| The serotonin transporter (SERT) is a 12-transmembrane domain protein that is responsible for clearance of serotonin from extraneuronal space. The gene responsible for encoding SERT is solute carrier 6 family A4 ( SLC6A4). A number of different genetic polymorphisms are hypothesized to regulate SERT function and expression, including the 43-base-pair insertion/deletion promoter polymorphism called the serotonin transporter linked-polymorphic region (5-HTTLPR). Being coded for in both peripheral cells and the brain by the same gene, SERT is the primary target for the selective serotonin reuptake inhibitor (SSRI) class of antidepressants and has been implicated in the etiology and treatment of a number of anxiety and depressive disorders. Major depressive disorder (MDD) is a highly prevalent disorder that is estimated to have an economic cost of approximately 83 billion dollars annually in the United States. While there are a number of effective treatments for MDD, not every treatment works for every patient. Recent studies have shown that initial SSRI treatment is successful in only ∼30% of patients. Furthermore, patients undergoing SSRI treatment often require a long duration of treatment (4-8 weeks) to achieve remission. With a low percentage of treatment success and delayed efficacy, a peripheral biomarker for treatment prediction would be highly advantageous. A biomarker is defined as an individual-specific factor or feature that delineates the presence of a disease or is predictive of treatment response. Pharmacogenetic studies relating SERT polymorphisms to MDD susceptibility and treatment response have yielded mixed results. To date, no peripheral biomarkers for predicting successful treatment of depression have been elucidated. Blood consists of a heterogeneous population of cells including erythrocytes, granulocytes, monocytes, lymphocytes, and platelets. While platelet SERT expression is well characterized, lymphocyte SERT expression is still debated. Lymphocytes that have been transformed with Epstein-Barr Virus (lymphoblasts; LCLs) for use in cell culture, however, have been shown to express SERT. In this thesis, we explore the use of peripheral blood cells in predicting SSRI treatment response in patients with MDD. First, we developed a flow cytometry assay using the fluorescent SERT substrate APP+ for assessing SERT function in mixed blood cell populations. Using this assay, we demonstrate that platelets, not lymphocytes, contribute to the majority of SERT function measured in peripheral blood mononuclear cell (PBMC) samples. Next, we evaluated the use of LCLs for studying SERT function with respect to complex SERT genotype. We report that LCLs express SERT, but overall SERT function is minimal. Third, we investigated using cell-phone based fluorescence microscopy to measure platelet SERT function. This novel approach has the potential to revolutionize the translatability of fluorescence-based assays as individuals will be able to make functional SERT measurements at home instead of having to access expensive clinical instrumentation. Finally, we conclude with results from a pilot study using electrochemical techniques to measure SERT function in PBCs from individuals diagnosed with MDD who are undergoing antidepressant treatment. We find that SERT function is highly variable across normal and depressed individuals, with serotonin uptake in PBCs slightly lower in the depressed population on the whole. Furthermore, we detect SSRI-specific treatment-related decreases in PBC SERT function as early as one week after initiating treatment with S-citalopram. Based on this pilot study, we have formulated a central hypothesis for a large-scale clinical trial. We theorize that we can predict which individuals will ultimately respond to SSRIs based on pretreatment SERT function in platelets. This research sets the stage for using peripheral blood cells to develop personalized predictive treatment paradigms for patients with MDD. |
