Supramolecular assembly of GSK3α as a cellular response to amino acid starvation.

The tolerance of amino acid starvation is fundamental to robust cellular fitness. Asparagine depletion is lethal to some cancer cells, a vulnerability that can be exploited clinically. We report that resistance to asparagine starvation is uniquely dependent on an N-terminal low-complexity domain of GSK3α, which its paralog GSK3ß lacks. In response to depletion of specific amino acids, including asparagine, leucine, and valine, this domain mediates supramolecular assembly of GSK3α with ubiquitin-proteasome system components in spatially sequestered cytoplasmic bodies. This effect is independent of mTORC1 or GCN2. In normal cells, GSK3α promotes survival during essential amino acid starvation. In human leukemia, GSK3α body formation predicts asparaginase resistance, and sensitivity to asparaginase combined with a GSK3α inhibitor. We propose that GSK3α body formation provides a cellular mechanism to maximize the catalytic efficiency of proteasomal protein degradation in response to amino acid starvation, an adaptive response co-opted by cancer cells for asparaginase resistance.

The interplay of serotonin 5-HT1A and 5-HT7 receptors in chronic stress.

Serotonin regulates multiple physiological and pathological processes in the brain, including mood and cognition. Serotonin receptors 5-HT1AR and 5-HT7R have emerged as key players in stress-related disorders, particularly depression. These receptors can form heterodimers, which influence their functions. Here we explored the developmental dynamics of 5-HT1AR and 5-HT7R expression and validated heterodimerization levels in the brain of control and stressed mice. In control animals, we obtained increase in 5-HT1AR expression over 5-HT7R in the prefrontal cortex (PFC) and hippocampus during development. Using a chronic unpredictable stress as a depression model, we found increase in 5-HT7R expression exclusively in the PFC of resilient animals, while no changes in 5-HT1AR expression between control and anhedonic mice were obtained. Quantitative in situ analysis of heterodimerization revealed the PFC as region exhibiting the highest abundance of 5-HT1AR/5-HT7R heterodimers. More importantly, upon chronic stress amount of heterodimers was significantly reduced only in PFC of anhedonic mice, while it was not affected in resilient animals. These results suggest an important role of brain-region specific 5-HT1AR/5-HT7R heterodimerization for establishing depressive-like behavior and for development of resiliency.

The spinal muscular atrophy gene product regulates actin dynamics.

Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by low levels of the Survival of Motoneuron (SMN) protein. SMN interacts with and regulates the actin-binding protein profilin2a, thereby influencing actin dynamics. Dysfunctional actin dynamics caused by SMN loss disrupts neurite outgrowth, axonal pathfinding, and formation of functional synapses in neurons. Whether the SMN protein directly interacts with and regulates filamentous (F-) and monomeric globular (G-) actin is still elusive. In a quantitative single cell approach, we show that SMN loss leads to dysregulated F-/G-actin fractions. Furthermore, quantitative assessment of cell morphology suggests an F-actin organizational defect. Interestingly, this is mediated by an interaction of SMN with G- and F-actin. In co-immunoprecipitation, in-vitro pulldown and co-localization assays, we elucidated that this interaction is independent of the SMN-profilin2a interaction. Therefore, we suggest two populations being relevant for functional actin dynamics in healthy neurons: SMN-profilin2a-actin and SMN-actin. Additionally, those two populations may influence each other and therefore regulate binding of SMN to actin. In SMA, we showed a dysregulated co-localization pattern of SMN-actin which could only partially rescued by SMN restoration. However, dysregulation of F-/G-actin fractions was reduced by SMN restoration. Taken together, our results suggest a novel molecular function of SMN in binding to actin independent from SMN-profilin2a interaction.

Structural determinants for activation of the Tau kinase CDK5 by the serotonin receptor 5-HT7R.

