Resistance of ?-synuclein null mice to the
parkinsonian neurotoxin MPTP
William Dauer*†‡, Nikolai Kholodilov*, Miquel Vila*, Anne-Cecile Trillat§, Rose Goodchild*, Kristin E. Larsen*,
Roland Staal*, Kim Tieu*, Yvonne Schmitz*, Chao Annie Yuan*, Marcelo Rocha§, Vernice Jackson-Lewis*,
Steven Hersch¶, David Sulzer*?**, Serge Przedborski*††, Robert Burke*††, and Rene Hen†§
Departments of *Neurology,††Pathology,?Psychiatry,†Pharmacology, and§Center for Neurobiology and Behavior, Columbia University,
New York, NY 10027; **Department of Neuroscience, New York State Psychiatric Institute, New York, NY 10027; and
¶Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114
Communicated by Gerald D. Fischbach, Columbia University College of Physicians and Surgeons, New York, NY, August 24, 2002 (received for review
July 19, 2002)
Parkinson’s disease (PD) is most commonly a sporadic illness, and
is characterized by degeneration of substantia nigra dopamine
Rarely, PD may be caused by missense mutations in ?-synuclein.
MPTP, a neurotoxin that inhibits mitochondrial complex I, is a
prototype for an environmental cause of PD because it produces a
pattern of DA neurodegeneration that closely resembles the neu-
ropathology of PD. Here we show that ?-synuclein null mice
display striking resistance to MPTP-induced degeneration of DA
neurons and DA release, and this resistance appears to result from
from in vitro data, this resistance is not due to abnormalities of the
DA transporter, which appears to function normally in ?-synuclein
null mice. Our results suggest that some genetic and environmen-
tal factors that increase susceptibility to PD may interact with a
common molecular pathway, and represent the first demonstra-
tion that normal ?-synuclein function may be important to DA
However, the relationship between genetic and environmental
factors is poorly understood; most models of disease focus on
single genes or toxins. A major challenge of postgenomic biology
will be to link the molecular pathways modified by disease-
associated alleles to the environmental factors implicated in
There is increasing evidence for genetic susceptibility to
Parkinson’s disease (PD) (1–3). Additionally, dysfunction of a
common molecular pathway has been implicated in the familial
and sporadic forms of PD. Mutations in the gene that encodes
?-synuclein cause a rare form of dominantly inherited PD, and
?-synuclein is an abundant protein in Lewy bodies, the protein-
aceous neuronal inclusions that are the pathological hallmark of
sporadic PD (4–6). The ?-synuclein pathway is also implicated
in an autosomal recessive form of PD caused by mutations in the
gene encoding parkin (7, 8). Epidemiological and twin studies
suggest that environmental factors alter susceptibility to PD (9).
The fact that exposure of humans to the environmental toxin
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes a
syndrome that mimics the core neurological symptoms and
relatively selective dopamine (DA) neuron degeneration of PD
lends support to this concept (10, 11).
We asked whether a model neurotoxin for an environmental
cause of PD might act on a molecular pathway implicated in
genetic and sporadic forms of the disease by generating
?-synuclein null mice, and testing whether they display altered
sensitivity to MPTP-induced degeneration of substantia nigra
(SN) DA neurons.
he concept of genetic predisposition to disease suggests that
one’s genes influence susceptibility to environmental insult.
Animal Generation. A 5.7-kb EcoRV mouse ?-synuclein fragment
(Fig. 1A) was used to generate the targeting construct. A DNA
fragment containing, in order, LoxP–phosphoglycerate kinase–
mutant human ?-synuclein cDNA (‘‘STOP Cassette,’’ Fig. 1A)
was cloned 7 bp upstream of the start ATG. The mutant human
?-synuclein cDNA contains a point mutation changing amino
acid 53 from alanine to threonine, but is not transcribed because
it is downstream of the ‘‘STOP’’ sequence (Fig. 1 D and E).
