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

© 2013 Dr. Myoung Suk SeoA previous study identified a group of subjects with schizophrenia, who have, on average, a 75% decrease in their cortical cholinergic receptor, muscarinic 1 (CHRM1). To begin to identify the potential factors responsible for the decreased CHRM1 expression, I investigated the DNA methylation level of CpG dinucleotides in the putative CHRM1 promoter region using the bisulphite-based Sequenom MassARRAY EpiTYPER in Brodmann’s area (BA9) from subjects with schizophrenia that have low levels of CHRM1 (SCZ Low [3H]PZP), subjects with schizophrenia that have normal levels of CHRM1 (SCZ Normal [3H]PZP) and control subjects. The levels of methylation were lower in both groups with schizophrenia compared to the control subjects. In addition, DNA methylation showed a strong positive correlation in the SCZ Low [3H]PZP but not those in SCZ Normal [3H]PZP, suggesting that the decreased level of CHRM1 in SCZ Low [3H]PZP was associated with a decrease rather than an increase in promoter DNA methylation, and they did not mediate the change in CHRM1 expression. Another aspect of my studies investigated the CHRMs levels in BA6 from subjects with schizophrenia. CHRM1, 3 and 4 levels were determined using in situ radioligand binding and Quantitative Real-time PCR (qPCR) in BA6 in the same groups. The results showed the levels of CHRM1 were significantly decreased in BA6 in SCZ low [3H]PZP but other CHRMs were not altered in either groups with schizophrenia. In further effort to discover the factors mediating the decreased CHRM1, levels of transcription factors, Sp1 transcription factor (SP1) and SP3 and miRNA-107, known to regulate CHRM1 expression, were determined using Western blot and qPCR. Levels of Sp1, Sp3 and miRNA-107 were not altered in either group of subjects with schizophrenia compared to those in control subjects. Finally, to identify potential regulatory systems, our laboratory conducted a microarray study to identify genes whose expression profile are changed in response to decreased CHRM1 in BA9. The mRNA levels of Ferredoxin 1 (FDX1) were differentially expressed in both groups of schizophrenia according to the microarray, and qPCR was used to validate this finding. FDX1 expression was increased in both groups of schizophrenia, validating the microarray finding. However, the levels of FDX1 were not changed in BA6 or in tissue from subjects with MDD (Major depressive disorder) or BPD (Bipolar disorder). Taken together, this study supports the existence of a subgroup within schizophrenia with dramatically decreased CHRM1 but not CHRM3 and 4 in the pre-frontal cortex. While the level of CHRM1 DNA methylation was changed in schizophrenia, the decreased CHRM1 expression was not simply regulated by local levels of transcription and post-transcription mechanism, such as Sp1, Sp3 and miRNA-107, suggesting that other mechanisms may contribute to the decreased CHRM1 in these subjects. Finally, the microarray validation study found the level of FDX1 mRNA was increased in subjects with schizophrenia. This finding is of particular interest given the metabolic dysfunction associated with the disorder