Role of palmitoylation of the serotonin 5-HT1A receptor in regulation of physiological and pathological receptor functions.

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Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter mediating a wide range of functional effects in the central nervous system via activation of heterogeneously expressed 5-HT receptors. The serotonergic system and in particular 5-HT1A receptor (5-HT1AR) are implicated not only into regulation of physiological functions in the brain, but also in major depression and anxiety. Besides the important role of 5-HT1AR in the pathogenesis of depressive disorders and in their clinical medications, underlying mechanisms are not completely understood. The 5-HT1AR undergoes post-translational palmitoylation which is essential for receptor-mediated activation of inhibitory Gi/o-proteins and down-stream effector signaling. Receptor palmitoylation is also responsible for the localization of the 5-HT1AR in lipid rafts, which plays an important role in regulation of receptor functions. In our preliminary experiments we have identified DHHC5, DHHC9 and DHHC21 proteins belonging to the DHHC (Asp-His-His-Cys) protein family as enzymes responsible for the 5-HT1AR palmitoylation. Knocking-down of these DHHCs by specific short hairpin (sh)RNAs leads to substantially reduced 5-HT1AR palmitoylation and strongly affects receptor-mediated signaling. In addition, we have demonstrated in vivo palmitoylation of 5-HT1AR in rodent and human brains, and also found reduced 5-HT1AR palmitoylation as well as decreased DHHC21 expression in the hippocampus of fear conditioned mice, suggesting involvement of palmitoylation in anxiogenesis.The major goal of this proposal is to molecularly define mechanisms regulating enzymatic 5-HT1AR palmitoylation and analyze involvement of receptor palmitoylation in modulation of neuronal properties under physiological and pathological conditions. The second major goal is to elucidate the functional role of 5-HT1AR palmitoylation for the developing, maintaining and pathogenesis depressive disorders. This also implies importance of the 5-HT1AR palmitoylation as a potential therapeutic target for the treatment of depression. To achieve these goals we will apply the synergistic combination of cutting edge techniques presented in the participant labs (e.g. analysis of palmitoylation in cultured neurons as well as in brain samples from mice and humans, quantitative molecular microscopy, FRET, electrophysiological recordings at the single cell level and neuronal network, application of different depression models in rodents, and behavioral analysis). The combined outcomes of these investigations will greatly advance our fundamental knowledge about the impact of palmitoylation for regulation of neuronal functions under physiological and pathological conditions, providing information about the possible therapeutic role of 5-HT1AR palmitoylation for treating depression and anxiety disorders.