Chimeric mice generated from this construct were bred with
129?Sv females and heterozygous mice were then bred to
generate ?-synuclein null mice. The null mutation was main-
tained on an inbred 129?Sv background for all experiments.
Mice were used in accordance with the National Institutes of
Health guidelines for the use of live animals and the animal
protocol was approved by the Institutional Animal Care and Use
Committee of Columbia University.
MPTP Studies. MPTP handling and safety measures were in
accordance with our published guidelines (13). Eight-week-old
?-synuclein null mice and littermate controls derived from
heterozygous matings were treated with the indicated MPTP
regimen, and after pentobarbital (35 mg/kg, i.p) anesthesia,
animals were perfused with cold 4% (wt/vol) paraformaldehyde
in 0.1M PBS (pH 7.1), and brains were postfixed in the same
buffer. Serial coronal sections (30 ?m) spanning the entire
midbrain and the mid striatum were cut and collected free
floating and processed for tyrosine hydroxylase (TH) immuno-
reactivity (polyclonal antibody, 1:1,000; Calbiochem) by using
standard methods. The total number of TH-positive SNpc
neurons was stereologically counted by using an unbiased optical
of reference (SNpc) or the size of the counted elements (neu-
rons). Densitometric analysis of striatal TH-immunostained
sections was performed by using a computerized image analysis
system as described (15). Differences were analyzed using one-
way ANOVA; where appropriate, Newman–Keuls post hoc
analysis was used to test pair-wise comparisons. The null hy-
pothesis was rejected at the 0.05 level.
Primary Neuronal Culture. Postnatal (P1–P3) ventral midbrain
neurons were cultured on rat astrocyte monolayers as described
(16). Briefly, ventral midbrain sections from wild-type or
?-synuclein pups were pooled, and incubated in papain (20
units/ml) with kynurenate (500 ?M) at 32°C under continuous
oxygenation with gentle agitation for 2 h. The tissue segments
medium, and plated onto 1-cm2astrocyte covered glass cover-
Abbreviations: PD, Parkinson’s disease; DA, dopamine; MPTP, 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine; SN, substantia nigra; DAT, DA transporter; MPP?, 1-methyl-4-
phenylpyridinium; VMAT, vesicular monoamine transporter; TH, tyrosine hydroxylase.
See commentary on page 13972.
‡To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
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slips at a density of 80,000 cells per dish. Cultures were main-
tained in serum-free media, and all experiments were conducted
in serum-free media on 14-day-old cultures. All TH? neurons
were counted on each plate. Within each genotype, the TH?
count from each experimental plate was compared with the
average value of the control plates to derive a ‘‘% control’’ value.
For all cell culture experiments, 2–5 plates per genotype were
used for each condition, and were repeated with independent
litters of mice. Experiments were pooled for statistical analysis.
Microdialysis. Mice were anesthetized with chloral hydrate (400
mg/kg i.p.) and placed in a stereotaxic frame. Dialysis probes
(membrane length, 2 mm; made as described in ref. 17) were
implanted into the right striatum. The stereotaxic coordinates
for implantation of microdialysis probes were: anterior–
posterior, ?0.6 mm; dorsal–ventral, ?4.2 mm; and lateral, 2.0
mm relative to bregma. Placement of the probe was verified by
histological examination subsequent to the experiments. After
surgery, animals were returned to their home cages with free
access to food and water. Twenty-four hours after surgery, the
dialysis probe was connected to a syringe pump and perfused at
1.5 ?l/min with ACSF composed of 147 mM NaCl, 3.5 mM KCl,
1.0 mM CaCl2, 1.2 mM MgCl2, 1.0 mM NaH2PO4, and 25.0 mM
NaHCO3(pH 7.2 ? 0.2). After a 1-h equilibration period, the
perfusates were collected every 15 min. Four control samples
were taken in the hour before the MPTP injection to determine
baseline. DA levels were measured using standard HPLC methods.
