immersion lens (Leica, NA 0.8). The confocal system
was controlled through Leica-supplied software running on a Windows
NT workstation.
[0133] Preparation of Detergent-Resistant Membrane (DRMs) Fractions
and Western Blot.
[0134] Preparation of Detergent-Resistant Membrane Fractions is One
of the Most widely used methods for studying lipid rafts. Two hours
after GFP-TTC injection (50 .mu.g), mouse gastrocnemius muscle
tissue was harvested, minced with scissors and homogenized in 2 ml
of MES-buffered saline containing 1% (v/v) Triton X-100.
Homogenization was carried out with a Polytron tissue grinder.
After centrifugation at low speed for 5 min, supernatant was
adjusted to 40% sucrose. A 5-30% linear sucrose gradient was formed
above the homogenate and centrifuged at 39,000 rpm for 18 h in a
SW41 rotor. Then, 11-12 fractions of 1 ml were collected from the
top of the gradient and precipitated with 6.5% trichloroacetic acid
in the presence of 0.05% sodium deoxycholate and washed with 80%
cold acetone. Samples were analyzed by Western Blot after
separating on a 4-15% SDS-PAGE followed by Western Blot. Membranes
were probed first with polyclonal anti-GFP and monoclonal
anti-caveolin 3 antibodies, and then incubated with goat
anti-rabbit IgGs and goat anti-mouse IgGs antibodies conjugated
with horseradish peroxydase. The SuperSignal (Pierce) was used to
visualize the reaction
[0135] Quantification of GFP-TTC Fluorescence Intensity at the
NMJ.
[0136] After intracardiac perfusion and fixation, LAL muscles were
harvested, washed in PBS for 20 min, stained with TRITC-conjugated
a-bungarotoxin (TRITC-a-BTX) (2 .mu.g/ml) for 45 min at 37.degree.
C. in PBS and washed twice in PBS. Images were acquired on an
Axiovert 200M laser scanning confocal microscope (LSM-510 Zeiss;
version 3.2) through a .times.40/1.2 water-immersion objective
using LP560 and BP505-550 filters. The pinhole aperture was set to
1 airy unit, and images were digitized at 8-bit resolution into a
512.times.512 pixel array. To be able to compare the intensity of
GFP staining between different experiments, laser illumination,
photomultiplier gain in regard of linear response, and other
acquisition variables were standardized. To quantify GFP-TTC
localization at the NMJ, series of "look-through" projection (of
MIP: Maximum Intensity Projection) was generated. Images from each
NMJ were processed identically: NMJ surface area (in .mu.m.sup.2)
was determined by TRITC-a-BTX labeling and GFP fluorescence global
intensity (sum of each pixel intensity) was then measured only in
this predefined area. This value, divided by the NMJ area yielded
GFP fluorescence intensity per square micrometer, which thus
defined the fluorescence level expressed as arbitrary units. For
each condition, .about.15 to 20 synapses were quantified and
results were expressed as the mean.+-.SD. Statistical significance
was defined as p<0.05 using a two-tailed t test. Each experiment
was repeated at least two or three times.
[0137] Analysis of Spinal Cord.
[0138] 24 hours after .beta.-gal-TTC or GFP-TTC and CT-b injection
into the gastrocnemius muscle (50 .mu.g each), mice deeply
anesthetized were perfused intracardially with 4% PFA. The spinal
cord was harvested and embedded in Tissue Tek embedding media after
overnight incubation in 25% sucrose in PBS 0.1M. Longitudinal
cryostat sections (30 .mu.m thickness) were cut and mounted onto
coated slides.
[0139] X-gal Reaction.
[0140] X-gal reaction was performed as previously described (Coen
et al., 1997).
