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Vol. 61, Issue 5, 1124-1131, May 2002
Molecular Neuropsychiatry Section, Intramural Research Program, National Institutes of Health/National Institute on Drug Abuse, Baltimore, Maryland
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
Summary
References
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The clinical abuse of methamphetamine (METH) is a major concern because it can cause long-lasting neurodegenerative effects in humans. Current concepts of the molecular mechanisms underlying these complications have centered on the formation of reactive oxygen species. Herein, we provide cDNA microarray evidence that METH administration caused the induction of c-Jun and of other members involved in the pathway leading to c-Jun activation [stress-activated protein kinase/Jun N-terminal kinase (JNK3), Crk-associated substrate-Cas and c-Src] after environmental stresses or cytokine stimulation. Reverse transcription-polymerase chain reaction analysis confirmed these increases and also showed that the expression of JNK1 and JNK3 but not JNK2 was also increased in the METH-treated mice. Western blot analysis showed that METH increased the expression of c-Jun phosphorylated at serine-63 and serine-73 residues. Other upstream members of the JNK pathway, including phosphorylated JNKs, mitogen-activated protein kinase kinase 4, mitogen-activated protein kinase kinase 7, Crk II, Cas, and c-Src were also increased at the protein level. These values returned to baseline by 1 week after drug treatment. These results are discussed in terms of their support for a possible role of the activation of the JNK/Jun pathway in the pathophysiological effects of METH.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Summary
References
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Methamphetamine (METH) is an
illicit drug; its use has substantially increased in several regions of
the United States and the world. The frequency of emergency department
admissions for acute intoxication with amphetamines has also increased
dramatically (Lan et al., 1998
; Perez et al., 1999). These admissions
are usually caused by the intake of large doses of METH, which can
amount to several grams of the drug (Connell, 1958; Kramer et al.,
1967). Acute intoxication with METH can be associated with acute
psychosis, belligerent behaviors, or multiple organ failures,
resembling clinical signs and symptoms caused by heatstroke (Lan et
al., 1998). In addition, myocardial infarction, stroke, and death have been reported (Perez et al., 1999). Moreover, long-term abuse of METH
can result in a paranoid-hallucinatory psychosis, which may be
indistinguishable from paranoid schizophrenia (Yui et al., 1999).
Reports that long-term METH abusers can also have cognitive deficits,
including memory loss (Simon et al., 2000) are of significant experimental and therapeutic interest. In addition to its
prolonged neurological and psychiatric effects, METH abuse may
be associated with persistent neurodegenerative indices in the human
brain (Ernst et al., 2000). For example, a marked reduction in striatal
dopamine transporters has been demonstrated in the brains of METH
abusers with the use of positron emission tomography scan (Volkow et
al., 2001) and postmortem human studies (Wilson et al., 1996). This suggests that in humans, METH abuse can cause functional alterations in
dopamine (DA)- (Volkow et al., 2001) and non-DA-innervated (Ernst et
al., 2000) brain regions. Some of these abnormalities might be caused
by large doses (0.3-1.0 g of METH taken 8-10 times daily for 3-10
days) of the drug used by some METH abusers (Connell, 1958; Kramer et
al., 1967). This binge administration is followed by a crash period,
which might be related in part to METH-induced monoamine depletion in
various brain regions.
Studies in animals have also documented that METH can indeed cause
substantial damage to various brain regions. These abnormalities include decreases in the striatal levels of DA, tyrosine hydroxylase activity, and loss of DA transporters (Cadet and Brannock, 1998). More
recently, studies from our laboratory have shown that subacute administration of METH can cause apoptosis in several brain regions of
mice, including the striatum, the cortex, the lateral habenula, and the
hippocampus (Deng et al., 1999, 2001). These models of short-term METH
injections might best represent phenomena associated with overdoses in
humans, whereas more long-term METH injections might be more akin to
the binging patterns observed in many METH abusers (Davidson et al.,
2001).
