Is the mTOR-signalling cascade disrupted in Schizophrenia?

JOURNAL OF NEUROCHEMISTRY

| 2013

doi: 10.1111/jnc.12622

,

*Behavioural Neuroscience Laboratory, The Florey Institute of Neuroscience & Mental Health, Parkville, Vic., Australia †Department of Pharmacology, University of Melbourne, University of Melbourne, Melbourne, Vic., Australia

Abstract The mammalian target of rapamycin (mTOR) signalling cascade is involved in the intracellular regulation of protein synthesis, specifically for proteins involved in controlling neuronal morphology and facilitating synaptic plasticity. Research has revealed that the activity of the mTOR cascade is influenced by several extracellular and environmental factors that have been implicated in schizophrenia. Therefore, there is reason to believe that one of the downstream consequences of dysfunction or hypofunction of these factors in schizophrenia is

disrupted mTOR signalling and hence impaired protein synthesis. This results in abnormal neurodevelopment and deficient synaptic plasticity, outcomes which could underlie some of the positive, negative and cognitive symptoms of schizophrenia. This review will discuss the functional roles of the mTOR cascade and present evidence in support of a novel mTORbased hypothesis of the neuropathology of schizophrenia. Keywords: BDNF, glutamate, mammalian target of rapamycin, Reelin, schizophrenia. J. Neurochem. (2013) 10.1111/jnc.12622

The aetiology of schizophrenia involves a significant neurodevelopmental component (Owen et al. 2011; Catts et al. 2013) which causes the central nervous system to become particularly susceptible to the effects of various extrinsic risk factors such as stress and drug abuse (McGrath et al. 2003). While it may be possible to mitigate the effects of extrinsic risk factors (Norman et al. 2002; Green et al. 2008), understanding and managing the neurodevelopmental causes is more difficult. They are characterized by complex neuromolecular mechanisms which affect processes crucial for normal growth and functioning of the brain. However, further investigations into these mechanisms could advance our understanding of how abnormal brain development increases the risk of developing schizophrenia. One mechanism that has been given limited attention in the context of schizophrenia is the mammalian target of rapamycin (mTOR)-signalling cascade. It is the aim of this review to highlight the functional roles of this pathway in the contexts of neurodevelopment and synaptic plasticity and how accumulated evidence supports its possible involvement in the pathology of schizophrenia.

of conserved domains each with specific functional roles (Fig. 1). At the N’-terminal end are, a series of HEAT (huntingtin, elongation factor 3, a subunit of PP2A, TOR1) repeats which provide the ability for the enzyme to interact with associated proteins Raptor (regulator associated protein of mTOR) and Rictor (rapamycin insensitive companion of mTOR protein). In the middle is a FAT (FKBP12-rapamycin-associated protein, ataxia-telangiectasia, transactivation/ transformation domain-associated protein) domain which is essential for mTOR catalytic activity. The FKBP-12-rapamycin-binding (FRB) domain is the binding site for the immunosuppressant drug, rapamycin which is a specific antagonist to mTOR activity (Sehgal 1995). Rapamycin does not directly impair mTOR catalytic activity but rather disrupts the association of mTOR with Raptor and Rictor which is essential for mTOR activity (Oshiro et al. 2004). At

The mTOR-signalling cascade Mammalian target of rapamycin is a 300-kDA serine/ threonine kinase which has a structure consisting of a series

Received September 9, 2013; revised manuscript received November 7, 2013; accepted November 10, 2013. Address correspondence and reprint requests to Anand Gururajan, Behavioural Neuroscience Laboratory, The Florey Institute of Neuroscience & Mental Health, 30 Royal Parade, Parkville, Vic. 3010, Australia. E-mail: anand.gururajan@florey.edu.au Abbreviations used: BDNF, brain-derived neurotrophic factor; HRM, heterozygous reelin mouse; LTD, long-term depression; LTP, long-term potentiation; mTOR, mammalian target of rapamycin.