BACKGROUND: Multiple neurodegenerative diseases are induced by the formation and deposition of protein aggregates. In particular, the microtubule-associated protein Tau leads to the development of so-called tauopathies characterized by the aggregation of hyperphosphorylated Tau within neurons. We recently showed that the constitutive activity of the serotonin receptor 7 (5-HT7R) is required for Tau hyperphosphorylation and aggregation through activation of the cyclin-dependent kinase 5 (CDK5). We also demonstrated physical interaction between 5-HT7R and CDK5 at the plasma membrane suggesting that the 5-HT7R/CDK5 complex is an integral part of the signaling network involved in Tau-mediated pathology. METHODS: Using biochemical, microscopic, molecular biological, computational and AI-based approaches, we investigated structural requirements for the formation of 5-HT7R/CDK5 complex. RESULTS: We demonstrated that 5-HT7R domains responsible for coupling to Gs proteins are not involved in receptor interaction with CDK5. We also created a structural model of the 5-HT7R/CDK5 complex and refined the interaction interface. The model predicted two conserved phenylalanine residues, F278 and F281, within the third intracellular loop of 5-HT7R to be potentially important for complex formation. While site-directed mutagenesis of these residues did not influence Gs protein-mediated receptor signaling, replacement of both phenylalanines by alanine residues significantly reduced 5-HT7R/CDK5 interaction and receptor-mediated CDK5 activation, leading to reduced Tau hyperphosphorylation and aggregation. Molecular dynamics simulations of 5-HT7R/CDK5 complex for wild-type and receptor mutants confirmed binding interface stability of the initial model. CONCLUSIONS: Our results provide a structural basis for the development of novel drugs targeting the 5-HT7R/CDK5 interaction interface for the selective treatment of Tau-related disorders, including frontotemporal dementia and Alzheimer's disease.

Nonsense mediated decay factor UPF3B is associated with cMyBP-C haploinsufficiency in hypertrophic cardiomyopathy patients.

Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited cardiac disease. Up to 40% of cases are associated with heterozygous mutations in myosin binding protein C (cMyBP-C, MYBPC3). Most of these mutations lead to premature termination codons (PTC) and patients show reduction of functional cMyBP-C. This so-called haploinsufficiency most likely contributes to disease development. We analyzed mechanisms underlying haploinsufficiency using cardiac tissue from HCM-patients with truncation mutations in MYBPC3 (MYBPC3trunc). We compared transcriptional activity, mRNA and protein expression to donor controls. To differentiate between HCM-specific and general hypertrophy-induced mechanisms we used patients with left ventricular hypertrophy due to aortic stenosis (AS) as an additional control. We show that cMyBP-C haploinsufficiency starts at the mRNA level, despite hypertrophy-induced increased transcriptional activity. Gene set enrichment analysis (GSEA) of RNA-sequencing data revealed an increased expression of NMD-components. Among them, Up-frameshift protein UPF3B, a regulator of NMD was upregulated in MYBPC3trunc patients and not in AS-patients. Strikingly, we show that in sarcomeres UPF3B but not UPF1 and UPF2 are localized to the Z-discs, the presumed location of sarcomeric protein translation. Our data suggest that cMyBP-C haploinsufficiency in HCM-patients is established by UPF3B-dependent NMD during the initial translation round at the Z-disc.

Palmitoylation of the small GTPase Cdc42 by DHHC5 modulates spine formation and gene transcription.