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Sequence CWU 1
1
1811600DNAClostridium tetaniCDS(88)..(1476) 1ggaaacagct atgaccatga
ttacgccaag ctcgaaatta accctcacta aagggaacaa 60aagctggagc tcggtacccg
ggccacc atg gtt ttt tca aca cca att cca ttt 114 Met Val Phe Ser Thr
Pro Ile Pro Phe 1 5tct tat tct aaa aat ctg gat tgt tgg gtt gat aat
gaa gaa gat ata 162Ser Tyr Ser Lys Asn Leu Asp Cys Trp Val Asp Asn
Glu Glu Asp Ile10 15 20 25gat gtt ata tta aaa aag agt aca att tta
aat tta gat att aat aat 210Asp Val Ile Leu Lys Lys Ser Thr Ile Leu
Asn Leu Asp Ile Asn Asn 30 35 40gat att ata tca gat ata tct ggg ttt
aat tca tct gta ata aca tat 258Asp Ile Ile Ser Asp Ile Ser Gly Phe
Asn Ser Ser Val Ile Thr Tyr 45 50 55cca gat gct caa ttg gtg ccc gga
ata aat ggc aaa gca ata cat tta 306Pro Asp Ala Gln Leu Val Pro Gly
Ile Asn Gly Lys Ala Ile His Leu 60 65 70gta aac aat gaa tct tct gaa
gtt ata gtg cat aaa gct atg gat att 354Val Asn Asn Glu Ser Ser Glu
Val Ile Val His Lys Ala Met Asp Ile 75 80 85gaa tat aat gat atg ttt
aat aat ttt acc gtt agc ttt tgg ttg agg 402Glu Tyr Asn Asp Met Phe
Asn Asn Phe Thr Val Ser Phe Trp Leu Arg90 95 100 105gtt cct aaa gta
tct gct agt cat tta gaa caa tat ggc aca aat gag 450Val Pro Lys Val
Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu 110 115 120tat tca
ata att agc tct atg aaa aaa cat agt cta tca ata gga tct 498Tyr Ser
Ile Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser 125 130
135ggt tgg agt gta tca ctt aaa ggt aat aac tta ata tgg act tta aaa
546Gly Trp Ser Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys
140 145 150gat tcc gcg gga gaa gtt aga caa ata act ttt agg gat tta
cct gat 594Asp Ser Ala Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu
Pro Asp 155 160 165aaa ttt aat gct tat tta gca aat aaa tgg gtt ttt
ata act att act 642Lys Phe Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe
Ile Thr Ile Thr170 175 180 185aat gat aga tta tct tct gct aat ttg
tat ata aat gga gta ctt atg 690Asn Asp Arg Leu Ser Ser Ala Asn Leu
Tyr Ile Asn Gly Val Leu Met 190 195 200gga agt gca gaa att act ggt
tta gga gct att aga gag gat aat aat 738Gly Ser Ala Glu Ile Thr Gly
Leu Gly Ala Ile Arg Glu Asp Asn Asn 205 210 215ata aca tta aaa cta
gat aga tgt aat aat aat aat caa tac gtt tct 786Ile Thr Leu Lys Leu
Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser 220 225 230att gat aaa
ttt agg ata ttt tgc aaa gca tta aat cca aaa gag att 834Ile Asp Lys
Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile 235 240 245gaa
aaa tta tac aca agt tat tta tct ata acc ttt tta aga gac ttc 882Glu
Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe250 255
260 265tgg gga aac cct tta cga tat gat aca gaa tat tat tta ata cca
gta 930Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro
Val 270 275 280gct tct agt tct aaa gat gtt caa ttg aaa aat ata aca
gat tat atg 978Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn Ile Thr
Asp Tyr Met 285 290 295tat ttg aca aat gcg cca tcg tat act aac gga
aaa ttg aat ata tat 1026Tyr Leu Thr Asn Ala Pro Ser Tyr Thr Asn Gly
Lys Leu Asn Ile Tyr 300 305 310tat aga agg tta tat aat gga cta aaa
ttt att ata aaa aga tat aca 1074Tyr Arg Arg Leu Tyr Asn Gly Leu Lys
Phe Ile Ile Lys Arg Tyr Thr 315 320 325cct aat aat gaa ata gat tct
ttt gtt aaa tca ggt gat ttt att aaa 1122Pro Asn Asn Glu Ile Asp Ser
Phe Val Lys Ser Gly Asp Phe Ile Lys330 335 340 345tta tat gta tca
