Plasma membrane norepinephrine transporters (NET) are the primary source for terminating noradrenergic synaptic transmission and are targets of several therapeutic agents for cognitive, affective, and motor disorders. Several proteins act in concert to regulate the function and trafficking of NET; however, a definitive endogenous mechanism for regulating NET has yet to be defined. We have identified a novel interaction between NET and the presynaptic cytosolic proteins alpha-synuclein (a-Syn) and gamma- synuclein (?-Syn). We demonstrate here that both a-Syn and ?-Syn down-regulate NET activity and trafficking. a-Syn modulation of NET is fully reversed by nocodazole, a microtubule (MT) destabilizing agent, while ?-Syn regulation of NET is insensitive to nocodazole. Insensitivity of ?-Syn-expressing cells to anti-MT agents has also been shown by others in breast cancer cells, suggesting that ?-Syn may regulate MT activity. A similar lack of sensitivity of NET to nocodazole treatment is observed in frontal cortex synaptosomes of WKY, a rat model of depression, but not in non-depressed controls, the Wistar and Sprague Dawley rat. Microarray studies show that v-Syn is overexpressed in the WKY rat brain. The antidepressant desipramine (DMI), a selective NET inhibitor, improved depressive behavior in the WKY rat and decreased expression of ?-Syn by 70%, with no changes in a-Syn or NET levels. We propose a novel role for ?-Syn as a prodepressant factor. We hypothesize that overexpression of ?-Syn in the WKY rat brain leads to abnormal regulation of NET function and trafficking, which leads to the development of depression in the WKY rat. We will treat WKY and Wistar rats with DMI or saline and examine NET interactions with a-Syn and ?-Syn, through co- immunoprecipitation assays. We will measure expression levels and colocalization of these proteins before and after DMI treatment, by cell fractionation, Western blots and immunohistochemistry. We will examine the ability of nocodazole to modulate NET in synaptosomes of WKY before and after DMI treatment. These studies will aid our understanding of brain chemistry and cellular processes, which underlie depression. We suggest the dysregulation and disruption of dynamic interactions between a- Syn and NET by ?-Syn may aid in the development of depression, and that these interactions are normalized upon treatment with a NET specific antidepressant, DMI. The proposed study will help elucidate a more chronic mechanism of NET regulation and provide insight into the neurochemical basis of depression. These studies are also likely to be important in the development of novel therapies which selectively target ?-Syn in the treatment of depression. [unreadable] [unreadable] [unreadable]