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, but had no effect on neurite growth in the context of a permissive laminin substrate. The ability of PhAPs to promote growth over glial-inhibitory molecules was dependent on antagonism of calmodulin, and at least one other calmodulin antagonist, W7, is able to mimic the growth-promoting abilities of PhAPs.
These results highlight the dual utility of the comparative microarray approach. The Connectivity Map is a hypothesis-generating tool that can be used to identify signaling pathways affected by a compound of interest as well as to discover new properties of clinically prescribed drugs. The comparative microarray findings presented here led to the hypothesis that inhibition of calmodulin signaling might allow neurons to alleviate substrate-derived neurite growth restriction. These findings suggest that calmodulin could be exploited as a novel target for promoting CNS regeneration in the face of environmental barriers. In addition, our results suggest a previously unrecognized potential for piperazine phenothiazine antipsychotics to induce axonal regeneration when neurons are challenged with glial-derived inhibitory molecules. Thus, although antipsychotics are primarily prescribed to alleviate psychosis, our work suggests that they may be repurposed to improve regrowth after CNS injury.
The possibility of repurposing PhAPs for treatment of CNS injury raises questions concerning dosage and therapeutic index. In our in vitro
assays, there was a narrow concentration window between efficacy (growth promotion) and toxicity (cell death), for the PhAPs tested. Presumably this reflects the existence of multiple targets for these drugs; future efforts could include structure-activity relationship studies to identify compounds that promote growth without killing cells. Alternatively, dose response studies in vivo
could determine an optimal window for promotion of regeneration by existing PhAPs. Prochlorperazine is typically prescribed at doses between 20 and 150 mg per day (~0.3–2.1 mg/kg for a 70 kg adult), depending on the indication and symptom severity. In one study, patients chronically treated with 120 mg trifluoperazine/day showed brain levels of 1.0 μg/mL (1.65 μM; Karson et al., 1992
). Thus, PhAP concentrations that we find to be effective for growth promotion in vitro
can be achieved with dosing schemes currently used in therapy. While PhAPs produce serious side effects, including extrapyramidal disorder, these would need to be balanced against the possibility of functional recovery from CNS trauma.
Other notable hits from the Connectivity Map include several agents that act on aspects of calcium signaling, including tetrandrine and fendiline. This reinforces our findings that targeting downstream calcium signaling pathways allows cells to overcome growth-inhibitory signals. In addition to PhAPs, there were several other clinically prescribed classes of drugs that emerged from the comparative microarray analysis. Antihistamines, vasodilators, antidepressants, glucocorticoid agonists, and antibiotics could all be studied further in assays of neuronal growth and regeneration. Future studies using any of these Connectivity Map hits could elucidate novel mechanisms of regenerative failure in the CNS and also point towards potential therapies for CNS injury.
Our studies show that PhAPs promote neurite growth from cells growing on a mixture of inhibitory CSPGs. We chose this mixture, which likely consists mainly of neurocan, phosphacan, versican, and aggrecan (Ernst et al., 1995
; Monnier et al., 2003
), in order to mimic the CSPGs that accumulate at lesion sites after CNS injury (Asher et al., 2000
; Levine 1994
; McKeon et al., 1999
; Monnier et al., 2003
). Future studies could assess whether PhAPs are more active against some individual CSPGs as opposed to others, which might provide additional insight into the mechanism of growth promotion and the effects of shared domains among different CSPGs (e.g., immunoglobulin domains, hyaluronic acid-binding domains, chondroitin sulfate side chains).
Previous studies have suggested that antipsychotics could modulate neurite outgrowth, but none have implicated piperazine phenothiazines as a class. Antipsychotics such as olanzapine, quetiapine, and clozapine have been shown to enhance growth factor induced neurite outgrowth in PC12 cells, at concentrations of 10–40 μM (Lu and Dwyer, 2005
). In neuroblastoma cells, high (100 μM) concentrations of haloperidol had the opposite effect, disrupting neuritic cytoskeletal organization (Benitez-King et al., 2010
). Interestingly, clozapine, and to some extent fluphenazine, were able to increase axon lengths from mechanosensory neurons in C. elegans
, when made available to larvae at high (160 μM) concentrations (Donohoe et al., 2008
). These drugs also inhibited neuronal migration, and it is unclear to what extent the effects on axons were direct or reflected abnormal positioning. In rats, haloperidol reduced the density of dopaminergic axon terminals when administered immediately after a lesion to the substantia nigra, but improved dopamine terminal sprouting when administered after a delay (Tripanichkul et al., 2003
), suggesting that the growth response of neurons to haloperidol can vary dramatically under slightly different circumstances. This parallels our observations that PhAPs can have either no effect or a robust growth-promoting effect when added to cells that are growing under permissive versus inhibitory conditions, respectively. The findings presented here are the first to show that a specific class of antipsychotics can improve growth of primary neurons in the face of inhibitory molecules that prevent CNS regeneration after injury, and do so at low micromolar concentrations.