The small GTPase Cdc42 exists in the form of two alternatively spliced variants that are modified by hydrophobic chains: the ubiquitously expressed Cdc42-prenyl and a brain-specific isoform that can be palmitoylated, Cdc42-palm. Our previous work demonstrated that Cdc42-palm can be palmitoylated at two cysteine residues, Cys188 and Cys189, while Cys188 can also be prenylated. We showed that palmitoylation of Cys188 is essential for the plasma membrane localization of Cdc42-palm and is critically involved in Cdc42-mediated regulation of gene transcription and neuronal morphology. However, the abundance and regulation of this modification was not investigated. In the present study, we found that only a minor fraction of Cdc42 undergoes monopalmitoylation in neuroblastoma cells and in hippocampal neurons. In addition, we identified DHHC5 as one of the major palmitoyl acyltransferases that could physically interact with Cdc42-palm. We demonstrate that overexpression of dominant negative DHHC5 mutant decreased palmitoylation and plasma membrane localization of Cdc42-palm. In addition, knockdown of DHHC5 significantly reduced Cdc42-palm palmitoylation, leading to a decrease of Cdc42-mediated gene transcription and spine formation in hippocampal neurons. We also found that the expression of DHHC5 in the brain is developmentally regulated. Taken together, these findings suggest that DHHC5-mediated palmitoylation of Cdc42 represents an important mechanism for the regulation of Cdc42 functions in hippocampus.

The 5-HT4 receptor interacts with adhesion molecule L1 to modulate morphogenic signaling in neurons.

Morphological remodeling of dendritic spines is critically involved in memory formation and depends on adhesion molecules. Serotonin receptors are also implicated in this remodeling, though the underlying mechanisms remain enigmatic. Here, we uncovered a signaling pathway involving the adhesion molecule L1CAM (L1) and serotonin receptor 5-HT4 (5-HT4R, encoded by HTR4). Using Förster resonance energy transfer (FRET) imaging, we demonstrated a physical interaction between 5-HT4R and L1, and found that 5-HT4R-L1 heterodimerization facilitates mitogen-activated protein kinase activation in a Gs-dependent manner. We also found that 5-HT4R-L1-mediated signaling is involved in G13-dependent modulation of cofilin-1 activity. In hippocampal neurons in vitro, the 5-HT4R-L1 pathway triggers maturation of dendritic spines. Thus, the 5-HT4R-L1 signaling module represents a previously unknown molecular pathway regulating synaptic remodeling.

Amisulpride as a potential disease-modifying drug in the treatment of tauopathies.

INTRODUCTION: Hyperphosphorylation and aggregation of the microtubule-associated protein tau cause the development of tauopathies, such as Alzheimer's disease and frontotemporal dementia (FTD). We recently uncovered a causal link between constitutive serotonin receptor 7 (5-HT7R) activity and pathological tau aggregation. Here, we evaluated 5-HT7R inverse agonists as novel drugs in the treatment of tauopathies. METHODS: Based on structural homology, we screened multiple approved drugs for their inverse agonism toward 5-HT7R. Therapeutic potential was validated using biochemical, pharmacological, microscopic, and behavioral approaches in different cellular models including tau aggregation cell line HEK293 tau bimolecular fluorescence complementation, primary mouse neurons, and human induced pluripotent stem cell-derived neurons carrying an FTD-associated tau mutation as well as in two mouse models of tauopathy. RESULTS: Antipsychotic drug amisulpride is a potent 5-HT7R inverse agonist. Amisulpride ameliorated tau hyperphosphorylation and aggregation in vitro. It further reduced tau pathology and abrogated memory impairment in mice. DISCUSSION: Amisulpride may be a disease-modifying drug for tauopathies.

DHHC7-mediated palmitoylation of the accessory protein barttin critically regulates the functions of ClC-K chloride channels.

Barttin is the accessory subunit of the human ClC-K chloride channels, which are expressed in both the kidney and inner ear. Barttin promotes trafficking of the complex it forms with ClC-K to the plasma membrane and is involved in activating this channel. Barttin undergoes post-translational palmitoylation that is essential for its functions, but the enzyme(s) catalyzing this post-translational modification is unknown. Here, we identified zinc finger DHHC-type containing 7 (DHHC7) protein as an important barttin palmitoyl acyltransferase, whose depletion affected barttin palmitoylation and ClC-K-barttin channel activation. We investigated the functional role of barttin palmitoylation in vivo in Zdhhc7-/- mice. Although palmitoylation of barttin in kidneys of Zdhhc7-/- animals was significantly decreased, it did not pathologically alter kidney structure and functions under physiological conditions. However, when Zdhhc7-/- mice were fed a low-salt diet, they developed hyponatremia and mild metabolic alkalosis, symptoms characteristic of human Bartter syndrome (BS) type IV. Of note, we also observed decreased palmitoylation of the disease-causing R8L barttin variant associated with human BS type IV. Our results indicate that dysregulated DHHC7-mediated barttin palmitoylation appears to play an important role in chloride channel dysfunction in certain BS variants, suggesting that targeting DHHC7 activity may offer a potential therapeutic strategy for reducing hypertension.