Several laboratories are actively seeking to decipher the cellular and
molecular mechanisms for the short- and long-term effects of the
amphetamines. For example, a growing consensus suggests that reactive
oxygen species (ROS) are important players in METH-induced neurodegeneration (Cadet and Brannock, 1998). Nevertheless, the complete picture of METH-induced neurodegeneration has yet to be drawn
fully. It is highly likely that the dissection of intracellular signals
elicited by METH-induced ROS will provide insights into the cellular
and molecular cascades that are involved in the actions of this illicit
stimulant. The available evidence has recently implicated the
activation of immediate early genes in METH-induced damage because METH
administration can cause increases in c-fos mRNA and in AP-1 DNA
binding activity in mouse brain (Sheng et al., 1996b; Asanuma and
Cadet, 1998). The induction in c-fos may serve a protective function
because c-fos null mice showed greater severity of METH-induced
apoptosis and dopaminergic toxicity (Deng et al., 1999).
Despite these observations, much remains to be done to elucidate the
role of AP-1-related genes (Herdegen and Waetzig, 2001) in this model
of neurodegeneration, because there is a dearth of evidence on the
possible role of the c-fos binding partner, c-Jun, in the physiological
or toxic effects of these illicit drugs. For example, although it has
been shown that d-amphetamine can cause increased expression
of c-Jun mRNA in rats (Persico et al., 1995) or that METH can cause
activation of phosphorylated Jun kinase in the striatum (Hebert and
O'Callaghan, 2000), there is a need to document whether other members
of the JNK/SAPK pathway are also activated by METH.
To tackle these issues, we used the cDNA array approach to identify
putative genes that might be induced by toxic doses of METH (Cadet et
al., 2001). We found that METH caused an early pattern of induction of
transcription factors and a delayed pattern of changes in genes related
to cell death and DNA repair (Cadet et al., 2001). The changes in genes
related to cell death and DNA repair could be related to METH-induced
apoptosis (Deng et al., 1999, 2001). Furthermore, we had reported that
several genes, including c-Jun and c-Src, were also up-regulated by the
drug (Cadet et al., 2001). Because some recent observations had shown a
very close relationship between ROS, Src, Cas, and JNK activation (Yoshizumi et al., 2000), we chose to test the idea that METH, which
can induce ROS production (Cadet and Brannock, 1998; Jayanthi et al.,
1998), might also cause the activation of molecular events similar to
those reported by Yoshizumi et al. (2000). Herein, we have extended our
cDNA array results (Cadet et al., 2001) by using RT-PCR and Western
blot analysis and have provided a detailed time course of the effects
of METH on the SAPK pathway.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Summary
References
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Animals and Drug Treatment.
Male CD-1 mice (Charles River
Laboratories, Inc., Raleigh, NC) weighing 30 to 45 g were used.
Mice received a single dose of 40.0 mg/kg i.p. METH or saline. Animals
showed no evidence of seizures, and all the mice survived this dose
regimen for the duration of the study, at least 7 days, when they were
sacrificed. The single METH dose approach is an acceptable regimen that
has been used by other investigators to cause marked reductions in the
levels of DA, 5-hydroxytryptamine, and tyrosine hydroxylase similar to
those observed with the multiple-dose schedule (Fukumura et al., 1998;
Barrett et al., 2001). This approach also causes astrogliosis, which is
also typical of METH toxicity (Fukumura et al., 1998). Recently, we
showed that METH, given in this fashion, can cause widespread apoptosis
in several brain regions (Deng et al., 2001; Jayanthi et al., 2001).
The mice were sacrificed at various times after drug treatment. Brain
tissues were processed for various assays as described below. All
procedures involving the use of animals were according to the National
Institutes of Health Guide for the Care and Use of Laboratory Animals
and were approved by the local Animal Care Committee.
