mGluR5 hypofunction is integral to glutamatergic dysregulation in schizophrenia.

Multiple lines of evidence point to glutamatergic signaling in the postsynaptic density (PSD) as a pathophysiologic mechanism in schizophrenia. Integral to PSD glutamatergic signaling is reciprocal interplay between GluN and mGluR5 signaling. We examined agonist-induced mGluR5 signaling in the postmortem dorsolateral prefrontal cortex (DLPFC) derived from 17 patients and age-matched and sex-matched controls. The patient group showed a striking reduction in mGluR5 signaling, manifested by decreases in Gq/11 coupling and association with PI3K and Homer compared to controls (p < 0.01 for all). This was accompanied by increases in serine and tyrosine phosphorylation of mGluR5, which can decrease mGluR5 activity via desensitization (p < 0.01). In addition, we find altered protein-protein interaction (PPI) of mGluR5 with RGS4, norbin, Preso 1 and tamalin, which can also attenuate mGluR5 activity. We previously reported molecular underpinnings of GluN hypofunction (decreased GluN2 phosphorylation) and here we show those of reduced mGluR5 signaling in schizophrenia. We find that reduced GluN2 phosphorylation can be precipitated by attenuated mGluR5 activity and that increased mGluR5 phosphorylation can result from decreased GluN function, suggesting a reciprocal interplay between the two pathways in schizophrenia. Interestingly, the patient group showed decreased mGluR5-GluN association (p < 0.01), a mechanistic basis for the reciprocal facilitation. In sum, we present the first direct evidence for mGluR5 hypoactivity, propose a reciprocal interplay between GluN and mGluR5 pathways as integral to glutamatergic dysregulation and suggest protein-protein interactions in mGluR5-GluN complexes as potential targets for intervention in schizophrenia.

Gradient doping - a case study with Ti-Fe2O3 towards an improved photoelectrochemical response.

The present study investigates the effect of gradient doping on modifying the photoelectrochemical response of Ti-doped Fe2O3 photoanodes for their use in sunlight based water splitting for hydrogen evolution. The deposition of a thin film over the ITO (tin doped indium oxide) substrate was carried out using a spray pyrolysis method. The concentration of dopant was varied from 0.5-8.0 at% and two sets of samples were also prepared with low to high (0.5-8%) and high to low (8-0.5%) dopant concentrations in the direction towards the substrate. The prepared thin films were characterized using X-ray Diffractometry (XRD), Scanning Electron Microscopy (SEM), Energy Dispersive X-ray (EDX) Spectroscopy, Secondary Ion Mass Spectroscopy (SIMS), X-ray Photoelectron Spectroscopy (XPS) and UV-visible Spectroscopy. The photoelectrochemical studies revealed that the deposition of dopant layers with a low to high concentration towards the substrate exhibited a highly improved photoresponse (200 times) in comparison to the pristine sample and a two fold enhancement in comparison to 2% Ti-doped Fe2O3. The improvement in the photoresponse has been attributed to the values of a high flat band potential, low resistance, high open circuit voltage, carrier separation efficiency, applied bias photon-to-current conversion efficiency (ABPE), and incident photon-to-current conversion efficiency (IPCE). A reduced charge transfer resistance has been demonstrated with Nyquist plots.

Biochemical fractionation and stable isotope dilution liquid chromatography-mass spectrometry for targeted and microdomain-specific protein quantification in human postmortem brain tissue.

