An investigation of the factors that regulate muscarinic receptor expression in schizophrenia

Abstract

We previously identified a group of subjects with schizophrenia who, on average, have a 75% decrease in cholinergic receptor, muscarinic 1 (CHRM1) in Brodmann's area (BA) 9. To extend this finding, we determined i) if the decrease in CHRM1 was present in another functionally related CNS region (BA6), ii) whether the marked decrease in CHRM1 was accompanied by changes in levels of other CHRMs and iii) potential factors responsible for the decreased CHRM1 expression. We measured CHRM1 and CHRM3 using in situ radioligand binding with [³H]pirenzepine and [³H]4-DAMP respectively in BA6 from 20 subjects with schizophrenia who had low levels of CHRM1 in BA9 (SzLow[³H]PZP), 18 subjects with schizophrenia whose levels of CHRM1 were similar to controls (SzNormal[³H]PZP) and 20 control subjects. Levels of CHRM1, 3 and 4 mRNA were measured using qPCR and levels of the transcription factors, SP1 and SP3, were determined using Western blots. In BA6, the density of [³H]pirenzepine binding was decreased in subjects with SzLow[³H]PZP (p < 0.001) compared to controls. The density of [³H]4-DAMP binding, levels of CHRM1, 3 and 4 mRNA and levels of SP1 and SP3 was not significantly different between the three groups. This study shows that the previously identified decrease in CHRM1 expression is not confined to the dorsolateral prefrontal cortex but is present in other cortical areas. The effect shows some specificity to CHRM1, with no change in levels of binding to CHRM3. Furthermore, this decrease in CHRM1 does not appear to be associated with low levels of CHRM1 mRNA or to simply be regulated by the transcription factors, SP1 and SP3, suggesting that other mechanisms are responsible for the decreased CHRM1 in these subjects.

Introduction

Schizophrenia is a neuropsychiatric disorder that most likely encompasses a syndrome of diseases with multiple underlying pathologies (Jablensky, 2006). Therefore, one approach to better understand the syndrome of schizophrenia is to study its component disorders (Raedler et al., 2007). We have reported a sub-group (25%) of people with schizophrenia that can be separated into a discrete population within schizophrenia because they have a marked loss (~ 70%) of the levels of [³H]pirenzepine binding in BA9 (Scarr et al., 2009), opening up the opportunity to separate people with schizophrenia into two populations based on cortical [³H]pirenzepine binding.

We have shown that, under the assay conditions we use, [³H]pirenzepine predominately binds to the CHRM1 (Scarr and Dean, 2008, Gibbons et al., 2013). Importantly, clinical and preclinical studies suggest that cortical CHRM1 is important in cognitive functions (Anagnostaras et al., 2003, Nathan et al., 2012). Thus, in this study, we choose to expand our studies on cortical CHRM1 to BA6, a region that has been shown to act in concert with BA9 when cognitive tasks such as attention, working memory, episodic memory retrieval and visual awareness are being undertaken (Naghavi and Nyberg, 2005). This is of particular interest because these processes are often affected in people with schizophrenia (Ranganath et al., 2003, Blasi et al., 2010). In addition, decreased levels of [³H]pirenzepine binding and CHRM1 mRNA in BA6 have been reported in people with schizophrenia (Mancama et al., 2003, Dean et al., 2008). Thus, in our current studies, we measured levels of [³H]pirenzepine binding in BA6 from people with schizophrenia who had low (SzLow[³H]PZP) and relatively unchanged (SzNormal[³H]PZP) levels of [³H]pirenzepine binding in BA9 and in age and sex matched controls. In addition, we determined if changes in CHRM1 may be somewhat selective by measuring levels of CHRM3 using a modified radioligand binding technique (Jeon et al., 2013) and of mRNA for CHRM1, 3 and 4.

Notably, we have shown that there was no change in CHRM1 gene sequence associated with a change in levels of CHRM1 gene expression (Scarr et al., 2009), suggesting that changes in levels of CHRM1 expression could be due to changes in transcriptional regulation. Importantly, assessing the putative promoter region of the human CHRM1 gene with Transcription Element Search System (TESS), which searches putative transcription binding sites in DNA sequence (http://www.cbil.upenn.edu/cgi-bin/tess/tess), identified a SP1 binding site. Furthermore, this SP1 site is reported to be capable of recruiting SP1 in vitro (Wood et al., 1999) and, by interacting with SP3, regulating levels of CHRM1 expression. One marker of a change in control of CHRM1 expression by this transcriptional regulation pathway would be changes in cellular localisation and ratio of SP1 and SP3 (Suske, 1999, Li et al., 2004, Chuang et al., 2008, Tan and Khachigian, 2009). Therefore, we measured the nuclear and cytosolic levels of SP1 and SP3 and their ratios as a surrogate measurement of whether changes in translocation of the transcription factors had occurred in SzLow[³H]PZP group.