Probing, Hybridization, and Analysis of cDNA Arrays. cDNA array analysis was performed by using Atlas Mouse Arrays (#7741-1; BD Biosciences Clontech, Palo Alto, CA) that contain 588 cDNA segments spotted in duplicate side by side on a nylon membrane. Probing of cDNA arrays was performed as described in the BD Biosciences Clontech Atlas cDNA Expression Arrays User Manual (PT3140-1). Briefly, total RNA was isolated from the frontal cortex of saline- and METH-treated mice sacrificed 2, 4, and 16 h after the single dose of saline or METH. All tissues were placed in denaturing solution, homogenized, and extracted with phenol-chloroform by using Atlas Pure RNA Isolation Kit (BD Biosciences Clontech) and subsequently digested with DNase-I to remove any trace of DNA. After confirming the integrity of total RNA on a denaturing formaldehyde gel, 50 µg of total RNA was used as a template in a 10-µl reverse transcription reaction. A pooled set of primers complementary to the genes represented on the array (7741-1; BD Biosciences Clontech) was used for the reverse transcription probe synthesis, which was radiolabeled with 32P-dATP and purified by passage over CHROMA SPIN-200 columns (BD Biosciences Clontech).
The cDNA expression array filters were prehybridized in ExpressHyb (BD Biosciences Clontech) for 30 min at 71°C and were hybridized with 32P-labeled first-strand cDNA probes overnight at 71°C. After a high-stringency wash, the membranes were exposed to a PhosphorImaging screen for 24 h at room temperature. The exposed screen was scanned on a Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA) at 100-µm resolution and stored as Molecular Dynamics .gel files. The array spots on the array images were analyzed using a theoretical pattern of template of Array Vision software for Windows NT (version 4; Imaging Research, St. Catherines, ON, Canada). The template elements were aligned over the true array spot, and the spot intensity value was quantified after the subtraction of set background. The signal for any given gene was calculated as the average of the signals from the two duplicate cDNA spots.Reverse Transcription (RT)-PCR and Detection of mRNA
Expression.
RT-PCR with gene-specific Custom Atlas Array primers
(BD Biosciences Clontech) were also used to analyze the levels of mRNAs of interest. These were carried out according to the manufacturer's protocol in both cortical and striatal tissue. RNA was extracted individually from three mice per time point. Total RNA (1 µg) was
reverse-transcribed with oligo(dT) primer. For PCR amplification of
cDNA, gene-specific Custom Atlas Array primers (BD Biosciences Clontech) were used to confirm changes in the levels of expression of
genes of interest. The sequences for these primers are described in
Table 1. The PCR reactions followed the
protocol for AmpliTaq Gold (Applied Biosystems, Foster City, CA):
preheating at 95°C for 10 min; repeating cycles of 95°C for 30 s, 64°C for 30 s, and 72°C for 30 s; and extending at
72°C for 7 min.
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Western Blot Analysis. Analysis of c-Jun, phospho c-Jun (ser63 and ser73), JNK, and phospho JNK (New England Biolabs, Beverly, MA); phospho SEK1/MKK 4 (Cell Signaling Technology Inc., Beverly, MA); Src (Upstate Biotechnology, Lake Placid, NY); Cas (Transduction Laboratories, Lexington, KY); Crk II, MKK4, and MKK7 (SantaCruz Biotechnology Inc., Santa Cruz, CA) protein concentration in the striatum of METH-treated CD-1 mice was performed by the use of Western blot. Briefly, mice striata were homogenized in a buffer containing 320 mM sucrose, 5 mM HEPES, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml pepstatin. Homogenates were centrifuged at 5000g for 5 min, and the supernatant fraction was subsequently centrifuged at 30,000g for 30 min. The resulting pellet was resuspended in the sample buffer (62.5 mM Tris-HCl, 10% glycerol, 2% SDS, 0.1% bromphenol blue, and 50 mM dithiothreitol) and subjected to SDS-polyacrylamide gel electrophoresis. Proteins were separated electrophoretically in SDS-12% polyacrylamide gels and transferred to Hybond nytran membrane. Membrane blocking, primary and secondary antibody incubations, and chemiluminescence reactions were conducted for each antibody individually according to the manufacturer's protocol. The intensity of each band was measured using an IS-1000 Digital Imaging System (Alpha Innotech) and quantitated using Flurochem version 2.0 software (AlphaEaseFC analysis software).
