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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 24, 2005 as doi:10.1096/fj.04-2881fje. |
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Molecular Neuropsychiatry Branch, NIDA-IRP, DHHS/NIH, Baltimore, Maryland, USA
1 Correspondence: Molecular Neuropsychiatry Branch, DHHS/NIH/NIDA, 5500 Nathan Shock Dr., Baltimore, MD 21224, USA. Email: jcadet{at}intra.nida.nih.gov
SPECIFIC AIMS
Amphetamine (AMPH) is a psychostimulant whose chronic abuse may cause impairment in attention and memory in humans. These cognitive deficits might be related to neurotoxic effects of the drug. A well-known toxic effect of AMPH is the degeneration of striatal dopamine (DA) terminals. The purpose of this study was to investigate the hypothesis that AMPH might cause neuronal apoptosis via activation of the mitochondria-dependent cell death pathway.
PRINCIPAL FINDINGS
1. AMPH causes apoptosis and reactive astrocytosis in the striatum
To clarify whether AMPH administration can cause apoptosis in the striatum, we used a TUNEL technique to assay for DNA fragmentation. Very few TUNEL-positive cells were seen in the striata of saline-treated mice. However, administration of the drug (10 mg/kg, 4 times, every 2 h, which causes significant damage to DA terminals) induced increases in TUNEL-positive staining in nondopaminergic cells in the striatum (Fig. 1
A). Quantification of these changes 1–7 days after AMPH injections revealed maximal cell death 4 days postdrug, reaching 9-fold increases in the number of TUNEL-positive cells (Fig. 1B
). The appearance of AMPH-induced cell death coincided with reactive astrocytosis, an indirect marker of neuronal injury. Few GFAP-positive astrocytes were observed in the striata of saline-treated mice; these cells had small cell bodies with very fine and short processes. In contrast, after AMPH injections, the astrocytes develop large, densely stained cell bodies as well as long and extensive processes (Fig. 1C
). Quantitative data show that the number of AMPH-induced glial cells had reached a maximum of 18-fold increases 4 days after drug treatment (Fig. 1D
).
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2. Cleaved caspase-3 is expressed in calbindin-positive striatal neurons
Activation of caspase-3 is a critical step in causing neuronal apoptosis after injury or chemical damage; we sought to examine whether this enzyme might be involved in AMPH-induced cell death. Since caspase-3 exists as a pro-enzyme until cleaved in response to apoptotic stimuli, we performed immunohistochemistry using an antibody selective against the cleaved fragment of caspase-3. No cleaved caspase-3-positive cells were observed in the striata of saline-treated mice. However, AMPH injections caused increases in cleaved caspase-3 staining 3 days after drug injections.
To identify striatal cell population that was affected by AMPH, we performed double label immunohistochemistry by using antibody against cleaved caspase-3 in conjunction with antibodies against markers of striatal neurons. Intrastriatal neurons consist of 
90% medium spiny projection neurons and 10% interneurons. First, we used antibody against calbindin, a calcium binding protein that is expressed mainly in medium spiny neurons of the striatum. Double label immunohistochemistry showed that cleaved caspase-3 was expressed in calbindin-positive neurons. We sought to determine whether cleaved caspase-3-positive cells contained DA and cAMP-regulated phosphoprotein, 32 kDa (DARPP-32) found in striatal projection neurons. Double label experiments revealed that all cleaved caspase-3-positive neurons contained DARPP-32. Interneurons in the striatum comprise three major classes: cholinergic neurons; GABA-ergic neurons containing parvalbumin and GABA-ergic neurons containing somatostatin, neuropeptide Y, and nitric oxide synthase. Double labeling showed that neither choline acetyltransferase (ChAT) -labeled cholinergic, parvalbumin nor somatostatin interneurons expressed cleaved caspase-3.
3. AMPH causes activation of mitochondria-dependent apoptotic signal transduction cascade in the striatum
To identify molecular bases for AMPH-induced neuronal apoptosis, we investigated the effects of this drug on the expression of intermediates of apoptotic signal transduction cascades at mRNA and protein levels. Because p53 is a known contributor to neuronal apoptosis, we measured p53 transcripts using real-time RT-PCR. Figure 2
A shows that p53 mRNA levels increased in the striatum up to +35% between 30 min and 16 h after AMPH treatment. The increases in p53 mRNA were reflected by changes in the expression of p53 protein, which reached +40% 4 h postdrug (Fig. 2A
').
