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The Journal of Neuroscience, August 1, 2000, 20(15):5587-5593
Complete and Long-Term Rescue of Lesioned Adult Motoneurons by
Lentiviral-Mediated Expression of Glial Cell Line-Derived Neurotrophic
Factor in the Facial Nucleus
Andreas F.
Hottinger,
Mimoun
Azzouz,
Nicole
Déglon,
Patrick
Aebischer, and
Anne D.
Zurn
Division of Surgical Research and Gene Therapy Center, Centre
Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland
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ABSTRACT |
To date, delivery of neurotrophic factors has only allowed to
transiently protect axotomized facial motoneurons against cell death.
In the present report, long-term protection of these neurons was
evaluated by continuously expressing the neurotrophic factor glial cell
line-derived neurotrophic factor (GDNF) within the facial
nucleus using a lentiviral vector system. The viral vector was injected
unilaterally into the facial nucleus of 4-month-old Balb/C mice. In
contrast to axotomy in other adult rodents, facial nerve lesion in
these animals leads to a progressive and sustained loss and/or atrophy
of >50% of the motoneurons. This model thus represents an attractive
model to evaluate potential protective effects of neurotrophic factors
for adult-onset motoneuron diseases, such as amyotrophic lateral
sclerosis. One month after unilateral lentiviral vector injection, the
facial nerve was sectioned, and the animals were killed 3 months
later. Viral delivery of the GDNF gene led to long-term expression and
extensive diffusion of GDNF within the brainstem. In addition,
axotomized motoneurons were completely protected against cell death,
because 95% of the motoneurons were present as demonstrated by both
Nissl staining and choline acetyltransferase immunoreactivity.
Furthermore, GDNF prevented lesion-induced neuronal atrophy and
maintained proximal motoneuron axons, despite the absence of target
cell reinnervation. This is the first evidence that viral-mediated
delivery of GDNF close to the motoneuron cell bodies of the facial
nucleus of adult mice can lead to complete and long-term protection
against lesion-induced cell death.
Key words:
axotomy; facial nerve; GDNF; gene expression; gene
therapy; gene transfer; lentiviral vector; motoneuron; neuroprotection; neurotrophic factor; Balb/C mice
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INTRODUCTION |
Amyotrophic lateral sclerosis (ALS)
is an adult-onset neurodegenerative disorder characterized by the loss
of motoneurons leading to progressive paralysis and death within 2-5
years. With the exception of mutations in the gene for superoxide
dismutase I (SOD) found in ~2% of all cases (Rosen, 1993 ), the
etiology of the disease is still unknown and no treatment is currently available. A number of animal models, such as facial or sciatic nerve
axotomy in neonatal rodents, have been developed to evaluate potential
protective effects of neurotrophic factors (Sendtner et al., 1990;
Koliatsos et al., 1993; Henderson et al., 1994; Zurn et al., 1994; Yan
et al., 1995). However, axotomy in neonates leads to a rapid cell death
(4-5 d) (Snider et al., 1992), whereas degeneration is progressive and
extends over several months in ALS. Second, ALS is an adult-onset
motoneuron disease, and mature motoneurons may be less dependent on
trophic support than developing neurons (Pollin et al., 1991). In the
present report, motoneuron axotomy was performed in adult Balb/C mice
because, in contrast to axotomy in other adult rodents, facial nerve
lesion in these animals leads to a progressive and long-term loss of
motoneurons (Kou et al., 1995; Ferri et al., 1998). This relatively
slow cell death more closely resembles the degeneration observed in
motoneuron diseases because it extends over a period of several weeks
(Kou et al., 1995). It thus represents a better model than axotomy in
neonatal animals to evaluate potential protective effects of neurotrophic factors for adult-onset motoneuron disorders. Spinal root
avulsion, another adult lesion model, is less appropriate because not
only motoneuron loss but also complete loss of associated Schwann cells
occurs (Koliatsos et al., 1994).