Generation of ?-Synuclein Null Mice.Wegenerated?-synucleinnull
mice by using homologous recombination to insert a transcrip-
tional blocking cassette (12) upstream of the start ATG of the
gene (Fig. 1). We confirmed that this ‘‘stop’’ cassette abolished
transcription and translation of ?-synuclein (Fig. 1). The
?-synuclein null mice are viable, fertile, and indistinguishable
from their wild-type littermates. Breeding of heterozygote
?-synuclein null mice produced all genotypes at the expected
Mendelian frequency. No gross or microscopic abnormalities
?-Synuclein Null Mice Are Resistant to MPTP. In mice, the regimen
of MPTP administration has been shown to determine the mode
of SN DA neuron cell death. An acute regimen of MPTP causes
necrotic death of DA neurons, whereas a chronic regimen causes
these cells to die via an apoptotic pathway (18, 19). We found
that ?-synuclein mutant mice display striking resistance to both
forms of MPTP-induced neurodegeneration (Fig. 2). In the
chronic regimen, MPTP-treated wild type mice display signifi-
cant losses of both TH-positive (TH?) SN cell bodies (29%
decrease) and striatal nerve terminals (40% decrease), whereas
and the recombined allele. A null allele was generated by blocking transcription from the ?-synuclein allele with a ‘‘STOP’’ cassette (12) inserted upstream of
the start ATG. The location of hybridization probes for Southern blot analysis (5? probe, 3? probe) are shown. (B) Southern blot analysis of embryonic stem cell
clones. DNA was digested with BglII and was hybridized with the 5? probe. The WT allele produced a 9.5-kb fragment, and the targeted allele produced a 6.5-kb
heterozygote mating. Tail DNA was isolated and analyzed as in B. (D) Northern blot analysis of whole brain mRNA. Total RNA was hybridized with ?-synuclein
brain protein blotted with an ?-synuclein antibody (Transduction Laboratories).
Dauer et al.
October 29, 2002 ?
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MPTP-treated ?-synuclein mutant mice do not differ signifi-
cantly from saline-treated controls on either measure. More
strikingly, wild-type mice treated with the acute MPTP regimen
exhibit an even greater loss of TH? SN cell bodies (58%
decrease) and striatal nerve terminals (65% decrease), but the
?-synuclein mutant mice again show no significant neurodegen-
eration in this paradigm. Because MPTP can down-regulate
phenotypic markers such as TH (20), we also counted Nissl
stained neurons to confirm that loss of TH staining represents
actual neuronal loss. Similar to the findings with TH staining,
wild-type mice display a significant loss of Nissl-stained neurons
in both MPTP regimens, whereas ?-synuclein mutant mice are
resistant to both paradigms: wild type: saline ? 12,805 ? 871,
chronic MPTP ? 10,589 ? 1,342, acute MPTP ? 7,716 ? 1,097;
?-synuclein null: saline ? 11,423 ? 534, chronic MPTP ?
11,132 ? 2,451, acute MPTP ? 9,751 ? 918 (for chronic and
acute MPTP, P ? 0.05 compared with saline injected mice,
Newman–Keuls post hoc. Data: means ? SEM for 4–9 mice per
MPTP Metabolism and Monoamine Transporter Function Are Normal
in ?-Synuclein Null Mice. Although the normal function of
?-synuclein is just beginning to be elucidated, much is known
about the molecular pathway through which MPTP exerts
neurotoxicity (11). Therefore, to determine the point of inter-
section between the pathways for ?-synuclein and MPTP,
we examined which aspect of the MPTP pathway was altered
in the ?-synuclein mutant mice. MPTP is a pro-toxin that is
converted to the active compound 1-methyl-4-phenylpyridinium
(MPP?) by monoamine oxidase B (predominantly glial) after
be transported into neurons by neurotransmitter transporters,
and once inside the neuron it is thought to act by inhibiting
mitochondrial complex I, resulting in cellular energy failure (21).