All clinically effective antipsychotics inhibit dopamine receptors, primarily D2 type receptors (Nord and Farde, 2011
). Dopamine receptor signaling has been linked to transcriptional regulation of axon guidance molecules (Jassen et al., 2006
), suggesting that modulation of dopamine pathways could affect neurite growth. Indeed, treatment with the D1 agonist SKF-38393 increased neurite length and arborization of dissociated rat striatal cells cultured on polyornithine (Schmidt et al., 1996
). The D2 agonist quinpirole can potentiate cortical neurite length and branching on a polylysine substrate (Todd, 1992
), and dopamine itself has been shown to increase neurite growth in striatal neurons (Schmidt et al., 1998
). Thus dopamine receptor agonism is generally associated with increases in neurite growth. Since antipsychotics act as dopamine antagonists, we hypothesized that their growth-promoting effects were independent of their effects on dopamine signaling. Accordingly, our results suggest that the growth-promoting effect of PhAPs did not depend on antagonism of dopamine receptors. Similarly, the ability of antipsychotics to cause excessive axon growth of mechanosensory axons in C. elegans
appears to be independent of dopamine receptor antagonism (Donohoe et al., 2008
). Overall, the results indicate that the ability of PhAPs to increase neurite growth is independent of their ability to antagonize dopamine receptors.
In addition to their effect on dopamine receptors, antipsychotics are known to target a variety of cell surface receptors and intracellular signaling cascades (Miyamoto et al., 2005
). Some of these targets, including serotonin receptors (Dudok et al., 2009
; Homma et al., 2006
), histamine receptors (Munis et al., 1998
), NMDA receptors (George et al., 2009
; Kuo et al., 2010
), adrenergic receptors (Kwon et al., 1996
), and muscarinic receptors (VanDeMark et al., 2009
), have been implicated in the modulation of neuronal growth and differentiation. We chose to investigate the calcium binding protein calmodulin, since phenothiazine-based compounds, and in particular the PhAP trifluoperazine, are known to bind to and inhibit calmodulin (Tanokura and Yamada, 1986
; Vandonselaar et al., 1994
Our results implicate calcium/calmodulin signaling in growth-inhibitory responses to extracellular cues after CNS injury. This is consistent with findings that both CSPGs and myelin proteins induce a local influx of calcium in growth cones that can affect turning behavior (Hasegawa et al., 2004
; Henley et al., 2004
; Snow et al., 1994
), and with studies in invertebrates linking calmodulin to growth inhibition (Polak et al., 1991
). However, since calcium influx can also potentiate neurite outgrowth (Homma et al., 2006
; Kater and Mills, 1991
), the relationship between calcium/calmodulin signaling and neurite growth is highly dynamic, and depends on the growth state of individual neurons. It is possible that the drop-off in neurite growth seen with high concentrations of both PhAPs and the calmodulin antagonist W7 reflects this dynamic relationship.
Since calmodulin appears to be a relevant target for the effects we observe, it is curious that pimozide, an antipsychotic known to inhibit calmodulin (Levin and Weiss, 1976
), did not promote neurite outgrowth on CSPGs. It may be that pimozide affects additional signaling pathways that negate potential growth promotion resulting from calmodulin inhibition. Alternatively, pimozide and PhAPs could have differing effects on calmodulin’s interaction with downstream effector proteins. These distinct possibilities should be addressed in future mechanistic studies.
In addition to the relevance of these findings to CNS regeneration, our results also have implications for the mechanisms through which antipsychotics alleviate symptoms of psychosis. Schizophrenia is characterized by deficits in neuronal growth and connectivity (Lynall et al., 2010
; Skudlarski et al., 2010
; Zalesky et al., 2011
), and genes associated with schizophrenia, such as Disrupted in Schizophrenia 1 (DISC1), are known to play significant roles in neurite growth (Hattori et al., 2010
; Miyoshi et al., 2003
; Ozeki et al., 2003
). In particular, there is evidence that glial-derived growth inhibitors are associated with schizophrenia. For example, levels of both CSPGs and the myelin-derived inhibitor Nogo are increased in postmortem schizophrenic brains (Novak et al., 2002
; Novak and Tallerico, 2006
; Pantazopoulos et al., 2010
). Therefore, the ability of antipsychotics to promote growth/sprouting in the presence of glial-derived inhibitory molecules may represent a mechanism for improving neuronal connectivity in schizophrenic patients. Similar considerations apply to other neurodevelopmental disorders for which antipsychotics are used, including autism and depression (McPheeters et al., 2011
; Pae et al., 2011
A major goal in both neuropsychiatric research and neuroregeneration studies is to understand and overcome the mechanisms that contribute to maladaptive pathology. Using a unique tool to compare gene expression profiles, we have uncovered a novel ability of PhAPs to improve neurite outgrowth from CNS neurons grown on glial-derived inhibitory molecules, and have identified calmodulin as a novel therapeutic target that could be manipulated to improve axonal regrowth. In addition, our results highlight an underappreciated link between CSPG and myelin inhibitory signaling and the developmental deficits associated with schizophrenia. Importantly, since piperazine antipsychotics are already clinically prescribed to alleviate psychosis, our work suggests that they could be repurposed to induce neuronal growth and restore connectivity after CNS injury.