To date, delivery of recombinant neurotrophic factors close to the cut
nerve in neonatal rats and mice has only allowed to transiently protect
axotomized motoneurons against cell death (Schmalbruch and Rosenthal,
1995; Vejsada et al., 1995). Long-term protection against degeneration
is therefore still a major unresolved issue. To obviate this problem,
adenoviral vectors have been used to deliver neurotrophic factors to
the cut nerve stump of lesioned motoneurons. However, <10% of the
motoneurons retrogradely transported and expressed the transgenes, and
only a partial rescue was observed (Baumgartner and Shine, 1997;
Gimenez y Ribotta et al., 1997; Gravel et al., 1997). We therefore
assessed whether continuous expression of glial cell line-derived
neurotrophic factor (GDNF) in the vicinity of the motoneuron cell
bodies of the facial nucleus would allow efficient and sustained
protection against cell death. GDNF was chosen because it is the most
potent neurotrophic factor for cultured embryonic motoneurons described
to date (Henderson et al., 1994), as well as the most efficient factor
to rescue axotomized neonatal motoneurons when delivered close to the
nerve stump (Yan et al., 1995; Vejsada et al., 1998). A lentiviral
(human immunodeficiency virus)-based vector system was used to
deliver GDNF directly into the brain. The core particles of this vector system are pseudotyped with vesicular stomatitis virus glycoprotein G,
which confers a broad tropism and allows to infect a large variety of
different cell types (Naldini et al., 1996b). Moreover, the vector has
been shown to transduce both dividing and nondividing cells such as
neurons and to integrate transgenes into the chromosomes of their
targets, resulting in the long-term expression of the transgene. This
efficient gene delivery system has been applied previously in
vivo in the brain of rats and monkeys (Naldini et al., 1996a;
Zufferey et al., 1997; Kordower et al., 1999; Déglon et al.,
2000).
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MATERIALS AND METHODS |
Production of recombinant lentiviral vectors. The
cDNA coding for a nuclear-localized  -galactosidase (LacZ), the human
GDNF containing a Kozak consensus sequence (a 636 bp fragment, position 1-151 and 1-485; GenBank accession numbers L19062 and L19063), or a
mutated GDNF (deletion of amino acids 74-85 of the mature GDNF,
leading to the absence of secretion of the trophic factor) (Choi-Lundberg et al., 1997) were cloned in the SIN-W-PGK transfer vector (Déglon et al., 2000). The packaging construct used in this study was the pCMVDR-8.92 (derived from the pCMVDR-8.91plasmid: destruction of the BamHI restriction site in the coding
region of the rev gene). To further decrease the risk of recombination and production of replication-competent retroviruses, the Rev gene was
inserted in the pRSV-Rev plasmid. The viral particles were pseudotyped
with the vesicular stomatitis virus G-protein encoded by the pMD.G
plasmids described previously (Naldini et al., 1996b). The viral
particles were produced by transient transfection of 293T cells
(Naldini et al., 1996b). Forty-eight hours later, the supernatant was
collected and filtered, and the particle content was determined by
ELISA assay (NEN, Boston, MA). High-titer stocks were obtained
by ultracentrifugation. The pellet was resuspended in PBS and 1% BSA
and stored frozen at  80°C. The batches of virus were tested for the
absence of replication-competent viral vectors (Naldini et al., 1996b).
The titers of the lentiviral vector encoding LacZ (LV-LacZ) stocks were
determined on 293T cells. The cells were plated at a density of 2 × 105 cells per well on six-well tissue
culture dishes (Costar, Cambridge, MA). Serial dilutions of the viral
stocks were added, and the number of LacZ-infected cells was analyzed
48 hr later. Titers were calculated by counting the number of blue foci
per well and dividing it by the dilution factor. LacZ-expressing
viruses with ranges from 3 to 7 × 108 transfection units/ml were
obtained. In all in vivo experiments, LacZ, GDNF, or mutated
GDNF-expressing virsuses (LV-LacZ, LV-GDNF, and LV-mGDNF, respectively)
were matched for particle content (200,000 ng p24 antigen/ml as
measured by ELISA assay). These experiments were performed in biosafety
level 2 laboratories.
