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The Journal of Neuroscience, April 1, 2002, 22(7):2780-2791
Parkinson-Like Neurodegeneration Induced by Targeted
Overexpression of  -Synuclein in the Nigrostriatal System
Deniz
Kirik1,
Carl
Rosenblad2,
Corinna
Burger3,
Cecilia
Lundberg1,
Teit E.
Johansen2,
Nicholas
Muzyczka3,
Ronald J.
Mandel4, and
Anders
Björklund1
1 Wallenberg Neuroscience Center, Department of
Physiological Sciences, Division of Neurobiology, Lund University, 221 84, Lund, Sweden, 2 NsGene A/S, 2750, Ballerup, Denmark,
and Departments of 3 Molecular Genetics and Microbiology
and 4 Neuroscience, McKnight Brain Institute and
Powell Gene Therapy Center, University of Florida, Gainesville, Florida
32610
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ABSTRACT |
Recombinant adeno-associated viral vectors display efficient
tropism for transduction of the dopamine neurons of the substantia nigra. Taking advantage of this unique property of recombinant adeno-associated viral vectors, we expressed wild-type and A53T mutated
human  -synuclein in the nigrostriatal dopamine neurons of adult rats
for up to 6 months. Cellular and axonal pathology, including
-synuclein-positive cytoplasmic inclusions and swollen, dystrophic
neurites similar to those seen in brains from patients with
Parkinson's disease, developed progressively over time. These pathological alterations occurred preferentially in the nigral dopamine
neurons and were not observed in other nondopaminergic neurons
transduced by the same vectors. The degenerative changes were
accompanied by a loss of 30-80% of the nigral dopamine neurons, a
40-50% reduction of striatal dopamine, and tyrosine hydroxylase levels that was fully developed by 8 weeks. Significant motor impairment developed in those animals in which dopamine neuron cell
loss exceeded a critical threshold of 50-60%. At 6 months, signs of
cell body and axonal pathology had subsided, suggesting that the
surviving neurons had recovered from the initial insult, despite the
fact that -synuclein expression was maintained at a high level.
These results show that nigral dopamine neurons are selectively
vulnerable to high levels of either wild-type or mutant -synuclein,
pointing to a key role for -synuclein in the pathogenesis of
Parkinson's disease. Targeted overexpression of -synuclein in the
nigrostriatal system may provide a new animal model of Parkinson's
disease that reproduces some of the cardinal pathological,
neurochemical, and behavioral features of the human disease.
Key words:
Parkinson's disease; adeno-associated virus vector; neurodegeneration; dopamine; tyrosine hydroxylase; nigral inclusion
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INTRODUCTION |
A growing body of evidence has
implicated -synuclein in the pathogenesis of Parkinson's disease
(PD) (Polymeropoulos et al., 1997 ; Kruger et al., 1998; Spillantini et
al., 1998; Takeda et al., 1998; Giasson et al., 2000b). The discovery
that point mutations in the -synuclein gene can be pathogenic in
rare cases of familial PD (Polymeropoulos et al., 1997; Kruger et al.,
1998) led to the identification of -synuclein as a major component
of Lewy bodies and dystrophic neurites, i.e., the pathological hall
marks of PD (Spillantini et al., 1997, 1998; Baba et al., 1998;
Irizarry et al., 1998; Takeda et al., 1998; Braak and Braak, 2000).
Subsequent in vitro studies have shown that -synuclein
may interact with ubiquitin-proteosomal processing, oxidative injury,
and/or mitochondrial dysfunction to induce neurodegeneration and cell
death (Ostrerova et al., 1999; Giasson et al., 2000b; Hsu et al., 2000;
Kanda et al., 2000; Tabrizi et al., 2000; McNaught et al., 2001;
Shimura et al., 2001). In support of this concept, recent in
vivo studies have shown that overexpression of wild-type (wt) or
mutated -synuclein in transgenic mice may lead to neuropathological
changes, axonal degeneration, and occasionally also cell death (Kahle
et al., 2000; Masliah et al., 2000; van der Putten et al., 2000).
However, in the transgenic mice generated to date, -synuclein
accumulation and signs of axonal pathology have been widespread and
most prominent in structures outside the nigrostriatal system. Although
Masliah et al. (2000) observed accumulation of -synuclein in neurons
of the substantia nigra (SN) and reduction in tyrosine hydroxylase
(TH)-positive axon terminals in the striatum (obtained only in the mice
with the highest transgene expression), no degeneration or loss of
nigrostriatal dopamine (DA) neurons was observed in any of these mice.
Indeed, subsequent studies of transgenic mice expressing either the
A30P (Matsuoka et al., 2001; Rathke-Hartlieb et al., 2001) or the A53T
(Matsuoka et al., 2001) mutated form of -synuclein have failed to
find any nigral neuropathology. It remains unclear, therefore, what
role the mutant forms of -synuclein play or to what extent
overexpression of -synuclein is sufficient to cause Parkinson-like
pathology and neurodegeneration in nigrostriatal DA neurons in the
context of the rodent. Other rare forms of familial PD have been
described that implicate the proteosomal processing pathway in the
etiology of PD. Thus, in addition to mutations in -synuclein,
missense mutations in parkin, an E3 ubiquitin ligase (Shimura et al.,
2000), and ubiquitin C-terminal-hydroxylase-L1, an E1
ubiquitin-activating enzyme (Leroy et al., 1998), all induce familial
forms of PD. Indeed, a 22 kDa O-glycosylated isoform of
-synuclein has been found to be a specific substrate for the E3
ubiquitin ligase, parkin (Shimura et al., 2001). Mutated forms of the
-synuclein protein (both A53T and A30P) could not bind to parkin,
thus abrogating their normal poly-ubiquitination and subsequent
proteosomal processing. These data suggest therefore that accumulation
of -synuclein, as occurs in Lewy bodies present in idiopathic PD,
either by inhibition of normal proteosomal processing, or by
overexpression may be pathogenic to nigral dopamine neurons.