Synaptic architecture and its adaptive changes require numerous molecular events that are both highly ordered and complex. A majority of neuropsychiatric illnesses are complex trait disorders, in which multiple etiologic factors converge at the synapse via many signaling pathways. Investigating the protein composition of synaptic microdomains from human patient brain tissues will yield valuable insights into the interactions of risk genes in many disorders. These types of studies in postmortem tissues have been limited by the lack of proper study paradigms. Thus, it is necessary not only to develop strategies to quantify protein and post-translational modifications at the synapse, but also to rigorously validate them for use in postmortem human brain tissues. In this study we describe the development of a liquid chromatography-selected reaction monitoring method, using a stable isotope-labeled neuronal proteome standard prepared from the brain tissue of a stable isotope-labeled mouse, for the multiplexed quantification of target synaptic proteins in mammalian samples. Additionally, we report the use of this method to validate a biochemical approach for the preparation of synaptic microdomain enrichments from human postmortem prefrontal cortex. Our data demonstrate that a targeted mass spectrometry approach with a true neuronal proteome standard facilitates accurate and precise quantification of over 100 synaptic proteins in mammalian samples, with the potential to quantify over 1000 proteins. Using this method, we found that protein enrichments in subcellular fractions prepared from human postmortem brain tissue were strikingly similar to those prepared from fresh mouse brain tissue. These findings demonstrate that biochemical fractionation methods paired with targeted proteomic strategies can be used in human brain tissues, with important implications for the study of neuropsychiatric disease.

Characterization of polycyclic aromatic hydrocarbons in emissions of different mosquito coils.

Emission rates and emission factors of particulate matter and polycyclic aromatic hydrocarbons (PAHs) were determined in the emissions of three prevalent mosquito coil brands available in the market in India, under controlled conditions in an environmental chamber. The emission rates varied between the brands and ranged between 2.0 ± 1.9 to 27.1 ± 1.5 µg h(-1) for brand A, 3.4 ± 1.1 to 32.3 ± 1.2 µg h(-1) for brand B and 3.1 ± 1.0 to 72.2 ± 1.1 µg h(-1) for brand C for different compounds. Five and six ring PAHs were not detected, however four and three ring PAHs dominated. Three ring PAHs contributed 24 %, 42 % and 29 %, while four ring compounds contributed 53 %, 35 % and 61 % to total PAHs in the three brands. Carcinogenic PAH compounds were also present in the emissions, indicating the potential risks associated with the inhalation of coil smoke.

Blocking Src-PSD-95 interaction rescues glutamatergic signaling dysregulation in schizophrenia.

The complex and heterogeneous genetic architecture of schizophrenia inspires us to look beyond individual risk genes for therapeutic strategies and target their interactive dynamics and convergence. Postsynaptic NMDA receptor (NMDAR) complexes are a site of such convergence. Src kinase is a molecular hub of NMDAR function, and its protein interaction subnetwork is enriched for risk-genes and altered protein associations in schizophrenia. Previously, Src activity was found to be decreased in post-mortem studies of schizophrenia, contributing to NMDAR hypofunction. PSD-95 suppresses Src via interacting with its SH2 domain. Here, we devised a strategy to suppress the inhibition of Src by PSD-95 via employing a cell penetrating and Src activating PSD-95 inhibitory peptide (TAT-SAPIP). TAT-SAPIP selectively increased post-synaptic Src activity in humans and mice, and enhanced synaptic NMDAR currents in mice. Chronic ICV injection of TAT-SAPIP rescued deficits in trace fear conditioning in Src hypomorphic mice. We propose blockade of the Src-PSD-95 interaction as a proof of concept for the use of interfering peptides as a therapeutic strategy to reverse NMDAR hypofunction in schizophrenia and other illnesses.

Evaluation of 2,6-difluoro-3-(oxazol-2-ylmethoxy)benzamide chemotypes as Gram-negative FtsZ inhibitors.

FtsZ inhibitors represent a new drug class as no drugs using this mode of action (MOA) have been approved by regulators. 3-alkoxy substituted 2,6-difluorobenzamide scaffold is one of the most studied FtsZ inhibitors among which the most promising anti-MRSA candidate TXA709 is in clinical trial. In this paper, we present the screening and evaluation of a benzamide class that is functionalized at the alkoxy fragment targeting Gram-negative bacteria. The variations in 3-alkoxy substitutions, specifically the hydroxylated alkyl residues to the secondary and stereogenic pseudo-benzylic carbon of their methyleneoxy linker, are particularly active against K. pneumoniae ATCC 10031 in marked contrast to the derivatives related to PC190723, all of which were inactive against Gram-negative bacteria. The two lead molecules TXA6101 and TXY6129 inhibit the polymerization of E. coli FtsZ in a concentration-dependent manner and induce changes in the morphology of E. coli and K. pneumoniae consistent with inhibition of cell division. These classes of compounds, however, were found to be substrates for efflux pumps in Gram-negative bacteria.