Animals and surgery. Adult BalB/C mice (4 months old; IFFA
Credo, Reims, France) were anesthetized with an intraperitoneal injection of 60 mg/kg pentobarbital and placed in a stereotaxic frame
(David Kopf Instruments, Tujunga, CA). The experiments were performed
in accordance with the European Community Council Directive (86/609/EEC) for care and use of laboratory animals. A midline incision
was performed on the skin covering the skull, and the bregma point was
determined. The skull was opened with a dental drill at a location 6.2 mm caudal and 1.2 mm lateral to the bregma, the coordinates for the
facial nucleus. One microliter of lentiviral vector expressing
either GDNF, mutated GDNF, or the reporter gene LacZ was injected at
the rate of 0.2 µl/min down to a depth of 5.6 mm. Other control
groups consisted of animals that received either a 1 µl PBS solution
at the same coordinates or no treatment. Thirty days later, the animals
were anesthetized with pentobarbital. The skin was incised below the
right ear. The right facial nerve was exposed, freed of connective
tissue, and transected at its exit from the stylomastoïd
foramen. A small segment of the nerve was removed to prevent
regeneration. The skin incision was sutured with a Prolene 6-0
filament (Johnson & Johnson). The success of the procedure was
confirmed by the absence of movement of the whiskers on the side of the
lesion. The animals were then returned to their cages and maintained in
a P2 facility. They were killed 30 or 90 d later by an overdose of
pentobarbital and perfused under RNase-free condition with PBS,
followed by a 4% paraformaldehyde solution. Thereafter, the brainstem
was removed and post-fixed in the same fixative overnight and
cryoprotected in PBS containing 25% sucrose. Frozen sections of the
brainstem (20-µm-thick) were cut on a Reichert Jung Cryocut 1800 at
24°C.
Immunohistochemistry. Brainstem sections from animals
injected with LV-LacZ were processed for -galactosidase
immunoreactivity and double stained for glial fibrillary acidic protein
(GFAP) or SMI-32, an antibody recognizing nonphosphorylated
neurofilament protein used to identify motoneurons and their central
processes. Free-floating sections were blocked in a solution containing
10% normal goat serum and 0.1% Triton X-100 in PBS for 3 hr. The
following primary antibodies were used: rabbit polyclonal
anti--galactosidase, 1:500 (5 Prime  3 Prime Inc., Boulder, CO),
mouse monoclonal anti-GFAP, 1:50 (Boehringer Mannheim, Mannheim,
Germany), and mouse anti-SMI-32, 1:4000 (Sternberger Monoclonals,
Lutherville, MD). Primary antibodies were diluted in the blocking
solution, and sections were incubated overnight at 4°C. Sections were
then incubated for 3 hr with the secondary goat anti-mouse
Cy3-conjugated antibody at a 1:400 dilution and goat anti-rabbit
FITC-conjugated antibody at a 1:100 dilution in PBS (Jackson
ImmunoResearch, West Grove, PA). Finally, sections were mounted onto
glass slides and coverslipped using Fluorosave (Calbiochem, La Jolla,
CA). For choline acetyltransferase (ChAT) immunohistochemistry,
sections were washed in PBS and incubated in 10% normal goat serum for 2 hr, followed by an overnight incubation at 4°C with a monoclonal ChAT antibody at a 1:400 dilution (generous gift of Dr. Cozzari, Istituto di Biologia Cellulare, Roma, Italy) (Umbriaco et al., 1994). Control sections were incubated without the primary antibody. After incubation with the secondary antibody, binding was detected using an Elite avidin-biotin-peroxidase kit (Vector Laboratories, Burlingame, CA). To evaluate diffusion of GDNF in the brainstem, immunohistochemistry using an anti-human GDNF antibody was performed. Briefly, 20-µm-thick sections were first quenched for 20 min in 0.1 M sodium periodate, followed by a 1 hr blocking
in 100 mM Tris/150 mM NaCl
(TBS) containing 2% BSA, 3% normal horse serum (NHS), and 0.5%
Triton X-100. They were then incubated for 48 hr at room temperature in
TBS containing biotinylated goat anti-GDNF antibody (R & D Systems,
Wiesbaden, Germany) at a 1:250 dilution, 1% BSA, 1% NHS, and
0.04%Triton X-100. The sections were then incubated for 1 hr at room
temperature in the same buffer containing biotinylated horse anti-goat
IgG (1:200) (Vector Laboratories), 1% NHS, and 1% BSA. Antibody
binding was revealed according to standard procedures using an Elite
avidin-biotin-peroxidase kit (Vector Laboratories) and DAB.
