Proinflammatory cytokines and serotonergic homeostasis have both been implicated in the pathophysiology of major psychiatric disorders. We have demonstrated that activation of p38 mitogen-activated protein kinase (MAPK) induces a catalytic activation of the serotonin transporter (SERT) arising from a reduction in the SERT K m for 5-hydroxytryptamine (5-HT). As inflammatory cytokines can activate p38 MAPK, we hypothesized that they might also activate neuronal SERT. Indeed, Interleukin-1beta (IL-1b) and tumor necrosis factor alpha (TNF-a) stimulated serotonin uptake in both the rat embryonic raphe cell line, RN46A, and in mouse midbrain and striatal synaptosomes. In RN46A cells, IL-1b stimulated 5-HT uptake in a dose-and time-dependent manner, peaking in 20 min at 100 ng/ml. This was abolished by IL-1ra (20 ng/ml), an antagonist of the IL-1 receptor, and by SB203580 (5 mM), a p38 MAPK inhibitor. TNF-a also dose-and time-dependently stimulated 5-HT uptake that was only partially blocked by SB203580. Western blots showed that IL-1b and TNF-a activated p38 MAPK, in an SB203580-sensitive manner. IL-1b induced an SB203580-sensitive decrease in 5-HT K m with no significant change in V max . In contrast, TNF-a stimulation decreased 5-HT K m and increased SERT V max . SB203580 selectively blocked the TNF-a-induced change in SERT K m . In mouse midbrain and striatal synaptosomes, maximal stimulatory effects on 5-HT uptake occurred at lower concentrations (IL-1b, 10 ng/ml; TNF-a, 20 ng/ml), and over shorter incubation times (10 min). As with RN46A cells, the effects of IL-1b and TNF-a were completely (IL-1b) or partially (TNF-a) blocked by SB203580. These results provide the first evidence that proinflammatory cytokines can acutely regulate neuronal SERT activity via p38 MAPK-linked pathways.

Serotonin (5-hydroxytryptamine, 5-HT) has long been implicated in regulation of mood. Medications that block the neuronal 5-HT transporter (SERT) are used as major pharmacological treatment for mood disorders. Conversely, stimuli that enhance SERT activity might be predicted to diminish synaptic 5-HT availability and increase the risk for 5-HT-related CNS disorders. We have shown that the inflammatory cytokines enhance brain SERT activity in cultured serotonergic cells and nerve terminal preparations in vitro. In this study, we establish that intraperitoneal injection of the cytokine-inducer lipopolysaccharide (LPS) stimulates brain SERT activity, acting at doses below those required to induce overt motor suppression. SERT stimulation by LPS is paralleled by increased immobility in both the tail suspension test (TST) and the forced swim test (FST); antidepressant-sensitive alterations are thought to model aspects of behavioral despair. Both the stimulation of SERT activity and induced immobility are absent when LPS is administered to interleukin-1 receptor (IL-1R)-deficient mice and in the presence of SB203580, an inhibitor of IL-1R-stimulated p38 MAPK. Moreover, the ability of LPS to enhance immobility in TST is lost in SERT knockout mice. These findings reveal an ability of peripheral inflammatory stimuli to enhance brain SERT activity through IL-1R and p38 MAPK pathways in vivo and identify a requirement for SERT expression in immune-system-modulated despair behaviors. Our studies identify IL-1R- and p38 MAPK-dependent regulation of SERT as one of the mechanisms by which environmentally driven immune system activation can trigger despair-like behavior in an animal model, encouraging future analysis of the pathway for risk factors in neuropsychiatric disorders.

The indoleamine 5-hydroxytryptamine (serotonin, 5-HT) 1 plays a pivotal, modulatory role in a variety of centrally controlled physiological processes, including respiration, arousal, aggression, and mood, and in the periphery supports gastrointestinal, platelet, and placental function (1). 5-HT is inactivated following vesicular release by a presynaptic, antidepressant-sensitive 5-HT transporter (SERT, 5-HTT), a member of the Na ϩ /Cl Ϫ -dependent solute transporter family (SLC6A4) (2-4). SERT knock-out mice display altered presynaptic 5-HT homeostasis, modified 5-HT receptor sensitivities, and stressdependent behavioral modulation as well as altered responses to psychostimulants (5). In humans, altered SERT gene expression and/or transport function have been linked to multiple disorders including autism, obsessive-compulsive disorder, depression, and suicide (6 -11).Previous studies have demonstrated that both genetic and posttranscriptional processes regulate SERT activity (12, 13). SERT activity in native cells and transfected models can be rapidly (in minutes) modulated by multiple signaling pathways (14, 15). Observations with transfected HEK cells expressing human SERT (hSERT) demonstrated that protein kinase C activators or protein phosphatase 1/2A inhibitors trigger hSERT phosphorylation and a parallel decrease in hSERT cell surface density, effects that can be attenuated by . Protein kinase A and protein kinase G (PKG) activation can also trigger SERT phosphorylation (17), although the functional consequences of these stimuli are only beginning to be appreciated. SERTs appear to form homomultimers at the plasma membrane (19) and also interact with a growing list of associated proteins, including syntaxin 1A (20 -22)

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