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 10.1111/jnc.12622

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A. Gururajan and M. van den Buuse

HEAT FAT

Rictor

Raptor

FRB

KIN

NRD

FATC

Rapamycin

Fig. 1 Structure of the mammalian target of rapamycin (mTOR) protein. At the N’-terminal end is the huntingtin, elongation factor 3, a subunit of PP2A, TOR1 domain which interacts with Raptor and Rictor. The FAT (FKBP12-rapamycin-associated protein, Ataxia-telangiectasia, Transactivation/transformation domain-associated protein) domain is essential for mTOR catalytic activity. Rapamycin binds to the FRB

domain and in doing so, inhibits mTORC1 complex formation. The negative regulator domain, within the KIN domain, contains phosphorylation sites which can regulate mTOR activity. The C’-terminal end contains the FATC domain and mutations within this domain abolish mTOR activity. See text for further details.

the C’-terminal end is the kinase domain (KIN) which itself contains a negative regulator domain. Phosphorylation of specific residues within this domain can lead to higher levels of mTOR activity. The C’-terminal end also contains a FAT domain (FATC) and mutations within this domain abolish mTOR activity (Takahashi et al. 2000). The functional effects of mTOR are mediated by two heteromeric complexes, mTORC1 and mTORC2 (Fig. 2). mTORC1, which is sensitive to rapamycin, is made up of mTOR, Raptor, GbL and proline-rich Akt/PKB substrate 40kD (PRAS40). GbL binds to the KIN domain and stimulates mTOR activity (Kim et al. 2003) and PRAS40 is an inhibitor as well as a substrate of mTORC1 activity (Wang et al. 2008). Phosphorylation of PRAS40 and dissociation from the complex is critical for mTORC1 activation (Vander Haar et al. 2007). mTORC2 contains mTOR, GbL and Rictor. Rictor is thought to bind to the N’-terminal end of mTOR but also occludes the FRB domain thus inhibiting the effect of rapamycin. However, chronic exposure to rapamycin eventually inhibits mTORC2 activity (Sarbassov et al. 2006). mTORC2 also consists of proteins SAPK interacting (Sin1) protein 1, which is essential for complex formation (Yang et al. 2006), and protein observed with Rictor (Protor) , the function of which is yet unknown (Pearce et al. 2007). mTORC1 activation is initiated by various factors including mitogens (e.g. insulin), trophic factors [e.g. brain-derived neurotrophic factor (BDNF)] and glutamate leading to activation of phosphatidyl inositol-3- kinase (PI3K). This in turn activates Akt (Protein Kinase B) which phosphorylates the tuberous sclerosis complex (TSC), a negative regulator of mTORC1 activity, and PRAS40 (Tee et al. 2003; Vander Haar et al. 2007). Phosphorylation of the TSC complex leads to disinhibition of the GTP-ase known as Rashomolog enriched in brain (Rheb) which positively regulates mTORC1 function (Zhang et al. 2003). mTORC1 activity can also be initiated by extracellular signal related kinases

that can directly phosphorylate the TSC complex (Ma et al. 2005). The two main cellular substrates for mTORC1 are the proteins that form the translation initiation machinery for protein synthesis, S6 kinase (S6K) and eukaryotic initiation factor (eIF) 4E binding protein, 4E-BP1 (Burnett et al. 1998). Phosphorylation of S6K leads to activation and biosynthesis of 40S ribosomal subunit S6 and also phosphorylation of eIF-4B, a cofactor of the RNA helicase, eIF4A. Increased eIF-4A activity is critical for the translation of mRNA substrates containing a terminal 5′-oligopyrimidine tract which code for proteins that form components of the translational machinery (Jefferies et al. 1997). S6K also regulates translation elongation via phosphorylation of eukaryotic elongation factor 2 (eEF2) kinase; phosphorylation of this kinase leads to reduced phosphorylation of the elongation factor eEF2, thus increasing the elongation rate (Proud 2009). Under basal conditions, 4E-BP1 binds to the eIF-4E cap-binding protein at the 5′ end of the mRNA, inhibiting translation but when 4E-BP1 is phosphorylated by mTORC1, eIF-4E is released and forms the eIF-4F complex with a range of other factors thus initiating mRNA translation (Beretta et al. 1996). Both S6K and 4E-BP1 contain a TOR signalling motif that is recognized by the Raptor component of mTORC1 and mutations within the motif inhibit mTORC1 activity (Schalm and Blenis 2002; Nojima et al. 2003). Although it is still unclear how mTORC2 is activated, it does play an important role in phosphorylating Akt which as an upstream activator of mTORC1 and this is an example of crosstalk between the two mTOR complexes (Jacinto et al. 2006). mTORC2, specifically the Sin1 subunit, is in turn a substrate for Akt (Humphrey et al. 2013). Other substrates of mTORC2 include protein kinase Ca which has roles in synaptic plasticity and actin dynamics (Sarbassov et al. 2004). mTORC2 was recently reported to have a significant role in the control of neuronal morphology. Thomanetz et al. (2013) generated mouse lines that had the Rictor protein