In the present study, we have used recombinant adeno-associated virus
(rAAV) vectors to express wt and mutated -synuclein at high levels
in the nigrostriatal DA neurons in adult rats. The rAAV vectors
integrate and stably express their transgene in nondividing cells,
mostly neurons in the CNS. Because 96% of the viral genome has
been removed, the vectors do not express any viral proteins, which
minimize the risk of host immune responses (Muzyczka, 1992). This
vector system has a particular affinity for the neurons of the SN pars
compacta, which makes it possible to express proteins stably, and at
high levels, in the nigrostriatal DA neurons in adult rats (Klein et
al., 1998; Kirik et al., 2000). Moreover, the transduction can be
restricted to one hemisphere, so that the contralateral side can serve
as a control.
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MATERIALS AND METHODS |
Animals and surgery. Young adult Sprague Dawley rats
(B & K Universal AB, Stockholm, Sweden) were housed six to a cage with ad libitum access to food and water during a 12 hr
light/dark cycle, according to the rules set by the Research Ethical
Committee at Lund University. Under halothane anesthesia, 2 µl of the
vector suspension was injected stereotaxically over the right SN at 5.2 mm posterior and 2.0 mm lateral to bregma, 7.2 mm ventral to dura. The
needle was kept in place for an additional 5 min before slowly being
withdrawn. rAAV-CBA--synuclein (8.2 × 1011 IU/ml),
rAAV-CBA-µ--synuclein (1.4 × 1012 IU/ml), and
rAAV-CBA-GFP (1.4 × 1011 IU/ml) were prepared and
titered as described (McLaughlin et al., 1988; Conway et al., 1997;
Zolotukhin et al., 1999). The CBA promoter used here is a chicken
 -actin promoter with enhancer elements from the cytomegalovirus
promoter, as previously described (Xu et al., 2001). For histological
analysis, the animals were killed at 1, 3, 8, and 27 weeks after
injection [green fluorescent protein (GFP) group,
n = 5; -synuclein group, n = 5-6;
and mutant -synuclein group, n = 5 for all time
points], and for biochemical analysis at 3, 8, and 27 weeks after
injection (n = 4-5 at 3 weeks and n = 5 per group at 8 and 27 weeks). A second group of animals (n = 18) were injected bilaterally with 0.2 µl of 2%
fluorogold (FG) solution in the striatum (1.0 mm anterior, 3.0 mm lateral, and 5.0 mm ventral) followed 5 d later by unilateral
vector injections, as above (n = 6 per group). These
animals were killed at 3 and 8 weeks after the virus injection
(n = 9 at each time point). A third group of animals
(n = 7) were injected with rAAV-CBA-GFP vector
bilaterally in the SN 5 d before a unilateral nigral injection of
rAAV-CBA--synuclein on the right side. These animals were killed at
3 (n = 3) and 8 weeks (n = 4) after injection.
A separate group of animals (n = 30) were injected with
the rAAV-CBA-GFP (n = 10), rAAV-CBA--synuclein
(n = 10), or rAAV-CBA-mutant µ--synuclein
(n = 10) vectors and were used in treatment with -methyl-D,L-para-tyrosine methyl
ester as described below.
Immunohistochemistry. Under pentobarbital anesthesia the
animals were perfused through the ascending aorta with physiological saline, followed by 4% ice-cold paraformaldehyde. The brains were post-fixed in the same solution for 2 hr, transferred to 25% sucrose, and sectioned on a freezing microtome at 40 µm in the coronal plane.
Immunohistochemical stainings were performed on free-floating sections
using antibodies raised against TH (mouse IgG, 1:2000; Chemicon,
Temecula, CA), GFP (chicken IgG, 1:5000; R & D systems, Minneapolis,
MN), vesicular monoamine transporter-2 (VMAT-2) (rabbit IgG, 1:5000; Chemicon), Hu (mouse IgG, 1:1000; courtesy of Dr. Steven
A. Goldman, Cornell University), and human -synuclein (mouse
IgG, 1:2000; courtesy of Dr. Virginia M. Lee, University of
Pennsylvania). Sections were rinsed three times in
potassium-phosphate buffer (KPBS) between each incubation period. All
incubation solutions contained 0.25% Triton X-100 in KPBS. The
sections were quenched for 10 min in 3%
H2O2/10% methanol. Two
hours of preincubation with 5% normal horse serum (NHS) was followed
by incubation with the primary antibody in 2% NHS at room temperature
and incubation with 1:200 dilution of biotinylated horse anti-mouse
antibody (BA2001; Vector Laboratories, Burlingame, CA) in 2% NHS,
followed with avidin-biotin-peroxidase complex (ABC Elite; Vector
Laboratories, Burlingame, CA), and visualized using
3,3-diaminobenzidine as a chromogen, mounted on chrome-alum-coated
glass slides, and coverslipped. For double fluorescent
immunohistochemical analysis of -synuclein and TH, Alexa 488 and
Cy3-conjugated secondary antibodies were used.
