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The Journal of Neuroscience, April 1, 2003, 23(7):2564
Cocaine Abusers Have an Overexpression of  -Synuclein in
Dopamine Neurons
Deborah C.
Mash1, 2,
Qinjie
Ouyang1,
John
Pablo1,
Margaret
Basile1,
Sari
Izenwasser1,
Abraham
Lieberman1, and
Richard J.
Perrin3
Departments of 1 Neurology and 2 Molecular
and Cellular Pharmacology, University of Miami School of Medicine,
Miami, Florida 33101, and 3 Department of Cell and
Structural Biology, University of Illinois at Urbana-Champaign, Urbana,
Illinois 61801
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ABSTRACT |
 -Synuclein is a presynaptic protein that has been implicated as
a possible causative agent in the pathogenesis of Parkinson's disease.
The native protein is a major component of nigral Lewy bodies in
Parkinson's disease, and full-length -synuclein accumulates in Lewy
neurites. Here we present evidence that -synuclein levels are
elevated in midbrain dopamine (DA) neurons of chronic cocaine abusers.
Western blot and immunoautoradiographic studies were conducted on
postmortem neuropathological specimens from cocaine users and
age-matched drug-free control subjects. The results demonstrated that
-synuclein levels in the DA cell groups of the substantia
nigra/ventral tegmental complex were elevated threefold in chronic
cocaine users compared with normal age-matched subjects. The increased
protein levels in chronic cocaine users were accompanied by changes in
the expression of -synuclein mRNA in the substantia nigra and
ventral tegmental area. Although -synuclein expression is prominent
in the hippocampus, there was no increase in protein expression in this
brain region. The levels of  -synuclein, a possible negative
regulator of -synuclein, also were not affected by cocaine exposure.
-Synuclein protein levels were increased in the ventral tegmental
area, but not the substantia nigra, in victims of excited cocaine
delirium who experienced paranoia, marked agitation, and hyperthermia
before death. The overexpression of -synuclein may occur as a
protective response to changes in DA turnover and increased oxidative
stress resulting from cocaine abuse. However, the accumulation of
-synuclein protein with long-term cocaine abuse may put addicts at
increased risk for developing the motor abnormalities of Parkinson's disease.
Key words:
cocaine; postmortem; brain; synucleins; DA; delirium
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Introduction |
The synucleins are a family of
soluble presynaptic proteins that are abundant in neurons and include
-synuclein, -synuclein, and  -synuclein (for review, see
Clayton and George, 1998 ; Lavedan, 1998, 1999). Although the functions
of the synucleins are poorly understood, it has been suggested that
synucleins are important regulatory elements of synaptic vesicle
transport processes (Jenco et al., 1998; Maroteaux and Scheller,
1999). An overexpression of human -synuclein has been
implicated in the etiology of two neurodegenerative diseases:
Parkinson's disease (Polymeropoulos et al., 1997; Kruger et al., 1998)
and Alzheimer's disease (Ueda et al., 1993). The native protein is a
major component of nigral Lewy bodies in sporadic Parkinson's disease
(Spillantini et al., 1997, 1998). The pathological hallmark of
Alzheimer's disease involves widespread deposition of -amyloid,
which leads to the death of hippocampal and cortical neurons. A
non-amyloidogenic fragment of -synuclein is an integral constituent
of amyloid plaques (Ueda et al., 1993), and this fragment facilitates
the aggregation of the 42 amino acid -amyloid peptide (Jensen
et al., 1997; Paik et al., 1998). Pathological inclusions of
-synuclein occur in several animal models (Betarbet et al., 2000;
Feany and Bender, 2000; Kowall et al., 2000; Masliah et al., 2000). The widespread ability of -synuclein to be toxic in various cells and
conditions suggests that some cells may not tolerate this gene product.
In Drosophila melanogaster, -synuclein overexpression resulted in degeneration of dopaminergic (DAergic) neurons and motor deficits (Feany and Bender, 2000). These studies suggest that
altered -synuclein function can trigger the neurodegeneration of
dopamine (DA) neurons.
The mesolimbic DAergic system is an important pathway mediating
reinforcement and addiction to psychostimulants (Self and Nestler,
1998). Cocaine potentiates DAergic neurotransmission by binding to the
DA transporter and blocking neurotransmitter uptake, leading to marked
elevations in synaptic DA (for review, see Giros and Caron, 1993).