Elucidating regulators of astrocytic Ca2+ signaling via multi-threshold event detection (MTED).

Recent achievements in indicator optimization and imaging techniques promote the advancement of functional imaging to decipher complex signaling processes in living cells, such as Ca2+ activity patterns. Astrocytes are important regulators of the brain network and well known for their highly complex morphology and spontaneous Ca2+ activity. However, the astrocyte community is lacking standardized methods to analyze and interpret Ca2+ activity recordings, hindering global comparisons. Here, we present a biophysically-based analytical concept for deciphering the complex spatio-temporal changes of Ca2+ biosensor fluorescence for understanding the underlying signaling mechanisms. We developed a pixel-based multi-threshold event detection (MTED) analysis of multidimensional data, which accounts for signal strength as an additional signaling dimension and provides the experimenter with a comprehensive toolbox for a differentiated and in-depth characterization of fluorescence signals. MTED was validated by analyzing astrocytic Ca2+ activity across Ca2+ indicators, imaging setups, and model systems from primary cell culture to awake, head-fixed mice. We identified extended Ca2+ activity at 25°C compared to 37°C physiological body temperature and dissected how neuronal activity shapes long-lasting astrocytic Ca2+ activity. Our MTED strategy, as a parameter-free approach, is easily transferrable to other fluorescent indicators and biosensors and embraces the additional dimensionality of signaling activity strength. It will also advance the definition of standardized procedures and parameters to improve comparability of research data and reports.

Serotonin receptor 4 regulates hippocampal astrocyte morphology and function.

Astrocytes are an important component of the multipartite synapse and crucial for proper neuronal network function. Although small GTPases of the Rho family are powerful regulators of cellular morphology, the signaling modules of Rho-mediated pathways in astrocytes remain enigmatic. Here we demonstrated that the serotonin receptor 4 (5-HT4 R) is expressed in hippocampal astrocytes, both in vitro and in vivo. Through fluorescence microscopy, we established that 5-HT4 R activation triggered RhoA activity via Gα13 -mediated signaling, which boosted filamentous actin assembly, leading to morphological changes in hippocampal astrocytes. We investigated the effects of these 5-HT4 R-mediated changes in mixed cultures and in acute slices, in which 5-HT4 R was expressed exclusively in astrocytes. In both systems, 5-HT4 R-RhoA signaling changed glutamatergic synaptic transmission: It increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in mixed cultures and reduced the paired-pulse-ratio (PPR) of field excitatory postsynaptic potentials (fEPSPs) in acute slices. Overall, our present findings demonstrate that astrocytic 5-HT4 R-Gα13 -RhoA signaling is a previously unrecognized molecular pathway involved in the functional regulation of excitatory synaptic circuits.

Brain microstructural changes in mice persist in adulthood and are modulated by the palmitoyl acyltransferase ZDHHC7.