Assessment of the total number of TH-positive neurons in the SN was
made according to the optical fractionator principle, using the Olympus
Denmark A/S (Albertslund, Denmark) CAST-Grid system, as described
(Kirik et al., 1998). Every eighth section covering the entire extent
of the SN was included in the counting procedure. A coefficient of
error of < 0.10 was accepted. Striatal TH-positive fiber
density was measured by densitometry at three coronal levels (+1.0,
0.0,  1.0) relative to bregma. Data are represented as the mean of the
three levels and expressed as a percentage of the intact control side.
Biochemical analyses. Tissue DA and DOPAC content (Schmidt
et al., 1982) and in vitro TH enzyme activity (Reinhard et
al., 1986) were determined on tissue samples from striatum and SN (at all time points) and also from the nucleus accumbens and prefrontal cortex from the animals in the 27 week group. The tissue pieces were
weighed and frozen separately on dry ice and kept at 80°C until assayed.
Behavioral testing. Drug-induced rotation was assessed at 8, 13, 19, and 27 weeks after vector injection and after a subcutaneous injection of 0.25 mg/kg apomorphine-HCl (Sigma, St. Louis, MO) and
monitored for 40 min in automated rotometers. Data are expressed as net
full turns per minute, with contralateral turns assigned a negative value.
Paw reaching in the staircase test was performed at 24-25 weeks after
vector injection, as described (Kirik et al., 1998). After 2 d of
food deprivation the animals were placed in the test boxes baited
bilaterally with 10 food pellets on each of the four steps (45 mg). The
test was conducted over 15 min for 9 consecutive days using the
standard platform (27 mm), and an additional 5 d using a wider
platform (34 mm). Average number of total successful pellet retrievals
in the last 3 d of testing with the wide platform constituted the
dependent variable.
The stepping test was performed as described (Kirik et al., 1998) at 8 weeks after injection of the viral vectors. The animals (n = 10 per group) were tested twice daily on 3 consecutive days to define the baseline performance. On the fourth day
the animals were tested once before an intraperitoneal injection of 60 mg/kg -methyl-DL-para-tyrosine
methyl ester (dissolved in 0.02% ascorbate-saline; Sigma); and again
6, 24, and 48 hr after injection. The number of adjusting steps was
counted while the rat was moved sideways along the table surface in the
forehand direction (90 cm in 5 sec).
Statistical analysis. A two-way repeated-measures ANOVA was
performed, and post hoc analysis was performed using simple
main-effects with a Bonferroni correction of the acceptable level
of p = 0.05. For comparisons between groups and
survival times for nigral cell numbers and striatal fiber density
measurements, two-way factorial ANOVAs and post hoc analysis
were performed using simple main effects with a Bonferroni correction
of the acceptable level of p = 0.05.
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RESULTS |
To investigate the effect of nigral overexpression of
-synuclein, the rats received a single 2 µl injection of a
high-titer rAAV vector, containing either the wt or the A53T mutant of
the human -synuclein gene, unilaterally in the SN pars compacta
(SNc). Control rats received identical injections of a rAAV vector
encoding GFP. Immunohistochemistry, using an antibody recognizing human (wt and mutant), but not rat, -synuclein (Giasson et al., 2000a), revealed expression of -synuclein protein in virtually all neurons within the SNc, in a large number of neurons in the SN pars reticulata (SNr), and in neurons located within the mesencephalic reticular formation, dorsal to the SN (Fig. 2E-L). A variable
number of cells in the ventral tegmental area (VTA) were also positive. A similar pattern of cellular expression was seen also in the rAAV-GFP-injected control animals (Fig.
1). Double TH/-synuclein immunofluorescence revealed that >90% of the TH-positive neurons in
the SNc expressed the -synuclein transgene (Fig.
2M-O).
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Figure 1.
Overview of the extent of expression of GFP in the
striatum (A-D) and SN
(E-L) at 1 (A, E,
I), 3 (B, F,
J), 8 (C, G,
K), and 27 (D, H,
L) weeks after injection. GFP protein was
detected at low levels at 1 week in the SN (B, C), with
no or very low levels present in the terminals
(A). At 3 weeks the immunoreactivity at the cell
bodies was increased to cover the entire pars compacta and their
dendritic tree in the pars reticulata (F, J)
while the fiber terminals were becoming visible
(B). At 8 weeks the expression was very high both
in the striatal terminals (C) and the nigra,
without any pathology (G, K). At the final end
point of this experiment, i.e., 27 weeks after transduction, the GFP
immunoreactivity was indistinguishable from the stainings obtained at 8 weeks, suggesting that the transgene expression was maintained at high
levels between these two time points. Scale bars: A, 1 mm (applies to A-C); E, 250 µm
(applies to E-H); I, 200 µm (applies
to I-L).
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Figure 2.
Overview of -synuclein expression in the
striatum (A-D) and SN
(E-O) at 1 (A, E, I), 3 (B, F, J, M-O), 8 (C, G, K), and
27 (D, H, I) weeks after injection. The nigral DA
cells were labeled with -synuclein at 1 (E, I)
and 3 (F, J) weeks after transduction. At 3 weeks, most of the TH-positive cell bodies in the pars compacta were
also positive for -synuclein (M-O).