Long-term cocaine abuse leads to neuroadaptive changes in the signaling
proteins that regulate DA homeostasis. DA transporter binding sites are
upregulated in vitro in the postmortem brain of cocaine
addicts (Little et al., 1993, 1998; Staley et al., 1994, 1995; Mash et
al., 2002) and in vivo in acutely abstinent cocaine-dependent individuals (Malison et al., 1995, 1998). The direct
binding and functional coupling of -synuclein to the DA transporter
has been shown to increase DA uptake and accelerate DA-induced
apoptosis (Lee et al., 2001). Because -synuclein binds to the DA
transporter and affects its activity, alterations in -synuclein
expression may occur as a neuroadaptive response to chronic cocaine exposure.
The issue of cocaine-induced DA neurotoxicity has not been resolved
(Bartzokis et al., 1999). Subclinical parkinsonian-like motor
abnormalities that persist over a 3 month period of abstinence have
been reported in cocaine-dependent subjects (Bauer, 1996). Cocaine-dependent subjects exhibited impaired performance on tests of
motor system functioning and showed significant resting hand tremor
that did not remit during a 3 month period of verified abstinence. The
possibility of long-lasting and possibly permanent brain changes is
supported by brain imaging studies showing structural as well as
functional abnormalities that persist after 4-9 months of abstinence
(Pascual-Leone et al., 1991; Volkow et al., 1992; Bartzokis et al.,
1996). We report here that -synuclein is overexpressed in midbrain
DA neurons from cocaine abusers. The overexpression of -synuclein is
a neuroadaptive response to cocaine exposure that may put cocaine
addicts at risk for degenerative changes in DA neurons, including the
motor abnormalities of Parkinson's disease.
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Materials and Methods |
All chemicals were obtained from Sigma (St. Louis,
MO). Iodine and C14 standards and
Hyperfilm were purchased from Amersham Biosciences (Piscataway, NJ).
Neuropathological tissue specimens. Postmortem
neuropathological specimens were obtained during routine autopsy from
age-matched drug-free control subjects. Medicolegal investigations of
the deaths were conducted by forensic pathologists. The circumstances of death and toxicology were reviewed carefully before classifying a
cocaine intoxication case with or without preterminal excited delirium
(ED) (Ruttenber et al., 1997). ED victims exhibited an acute onset of
bizarre and violent behavior, which was characterized by one or more of
the following: aggression, combativeness, hyperactivity, extreme
paranoia, demonstration of unexpected strength, or incoherent shouting
(Wetli and Fishbain, 1985; Wetli et al., 1996). The syndrome of fatal
ED is defined as accidental cocaine toxicity in subjects who exhibited
bizarre and violent behavior (as described above) followed by sudden
death (Ruttenber et al., 1997). All cocaine cases (n = 21) were evaluated for common drugs of abuse and alcohol, and positive
urine screens were confirmed by quantitative analysis of blood. Blood
cocaine was quantified using gas-liquid chromatography with a nitrogen
detector. Drug-free age-matched control subjects (n = 13) were selected from accidental or cardiac sudden deaths with
negative urine screens for all common drugs and where there was no
history of licit or illicit drug use before death.
Immunoautoradiography and Western blotting. Serial coronal
sections (30 µm) from fresh-frozen blocks of human brain were cut on
a cryostat, thaw-mounted on subbed slides, and dried under reduced
pressure at 4°C. Adjacent sections were stained for Nissl substance
to delineate cytoarchitecture. Slide-mounted sections of the midbrain
were postfixed in 4% paraformaldehyde, 20% ethanol, 20% ethylene
glycol, 10% glycerol, and 0.32 M sucrose in PBS, pH 7.4, and then blocked for 2 hr in 0.3% Tween 20, 3% bovine serum
albumin, 3% goat serum, and 0.05% NaN3 in PBS,
pH 7.4, and incubated overnight at 4°C with a polyclonal
anti--synuclein-peptide (amino acid 111-131) antibody (AB5038;
Chemicon International, Temecula, CA) diluted 1:1000.
After a 1 hr incubation with 125I-goat
anti-rabbit IgG (PerkinElmer Life Sciences, Boston, MA) secondary antibody, the sections were rinsed eight times for 10 min
each and dried. The slide-mounted tissue was apposed to Hyperfilm along
with [125I] standard for 7 d at
 80°C. To establish antigen specificity for the -synuclein
antibody, controls included no primary antibody or an irrelevant IgG.
In some initial experiments, sections from control subjects and cocaine
users were labeled with purified IgG1 isotype raised against amino acid
residues 98-115 of human -synuclein (Dr. Matthew Farrer, Mayo
Clinic, Jacksonville, FL) that verified a comparable pattern of
expression and distribution of the protein. All sections were
immunolabeled at the same time and in the same batch of chemicals to
minimize variability.
