A chemical genetic approach identifies piperazine antipsychotics as promoters of CNS neurite growth on inhibitory substrates
Introduction
A striking feature of the adult mammalian central nervous system (CNS) is that severed axons often fail to regrow and reform lost connections after injury. As a consequence, spinal cord injury (SCI) typically results in a lifelong loss of sensory and motor function below the level of the lesion. Unfortunately, there are currently no proven clinical therapies that can stimulate long distance regeneration and improve functional recovery for SCI patients.
CNS regenerative failure results from the poor intrinsic growth capacity of mature CNS neurons (Goldberg et al., 2002), together with the inhibitory environment of CNS lesions (Yiu and He, 2003). Genetic perturbations that stimulate intrinsic growth programs can improve regeneration (Liu et al., 2010, Moore et al., 2009, Park et al., 2008); however, environmental obstacles, such as those found in myelin and the glial scar, still limit axon growth through the lesion site (Yiu and He, 2003). Inhibitory proteins in myelin include Nogo (Huber and Schwab, 2000), myelin-associated glycoprotein (MAG; McKerracher et al., 1994), and oligodendrocyte myelin glycoprotein (Wang et al., 2002). Manipulations designed to overcome these myelin-derived molecules have resulted in limited improvements in regeneration (Atwal et al., 2008, Cafferty et al., 2010, Kubo et al., 2008, Lee et al., 2010, Walmsley and Mir, 2007). Similarly, glial scar proteins such as chondroitin sulfate proteoglycans (CSPGs) inhibit growth in vitro (Monnier et al., 2003, Snow et al., 1990, Ughrin et al., 2003), and their expression correlates with regenerative failure in vivo (Davies et al., 1999, McKeon et al., 1991). Reducing expression of these CSPGs (Bradbury et al., 2002) or targeting their downstream signaling pathways (Koprivica et al., 2005, Shen et al., 2009, Sivasankaran et al., 2004) has led to a modest degree of regeneration and functional recovery after injury. Despite these successes, the signaling pathways responsible for growth inhibition by myelin proteins and CSPGs are not fully understood and regeneration-promoting therapies have yet to reach clinical practice.
The translation of novel therapies from the lab to the clinic is rife with obstacles. Failure rates for new drugs can be as high as 95%, and Food and Drug Administration (FDA)-approved new drugs spend an average of 13 years in testing at a cost of around $1 billion (Collins, 2011). An increasingly attractive strategy to circumvent these problems is to discover new applications for drugs that are already clinically approved. One approach to achieve this “drug repurposing” is to compare the gene expression profiles of clinically approved compounds to profiles of perturbations that induce a phenotype of interest. Such strategies have been previously used to identify potentially novel therapies for hepatocellular carcinoma (Braconi et al., 2009, Chen et al., 2011), gastric cancer (Claerhout et al., 2011), ovarian cancer (Gullbo et al., 2011), lung cancer (Wang et al., 2011), colorectal cancer (Vilar et al., 2009), neuroblastoma (De Preter et al., 2009), and influenza (Josset et al., 2010). Here, we apply this comparative microarray approach to implicate novel strategies for improving regeneration after CNS injury.
Previously, we identified four novel compounds that promote CNS neurite outgrowth in the presence of an inhibitory myelin substrate in vitro. All four compounds also promoted growth on CSPG-derived substrates and on an in vitro model of the glial scar, but did not affect growth on permissive substrates (Usher et al., 2010). The signaling mechanisms through which these compounds act are not known, though they perturb growth cone microtubule dynamics. One compound, F05, promoted regeneration in vivo after acute transection of dorsal column sensory axons, as well as regrowth of retinal ganglion cell axons after optic nerve crush (Usher et al., 2010). These results suggest that the compounds can be exploited to identify convergent mechanisms of inhibitory environmental signaling and to develop treatment strategies for SCI.