Disrupted mTOR signaling in schizophrenia

mGluR

NMDAR

5-HT6

3

ApoER2 VLDLR

TrkB

PI3K

?

mTORC2 Protor

GβL

Akt

Rictor mTOR

Sin1

TSC1 TSC2 mTORC1

PKCα

Rheb GβL Fig. 2 Flow chart which shows the individual components of mTORC1 and mTORC2 as well as the various upstream activators and downstream substrates of these two complexes. Dashed and solid arrow lines refer to multi- or direct pathways of activation. Solid lines with black circles at their ends refer to inhibitory pathways. See text for abbreviations and further details.

conditionally knocked out of the central nervous system thus inhibiting the formation of mTORC2. They found these mice to have smaller neurons, reduced brain weight and volume at birth and adulthood. Moreover, these morphological changes occurred in the absence of effects on the downstream targets of mTORC1, S6K and 4E-BP1.

mTOR signalling in neurodevelopmental architecture Axonal growth is a tightly regulated process dependent on the presence of neurochemical cues, chemoattractants and chemorepellants, which ‘push, pull and hem’ the axon, guiding it towards its target. Response to these cues is dependent on localized protein synthesis and degradation in the growth cone, thus allowing the axon to regulate its own structure and function independently of the rest of the neuron (Martin 2004). The role of mTOR in this process was first identified in a study by Campbell and Holt (2001) who demonstrated that in Xenopus neurons, rapamycin inhibited the chemoattractant and chemorepellant effects of netrin-1

PRAS40

ERKs

Raptor mTOR

DISC1 S6K

4E-BP1

and Semaphorin3A respectively. Piper et al. (2006) further showed, also using Xenopus neurons that Slit, another chemorepellant, induced rapamycin-sensitive protein synthesis of cofilin, an actin-depolymerizing protein, which resulted in growth cone collapse. Synaptic input into the neuron and subsequently signal integration is dependent on the shape, size and branching pattern of the dendritic arbour. Like axonal growth, arbour development is also dependent on external cues as well as internal mechanisms that activate transcription factors and various kinases relevant to this process (Jan and Jan 2003). Studies have shown that chronic treatment with rapamycin reduced the number of branches and arbour field size of neurons cultured in vitro (Jaworski et al. 2005; Kumar et al. 2005; Grider et al. 2009). Several proteins that are synthesized in response to mTOR signalling include Glutamate recepor interacting protein PSD-95: post-synaptic density 95, glutamate receptor subunits, PSD-95 and Shank which are involved in synapse formation (Swiech et al. 2008). Related to the process of dendrite arbourization is also the formation of filopodia and their conversion into dendritic spines which


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form synapses with pre-synaptic axons (Ziv and Smith 1996). To demonstrate a role for mTOR in this process, Kumar et al. (2005) showed that chronic rapamycin treatment reduced the number of filopodia and spines. Others have shown that deletion of TSC proteins or over-expression of PI3K and Akt cause an increase in spine head width (Jaworski et al. 2005; Tavazoie et al. 2005). Recently, Urbanska et al. (2012) showed using RNAi to selectively knock down Raptor and Rictor that both mTORC1 and mTORC2 were involved in controlling dendrite morphology of hippocampal neurons. Furthermore, and in contrast to the findings by Thomanetz et al. (2013), knockdown of Rictor induced a decrease in S6K phosphorylation, which led the authors to suggest that the effects of mTORC1 in this context are under the control of mTORC2.

mTOR signalling in synaptic plasticity Synaptic plasticity is defined as the ability for interneuronal connections to be modified in terms of their strength and is thought to form the basis for learning and memory (Martin et al. 2000). The two main forms of long-term synaptic plasticity are long-term potentiation (LTP), which increases synaptic strength, and long-term depression (LTD), which is the weakening of synaptic strength. Under experimental settings both can be induced by tetanic pre-synaptic stimulation of opposing frequencies (Malenka 1994). LTP can be divided into two phases which are defined by specific postsynaptic events and each phase may be involved in stages of memory formation and consolidation (Fig. 3). Early phase