Statistical Analysis. Data were analyzed using statistical software (StatView 4.02; SAS Institute, Cary, NC). Statistical analysis was performed using analysis of variance (ANOVA) followed by Fisher's protected least significant difference (PLSD). The null hypothesis was rejected at the 0.05 level.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Summary
References
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METH Causes Up-Regulation of c-Jun and c-Src.
The single-dose
approach has recently been used to cause neurotoxic damage in rats
(Fukumura et al., 1998) and neuronal cell death in several brain areas
of mice (Deng et al., 2001; Jayanthi et al., 2001). Using this
approach, we had performed gene expression analysis by using the mouse
Atlas cDNA Array (BD Biosciences Clontech) to determine whether
METH-induced adverse actions were associated with changes in specific
genes that might be involved in the initiation and progression of
METH-induced transcriptional effects (Cadet et al., 2001). Cluster
analysis helped to identify an early pattern of up-regulation of
transcription factors, including members of c-Jun family (Cadet et al.,
2001), and a delayed pattern of up-regulation of genes related to cell
death and DNA repair (Cadet et al., 2001; Jayanthi et al., 2001).
Specifically, we found that METH caused differential regulation of
several Bcl-2 family genes, which resulted in two distinct clusters
consisting of up-regulation of pro-death gene expression such as
Bax and down-regulation of anti-death gene expression such as Bcl-2
(Jayanthi et al., 2001). In what follows, we report further
characterization of the pattern of changes observed in the c-Jun family
and related genes incorporated in the array (Cadet et al., 2001) by
providing RT-PCR confirmation and extension of the time course of these
changes. We also provide detailed measurements of proteins encoded by
these genes. As stated above, we chose to do so because the JNK/SAPK
pathway has been implicated in several models of neurotoxic processes
in the central nervous system (Herdegen and Waetzig, 2001).
). Figure 1A
shows that there was little variability between transcript levels
measured by duplicate spots corresponding to a given gene
(r2 = 0.951, p < 0.0001). The increased expression of the genes of interest found on the
BD Biosciences Clontech array (#7741-1) was confirmed by RT-PCR
analysis (Fig. 1B). Because of the possible involvement of the AP-1
family of genes in METH neurotoxicity (Sheng et al., 1996b; Deng et
al., 1999) and of the reported participation of the JNK/SAPK pathway in
neuronal apoptosis (Herdegen et al., 1998; Watson et al., 1998), we
chose to further characterize the JNK cascade in this model by using
RT-PCR and Western blot analyses while extending the time course of
observations.
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RT-PCR Evidence for METH-Induced Increased Transcript Levels for
Downstream and Upstream Members of the JNK/SAPK Cascade in the Cortex
and the Striatum.
Figure 2 shows a
representative RT-PCR photomicrograph of METH-mediated induction in the
expression of members located downstream and upstream of JNK in the
JNK/SAPK pathway in the cortex. The quantification of the RT-PCR
changes is provided in Fig. 3, A through
D and E through H, for the cortex and the striatum, respectively. Although only JNK3 was incorporated in the BD Biosciences Clontech array (#7741-1) used in our array experiments (Cadet et al., 2001), we
also ran RT-PCR of two other isoforms of JNKs, namely JNK1 and JNK2
(Figs. 2, 3C, and 3F). Expression of JNK1 and JNK3 but not JNK2
transcripts was higher in METH-treated mice compared with
saline-treated mice (Figs. 2 and 3C). In the frontal cortex (Fig. 3, A
through D), the increases in the expression of the various members of
JNK cascade peaked at 8 h. In contrast, Fig. 3, E through H,
showed that the METH induction up-regulation of these transcripts in
the mouse striatum occurred somewhat earlier (2-4 h) than did those
observed in the cortex (8 h) (compare Fig. 3, A through D, with Fig. 3,
E through H, respectively).
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METH Causes Increases in Protein Levels and in Protein
Phosphorylation of Members of the JNK/SAPK Pathway in the
Striatum.
MAP kinase cascades are believed to be among the most
important intracellular signaling pathways transmitting signals from the cell membrane to the nucleus, and their most prominent function is
believed to be the regulation of cellular gene expression via phosphorylation of transcription factors (Fukunaga and Miyamoto, 1998).
c-Jun activity is regulated via phosphorylation at serines 63 and 73 located in its activation domain. This is mediated by members of
JNK/SAPKs family, namely JNK1, JNK2, and JNK3 (Derijard et al., 1994).