Evaluation of a Conformationally Constrained Indole Carboxamide as a Potential Efflux Pump Inhibitor in Pseudomonas aeruginosa

Efflux pumps in Gram-negative bacteria such as Pseudomonas aeruginosa provide intrinsic antimicrobial resistance by facilitating the extrusion of a wide range of antimicrobials. Approaches for combating efflux-mediated multidrug resistance involve, in part, developing indirect antimicrobial agents capable of inhibiting efflux, thus rescuing the activity of antimicrobials previously rendered inactive by efflux. Herein, TXA09155 is presented as a novel efflux pump inhibitor (EPI) formed by conformationally constraining our previously reported EPI TXA01182. TXA09155 demonstrates strong potentiation in combination with multiple antibiotics with efflux liabilities against wild-type and multidrug-resistant (MDR) P. aeruginosa. At 6.25 µg/mL, TXA09155, showed ≥8-fold potentiation of levofloxacin, moxifloxacin, doxycycline, minocycline, cefpirome, chloramphenicol, and cotrimoxazole. Several biophysical and genetic studies rule out membrane disruption and support efflux inhibition as the mechanism of action (MOA) of TXA09155. TXA09155 was determined to lower the frequency of resistance (FoR) to levofloxacin and enhance the killing kinetics of moxifloxacin. Most importantly, TXA09155 outperformed the levofloxacin-potentiation activity of EPIs TXA01182 and MC-04,124 against a CDC/FDA panel of MDR clinical isolates of P. aeruginosa. TXA09155 possesses favorable physiochemical and ADME properties that warrant its optimization and further development.

Diagnostic dilemma of Hodgkin's lymphoma versus tuberculosis: a case report and review of the literature.

BACKGROUND: Hodgkin's Lymphoma (HL) is a rare malignancy characterised histologically by the presence of Reed-Sternberg cells. Diagnosis of lymphomas can be difficult due to broad, non-specific presentations of disease, which can be similar to several other conditions ranging from infective, inflammatory or malignant causes, with one of the most common differentials being tuberculosis (TB). We aim to highlight the diagnostic dilemma of TB versus lymphoma with an atypical presentation of HL and explored areas for further research and improvement with a non-systematic literature review using MEDLINE database and Google Scholar. Written consent was obtained from the patient in compliance with ethical guidelines. CASE PRESENTATION: A 23-year-old Asian female initially presented to rheumatology with over a one-year history of neuropathic pain, alongside abnormal white cell count and inflammatory markers. This was investigated with magnetic resonance imaging resulting in an incidental finding of mediastinal mass and pulmonary infiltrates. An initial diagnosis of TB was made despite testing negative for acid-fast bacilli and anti-tubercular treatment was commenced. Four months later, following clinical deterioration and further investigations, a mediastinal biopsy assisted in diagnosing Stage IV HL. CONCLUSIONS: Lymphoma is often misdiagnosed as TB, prolonging time to treatment and may adversely impact patient prognosis due to disease progression. Existing TB guidelines for smear-negative cases are not clear when to consider alternative diagnoses. In smear-negative TB, lymphoma should be considered as a differential and definitive diagnostic tests such as molecular testing and histological examination of biopsies should be considered earlier in the diagnostic work-up to prevent diagnostic delay.

Evaluation of Heterocyclic Carboxamides as Potential Efflux Pump Inhibitors in Pseudomonas aeruginosa.

