RGS proteins: impact on the treatment of depression and anxiety

This is the publisher's version, also available electronically from "http://ijnp.oxfordjournals.org/".No abstract available

Estradiol potentiates 8-OH-DPAT-induced sumoylation of 5-HT1A receptor: characterization and subcellular distribution of sumoylated 5-HT1A receptors

Sumoylation is a recently described post-translational modification and only a few sumoylated neurotransmitter receptors are known. Through the present studies, we discovered that serotonin1A receptors (5-HT1A-Rs) can be sumoylated by SUMO1 (Small-Ubiquitin-related modifier 1) protein. The SUMO1-5-HT1A-R is ∼ 55kD, is located in the membrane fraction, but not the cytosol, and is distributed in all of the brain regions expressing 5-HT1A-Rs examined. Acute stimulation of 5-HT1A-Rs significantly increased SUMO1-5-HT1A-R in rat hypothalamus. Pre-treatment with estradiol for 2 days, which causes a partial desensitization of 5-HT1A-R signaling, potentiated agonistinduced increases in SUMO1-5-HT1A-Rs in the hypothalamus of ovariectomized rats. Using discontinuous gradient centrifugation followed by digitonin treatment, we found that the majority of SUMO1-5-HT1A-Rs is co-localized with endoplasmic-reticulum and trans-Golgi-network markers. Although a small proportion of SUMO1-5-HT1A-Rs are located in the detergent resistant microdomain (DRM) that contain active G-protein coupled receptors, their distribution was different from that of the Gαz protein that couples to the receptors. These data suggest that the SUMO1-5-HT1A-Rs are an inactive form of 5-HT1A-Rs, a finding further supported by results showing minimal 5-HT1A-R agonist binding to SUMO1-5-HT1A-Rs. Furthermore, SUMO1-5-HT1A-Rs in the DRM were increased by treatment with a 5-HT1A-R agonist, 8-OH-DPAT ((+)8-hydroxy-2-dipropylaminotetralin). Together, these data suggest that sumoylation of 5-HT1A-Rs may be related to 5-HT1A-R trafficking and internalization, which may contribute to 5-HT1A-R desensitization. Since 5-HT1A-Rs play an important role in mood regulation, the present results significantly impact on the understanding of the pathogenesis of affective disorders and development of better therapeutic approaches for these diseases

Neuroprotective Effects of Calmodulin Peptide 76-121aa: Disruption of Calmodulin Binding to Mutant Huntingtin

Huntington's disease (HD) is a neurodegenerative disease caused by mutant huntingtin protein containing an expanded polyglutamine tract, which may cause abnormal protein–protein interactions such as increased association with calmodulin (CaM). We previously demonstrated in HEK293 cells that a peptide containing amino acids 76-121 of CaM (CaM-peptide) interrupted the interaction between CaM and mutant huntingtin, reduced mutant huntingtin-induced cytotoxicity and reduced transglutaminase (TG)-modified mutant huntingtin. We now report that adeno-associated virus (AAV)-mediated expression of CaM-peptide in differentiated neuroblastoma SH-SY5Y cells, stably expressing an N-terminal fragment of huntingtin containing 148 glutamine repeats, significantly decreases the amount of TG-modified huntingtin and attenuates cytotoxicity. Importantly, the effect of the CaM-peptide shows selectivity, such that total TG activity is not significantly altered by expression of CaM-peptide nor is the activity of another CaM-dependent enzyme, CaM kinase II. In vitro, recombinant exon 1 of huntingtin with 44 glutamines (htt-exon1-44Q) binds to CaM-agarose; the addition of 10 µM of CaM-peptide significantly decreases the interaction of htt-exon1-44Q and CaM but not the binding between CaM and calcineurin, another CaM-binding protein. These data support the hypothesis that CaM regulates TG-catalyzed modifications of mutant huntingtin and that specific and selective disruption of the CaM-huntingtin interaction is potentially a new target for therapeutic intervention in HD

Phospholipase C, Ca2+, and calmodulin signaling are required for 5-HT2A receptor-mediated transamidation of Rac1 by transglutaminase