Brain samples from the substantia nigra and hippocampus (100 mg tissue
punch) were sonicated in lysis buffer containing protease inhibitors
(25 mM Tris, pH 7.4, with 300 µg/ml phenylmethanesulfonyl fluoride, 2 µg/ml leupeptin, 16 µg/ml benzamidine, 2 µg/ml
pepstatin A, and 50 µg/ml lima bean trypsin inhibitor) and
centrifuged at 12,000 × g for 10 min at 4°C
(Langston et al., 1998). The supernatants were collected and the
proteins were measured by the bicinchoninic acid assay (Pierce
Chemical, Rockford, IL). Protein extracts (0.01-0.2 µg) were
processed on SDS-PAGE on 15% separating and 4% stacking gel
and transferred to Immobilon-P nitrocellulose (Millipore, Bedford, MA). Blots were blocked in 100% methanol for 30 sec and then
incubated for 2 hr at room temperature with either anti--synuclein (1:1000) or anti--synuclein (1:1000) antibodies (Chemicon
International) diluted with PBS containing 1% nonfat dry milk
and 0.04% Tween 20, followed by 30 min in a donkey anti-rabbit
horseradish peroxidase-conjugated secondary antibody (Amersham
Biosciences) diluted 1:10,000. Blots were stripped and reprobed
with a monoclonal anti--tubulin (1:1000) in PBS containing 1%
nonfat dry milk and 0.04% Tween 20 to confirm that equal amounts of
protein were loaded for each case. Proteins were visualized by
SuperSignal West Pico Chemiluminescent Substrate (Pierce
Chemical). Exposures with maximal signal yet below the photographic saturation point were quantitatively analyzed by densitometry and compared with dilutional standards of recombinant -synuclein protein (gift from Dr. Euijung Jo, Centre for
Neurodegenerative Diseases, University of Toronto). Optical densities
were determined using IMAGE (version 1.44, NIH Shareware) and expressed
as arbitrary units.
In situ hybridization in human brain tissue.
Full-length human -synuclein cDNA containing the entire coding
region and 290 bp of the 3' untranslated region was cloned into pGEM-3
vector. The integrity of the construct was confirmed by sequencing. A 400 base riboprobe of the 3' end of -synuclein cDNA (352-752 bp)
was determined to be specific for the -synuclein target gene transcript (National Center for Biotechnical Information Database; BLAST Search). The 35S-labeled RNA probe
was prepared using a ScriptEase Probe Kit (Novagen,
Madison, WI). In situ hybridization was done by a
modification of the method of Kholodilov et al. (1999). Slide-mounted
brain tissue sections were fixed by immersion in 4%
paraformaldehyde-PBS, pH 7.4, for 5 min, rinsed with PBS, and
incubated in triethanolamine acetic anhydride solution for 10 min. The
sections were defatted in a series of graded ethanol washes and then
chloroform and were dried at 37°C for 1-2 hr. Sections were
prehybridized at 40°C for 2 hr with 1:1 formamide/prehybridization
mix. Hybridization was performed in a 1:1 formamide/hybridization mix
at 55°C overnight. Labeled riboprobe was added to a final activity of
~3000 cpm/µl of the 1:1 formamide/hybridization solution. Sections
were washed in 2× saline sodium citrate (SSC) and then treated with
RNase at 37°C for 45 min. Sections were then washed in 2× SSC for 60 min at 40°C, followed by immersion in 4 l of 0.1× SSC
containing 0.05% sodium pyrophosphate and 14 mM
2-mercaptoethanol at 40°C for 3 hr with gentle stirring, dehydrated
in ethanol, vacuum-dried, and apposed to Hyperfilm at 80°C for 2 weeks together with brain paste and 14C
standards (Amersham Biosciences). The specificity of
riboprobe hybridization was confirmed by comparing sections hybridized
with antisense riboprobes with two control conditions: serial sections hybridized with sense strand probes and antisense-hybridized sections that were predigested with RNase A. In both of these conditions, there
was no detectable hybridization signal.
Data analysis. Slide-mounted sections of the midbrain were
apposed with radioactive standards to Hyperfilm for the times
indicated. Films were scanned using a Howtek Scanmaster 3 at 400 dots
per inch using a transparency illuminator. After background
subtraction, two-dimensional maps were created to allow specific
radioactivity levels to be superimposed on the sections. The midbrain
sections were compared with adjacent Nissl-stained sections along with a delineated map of observable tracts and nuclei at a resolution of
150 × 150 × 500 µm. The region-of-interest measurements
were exported in Brain (version 3.0) to PICT files readable by the Canvas program (version 3.5). The resulting tagged image file format
for RGB color files was converted to pseudocolor format in specific
activity units. ANOVA followed by Dunnett's post hoc comparisons for significance at p < 0.05 or better was
performed using Prism (Graphpad, San Diego, CA).
