An investigation of the factors that regulate muscarinic receptor expression in schizophrenia
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.
Section snippets
Human tissue collection, clinical follow up and diagnosis
All human tissues were sourced through the Victorian Brain Bank Network, held at the Mental Health Research Institute. Approval for the study was obtained from both the Ethics Committee of the Victorian Institute of Forensic Medicine and the Mental Health Research and Ethics Committee of Melbourne Health. In order for the diagnosis of a psychiatric illness to be accepted, clinical case histories were used to reach a diagnostic consensus using the Diagnostic Instrument for Brain Studies (DIBS) (
CNS collection, clinical and pharmacological data
Due to our experimental design, there were no significant differences between subjects with schizophrenia and control subjects for age (F = 0.02, d.f. = 1,56, p = 0.88), brain pH (F = 2.83, d.f. = 1,56, p = 0.10), PMI (F = 0.45, d.f. = 1,56, p = 0.50) or gender (p = 1, Fisher's exact test). When subjects with schizophrenia were divided into groups according to their levels of [3H]pirenzepine binding, age (F = 0.22, d.f. = 2,55, p = 0.98), PMI (F = 0.37, d.f. = 2,55, p = 0.69), brain pH (F = 1.95, d.f. = 2,55, p = 0.15) and gender
Discussion
This study shows that the density of [3H]pirenzepine binding is decreased in subjects with schizophrenia compared to control subjects and that the loss of radioligand binding that defined SzLow[3H]PZP in BA9 is present in BA6. Although the group with schizophrenia that have normal levels of CHRM1 in BA9 also showed a decrease in CHRM1 in BA6, the magnitude of the decrease was much smaller. Importantly, there was no relationship between experimental data and any potential confounding factor
Role of funding source
The study sponsors had no role in the study design, data, collection, writing the report or decisions on journal submission.
Contributors
Author Myoung Suk Seo had a major role in data collection and analyses and contributed to the preparation of the final manuscript. Author Brian Dean was involved in the conceptual design of the study and took the lead role in preparing the manuscript for publication. Author Elizabeth Scarr was involved in data collection and analyses as well as playing a significant role in preparation of the manuscript for submission.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The authors gratefully acknowledge the assistance of Geoffrey Pavey for the preparation of post-mortem tissue for their roles in tissue collection and diagnostic confirmation. Tissues were received from the Victorian Brain Bank Network, supported by the Mental Health Research Institute, Alfred Hospital, Victorian Forensic Institute of Medicine, The University of Melbourne and funded by Australia's National Health & Medical Research Council, Helen Macpherson Smith Trust, Parkinson's Victoria and
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