RATIONALE: Serotonin and especially serotonin 2A (5-HT2A) receptor signaling are important in the etiology and treatment of schizophrenia and affective disorders. We previously reported a novel 5-HT2A receptor effector, increased transglutaminase (TGase)-catalyzed transamidation, and activation of the small G protein Rac1 in A1A1v cells, a rat embryonic cortical cell line. OBJECTIVES: In this study, we explore the signaling pathway involved in 5-HT2A receptor-mediated Rac1 transamidation. METHODS: A1A1v cells were pretreated with pharmacological inhibitors of phospholipase C (PLC) or calmodulin (CaM), and then stimulated by the 5-HT2A receptor agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI). Intracellular Ca2+ concentration and TGase-modified Rac1 transamidation were monitored. The effect of manipulation of intracellular Ca2+ by a Ca2+ ionophore or a chelating agent on Rac1 transamidation was also evaluated. RESULTS: In cells pretreated with a PLC inhibitor U73122, DOI-stimulated increases in the intracellular Ca2+ concentration and TGase-modified Rac1 were significantly attenuated as compared to those pretreated with U73343, an inactive analog. The membrane-permeant Ca2+ chelator, BAPTA-AM strongly reduced TGase-catalyzed Rac1 transamidation upon DOI stimulation. Conversely, the Ca2+ ionophore ionomycin, at a concentration that induced an elevation of cytosolic Ca2+ to a level comparable to cells treated with DOI, produced an increase in TGase-modified Rac1 without 5-HT2A receptor activation. Moreover, the CaM inhibitor W-7, significantly decreased Rac1 transamidation in a dose-dependent manner in DOI-treated cells. CONCLUSIONS: These results indicate that 5-HT2A receptorcoupled PLC activation and subsequent Ca2+ and CaM signaling are necessary for TGase-catalyzed Rac1 transamidation, and an increase in intracellular Ca2+ is sufficient to induce Rac1 transamidation

Regulation of Serotonin 1A Receptor SUMOylation by SENP2 and PIASxα

Serotonin 1A receptors (5-HT1ARs) are implicated in the control of mood, cognition, and memory and in various neuropsychiatric disorders such as depression and anxiety. As such, understanding the regulation of 5-HT1ARs will inform the development of better treatment approaches. We previously demonstrated 5-HT1ARs are SUMOylated by SUMO1 in the rat brain. Agonist stimulation increased SUMOylation and was further enhanced when combined with 17β-estradiol-3-benzoate (EB), which are treatments that cause the transient and prolonged desensitization of 5-HT1AR signaling, respectively. In the current study, we identified the protein inhibitor of activated STAT (PIAS)xα as the enzyme that facilitates SUMOylation, and SENP2 as the protein that catalyzes the deSUMOylation of 5-HT1ARs. We demonstrated that PIASxα significantly increased in the membrane fraction of rats co-treated with EB and an agonist, compared to either the EB-treated or vehicle-treated groups. The acute treatment with an agonist alone shifted the location of SENP2 from the membrane to the cytoplasmic fraction, but it has little effect on PIASxα. Hence, two separate mechanisms regulate SUMOylation and the activity of 5-HT1ARs by an agonist and EB. The effects of EB on 5-HT1AR SUMOylation and signaling may be related to the higher incidence of mood disorders in women during times with large fluctuations in estrogens. Targeting the SUMOylation of 5-HT1ARs could have important clinical relevance for the therapy for several neuropsychiatric disorders in which 5-HT1ARs are implicated

Activation of the JAK-STAT pathway is necessary for desensitization of 5-HT2A receptor-stimulated phospholipase C signalling by olanzapine, clozapine and MDL 100907