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Results |
Cocaine-related fatalities were identified and classified as part
of an ongoing case-control study of the toxicology reports, scene
descriptions, supplemental background information, and autopsy findings
(Escobedo et al., 1991; Ruttenber et al., 1997). The cocaine users were
selected for the present study on the basis of evidence of a number of
surrogate variables of chronicity, including the review of previous
arrest records, hospital and previous substance abuse treatment
admissions, and pathological signs determined at autopsy (e.g.,
perforation of the nasal septum, needle track marks, cardiomegaly, and
"crack" lung). Most of the cocaine cases had informant reports of
binge cocaine use in the days immediately before death. Review of
hospital records and interviews with next-of-kin informants were done
to confirm pattern and history of illicit drug use. The cocaine users
(n = 13; 12 male/1 female), ED victims
(n = 8; 8 male), and control subjects (n = 13; 11 male/2 female) were not significantly
different on demographic characteristics. Their mean (±SEM) ages were
36.6 ± 2.2, 32.3 ± 1.9, and 33.1 ± 2.6, respectively.
The postmortem intervals did not differ significantly across groups
(cocaine users = 12.7 ± 1.70; ED = 13.1 ± 1.1;
control subjects = 15.9 ± 1.5). Excited cocaine delirium
cases have been included in this study as a comparison group. This
psychiatric syndrome comprises delirium with marked agitation,
respiratory depression, hyperthermia, and sudden death (Wetli et al.,
1996; Ruttenber et al., 1997). The mode of death and agonal state are
important variables when investigating postmortem human brain (Wester
et al., 1985). All of the cocaine deaths were sudden because of cocaine
intoxication. ED victims survived longer, but all of the cases included
in this study had cocaine measured in blood, suggesting that these
subjects died only a few hours after their cocaine binge (Ruttenber et al., 1997). Four of the control cases were homicide victims of gunshot
wounds, one was a blunt trauma death, and the remaining eight cases
died from cardiac sudden death. Thus, all of the cocaine users and
control subjects died suddenly.
Cocaine and benzoylecgonine (BE) were detected in blood and urine at
the time of death for all cocaine intoxication cases and ED victims. No
other illicit drugs were detected in urine screens, suggesting that
none of the subjects had recent poly-drug abuse. Two of the cocaine
cases and three of the ED victims had alcohol in postmortem blood at
low levels (blood alcohol concentration <0.08%). The
concentrations of cocaine and its main metabolite BE were measured in
blood samples obtained at autopsy. The average (mean ± SEM) blood
levels of cocaine and BE were 3.7 ± 0.6 and 4.8 ± 1.0 mg/l
in the cocaine users. The ED victims had lower levels of cocaine
(1.9 ± 0.7 mg/l) and comparable levels of BE (4.4 ± 1.2 mg/l) in blood. In cases that had alcohol measured in blood,
cocaethylene levels were 0.2 ± 0.1 for cocaine users and 0.3 ± 0.1 for ED victims. None of the control cases tested positive for
any neuroactive drug or metabolite. None of the cases selected for this
study tested positive for opiates in blood or in urine toxicology screens.
Regulation of -synuclein levels by cocaine exposure
The effect of cocaine exposure on -synuclein protein expression
was examined by immunoautoradiography with an anti--synuclein antibody. The substantia nigra was faintly labeled in drug-free control
subjects (Fig. 1B). In
contrast, immunolabeling was intense over the substantia nigra and
ventral tegmental area (VTA) in cocaine users (Fig.
1D). Quantitative region-of-interest measurements of
-synuclein immunolabeling were taken to assess the regulatory effects of cocaine on protein expression in cocaine users with and
without preterminal excited delirium. Densitometric measurements demonstrated a threefold elevation in the substantia nigra and ventral
tegmental area of the cocaine users as compared with drug-free age-matched control subjects (p < 0.01) (Fig.
2). Interestingly, in the brains of ED
victims there was a significant but smaller increase in -synuclein
immunolabeling in the ventral tegmental area (p < 0.05) (Fig. 2). However, unlike in other cocaine users, the
densities of -synuclein immunolabeling in the substantia nigra in ED
victims were not elevated but were comparable with the levels measured
in brains of the age-matched drug-free control subjects.
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Figure 1.
Cocaine-induced -synuclein upregulation in
substantia nigra/ventral tegmental complex. Nissl-stained sections from
a representative control subject (A) and cocaine
user (C) show the location of the substantia
nigra (SN) and ventral tegmental area
(Vt). B, D, -Synuclein
immunoautoradiography in adjacent sections from a control subject
(B) and cocaine user (D).
Pseudocolor codes represent a rainbow scale (red = highest densities; yellow to green = intermediate densities; blue to
purple = low densities). RN, Red
nucleus; lgn, lateral geniculate nucleus.
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Figure 2.
