Regulation of CRFR1 and 5-HT2AR by PDZ Domain-Containing Proteins SAP97 and PSD-95

Previous studies identified a crosstalk mechanism whereby CRFR1 sensitized 5-HT2AR-mediated signaling via interactions with PDZ domain-containing proteins: a mechanism that may underlie stress-induced anxiety and depression. This prompted an investigation into uncovering which PDZ domain-containing proteins could regulate the crosstalk between these two receptors, and how they could be regulated individually. In the current studies, a subset of PDZ domain-containing proteins were identified that may interact with CRFR1 and 5-HT2AR. The focus narrowed to two candidates previously implicated in psychiatric disease: SAP97 and PSD-95. We confirmed SAP97 and PSD-95 as interacting partners of CRFR1 in adult mouse cortex via co-immunoprecipitation. Both proteins exhibited functional regulation of CRFR1 by antagonizing CRFR1 endocytosis in HEK293 cells, measured by flow cytometry. Additionally, PSD-95 suppressed β-arrestin2 recruitment, thereby providing a potential mechanism for antagonizing endocytosis. Although neither SAP97 nor PSD-95 appeared to play a significant role in CRFR1-mediated cAMP signaling, endogenous SAP97 was integral for CRF-mediated ERK1/2 phosphorylation in HEK293 and AtT20 cells. Despite extensive sequence homology between SAP97 and PSD-95, PSD-95 did not appear to play a significant role in CRF-mediated ERK1/2 phosphorylation. Thus, we begin to understand subtle signaling biases between these two proteins. As PSD-95 was already documented to regulate 5-HT2AR, we investigated if SAP97 could play a role in regulating 5-HT2AR function. The interaction between SAP97 and 5-HT2AR was confirmed in adult mouse cortex. As was seen with CRFR1, SAP97 antagonized 5-HT2AR endocytosis. Although SAP97 did not appear to significantly modulate Gs-coupled signaling via CRFR1, the endogenous expression of SAP97 was integral for maximal Gq-coupled signaling via 5-HT2AR. Endogenous SAP97 was also required for ERK1/2 phosphorylation, and this regulatory role appears to be downstream of receptor interactions. Finally, we were unable to prevent the CRFR1-mediated sensitization of 5-HT2AR-mediated signaling by knocking down either SAP97 or PSD-95 using shRNA. Therefore, neither SAP97 nor PSD-95 appear to be exclusively involved in this heterologous crosstalk mechanism. Nevertheless, we have identified SAP97 and PSD-95 as novel regulators of CRFR1 function, and SAP97 as a novel regulator of 5-HT2AR function. These functional interactions may be targeted for the treatment of CRFR1- and 5-HT2AR-mediated mood disorders

Alien Registration- Dunn, Henry A. (Dexter, Penobscot County)

https://digitalmaine.com/alien_docs/11388/thumbnail.jp

“Mirror, mirror…” A preliminary investigation of skin tone dissatisfaction and its impact among British adults

This study examined skin tone dissatisfaction, measured using a skin tone chart, among a multiethnic sample of British adults. A total of 648 British White individuals, 292 British South Asians, and 260 British African Caribbean participants completed a visual task in which they were asked to indicate their actual and ideal skin tones. They also completed measures of body appreciation, self-esteem, and ethnic identity attachment. Results showed that Asians had a lighter skin tone ideal than White and African Caribbean participants. Conversely, White participants had higher skin tone dissatisfaction (preferring a darker skin tone) than Asian and African Caribbean participants, who preferred a lighter skin tone. Results also showed that skin tone dissatisfaction predicted body appreciation once the effects of participant ethnicity, age, ethnic identity attachment, and self-esteem had been accounted for. Implications of our findings and suggestions for future research are discussed

Inactivation of cloned Na channels expressed in Xenopus oocytes

This study investigates the inactivation properties of Na channels expressed in Xenopus oocytes from two rat IIA Na channel cDNA clones differing by a single amino acid residue. Although the two cDNAs encode Na channels with substantially different activation properties (Auld, V. J., A. L. Goldin, D. S. Krafte, J. Marshall, J. M. Dunn, W. A. Catterall, H. A. Lester, N. Davidson, and R. J. Dunn. 1988. Neuron. 1:449-461), their inactivation properties resemble each other strongly but differ markedly from channels induced by poly(A+) rat brain RNA. Rat IIA currents inactivate more slowly, recover from inactivation more slowly, and display a steady-state voltage dependence that is shifted to more positive potentials. The macroscopic inactivation process for poly(A+) Na channels is defined by a single exponential time course; that for rat IIA channels displays two exponential components. At the single-channel level these differences in inactivation occur because rat IIA channels reopen several times during a depolarizing pulse; poly(A+) channels do not. Repetitive stimulation (greater than 1 Hz) produces a marked decrement in the rat IIA peak current and changes the waveform of the currents. When low molecular weight RNA is coinjected with rat IIA RNA, these inactivation properties are restored to those that characterize poly(A+) channels. Slow inactivation is similar for rat IIA and poly(A+) channels, however. The data suggest that activation and inactivation involve at least partially distinct regions of the channel protein