This is the publisher's version, also available electronically from http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=5564880&fileId=S1461145708009590We have previously demonstrated that olanzapine-induced desensitization of 5-HT2A receptor-stimulated phospholipase C (PLC) activity is associated with increases in RGS7 protein levels both in vivo and in cells in culture, and the increase in RGS7 is dependent on activation of the JAK-STAT pathway in cells in culture. In the present study, we found that desensitization of 5-HT2A receptor-stimulated PLC activity induced by olanzapine is dependent on activation of the JAK-STAT pathway. Similar to olanzapine, clozapine-induced desensitization of 5-HT2A receptor signalling is accompanied by increases in RGS7 and activation of JAK2. Treatment with the selective 5-HT2A receptor antagonist MDL 100907 also increased RGS7 protein levels and JAK2 activation. Using a JAK2 inhibitor AG490, we found that clozapine and MDL 100907-induced increases in RGS7 are dependent on activation of the JAK-STAT pathway. Olanzapine, clozapine, and MDL 100907 treatment increased mRNA levels of RGS7. Using a chromatin immunoprecipitation assay we found STAT3 binding to the putative RGS7 promoter region. Taken together, olanzapine-induced activation of the JAK-STAT pathway, and STAT3 binding to the RGS7 gene could underlie the increase in RGS7 mRNA which could subsequently increase protein expression. Furthermore, the increase in RGS7 protein could play a role in the desensitization of 5-HT2A receptor signalling by terminating the activated Gαq/11 proteins more rapidly. Overall, our data suggest that the complete desensitization of 5-HT2A receptor-stimulated PLC activity by olanzapine, clozapine and MDL 100907 requires activation of the JAK-STAT pathway, which in turn increases RGS7 expression probably by direct transcriptional activity of STAT3

Tau protein is cross-linked by transglutaminase in P301L tau transgenic mice

This is the publisher's version, also available electronically from "http://www.jneurosci.org".The microtubule-associated protein tau is highly soluble under physiological conditions. However, in tauopathies, tau protein aggregates into insoluble filaments and neurofibrillary tangles (NFTs). The mechanisms underlying the formation of tau filaments and NFTs in tauopathies remain unclear. Several lines of evidence suggest that transglutaminase may cross-link tau into stable, insoluble aggregates, leading to the formation of NFTs in Alzheimer's disease and progressive supranuclear palsy. To further determine the contribution of transglutaminase in the formation of NFTs, we compared the levels of cross-linked tau protein from P301L tau transgenic mice that develop NFTs to four-repeat wild-type (4RWT) tau transgenic and nontransgenic mice that do not develop NFT pathology. Immunoprecipitation and immunoblotting experiments show that transglutaminase cross-links phosphorylated tau in the hindbrain of P301L tau transgenic mice but not in mice overexpressing 4RWT tau and nontransgenic mice. Cross-linked, phosphorylated tau from P301L tau transgenic mice runs as high-molecular mass aggregates on Western blots, similar to cross-linked tau from paired helical filaments of Alzheimer's disease. We also used double-label immunofluorescence to demonstrate colocalization of PHF-1-immunoreactive tau and the transglutaminase-catalyzed cross-link in the hindbrain, spinal cord, and cortex of P301L tau transgenic mice. In the spinal cord, 87% of PHF-1-labeled cells colocalize with the transglutaminase-catalyzed cross-link. Additionally, transglutaminase enzymatic activity is significantly elevated in the spinal cord of P301L tau transgenic mice. These studies further implicate transglutaminase in the formation and/or stabilization of NFT and paired helical filaments and provide a model system to investigate the therapeutic potential of transglutaminase inhibitors in tauopathies

GPER1 stimulation alters posttranslational modification of RGSz1 and induces desensitization of 5-HT1A receptor signaling in the rat hypothalamus

The final, published version of this article is available at http://www.karger.com/?doi=10.1159/000369467.Hyperactivity of the hypothalamic-pituitary-adrenal axis is a consistent biological characteristic of depression and response normalization coincides with clinical responsiveness to antidepressant medications. Desensitization of serotonin 1A receptor (5-HT1AR) signaling in the hypothalamic paraventricular nucleus (PVN) follows selective serotonin reuptake inhibitor (SSRI) antidepressant treatment and contributes to the antidepressant response. Estradiol alone produces a partial desensitization of 5-HT1AR signaling, and synergizes with SSRIs to result in a complete and more rapid desensitization than with SSRIs alone as measured by a decrease in the oxytocin and adrenocorticotrophic hormone(ACTH) responses to 5-HT1AR stimulation. G protein-coupled estrogen receptor1 (GPER1) is necessary for estradiol-induced desensitization of 5-HT1AR signaling, although the underlying mechanisms are still unclear. We now find that stimulation of GPER1 with the selective agonist G-1 and non-selective stimulation of estrogen receptors dramatically alter isoform expression of a key component of the 5-HT1AR signaling pathway, RGSz1, a GTPase activating protein selective for Gαz, the Gα subunit necessary for 5-HT1AR-mediated hormone release. RGSz1 isoforms are differentially glycosylated, SUMOylated, and phosphorylated, and differentially distributed in subcellular organelles. High molecular weight RGSz1 is SUMOylated and glycosylated, localized to the detergent-resistant microdomain (DRM) of the cell membrane, and increased by estradiol and G-1 treatment. Because activated Gαz also localizes to the DRM, increased DRM-localized RGSz1 by estradiol and G-1could reduce Gαz activity, functionally uncoupling 5-HT1AR signaling. Peripheral G-1 treatment produced partial reduction in oxytocin and ACTH responses to 5-HT1AR-stimulation similar to direct injections into the PVN. Together, these results identify GPER1 and RGSz1 as novel targets for the treatment of depression