The ability to rescue the activity of antimicrobials that are no longer effective against bacterial pathogens such as Pseudomonas aeruginosa is an attractive strategy to combat antimicrobial drug resistance. Herein, novel efflux pump inhibitors (EPIs) demonstrating strong potentiation in combination with levofloxacin against wild-type P. aeruginosa ATCC 27853 are presented. A structure activity relationship of aryl substituted heterocyclic carboxamides containing a pentane diamine side chain is described. Out of several classes of fused heterocyclic carboxamides, aryl indole carboxamide compound 6j (TXA01182) at 6.25 µg/mL showed 8-fold potentiation of levofloxacin. TXA01182 was found to have equally synergistic activities with other antimicrobial classes (monobactam, fluoroquinolones, sulfonamide and tetracyclines) against P. aeruginosa. Several biophysical and genetic studies rule out membrane disruption and support efflux inhibition as the mechanism of action (MOA) of TXA01182. TXA01182 was determined to lower the frequency of resistance (FoR) of the partner antimicrobials and enhance the killing kinetics of levofloxacin. Furthermore, TXA01182 demonstrated a synergistic effect with levofloxacin against several multidrug resistant P. aeruginosa clinical isolates.

Transcriptional corepressor SIN3A regulates hippocampal synaptic plasticity via Homer1/mGluR5 signaling.

Long-term memory depends on the control of activity-dependent neuronal gene expression, which is regulated by epigenetic modifications. The epigenetic modification of histones is orchestrated by the opposing activities of 2 classes of regulatory complexes: permissive coactivators and silencing corepressors. Much work has focused on coactivator complexes, but little is known about the corepressor complexes that suppress the expression of plasticity-related genes. Here, we define a critical role for the corepressor SIN3A in memory and synaptic plasticity, showing that postnatal neuronal deletion of Sin3a enhances hippocampal long-term potentiation and long-term contextual fear memory. SIN3A regulates the expression of genes encoding proteins in the postsynaptic density. Loss of SIN3A increases expression of the synaptic scaffold Homer1, alters the metabotropic glutamate receptor 1α (mGluR1α) and mGluR5 dependence of long-term potentiation, and increases activation of ERK in the hippocampus after learning. Our studies define a critical role for corepressors in modulating neural plasticity and memory consolidation and reveal that Homer1/mGluR signaling pathways may be central molecular mechanisms for memory enhancement.

Novel environment and GABA agonists alter event-related potentials in N-methyl-D-aspartate NR1 hypomorphic and wild-type mice.

Clinical and experimental data suggest dysregulation of N-methyl-d-aspartate receptor (NMDAR)-mediated glutamatergic pathways in schizophrenia. The interaction between NMDAR-mediated abnormalities and the response to novel environment has not been studied. Mice expressing 5 to 10% of normal N-methyl-d-aspartate receptor subunit 1 (NR1) subunits [NR1(neo)(-/-)] were compared with wild-type littermates for positive deflection at 20 ms (P20) and negative deflection at 40 ms (N40) auditory event-related potentials (ERPs). Groups were tested for habituation within and across five testing sessions, with novel environment tested during a sixth session. Subsequently, we examined the effects of a GABA(A) positive allosteric modulator (chlordiazepoxide) and a GABA(B) receptor agonist (baclofen) as potential interventions to normalize aberrant responses. There was a reduction in P20, but not N40 amplitude within each habituation day. Although there was no amplitude or gating change across habituation days, there was a reduction in P20 and N40 amplitude and gating in the novel environment. There was no difference between genotypes for N40. Only NR1(neo)(-/-) mice had reduced P20 in the novel environment. Chlordiazepoxide increased N40 amplitude in wild-type mice, whereas baclofen increased P20 amplitude in NR1(neo)(-/-) mice. As noted in previous publications, the pattern of ERPs in NR1(neo)(-/-) mice does not recapitulate abnormalities in schizophrenia. In addition, reduced NR1 expression does not influence N40 habituation but does affect P20 in a novel environment. Thus, the pattern of P50 (positive deflection at 50 ms) but not N100 (negative deflection at 100 ms) in human studies may relate to subjects' reactions to unfamiliar environments. In addition, NR1 reduction decreased GABA(A) receptor-mediated effects on ERPs while causing increased GABA(B) receptor-mediated effects. Future studies will examine changes in GABA receptor subunits after reductions in NR1 expression.

Src deficient mice demonstrate behavioral and electrophysiological alterations relevant to psychiatric and developmental disease.