Phosphorylation of c-Jun potentiates its ability to activate the
transcription of AP1 target genes (Herdegen and Waetzig, 2001).
; Deng et al., 1999; Volkow et al.,
2001). Moreover, DA-induced cell death, both in vivo (Luo et al.,
1999a) and in vitro (Luo et al., 1998, 1999b), seems to occur via
activation of the JNK pathway. Figure 5D shows that there was an almost
immediate increase in c-Jun protein after the METH injection. These
changes were first observed at 15 min (p < 0.01),
peaked at 4 h (p < 0.001), and then returned to
basal level by 7 days (p < 0.05) after drug administration. Phosphorylation of c-Jun at ser73 was very intense from
4 to 16 h (p < 0.001), whereas phosphorylation at
ser63 was greatest approximately 2 to 4 h (p < 0.001) after METH treatment (Fig. 5D).
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) or after transient forebrain
ischemia in the gerbil (Ferrer et al., 1997a). Because the activity of
JNK is increased after their phosphorylation at threonine 183 and
tyrosine 185 (Tournier et al., 1997), we also sought to determine
whether there were any changes in JNK phosphorylation after METH
treatment. Figure 5C also shows that there was indeed a substantial
increase in the phosphorylation of the p46 and p54 bands
(p < 0.001). According to the manufacturer's
literature (New England Biolabs), the antibody against phospho-JNK
recognizes the dually phosphorylated isoforms of JNK1, JNK2, and JNK3.
Western blot analysis was also used to characterize more upstream
members of the JNK pathway (Fig. 5, A and B). The SH2 and SH3 domains
of Crk II have been shown to bind to JNK (Yoshizumi et al., 2000) and
to activate it via the tyrosine-phosphorylated effector molecule, Cas.
Alternatively, JNK activation is accomplished upon its phosphorylation
by upstream kinases, MKK4 (Derijard et al., 1995) or MKK7 (Moriguchi et
al., 1997). As shown in Figs. 4 and 5A, c-Src, Cas, and Crk II showed
increases after treatment with METH and returned to basal levels after
1 week. MKK4, also known as MEK4, showed an increase at the 4-h time
point (p < 0.05, Fig. 5B). MKK4 did not show any
significant increases in its phosphorylated state after METH treatment
(Fig. 5B). In contrast, MKK7 showed significant increases from the 1-h
time point (p < 0.001), with subsequent slow reversal
to normal over a 7-day period (Fig. 5B).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Summary
References
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The major findings of our study show that METH-induced c-Jun
expression is associated with JNK-induced c-Jun phosphorylation and
that JNK activation occurs via stimulation of the Src-Cas-Crk upstream
pathway. We discuss the possible involvement of that pathway in the
pathobiological events associated with the use of METH from the wealth
of information that links that pathway to a number of pathobiological
states (Herdegen and Waetzig, 2001).
Possible Mechanisms for METH-Induced Activation of the JNK
Pathway.
In this study, we found that a single injection of METH
can cause increases in the levels of c-Jun mRNA and its protein in the
mouse brain. These findings extend those of Persico et al. (1995), who
reported that d-amphetamine can cause increases in c-Jun
mRNA in rats. Because the ability of c-Jun to activate gene transcription is potentiated by phosphorylation at serine residues 63 and 73 of the c-Jun activation domain (Derijard et al., 1994), the
increase in phosphorylated ser63 and ser73 c-Jun observed after METH
administration may be of significance to METH-induced cellular damage.
This idea is supported by the observations of increases in c-Jun
phosphorylated at ser63 in models of neuronal death caused by trophic
factor withdrawal (Watson et al., 1998) and by the report of an
involvement of c-Jun phosphorylated at ser73 in ischemia-induced
neuronal damage (Herdegen et al., 1998).
