Diadenosine tetraphosphate reduces toxicity caused by high-dose methamphetamine administration

Abstract

Diadenosine tetraphosphate (AP(4)A), two adenosine moieties bridged by four phosphates, is an endogenous purinergic ligand found in brain. Previous studies have shown that AP(4)A reduced neurodegeneration caused by the dopaminergic neurotoxin 6-hydroxydopamine in rat striatum and substantia nigra. The purpose of this study was to determine whether AP(4)A is protective against methamphetamine (MA)-mediated toxicity. Primary neuronal cultures were prepared from rat embryonic (E14-E15) ventral mesencephalic tissue. Cultures treated with 2mM MA exhibited decreased tyrosine hydroxylase (TH) immunoreactivity and increased cleaved caspase-3 immunoreactivity and TUNEL labeling. All these changes were lessened by pretreatment with AP(4)A. The protective effect of AP(4)A was also found in vivo. Adult Sprague-Dawley rats were injected with AP(4)A (25 microg/20 microl) or vehicle intracerebroventricularly followed by 4 doses of MA (5 or 10 mg/kg), given subcutaneously every 2h. Administration of MA reduced locomotor activity 1 day after injection, which was significantly antagonized by the pretreatment with AP(4)A. Using immunohistochemical analysis, TH fiber density at the substantia nigra pars reticulata was found reduced while cleaved caspase-3 immunoreactivity in striatum was increased after MA treatment; these responses were also significantly antagonized by AP(4)A. Taken together, our data show that AP(4)A has protective effects against MA-mediated toxicity both in vitro and in vivo. The mechanism of action involves suppression of MA-induced apoptosis.

Figure 1

AP₄A reduces methamphetamine (MA) toxicity in primary VM cultures. (A) Density of TH (+) cells. MA decreased the density of TH (+) neurons. AP₄A (100 μM) antagonized the decrease in TH (+) cell density induced by 2 mM MA. (B) Density of GFAP immunoreactivity. AP₄A did not alter GFAP immunoreactivity in cultures treated with MA. (C) TUNEL labeling. Pretreatment with AP₄A reduced MA -mediated TUNEL labeling. (D) Activation of caspase-3. Density of activated caspase-3 positive cells was enhanced by MA, which was significantly reduced by AP₄A. All data were normalized to the mean of vehicle/no MA controls in each experiment. * p<0.05, 1- or 2-Way ANOVA.

Figure 2

Photomicrographs of culture VM cells treated with MA and AP₄A. MA reduced the density of TH (+), and increased the density of TUNEL (+) and activated caspase-3 cells. Pretreatment with AP₄A antagonized these MA –induced changes. AP₄A and MA did not alter GFAP immunoreactivity. Calibration: TH, GFAP, TUNEL: 80 μm; activated caspase 3: 160 μm.

Figure 3

MA decreased TH-immmunoreactivity and increased activated caspase-3 immunoreactivity in VM cell culture. Red: activated caspase-3 cells, green: TH cells. MA reduced TH, but increased activated caspase-3 immunoreactivity (A: vehicle; B: MA). There is little colocalization of TH and activated caspase-3. (C) AP₄A alone did not alter caspase-3 expression, but (D) reduced the density of activated caspase-3 positive cells in the presence of MA. Calibration= 80 μm.

Figure 4

Interactions of AP4A and MA in the first 10-min exploratory phase of locomotor activity. Locomotor parameters were monitored at one day after systemic MA or saline injection in adult rats. In animals receiving i.c.v. vehicle pretreatment, MA dose-dependently reduced (A) movement number, (B) vertical activity, (C) horizontal movement time, and (D) vertical movement time. Pretreatment with AP₄A significantly enhanced movement number, vertical activity and vertical movement in MA –treated rats. A marginal increase in horizontal movement time was found in animals treated with AP4A (p=0.078, 2-way ANOVA).

Fig 5. TH immunoactivity in striatum, SNpc, and SNpr

Animals were treated with veh or AP4A (i.c.v.) and then saline or MA (s.c.). THir was analyzed 4 days after injection. (A) In striatum, parenteral administration of MA significantly reduced density of striatal THir. Administration of AP₄A did not significantly reduce the loss of THir density in MA – treated rats. (B) In SNpr, MA administration significantly reduced THir density. AP4A significantly increased THir in SNpr in animals receiving MA. (C) In SNpc, MA did not alter the density of TH neurons. There is no significant interaction in TH neuronal density between treatments with AP4A and MA. (D) Typical immunostaining in SNpr. (D1: vehicle+saline; D2: vehicle+MA; D3: AP₄A +saline; D4: AP₄A +MA). Calibration = 500 μm.

Figure 6

MA- induced caspase-3 activation in rat striatum is suppressed by AP₄A. Animals were injected with AP₄A or vehicle into the left lateral cerebral ventricle followed by systemic administration of MA. Activated caspase-3 immunoreactivity was analyzed 2 days after MA injection. Examples of striatal immunoreactivity for activated caspase 3 in MA-injected pretreated with vehicle (A) or AP₄A (B). MA increased activated caspase-3 immunoreactivity in striatum in a dose-dependent manner (p<0.001, 2-way ANOVA). (C) Pretreatment with AP₄A significantly antagonized MA – mediated caspase-3 activation (*p<0.001, 2-way ANOVA). AP₄A significantly reduced striatal caspase-3 immunoreactivity induced by MA at 4×5 mg/ kg or 4×10 mg/ kg (#p<0.05, 2-way ANOVA, SNK post-hoc analysis).

Figure 7

AP₄A did not alter basal and MA -evoked dopamine release from dorsal striatum. Animals were perfused with AP₄A (25 μg/μl, 2 μl/min) or vehicle through the microdialysis cannula. Systemic injection of MA at 10 mg/ kg, (arrow) induced dopamine overflow (dashed line). The presence of AP₄A did not alter the peak or the duration of dopamine overflow induced by MA (solid line).