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 compounds that activate gene expression of glutamate transporters, proteins that are known to become dysfunctional and downregulated during ALS disease progression, as well as in other neurological disorders (Gegelashvili et al., 2001
) (Lepore et al., 2007
; Rothstein, 2009
; Rothstein et al., 2005
This assay was designed to be readily translated into a high-throughput screening (HTS) platform to screen large chemical libraries (e.g. 105-106 compounds). A pilot screen was successfully performed with an external HTS facility screening a library of about 30,000 compounds (R. Sattler and J. Rothstein, unpublished observation). In addition, this cell-based assay was used to perform a combinatorial screen of the Microsource library used in this publication (Li et al., manuscript in preparation).
Our laboratory had performed a recent screen for glutamate transporter activators using primary organotypic spinal cord slice cultures (Rothstein et al., 2005
). In this screen, we identified numerous beta lactam antibiotics, such as ceftriaxone, as effective small-molecule compounds that upregulated GLT-1 gene expression and observed that it protected against motor neuron cell death. Since primary cultures cannot be used for high throughput screening, we developed the current screening assay and validated it by testing the newly updated Microsource library. A recent report shows the development of a HTS for translational activators of glutamate transporter EAAT2 (Colton et al., 2010
). Using a cell-based enzyme-linked immunosorbent assay in a stably transfected astrocyte cell line, Colton and colleagues developed a screening assay to specifically screen small molecule compounds that activate EAAT2 translation, although the active compounds in that assay were not shown to actually increase endogenous protein in vivo
. That assay differs from our assay in that we screen for compounds acting on a transcriptional level, which means we screen for the formation of new mRNA followed by new protein synthesis. Studies have shown that mRNA levels of GLT-1 are downregulated even before disease onset (Yang et al., 2009
), hence, it will be crucial to boost new mRNA synthesis to overcome the loss of GLT-1/EAAT2 protein during disease.
The use of luciferase-based reporter gene assays has to be taken with care, as some compound structures can lead to false positive hits by stabilizing the luciferase protein instead of modulating gene transcription (Auld et al., 2008
). In our study, we focused on only a few of the positive hits we obtained from our screening assay. While most compounds showed specific activity towards the EAAT2 promoter, some compounds with increased luciferase activity failed to be blocked with actinomycin treatment and consequently failed to increase mRNA levels of GLT-1 in vitro
. None of these compounds were further tested for transporter activity.
With the newly developed assay, we identified harmine, a naturally occurring small molecule, as an effective compound to induce transcriptional activation of astrocytic glutamate transporters in cultured astroglia- but more significantly, in vivo following repeated administration. Harmine is a beta-carboline alkaloid that was first isolated in 1847 from seeds of Peganum harmala
(Syrian rue) and Banisteriopsis caapi
, both of which have traditionally been used for ritual and medicinal preparations in the Middle East, Central Asia, and South America (Sourkes, 1999
). It is also present in common plant-derived foods and in human tissues (Guan et al., 2001
). Known pharmacologic effects of harmine include hallucinogenesis (Sourkes, 1999
), convulsive or anticonvulsive actions (Aricioglu et al., 2003
), and tremor (Guan et al., 2001
; Lutes et al., 1988
). Several potential molecular targets for these central pharmacologic effects of harmine have been identified. These include MAO-A (Kim et al., 1997
), 5-HT2A (Glennon et al., 2000
), imidazoline receptors (I1 and I2 sites) (Husbands et al., 2001
), and cyclin-dependent kinases (CDK1, 2, and 5) (Song et al., 2004
). To our knowledge, activation of any of these targets does not lead to increased glutamate transporter expression.
Few compounds have been shown to activate increased glutamate transporter-EAAT2/GLT1 protein expression and function in vivo. Our new cellular screen has provided insight into a class of compounds that could serve as a starting point for new GLT1 activators. Given harmine’s CNS side effects and its multi-target activities, harmine itself is not a suitable candidate for immediate clinical applications. However, it may eventually be possible to separate its effects on GLT-1 upregulation and on CNS pharmacology through optimization of its chemical structure. Medicinal chemistry efforts to do so are ongoing, which will hopefully lead to a more potent and selective drug candidate that can be moved through the drug development process reaching from preclinical studies to clinical trials. These trials may include clinical applications such as ALS, but also other neurodegenerative disorders as well as psychiatric diseases.