Introduction: Bipolar disorder (BD) is a debilitating mental illness proven to be very difficult to treat. By studying the molecular mechanisms that underlie BD, we can learn how to improve treatments. The CLOCK protein is a key transcription factor involved in the persistence of circadian rhythms. Studies have found that manipulations of the Clock gene are sufficient to produce manic-like behavior in mice. The Clock 3111T/C single-nucleotide polymorphism (SNP) is a variation of the human Clock gene that is associated with increased frequency of mania in BD patients. In this study, we sought to examine the functional implications of the Clock 3111T/C SNP on Clock and Per2 expression over 24 hours.
Methods and Materials: Plasmid construction: The human Clock gene was cloned and site-directed mutagenesis was performed to produce the 3111T and 3111C versions of the gene. An Npas2 shRNA was also constructed in order to knockdown Npas2 expression, which has been shown to be significantly upregulated in Clock KO MEFs. MEFs: Mouse embryonic fibroblasts (MEFs) were collected from Clock Knockout (KO) mice. Clock KO MEFs were used to avoid confounds of endogenous Clock expression. Transfections: MEFs were transfected with either the Clock T or C constructs, in addition with an Npas2 shRNA. Quantitative PCR: RNA from transfected MEFs was isolated, and then reverse-transcribed to cDNA. Quantitative polymerase chain reaction (qPCR) was performed over a 24 hour period.
Results: Quantitative PCR revealed significantly increased levels of Clock and Per2 mRNA over a 24-hour time course in MEFs expressing the 3111C SNP, as compared with 3111T.
Conclusions: The CLOCK protein controls expression of genes beyond the circadian clock. These include dopamine regulators, which are known to be involved in decreased depression-like behaviors and increased impulsivity/drug intake. From this, it is clear that altered Clock expression may have tremendous implications for a variety of diseases, including BD