E-LTP

LTP (E-LTP), thought to be involved in short-term memory, describes the immediate and brief enhancement in synaptic neurotransmission by stimulation of kinase activity, whereas late phase L-LTP, thought to be responsible for long-term memory, is delayed in onset, persists longer and involves gene expression and protein synthesis that is localized to the pre- and post-synaptic terminals (Huang 1998). LTD can also be divided into early and late phases which correspond to the activation of phosphatases that deactivate target proteins activated by LTP-sensitive kinases and transcription and translation, respectively. The role of LTD in learning and memory is thought to be a fine-tuning mechanism to ensure precision storage of information (Kauderer and Kandel 2000). Tang et al. (2002) observed in hippocampal slices the postsynaptic existence of 4E-BP1 and reported that while L-LTP was disrupted by rapamycin, E-LTP was unaffected. Stoica et al. (2011) found that a dose of rapamycin that was inactive in hippocampal neurons from wild-type mice was able to disrupt L-LTP in hippocampal neurons obtained from mTOR heterozygote mice. In a study by Cammalleri et al. (2003), it was observed that rapamycin inhibition of L-LTP was specific to the induction phase and that NMDA- and PI3K-induced phosphorylation of S6K was localized to the dendrites of CA1 neurons in hippocampal slices. To further highlight that mTORC1-dependent synthesis of synaptic proteins is a localized phenomenon, two studies using hippocampal slices, reported that there was no difference or loss in induction of LLTP when dendrites were physically isolated from the cell bodies (Cracco et al. 2005; Vickers et al. 2005). Recently

L-LTP

Post-synaptic potential

Kinase activity

HFS Gene transcription, translation

Time

LFS

Phosphatase activity E-LTD

L-LTD

Fig. 3 The two main forms of long-term synaptic plasticity are long-term potentiation (LTP), induced by high-frequency stimulation (HFS), and longterm depression (LTD), induced by low-frequency stimulation (LFS), which increase and decrease synaptic connection strength respectively. LTP and LTD can be divided into early and late phases which are defined by different pre- and post-synaptic events. See text for further details.



Huang et al. (2013) have shown that mTORC2 also has a role in L-LTP and the conversion from E-LTP to L-LTP via its stimulatory effects on actin polymerization. Several studies have reported that LTD also involves the mTOR-signalling cascade. Particularly, LTD evoked by the stimulation of metabotropic glutamate receptors (mGlu-LTD) with the agonist dihydroxyphenylglycine was associated with increased phosphorylation of mTOR, 4E-BP1, S6K and blocked by rapamycin (Hou and Klann 2004; Banko et al. 2006; Antion et al. 2008a). It was also demonstrated that conversion of LTP into LTD (depotentiation) by application of dihydroxyphenylglycine 3 min after induction of LTP was inhibited by rapamycin (Zho et al. 2002). From these studies, it would appear that the mTORsignalling cascade has a specific function in L-LTP and formation of long-term memories which is consistent with its function in controlling synthesis of proteins which facilitate but not necessarily induce synaptic plasticity (Graber et al. 2013).

Disrupted mTOR signalling in schizophrenia Evidence which supports a role of disrupted mTOR signalling in the neuropathology of schizophrenia is based on a range of studies which have reported that dysfunction of diverse upstream activators and environmental stressors, that have been previously implicated in schizophrenia, can lead to either over-activation or inhibition of the signalling pathway (Fig. 1). Here, we review several of these studies and discuss the details which could explain the nature and direction of disruption. Reelin is a glycoprotein which binds to apolipoprotein E receptor 2 and very low density lipoprotein receptors. It is secreted by Cajal-Retzius cells during brain development to control cortical layering and by hippocampal GABA-ergic and cerebellar glutaminergic granular cells in the adult brain to maintain neural networks (Folsom and Fatemi 2013). There is significant down-regulation of reelin expression in schizophrenia (~50%) by an epigenetic phenomenon (Tamura et al. 2007) and preclinical studies in heterozygous reelin mice (HRM) have revealed a schizophrenia-like behavioural phenotype (van den Buuse et al. 2012; Rogers et al. 2013). Several studies have reported reelin-induced recruitment of Akt and PI3K via phosphorylation of Disabled-1 (Bock et al. 2003; Jossin and Goffinet 2007). Specifically, Jossin and Goffinet (2007) showed that while the mTOR-signalling cascade has no role in cortical development, it does mediate the trophic effects of reelin on hippocampal dendrite growth and branching via phosphorylation of S6K, effects which may influence synaptic plasticity. Most recently, HRM have been tested using the fear conditioning and extinction paradigm. In a clinical context, this paradigm has detected deficits in the emotional memory of patients with post-traumatic stress or other

