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Although lithium is widely used to treat Bipolar disorder (BD), its therapeutic role in BD is unclear. To gain insights into its mechanism of action we have used proteomic analysis to identify differentially expressed proteins in rat Prefrontal cortex (PFC), a region specifically affected in BD, after six weeks of lithium treatment. Proteins from control and lithium treated rat PFCs were separated by 2 Dimensional - Differential In-Gel Electrophoresis (2D-DIGE) and identified by mass spectrometry. Of the 2198 protein spots resolved, the abundance of 19 proteins was found to be significantly altered in the lithium treated group (with the levels of 5 proteins increasing and those of 14 decreasing). The levels of two protein spots exhibiting significant alteration after chronic lithium exposure were verified by Western blot analysis of rat PFC extracts. The 19 identified proteins represent novel targets for lithium action and participate in diverse functions that converge on a biological network that is specifically related to brain cell survival, prevention of neurodegeneration, and/or suppression of hyperactivity related signaling pathways. The identification of these targets should facilitate a better understanding of lithium’s overall effect on mood control.