Similarly, nigrostriatal fiber terminals in the striatum were filled
with transgenic human -synuclein (compare A, B). In
contrast to the rAAV-GFP-injected animals, the expression of
-synuclein led to the appearance of degenerative changes both in the
nigral cell bodies (G, K) and in the striatal
terminals (C). Degeneration of cell bodies in the
pars compacta was seen as a reduction in the intensity of
-synuclein-immunoreactive cell bodies in the SN pars compacta at 8 weeks (K). The reduction in
-synuclein-positive cell bodies remained low at the 27 week time
point (H, L) while some degree of recovery was seen at
the striatal level (D). Scale bars:
A, 1 mm (applies to A-C);
E, 250 µm (applies to E-H);
I, 200 µm (applies to I-L);
M, 30 µm (applies to M-O).
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Both types of -synuclein, wt and mutant, were effectively
transported intra-axonally to their respective terminal fields: from
SNc to striatum, from VTA to limbic and cortical forebrain areas, from
SNr to superior colliculus and thalamus, and from the reticular
formation to widespread diencephalic regions. In the nigrostriatal
pathway, the cell bodies and axons, including a portion of the striatal
terminals, expressed a diffuse, cytoplasmic -synuclein
immunoreactivity at 1 week (Fig. 2A,E). At 3 weeks the entire striatal terminal network was filled (Fig.
2B), including variable portions of the VTA
projections to the olfactory tubercle, nucleus accumbens, lateral
septum, and the anterior cingulate cortex. The transduction and
transport of GFP in the rAAV-GFP-injected control animals was equally
efficient, completely filling out the cell bodies, dendrites, axons,
and axon terminals of the SNc neurons (Fig. 1). Expression increased
progressively over the first few weeks; maximal expression of the GFP
or -synuclein proteins was observed at 3-8 weeks (Figs.
1E-G, 2E-G) and was maintained at
a high level in the 27 week animals (Figs. 1D,H,L, 2D,H,L). Consistent with previous observations (cf.
Kirik et al., 2000) no signs of toxic or inflammatory reactions were
observed in the cresyl violet and hematoxylin-stained sections at any
time point, with either of the vectors used.
-Synuclein-induced neuropathology and cell death
Signs of neuronal pathology developed progressively over the first
2 months after vector injection. This included the appearance of
-synuclein-positive cytoplasmic inclusions and granular deposits (Fig. 3A-C, arrowheads),
swollen -synuclein-positive dystrophic and fragmented neurites (Fig.
3B,D-F), and dense, shrunken neuronal perikarya with
a pyknotic appearance and strong -synuclein immunoreactivity in the
cytoplasm (Fig. 3F). Some of the larger inclusions
had a clear immunonegative core surrounded by a -synuclein-positive halo (Fig. 3C,D). These changes were first observed in the
SNc neurons at 3 weeks and were further increased in magnitude at 8 weeks after injection, and resembled the -synuclein inclusions and
dystrophic neurites seen in idiopathic PD (Hayashida et al., 1993;
Arima et al., 1998; Braak and Braak, 2000; Duda et al., 2000; Giasson
et al., 2000b). None of them, however, stained positively with the
ubiquitin antibody.
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Figure 3.
Eight weeks after injection -synuclein-positive
granular inclusions (A, B, arrowheads) and discrete
intracytoplasmic inclusions where -synuclein immunoreactivity
occurred as a halo around a pale core (C, D, arrowheads)
appear in large numbers in the cells of SNc, associated with swollen
dystrophic -synuclein-positive neurites (B,
E, F) and atrophic perikarya
(F). Arrows in B,
E, F, and G indicate
intact neurites of normal size; G, H,
none of these changes occurred in GFP overexpressing SNc neurons;
I-K, the -synuclein-immunoreactive terminals in the
striatum had a normal morphology at 3 weeks
(I) but were reduced in number and
displayed large numbers of swollen, dystrophic profiles at 8 weeks
(J, P, arrowheads). At 27 weeks, fewer dystrophic fibers
were seen, but the density of -synuclein-positive terminals remained
reduced (K); L, none of these
axonal changes were observed in the GFP transduced controls, although
GFP expression was maintained at high levels still by 27 weeks;
M-Q, examples of swollen, dystrophic axon terminals in
the striatum, revealed by TH (M-O) and
-synuclein antibodies (P, Q); R,
S, expression of -synuclein did not induce any
pathological changes in nondopaminergic projections to the superior
colliculus (R) and thalamus
(S) (8 week survival). Scale bars: A,
10 µm (applies to A-H, O-Q); I, 50 µm
(applies to I-L, R, S); M, 20 µm (applies to
M, N).
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In the striatum, single -synuclein-positive swollen axons occurred
at 3 weeks after injection, and by 8 weeks abundant
-synuclein-positive dystrophic neurites were scattered throughout
the striatum, most densely in the dorsolateral part (Fig. 3J,P,Q,
arrowheads), accompanied by a marked reduction in the density of
-synuclein-positive striatal axon terminals (Figs.
2B,C, 3I,J). Distorted axonal
profiles also occurred in varying numbers in the olfactory tubercle and
nucleus accumbens, and occasionally in septum and anterior cingulate
cortex, i.e., in areas that receive projections from DA neurons in the VTA. Virtually all dystrophic neurites were positive for the
DA-synthesizing enzyme TH (Fig. 3M-O), but they did not
stain for either ubiquitin or the VMAT-2.