In situ hybridization. To confirm the expression of the
transgene close to the facial nucleus, in situ hybridization
was performed with a woodchuck hepatitis virus (WHV) digoxigenin (DIG)
riboprobe. The WHV regulatory element is present on all lentiviral
constructs used in this study (Déglon et al., 2000). A 591 bp
fragment of the WHV post-transcriptional element (position 1093-1684
of the WHV complete genome; GenBank accession number J04514) was cloned in the pBluescript SK+ plasmid
(Stratagene, La Jolla, CA). The antisense riboprobe was synthesized
from a BamHI linearized pBluescript-WHV vector according to
the supplier's manual (DIG RNA labeling kit; Roche Products, Rotkreuz,
Switzerland). The concentration of the digoxigenin-11 UTP- labeled
probe was measured by dot blot using a DIG-labeled control RNA as
internal standard. Frozen brain tissue obtained as described above was
embedded in RNase-free Tissue Tek OCT Compound (Bayer, Zurich,
Switzerland) and 20 µm sections were cut at 24°C using a
cryostat. Free-floating sections were washed in 0.1% DEPC in PBS twice
for 15 min, treated with HCl 0.02 N for 10 min, and equilibrated in 5×
SSC (0.75 mM NaCl and 0.075 mM Na3 citrate) for 15 min.
The sections were then prehybridized in 50% formamide, 5× SSC, 5%
dextran sulfate, 1× Denhardt's solution (1% bovine serum albumin,
1% polyvinylpyrrolidone, and 1% Ficoll 400), 250 µg/ml
heat-denatured sheared salmon sperm DNA, and 100 µg/ml yeast t-RNA
for 2 hr at 48°C. Hybridization was performed overnight at 48°C
with the DIG riboprobe in the prehybridization buffer. Sections were
then washed in 2× SSC for 30 min at room temperature, followed by 2×
SSC for 1 hr and 0.1× SSC for 1 hr at 48°C. The hybridized DIG
riboprobe was revealed as described by the manufacturer (Boehringer Mannheim).
GDNF ELISA assay. To evaluate the level of GDNF expression
in the brainstem, 4-month-old Balb/C mice received intrafacial nucleus
injections of either 1 µl of lentivirus carrying the GDNF or LacZ
gene, or the carrier solution PBS (see below). Sixty days later, the
animals were killed with an overdose of pentobarbital and perfused with
an ice-cold solution of PBS containing 0.2% ascorbic acid. The
brainstem was immediately removed and frozen at 70°C on dry ice. It
was then cut in the midline and resuspended in 500 µl of PBS
containing 0.5% Triton X-100 and protease inhibitors [chymotrypsin,
papain, pronase, thermolysin, and trypsin (Complete Protease Inhibitor;
Boehringer Mannheim)]. The proteins were extracted by sonication and
centrifuged at 10,000 rpm for 10 min. The supernatants were frozen to
70°C before further use. The amount of human GDNF present in the
sample was determined using an ELISA assay (Promega, Madison, WI).
Because of the low recovery rate of GDNF from mouse brain, an
internal standard curve was performed by adding increasing doses of
recombinant human GDNF to brainstem tissue of control mice before extraction.
Motoneuron counts. To evaluate the number of motoneurons in
the facial nucleus, adjacent sections stained for Nissl, and ChAT and
SMI-32 immunoreactivity, were counted in every fifth section on both
the lesioned and control sides. Motoneuron survival was expressed as
the percentage of cells surviving on the lesioned side compared with
the nonlesioned side. With Nissl staining, only neurons with clearly
visible nucleoli and a large soma diameter (~20 µm) were counted.
All ChAT-immunopositive cells were counted. SMI-32-positive cells were
counted as motoneurons when they presented a large soma diameter. The
number of cells expressing the GDNF construct was evaluated by in
situ hybridization for the WHV post-transcriptional enhancer
element in randomly selected animals.
Motoneuron soma size. Motoneuron soma size was measured by
digitizing the most central section of the facial nucleus of all animals in each group (~350 cells per group) with a Panasonic CCD
camera and by drawing the perimeter of each motoneuron at a
magnification of 200×. Results were analyzed using NIH Image software
(version 1.47).
Statistical analysis. All data were expressed as the
mean ± SEM. For statistical evaluation of motoneuron numbers
identified by either Nissl staining or ChAT immunoreactivity, the
number of cells present in each facial nucleus was counted, multiplied by five (every fifth section was counted), and then multiplied by the Abercrombie correction factor, which compensates for double counting in adjacent sections (Abercrombie, 1946). Standard one-way ANOVA followed by a Scheffe's PLSD post hoc test was
performed using the statistical software Statview 4.0. Statistical
analyses of the square areas of the cell bodies were performed using
one-way ANOVA followed by a Scheffe's PLSD post hoc test.
