Elsevier

Neuropharmacology

Volume 60, Issues 7–8, June 2011, Pages 1168-1175
Neuropharmacology

Harmine, a natural beta-carboline alkaloid, upregulates astroglial glutamate transporter expression

https://doi.org/10.1016/j.neuropharm.2010.10.016Get rights and content

Abstract

Glutamate is the predominant excitatory amino acid neurotransmitter in the mammalian central nervous system (CNS). Glutamate transporter EAAT2/GLT-1 is the physiologically dominant astroglial protein that inactivates synaptic glutamate. Previous studies have shown that EAAT2 dysfunction leads to excessive extracellular glutamate and may contribute to various neurological disorders including amyotrophic lateral sclerosis (ALS). The recent discovery of the neuroprotective properties of ceftriaxone, a beta lactam antibiotic, suggested that increasing EAAT2/GLT-1 gene expression might be beneficial in ALS and other neurological/psychiatric disorders by augmenting astrocytic glutamate uptake. Here we report our efforts to develop a new screening assay for identifying compounds that activate EAAT2 gene expression. We generated fetal derived-human immortalized astroglial cells that are stably expressing a firefly luciferase reporter under the control of the human EAAT2 promoter. When screening a library of 1040 FDA approved compounds and natural products, we identified harmine, a naturally occurring beta-carboline alkaloid, as one of the top hits for activating the EAAT2 promoter. We further tested harmine in our in vitro cell culture systems and confirmed its ability to increase EAAT2/GLT1 gene expression and functional glutamate uptake activity. We next tested its efficacy in both wild type animals and in an ALS animal model of disease and demonstrated that harmine effectively increased GLT-1 protein and glutamate transporter activity in vivo. Our studies provide potential novel neurotherapeutics by modulating the activity of glutamate transporters via gene activation.

This article is part of a Special Issue entitled ‘Trends in Neuropharmacology: In Memory of Erminio Costa’.

Introduction

Glutamate is the predominant excitatory amino acid (EAA) neurotransmitter in the mammalian CNS. It activates ligand-gated ion channels that are named after their agonists N-methyl-d-aspartate (NMDA), amino-3-hydroxy-5- methyl-4-isoxazolepropionate (AMPA), kainate and G-protein-coupled metabotropic receptors. Extracellular accumulation of EAAs and excessive activation of EAA receptors contribute to neuronal cell death observed both in acute insults to the CNS and in chronic neurodegenerative diseases, including ALS, Huntington’s disease, Alzheimer disease and behavioral disorders such as depression (Gegelashvili et al., 2001).

Low extracellular levels of glutamate are maintained by transport into neurons and astrocytes. Five distinct glutamate transporters have been cloned that allow for sodium-dependent high-affinity glutamate transport, designated GLT1/EAAT2, EAAC1/EAAT3, GLAST/EAAT1, EAAT4, and EAAT5 (Danbolt, 2001). The EAAT (excitatory amino acid transporter) nomenclature refers to the human transporter species while GLT refers to the rodent transporter homologue of EAAT. Expression of EAAT1 and EAAT2 is generally restricted to astroglia; other EAATs are neuronal. The astroglial transporter EAAT2 is the dominant transporter in brain and spinal cord, accounting for up to 95% of all activity (Danbolt, 2001).

Excess levels of glutamate in the cerebral spinal fluid (CSF) (10 fold increase) are found in up to 40% of ALS patients (Rothstein et al., 1990). Reduced functional transport of glutamate has been observed in postmortem tissue from patients with ALS (Rothstein et al., 1992). This is due to a selective loss of EAAT2, whose immunoreactivity is dramatically reduced in ALS (Rothstein et al., 1995). At least 40% (and as much as 75%) of all sporadic ALS patients appear to have defects in glutamate transport and excessive extracellular glutamate levels (Spreux-Varoquaux et al., 2002). SOD1G93A transgenic ALS rats also show reduced synaptosomal glutamate uptake activity as well as increased extracellular levels of glutamate and reduced clearance of glutamate (Dunlop et al., 2003, Howland et al., 2002). All SOD1 mutant rodent animal models of ALS show reduced levels of GLT1 (Dunlop et al., 2003, Howland et al., 2002). Alterations of glutamate transport protein expression have also been reported in other neurodegenerative and demyelinating diseases including Huntington’s disease, Alzheimer’s disease, and multiple sclerosis.

Genetic overexpression of the EAAT2/GLT1 protein is protective in vitro and delays disease in vivo (Guo et al., 2003). A recent unbiased small molecule screen employing low throughput spinal cord tissue slices revealed that beta lactam antibiotics, including ceftriaxone, increase GLT1/EAAT2 expression, protect against neural injury and delay disease in ALS mice (Rothstein et al., 2005).

Here we report our effort to employ a new drug discovery approach by developing a cell-based luciferase reporter screening system for identifying EAAT2 promoter activators. By screening a library of up to 1040 FDA approved compounds and natural products, we identified harmine, a naturally occurring beta-carboline alkaloid, as one of the top hits for turning on the EAAT2 promoter. We demonstrated that harmine effectively increased GLT-1 expression both in vitro and in vivo. Our studies provide a potential new neurotherapeutic strategy by modulating the activity of glutamate transporters via gene activation.

Section snippets

Generation of CC4 line and luminescence based screening assay

We obtained a human immortalized astroglial-fetal derived cell (HIA) line from Dr. Ahmet Hoke at Johns Hopkins University (Hoke et al., manuscript in preparation). To be able to use this line in our screening assay, we stably transfected HIA cells with a plasmid encoding firefly luciferase reporter under the control of a short fragment of the human EAAT2 promoter (2.5 kb) as described previously (Rothstein et al., 2005). The obtained cell line (CC4 line) was then validated to see if it is

Astroglial-based EAAT2 promoter reporter screening assay reveals Harmine as potent activator of gene expression

A reporter cell line (CC4) was generated by stably expressing a firefly luciferase reporter under the control of a 2.5 kb human EAAT2 (GLT-1) promoter fragment in human immortalized astroglial-fetal derived cells (Rothstein et al., 2005, Yang et al., 2009). Compared to other reporter systems, luminescence based assays are most sensitive therefore suitable to develop a screening assay that can be converted into a high throughput screening (HTS) assay to screen large chemical libraries (100,000s

Discussion

The discovery and development of new drugs that could modulate glutamate transporters could be a novel approach to multiple neurological and psychiatric disorders. Dysfunctional glutamate transmission and consequent accumulation of extracellular glutamate has been a target for therapy in ALS, Huntington’s disease, epilepsy, multiple sclerosis as well as depression and schizophrenia. In this paper, we have outlined a new cell-based screening assay for the identification of small molecule

Acknowledgements

We thank Ahmet Hoke for HIA cells and Clive Svendsen for HSC line. This work was supported by the Cinque Foundation and the Maryland Stem Cell Research Foundation. The findings from this study are dedicated to the memory of Steven Cinque.

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