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anxiety-related disorders and recently in patients with schizophrenia (LeDoux 2000; Holt et al. 2009). In the preclinical version of this test performed by Iafrati et al. (2013), mice were first tested in Context A where they were exposed to a conditioning stimulus (CS, auditory) that coterminated with an unconditioned stimulus (US, footshock). The next 2 days (extinction training), mice were placed in Context B which was different to Context A visually and also had a different odour. They were exposed to the CS and their freezing behaviour was recorded. Seven days after the last extinction training session, they were placed back in Context A and exposed again to the CS while their freezing behaviour was recorded. Iafrati et al. have shown that while HRM display normal extinction behaviour, during fear renewal testing several days later there was a significant reduction in freezing behaviour suggestive of a deficit in long-term fear conditioned memory. This deficit was correlated with an absence of LTP in the prefrontal cortex (PFC) as well as defects in dendritic spine morphology and density in HRM. The authors proposed from these findings that reelin functions to ensure proper maturation of synaptic networks which facilitate consolidation of long-term fear memory even after a period of extinction training. Interestingly, the observed HRM deficit in fear memory, PFC LTP and defective spine morphology was rescued by injections with ketamine, an NMDA-receptor (NMDAR) antagonist and this effect was blocked by rapamycin. Independent of its interactions with reelin on mTOR signalling, glutamate has direct effects on the mTORsignalling pathway as described in the next section. Glutamate is the main excitatory neurotransmitter in the brain and exerts its effects via a myriad of ionotropic and metabotropic receptors. ‘Glutamatergic dysfunction’ is a well-known aspect of schizophrenia neuropathology (Moghaddam and Javitt 2012) and most recently a metaanalysis of imaging studies by Marsman et al. (2013) reported that in schizophrenia patients there was a significant reduction in medial prefrontal glutamate levels and an increase in its metabolite, glutamine. Furthermore, reductions in both glutamate and glutamine in patients decreased at a much faster rate with age compared with healthy controls. Gong et al. (2006), in their experiments with mouse embryonic neurons, showed that rapamycin inhibited hippocampal dendritic protein synthesis induced by the activation of NMDAR and metabotropic glutamate (mGlu-Group 1 and II) receptors. They also showed that the effects of NMDAR activation were in particular mediated by recruitment of PI3K and Akt. The fact that both NMDA and mGlu receptors can initiate mTOR-dependent protein synthesis either suggests a yet to be determined cooperative and receptor-dependent differential activation. What is something of a paradox, however, is that in the HRM, NMDAR inhibition activated the mTOR-signalling cascade and restored synthesis of synaptic proteins required for formation of long-term


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memories (Iafrati et al. 2013). Our explanation for this inconsistency is based on three separate reports; our group has shown that the stoichiometry of NMDA-receptor subunit expression is significantly altered in the hippocampus of HRM (van den Buuse et al. 2012) which could lead to increased NMDA-receptor-mediated excitotoxicity and cell death (Liu et al. 2007). Moreover, Autry et al. (2011) have shown that ketamine triggers an increase in levels of BDNF in the hippocampus. Thus, in HRM an inhibition by ketamine would block NMDA-receptor-mediated neurotransmission, trigger BDNF production in the hippocampus which reactivates mTOR signalling. This would suggest that HRM have reduced BDNF levels which is not the case; there is an inverse (and sex-specific) relationship between the two (Pillai and Mahadik 2008; Hill et al. 2013). Further experiments are clearly warranted to more carefully examine interactions between glutamatergic neurotransmission and BDNF and their effects on mTOR signalling in HRM. Brain-derived neurotrophic factor, like reelin, has key roles in neurodevelopment and synaptic plasticity in the brain that are mediated by binding to the tropomysin-related kinase B (TrkB) receptor and activation of various signalling cascades (Leal et al. 2013). Notably, our group has reported that the effects of BDNF are highly regulated in a sex- and regionally specific manner (Wu et al. 2012). Several clinical studies have reported reductions in brain BDNF levels, reduced expression of TrkB in schizophrenic patients and polymorphisms to the gene encoding BDNF causes resistance to treatment with anti-psychotics (Issa et al. 2010; Zhang et al. 2013). In a microarray study of rat cortical and hippocampal neurons, Schratt et al. (2004) found that BDNF induced the translation of 79 mRNAs at 4 days in vitro and 48 mRNAs at 14 days in vitro in a rapamycin-sensitive manner. Translation of mRNAs at the earlier time point coded for proteins with roles in axonal growth (e.g. Limk1), whereas translation of the ones at 14 days in vitro coded for proteins that have roles in synaptic plasticity (Homer2, NMDAR subunit 1). Others have shown that BDNF-induced local synthesis of synaptic proteins in dendrites is via the recruitment of eukaryotic elongation factors, eEF1A and eEF2 and also that BDNF altered the spine to filopodia ratio in a rapamycinsensitive manner (Inamura et al. 2005; Kumar et al. 2005). The expression of the a-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor subunit, GluR1, has emerged as one of the proteins that is dependent on BDNF-induced activation of the mTOR-signalling pathway and is crucial for the formation of long-term memories (Slipczuk et al. 2009). Although all these authors implicated mTORC1 as the main downstream target following TrkB activation, based on the findings by Huang et al. (2013), it is quite possible that mTORC2 may also mediate some of the effects of BDNF on neuronal morphology and therefore synaptic plasticity. Thus far, the studies we have reviewed have broadly implicated depressed mTOR signalling as having a