The selective toxicity of MPP?for DA neurons derives, at least
in part, from its high affinity for the DA transporter (DAT) (22).
MPP?is also sequestered into synaptic vesicles by the vesicular
monoamine transporter (VMAT), and sequestration into vesi-
controls were treated with a chronic (30 mg/kg/d for 5 d) or acute (80 mg/kg for 1 d) MPTP regimen, and were killed 21 d after the final chronic dose or 7 d after
the acute dose. n ? 4–9 mice per group. (A) Stereological counts of DA TH? cell bodies after saline (gray bar), chronic MPTP (white bar), or acute MPTP (black
bar). (B) TH antibody staining of the SN pars compacta (DA) after the two different MPTP regimens. (C) Quantitation (OD) of the intensity of striatal TH staining;
legend as in A. (D) TH antibody staining of the striatum after two different MPTP regimens. (Error bars ? SEM.) Both MPTP regimens produced significant reductions
of TH? DA neurons and striatal immunostaining in wild-type mice, but no significant changes in ?-synuclein?/? mice (P ? 0.05, Newman–Keuls post hoc).
?-Synuclein?/? dopaminergic neurons are strikingly resistant to MPTP-induced neurodegeneration. ?-Synuclein?/? mice and wild-type littermate
www.pnas.org?cgi?doi?10.1073?pnas.172514599Dauer et al.
cles decreases MPP?toxicity by preventing its interaction with
mitochondria (23, 24). According to this framework, cellular
resistance occurs if synaptic alterations prevent MPP?from
reaching the mitochondria (‘‘pre-complex I’’) or if the cell is
better able to withstand the consequences of complex I inhibi-
tion (‘‘post-complex I’’).
We first determined that normal levels of MPP?were reach-
ing the DA neurons of the mutant mice by assessing striatal
MPP?levels in both MPTP regimens; these studies show that
there is no significant genotype difference in the amount of
MPP?reaching the striatum in either MPTP regimen. For the
acute regimen, MPP?levels 90 min after the final 20 mg/kg
injection of MPTP were: wild type ? 18.5 ? 0.4, KO ? 19.1 ?
0.4 ?g/g striatal tissue (n ? 6 wild type, 7 knockout). For the
chronic regimen, MPP?levels 90 min after single i.p. injection
of 30 mg/kg MPTP were: wild type ? 10.6 ? 0.9, knockout ?
9.2 ? 0.9 ?g/g striatal tissue (n ? 10 wild type, 11 knockout). We
then asked whether abnormalities of the pre-complex I pathway
might be responsible for the MPTP resistance of ?-synuclein
mutant mice, by analyzing the level and activity of DAT and
VMAT in the mutant mice. Although it has been suggested that
quantification of DAT by using immunofluorescence or receptor
autoradiography demonstrated that DAT levels are normal in
the striatum of ?-synuclein mutant mice (Fig. 3 A and B). DAT
function, as measured by [3H]DA uptake, was normal in primary
cultures of mutant DA neurons (Fig. 3C). Additionally, no
abnormality in the kinetics of DAT or VMAT were found in
experiments in striatal synaptosomes or isolated synaptic vesicles
?-Synuclein Null DA Neurons Have a Pre-Complex I Alteration That
Prevents MPP?from Reaching Mitochondrial Complex I. To further
probe the mechanism for the MPTP resistance of ?-synuclein
null mice, we used primary cultures of postnatal midbrain
neurons. Because these cultures are prepared by plating wild-
type or ?-synuclein null neurons onto wild-type rat astrocytes,
only the neuronal genotype differs between the cultures. There-
fore, in addition to its tractability, a particular advantage of this
system is that it can be used to identify cell autonomous effects
in the plated neurons.