These pathological changes were similar, both quantitatively and
qualitatively, in animals given either wt or mutant -synuclein. No
cellular or axonal pathology was seen at any time point in the
rAAV-GFP-expressing animals (Figs. 1, 3G,H,L), showing that neither nonspecific impact of viral infection nor high expression of
any cytoplasmic protein were sufficient to induce pathological changes
of the kind seen in the -synuclein overexpressing SNc neurons.
To analyze the impact of -synuclein overexpression on
nondopaminergic neurons, we studied three other projection systems that
were efficiently transduced in the vector-injected animals: the
nigrotectal and nigrothalamic projections, and the projections from the
mesencephalic reticular formation to the diencephalon. -Synuclein
(both types) was diffusely expressed in cell bodies, axons, and
terminals in all three projection systems, but apart from single
swollen axonal profiles in the thalamus, -synuclein-positive inclusions or dystrophic neurites were conspicuously absent in these
nondopaminergic systems (Fig. 3R,S).
TH immunohistochemistry demonstrated a gradual loss of TH-positive cell
bodies in the SN, similar for both wt and mutant -synuclein, amounting to an average of 23% at 3 weeks and 55% at 8 weeks (Fig. 4A,D-H)
(data from the two -synuclein groups combined). The TH-positive cell
loss was notably variable, however, from 30-40% in some animals (Fig.
4G) to 70-80% in others (Fig. 4H).
Striatal TH-positive innervation was only marginally affected at 3 weeks after transduction (approximately 10%) (Fig.
5B) but substantially reduced
throughout the striatum (by ~50%) at 8 weeks (Fig.
5C,F). A similar reduction in the nigral DA neurons
and striatal DA innervation was revealed by the VMAT-2 antibody (data
not shown), as well as by the -synuclein staining (Fig.
2C). Analysis of adjacent sections stained for TH and
-synuclein revealed that part of the remaining fibers (identified
through their -synuclein content) had a very weak TH
immunoreactivity. Some variable TH-positive cell loss occurred also in
the VTA, and this was accompanied by a similarly variable reduction in
the TH-positive innervation in nucleus accumbens and the olfactory
tubercle (data not shown). TH-positive cell numbers and striatal
TH-positive innervation were not significantly affected in the
rAAV-GFP-injected animals at any time point, as observed in both the
TH- and GFP-immunostained sections (Figs. 1C,D,
4B,I, 5E,F).
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Figure 4.
Cell loss in SN. A,
B, Changes in total number of TH-positive cells in SNc,
as determined by stereology in the rAAV--synuclein
(A; n = 10-11 per time point) and
rAAV-GFP-injected rats (B; n = 5 per
time point); C, the number of SNc cells labeled with FG
was reduced by ~50% at 8 weeks in the rAAV--synuclein transduced
animals but remained unchanged in the GFP transduced controls
(M, injected side; L, contralateral
intact side); D-H, TH immunohistochemistry showing the
progressive loss of TH-positive cells in SNc over time, varying from
~30-40% (G) to 70-80%
(H); I, no TH-positive cell
loss was observed in the rAAV-GFP-treated animals; J,
K, the degeneration of neurons in the injected SNc
(K) was confirmed by using the neuron specific
antibody Hu. Scale bars: D, J, 250 µm
(apply to D-I and J-M, respectively).
 p < 0.0125 compared with intact side;
*p < 0.05 compared with rAAV-GFP group.
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Figure 5.
Loss of TH-positive innervation in the
striatum. A-E, TH-immunostained sections showing the
extent of loss of TH-positive striatal innervation at 3 (B), 8 (C), and 27 weeks
(D) after intranigral rAAV--synuclein
injection, as compared with the intact control side
(A), and the innervation in the rAAV-GFP injected
animals, which was unaffected (E).
F, Densitometry revealed a significant reduction in
TH-positive striatal innervation in the -synuclein transduced
animals at 8 and 27 weeks after injection ( significant difference
from both 1 week value and the GFP transduced control group at
p < 0.0125). The recovery between 8 and 27 weeks
in the -synuclein group was significant (*p < 0.0125); G, further analysis of the correlation between
nigral TH-positive cell numbers and striatal TH-positive fiber density
(% of control side) showed that the best fit was obtained with a
simple-linear regression analysis. At 8 weeks, the regression line
(dashed) had a slope of 0.695 and
y-intercept of 22.905 (p = 0.0003). However, at 27 weeks the slope of the line
(solid) was reduced to 0.478, and y-axis
intercept increased to 55.167 (p = 0.0016),
suggesting that the loss of nigrostriatal projection neurons was in
part compensated by increased innervation from the remaining cells.
Scale bar: A, 1 mm (applies to
A-E).
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To determine whether the reduced number of TH-positive cells in the SN
was attributable to an actual loss of cells or to a downregulation of
the cellular markers used, i.e., the TH enzyme and the VMAT protein, we
used three independent ways to label the nigral neurons. First, we used
a retrograde tracer, FG, injected bilaterally in the striatum 5 d
before the injection of the rAAV--synuclein vector, to label the
cells in the SNc before the insult. The number of surviving FG-positive
cells in the SNc was reduced by 32% at 3 weeks and by 52% at 8 weeks
(Fig. 4C,L,M). Cell loss was similar in the wt and
mutant -synuclein groups (p > 0.05). Second,
we used an antibody to the neuron-specific protein, Hu, as an
independent marker of surviving SNc neurons. Consistent with the FG
data, the Hu marker revealed a marked loss of SNc cells on the
rAAV--synuclein-injected side that was fully developed at 8 weeks
(Fig. 4J,K). Third, we used the rAAV-GFP
vector to prelabel the SNc neurons before the -synuclein vector
injection. These animals received bilateral rAAV-GFP injections over
the SN 5 d before the unilateral rAAV--synuclein injection. The
number of GFP-labeled cells in the SNc (as assessed 8 weeks after
injection) was markedly reduced on the -synuclein transduced side
(data not shown).