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RESULTS |
Transgene expression in unlesioned animals
-galactosidase expression
Two months after LV-LacZ injection, -galactosidase was found to
be expressed in 20,595 ± 2737 cells (n = 4) in
brainstem sections of nonlesioned animals. Labeling was observed over a rostrocaudal and lateral distance of 2.5 ± 0.13 and 0.9 ± 0.11 mm (n = 4), respectively, including the whole
extent of the facial nucleus (Fig.
1). Long-term expression of the
transgene was confirmed by the presence of 24,213 ± 3130 (n = 4) -galactosidase-positive cells 4 months after
the injection. Because of the small size and the location of the facial
nucleus, the injection needle could be placed within the facial nucleus
itself in approximately half of the cases, as evidenced by the
localization of the needle tract by Nissl staining. In the latter case,
6293 ± 753 (n = 2) cells were infected within the
facial nucleus itself. Double-labeling with anti-SMI-32
antibodies (used as a marker for motoneurons) showed that 35 ± 7.7% (n = 2) of the motoneurons expressed
-galactosidase. When the injection was performed above the nucleus,
10.9 ± 5.1% (n = 2) of the SMI-32-positive
motoneurons were labeled. Using the glial cell marker GFAP, 17.27 ± 1.2% (n = 4) of the infected cells were found to be
astrocytes.
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Figure 1.
-galactosidase expression in the brainstem of
nonlesioned mice 60 d after the injection of LV-LacZ. The number
of -galactosidase-positive cells per 20 µm section was evaluated
every 100 µm over a rostrocaudal distance of 3 mm. The center of the
facial nucleus was determined and used as a reference point in the four
animals (number of cells per section ± SEM).
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Localization of the GDNF construct
The GDNF construct itself was localized by in situ
hybridization for the post-transcriptional enhancer element of the WHV (Fig. 2). Sixty days after viral
infection, the mRNA was found to be present in 17,045 ± 981 (n = 4) cells covering a distance of 2.4 ± 0.2 mm
rostrocaudally. In the cases in which the injection was directly within
the facial nucleus, 41.6 ± 2.1% (n = 3) of the
motoneurons expressed the transgene, as verified on adjacent Nissl-stained sections.
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Figure 2.
In situ hybridization for the WHV
element. A, Nonlesioned mice 60 d after LV-GDNF
injection in the facial nucleus. The region of the facial nucleus is
indicated by small dots. Scale bar, 500 µm.
B, Close-up view showing motoneurons in the facial
nucleus (arrows) and other infected cells. Scale bar,
100 µm.
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GDNF expression and diffusion
In vitro infection of 293T cells with LV-GDNF allows
the synthesis and secretion of 319 ± 46 ng
GDNF/106 cells per day, as assayed by
ELISA (Déglon et al., 2000). In addition, the released GDNF is
bioactive because it increases ChAT activity twofold to threefold in
embryonic day 14 spinal cord cultures (Déglon et al., 2000).
Infection with LV-mGDNF resulted in no detectable GDNF production. An
ELISA assay for GDNF was performed 60 d after viral infection on
brainstem tissue punches of 0.8 mm diameter and 2 mm depth. Mice that
had received injections of LV-GDNF expressed 1.69 ± 0.51 ng of
GDNF per punch (n = 4), whereas GDNF levels were below
the detection limit on the uninjected side or in control animals that
had received LV-LacZ (n = 4). To assess long-term
expression of GDNF in and around the facial nucleus, GDNF production
and diffusion were evaluated by immunohistochemistry. The micrograph in
Figure 3 reveals that GDNF diffused over
long distances, beyond the location of the transduced cells (Fig.
2).
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Figure 3.
Immunodetection of GDNF in the brainstem.
A, GDNF immunoreactivity 4 months after injection of
LV-GDNF close to the facial nucleus. The region of the facial nucleus
is indicated by small dots. Animals injected with
LV-LacZ (B) or LV-mGDNF
(C). Scale bar, 500 µm.
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Protective effects of GDNF in lesioned animals
Facial nerve axotomy in 5-month-old Balb/C mice led to a severe
loss of motoneurons, with only 40.2 ± 2.1 (n = 4)
and 46.3 ± 2.5% (n = 6) of the neurons surviving
compared with the nonlesioned side 30 and 90 d after the lesion,
respectively (no statistical difference between these two groups;
p > 0.1) (Fig.