We first confirmed that cultured ?-synuclein null DA neurons
retain resistance to MPP?(Fig. 4A), suggesting that a cell
autonomous effect of ?-synuclein null DA neurons makes them
resistant to MPP?. The fact that the degree of resistance
observed in vitro is less striking than observed in vivo suggests
that additional factors contributing to resistance in the animals
may not be captured in the in vitro setting. The presence of
exogenous neuroprotectants (superoxide dismutase 1, glial-
derived neurotrophic factor) in the cultures (added as part of the
standard primary culture protocol) also likely contribute to this
We next tested the sensitivity of cultured DA neurons to
rotenone, a mitochondrial toxin that selectively inhibits complex
I at the same site as MPP?. In contrast to MPP?, rotenone is
highly lipophilic, does not depend on DAT for cellular entry, and
is not sequestered into synaptic vesicles. Therefore, rotenone
would bypass a pre-complex I site of resistance, but if a post-
complex I mechanism accounts for the MPTP resistance, the
mutant neurons should also be resistant to rotenone. Rotenone
was found to bypass the resistance of ?-synuclein mutant DA
neurons (Fig. 4B), and in fact, ?-synuclein null DA neurons were
significantly more sensitive to 20 nM rotenone. Interestingly, the
enhanced sensitivity to rotenone may reflect a synergistic effect
of complex I inhibition and DA toxicity (26–28), as ?-synuclein
null mice display abnormalities in the synaptic handling of DA
(29). The clear dissociation between the effects of MPP?and
rotenone suggests that the MPTP resistance of ?-synuclein null
mice is caused by a pre-complex I alteration that prevents MPP?
from inhibiting complex I.
To further probe the pre-complex I pathway, we studied an
acute in vivo effect of MPTP, MPP?-induced efflux of striatal
dopamine. This response is believed to result from complex
I-related energy failure, because it occurs with the same time
course as the lactate elevation that occurs secondary to MPP?-
induced complex I inhibition, and other interventions that
inhibit mitochondrial respiration also produce this effect (30–
32). We reasoned that if a pre-complex I abnormality was
preventing MPP?from accessing and inhibiting mitochondrial
complex I, as suggested by the rotenone experiment, there
should be deficient MPTP-induced DA efflux in the ?-synuclein
null mice. Using in vivo microdialysis in awake freely moving
animals, we observed a greater than 60% decrease in the efflux
mice. (A and B) Analysis of DAT in the striatum of wild type and ?-synuclein
mutant mice using DAT antibody staining (A) and [3H]mazindol (30 nM)
autoradiography (B) revealed no genotype-related differences. Immunoflu-
orescence was measured by obtaining all images at the same empirically
defined exposure that contained no saturated pixels, and all images were
taken at the same rostrocaudal striatal plane. (C) [3H]DA uptake in ventral
The amount and activity of the DAT is normal in ?-synuclein mutant
Table 1. Kinetics of [3H]DA and [3H]MPP?handling in striatal
synaptosomes and synaptic vesicles
80 ? 6
2.4 ? 1.2
72 ? 10
2.1 ? 1.1
302 ? 60
1.7 ? 0.9
298 ? 73
1.9 ? 1.3
3.4 ? 0.6
8.9 ? 0.5
3.6 ? 0.6
8.9 ? 0.8
0.64 ? 0.20.85 ? 0.3
All values are average ? standard deviation. Values are the average of 2–3
independent experiments. Kmand Kdvalues are nM; Vmaxunits are pmol?mg
of protein per min. Bmaxunits are pmol?mg protein. Transport rate is counts
per min (Va?B).
Dauer et al.
October 29, 2002 ?
vol. 99 ?
no. 22 ?
of striatal dopamine in ?-synuclein mutant mice (Fig. 5A). Like
amphetamine, MPTP-induced efflux of DA is thought to occur
via reversal of the DAT. Therefore, to control for the possibility
that deficient MPTP-induced DA efflux might reflect an inabil-
ity of DAT to mediate reverse transport in ?-synuclein mutants,
we measured amphetamine-induced DA release, and found no
difference between mutant and wild-type mice (Fig. 5B). This
result further suggests that DAT function is normal in
?-synuclein mutant mice, and is consistent with the concept that
the differences observed in MPTP-induced DA efflux relate to
complex I inhibition.