Much evidence suggests that hypofunction of the N-methyl-d-aspartate glutamate receptor (NMDAR) may contribute broadly towards a subset of molecular, cognitive and behavioral abnormalities common among psychiatric and developmental diseases. However, little is known about the specific molecular changes that lead to NMDAR dysfunction. As such, personalized approaches to remediating NMDAR dysfunction based on a specific etiology remains a challenge. Sarcoma tyrosine kinase (Src) serves as a hub for multiple signaling mechanisms affecting GluN2 phosphorylation and can be disrupted by convergent alterations of various signaling pathways. We recently showed reduced Src signaling in post mortem tissue from schizophrenia patients, despite increased MK-801 binding and NMDA receptor complex expression in the postsynaptic density (PSD). These data suggest that Src dysregulation may be an important underlying mechanism responsible for reduced glutamate signaling. Despite this evidence for a central role of Src in NMDAR signaling, little is known about how reductions in Src activity might regulate phenotypic changes in cognition and behavior. As such, the current study sought to characterize behavioral and electrophysiological phenotypes in mice heterozygous for the Src Acl gene (Src+/- mice). Src+/- mice demonstrated decreased sociability and working memory relative to Src+/+ (WT) mice while no significant differences were seen on locomotive activity and anxiety-related behavior. In relation to WT mice, Src+/- mice showed decreased mid-latency P20 auditory event related potential (aERP) amplitudes, decreased mismatch negativity (MMN) and decreased evoked gamma power, which was only present in males. These data indicate that Src+/- mice are a promising new model to help understand the pathophysiology of these electrophysiological, behavioral and cognitive changes. As such, we propose that Src+/- mice can be used in the future to evaluate potential therapeutic approaches by targeting increased Src activity as a common final pathway for multiple etiologies of SCZ and other diseases characterized by reduced glutamate function.

Ligand-independent pathway that controls stability of interferon alpha receptor.

Ligand-specific negative regulation of cytokine-induced signaling relies on down regulation of the cytokine receptors. Down regulation of the IFNAR1 sub-unit of the Type I interferon (IFN) receptor proceeds via lysosomal receptor proteolysis, which is triggered by ubiquitination that depends on IFNAR1 serine phosphorylation. While IFN-inducible phosphorylation, ubiquitination, and degradation requires the catalytic activity of the Tyk2 Janus kinase, here we found the ligand- and Tyk2-independent pathway that promotes IFNAR1 phosphorylation, ubiquitination, and degradation when IFNAR1 is expressed at high levels. A major cellular kinase activity that is responsible for IFNAR1 phosphorylation in vitro does not depend on either ligand or Tyk2 activity. Inhibition of ligand-independent IFNAR1 degradation suppresses cell proliferation. We discuss the signaling events that might lead to ubiquitination and degradation of IFNAR1 via ligand-dependent and independent pathways and their potential physiologic significance.

RNA Interference: A Novel Source of Resistance to Combat Plant Parasitic Nematodes.

Plant parasitic nematodes cause severe damage and yield loss in major crops all over the world. Available control strategies include use of insecticides/nematicides but these have proved detrimental to the environment, while other strategies like crop rotation and resistant cultivars have serious limitations. This scenario provides an opportunity for the utilization of technological advances like RNA interference (RNAi) to engineer resistance against these devastating parasites. First demonstrated in the model free living nematode, Caenorhabtidis elegans; the phenomenon of RNAi has been successfully used to suppress essential genes of plant parasitic nematodes involved in parasitism, nematode development and mRNA metabolism. Synthetic neurotransmitants mixed with dsRNA solutions are used for in vitro RNAi in plant parasitic nematodes with significant success. However, host delivered in planta RNAi has proved to be a pioneering phenomenon to deliver dsRNAs to feeding nematodes and silence the target genes to achieve resistance. Highly enriched genomic databases are exploited to limit off target effects and ensure sequence specific silencing. Technological advances like gene stacking and use of nematode inducible and tissue specific promoters can further enhance the utility of RNAi based transgenics against plant parasitic nematodes.

Sociability Deficits and Altered Amygdala Circuits in Mice Lacking Pcdh10, an Autism Associated Gene.