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Because p53 can cause up-regulation of the mitochondrial proapoptotic protein Bax, which plays an essential role in p53-mediated apoptosis, we examined whether increase in p53 levels was associated with changes in Bax expression. RT-PCR showed that AMPH caused up-regulation of Bax mRNA 30 min–2 h after treatment (+20%) (Fig. 2B
). However, Bax protein expression was of greater magnitude reaching about +80% 16 h postdrug (Fig. 2B
'). The proapoptotic effects of Bax are known to depend on its heterodimerization with Bcl-2 in mitochondria, with the ratio of Bax/Bcl-2 determining whether a cell survives or dies in response to a toxic stimulus. In addition to its effects on Bax expression, p53 can cause down-regulation of the antiapoptotic protein, Bcl-2. As shown in Fig. 2C
, Bcl-2 mRNA was decreased in the striata of AMPH-treated animals (–26%) between 16 h to 7 days after drug injections. AMPH caused progressive decreases in the levels of Bcl-2 protein starting at 4 h, reaching a minimum at 2 days (–68%), and staying low for up to 7 days after treatment (Fig. 2C
').
4. Bax deficiency attenuates AMPH-induced cell death
The experiments reported above suggested that increased Bax expression might play a crucial role in AMPH-induced apoptosis in the striatum. We administered AMPH to wild-type and Bax null mice. These injections caused the appearance of TUNEL-positive cells in the striatum of both genotypes. However, there was a substantial reduction (–57%) in the number of drug-induced TUNEL-positive cells in the Bax knockout mice.
CONCLUSIONS AND SIGNIFICANCE
The main findings of this study are that 1) AMPH can cause apoptosis in nondopaminergic striatal neurons, which is linked to the appearance of reactive astrocytosis; 2) medium spiny projection neurons are susceptible to stimulant-induced cell death; and 3) AMPH-induced apoptosis is mediated, in part, by mitochondria-dependent mechanisms.
Projection neurons comprise 90% of striatal neurons and receive the majority of inputs into that structure. Interneurons, on the other hand, comprise 10% of striatal cells and are implicated in regulating the function of striatal projection neurons. We found that calbindin- and DARPP-32-positive medium spiny projection neurons, but not ChAT, parvalbumin, or somatostatin interneurons, underwent apoptosis after AMPH injections. Although the reason for the vulnerability of striatal projection neurons to AMPH remains to be clarified, oxidative stress, mitochondrial dysfunction, disturbance of calcium homeostasis, toxic actions of glutamate, and neurotoxic effects of activated glia might contribute to these observations.
The effects of AMPH are thought to be secondary to drug-induced redistribution of DA from synaptic vesicles to the cytosol, followed by its release to the extracellular space by reverse transport through DA transporters, resulting in increased DA levels in the synaptic cleft. This is associated with the production of hydroxyl and superoxide radicals that may participate in the toxic effects of AMPH via free radical-mediated protein oxidation and DNA damage. The latter events are associated with p53 accumulation in vitro, which itself has been linked to neuronal apoptosis. P53-mediated apoptosis might be related to its effects on Bcl-2 family of mitochondrial proteins, such as proapoptotic Bax and antiapoptotic Bcl-2. Activation of a caspase cascade might be involved in p53-mediated apoptosis. These ideas are consistent with the up-regulation of Bax and persistent reduction of Bcl-2 that were observed in the present study. The balance between Bcl-2 and Bax might have been disturbed in the striatum, leading to a shift toward cell death due to excessive Bax protein. These observations suggest that p53 accumulation may be responsible for Bax activation in this AMPH neurodegeneration model. Activation of Bax results in its intramembranous homooligomerization, followed by permeabilization of the outer mitochondrial membrane and release of cytochrome c. Translocation of mitochondrial cytochrome c to the cytosol is a critical event in mitochondria-dependent activation of caspase-3. Consistent with the involvement of Bax in apoptotic process is our finding of partial resistance of Bax knockout mice to AMPH-induced cell death. Our findings suggest that AMPH can cause neuronal apoptosis in the striatum via mitochondria-dependent mechanisms (Fig. 3
). These results suggest that AMPH-induced activation of p53 may trigger a Bax-dependent, mitochondria-mediated caspase death cascade within a subclass of striatal projection neurons.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2881fje;
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