CaMKIIα expression in a mouse model of NMDAR hypofunction schizophrenia: Putative roles for IGF-1R and TLR4

Highlights

• Induced NMDAR hypofunction caused hippocampal CaMKIIα loss.

• Depletion of hippocampal IGF-1R was linked with CaMKIIα loss in NMDAR hypofunction.

• Cre-lox knockdown of TLR4 rescued IGF-1R and CaMKIIα loss after an induced NMDAR hypofunction.

Abstract

Schizophrenia (SCZ) is a neuropsychiatric disorder that is linked to social behavioral deficits and other negative symptoms associated with hippocampal synaptic dysfunction. Synaptic mechanism of schizophrenia is characterized by loss of hippocampal N-Methyl-d-Aspartate Receptor (NMDAR) activity (NMDAR hypofunction) and dendritic spines. Previous studies show that genetic deletion of hippocampal synaptic regulatory calcium-calmodulin dependent kinase II alpha (CaMKIIα) cause synaptic and behavioral defects associated with schizophrenia in mice. Although CaMKIIα is involved in modulation of NMDAR activity, it is equally linked to inflammatory and neurotropin signaling in neurons. Based on these propositions, we speculate that non-neurotransmitter upstream receptors associated with neurotropic and inflammatory signaling activities of CaMKIIα may alter its synaptic function. Besides, how these receptors (i.e. inflammatory and neurotropic receptors) alter CaMKIIα function (phosphorylation) relative to hippocampal NMDAR activity in schizophrenia is poorly understood. Here, we examined the relationship between toll-like receptor (TLR4; inflammatory), insulin-like growth factor receptor 1 (IGF-1R; neurotropic) and CaMKIIα expression in the hippocampus of behaviorally deficient schizophrenic mice after we induced schizophrenia through NMDAR inhibition.

Schizophrenia was induced in WT (C57BL/6) mice through intraperitoneal administration of 30 mg/Kg ketamine (NMDAR antagonist) for 5 days (WT/SCZ). Five days after the last ketamine treatment, wild type schizophrenic mice show deficiencies in sociability and social novelty behavior. Furthermore, there was a significant decrease in hippocampal CaMKIIα (p < 0.001) and IGF-1R (p < 0.001) expression when assessed through immunoblotting and confocal immunofluorescence microscopy. Additionally, WT schizophrenic mice show an increased percentage of phosphorylated CaMKIIα in addition to upregulated TLR4 signaling (TLR4, NF-κB, and MAPK/ErK) in the hippocampus. To ascertain the functional link between TLR4, IGF-1R and CaMKIIα relative to NMDAR hypofunction in schizophrenia, we created hippocampal-specific TLR4 knockdown mouse using AAV-driven Cre-lox technique (TLR4 KD). Subsequently, we inhibited NMDAR function in TLR4 KD mice in an attempt to induce schizophrenia (TLR4 KD SCZ). Interestingly, IGF-1R and CaMKIIα expressions were preserved in the TLR4 KD hippocampus after attenuation of NMDAR function. Furthermore, TLR4 KD SCZ mice showed no prominent defects in sociability and social novelty behavior when compared with the control (WT).

Our results show that a sustained IGF-1R expression may preserve the synaptic activity of CaMKIIα while TLR4 signaling ablates hippocampal CaMKIIα expression in NMDAR hypofunction schizophrenia. Together, we infer that IGF-1R depletion and increased TLR4 signaling are non-neurotransmitter pro-schizophrenic cues that can reduce synaptic CaMKIIα activity in a pharmacologic mouse model of schizophrenia.