BACKGROUND: Behavioral symptoms in individuals with autism spectrum disorder (ASD) have been attributed to abnormal neuronal connectivity, but the molecular bases of these behavioral and brain phenotypes are largely unknown. Human genetic studies have implicated PCDH10, a member of the δ2 subfamily of nonclustered protocadherin genes, in ASD. PCDH10 expression is enriched in the basolateral amygdala, a brain region implicated in the social deficits of ASD. Previous reports indicate that Pcdh10 plays a role in axon outgrowth and glutamatergic synapse elimination, but its roles in social behaviors and amygdala neuronal connectivity are unknown. We hypothesized that haploinsufficiency of Pcdh10 would reduce social approach behavior and alter the structure and function of amygdala circuits. METHODS: Mice lacking one copy of Pcdh10 (Pcdh10+/-) and wild-type littermates were assessed for social approach and other behaviors. The lateral/basolateral amygdala was assessed for dendritic spine number and morphology, and amygdala circuit function was studied using voltage-sensitive dye imaging. Expression of Pcdh10 and N-methyl-D-aspartate receptor (NMDAR) subunits was assessed in postsynaptic density fractions of the amygdala. RESULTS: Male Pcdh10+/- mice have reduced social approach behavior, as well as impaired gamma synchronization, abnormal spine morphology, and reduced levels of NMDAR subunits in the amygdala. Social approach deficits in Pcdh10+/- male mice were rescued with acute treatment with the NMDAR partial agonist d-cycloserine. CONCLUSIONS: Our studies reveal that male Pcdh10+/- mice have synaptic and behavioral deficits, and establish Pcdh10+/- mice as a novel genetic model for investigating neural circuitry and behavioral changes relevant to ASD.

Pyramidal cell selective ablation of N-methyl-D-aspartate receptor 1 causes increase in cellular and network excitability.

BACKGROUND: Neuronal activity at gamma frequency is impaired in schizophrenia (SZ) and is considered critical for cognitive performance. Such impairments are thought to be due to reduced N-methyl-D-aspartate receptor (NMDAR)-mediated inhibition from parvalbumin interneurons, rather than a direct role of impaired NMDAR signaling on pyramidal neurons. However, recent studies suggest a direct role of pyramidal neurons in regulating gamma oscillations. In particular, a computational model has been proposed in which phasic currents from pyramidal cells could drive synchronized feedback inhibition from interneurons. As such, impairments in pyramidal neuron activity could lead to abnormal gamma oscillations. However, this computational model has not been tested experimentally and the molecular mechanisms underlying pyramidal neuron dysfunction in SZ remain unclear. METHODS: In the present study, we tested the hypothesis that SZ-related phenotypes could arise from reduced NMDAR signaling in pyramidal neurons using forebrain pyramidal neuron specific NMDA receptor 1 knockout mice. RESULTS: The mice displayed increased baseline gamma power, as well as sociocognitive impairments. These phenotypes were associated with increased pyramidal cell excitability due to changes in inherent membrane properties. Interestingly, mutant mice showed decreased expression of GIRK2 channels, which has been linked to increased neuronal excitability. CONCLUSIONS: Our data demonstrate for the first time that NMDAR hypofunction in pyramidal cells is sufficient to cause electrophysiological, molecular, neuropathological, and behavioral changes related to SZ.

Neuregulin 1-erbB4 pathway in schizophrenia: From genes to an interactome.

