Changes in gene expression linked to methamphetamine-induced dopaminergic neurotoxicity

Abstract

The purpose of these studies was to examine the role of gene expression in methamphetamine (METH)-induced dopamine (DA) neurotoxicity. First, the effects of the mRNA synthesis inhibitor, actinomycin-D, and the protein synthesis inhibitor, cycloheximide, were examined. Both agents afforded complete protection against METH-induced DA neurotoxicity and did so independently of effects on core temperature, DA transporter function, or METH brain levels, suggesting that gene transcription and mRNA translation play a role in METH neurotoxicity. Next, microarray technology, in combination with an experimental approach designed to facilitate recognition of relevant gene expression patterns, was used to identify gene products linked to METH-induced DA neurotoxicity. This led to the identification of several genes in the ventral midbrain associated with the neurotoxic process, including genes for energy metabolism [cytochrome c oxidase subunit 1 (COX1), reduced nicotinamide adenine dinucleotide ubiquinone oxidoreductase chain 2, and phosphoglycerate mutase B], ion regulation (members of sodium/hydrogen exchanger and sodium/bile acid cotransporter family), signal transduction (adenylyl cyclase III), and cell differentiation and degeneration (N-myc downstream-regulated gene 3 and tau protein). Of these differentially expressed genes, we elected to further examine the increase in COX1 expression, because of data implicating energy utilization in METH neurotoxicity and the known role of COX1 in energy metabolism. On the basis of time course studies, Northern blot analyses, in situ hybridization results, and temperature studies, we now report that increased COX1 expression in the ventral midbrain is linked to METH-induced DA neuronal injury. The precise role of COX1 and other genes in METH neurotoxicity remains to be elucidated.

Fig. 1.

Effect of cycloheximide and METH, alone and in combination, on striatal DA (a), DOPAC (b), and core temperature (c). Mice received either vehicle (saline), METH (45 mg/kg, s.c.), cycloheximide (150 mg/kg, s.c.), or METH (45 mg/kg, s.c.) plus cycloheximide (150 mg/kg, s.c., 0.5 hr before METH). Core temperature was measured during drug treatment, as described in Materials and Methods. For DA and DOPAC determinations, mice were killed 1 week after drug treatment. Results shown represent the mean ± SEM for each group (n = 5 per group). * designates significant difference from control (p < 0.05).

Fig. 2.

Effect of actinomycin-D and METH, alone or in combination, on striatal DA (a), DOPAC (b), and core temperature (c). Mice received vehicle (saline), METH (45 mg/kg, s.c.), actinomycin-D (0.5 mg/kg, i.p.), or METH (45 mg/kg, s.c.) plus cycloheximide (0.5 mg/kg, i.p., 2 hr before METH). Core temperature was measured during drug treatment, as described in Materials and Methods. For DA and DOPAC determinations, mice were killed 1 week after drug treatment. Results shown represent the mean ± SEM for each group (n = 5 per group). * designates significant difference from control (p < 0.05).

Fig. 3.

Effect of actinomycin-D on [³H] DA uptake (a) and [³H] WIN35,428 binding (b). Uptake and binding studies were performed as described in Materials and Methods. Results shown represent mean ± SEM for each group (n = 3 per group). * designates significant difference from control (p < 0.05).

Fig. 4.

WIN35,428 blockade of METH-induced DA neurotoxicity. Mice were treated with METH (45 mg/kg, s.c.), WIN35,428 (12.5 mg/kg, i.p.), WIN35,428 (12.5 mg/kg, i.p., immediately before METH) plus METH (45 mg/kg, s.c.), or saline at room temperature. Rectal temperatures were measured during drug treatment, and mice were killed 1 week later for measurement of striatal DA and DOPAC levels, as described in Materials and Methods. Results shown represent the mean ± SEM for each group (n = 6 per group).

Fig. 5.

Representative scatter plots demonstrating the strategy used to identify genes specifically involved in METH-induced DA neurotoxicity. Neuronal array hybridization data 3 hr after various treatments are shown. Methods are as described in Materials and Methods. Each panel contains 1100 genes. The red dots represent genes with expressions that have been either upregulated or downregulated by at least a factor of 2 compared with control. Listed in the y-axis are the log-transformed intensity data for the genes expressed after being treated with METH alone, METH plus WIN35,428, or WIN35,428 alone. Listed in the x-axis are the log-transformed intensity data for genes expressed after saline treatment as control. There are 11 serial density reads ranging from 2000 to 20,000 on thex- and y-axis; only the first and last reads on the figures are labeled because of the limit in space. Only those genes upregulated or downregulated by at least a factor of 2 in the METH group but not in the other groups (METH plus WIN35,428 and WIN35,428 alone) were viewed as being specifically involved in METH neurotoxicity.

Fig. 6.

Representative image for Northern blot analysis of COX1 mRNA levels 12 hr after various treatments. The method was as described under Materials and Methods. β-actin cDNA probe was used as internal control. Note increase in COX1 mRNA level after METH.

Fig. 7.

Representative of in situhybridization image of COX1 mRNA in the substantia nigra pars reticulata, of mice treated with METH or saline 24 hr previously. The method was as described in Materials and Methods. The orientation of the substantia nigra pars reticulata and the area of interest (box) amplified for quantitative analysis are shown in the top panel (amplification 60×). Increased mRNA levels were observed in neurons after METH (middle panel, top left corner; amplification 250×) compared with saline control (middle panel, top right corner) using COX1 antisense cRNA probes. Sense probe (middle panel, bottom left corner) and zero probe (without any probe; middle panel,bottom right corner) were also used in the METH sections as negative controls. Quantitative study of the COX1 mRNA expression showed a twofold increase in METH-treated mice compared with control, with p < 0.01 (bottom panel, shown as *). Results shown represent the mean ± SEM for each group (n = 20 neurons per group).