Introduction

Schizophrenia affects approximately 1.1% (∼83 Million people) of the world’s population across all sex, age and background (NIH-NIMH, 2017, SARDAA, 2017). About 3.5 Million people are affected in the United States and it is one of the leading causes of disability (NIH-NIMH, 2017, SARDAA, 2017). While the disorder is partially genetic, stress and drugs are predominant causes of schizophrenia. Importantly, 75% of individuals that become schizophrenic show the symptoms in late adolescence to early adulthood (age 16–25yrs) (NIH-NIMH, 2017, SARDAA, 2017, Arain et al., 2013). This coincides with the peak of brain wiring, then re-wiring (age 22–25yrs) required for transitioning from childhood to adult brain (Arain et al., 2013, Owens et al., 2012, Owen and O'Donovan, 2012, Eckfeld et al., 2017). In schizophrenia, this generally involves changes in expression of neurotransmitters (glutamate and dopamine), receptors (NMDAR; N-Methyl-d-Aspartate receptor and D₂R; Dopaminergic D₂ receptor), and proteins involved in the regulation of their synaptic activities (Nakazawa et al., 2017, Cohen et al., 2015, Dias, 2012, Barkus et al., 2012, Seillier and Giuffrida, 2009). As such, most available treatment methods involve the use of antipsychotics that target these receptors (i.e. NMDAR and D₂R). Side effects of these drugs are a limitation as it often leads to depression, movement disorders and catalepsy (Amodeo et al., 2017, Nakata et al., 2017, Nikvarz et al., 2017, Freyberg et al., 2017, Ibi et al., 2017, Meltzer, 2017, Ostinelli et al., 2017, Su et al., 2017). Therefore, there is a need for assessment of other synaptic targets that can reduce synaptic and behavioral defects of schizophrenia while circumventing neurotransmitter receptor sensitivity (Yang and Tsai, 2017).

Despite the limitations, treatment methods that target dopamine and glutamate receptors are laudable based on vastly studied dopamine and glutamate hypothesis of schizophrenia (Yang and Tsai, 2017, Purves-Tyson et al., 2017, Howes et al., 2017). However, the significance of synaptic regulatory calcium-calmodulin-depended kinase 2 alpha (CaMKIIα) − linked with synaptic function of NMDAR (Hagihara et al., 2014, Hagihara et al., 2013, Purkayastha et al., 2012, Dhavan et al., 2002, Robison et al., 2005) and D₂R (Ng et al., 2010, Takeuchi et al., 2002, Vekshina et al., 2017) − cannot be over emphasized in schizophrenia. Substantive evidence now exists to confirm that genetic deletion of CaMKIIα is a cause of synaptic and behavioral changes that are characteristic of schizophrenia (Frankland et al., 2008, Matsuo et al., 2009, Yamasaki et al., 2008). Roles of CaMKIIα in neurons are wide and varied. CaMKIIα is involved in inflammatory signaling that is linked with toll-like receptor 4 (TLR4/NF-κB) (Kaltschmidt et al., 2005, Mémet, 2006). Additionally, it acts downstream of IGF-1R-Ras/Raf pathway to regulate calcium-mediated synaptic activity of NMDAR (Hu et al., 2016a, Hu et al., 2016b, Le Grevès et al., 2005, Dou et al., 2005). Based on these propositions, it is logical to speculate that synaptic and inflammatory activity of CaMKIIα are not mutually exclusive. Although CaMKIIα depletion has been implicated in schizophrenia (Matsuo et al., 2009, Yamasaki et al., 2008), the mechanism through which IGF-1R and TLR4 can putatively affect synaptic CaMKIIα expression in pathophysiology of schizophrenia is poorly understood.

Localization of CaMKIIα at post-synaptic sites enables interactions with neurotrophic factors and receptors involved in synaptic development and maintenance (Hami et al., 2014, Kiray et al., 2014, Sun, 2006, Gunnell et al., 2007, Venkatasubramanian et al., 2010, Demirel et al., 2014). Moreover, loss of these proteins have been implicated in several developmental neuropsychiatric disorders including schizophrenia (Nelson et al., 2006, Ribasés et al., 2008). Among these, IGF-1R may alter CaMKIIα expression through Wnt/GSK3 and Ras/Raf signaling in growth, synaptic function, inflammation, and cell death (Pereira et al., 2008). Furthermore, both IGF-1R and CaMKIIα promotes pre- and post-synaptic calcium movement linked to NMDAR function in the hippocampus during long-term potentiation (LTP) and cognition (Cassilhas et al., 2012, Hu et al., 2016a, Hu et al., 2016b). In addition to the control of growth and aging, a recent study demonstrates the significance of IGF-1R signaling in presynaptic calcium release and glutamatergic neurotransmission. In their study, Gazit and co-workers show that IGF-1R modulates calcium release from the mitochondria and regulate presynaptic activity of synaptophysin (Gazit et al., 2016). Equally, IGF-1R signaling alters protein kinase systems (Akt/mTOR and MAPK/ErK) (Dyer et al., 2016, Netchine et al., 2011, Salani et al., 2015) involved in the regulation of neurodevelopment and LTP (Zheng et al., 2012, Kéri et al., 2011, Romanelli et al., 2007). Owing to its effect on synaptic calcium currents (Gazit et al., 2016) and gene expression (Le Grevès et al., 2005, Le Grevès et al., 2006), IGF-1R may significantly affect the activity of ionotropic glutamate receptors in normal LTP and disease conditions (Jiang et al., 2015, Demirel et al., 2014, Gunnell et al., 2007).

