Elsevier

Neuroscience Research

Volume 64, Issue 4, August 2009, Pages 413-420
Neuroscience Research

Effects of lithium chloride on the gene expression profiles in Drosophila heads

https://doi.org/10.1016/j.neures.2009.04.015Get rights and content

Abstract

To gain insight into the basic neurobiological processes regulated by lithium—an effective drug for bipolar disorder—we used Affymetrix Genome Arrays to examine lithium-induced changes in genome-wide gene expression profiles of head mRNA from the genetic model organism Drosophila melanogaster. First, to identify the individual genes whose transcript levels are most significantly altered by lithium, we analyzed the microarray data with stringent criteria (fold change > 2; p < 0.001) and evaluated the results by RT-PCR. This analysis identified 12 genes that encode proteins with various biological functions, including an enzyme responsible for amino acid metabolism and a putative amino acid transporter. Second, to uncover the biological pathways involved in lithium's action in the nervous system, we used less stringent criteria (fold change > 1.2; FDR < 0.05) and assigned the identified 66 lithium-responsive genes to biological pathways using DAVID (Database for Annotation, Visualization and Integrated Discovery). The gene ontology categories most significantly affected by lithium were amino acid metabolic processes. Taken together, these data suggest that amino acid metabolism is important for lithium's actions in the nervous system, and lay a foundation for future functional studies of lithium-responsive neurobiological processes using the versatile molecular and genetic tools that are available in Drosophila.

Introduction

The alkaline metal lithium affects various developmental and physiological processes in evolutionarily diverse organisms (Phiel and Klein, 2001). In particular, lithium's actions in the nervous system have attracted special attention because lithium is highly effective in the prophylaxis and treatment of bipolar affective disorder (Schou, 2001). In addition, recent studies suggest that chronic lithium treatment is efficacious in preventing apoptosis-dependent neuronal death (Chuang, 2004), which raises the interesting possibility that lithium might be effective in treating or preventing brain damage, either following injury or over the course of progression of neurodegenerative diseases. Despite the proven clinical usefulness and the further potential of this drug, the molecular mechanisms underlying its actions in the nervous system are still only poorly understood.

At therapeutically relevant concentrations, lithium inhibits several enzymes, including glycogen synthase kinase 3β (GSK3β) as well as inositol monophosphatase (IMPase) and related enzymes (Berridge et al., 1989, Klein and Melton, 1996). These lithium-sensitive enzymes are intimately involved in the regulation of various intracellular molecular cascades, such as the Wnt and inositol phosphate signaling pathways (Chen et al., 2000, Ding et al., 2000). Lithium-based perturbation of these signaling pathways has a significant impact on global gene expression profiles (Rowe and Chuang, 2004), and this may contribute to the therapeutic as well as toxic actions of lithium. In order to elucidate the importance of gene regulation in lithium's actions in the nervous system, we need to identify genes whose expression is influenced by therapeutic concentrations of lithium, and to study their roles in the lithium-responsive neurobiological processes at the molecular, cellular and organismal levels.

The fruit fly Drosophila melanogaster has been a valuable genetic model system for examining fundamental problems in neurobiology. In part, this is due to the fact that Drosophila and higher vertebrates share genetic pathways for cellular signaling (Miklos and Rubin, 1996, Rubin et al., 2000). In addition, many human genes involved in brain functions and neurological disorders have fly counterparts (Reiter et al., 2001, Davis, 2005, Hamet and Tremblay, 2006). Importantly, the genetic pathways involved in lithium's actions in the nervous system appear to be shared by Drosophila and vertebrates. For example, the administration of lithium to fruit flies and vertebrates has a similar effect on circadian clocks, and in both cases this effect involves the inhibition of glycogen synthase kinase 3β (GSK3β) (Padiath et al., 2004, Dokucu et al., 2005, Iitaka et al., 2005). Additionally, as in vertebrates, lithium has neuroprotective effects in transgenic flies that over-express either human tau proteins or a mutant form of huntingtin (Mudher et al., 2004, Berger et al., 2005). Furthermore, lithium improves the physiological, behavioral and developmental mutant phenotypes characteristic of a mouse model of Fragile X syndrome (Min et al., 2009), and likewise rescues such defects in a Drosophila model of this disease (Mcbride et al., 2005). These results strongly suggest that studies of the genes responsible for lithium's actions in the Drosophila nervous system would provide important insights into the basis of lithium's neurobiological effects in vertebrates.

In this study, we carried out a microarray-based gene expression profiling analysis of Drosophila head mRNA, to identify the genes and biological pathways of the nervous system that are significantly influenced by lithium treatment in adult animals. This study lays the foundation for future functional studies using the versatile molecular and genetic tools available in Drosophila to understand the lithium-responsive neurobiological processes.

Section snippets

Drosophila stock

Flies were reared at 25 °C at 65% humidity, in a 12:12 h light:dark cycle, on a conventional cornmeal-based medium containing glucose, yeast and agar supplemented with the mold inhibitor methyl 4-hydroxybenzoate (0.05%). The Canton-S (CS) strain was used as the wild-type control.

RNA extraction and microarray experiment

Newly eclosed 0 to 1 day-old wild-type female flies were grouped into sets of 20 and placed into a vial containing regular fly food with or without 50 mM LiCl. Flies in five vials (total of 100 flies) were combined as one

Results

In order to determine the transcriptome response to lithium treatment in Drosophila heads, wild-type female flies were treated for 24 h with or without 50 mM LiCl. We chose this concentration of lithium because it results in an internal lithium level that is comparable to the therapeutic serum concentrations (μM-mM) in bipolar disorder patients treated with lithium (Padiath et al., 2004, Dokucu et al., 2005). These lithium concentrations do not cause obvious toxic effects on adult wild-type flies

Characteristic features of the individual genes whose expression is significantly affected by lithium

In this study, we examined the lithium-induced alterations in genome-wide gene expression profiles in the adult head of the genetic model organism, D. melanogaster. Using stringent analysis criteria, we identified 12 genes whose transcript levels are most significantly altered by lithium (Table 2). These genes can be categorized into four groups based on their biological function or characteristic features: (i) amino acid transport and metabolism, (ii) detoxication, stress response or

Acknowledgements

We would like to thank Dr. Tom Bair (DNA core facility, University of Iowa) for his assistance with the statistical analyses of microarray data. This study was supported by grants from the NIH (R03 MH078271), American Parkinson's Disease Association Research and National Alliance for Research on Schizophrenia and Depression (NARSAD) to T.K.

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