In the long-term surviving animals, analyzed 6 months after vector
injection, the signs of cell body and axonal pathology were greatly
reduced (Fig. 3K), suggesting that the damaged nigral cell bodies and axons had either degenerated (and disappeared), or recovered from the initial insult. -Synuclein protein expression, however, was still maintained at a high level in the cell bodies and
axons of the surviving nigrostriatal neurons after 6 months (Fig.
3K). Signs of recovery were also observed in the
TH-immunostained sections: the density of TH-positive fibers in the
striatum had increased, from ~50% of normal at 8 weeks to ~80% of
normal at 6 months (p < 0.0125) (Fig.
5C,D,F). A similar degree of recovery was also
observed in the VMAT-2-stained sections (data not shown). Further
analysis indicated that the recovery of striatal TH-positive fibers, in
part at least, was attributable to recovery of TH expression in a
portion of the striatal axon terminals that were weakly TH immunoreactive at 8 weeks. Although the number of TH-positive neurons
in the SNc showed a trend to an increase (from 46 to 65% of normal)
between 8 and 27 weeks (Fig. 4A), this did not reach significance. Thus, the recovery of striatal TH-positive innervation was not matched by a similar recovery of TH-positive neurons in SNc,
suggesting that axonal sprouting may have contributed to the recovery
of striatal TH-positive innervation seen in the long-term survival
animals (Fig. 5G).
Functional decline in the nigrostriatal dopamine system
The impact of -synuclein overexpression on DA synthesis,
storage, and turnover was assessed neurochemically in tissue samples from striatum and SN. Striatal DA levels (Fig.
6A) and striatal TH
enzyme activity (Fig. 6B) were reduced by 40-50% at
3 weeks, and these reductions remained unchanged at 8 and 27 weeks
after -synuclein transduction, similar for both wt and mutated
-synuclein (p > 0.05). No significant
changes were seen in the rAAV-GFP-injected control rats (Fig.
6A,B). DA levels in SN were reduced by 27.3 ± 7.9% at 3 weeks, 41.8 ± 7.0% at 8 weeks, and 28.2 ± 10.1% at 27 weeks in the rAAV--synuclein animals, whereas DA levels
in other forebrain areas (nucleus accumbens and prefrontal cortex) were
unchanged (data not shown). The reductions in striatal DA and TH
activity levels were accompanied by a significant increase in DA
turnover, measured as the DOPAC/DA ratio (Fig. 6C). This increase was only transient in the rAAV-GFP-treated animals, but was
maintained at a level of ~160% of normal at 8 and 27 weeks after
injection in the -synuclein-treated animals. No change in DA
turnover was observed in either nucleus accumbens or prefrontal cortex.
View larger version (12K):
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|
Figure 6.
Reductions in striatal DA and DOPAC and TH
activity levels over time. A, B, Striatal
DA content (A) and TH activity
(B) were reduced by ~40-50% at all time
points; C, striatal DA turnover, as assessed by the
DOPAC/DA ratio, was increased by ~60% at all time points in the
-synuclein transduced animals (filled circles)
but only transiently increased in the GFP group (open
circles). p < 0.05 (effect of group,
two-way repeated measures ANOVA).
|
|
The long-lasting reductions in striatal DA and TH activity levels in
the -synuclein transduced animals are consistent with the observed
cell loss in the SNc. However, the fact that striatal DA levels and TH
activity was already maximally reduced at 3 weeks, i.e., at a time when
the loss of TH-positive cells in the SNc was only partial (~20%),
suggests that elevated cytoplasmic levels of -synuclein (both wt and
mutant) may exert an inhibitory effect on DA synthesis and storage,
independent of its neurodegenerative action. Such an inhibitory effect
on neurotransmitter function may also explain why striatal DA and TH
enzyme activity remained low (40 to 50%) at 6 months, despite that
TH-positive fiber density in the striatum had recovered to ~75% of
normal at this time point.
Impairments in motor behavior
Motor behavior, as monitored in a battery of
drug-induced and spontaneous behavioral tests, was overall only
marginally affected in the -synuclein-treated animals at
all time points. This suggests that the observed magnitude of DA neuron
cell loss, and striatal DA depletion, was insufficient to induce easily
detectable behavioral impairments. This is in agreement with previous
studies, which have shown that significant impairments in drug-induced
and spontaneous motor behavior will appear only in animals in which the
nigral DA neurons and striatal DA levels are reduced by >50-60% (Lee et al., 1996; Kirik et al., 1998). Closer inspection of the individual animals, however, revealed a marked variability in the behavioral performance among the animals in the -synuclein-treated group (Fig.