The function of CaMKIIα is not restricted to neurotropic regulation of growth, and synaptic function. Downstream of IGF-1R, CaMKIIα can reduce the activity of proinflammatory proteins like NF-κB and MAPK/ErK, and purigenic receptors such as TLR4 and P2RX₇ (Meffert and Baltimore, 2005, Luo et al., 2008, Gómez-Villafuertes et al., 2015, Huang et al., 2009). Therefore, a reduction of CaMKIIα activity can lead to inflammation and progression of synaptic dysfunction associated with schizophrenia. Evidently, the expression of TLR4 and associated pro-inflammatory proteins increases in the blood of clinically-diagnosed schizophrenia patients (Venkatasubramanian and Debnath, 2013, Chang et al., 2011, Hansen et al., 2008, McQuillin et al., 2009). In addition to their involvement in inflammation, proteins like NF-κB and MAPK/ErK − in the TLR4 signaling pathway − are known to reduce synaptic activity of CaMKIIα by increasing CaMKIIα phosphorylation (Meffert and Baltimore, 2005, Han et al., 2009). Furthermore, MAPK/ErK and CaMKIIα occupy similar sites at post-synaptic densities where MAPK/ErK phosphorylates CaMKIIα during early and late LTP phases (Du et al., 2004, Giovannini et al., 2001, Tsui et al., 2005, Derkach et al., 2007). Although primarily inflammatory, TLR4 is involved in synaptogenesis as it increases glutamatergic function in the developing brain (Shen et al., 2016, Henneberger and Steinhäuser, 2016). This is required for the development and formation of synapses by migrating neurons (Shen et al., 2016, Venkatasubramanian and Debnath, 2013). However, in excess, TLR4-linked glutamate release may induce epileptogenic effects and other forms of excitotoxicity through an upregulated MAPK/ErK signaling (Jiang et al., 2015, Shen et al., 2016, Henneberger and Steinhäuser, 2016). Since MAPK/ErK also act downstream of IGF-1R (Xing et al., 2016, Dyer et al., 2016, Vahdatpour and Dyer, 2016), neural activity of TLR4 may overlap with neurotropic cues of IGF-1R and other kinase receptors involved in nervous system development and neural circuit formation (Fig. 1). Thus, an increased TLR4 signaling may upregulate MAPK/ErK which can abolish IGF-1R signaling and CaMKIIα activities in the brain.

Based on these propositions, we hypothesize that NMDAR-linked CaMKIIα activity in synaptic function and neurotropic signaling are not exclusive of its fate in inflammation. In brief, CaMKIIα modulates synaptic NMDAR function (Mao et al., 2014, Ma et al., 2015, Park et al., 2008, Johnston and Morris, 1995), and itself (i.e CaMKIIα) can be altered through TLR4 and IGF-1R signaling. Through upregulated MAPK/ErK, TLR4 may alter synaptic function by reducing IGF-1R signaling. This may lead to a reduced CaMKIIα activation downstream of IGF-1R (Ras/Raf), and increased CaMKIIα phosphorylation (inactivation) by MAPK/ErK. Therefore, decreased TLR4 and upregulated IGF-1R signaling are possible mechanisms through which CaMKIIα activity can be preserved in NMDAR hypofunction. Here, we show that induced loss of NMDAR function can cause hippocampal CaMKIIα loss and decrease IGF-1R signaling. Furthermore, we tested the effectiveness of hippocampal-specific TLR4 knockdown as an intervention method to preserve IGF-1R and CaMKIIα in mice after a persistent NMDAR inhibition.