For a long time, mice have been classified as adults with completely mature brains at 8 weeks of age, but recent research suggests that developmental brain changes occur for up to 6 months. In particular, adolescence coincides with dramatic changes of neuronal structure and function in the brain that influence the connectivity between areas like hippocampus and medial prefrontal cortex (mPFC). Neuronal development and plasticity are regulated in part by the palmitoyl acyltransferase ZDHHC7, which modulates structural connectivity between hippocampus and mPFC. The aim of the current study was to investigate whether developmental changes take place in hippocampus and mPFC microstructure even after 8 weeks of age and whether deficiency of ZDHHC7 impacts such age-dependent alterations. Altogether, 46 mice at 11, 14 or 17 weeks of age with a genetic Zdhhc7 knockout (KO) or wild type (WT) were analysed with neuroimaging and diffusion tensor-based fibre tractography. The hippocampus and mPFC regions were compared regarding fibre metrics, supplemented by volumetric and immunohistological analyses of the hippocampus. In WT animals, we identified age-dependent changes in hippocampal fibre lengths that followed a U-shaped pattern, whereas in mPFC, changes were linear. In Zdhhc7-deficient animals, the fibre statistics were reduced in both regions, whereas the hippocampus volume and the intensities of myelin and neurofilament were higher in 11-week-old KO mice compared to WTs. Our results confirmed ongoing changes of microstructure in mice up to 17 weeks old and demonstrate that deleting the Zdhhc7 gene impairs fibre development, suggesting that palmitoylation is important in this process.

Serotonin receptor oligomerization regulates cAMP-based signaling.

Protein-protein interaction is often investigated using quantitative molecular microscopy with Förster resonant energy transfer (FRET). Here, we combined 'linear unmixing FRET' (lux-FRET) with the simultaneous application of a FRET-based biosensor for cAMP to investigate the oligomerization between the 5-HT7 receptor (5-HT7R, also known as HTR7) and the 5-HT1A receptor (5-HT1AR, also known as HTR1A) and its importance for cAMP signaling. We found that the 5-HT7R not only stimulates cAMP production, but also forms hetero-oligomers with 5-HT1AR, which blocks the inhibitory effect of the latter. 5-HT7R signaling, however, is not affected by this hetero-oligomerization. By modeling the kinetics of intracellular cAMP level changes in relation to the 5-HT7R:5-HT1AR stoichiometry, we were able to decipher the complex signaling characteristics of endogenous serotonin receptors in cultured hippocampal neurons. Our findings indicate that serotonergic signaling is not only modulated by the concentration of an individual receptor but also by its specific interaction with other receptors in endogenous systems. We conclude that the regulated ratio of serotonin receptors in immature and mature neurons may be critically involved in both the onset and response to treatments of psychiatric diseases, such as anxiety and depression.

S-Palmitoylation of Synaptic Proteins as a Novel Mechanism Underlying Sex-Dependent Differences in Neuronal Plasticity.

Although sex differences in the brain are prevalent, the knowledge about mechanisms underlying sex-related effects on normal and pathological brain functioning is rather poor. It is known that female and male brains differ in size and connectivity. Moreover, those differences are related to neuronal morphology, synaptic plasticity, and molecular signaling pathways. Among different processes assuring proper synapse functions are posttranslational modifications, and among them, S-palmitoylation (S-PALM) emerges as a crucial mechanism regulating synaptic integrity. Protein S-PALM is governed by a family of palmitoyl acyltransferases, also known as DHHC proteins. Here we focused on the sex-related functional importance of DHHC7 acyltransferase because of its S-PALM action over different synaptic proteins as well as sex steroid receptors. Using the mass spectrometry-based PANIMoni method, we identified sex-dependent differences in the S-PALM of synaptic proteins potentially involved in the regulation of membrane excitability and synaptic transmission as well as in the signaling of proteins involved in the structural plasticity of dendritic spines. To determine a mechanistic source for obtained sex-dependent changes in protein S-PALM, we analyzed synaptoneurosomes isolated from DHHC7-/- (DHHC7KO) female and male mice. Our data showed sex-dependent action of DHHC7 acyltransferase. Furthermore, we revealed that different S-PALM proteins control the same biological processes in male and female synapses.

Neuronal branching of sensory neurons is associated with BDNF-positive eosinophils in atopic dermatitis.