Densitometric measurements of -synuclein
immunolabeling in cocaine users and control subjects. -Synuclein
levels were increased in the substantia nigra and ventral tegmental
area in the cocaine users (COC; n = 13) but not in victims of excited delirium (ED;
n = 8). No change in -synuclein was observed in
the hippocampus. Significant differences from control values:
*p < 0.05; **p < 0.01.
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There was a trend toward reduced -synuclein labeling for some ED
cases, but the average protein levels were not significantly different
from control subjects, in agreement with the results of Western
immunoblot analysis (Fig. 3). Victims of
fatal ED failed to demonstrate an increase in -synuclein protein,
although these cases had comparable premorbid histories of cocaine
abuse and were positive at autopsy for cocaine and benzoylecgonine in
both blood and urine. Specific -synuclein immunolabeling was seen in
the hippocampus over the pyramidal layer of the CA1 sector (data not
shown). Region-of-interest measurements gave comparable values across
cocaine users, ED victims, and age-matched drug-free control subjects
(Fig. 2). There was no increase seen in the entorhinal cortex or
adjacent deep layers of the neocortex with cocaine exposure (data not
shown). There was no labeling under control conditions, using either no
primary antibody or IgG isotype.
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Figure 3.
Western blotting of -synuclein protein.
A, Dilutional standards of recombinant -synuclein
protein (0.01-0.2 µg). Serial dilution of human -synuclein was
loaded across the lanes and probed by Western blotting with enhanced
chemiluminescence. B, Standard curve generated from the
densitometric values obtained from computer analysis of digitized film
from immunoblot. A linear relationship between optical density and the
amount of recombinant -synuclein protein was observed with a
correlation coefficient of 0.98. C, Representative blots
with an antibody against -synuclein show 19 kDa bands in the human
substantia nigra from control subjects, cocaine users
(COC), and excited delirium (ED) victims.
D, Representative blots with an antibody against
-synuclein show 14 kDa bands in the human substantia nigra and
illustrate no change in -synuclein protein expression with cocaine
exposure.
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- and -synuclein-immunoreactive proteins in
cocaine users
-Synuclein expression was examined by Western immunoblot
analysis with the anti-human -synuclein antibody in the substantia nigra from cocaine users, ED victims, and control subjects and compared
with dilutional standards of recombinant -synuclein (Fig. 3). A
single band was observed at the expected molecular mass of 19 kDa for
all cases (Fig. 3C). Denser -synuclein-positive bands
were consistently observed in cocaine users as compared with control
subjects. There was a significant increase in protein expression
observed for the cocaine users (p < 0.01) (Fig.
4). The amount of -synuclein
(nanograms per microgram of total protein) measured in the substantia
nigra was 48.1 ± 2.8 in cocaine users, 11.3 ± 1.8 for ED
victims, and 14.4 ± 2.0 in control subjects. These results demonstrate
that the protein levels were the same for ED victims as compared with
control subjects. The marked increase in the heat-soluble fraction of
-synuclein protein determined by Western immunoblot analysis was
comparable in magnitude with the regional densitometric analysis of
total protein levels measured in slide-mounted midbrain sections from
cocaine users. In keeping with immunoautoradiographic analysis, there
was no change in -synuclein protein expression within the human
hippocampus with cocaine exposure (Fig. 4).
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Figure 4.
Densitometric analysis of -synuclein blots in
substantia nigra and hippocampus. Results demonstrate upregulation of
-synuclein protein in cocaine users (COC)
but not excited delirium (ED) cases in the substantia
nigra. There was no change in -synuclein protein expression in the
hippocampus. Significant differences from control values:
**p < 0.01.
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Immunoblot analysis with a -synuclein-specific antibody was done in
human midbrain from cocaine users and control subjects. -Synuclein
is the 34 amino acid non-amyloidogenic homolog of -synuclein. A
single band was observed for -synuclein at the expected molecular
mass of 14 kDa in cocaine users with and without preterminal excited
delirium and in control subjects (Fig. 3D). The upregulation
of -synuclein in the substantia nigra (Fig. 3C)
contrasted with the lack of an increase in -synuclein (Fig. 3D) and -tubulin (50 kDa; data not shown). Densitometric
analysis of -synuclein immunoblots gave values for cocaine users and
ED victims that were not different from control subjects (data not shown). These results demonstrate that the cocaine-induced upregulation of -synuclein was not accompanied by changes in -synuclein.
Expression of -synuclein mRNA in DA neurons
A specific hybridization signal was observed in young control
subjects for the -synuclein gene in the DA cells of the midbrain (Fig. 5A). In the midbrain,
the label was clearly localized to the substantia nigra. Increased
expression was seen in the ventral tegmental area in cocaine users as
compared with control subjects (Fig. 5B). Within the pars
compacta, the ventral tier exhibited the strongest hybridization signal
in cocaine users (Fig. 5B). Increased expression was
confirmed by image analysis, which revealed a significant increase in
the substantia nigra and ventral tegmental area in cocaine users as
compared with control subjects (p < 0.01) (Fig.