pathological role to play in schizophrenia. However, two recent studies have suggested that overactive mTOR-signalling activity may have an equally significant role. The disrupted-in-schizophrenia 1 (DISC1) gene codes for a scaffolding protein which interacts with a range of other cellular proteins to modulate their functional activities at different stages of neurodevelopment (Bradshaw and Porteous 2012). Of all the genetic factors that are thought to contribute to the risk of developing schizophrenia, mutations in DISC1 have been particularly relevant (Roberts 2007; Bradshaw and Porteous 2012) with several DISC1 mouse models displaying a range of behaviours of clinical relevance (Jaaro-Peled 2009). Zhou et al. (2013) showed that DISC1 knockdown in the dentate gyrus of adult mice induced abnormal morphology and excitability of neuronal networks, deficits in aspects of cognition, depressive as well as anxietylike behaviours. Importantly, injections of rapamycin normalized excitability thresholds and rescued selected behavioural phenotypes without affecting morphological defects. The results of this study would support the unique hypothesis that mTOR signalling is negatively regulated by DISC1. The role of serotonin (5-HT) in schizophrenia pathology is now recognized with equal importance to the roles of other neurotransmitters such as dopamine and glutamate (AbiDargham et al. 1997). In particular, 5-HT6 receptor antagonists have shown promise to treat cognitive dysfunction in schizophrenia (Rosse and Schaffhauser 2010) which otherwise is still a significant challenge to address with currently available pharmacotherapies (Barch and Ceaser 2012; Meffre et al. 2012). Recently, Meffre et al. (2012) observed that 5HT6 receptor forms a complex with mTORC1 and ligand binding initiates activation of downstream substrates, S6K and 4E-BP1, in the prefrontal cortex an effect which was inhibited by pre-treatment with rapamycin. They also showed that acute impairments of social recognition and episodic-like memories induced by 5-HT6 receptor activation were reversed by rapamycin. But perhaps one of the most significant findings from their work was that in two neurodevelopmental models, neonatal phencyclidine treatment and post-weaning social isolation, chronic impairments in these same aspects of cognition were effectively rescued by rapamycin acutely administered at adulthood. While the effects of chronic phencyclidine treatment could possibly be explained by effects on glutamatergic control over the mTOR signalling pathway, the implication that social isolation during critical stages of growth and development can also influence mTOR signalling is suggestive of an epigenetic vulnerability to this form of early life stress (Nestler 2012). We have reviewed several scenarios related to schizophrenia pathology that can lead to either a suppression or overstimulation of mTOR signalling. Aside from the numerous methodological differences between the reviewed studies that may offer one explanation for this differential activation, there may more complex reasons. Firstly, when considering



the role of neurotransmitters such as serotonin, glutamate and their receptors in schizophrenia, it is never a simple case of a brain-wide unidirectional reduction or increase that causes the various symptoms but rather, abnormal fluctuations specific to regions which control particular behaviours (AbiDargham et al. 1997; Tuominen et al. 2005). Consequently, mTOR signalling under these conditions may vary between and possibly within brain regions as well. Secondly, some neuronal populations may be more sensitive than others to pathogenic effects on mTOR signalling. Weston et al. (2012) have shown that chronic suppression of mTOR activity selectively affects only glutamatergic neurons in hippocampal preparations but not GABA-ergic neurons in striatal preparations. A third possibility that needs to be considered is how the mTOR-signalling cascade is influenced by environmental factors at various stages of growth and development. Indeed, as described above (Meffre et al. 2012), early-life stress induces an over-activation of mTOR signalling perhaps as a compensatory response to the wellknown morphological effects of stress on neurons (Watanabe et al. 1992; Cook and Wellman 2004; Polman et al. 2012). Conversely, stress also has an attenuating effect on BDNF release and function (Suri and Vaidya 2013) and so it is possible that this could lead to suppression of mTOR signalling. Again, these changes could vary between brain regions so mTOR signalling could potentially be both increased and decreased in distinct brain regions. Such variability could underlie the complex clinical phenotype of schizophrenia. In the next section, we will review evidence from other animal models with disrupted mTOR signalling some of which have yet to be confirmed demonstrable links with schizophrenia but display strong schizophrenia-like behaviours.

