Neuropharmacology and Analgesia
Chronic morphine administration induces over-expression of aldolase C with reduction of CREB phosphorylation in the mouse hippocampus

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Abstract

In recent studies, alterations in the activity and expression of metabolic enzymes, such as those involved in glycolysis, have been detected in morphine-dependent patients and animals. Increasing evidence demonstrates that the hippocampus is an important brain region associated with morphine dependence, but the molecular events occurring in the hippocampus following chronic exposure to morphine are poorly understood. Aldolase C is the brain-specific isoform of fructose-1, 6-bisphosphate aldolase which is a glycolytic enzyme catalyzing reactions in the glycolytic, gluconeogenic, and fructose metabolic pathways. Using Western blot and immunofluorescence assays, we found the expression of aldolase C was markedly increased in the mouse hippocampus following chronic morphine treatment. Naloxone pretreatment before morphine administration suppressed withdrawal jumping, weight loss, and overexpression of aldolase C. CREB is a transcription factor regulated through phosphorylation on Ser133, which is known to play a key role in the mechanism of morphine dependence. When detecting the expression of phosphorylated CREB (p-CREB) in the mouse hippocampus using Western blot and immunohistochemistry, we found CREB phosphorylation was clearly decreased following chronic morphine treatment. Interestingly, laser-confocal microscopy showed that overexpression of aldolase C in mouse hippocampal neurons was concomitant with the decreased immunoreactivity of p-CREB. The results suggest potential links between the morphine-induced alteration of aldolase C and the regulation of CREB phosphorylation, a possible mechanism of morphine dependence.

Introduction

In recent studies, alterations of the activity and expression of enzymes in cell metabolism, such as glycolysis, were detected in morphine-dependent patients and animals, indicating that a disturbance of glycometabolism may be involved in the molecular events of morphine dependence (Sharma et al., 2003, Xiang et al., 2006, Serres et al., 2005, Chen et al., 2007). Aldolase catalyzes a step of the glycolytic pathway, the aldolhydrolysis of fructose-1, 6-biphosphate into dihydroxyacetone phosphate and glycerol-3-phosphate, and the two substrates play key roles in producing ATP through the tricarboxylic acid cycle and other energy metabolism pathways. Vertebrate aldolases exist as three isozymes with different tissue distribution and kinetics: A, B, and C. Aldolase C is the brain-specific isoform of fructose-1, 6-bisphosphate aldolase (Lebherz and Rutter, 1969). Recently, several proteomics studies have reported altered expression levels of aldolase C in different brain regions of chronic morphine-administered animals (Li et al., 2006, Kim et al., 2005).

Increasing evidence demonstrates that the hippocampus is an important region associated with morphine dependence (Nestler, 2002, Morón et al., 2007). The rodent hippocampus is functionally involved in morphine-induced conditioned place preference (Rezayof et al., 2003, Zarrindast et al., 2006) and withdrawal behavior (Done et al., 1992, Lu et al., 2000, Dong et al., 2008). One recent study found downregulation of some energy metabolism enzymes, lower ATP levels, and an impaired ability to convert glucose into ATP in the mouse hippocampus following chronic morphine treatment, which indicates that an abnormality in hippocampal energy metabolism may contribute to morphine dependence (Chen et al., 2007). However, whether morphine treatment induces alterations in the expression of aldolase C in the hippocampus remains unclear.

Modulation of transcription factors such as the cAMP response element binding protein (CREB) is an important mechanism underlying the development of morphine dependence (Williams et al., 2001, Deisseroth et al., 1996). CREB is regulated through phosphorylation of Ser133, which modulates the transcription of genes containing cAMP response elements (CRE) in their promoters (Lonze and Ginty, 2002). Evidence suggests that chronic morphine administration induces changes in the expression and function of CREB in several brain regions such as the nucleus accumbens and locus coeruleus, which may contribute to withdrawal behaviors and neural adaptations associated with morphine dependence (Guitart et al., 1992, Lane-Ladd et al., 1997, Shaw-Lutchman et al., 2002). However, few studies have reported regulation of CREB phosphorylation in the hippocampus associated with morphine dependence.

In this study, we used Western blot and immunofluorescence to evaluate the effect of morphine on the regulation of aldolase C in the mouse hippocampus. In addition, we examined the expression of phosphorylated CREB (p-CREB) by Western blot and immunohistochemistry, hypothesizing that chronic morphine administration would cause alterations in CREB phosphorylation. Finally, we examined the co-expression of aldolase C and p-CREB in hippocampal neurons using laser-scanning confocal microscopy in order to study changes in aldolase C expression associated with changes in the regulation of morphine-mediated CREB phosphorylation.

Section snippets

Animals and treatment

Thirty-two male ICR mice (20–25 g; Experimental Animal Center of Peking University Health Science Center, Beijing, China) were maintained in a colony room at 24 ± 2 °C on a 12 h light/dark cycle with food and water available freely. One week after arrival, mice were randomly divided into the following 4 groups, containing 8 mice per group: a control group receiving saline (Sal), a morphine-treated group (Mor), a naloxone-precipitated withdrawal group (Mor + Nal), and a naloxone-pretreated morphine

The assessment of morphine withdrawal

We found that naloxone precipitation induced robust withdrawal jumping in morphine-dependent mice (Fig. 1A). The effect was not observed with naloxone pretreatment before morphine administration in mice (P < 0.05). To observe the effect of chronic morphine treatment on weight loss, the weight of each animal was measured before injection with morphine or saline each day. We found that mice treated with morphine showed significantly less weight gain than the control group from day 2 to day 5 (all P <

Discussion

Recently, several studies have measured the concentrations of glycolytic intermediates in rodent brains following chronic exposure to morphine using nuclear magnetic resonance spectroscopy. They found that the rate of glycolysis was increased and related metabolic enzymes were coordinately activated, which indicates that an abnormality of glycolysis may be involved in the molecular mechanism of morphine dependence (Sharma et al., 2003, Xiang et al., 2006, Serres et al., 2005). The hippocampus

Acknowledgements

We thank Prof. Qi-Hua He (The Medicine and Health Analysis Center of Peking University) for the gifts of FITC-conjugated and TRITC-conjugated antibodies and her help with laser-confocal microscopy analysis. We also thank Prof. Xiao-Yan Qiu and Prof. Jing-Rong Zhang (The Human Disease Genomics Research Center of Peking University) for their technical assistance. We are grateful to Dr. Quan Li and Dr. Xin Zhao for their helpful comments. This work was supported by a grant from the National

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