6). In contrast to these findings,
-synuclein mRNA measured over the DA cells of the substantia
nigra/ventral tegmental area complex was not increased in ED victims as
compared with cocaine users (Fig. 6).
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Figure 5.
Expression of -synuclein in substantia nigra
and ventral tegmental area. A shows a representative
age-matched control subject (white, male, age 34), and B
shows a cocaine user who died suddenly (black, male, age 31). Film
autoradiograms of midbrain sections reveal intense expression for
-synuclein mRNA, particularly in the ventral tier of the substantia
nigra pars compacta. Hipp, Hippocampus;
RN, red nucleus; SN, substantia nigra;
Vt, ventral tegmental area.
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Figure 6.
Quantitative analysis of -synuclein mRNA
expression. Measurements were made in the substantia nigra and ventral
tegmental area of cocaine users (COC) and
excited delirium (ED) victims. Significant differences
from control values: **p < 0.01.
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Discussion |
We have investigated the effect of cocaine abuse on the expression
of -synuclein protein and mRNA in postmortem human brain. These
findings provide the first demonstration of adaptations in
-synuclein expression with cocaine exposure in midbrain DA neurons.
In cocaine users, -synuclein mRNA was elevated in the substantia
nigra and ventral tegmental area compared with age-matched drug-free
control subjects. The functional relevance of this increase was
confirmed by robust increases in the levels of -synuclein protein.
Victims of excited cocaine delirium failed to show an upregulation of
-synuclein protein or mRNA levels in the substantia nigra.
Significant albeit smaller increases in the levels of -synuclein protein were measured in the ventral tegmental area in ED victims. The
failure of -synuclein to upregulate in the substantia nigra in these
subjects is in keeping with the lack of an increase in DA transporter
function and binding sites (Mash et al., 2002). ED victims exhibit
profound neuropsychiatric complications and hyperthermia before death,
suggesting that there may be a different pattern of -synuclein
regulation in this subgroup of chronic cocaine users.
Previous high resolution in situ hybridization histochemical
studies have demonstrated that -synuclein is expressed in
melanin-containing neurons of the human substantia nigra (Solano et
al., 2000). Non-melanized cells in the substantia nigra do not contain
-synuclein. Thus, the increased levels of immunoreactivity in the
substantia nigra and ventral tegmental area measured in cocaine users
likely reflect increases in neuronal expression of the protein in DA
neurons. -Synuclein levels were unchanged in the hippocampus or
adjacent temporal neocortex, brain regions that have relatively high
protein expression in normal subjects (Murphy et al., 2000). Because
the increase in -synuclein with cocaine exposure was confined to DA
cell groups and was not observed over the hippocampus or neocortex, the
effect of cocaine on -synuclein expression appears to be specific
for DA-containing cells of the midbrain.
Lewy bodies are aggregates of -synuclein, ubiquitin, neurofilaments,
and other proteins (Spillantini et al., 1998). In the course of
neurodegeneration in Parkinson's disease, susceptible regions and
vulnerable nerve cell populations become progressively impaired because
of the extensive presence of Lewy neurites and Lewy bodies (Neystat et
al., 1999; Del Tredici et al., 2002). Brain regions in which Lewy
bodies have been reported in Parkinson's disease (substantia nigra and
locus coeruleus) and diffuse Lewy body disease (hippocampus and deep
layers of the entorhinal and neocortex) (Forno, 1996; McKeith et al.,
1996) express the -synuclein gene. In cocaine users, none of the
extranigral regions expressing -synuclein had elevations in the
levels of the protein. This observation suggests that the substantia
nigra may be an induction site in brain for cocaine-induced increases
in -synuclein. However, Lewy bodies are also found in monoaminergic
neurons of the coeruleus/subcoeruleus complex and caudal raphe nuclei
(Forno, 1996; Del Tredici et al., 2002). Because cocaine also blocks
the reuptake of norepinephrine and serotonin, it will be important to
determine in future studies whether cocaine affects -synuclein
expression in brainstem monoamine neurons.
The synucleins are enriched in presynaptic terminals, as shown by
combined immunocytochemistry and subcellular fractionation studies in
the songbird, rat, and human brain (for review, see Clayton and George,
1998). A contribution of brain -synuclein to regulation or support
of synaptic plasticity is suggested by early studies implicating
synelfin in the canary song-learning process (George et al., 1995).