tat aac aat aat gag cac att gta ggt tat ccg aaa 1170Leu Tyr Val Ser
Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys 350 355 360gat gga
aat gcc ttt aat aat ctt gat aga att cta aga gta ggt tat 1218Asp Gly
Asn Ala Phe Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr 365 370
375aat gcc cca ggt atc cct ctt tat aaa aaa atg gaa gca gta aaa ttg
1266Asn Ala Pro Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu
380 385 390cgt gat tta aaa acc tat tct gta caa ctt aaa tta tat gat
gat aaa 1314Arg Asp Leu Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp
Asp Lys 395 400 405aat gca tct tta gga cta gta ggt acc cat aat ggt
caa ata ggc aac 1362Asn Ala Ser Leu Gly Leu Val Gly Thr His Asn Gly
Gln Ile Gly Asn410 415 420 425gat cca aat agg gat ata tta att gca
agc aac tgg tac ttt aat cat 1410Asp Pro Asn Arg Asp Ile Leu Ile Ala
Ser Asn Trp Tyr Phe Asn His 430 435 440tta aaa gat aaa att tta gga
tgt gat tgg tac ttt gta cct aca gat 1458Leu Lys Asp Lys Ile Leu Gly
Cys Asp Trp Tyr Phe Val Pro Thr Asp 445 450 455gag gga tgg aca aat
gat taaacagatt gatatgttca tgacatatgc 1506Glu Gly Trp Thr Asn Asp
460ccgggatcct ctagagtcga cctcgagggg gggcccggta cccaattcgc
cctatagtga 1566gtcgtattac aattcactgg ccgtcgtttt acaa
16002463PRTClostridium tetani 2Met Val Phe Ser Thr Pro Ile Pro Phe
Ser Tyr Ser Lys Asn Leu Asp1 5 10 15Cys Trp Val Asp Asn Glu Glu Asp
Ile Asp Val Ile Leu Lys Lys Ser 20 25 30Thr Ile Leu Asn Leu Asp Ile
Asn Asn Asp Ile Ile Ser Asp Ile Ser 35 40 45Gly Phe Asn Ser Ser Val
Ile Thr Tyr Pro Asp Ala Gln Leu Val Pro 50 55 60Gly Ile Asn Gly Lys
Ala Ile His Leu Val Asn Asn Glu Ser Ser Glu65 70 75 80Val Ile Val
His Lys Ala Met Asp Ile Glu Tyr Asn Asp Met Phe Asn 85 90 95Asn Phe
Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser 100 105
110His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile Ser Ser Met
115 120 125Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val Ser
Leu Lys 130 135 140Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala
Gly Glu Val Arg145 150 155 160Gln Ile Thr Phe Arg Asp Leu Pro Asp
Lys Phe Asn Ala Tyr Leu Ala 165 170 175Asn Lys Trp Val Phe Ile Thr
Ile Thr Asn Asp Arg Leu Ser Ser Ala 180 185 190Asn Leu Tyr Ile Asn
Gly Val Leu Met Gly Ser Ala Glu Ile Thr Gly 195 200 205Leu Gly Ala
Ile Arg Glu Asp Asn Asn Ile Thr Leu Lys Leu Asp Arg 210 215 220Cys
Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys Phe Arg Ile Phe225 230
235 240Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu Tyr Thr Ser
Tyr 245 250 255Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn Pro
Leu Arg Tyr 260 265 270Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser
Ser Ser Lys Asp Val 275 280 285Gln Leu Lys Asn Ile Thr Asp Tyr Met
Tyr Leu Thr Asn Ala Pro Ser 290 295 300Tyr Thr Asn Gly Lys Leu Asn
Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly305 310 315 320Leu Lys Phe Ile
Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser 325 330 335Phe Val
Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn 340 345
350Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn Asn
355 360 365Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile
Pro Leu 370 375 380Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu
Lys Thr Tyr Ser385 390 395 400Val Gln Leu Lys Leu Tyr Asp Asp Lys
Asn Ala Ser Leu Gly Leu Val 405 410 415Gly Thr His Asn Gly Gln Ile