). For example, JNK1 and JNK2 are widely
expressed in most cell types (Mielke et al., 2000), whereas JNK3 is
mainly expressed in the nervous system (Gupta et al., 1996). Moreover,
Jun-JNK complexes formed in response to tumor necrosis factor seem to
consist mostly of JNK1-c-Jun instead of JNK2-c-Jun (Zhang et al.,
1998). In any case, given the observations that JNK seems to be
involved in several models of neuronal death (Herdegen et al., 1998;
Watson et al., 1998), it is not farfetched to suggest that the
METH-induced increases in phosphorylated c-Jun and its kinases might
play important roles in METH-induced apoptotic events (Deng et al.,
2001). This suggestion is supported by observations that JNK3 null mice
are resistant to kainate-induced cell death (Yang et al., 1997) and by
our recent demonstration that c-Jun knockout mice are protected against
METH-induced apoptosis (unpublished observations). Other central
nervous system active drugs such as morphine (Fuchs and Pruett, 1993)
and haloperidol (Noh et al., 2000), which can cause apoptosis, have
also been reported to activate the JNK pathway (Noh et al., 2000; Ma et
al., 2001).
The manner by which METH might cause activation of the JNKs is not
clear. However, our results showing that c-Src mRNA and protein
expression increased proportionally with c-Jun and JNK expression
suggest that METH might mediate JNK activation via Src because Src can
stimulate JNK activity (Feng et al., 2001). This is supported by the
recent demonstration that adenoviral transfection of a
dominant-negative form of Src can abrogate
H2O2-induced JNK activation
(Chen et al., 2001). Src-dependent signal events are mediated by the
assembly of signal transduction complex that involves Cas and Crk via
SH2 binding motifs (Burnham et al., 1996). Cas is the major
tyrosine-phosphorylated protein in cells transformed by Src (Sakai et
al., 1994). The unique structure of this protein, with amino-terminal
SH3 domain, followed by a cluster of SH2 binding motifs and a
carboxyl-terminal domain containing Src SH3 and SH2 binding regions,
provides an important scaffold for the assembly of a multiprotein
signaling complex (Sakai et al., 1994). Thus, our observations of
proportional increases in Cas and Crk protein expression in conjunction
with Src suggest that such an assembly may mediate Src-dependent
signaling events. Recent studies by Yoshizumi et al. (2000) in
H2O2-treated cells have
indeed demonstrated Src-dependent Cas tyrosine phosphorylation, Cas-Crk
complex formation, and binding of the SH3 domain of Crk to JNK with
associated activation of JNK. Taken together, these observations
suggest a novel redox-sensitive pathway for METH-induced JNK activation.
JNK activation can also be accomplished via its phosphorylation by
upstream kinases MKK4 (Derijard et al., 1995) or MKK7 (Tournier et al.,
1997). Ward and Hagg (2000) showed a disjunction between the activation
patterns of MKK4 and c-Jun during injury by reporting the presence of
c-Jun phosphorylation in the absence of MKK4 phosphorylation. In the
present study, we failed to detect substantial increases in
phospho-MKK4 even though there was prominent METH-induced c-Jun phosphorylation. These results suggest that MKK7 might be a more important factor in METH-induced JNK activation. Nevertheless, given
the long time course of the increases observed in MKK7 and the similar
time-course of METH-induced phosphorylated JNKs, our results suggest
that the Src-Cas-Crk connection via activation of MKK7 might play a
very prominent role in the prolonged JNK phosphorylation.
Possible Relationships between METH, Oxidative Stress, and JNK
Activation.
As stated earlier, the long-term deleterious effects
of amphetamine analogs are believed to be related to increased
production of ROS (Cadet and Brannock, 1998; Jayanthi et al., 1998).
This is believed to occur via increased METH-induced release of
dopamine within DA terminals or in the synaptic cleft, with subsequent formation of quinone, superoxide anions,
H2O2, and hydroxyl radicals (Cadet and Brannock, 1998). These DA-associated reactive species might
act in concert to cause the induction and phosphorylation of c-Jun via
activation of JNK/SAPKs (Fei et al., 2000). This idea is supported by
recent reports that DA can cause activation of the JNK pathway both in
vitro (Luo et al., 1998) and in vivo (Luo et al., 1999a). These
suggestions are supported by reports that JNK activation paralleled the
course of ROS formation in endothelin-stimulated smooth muscle cells
(Fei et al., 2000). Further support for this notion is also provided by
the observations that the superoxide generator menadione and
H2O2 can activate the
Src-dependent JNK pathway (Yoshizumi et al., 2000; Chen et al., 2001).