Animal models of disrupted mTOR signalling – relevance to schizophrenia The role of the mTOR-signalling pathway in cognition has been elucidated by determining the effect of rapamycin using different testing paradigms. A study by Parsons et al. (2006) investigated the role of the mTOR pathway in the formation of long-term fear memory in amygdalar neurons. Using a fear conditioning paradigm they showed that 24 h after trained rats had received bilateral infusions of rapamycin into the amygdala, there was a reduction in the time spent freezing when exposed to Context A or Context B with the CS but without the US. They also observed that 30–60 min after the training procedure, there was a significant increase in the phosphorylation of S6K in the amygdala which was effectively inhibited by rapamycin. To assess memory retrieval and consolidation in another group of rats, the day after training they were exposed to the CS in Context B before receiving infusions of rapamycin. Twenty-four hour later they were tested in the same context but with modified

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exposure to the CS. These rats showed less freezing behaviour which led to the suggestion that even after a stimulus reminder, inhibition of the mTOR pathway led to an inability to consolidate their fear memory. The inhibitory avoidance test is another behavioural paradigm used to assess fear memory and several studies using it have reported deficits in animal models of relevance to schizophrenia (Alonso et al. 2002; Savonenko et al. 2008). In this test, rodents are placed onto an elevated platform adjacent to a shock grid. During the training phase, as the rodents step down onto the grid, they receive a shock. During the test session, they are placed back onto the platform and latency to step down on the grid (no shock) is measured. Bekinschtein et al. (2007) showed that there was a rapid and transient increase in mTOR phosphorylation in the dorsal hippocampus during the training phase and an increase in S6K phosphorylation 15 min after the training phase. Bilateral infusions of rapamycin into the CA1 region of the dorsal hippocampus prior to training did not affect step-down latency onto the shock grid when rats were tested 3 h later but caused long-lasting retrograde amnesia (decreased latency to step down on shock grid) when tested 24 h to 7 days later. This was accompanied by a decrease in S6K phosphorylation. To ensure that these effects were not because of any non-specific effects of rapamycin, rats with rapamycin-induced memory deficits underwent another training session 24 h after the initial one after which they were able to learn and form avoidance normally. The authors concluded that mTOR signalling in the hippocampus was significantly involved in the formation of long-term as opposed to short-term memory. There are two mammalian genes which code for S6K– S6K1 and S6K2. Antion et al. (2008b) examined the behavioural phenotype of the S6K-KO mice in the context of learning and memory. Using the fear conditioning paradigm, they showed that S6K1-KO mice had memory deficits as early as 1 h after the training phase, whereas in S6K2-KO mice, memory deficits were mild and seen only 7 days after. This was indicative of the differential roles of S6K1 and S6K2 in the formation of short-term and long-term memories respectively. In the conditioned taste aversion test, S6K1-KO mice were less averse to the pairing of the novel tasting saccharin with the LiCl injection over multiple sessions. In contrast, the S6K2-KO mice exhibited normal taste aversion. However, in a latent inhibition paradigm (Weiner 2003) when S6K2-KO mice were pre-exposed twice to the saccharin before pairing with the LiCl injection, their taste aversion was unaffected compared to WTs in which it was reduced. Interestingly, L-LTP in hippocampal slices from both S6K1-KO and S6K2-KO mice was unaffected, but E-LTP was impaired only in S6K1-KO mice, which meant that while neither S6K1 nor S6K2 were required for facilitation of L-LTP, for S6K1-KO mice another substrate of the mTOR-signalling pathway must be involved in


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At birth neuronal dysfunction