4A,B,
Nissl staining). Note that motoneuron loss does not seem to occur
naturally in these mice with age, because 2947 ± 30 and 2929 ± 200 (n = 4) facial motoneurons were present at 2 and
8 months of age, respectively, and because ChAT levels were also stable
with age (data not shown). Injection of PBS, LV-LacZ, or LV-mGDNF had
no significant effect on cell loss, with 44.95 ± 0.8 (n = 4), 51.8 ± 7.2 (n = 4), and 44 ± 6.6% (n = 6) of the motoneurons remaining
after 30 d, respectively (Fig. 4A) (no
statistical difference between these groups and untreated animals).
Similar results were obtained in the different control groups 90 d
after the lesion (Fig. 4B). In contrast, animals pretreated with LV-GDNF showed a complete rescue of the axotomized motoneurons, with 109.4 ± 4.3 (n = 7, p < 0.0001) and 95 ± 4.9% (n = 8, p < 0.0001) of the cells remaining after 30 and
90 d, respectively (Fig.
4A,B). Evaluation of the number of
ChAT-positive cells 30 d after the lesion showed that only
26.2 ± 4.8 (n = 4) and 23.7 ± 8.9%
(n = 4) of the cells were present in PBS- and LV-mGDNF-treated animals, respectively, whereas in the presence of
GDNF, 77.9 ± 6.1% of the cells expressed ChAT (n = 4, p < 0.001) (Fig. 4C). Protective
effects were sustained for several months, with 95.3 ± 2.5%
(n = 8) of the cells expressing ChAT at 3 months in the
presence of GDNF but only 29.9 ± 5.2 (n = 6),
35.4 ± 5.2 (n = 6), 38.37 ± 6.7 (n = 6), and 45.5 ± 4.5% (n = 6)
in animals treated with LV-LacZ, LV-mGDNF, PBS, or no treatment,
respectively (Fig. 4D). Photomicrographs of Nissl-
and ChAT-stained sections 3 months after the lesion are shown in
Figures 5 and
6, respectively. Importantly, not only
cell bodies but also central processes were maintained in the brainstem
in the presence of GDNF, as evidenced by immunostaining with
anti-neurofilament antibody (Fig. 7).
(Note that SMI-32 does not distinguish between axons and dendrites.) Thus, motoneuron cell bodies as well as proximal processes were present
with GDNF despite the absence of target cell reinnervation, as
evidenced by the absence of whisker movement.
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Figure 4.
Number of motoneurons in mice injected with PBS,
LV-LacZ, LV-mGDNF, or LV-GDNF 30 (A, C)
and 90 (B, D) d after transection of the
facial nerve. A, B, Nissl staining.
C, D, ChAT immunostaining. Motoneuron
survival is expressed as the percentage of cells surviving on the
lesioned compared with the nonlesioned side. Only GDNF is statistically
different from PBS, LacZ, and mGDNF or untreated mice;
***p < 0.001.
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Figure 5.
Nissl stains of cross-sections through the facial
nucleus from lesioned animals injected with LV-GDNF (A,
B) or LV-mGDNF (C, D)
90 d after nerve transection (Nissl staining). A,
C, Nonlesioned side. B, D,
Lesioned side. Scale bar, 200 µm.
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Figure 6.
ChAT immunostaining of cross-sections through the
facial nucleus from lesioned animals injected with LV-GDNF
(A) and LV-LacZ (B) 90 d after nerve transection. Left, Nonlesioned side.
Right, Lesioned side. Scale bar, 500 µm.
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Figure 7.
Central processes of facial motoneurons stained
with anti-neurofilament antibody (SMI-32). A,
Nonlesioned, noninjected side. B, Lesioned side injected
with lenti-mutated GDNF. C, Lesioned side injected with
lenti-GDNF. Scale bar, 40 µm.
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Assessment of the distribution of motoneuron soma sizes on the lesioned
and the nonlesioned side showed that there was a decrease in the
percentage of cells in the 400-500 µm2
size and an increase in the 250-350 µm2
size 3 months after axotomy (Fig.