Transient band keratopathy associated with ocular inflammation and systemic hypercalcemia

Anat Galor, Henry A Leder, Jennifer E Thorne, James P DunnThe Wilmer Eye Institute, Department of Ophthalmology, the Johns Hopkins University School of MedicinePurpose: To report a case of visually significant band keratopathy associated with ocular inflammation and systemic hypercalcemia which markedly decreased in severity after treatment of these underlying factors.Methods: Retrospective case report.Results: A 53-year-old Asian female with granulomatous panuveitis in the left eye presented with diffuse band keratopathy through the central cornea. The serum calcium was elevated. The patient was treated with topical prednisolone acetate 1% and oral prednisone with marked improvement in inflammation. The band keratopathy lessened in severity with clearing of the central cornea and improvement in visual acuity.Conclusions: Early medical treatment of underlying factors may allow reversal of band keratopathy.Keywords: ocular inflammation, transient band keratopath

Suppression of piriform cortex activity in rat by corticotropin-releasing factor 1 and serotonin 2A/C receptors

The piriform cortex (PC) is richly innervated by Corticotropin-releasing factor (CRF) and Serotonin (5-HT) containing axons arising from central amygdala and Raphe nucleus. CRFR1 and 5-HT2A/2CRs have been shown to interact in manner where CRFR activation subsequently potentiates the activity of 5-HT2A/2CRs. The purpose of this study was to determine how the activation of CRFR1 and/or 5-HT2Rs modulates PC activity at both the circuit and cellular level. Voltage sensitive dye imaging showed that CRF acting through CRFR1 dampened activation of the layer II of PC and interneurons of endopiriform nucleus. Application of the selective 5-HT2A/CR agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) following CRFR1 activation potentiated this effect. Blocking the interaction between CRFR1 and 5-HT2R with a Tat-CRFR1-CT peptide abolished this potentiation. Application of forskolin did not mimic CRFR1 activity but instead blocked it, while a protein kinase A antagonist had no effect. However, activation and antagonism of protein kinase C (PKC) either mimicked or blocked CRF modulation respectively. DOI had no effect when applied alone indicating that the prior activation of CRFR1 receptors was critical for DOI to show significant effects similar to CRF. Patch clamp recordings showed that both CRF and DOI reduced the synaptic responsiveness of layer II pyramidal neurons. CRF had highly variable effects on interneurons within layer III, both increasing and decreasing their excitability, but DOI had no effect on the excitability of this group of neurons. These data show that CRF and serotonin, acting through both CRFR1 and 5-HT2A/CRs, reduce the activation of the PC. This modulation may be an important blunting mechanism of stressor behaviours mediated through the olfactory cortex

Messenger RNA coding for only the alpha subunit of the rat brain Na channel is sufficient for expression of functional channels in Xenopus oocytes

Several cDNA clones coding for the high molecular weight (alpha) subunit of the voltage-sensitive Na channel have been selected by immunoscreening a rat brain cDNA library constructed in the expression vector lambda gt11. As will be reported elsewhere, the amino acid sequence translated from the DNA sequence shows considerable homology to that reported for the Electrophorus electricus electroplax Na channel. Several of the cDNA inserts hybridized with a low-abundance 9-kilobase RNA species from rat brain, muscle, and heart. Sucrose-gradient fractionation of rat brain poly(A) RNA yielded a high molecular weight fraction containing this mRNA, which resulted in functional Na channels when injected into oocytes. This fraction contained undetectable amounts of low molecular weight RNA. The high molecular weight Na channel RNA was selected from rat brain poly(A) RNA by hybridization to a single-strand antisense cDNA clone. Translation of this RNA in Xenopus oocytes resulted in the appearance of tetrodotoxin-sensitive voltage-sensitive Na channels in the oocyte membrane. These results demonstrate that mRNA encoding the alpha subunit of the rat brain Na channel, in the absence of any beta-subunit mRNA, is sufficient for translation to give functional channels in oocytes

IRG and GBP host resistance factors target aberrant, ‘‘Non-self’’ vacuoles characterized by the missing of ‘‘Self’’ IRGM proteins