In this study, we sought to uncover the signaling pathways affected by F05 and to identify clinically relevant regeneration-promoting compounds. Our approach makes use of the Broad Institute “Connectivity Map”, a repository of gene expression signatures for over 1300 small molecules (Lamb et al., 2006). Importantly, the database derives signatures from both nondrug bioactive compounds as well as a variety of FDA-approved compounds, thereby allowing the opportunity to suggest novel uses for currently prescribed drugs. Our analysis identified a subclass of antipsychotics (piperazine phenothiazines) that induce remarkably similar changes in gene expression to those seen with F05. We found that this structural class of antipsychotics promotes neurite growth in different types of cultured CNS neurons challenged with either myelin proteins or CSPGs through a mechanism dependent on antagonism of calmodulin signaling.
Section snippets
The novel regeneration-promoting compound F05 induces changes in gene expression similar to those induced by piperazine phenothiazine antipsychotics (PhAPs)
The novel compound F05 can promote growth in a variety of in vitro assays in which neurons encounter glial inhibitory molecules, and it also promotes regeneration in vivo, but its mechanism of action is unclear (Usher et al., 2010). To learn more about mechanisms of growth inhibitory signaling, we took advantage of the Broad Institute Connectivity Map, which allows users to query a database containing gene expression signatures from human cells treated with FDA-approved drugs and other
Discussion
Using a comparative microarray analysis of compound-induced changes in gene expression, we have demonstrated an unexpected similarity between piperazine phenothiazine antipsychotics and F05, a novel regeneration-promoting compound. PhAPs, but not antipsychotics of other structural classes, shared F05's ability to enhance CNS neuronal outgrowth in an assay in which growth was restricted by CSPGs. PhAPs were also able to overcome growth inhibition in response to the myelin-derived inhibitor MAG,
Microarray
Microarray data from F05 and vehicle treated samples were obtained using methods similar to those employed by the Broad Institute to generate gene expression signatures for the Connectivity Map (Lamb et al., 2006). Human MCF7 breast adenocarcinoma cells (American Type Culture Collection) were grown to ~ 70% confluency over a period of 24 h in DMEM media containing 10% fetal bovine serum and 1% penicillin–streptomycin–glutamine (Gibco). The cells were then treated for 6 h with F05 (5 μM) or vehicle
Acknowledgments
We thank Dr. Stan Hoffman for generously donating CSPGs, and Dr. Roman Giger for the gift of MAG-expressing CHO cells. We also thank Dr. Justin Lamb (Broad Institute) for helping to determine the experimental conditions for optimizing the microarray experiments. We acknowledge the support of the NINDS/NIMH Microarray Consortium in performing the microarray experiments. We thank Tania Slepak, Dr. Anthony Oliva, Guerline Lambert, Dr. Hassan Al-Ali, Dr. Michael Steketee, and Ephraim Trakhtenberg
References (98)
- et al.
Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning
Neuron
(1988) - et al.
Haloperidol causes cytoskeletal collapse in N1E-115 cells through tau hyperphosphorylation induced by oxidative stress: Implications for neurodevelopment
Eur. J. Pharmacol.
(2010) - et al.
High content screening of cortical neurons identifies novel regulators of axon growth
Mol. Cell. Neurosci.
(2010) - et al.
Neuronal polarity: vectorial cytoplasmic flow precedes axon formation
Neuron
(1997) Regeneration in the spinal cord
Curr. Opin. Neurobiol.
(1998)- et al.
Prior exposure to neurotrophins blocks inhibition of axonal regeneration by MAG and myelin via a cAMP-dependent mechanism
Neuron
(1999) - et al.
Overcoming inhibitors in myelin to promote axonal regeneration
J. Neurol. Sci.
(2005) - et al.
Antipsychotic drugs alter neuronal development including ALM neuroblast migration and PLM axonal outgrowth in Caenorhabditis elegans
Int. J. Dev. Neurosci.
(2008) - et al.
Chronic activation of the 5-HT(2) receptor reduces 5-HT neurite density as studied in organotypic slice cultures
Brain Res.
(2009) - et al.
Phenotype-based drug screening in primary ovarian carcinoma cultures identifies intracellular iron depletion as a promising strategy for cancer treatment
Biochem. Pharmacol.
(2011)