Our experiments suggest that in the absence of ?-synuclein
function, MPP?is unable to inhibit mitochondrial complex I.
This conclusion is supported by three observations: (i) rotenone
can bypass the resistance of ?-synuclein mutant DA neurons,
the ?-synuclein mutant mice, and (iii) ?-synuclein mutant mice
are resistant to regimens of MPTP that lead to different modes
of cell death, a finding inconsistent with a role for ?-synuclein in
a discrete post-complex I cell death-related pathway (Fig. 2).
Pre-complex I-mediated MPTP resistance could result from
either decreased DAT-mediated cellular entry of MPP?or
enhanced vesicular storage of MPP?. Although a report using
transient transfection suggests that ?-synuclein interacts with
DAT and is involved in trafficking it to the plasma membrane
(25), our studies in whole animals, striatal tissue, cultured DA
neurons, and synaptosomes suggest that DAT function is normal
in ?-synuclein null mice (Figs. 3 and 5B, and Table 1).
Vesicular sequestration of MPP?, the second component of
the pre-complex I pathway, promotes resistance to MPTP (23,
24). Although our data on isolated vesicles suggest that VMAT
kinetics are normal in ?-synuclein mutant mice, increased
vesicular sequestration of MPP?may be related to a change in
?-synuclein null DA neurons. Ventral midbrain cultures were treated for two
days with 10 or 50 ?M MPP?(A) and 20 or 100 nM rotenone (B). ?-Synuclein
mutant DA neurons displayed significant resistance to death at both concen-
trations of MPP?, but did not show resistance to rotenone; there were
significantly fewer ?-synuclein?/? TH? neurons at the 20 nM rotenone dose
(P ? 0.05, Newman–Keuls post hoc). For each condition, n ? 5–10 plates per
genotype. Values are represented as a percentage of untreated controls.
(Error bars ? SEM.)
Dissociation between MPTP- and rotenone-induced death of
vivo microdialysis of striatal dopamine efflux after a single i.p injection of 30
mg/kg MPTP. Gray circle, wild type; black circle, ?-synuclein?/?. (B)
?-Synuclein mutant mice display normal amphetamine-induced DA release.
Legend as in A. Microdialysis values are represented as a percentage of the
average of four baseline measurements taken in the hour preceding MPTP
injection; each animal was used as its own baseline control. There were no
significant differences in baseline values. n ? 3–8 mice per genotype.
MPTP-induced DA efflux is deficient in ?-synuclein null mice. (A) In
www.pnas.org?cgi?doi?10.1073?pnas.172514599 Dauer et al.
a dynamic property of vesicles not measured in an in vitro Download full-text
preparation, such as movement of vesicles between different
pools. A number of observations are consistent with a role for
?-synuclein in vesicular dynamics (29, 33, 34). Alternatively,
there may be a direct requirement for ?-synuclein in MPTP-
mediated neurotoxicity, consistent with the suggestion that
?-synuclein-dopamine adducts may be an important mediator of
toxicity in DA neurons (35).
It is not known whether the mutations in ?-synuclein that
cause dominantly inherited PD alter the normal function of the
protein, or endow it with a novel property, such as the ability to
aggregate. Findings that overexpression of wild-type ?-synuclein
in mice and flies is toxic, and that ?-synuclein accumulates but
does not aggregate in parkin-related PD, are consistent with the
possibility that an increase in normal ?-synuclein function leads
to cellular toxicity (7, 36, 37). The current study lends further
support to the hypothesis that the normal function of ?-synuclein
is important to DA neuron viability, and raises the possibility
that alterations in normal ?-synuclein function may modify the
vulnerability of DA neurons to an environmental toxin.