Interferon-inducible GTPases of the Immunity Related GTPase (IRG) and Guanylate Binding Protein (GBP) families provide resistance to intracellular pathogenic microbes. IRGs and GBPs stably associate with pathogen-containing vacuoles (PVs) and elicit immune pathways directed at the targeted vacuoles. Targeting of Interferon-inducible GTPases to PVs requires the formation of higher-order protein oligomers, a process negatively regulated by a subclass of IRG proteins called IRGMs. We found that the paralogous IRGM proteins Irgm1 and Irgm3 fail to robustly associate with ‘‘non-self’’ PVs containing either the bacterial pathogen Chlamydia trachomatis or the protozoan pathogen Toxoplasma gondii. Instead, Irgm1 and Irgm3 reside on ‘‘self’’ organelles including lipid droplets (LDs). Whereas IRGM-positive LDs are guarded against the stable association with other IRGs and GBPs, we demonstrate that IRGM-stripped LDs become high affinity binding substrates for IRG and GBP proteins. These data reveal that intracellular immune recognition of organelle-like structures by IRG and GBP proteins is partly dictated by the missing of ‘‘self’’ IRGM proteins from these structures.Fil: Haldar, Arun K.. University Of Duke; Estados UnidosFil: Saka, Hector Alex. University Of Duke; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Piro, Anthony S.. University Of Duke; Estados UnidosFil: Dunn, Joe Dan. University Of Duke; Estados UnidosFil: Henry, Stanley C.. University Of Duke; Estados Unidos. Veteran Affairs Medical Center; Estados UnidosFil: Taylor, Gregory A.. University Of Duke; Estados Unidos. Veteran Affairs Medical Center; Estados UnidosFil: Frickel, Eva M.. National Institute for Medical Research; Reino UnidoFil: Valdivia, Raphael H.. University Of Duke; Estados UnidosFil: Coers, Jörn. University Of Duke; Estados Unido

Role of Spinophilin in Group I Metabotropic Glutamate Receptor Endocytosis, Signaling, and Synaptic Plasticity

Activation of Group I metabotropic glutamate receptors (mGluRs) activates signaling cascades, resulting in calcium release from intracellular stores, ERK1/2 activation, and long term changes in synaptic activity that are implicated in learning, memory, and neurodegenerative diseases. As such, elucidating the molecular mechanisms underlying Group I mGluR signaling is important for understanding physiological responses initiated by the activation of these receptors. In the current study, we identify the multifunctional scaffolding protein spinophilin as a novel Group I mGluR-interacting protein. We demonstrate that spinophilin interacts with the C-terminal tail and second intracellular loop of Group I mGluRs. Furthermore, we show that interaction of spinophilin with Group I mGluRs attenuates receptor endocytosis and phosphorylation of ERK1/2, an effect that is dependent upon the interaction of spinophilin with the C-terminal PDZ binding motif encoded by Group I mGluRs. Spinophilin knock-out results in enhanced mGluR5 endocytosis as well as increased ERK1/2, AKT, and Ca2+ signaling in primary cortical neurons. In addition, the loss of spinophilin expression results in impaired mGluR5-stimulated LTD. Our results indicate that spinophilin plays an important role in regulating the activity of Group I mGluRs as well as their influence on synaptic activity

Extreme AGN Feedback and Cool Core Destruction in the X-ray Luminous Galaxy Cluster MACS J1931.8-2634

We report on a deep, multiwavelength study of the galaxy cluster MACS J1931.8-2634 using Chandra X-ray, Subaru optical, and VLA 1.4 GHz radio data. This cluster (z=0.352) harbors one of the most X-ray luminous cool cores yet discovered, with an equivalent mass cooling rate within the central 50 kpc is approximately 700 solar masses/yr. Unique features observed in the central core of MACSJ1931.8-2634 hint to a wealth of past activity that has greatly disrupted the original cool core. We observe a spiral of relatively cool, dense, X-ray emitting gas connected to the cool core, as well as highly elongated intracluster light (ICL) surrounding the cD galaxy. Extended radio emission is observed surrounding the central AGN, elongated in the east-west direction, spatially coincident with X-ray cavities. The power input required to inflate these `bubbles' is estimated from both the X-ray and radio emission to reside between 4 and 14e45 erg/s, putting it among the most powerful jets ever observed. This combination of a powerful AGN outburst and bulk motion of the cool core have resulted in two X-ray bright ridges to form to the north and south of the central AGN at a distance of approximately 25 kpc. The northern ridge has spectral characteristics typical of cool cores and is consistent with being a remnant of the cool core after it was disrupted by the AGN and bulk motions. It is also the site of H-alpha filaments and young stars. The X-ray spectroscopic cooling rate associated with this ridge is approximately 165 solar masses/yr, which agrees with the estimate of the star formation rate from broad-band optical imaging (170 solar masses/yr). MACS J1931.8-2634 appears to harbor one of most profoundly disrupted low entropy cores observed in a cluster, and offers new insights into the survivability of cool cores in the context of hierarchical structure formation.Comment: 19 pages, 15 figures, 5 tables. Accepted by MNRAS for publication September 30 201