7A,B). Thus, in both the
apomorphine rotation and the paw-reaching tests, ~25% of the
-synuclein transduced animals were significantly impaired compared
with the GFP controls (Fig. 7A,B, filled circles). This
level of impairment (10-20 pellets retrieved in the paw reaching test,
compared with 30 ± 4 pellets in the rAAV-GFP control group) is
compatible with a loss of 60-80% of the nigral DA neurons (Lee et
al., 1996; Kirik et al., 1998).
View larger version (14K):
[in this window]
[in a new window]
|
Figure 7.
Changes in motor behavior. In all three tests the
overall performance of the -synuclein transduced animals did not
differ from the GFP group (p > 0.05 for all
contrasts). However, in both the apomorphine rotation test
(A; data from the 8 week test) and the paw reaching test
(B; performed at 24 weeks) 25% of the -synuclein
transduced animals were clearly impaired in that they scored lower than
any animal in the GFP control group (filled
circles). Bars in A and B give
means ± 1 SD. C, A single low dose of the DA
synthesis inhibitor
-methyl-DL-p-tyrosine, given on the
fourth day of testing, produced a significant impairment in the
stepping test. Different from both predrug test and GFP group at
p < 0.0125.
|
|
These data suggest that behavioral impairment developed in those
-synuclein-treated rats where TH-positive nigral cell loss exceeded
a critical threshold of ~60%. It is known that partial lesions of
the nigrostriatal DA projection may be partly compensated for by an
increased DA turnover in the residual afferents (Zigmond et al., 1990),
which explains why normal striatal function can be maintained in
animals where 50-60% of the striatal DA innervation is lost. Such
compensated animals, however, are sensitive to blockers of DA
synthesis, which will induce motor impairments at dose levels that have
no effect in intact animals (Heffner et al., 1977; Marshall, 1979).
This approach, therefore, can be used to reveal the underlying functional deficit in neurochemically compensated partially lesioned animals.
The effect of low-dose DA synthesis blockade was investigated 8 weeks after injection in rats given wt rAAV--synuclein
(n = 10), mutant rAAV--synuclein (n = 10), or rAAV-GFP (n = 10), using the forelimb
stepping test (Fig. 7C). In the 3 d predrug test, all
groups performed at normal levels (~11 steps). On the fourth day, the
rats were given a single low dose of the TH enzyme inhibitor,
-methyl-p-tyrosine. Six hours later, when TH inhibition was maximal, the -synuclein transduced animals were significantly impaired both relative to the rAAV-GFP-treated controls and their own
predrug baseline (p < 0.01). The effect was
similar in both -synuclein groups (p > 0.05)
(combined in Fig. 7C). Gradual recovery of normal motor
performance was seen over the subsequent 2 d.
| |
DISCUSSION |
These results show that overexpression of wt or mutant
-synuclein can induce a progressive neurodegenerative pathology in the nigrostriatal DA neurons, characterized by -synuclein-positive cytoplasmic and axonal inclusions, dystrophic and fragmented neurites, and cell death. These degenerative changes were specific for the nigral
DA neurons, and were not seen in any of the three mesencephalic nondopaminergic neuron systems that were efficiently transduced by the
rAAV--synuclein vectors. The impact of -synuclein expression appeared to be twofold: first, a suppression of TH enzyme activity and
striatal DA levels (by ~40-50%) at a stage when -synuclein had a
diffuse cytoplasmic distribution (3 weeks after injection), followed by
a loss of 30-80% of the nigral DA neurons, coincident with the
appearance of cytoplasmic inclusions and dystrophic neurites, and the
development of significant motor impairment in those animals in which
DA neuron cell loss exceeded a critical threshold of 50-60%. More
than 90% of the TH-positive neurons in the SNc were transduced by the
rAAV vector. Consistent with previous studies using this vector (Mandel
et al., 1998; Bjorklund et al., 2000; Kirik et al., 2000) the
-synuclein and GFP transgenes showed stable, long-term expression in
the nigrostriatal neurons and high expression was maintained 27 weeks
after vector injection. Because rAAV is a single-stranded DNA virus, it
is possible that two virions with the cDNA in complementary directions
must infect a cell for successful transduction (Ferrari et al., 1996).
This opens the possibility that widely varying numbers of genome copies are inserted into a given cell. The final expression level, therefore, is likely to vary among the transduced cells. This may readily explain
why only some of the transduced SNc neurons degenerated and why cell
loss was variable from animal to animal.
Previous in vitro studies suggest that overexpression of
-synuclein can be toxic to cells and that this protein can interact with oxidative stress and mitochondrial damage to exert its cytotoxic actions (Ostrerova et al., 1999; Hsu et al., 2000; Kanda et al., 2000;
Tabrizi et al., 2000). Thus, oxidative stress, as well as increased
cellular -synuclein levels, may promote -synuclein aggregation
(Giasson et al., 2000b; Hsu et al., 2000), which in turn may lead to
mitochondrial damage and increased production of toxic free radicals
(Ostrerova et al., 1999; Hsu et al., 2000). Moreover, Giasson et al.
(2000b) have reported that -synuclein is a target for oxidative
damage and that aggregation of -synuclein into toxic inclusions may
be caused by nitration of the protein by reactive oxygen and nitrogen
species. Our results, as well as those of a recent study in
Drosophila (Feany and Bender, 2000), indicate that DA
neurons may be particularly vulnerable to -synuclein overexpression.
This may be explained by the fact that free radical production is high
in these types of cells, because of their content of both DA and iron.