8A-E). This indicates
that, in addition to cell loss, there is also atrophy in neurons
remaining after the lesion. Furthermore, there is an increase in the
percentage of cells in the 400-700 µm2
size in the presence of GDNF on the lesioned but also on the nonlesioned side (Fig. 8C,F), indicating
hypertrophy induced by the neurotrophic factor. Similar changes were
observed at 1 month. Continuous expression of GDNF in the facial
nucleus region induced a sustained weight loss starting day 3 after
injection and reaching 31.2 ± 2.4% (n = 8) loss
at 40 d, which it maintained for 4 months. Despite this important
loss and a 27.3 ± 3.1% (n = 8) decrease in food
intake, these animals have unchanged activity levels as evaluated using
a monitoring box (data not shown).
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Figure 8.
Effect of GDNF on the motoneuron size distribution
3 months after the lesion. A-C, Size distribution (in
square micrometers) of facial motoneurons on the nonlesioned side:
A, untreated; B, LacZ; and
C, GDNF. D-F, Soma size distribution on
the lesioned side: D, Noninfected; E,
LacZ; and F, GDNF-infected animals.
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DISCUSSION |
The present report demonstrates that, using facial nerve lesion in
adult Balb/C mice, expression of the neurotrophic factor GDNF close to
the motoneuron cell bodies of the facial nucleus using a lentiviral
vector system leads to extensive diffusion of GDNF in the brainstem and
to a complete and sustained protection against lesion-induced cell death.
Infection of the brainstem with a lentiviral vector encoding the
reporter gene LacZ leads to -galactosidase expression in >20,000
cells within and surrounding the facial nucleus. A comparable number of
transduced cells has been obtained previously in rat striatum using a
similar vector (Zufferey et al., 1997). Viral infection allows to
stably express the transgene for up to at least 4 months. Only a mild
inflammation consisting in minor perivascular cuffing and in
aggregation of lymphocytes around the injection tract is observed. Both
neurons and astrocytes are transduced, with up to 45% of the
SMI-32-positive facial motoneurons expressing the reporter
gene in the best experiments. In comparison, only 5-10% of the
axotomized facial motoneurons express -galactosidase after injection
of recombinant adenovirus into lesioned nerves or facial muscles of
neonatal rats (Baumgartner and Shine, 1997; Gravel et al., 1997).
In contrast to nerve axotomy in other strains of adult mice and in
rats, which only results in the downregulation of a number of proteins,
including ChAT, the enzyme responsible for the synthesis of the
neurotransmitter acetylcholine (Lams et al., 1988; Yan et al., 1994),
facial nerve axotomy in 2-month-old Balb/C mice has been shown to lead
to 20-30% cell loss (Kou et al., 1995; Ferri et al., 1998). In our
hands, facial nerve lesion leads to the sustained loss of as much as
60% of the motoneurons, probably because of the older age of the mice
at the time of the lesion (5 months). However, the difference may also
in part be attributable to our counting method because we considered
only clearly identifiable motoneurons with a large soma diameter (~20
µm). Therefore, part of the cells not included in our counts may have
been counted by others. In addition, some of the motoneurons that were
not counted were probably atrophic and not lost (Fig. 8).
Intraparenchymal delivery of the GDNF gene in adult Balb/C mice leads
to sustained expression and extensive diffusion of GDNF within the
brainstem. This viral gene delivery system thus allows much better
diffusion of the neurotrophic factor within the brain parenchyma than
intraventricular bolus injections of GDNF (Lapchak et al., 1998). We
also demonstrate that GDNF expression within and close to the facial
nucleus allows to completely protect facial motoneurons against
axotomy-induced cell death and/or atrophy, for as long as 3 months.
