Conte Center

Vanderbilt / NIMH Silvio O. Conte Center for Neuroscience Research

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Vanderbilt University
465 21st Avenue South
Nashville, TN 37232
For more information,
please contact Denise Malone.

2010 Pilot Grant Recipients

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Kevin Currie, Ph.D.

A novel SERT-dependent mechanism that controls neuroendocrine hormone release

The serotonin transporter (SERT) is prominently expressed in adrenal chromaffin cells. Our preliminary data in bovine chromaffin cells indicate that this leads to uptake and intracellular accumulation of 5HT. Moreover, we show that extracellular 5HT potentiates catecholamine release in a citalopram sensitive manner. We postulate that rather than binding to cell surface G protein coupled receptors, 5HT acts at an intracellular site(s) following acute uptake by SERT. One possible downstream mechanism might involve covalent binding of 5HT to cytosolic proteins by transglutaminase (dubbed “serotonylation”) as has been proposed recently for pancreatic β- cells. We will use patch-clamp electrophysiology, carbon fiber amperometry and calcium imaging approaches to dissect the effects on 5HT on catecholamine release from mouse adrenal chromaffin cells. This will allow us to exploit powerful genetic mouse models available through collaboration with Dr Randy Blakely (SERT knockout mice and knock-in mice that lack the recognition site for citalopram on SERT). Characterization of this mechanism will provide new insight into the therapeutic mechanisms of SSRI’s and other drugs that target SERT function. It will also help define a novel cellular mechanism by which exocytotic transmitter release is controlled. Chromaffin cells are part of the wider sympathoadrenal system and exert profound effects on the cardiovascular, endocrine, and nervous systems by release of catecholamines and other transmitters. Furthermore, chromaffin cells are used widely as a neurosecretory model, so our findings will be more broadly applicable to the understanding of neurotransmitter release and synaptic transmission.

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Elizabeth Hammock, Ph.D.

Mechanisms of developmental programming of the serotonergic system by life experience

Developmental exposure to stress has long lasting consequences on the organization and maintenance of serotonergic tone in the central nervous system and is associated with increased anxiety-like traits and risk for major depression. Epigenetic events at the Pet-1 promoter may mediate such effects. Because of its role as a transcription factor regulating several key players in the serotonin system, PET-1 is poised to set the gain on serotonin signaling and could be a key molecular target of early life experience. The aims of this pilot grant are to establish a robust model of early life stress at Vanderbilt and to identify the effects of such manipulation on serotonergic tone in the brain of male and female C57Bl6J mice. Additionally, we are interested in the potential effects of early life stress on methylation state of the Pet-1 promoter. We hypothesize that early life stress will be associated with increased methylation of the Pet-1 promoter, which is then responsible for maintained and long lasting reductions of levels of serotonin signaling components including serotonin and the serotonin transporter.

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David Miller, Ph.D.

Gene expression profiles of C. elegans serotonergic neurons

Serotonin regulates key behaviors such as mood, learning and satiety. Deficits in serotonin signaling are linked to mental disorders, including depression and impulsivity and may alter development of brain circuits that govern adult behavior. Although pharmalogical treatments are widely employed to ameliorate serotonin-dependent behaviors, new, more efficacious therapies are desirable and could emerge from a comprehensive understanding of serotonergic signaling pathways. Studies in the nematode C. elegans suggest that the roles of serotonin are evolutionary ancient and thus that basic mechanisms that govern serotonin function are likely conserved. With its simple, well-defined nervous system and facile genetics, C. elegans is particularly amenable to studies of the serotonin pathway. The C. elegans nervous system includes nine serotonergic neurons that are grouped in five distinct classes. Each of these neuron types has been linked to specific serotonin dependent behaviors. Here we propose to use powerful new expression profiling strategies to identify all genes expressed in two of these serotonergic neurons, NSM and ADF. We hypothesize that the NSM and ADF transcripts revealed by this strategy will identify a core group of genes that are also required for the differentiation or function of mammalian serotonergic neurons. To test this idea we will 1) produce a gene expression profile of developing embryonic NSM and ADF neurons; 2) obtain gene expression profiles of functional NSM and ADF serotonergic neurons in larval animals; 3) use bioinformatics approaches and experiments with GFP reporter genes to validate this data set for comparison to previously reported expression profiles of mammalian serotonergic neurons. The outcome of this work is expected to identify a fundamental gene set that defines serotonergic phenotype.

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Randi Ulbricht, Ph.D.

Post-Transcriptional Modification of Tph2 as a Region-Specific Regulator of Serotonin Biosynthesis

Tryptophan hydroxylase (TPH) is responsible for the rate-limiting catalytic step in serotonin biosynthesis. Two TPH genes are encoded in the mammalian genome, TPH1 and TPH2. TPH2 is the main contributor to serotonin synthesis in the brain and its functional polymorphisms have been linked to suicide, attention deficit/hyperactivity disorder and major depression. A recent study identified TPH2 as a target for multiple mRNA processing events, leading to the expression of multiple protein isoforms in human tissue. Two possible mRNA isoforms are created as a result of alternative splicing and four different classes of RNA editing events change up to eight nucleotides in the coding region of the transcript. Editing at four of these sites leads to changes in amino acid sequence, while one editing event creates a premature stop codon and thus, a null allele. Functional analyses of proteins encoded by edited TPH2 mRNA isoforms suggest that editing significantly decreases catalytic activity; thus mRNA editing may represent a post-transcriptional mechanism to modulate serotonin biosynthesis and availability. We hypothesize that TPH2 mRNA processing is a dynamic regulator of TPH2 expression and protein function. To address this hypothesis, we propose to quantitatively compare TPH2 alternative splicing and mRNA editing in dissected human brain regions. We also propose to analyze TPH2 mRNA processing in rodent model systems with the expectation that the identification of analogous editing events will allow for an expanded analysis of regional editing profiles in these species. We anticipate that these studies will further our understanding of the role that RNA processing plays in modulating TPH2 expression to control serotonergic function in health and human disease.