Gly Asn Asp Pro Asn Arg Asp Ile Leu 420 425 430Ile Ala Ser Asn Trp
Tyr Phe Asn His Leu Lys Asp Lys Ile Leu Gly 435 440 445Cys Asp Trp
Tyr Phe Val Pro Thr Asp Glu Gly Trp Thr Asn Asp 450 455
46031392DNAClostridium tetani 3atggtttttt caacaccaat tccattttct
tattctaaaa atctggattg ttgggttgat 60aatgaagaag atatagatgt tatattaaaa
aagagtacaa ttttaaattt agatattaat 120aatgatatta tatcagatat
atctgggttt aattcatctg taataacata tccagatgct 180caattggtgc
ccggaataaa tggcaaagca atacatttag taaacaatga atcttctgaa
240gttatagtgc ataaagctat ggatattgaa tataatgata tgtttaataa
ttttaccgtt 300agcttttggt tgagggttcc taaagtatct gctagtcatt
tagaacaata tggcacaaat 360gagtattcaa taattagctc tatgaaaaaa
catagtctat caataggatc tggttggagt 420gtatcactta aaggtaataa
cttaatatgg actttaaaag attccgcggg agaagttaga 480caaataactt
ttagggattt acctgataaa tttaatgctt atttagcaaa taaatgggtt
540tttataacta ttactaatga tagattatct tctgctaatt tgtatataaa
tggagtactt 600atgggaagtg cagaaattac tggtttagga gctattagag
aggataataa tataacatta 660aaactagata gatgtaataa taataatcaa
tacgtttcta ttgataaatt taggatattt 720tgcaaagcat taaatccaaa
agagattgaa aaattataca caagttattt atctataacc 780tttttaagag
acttctgggg aaacccttta cgatatgata cagaatatta tttaatacca
840gtagcttcta gttctaaaga tgttcaattg aaaaatataa cagattatat
gtatttgaca 900aatgcgccat cgtatactaa cggaaaattg aatatatatt
atagaaggtt atataatgga 960ctaaaattta ttataaaaag atatacacct
aataatgaaa tagattcttt tgttaaatca 1020ggtgatttta ttaaattata
tgtatcatat aacaataatg agcacattgt aggttatccg 1080aaagatggaa
atgcctttaa taatcttgat agaattctaa gagtaggtta taatgcccca
1140ggtatccctc tttataaaaa aatggaagca gtaaaattgc gtgatttaaa
aacctattct 1200gtacaactta aattatatga tgataaaaat gcatctttag
gactagtagg tacccataat 1260ggtcaaatag gcaacgatcc aaatagggat
atattaattg caagcaactg gtactttaat 1320catttaaaag ataaaatttt
aggatgtgat tggtactttg tacctacaga tgagggatgg 1380acaaatgatt aa
1392449DNAArtificial SequenceDescription of Artificial Primer
4ccccccgggc caccatggtt ttttcaacac caattccatt ttcttattc
49518DNAArtificial SequenceDescription of Artificial Primer
5ctaaaccagt aatttctg 18625DNAArtificial SequenceDescription of
Artificial Primer 6aattatggac tttaaaagat tccgc 25724DNAArtificial
SequenceDescription of Artificial Primer 7ggcattataa cctactctta
gaat 24827DNAArtificial SequenceDescription of Artificial Primer
8aatgccttta ataatcttga tagaaat 27941DNAArtificial
SequenceDescription of Artificial Primer 9ccccccgggc atatgtcatg
aacatatcaa tctgtttaat c 411024DNAArtificial SequenceDescription of
Artificial Primer 10ctgaatatcg acggtttcca tatg 241140DNAArtificial
SequenceDescription of Artificial Primer 11ggcagtctcg agtctagacc
atggcttttt gacaccagac 401220DNAArtificial SequenceDescription of
Artificial Synthetic oligonucleotide linker 12catgactggg gatccccagt
201324DNAArtificial SequenceDescription of Artificial Primer
13tatgataaaa atgcatcttt agga 241437DNAArtificial
SequenceDescription of Artificial Primer 14tggagtcgac gctagcagga
tcatttgtcc atccttc 37158DNAArtificial SequenceDescription of
Artificial Synthetic oligonucleotide linker 15gctagcgc
81617DNAArtificial SequenceDescription of Artificial Synthetic
oligonucleotide linker 16gatatcggcg cgccagc 171717DNAArtificial
SequenceDescription of Artificial Synthetic oligonucleotide linker
17tggcgcgccg atatcgc 171814DNAArtificial SequenceDescription of
Artificial Synthetic oligonucleotide linker 18tcgatggcgc gcca
14
* * * * *