BACKGROUND: Pruritus is a major symptom of atopic dermatitis (AD) and is transmitted by a subpopulation of non-myelinated C-type free nerve endings in the epidermis and upper dermis. Stimulation of these nerve terminals is affected by histamine, neurotrophins and physical factors. Eosinophils of patients with AD are a source of neurotrophins, including brain-derived neurotrophic factor (BDNF), levels of which correlate with disease severity. OBJECTIVE: The purpose of this study was to determine the anatomical localization of eosinophils in the skin of patients with AD with regard to peripheral nerves and to investigate whether eosinophils induce sprouting and neurite outgrowth in murine sensory neurons. METHODS: Cryosections of skin derived from AD and control (NA) patients were subjected to immunofluorescence analysis with markers for eosinophils, BDNF and neuronal cells. Stimulated eosinophil supernatants were used for the treatment of cultured peripheral mouse dorsal root ganglia (DRG) neurons followed by morphometric analysis. RESULTS: Dermal axon density and the proximity of eosinophils to nerve fibres were significantly higher in AD patients vs NA. Both neuronal projections and eosinophils expressed BDNF. Furthermore, activated eosinophil supernatants induced BDNF-dependent mouse DRG neuron branching. CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that BDNF-positive eosinophils are also localized in close proximity with nerve fibres in AD, suggesting a functional relationship between BDNF-expressing eosinophils and neuronal projections. These observations suggest that eosinophils may have considerable impact on pruritus by supporting sensory nerve branching.

The guanine nucleotide exchange factor Vav3 modulates oligodendrocyte precursor differentiation and supports remyelination in white matter lesions.

The tightly controlled processes of myelination and remyelination require the participation of the cytoskeleton. The reorganization of the cytoskeleton is controlled by small GTPases of the RhoA family. Here, we report that Vav3, a Rho GTPase regulating guanine nucleotide exchange factor (GEF) is involved in oligodendrocyte maturation, myelination and remyelination. When Vav3 was eliminated by genetic recombination, oligodendrocyte precursor cell (OPC) differentiation toward mature oligodendrocytes was accelerated. In contrast, Vav3-deficient oligodendrocytes displayed a reduced capacity to myelinate synthetic microfibers in vitro. Furthermore, remyelination was impaired in Vav3 knockout cerebellar slice cultures that were demyelinated by the addition of lysolecithin. In agreement with these observations, remyelination was compromised when the cuprizone model of myelin lesion was performed in Vav3-deficient mice. When Vav3-deficient oligodendrocytes were examined with Förster resonance energy transfer (FRET)-based biosensors, an altered activation profile of RhoA GTPases was revealed on the cellular level, which could be responsible for an impaired remyelination. Taken together, this study highlights Vav3 as a novel regulator of oligodendrocyte maturation and remyelination, suggesting that manipulation of the Vav3-dependent signaling pathway could help to improve myelin repair.

Palmitoylation as a Functional Regulator of Neurotransmitter Receptors.

The majority of neuronal proteins involved in cellular signaling undergo different posttranslational modifications significantly affecting their functions. One of these modifications is a covalent attachment of a 16-C palmitic acid to one or more cysteine residues (S-palmitoylation) within the target protein. Palmitoylation is a reversible modification, and repeated cycles of palmitoylation/depalmitoylation might be critically involved in the regulation of multiple signaling processes. Palmitoylation also represents a common posttranslational modification of the neurotransmitter receptors, including G protein-coupled receptors (GPCRs) and ligand-gated ion channels (LICs). From the functional point of view, palmitoylation affects a wide span of neurotransmitter receptors activities including their trafficking, sorting, stability, residence lifetime at the cell surface, endocytosis, recycling, and synaptic clustering. This review summarizes the current knowledge on the palmitoylation of neurotransmitter receptors and its role in the regulation of receptors functions as well as in the control of different kinds of physiological and pathological behavior.

Serotonin 5-HT7 receptor increases the density of dendritic spines and facilitates synaptogenesis in forebrain neurons.