Similar observations have been made with other agents that cause
oxidative stress (Verheij et al., 1996) and agents that can cause
oxidative injury by depleting cellular glutathione (Wilhelm et al.,
1997).
). The
released glutamate could activate glutamate receptors with subsequent
changes in intracellular calcium and secondary increases in the
production of the physiological regulator nitric oxide (Radi et al.,
1991). Thus, METH administration might also activate the JNK pathway
via the actions of glutamate on striatal cells, as demonstrated by
Schwarzschild et al. (1997) using primary cultures of the striatum.
This idea is consistent with the demonstration that nitric oxide is
involved in the neurodegenerative effects of METH (Sheng et al.,
1996a).
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Summary |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Summary
References
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In summary, administration of a single moderate dose of METH caused persistent JNK activation with increases in c-Jun expression and phosphorylation. These results hint to the possibility that METH-induced DA and glutamate release, with subsequent ROS and nitric oxide production, might work in concert to activate the Src-JNK-Jun cascade. Activation of this cascade could then lead to increases in the expression of Jun effector genes that might be involved in causing neurodegeneration. In any case, the identification of c-Jun target genes should provide substantial insights into the molecular neurotoxicology of this stimulant. These studies are underway in our laboratory.
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Footnotes |
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Received October 25, 2001; Accepted January 24, 2002
Address correspondence to: Jean Lud Cadet, M.D., Molecular Neuropsychiatry Section, NIH/NIDA Intramural Research Program, 5500 Nathan Shock Drive, Baltimore, MD 21224. E-mail: jcadet{at}intra.nida.nih.gov
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Abbreviations |
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METH, methamphetamine; DA, dopamine; ROS, reactive oxygen species; Src, Rous sarcoma oncogene; Cas, Crk-associated substrate; JNK, Jun N-terminal kinase; RT-PCR, reverse transcription-polymerase chain reaction; MKK4, mitogen-activated protein kinase kinase 4; MKK7, mitogen-activated protein kinase kinase 7; Crk II, avian sarcoma CT10 oncogene homolog; SAPK, stress-activated protein kinase; AP-1, activator protein 1; PLSD, protected least significant difference; ANOVA, analysis of variance.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Summary
References
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 S. Jayanthi, X. Deng, B. Ladenheim, M. T. McCoy, A. Cluster, N.-s. Cai, and J. L. Cadet Calcineurin/NFAT-induced up-regulation of the Fas ligand/Fas death pathway is involved in methamphetamine-induced neuronal apoptosis PNAS, January 18, 2005; 102(3): 868 - 873. [Abstract] [Full Text] [PDF]
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 S. JAYANTHI, X. DENG, P.-A. H. NOAILLES, B. LADENHEIM, and J. L. CADET Methamphetamine induces neuronal apoptosis via cross-talks between endoplasmic reticulum and mitochondria-dependent death cascades FASEB J, February 1, 2004; 18(2): 238 - 251. [Abstract] [Full Text] [PDF]
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J. L. CADET, S. JAYANTHI, and X. DENG Speed kills: cellular and molecular bases of methamphetamine-induced nerve terminal degeneration and neuronal apoptosis FASEB J, October 1, 2003; 17(13): 1775 - 1788. [Abstract] [Full Text] [PDF]
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 X. Deng, S. Jayanthi, B. Ladenheim, I. N. Krasnova, and J. L. Cadet Mice with Partial Deficiency of c-Jun Show Attenuation of Methamphetamine-Induced Neuronal Apoptosis Mol. Pharmacol., November 1, 2002; 62(5): 993 - 1000. [Abstract] [Full Text] [PDF]
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-Actin is
included as a control and shows no changes during the course of the
experiments.
