Pharmacological Properties of Chromobacterium violaceum Violacein at the Human Serotonin 2C Receptor.

The monoamine neurotransmitter serotonin (5-HT) plays a role in many physiological responses by interacting with various receptor subtypes. The 5-HT2C receptor subtype is a 7-transmembrane, G protein-coupled receptor (GPCR) that is involved in neuronal excitability, spatial learning, mood, and appetite. The microorganism Chromobacterium violaceum produces a purple pigment, violacein, which can be extracted and purified. Violacein has antibiotic, antileishmanial, antifungal and antitumoral properties in various cancer cell lines. Violacein is derived from the amino acid tryptophan as is 5-HT and therefore, the two have similar chemical structures. However, no one has reported the activity of violacein at 5-HT receptors. Therefore the Fentress lab decided to investigate whether or not violacein had an effect on 5-HT2C receptor trafficking. Human Embryonic Kidney (HEK) 293 cells expressing fluorescently-tagged 5-HT2C receptor were treated with 5-HT, violacein, water or vehicle and then cells were fixed and visualized with fluorescent microscopy. Violacein treatment did not cause receptor internalization. Recent studies suggest that the 5-HT2C receptor can activate the JAK/STAT pathway. To see if violacein can modulate this pathway, HEK 293 cells expressing 5-HT2C receptor were treated with either 5-HT, violacein, or pretreated with violacein followed by incubation with 5-HT. Phosphorylation states of JAK2 and STAT3 were examined by immunoblotting. Results determined that 5-HT2C receptor activation had no effect on JAK2 phosphorylation and that violacein blocked STAT3 phosphorylation. Primary radioligand binding determined that violacein has a low affinity for 5-HT2C receptor but has a higher affinity for adrenergic receptors. Future studies will examine G protein-coupling by measuring phosphoinositide hydrolysis and cAMP assay to investigate adrenergic pathways.

Molecular Mechanisms of Obesity-Induced Osteoporosis and Muscle Atrophy.

Obesity and osteoporosis are two alarming health disorders prominent among middle and old age populations, and the numbers of those affected by these two disorders are increasing. It is estimated that more than 600 million adults are obese and over 200 million people have osteoporosis worldwide. Interestingly, both of these abnormalities share some common features including a genetic predisposition, and a common origin: bone marrow mesenchymal stromal cells. Obesity is characterized by the expression of leptin, adiponectin, interleukin 6 (IL-6), interleukin 10 (IL-10), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), macrophage colony stimulating factor (M-CSF), growth hormone (GH), parathyroid hormone (PTH), angiotensin II (Ang II), 5-hydroxy-tryptamine (5-HT), Advance glycation end products (AGE), and myostatin, which exert their effects by modulating the signaling pathways within bone and muscle. Chemical messengers (e.g., TNF-α, IL-6, AGE, leptins) that are upregulated or downregulated as a result of obesity have been shown to act as negative regulators of osteoblasts, osteocytes and muscles, as well as positive regulators of osteoclasts. These additive effects of obesity ultimately increase the risk for osteoporosis and muscle atrophy. The aim of this review is to identify the potential cellular mechanisms through which obesity may facilitate osteoporosis, muscle atrophy and bone fractures.

Norepinephrine transporter variant A457P knock-in mice display key features of human postural orthostatic tachycardia syndrome.

Postural orthostatic tachycardia syndrome (POTS) is a common autonomic disorder of largely unknown etiology that presents with sustained tachycardia on standing, syncope and elevated norepinephrine spillover. Some individuals with POTS experience anxiety, depression and cognitive dysfunction. Previously, we identified a mutation, A457P, in the norepinephrine (NE; also known as noradrenaline) transporter (NET; encoded by SLC6A2) in POTS patients. NET is expressed at presynaptic sites in NE neurons and plays a crucial role in regulating NE signaling and homeostasis through NE reuptake into noradrenergic nerve terminals. Our in vitro studies demonstrate that A457P reduces both NET surface trafficking and NE transport and exerts a dominant-negative impact on wild-type NET proteins. Here we report the generation and characterization of NET A457P mice, demonstrating the ability of A457P to drive the POTS phenotype and behaviors that are consistent with reported comorbidities. Mice carrying one A457P allele (NET(+/P)) exhibited reduced brain and sympathetic NE transport levels compared with wild-type (NET(+/+)) mice, whereas transport activity in mice carrying two A457P alleles (NET(P/P)) was nearly abolished. NET(+/P) and NET(P/P) mice exhibited elevations in plasma and urine NE levels, reduced 3,4-dihydroxyphenylglycol (DHPG), and reduced DHPG:NE ratios, consistent with a decrease in sympathetic nerve terminal NE reuptake. Radiotelemetry in unanesthetized mice revealed tachycardia in NET(+/P) mice without a change in blood pressure or baroreceptor sensitivity, consistent with studies of human NET A457P carriers. NET(+/P) mice also demonstrated behavioral changes consistent with CNS NET dysfunction. Our findings support that NET dysfunction is sufficient to produce a POTS phenotype and introduces the first genetic model suitable for more detailed mechanistic studies of the disorder and its comorbidities.

Early-life forebrain glucocorticoid receptor overexpression increases anxiety behavior and cocaine sensitization.

BACKGROUND: Genetic factors and early-life adversity are critical in the etiology of mood disorders and substance abuse. Because of their role in the transduction of stress responses, glucocorticoid hormones and their receptors could serve as both genetic factors and mediators of environmental influences. We have shown that constitutive overexpression of the glucocorticoid receptor (GR) in forebrain results in increased emotional reactivity and lability in mice. Here, we asked whether there was a critical period for the emergence of this phenotype. METHODS: We generated a mouse line with inducible GR overexpression specifically in forebrain. Anxiety-like behaviors and cocaine-induced sensitization were assessed in adult mice following GR overexpression during different periods in development. The molecular basis of the behavioral phenotype was examined using microarray analyses of dentate gyrus and nucleus accumbens. RESULTS: Transient overexpression of GR during early life led to increased anxiety and cocaine sensitization, paralleling the phenotype of lifelong GR overexpression. This increased emotional reactivity was not observed when GR overexpression was induced after weaning. Glucocorticoid receptor overexpression in early life is sufficient to alter gene expression patterns for the rest of the animal's life, with dentate gyrus being more responsive than nucleus accumbens. The altered transcripts are implicated in GR and axonal guidance signaling in dentate gyrus and dopamine receptor signaling in nucleus accumbens. CONCLUSIONS: Transient overexpression of GR early in life is both necessary and sufficient for inducing transcriptome-wide changes in the brain and producing a lifelong increase in vulnerability to anxiety and drugs of abuse.