Precise control of dendritic spine density and synapse formation is critical for normal and pathological brain functions. Therefore, signaling pathways influencing dendrite outgrowth and remodeling remain a subject of extensive investigations. Here, we report that prolonged activation of the serotonin 5-HT7 receptor (5-HT7R) with selective agonist LP-211 promotes formation of dendritic spines and facilitates synaptogenesis in postnatal cortical and striatal neurons. Critical role of 5-HT7R in neuronal morphogenesis was confirmed by analysis of neurons isolated from 5-HT7R-deficient mice and by pharmacological inactivation of the receptor. Acute activation of 5-HT7R results in pronounced neurite elongation in postnatal striatal and cortical neurons, thus extending previous data on the morphogenic role of 5-HT7R in embryonic and hippocampal neurons. We also observed decreased number of spines in neurons with either genetically (i.e. 5-HT7R-knock-out) or pharmacologically (i.e. antagonist treatment) blocked 5-HT7R, suggesting that constitutive 5-HT7R activity is critically involved in the spinogenesis. Moreover, cyclin-dependent kinase 5 and small GTPase Cdc42 were identified as important downstream effectors mediating morphogenic effects of 5-HT7R in neurons. Altogether, our data suggest that the 5-HT7R-mediated structural reorganization during the postnatal development might have a crucial role for the development and plasticity of forebrain areas such as cortex and striatum, and thereby can be implicated in regulation of the higher cognitive functions. Read the Editorial Highlight for this article on page 644.

The serotonin receptor 5-HT7R regulates the morphology and migratory properties of dendritic cells.

Dendritic cells are potent antigen-presenting cells endowed with the unique ability to initiate adaptive immune responses upon inflammation. Inflammatory processes are often associated with an increased production of serotonin, which operates by activating specific receptors. However, the functional role of serotonin receptors in regulation of dendritic cell functions is poorly understood. Here, we demonstrate that expression of serotonin receptor 5-HT7 (5-HT7R) as well as its downstream effector Cdc42 is upregulated in dendritic cells upon maturation. Although dendritic cell maturation was independent of 5-HT7R, receptor stimulation affected dendritic cell morphology through Cdc42-mediated signaling. In addition, basal activity of 5-HT7R was required for the proper expression of the chemokine receptor CCR7, which is a key factor that controls dendritic cell migration. Consistent with this, we observed that 5-HT7R enhances chemotactic motility of dendritic cells in vitro by modulating their directionality and migration velocity. Accordingly, migration of dendritic cells in murine colon explants was abolished after pharmacological receptor inhibition. Our results indicate that there is a crucial role for 5-HT7R-Cdc42-mediated signaling in the regulation of dendritic cell morphology and motility, suggesting that 5-HT7R could be a new target for treatment of a variety of inflammatory and immune disorders.

Human CLC-K Channels Require Palmitoylation of Their Accessory Subunit Barttin to Be Functional.

CLC-K/barttin chloride channels are essential for NaCl re-absorption in Henle's loop and for potassium secretion by the stria vascularis in the inner ear. Here, we studied the posttranslational modification of such channels by palmitoylation of their accessory subunit barttin. We found that barttin is palmitoylated in vivo and in vitro and identified two conserved cysteine residues at positions 54 and 56 as palmitoylation sites. Point mutations at these two residues reduce the macroscopic current amplitudes in cells expressing CLC-K/barttin channels proportionally to the relative reduction in palmitoylated barttin. CLC-K/barttin expression, plasma membrane insertion, and single channel properties remain unaffected, indicating that these mutations decrease the number of active channels. R8W and G47R, two naturally occurring barttin mutations identified in patients with Bartter syndrome type IV, reduce barttin palmitoylation and CLC-K/barttin channel activity. Palmitoylation of the accessory subunit barttin might thus play a role in chloride channel dysfunction in certain variants of Bartter syndrome. We did not observe pronounced alteration of barttin palmitoylation upon increased salt and water intake or water deprivation, indicating that this posttranslational modification does not contribute to long term adaptation to variable water intake. Our results identify barttin palmitoylation as a novel posttranslational modification of CLC-K/barttin chloride channels.