The finding that ?-synuclein null mice are resistant to MPTP-
induced neurodegeneration provides an example of how a
disease-related gene can modulate susceptibility to a relevant
environmental insult. Together with the fact that the ?-synuclein
pathway has also been implicated in sporadic and parkin-related
PD, this finding suggests that ?-synuclein participates in a
pathway of central importance to the survival of dopaminergic
We thank Ms. N. Romero and Ms. L. Zhang for expert animal care,
Leonidas Stefanis for providing a synuclein cDNA, and Luca Santarelli,
Brett Lauring, and Eric Schon for helpful comments on the manuscript.
Thanks to Stanley Fahn for his invaluable mentorship and support.
Thanks also to Michio Hirano and Ramon Marti for HPLC analysis and
Monica Mendelsohn for ES cell injection. This work was supported by
the Parkinson’s Disease Foundation, Lowenstein Foundation, Goldman
Foundation, National Institute of Neurological Disorders and Stroke,
and National Institute on Drug Abuse. W.D. is the recipient of a
Howard Hughes Medical Institute Postdoctoral Fellowship for Physi-
cians, and M.V. is the recipient of the Human Frontier Science Program
1. Scott, W. K., Nance, M. A., Watts, R. L., Hubble, J. P., Koller, W. C., Lyons,
K., Pahwa, R., Stern, M. B., Colcher, A., Hiner, B. C., et al. (2001) J. Am. Med.
Assoc. 286, 2239–2244.
2. Kruger, R., Vieira-Saecker, A. M., Kuhn, W., Berg, D., Muller, T., Kuhnl, N.,
Fuchs, G. A., Storch, A., Hungs, M., Woitalla, D., et al. (1999) Ann. Neurol. 45,
3. Martin, E. R., Scott, W. K., Nance, M. A., Watts, R. L., Hubble, J. P., Koller,
W. C., Lyons, K., Pahwa, R., Stern, M. B., Colcher, A., et al. (2001) J. Am. Med.
Assoc. 286, 2245–2250.
4. Polymeropoulos, M. H., Lavedan, C., Leroy, E., Ide, S. E., Dehejia, A., Dutra,
A., Pike, B., Root, H., Rubenstein, J., Boyer, R., et al. (1997) Science 276,
5. Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S.,
Przuntek, H., Epplen, J. T., Schols, L. & Riess, O. (1998) Nat. Genet. 18,
6. Spillantini, M. G., Schmidt, M. L., Lee, V. M., Trojanowski, J. Q., Jakes, R. &
Goedert, M. (1997) Nature 388, 839–840.
7. Shimura, H., Schlossmacher, M. G., Hattori, N., Frosch, M. P., Trockenbacher,
A., Schneider, R., Mizuno, Y., Kosik, K. S. & Selkoe, D. J. (2001) Science 293,
8. Chung, K. K., Zhang, Y., Lim, K. L., Tanaka, Y., Huang, H., Gao, J., Ross,
C. A., Dawson, V. L. & Dawson, T. M. (2001) Nat. Med. 7, 1144–1150.
9. Marion, S. A. (2001) Adv. Neurol. 86, 163–172.
10. Langston, J. W., Ballard, P., Tetrud, J. W. & Irwin, I. (1983) Science 219,
11. Przedborski, S. & Vila, M. (2001) Clin. Neurosci. Res. 1, 407–418.
12. Lakso, M., Sauer, B., Mosinger, B., Jr., Lee, E. J., Manning, R. W., Yu, S. H.,
Mulder, K. L. & Westphal, H. (1992) Proc. Natl. Acad. Sci. USA 89, 6232–6236.
13. Przedborski, S., Jackson-Lewis, V., Naini, A. B., Jakowec, M., Petzinger, G.,
Miller, R. & Akram, M. (2001) J. Neurochem. 76, 1265–1274.