Synelfin is a 143 amino acid homolog of the human -synuclein protein
that is highly expressed during critical periods of song plasticity in
birds (for review, see Clayton and George, 1998, 1999). Whether the
change in gene expression is a cause or consequence of synaptic
reorganization is unknown. However, evidence exists for changes in
synuclein gene expression and protein localization after synaptic
activity or metabolic stress (Maroteaux et al., 1988; Maroteaux
and Scheller, 1991; Clayton and George, 1998). Synuclein mRNA is
upregulated in the substantia nigra when nigrostriatal neurons are
developing target contacts, sprouting, and forming synapses (Hsu et
al., 1998). Graybiel (Canales and Graybiel, 2000) has suggested that
different neural circuits become activated in response to cocaine as a
result of repeated administrations and involve DA and glutamate as key co-players in regulating basal ganglia loops that affect both locomotion and stereotypy. Thus, changes in the expression of -synuclein protein may be an adaptive response to cocaine in reward-related neurons of the nigral/ventral tegmental area complex.
Cocaine inhibits the activity of the DA transporter (Ritz et al., 1987;
Madras et al., 1989; Reith and Selmeci, 1992) and increases vesicular
DA uptake (Brown et al., 2001). Repeated exposure to cocaine may shift
the normal balance of DA signaling through modifications of vesicular
release and recycling of the neurotransmitter. These changes are likely
linked to altered DA uptake function, which is upregulated in human
cocaine addicts (Mash et al., 2002). The regulatory effects of cocaine
on DA transporter binding site densities have been studied in
vitro in the postmortem brain of cocaine addicts and in
vivo in acutely abstinent cocaine-dependent individuals. Some of
the previous studies (Little et al., 1993, 1998; Staley et al.,
1994, 1995), but not all (Hurd and Herkenham, 1993; Wilson et
al., 1996), have reported increased numbers of DA transporters using
radiolabeled cocaine congeners. One possible explanation for
conflicting results across studies is a loss of DA nerve terminals in
more advanced and severely dependent cocaine users (Wilson et al.,
1996). -Synuclein complexes with the human DA transporter through
the direct binding of the non-A amyloid component of -synuclein
to the C-terminal tail of the DA transporter (Lee et al., 2001).
-Synuclein-DA transporter complexes facilitate the membrane
clustering of the DA transporter, thereby accelerating DA uptake
in vitro (Lee et al., 2001). Concomitant increases in -synuclein and DA transporter numbers and function in cocaine abusers provide additional support for a role of -synuclein in regulating DAergic tone.
Transgenic mice deficient in -synuclein demonstrate an attenuated
locomotor response to amphetamine (Abeliovich et al., 2000). Because
amphetamine is known to exert its psychostimulant effects through the
DA transporter, it is possible that this effect may be caused by a
change in trafficking of the DA transporter to the cell surface
membrane in -synuclein-deficient mice. However, a recent study
suggests that the DA transporter densities were not lower in
-synuclein null mice as compared with wild-type mice (Dauer et al.,
2002). In contrast to these results, overexpression of wild-type
-synuclein in mice leads to increased densities of the DA
transporter (Richfield et al., 2002), suggesting a
concentration-dependent effect of the protein on the trafficking of the
DA transporter. Although -synuclein-deficient mice appear to have a
normal complement of DA neurons and terminals, they display
abnormalities in the synaptic handling of DA (Abeliovich et al., 2000).
The recovery of peak DA release after an initial stimulus is more rapid
in -synuclein-deficient mice, in keeping with an inhibitory role for
-synuclein in activity-dependent modulation of neurotransmitter release. These observations suggest that the protein is an essential presynaptic, activity-dependent negative regulator of DA
neurotransmission (Abeliovich et al., 2000). Unlike in other chronic
cocaine users, compensatory increases in DA transporter densities or
uptake function do not occur in ED victims, although their severity and
amount of cocaine abuse are the same (Wetli et al., 1996; Mash et al., 2002). We have speculated that the lack of neuroadaptive increase in DA
uptake function may contribute to the persistence of a
hyperdopaminergic state. Within the DA cell body fields, -synuclein
protein levels were elevated only in the VTA, but not the substantia
nigra, in cocaine users presenting with preterminal ED. This pattern of differential protein expression may reflect a progressive change in
-synuclein levels that occurs in the nigral/VTA complex depending on
the duration and intensity of cocaine misuse. Because disruption of the
synthesis, function, or possible aggregation of -synuclein protein
is predicted to increase DA release from DA neurons (Abeliovich et al.,
2002), the lack of a compensatory increase in -synuclein in the
substantia nigra may augment DA release in particular striatal regions
during a cocaine "binge" in ED subjects. Further studies are needed
to link coordinated regulation of the DA transporter or other
presynaptic proteins with -synuclein expression to the progression
of habitual drug-seeking and the occurrence of cocaine delirium. The
adaptive change in -synclein in DA neurons from human cocaine users
is another example of the extreme neuronal plasticity that occurs in
response to altered DA homeostasis with long-term cocaine abuse.