Striatal expression of a calmodulin fragment improved motor function, weight loss and neuropathology in the R6/2 mouse model of Huntington's disease

This is the published version, also available here: http://dx.doi.org/10.1523/JNEUROSCI.3307-09.2009.Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, caused by a polyglutamine expansion in the huntingtin protein (htt). Increasing evidence suggests that transglutaminase (TGase) plays a critical role in the pathophysiology of HD possibly by stabilizing monomeric, polymeric and aggregated htt. We previously reported that in HEK293 and SH-SY5Y cells expression of a calmodulin (CaM)-fragment, consisting of amino acids 76-121 of CaM, decreased binding of CaM to mutant htt, TGase-modified htt and cytotoxicity associated with mutant htt and normalized intracellular calcium release. In this study, an adeno-associated virus (AAV) that expresses the CaM-fragment was injected into the striatum of HD transgenic R6/2 mice. The CaM-fragment significantly reduced body weight loss and improved motor function as indicated by improved rotarod performance, longer stride length, lower stride frequency, fewer low mobility bouts and longer travel distance than HD controls. A small but insignificant increase in survival was observed in R6/2 mice with CaM-fragment expression. Immunoprecipitation studies show that expression of the CaM-fragment reduced TGase-modified htt in the striatum of R6/2 mice. The percentage of htt-positive nuclei and the size of intranuclear htt aggregates were reduced by the CaM-fragment without striatal volume changes. The effects of CaM-fragment appear to be selective, as activity of another CaM-dependent enzyme, CaM-dependent kinase II, was not altered. Moreover, inhibition of TGase-modified htt was substrate-specific since overall TGase activity in the striatum was not altered by treatment with the CaM-fragment. Together, these results suggest that disrupting CaM–htt interaction may provide a new therapeutic strategy for HD

GPR30 is necessary for estradiol-induced desensitization of 5- HT1A receptor signaling in the paraventricular nucleus of the rat hypothalamus

Estrogen therapy used in combination with selective serotonin reuptake inhibitor (SSRI) treatment improves SSRI efficacy for the treatment of mood disorders. Desensitization of serotonin 1A (5-HT1A) receptors, which takes one to two weeks to develop in animals, is necessary for SSRI therapeutic efficacy. Estradiol modifies 5-HT1A receptor signaling and induces a partial desensitization in the paraventricular nucleus (PVN) of the rat within two days, but the mechanisms underlying this effect are currently unknown. The purpose of this study was to identify the estrogen receptor necessary for estradiol-induced 5-HT1A receptor desensitization. We previously showed that estrogen receptor β is not necessary for 5-HT1A receptor desensitization and that selective activation of estrogen receptor GPR30 mimics the effects of estradiol in rat PVN. Here, we used a recombinant adenovirus containing GPR30 siRNAs to decrease GPR30 expression in the PVN. Reduction of GPR30 prevented estradiol-induced desensitization of 5-HT1A receptor as measured by hormonal responses to the selective 5-HT1A receptor agonist, (+)8-OH-DPAT. To determine the possible mechanisms underlying these effects, we investigated protein and mRNA levels of 5-HT1A receptor signaling components including 5-HT1A receptor, Gαz, and RGSz1. We found that two days of estradiol increased protein and mRNA expression of RGSz1, and decreased 5-HT1A receptor protein but increased 5-HT1A mRNA; GPR30 knockdown prevented the estradiol-induced changes in 5-HT1A receptor protein in the PVN. Taken together, these data demonstrate that GPR30 is necessary for estradiol-induced changes in the 5-HT1A receptor signaling pathway and desensitization of 5-HT1A receptor signaling