Similarly, oxidative stress in combination with reduced levels of
mitochondrial complex I and -synuclein aggregation have been
proposed to underlie the selective death of nigral DA neurons in human
PD (Hsu et al., 2000; Tabrizi et al., 2000).
This "double-hit" model may account for the fact that -synuclein
overexpression in the present study induced pathological changes and
cell death in some, but not all nigral DA neurons. The signs of ongoing
pathology, including -synuclein-positive inclusions and dystrophic
neurites, subsided over time, despite the fact that the cellular
expression of wt or mutant -synuclein protein was maintained at a
high level. This suggests that those neurons that survived the initial
impact of -synuclein overexpression could survive and function even
in the presence of a maintained, increased cellular level of the
transgene product. -Synuclein per se, therefore, may not be toxic to
the cells as long as they are not exposed to excessive oxidative stress
or other events that may impair mitochondrial function. The
neurochemical data, however, suggest that -synuclein may have a
negative effect on DA neuron function, even in the absence of any overt
pathological signs. Thus, in the long-term surviving animals, 6 months
after vector injection, striatal DA levels and striatal TH enzyme
activity was reduced by an average of 40-50%, whereas the striatal
TH-positive innervation showed a partial recovery at this time point
(to ~75% of normal). Consistent with the observations in the
short-term surviving animals, i.e., 3 weeks after vector injection,
these data suggest that increased intracellular levels of -synuclein may have a direct inhibitory effect on DA synthesis and storage.
Although oxidative stress has long been hypothesized to be involved in
the pathogenesis of PD, dysfunction of protein degradation by the
ubiquitin-proteosome processing system has only recently been
implicated as a major pathogenic mechanism. The interest in this
protein degradation mechanism is attributable to the identification of
multiple forms of familial PD in which the mutated genes:
-synuclein, parkin, and ubiquitin C-terminal hydrolase L1 (UCHL1)
are all related to the proteosome degradation system (Shimura et al., 2000). Thus, overexpression of -synuclein in nigral DA neurons, at
the levels achieved in this study, may overwhelm the ability of the
proteosomal pathway to successfully process and remove excess
-synuclein from the cell. Moreover, -synuclein overexpression may
lead to increased levels of the 22 kDa form of -synuclein in the
cell (Shimura et al., 2001). Whereas this glycosylated -synuclein
isoform has not been well characterized, it may be toxic to some
neurons but not others (Shimura et al., 2000). According to this model,
all forms of -synuclein, wt or mutant, may be toxic if they are
expressed at sufficiently high levels, or if they are subjected to
oxidative damage (e.g., by oxidative metabolism of DA in nigral
neurons), or because the cellular handling of the -synuclein protein
is impaired (as may be the case in patients with mutations in the
parkin gene; Shimura et al. 2001). This contention would further
suggest that nondopaminergic neurons, outside the substantia nigra, or
the resistant DA neurons within the substantia nigra, may be protected
by more efficient handling of excess -synuclein. The fact that the
-synuclein-positive inclusions seen here did not stain for ubiquitin
would be consistent with the fact that -synuclein itself may not be
a substrate for ubiquitination (Tofaris et al., 2001). The speed by
which neurodegeneration occurs (3-8 weeks in the overexpression model
studied here, in contrast to months or years in PD patients),
furthermore, may be explained, at least in part, by the level of
overexpression of -synuclein within the cells and the ability of the
cells to handle degradation of this protein.
The technique for targeted -synuclein overexpression in the
nigrostriatal system using rAAV vectors provides a new model of PD that
reproduces some of the cardinal pathological, neurochemical, and
behavioral features of the human disease, and may, therefore provide an
interesting tool to elucidate the pathogenetic mechanisms underlying
neurodegeneration in familial or idiopathic PD. The use of viral
vectors to overexpress putative toxic proteins, such as -synuclein,
may offer distinct advantages over standard transgenic mice. First,
viral vectors may be administered at any time during the life span of
the animal to any specific anatomical location in the brain. Second,
viral vectors can be administered in one hemisphere, allowing the other
hemisphere to serve as a control, and in the model system used here,
allow the use of functional tests that rely on behavioral asymmetries.
Third, viral vector transduction has the advantage that it can be used
in rats, i.e., in a species that is more useful for behavioral studies
than mice. Finally, the use of the viral vector strategy is also
applicable to other species and may thus also allow studies on the
effects of -synuclein overexpression in primates. Indeed,
-synuclein overexpression using the viral vector strategy resulted
in more pronounced neuropathology and cell death in the nigrostriatal DA system than has been achieved so far using transgenic technology in
mice. The viral vector strategy will thus add an important research
tool to complement other, more well known, in vivo gene modification strategies.
| |
FOOTNOTES |
Received Oct. 1, 2001; revised Jan. 10, 2002; accepted Jan. 16, 2002.
This work was supported by Swedish Medical Research Council Grants
04X-3874 and 99-XG-13285, NsGene A/S (Denmark), and National Institutes
of Health Grant PO1 NS36302 (N.M.). We thank Kerstin Fogelström
and Ulla Jarl for expert technical assistance, the Powell Gene Therapy
Center Vector Core Laboratory for production of the vectors, and Dr.
Virginia M. Lee for generous supply of the -synuclein antibody.
Correspondence should be addressed to Anders Björklund,
Wallenberg Neuroscience Center, BMC A11, S-221 84, Lund, Sweden. E-mail: Anders.Bjorklund{at}mphy.lu.se.
| |
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