Overexpression of wild-type or mutant forms of -synuclein in
cultured human DA neurons leads to apoptosis, an effect that is blocked
by the addition of a tyrosine hydroxylase inhibitor (Xu et al., 2002).
These observations suggest that it is the combination of -synuclein
and DA that causes cell death. The pattern of cocaine-induced increases
in -synuclein expression described here suggests that increased
protein expression is part of a neuronal response to chronic cocaine
exposure. The upregulation of -synuclein alone by chronic cocaine
abuse is not likely to lead to increased protein aggregation in
neurites and intracytoplasmic bodies. However, one consequence of the
ability of cocaine to inhibit DA reuptake is marked elevations in
extracellular DA. Also, cocaine causes a redistribution of plasmalemma
vesicles and increases vesicular DA uptake (Brown et al., 2001). Rapid
vesicular sequestration of the neurotransmitter will limit the
formation of reactive oxygen species such as DA-quinone (Cubells et
al., 1994; Hastings et. al., 1996; Stokes et al., 1999). Because excess
-synuclein potentiates production of reactive oxygen species by DA
(Zhou et al., 2000; Xu et al., 2002) and the mutant protein causes
increased susceptibility to DA toxicity (Tabrizi et al., 2000),
alterations in DA turnover by cocaine may accelerate the formation of
toxic forms of the protein. The protofibrillar conformation of
-synuclein undergoes kinetic stabilization in the cell by
catecholamines, including DA and norepinephrine (Conway et al., 2001).
DA in the oxidized form appears to sustain the toxic protein within the
cell as a DA--synuclein adduct (Sulzer, 2001). The Lewy neurites
and inclusions of Parkinson's disease are made up of fibrillar
-synuclein protein, as opposed to the unfolded form measured in
normal brain (Giasson et al., 2000). Thus, the cocaine-induced
upregulation of -synuclein may be initially an adaptive response
that could turn toxic depending on the local cellular milieu.
The effects of cocaine on -synuclein may occur only with long-term
cocaine abuse. We have compared cocaine users that came to autopsy with
documented histories of the highest patterns of cocaine use with
individuals with no exposure. This is the case also for ED victims, who
only demonstrated elevations in -synuclein expression within the
ventral tegmental area, but not the substantia nigra. Although every
attempt is made to obtain information about the premortem pattern of
cocaine use (amount, duration, and total lifetime use), it is more
difficult to collect absolute exposure measures from interviews with
informants and next-of-kin. However, a cocaine intoxication death in a
recreational user is an extremely rare occurrence, and most of the
cases that come to autopsy have many surrogate variables of chronic
cocaine use, including crack lung and perforation of the nasal septum.
Whether chronic cocaine use is neurotoxic to DA neurons remains
uncertain, and animal data suggest that amphetamines are more likely to
cause damage to these cells than cocaine (Bennett et al., 1993; Ellison
and Switzer, 1993). However, many cocaine-dependent subjects show signs
of subclinical parkinsonism that are reversible with protracted periods
of abstinence (Bauer, 1996). Young cocaine-dependent subjects have
significant resting hand tremor that does not remit during a 3 month
period of verified abstinence, suggesting the possibility of neurotoxic
damage to DA terminals.
The epidemic of crack cocaine use began in the United States
around 1986 (Escobedo et al., 1991). Many crack cocaine-addicted individuals continue to misuse the drug for decades despite attempts at
abstinence. We speculate that abnormal -synuclein expression may be
a risk factor for the development of cocaine-related brain changes
involving cognitive and motor systems. Overexpression of -synuclein
may be a toxic gain that puts cocaine addicts at risk for degenerative
changes in DA neurons, including the motor abnormalities of
Parkinson's disease.
| |
FOOTNOTES |
Received July 29, 2002; revised Dec. 23, 2002; accepted Jan. 13, 2003.
This study was supported by National Institutes of Health Grant
DA-06227 and the National Parkinson Foundation, Miami, FL. We thank the
members of the Clayton lab for helpful discussions, Dr. Euijung Jo for
providing recombinant -synuclein protein, and Dr. Matthew Farrer for
providing anti--synuclein antibody that was developed in the
laboratory of Dr. John Hardy.
Correspondence should be addressed to Dr. Deborah C. Mash, Department
of Neurology (D4-5), 1501 Northwest Ninth Avenue, Miami, FL 33136. E-mail: dmash{at}newssun.med.miami.edu.
| |
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TOP
ABSTRACT
Introduction