Recombinant GDNF or GDNF released by encapsulated genetically
engineered cells applied in the vicinity of the cut nerve in neonatal
rodents has also been shown to be protective (Henderson et al., 1994;
Zurn et al., 1994; Oppenheim et al., 1995; Yan et al., 1995; Houenou et
al., 1996). However, only a transient, 1-2 week rescue could be
obtained (Vejsada et al., 1998). Protection lasting for 28 d was
obtained in neonatal rats by repeated application of GDNF at days 0 and
14 after axotomy. However, although rescue was complete at 14 d,
it was only partial (40%) at 28 d (Matheson et al., 1997). In an
attempt to prolong the rescue period, adenoviral vectors have been used
recently to deliver neurotrophic factors either to denervated muscles
(Baumgartner and Shine, 1997; Gimenez y Ribotta et al., 1997) or
directly into the lesioned nerves (Gravel et al., 1997). Although
survival of axotomized motoneurons was maintained for at least 5 weeks
under those conditions (Gravel et al., 1997), only a 40-60% rescue
was observed (Baumgartner and Shine, 1997; Gimenez y Ribotta et al., 1997; Gravel et al., 1997). This may be attributable to the fact that
only 5-10% of the motoneurons were transduced. In contrast, direct
delivery and continuous expression of GDNF via viral gene transfer
within and surrounding the facial motoneuron cell bodies leads to
sustained and complete neuroprotection as evaluated both by Nissl
staining and ChAT immunohistochemistry. It is interesting to note that,
despite the variation in the number of motoneurons infected by the
lentiviral vector, we observe no correlation between the percentage of
motoneurons infected by LV-GDNF and the extent of rescue. Thus, GDNF
released by neurons and astrocytes at a distance can reach motoneuron
cell bodies of the facial nucleus and protect them against
degeneration. This indicates that both autocrine and paracrine
mechanisms are involved. Evidence for a paracrine mechanism of action
of GDNF has been obtained previously in neonatal rats in which a larger
number of motoneurons survived after facial nerve axotomy than were
transduced (Baumgartner and Shine, 1997; Gravel et al., 1997).
Upregulation of the signaling component of the GDNF receptor
c-ret and the ligand binding subunit GDNFR- has been
described to occur after facial nerve lesion in the rat (Burazin and
Gundlach, 1998). Such increased expression, combined with long-distance
diffusion of GDNF and action both via an autocrine and paracrine
mechanism, may explain the efficient neuroprotection obtained in adult
Balb/C mice.
GDNF does not only protect facial motoneuron cell bodies against cell
death, but it also maintains the proximal processes of these cells.
This occurs despite the lack of target cell reinnervation as evidenced
by the absence of whisker movement on the lesioned side. In addition,
GDNF also prevents atrophy and/or induces hypertrophy in the lesioned
motoneurons. Motoneuron hypertrophy has also been described to occur
with GDNF after neonatal motor nerve axotomy and ventral root avulsion
(Henderson et al., 1994; Li et al., 1995; Oppenheim et al., 1995). The
mechanism for this hypertrophy is unknown but may resemble the
mechanism observed with nerve growth factor on sympathetic and sensory
neurons (Levi-Montalcini, 1987).
Mice treated with GDNF show a sustained weight loss. This effect
appears to be specific to GDNF delivery close to the facial nucleus,
because intrastriatal and intranigral injection of identical amounts of
LV-GDNF in rat and mouse does not induce weight loss (Déglon et
al., 2000; data not shown). A 10-15% weight reduction has been
described previously to occur after intraventricular injection of
microgram quantities of GDNF (Lapchak et al., 1997; Giehl et al.,
1998). The mechanisms responsible for this weight loss are unknown. It
is possible that indirect activation of higher brain centers involved
in the control of feeding occurs in the present study (Kupfermann,
1994).
In conclusion, the present report provides the first evidence that
continuous delivery of nanogram quantities of GDNF close to the facial
nucleus of adult mice via a lentiviral vector system can lead to (1)
sustained expression of the transgene, (2) extensive diffusion of the
molecule, and (3) complete and long-term protection of motoneurons and
their central processes against lesion-induced cell death and atrophy.
Delivery of trophic factors close to the degenerating motoneuron cell
bodies may be a prerequisite to obtain significant neuroprotection in
SOD transgenic mice, currently the best model of ALS (Gurney et al.,
1994), as well as in ALS patients, because application in the periphery
has not given any positive results so far.
| |
FOOTNOTES |
Received Dec. 28, 1999; revised April 21, 2000; accepted April 26, 2000.
This work was supported by the Swiss National Science Foundation.
A.F.H. is supported by the Roche Research Foundation MD-PhD program. We
thank Anne Maillard, Fabienne Pidoux, Maria Rey, and Dana Hornfeld for
their technical assistance and helpful comments.
Correspondence should be addressed to Dr. Anne D. Zurn, Division of
Surgical Research and Gene Therapy Center, Pavillon 4, Centre
Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland. E-mail:
anne.zurn{at}chuv.hospvd.ch.
| |
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ABSTRACT
INTRODUCTION