Epigenetic factors

Disrupted neurodevelopment

ensuring continuity of protein synthesis, such as 4E-BP1, for E-LTP. Differences in the behavioural phenotypes of S6K1 and S6K2 KO mice were suggested to specific localization of the different kinases; S6K1 is localized in the nucleus and cytosol and S6K2 is restricted to the nucleus. In a more recent study, Siuta et al. (2010) generated mice with neuronal deletion of Rictor, which as described above is an activator of Akt. Aside from the expected reduction in Akt phosphorylation, Rictor null mice displayed deficits in sensorimotor gating, an endophenotype of schizophrenia (van den Buuse 2010). This deficit was accompanied by cortical and striatal hypodopaminergia as well as an increase in noradrenaline content and expression of the noradrenaline transporter. The authors stated that bidirectional effects on dopamine and noradrenaline may be because of the higher affinity for dopamine than noradrenaline by this transporter. They also observed that direct inhibition of Akt using an in vivo preparation produced a similar increase in transporter expression. This led to the suggestion that Rictor/Aktinduced regulation of transporter expression was crucial in maintaining monoamine homeostasis and consequently the regulation of sensorimotor gating as evidenced by the ability of the transport blocker nisoxetine to reverse deficits. In summary, under homeostatic conditions rapamycin could serve as a useful compound to induce deficits in cognition in rodents. Alternatively, specific genetic mutations to mTOR complex components (Rictor) and downstream substrates (S6K) lead to similar and additional behaviours of clinical relevance. Collectively, these animal models could prove extremely useful for the screening of novel pharmacotherapies.

Disrupted mTOR signaling

Disrupted protein synthesis

Abnormal neuronal morphology

Poor brain network connectivity

Reduced synaptic plasticity

Environmental risk factors

Schizophrenia Fig. 4 Modified ‘two-hit’ hypothesis of schizophrenia which incorporates aspects of disrupted mammalian target of rapamycin (mTOR) signalling, owing to at birth neuronal dysfunction or epigenetic factors, as a core feature of neurodevelopmental pathology. This leads to deficits in neuronal morphology and synaptic plasticity which increases the vulnerability to the effects of environmental risk factors, ultimately leading to schizophrenia onset.

To test a new hypothesis Reviewing the data, one of the main functions of the mTORsignalling cascade is the control of protein synthesis which underlies aspects of neurodevelopment and synaptic plasticity. The cascade is in turn under the control of extracellular and environmental factors which have been previously implicated in schizophrenia neuropathology. The fact that these factors influence the activation of the mTOR-signalling cascade as a common consequence suggests that disrupted protein synthesis could have a significant role in schizophrenia neuropathology. More specifically, disrupted protein synthesis during crucial stages of neurodevelopment could render the brain vulnerable to subsequent exposure to secondary environmental risk factors which inflict further damage, causing schizophrenia (Fig. 4). This is essentially a pathway hypothesis which seeks to combine various known aspects of schizophrenia neuropathology, thus making it unique and at the same time a rather complex challenge to prove. To test this new hypothesis, there are several possible avenues of experimentation. First and foremost, a

neuroproteomics approach would be needed to look more closely into the roles of mTORC2, how it interacts with mTORC1 and also the types of proteins that are synthesized in response to activation of the mTOR-signalling cascade and their functions. Secondly, we would need to study further the various isoforms of the upstream activators and downstream substrates of the mTOR-signalling cascade as we all as components which make-up the mTORC1 and mTORC2 complexes. This would guide the development of genetically modified rodents which could be used in the creation of socalled ‘two-hit’ models which incorporate disrupted mTOR signalling as well as other risk factors (e.g. maternal infection, chronic cannabinoid exposure) to then be phenotyped using a range of different behavioural testing paradigms of relevance to schizophrenia (e.g. fear condition, latent inhibition, sensorimotor gating, social interaction). Thirdly, these animal models could be used to identify possible early intervention points and design therapeutic strategies to stop disease progression and provide symptomatic relief. Finally, we need to establish that mTOR signalling



is impaired clinically by examining post-mortem brain specimens from patients with schizophrenia and other psychotic disorders. This study should also incorporate both drug-naive and medicated patients to examine whether currently prescribed medication serves to reverse any deficits in mTOR-dependent protein synthesis.

Conclusion We are currently limited in terms of pharmacotherapeutic options for the treatment of schizophrenia and this may reflect either the poor validity of existing animal models or an incomplete understanding of schizophrenia neuropathology. Either way, a renewed focus is necessary, perhaps on the basic processes of neuronal physiology that have gone awry in schizophrenia. The mTOR-signalling cascade is one such neuronal mechanism for which we have reviewed the evidence to justify an aetiological role it may have. Future investigations will reveal the strengths and weaknesses of the proposed hypothesis and produce exciting, novel insights that could advance our understanding of schizophrenia and how we treat it.

Acknowledgements AG conceived the hypothesis based on literature and prepared drafts of the manuscript. MvdB provided critical insight and approved the final draft for submission. MvdB is a senior research fellow of the Australian National Health and Medical Research Council. The authors have no conflict of interest to declare.

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