14. Liberatore, G. T., Jackson-Lewis, V., Vukosavic, S., Mandir, A. S., Vila, M.,
McAuliffe, W. G., Dawson, V. L., Dawson, T. M. & Przedborski, S. (1999) Nat.
Med. 5, 1403–1409.
15. Burke, R. E., Cadet, J. L., Kent, J. D., Karanas, A. L. & Jackson-Lewis, V.
(1990) J. Neurosci. Methods 35, 63–73.
16. Przedborski, S., Khan, U., Kostic, V., Carlson, E., Epstein, C. J. & Sulzer, D.
(1996) J. Neurochem. 67, 1383–1392.
17. Trillat, A. C., Malagie, I., Scearce, K., Pons, D., Anmella, M. C., Jacquot, C.,
Hen, R. & Gardier, A. M. (1997) J. Neurochem. 69, 2019–2025.
18. Tatton, N. A. & Kish, S. J. (1997) Neuroscience 77, 1037–1048.
19. Jackson-Lewis, V., Jakowec, M., Burke, R. E. & Przedborski, S. (1995)
Neurodegeneration 4, 257–269.
20. Tatton, W. G., Kwan, M. M., Verrier, M. C., Seniuk, N. A. & Theriault, E.
(1990) Brain Res. 527, 21–31.
21. Nicklas, W. J., Youngster, S. K., Kindt, M. V. & Heikkila, R. E. (1987) Life Sci.
22. Javitch, J. A. & Snyder, S. H. (1984) Eur. J. Pharmacol. 106, 455–456.
23. Reinhard, J. F., Jr., Diliberto, E. J., Jr., Viveros, O. H. & Daniels, A. J. (1987)
Proc. Natl. Acad. Sci. USA 84, 8160–8164.
24. Liu, Y., Peter, D., Roghani, A., Schuldiner, S., Prive, G. G., Eisenberg, D.,
Brecha, N. & Edwards, R. H. (1992) Cell 70, 539–551.
25. Lee, F. J., Liu, F., Pristupa, Z. B. & Niznik, H. B. (2001) FASEB J. 15, 916–926.
26. Nakao, N., Nakai, K. & Itakura, T. (1997) Brain Res. 777, 202–209.
27. McLaughlin, B. A., Nelson, D., Erecinska, M. & Chesselet, M. F. (1998)
J. Neurochem. 70, 2406–2415.
28. Burrows, K. B., Nixdorf, W. L. & Yamamoto, B. K. (2000) J. Pharmacol. Exp.
Ther. 292, 853–860.
P. E., Shinsky, N., Verdugo, J. M., Armanini, M., Ryan, A., et al. (2000) Neuron
30. Ferger, B., Eberhardt, O., Teismann, P., de Groote, C. & Schulz, J. B. (1999)
J. Neurochem. 73, 1329–1332.
31. Rollema, H., Kuhr, W. G., Kranenborg, G., De Vries, J. & Van den Berg, C.
(1988) J. Pharmacol. Exp. Ther. 245, 858–866.
32. Buyukuysal, R. L. & Mete, B. (1999) J. Neurochem. 72, 1507–1515.
33. Murphy, D. D., Rueter, S. M., Trojanowski, J. Q. & Lee, V. M. (2000)
J. Neurosci. 20, 3214–3220.
34. Lotharius, J., Barg, S., Wiekop, P., Lundberg, C., Raymon, H. K. & Brundin,
P. (2002) J. Biol. Chem., ePub, M205518200v1.
35. Conway, K. A., Rochet, J. C., Bieganski, R. M. & Lansbury, P. T., Jr. (2001)
Science 294, 1346–1349.
36. Masliah, E., Rockenstein, E., Veinbergs, I., Mallory, M., Hashimoto, M.,
Takeda, A., Sagara, Y., Sisk, A. & Mucke, L. (2000) Science 287, 1265–1269.
37. Feany, M. B. & Bender, W. W. (2000) Nature 404, 394–398.
Dauer et al.
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