Recently identified candidate susceptibility genes for schizophrenia are likely to play, important roles in the pathophysiology of the illness. It is also clear, however, that the etiologic, contribution of these genes is not only via their own functions but also through interactions with other, genes and environmental factors. Genetic, transgenic and postmortem brain studies support a, potential role for NRG1-erbB4 signaling in schizophrenia. Embedded in the results of these studies, however, are clues to the notion that NRG1-erbB4 signaling does not act alone but in conjunction with, other pathways. This article aims to re-evaluate the evidence for the role of neuregulin 1 (NRG1)-erbB4 signaling in schizophrenia by focusing on its interactions with other candidate susceptibility, pathways. In addition, we consider molecular substrates upon which the NRG1-erbB4 and other, candidate pathways converge contributing to susceptibility for the illness (schizophrenia interactome). Glutamatergic signaling can be an interesting candidate for schizophrenia interactome. Schizophrenia is associated with NMDA receptor hypofunction and moreover, several susceptibility genes for, schizophrenia converge on NMDA receptor signaling. These candidate genes influence NMDA receptor, signaling via diverse mechanisms, yet all eventually impact on protein composition of NMDA receptor, complexes. Likewise, the protein associations in the receptor complexes can themselves modulate, signaling molecules of candidate genes and their pathways. Therefore, protein-protein interactions in the NMDA receptor complexes can mediate reciprocal interactions between NMDA receptor function, and susceptibility candidate pathways including NRG1-erbB4 signaling and thus can be a, schizophrenia interactome.

The post-synaptic density of human postmortem brain tissues: an experimental study paradigm for neuropsychiatric illnesses.

Recent molecular genetics studies have suggested various trans-synaptic processes for pathophysiologic mechanisms of neuropsychiatric illnesses. Examination of pre- and post-synaptic scaffolds in the brains of patients would greatly aid further investigation, yet such an approach in human postmortem tissue has yet to be tested. We have examined three methods using density gradient based purification of synaptosomes followed by detergent extraction (Method 1) and the pH based differential extraction of synaptic membranes (Methods 2 and 3). All three methods separated fractions from human postmortem brains that were highly enriched in typical PSD proteins, almost to the exclusion of pre-synaptic proteins. We examined these fractions using electron microscopy (EM) and verified the integrity of the synaptic membrane and PSD fractions derived from human postmortem brain tissues. We analyzed protein composition of the PSD fractions using two dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) and observed known PSD proteins by mass spectrometry. Immunoprecipitation and immunoblot studies revealed that expected protein-protein interactions and certain posttranscriptional modulations were maintained in PSD fractions. Our results demonstrate that PSD fractions can be isolated from human postmortem brain tissues with a reasonable degree of integrity. This approach may foster novel postmortem brain research paradigms in which the stoichiometry and protein composition of specific microdomains are examined.

Basal ubiquitin-independent internalization of interferon alpha receptor is prevented by Tyk2-mediated masking of a linear endocytic motif.

Linear endocytic motifs of signaling receptors as well as their ubiquitination determine the rate of ligand-induced endocytosis that mediates down-regulation of these receptors and restricts the magnitude and duration of their respective signal transduction pathways. We previously hypothesized that, in the absence of its cognate ligand, type I interferon (IFN), the IFNalpha receptor chain 1 (IFNAR1) receptor chain is protected from basal endocytosis by a hypothetical masking complex that prevents the Tyr-based endocytic motif within IFNAR1 from interacting with components of the adaptin protein complex 2 (AP2). Here we identify a member of the Janus kinase (Jak) family, Tyk2, as a component of such a masking complex. In the absence of ligand or of receptor chain ubiquitination, binding of Janus kinase Tyk2 within the proximity of the Tyr-based linear motif of IFNAR1 is required to prevent IFNAR1 internalization and to maintain its cell surface expression. Furthermore, interaction experiments revealed that Tyk2 physically shields this Tyr-based motif from the recognition by the AP50 subunit of AP2. These data delineate a long-sought ligand- and ubiquitin-independent mechanism by which Tyk2 contributes to both the regulation of total IFNAR1 levels as well as the regulation of the cell surface density of this receptor chain.

Polyubiquitination of prolactin receptor stimulates its internalization, postinternalization sorting, and degradation via the lysosomal pathway.

The ubiquitination of the receptor that mediates signaling induced by the polypeptide pituitary hormone prolactin (PRL) has been shown to lead to the degradation of this receptor and to the ensuing negative regulation of cellular responses to PRL. However, the mechanisms of PRL receptor (PRLr) proteolysis remain largely to be determined. Here we provide evidence that PRLr is internalized and primarily degraded via the lysosomal pathway. Ubiquitination of PRLr is essential for the rapid internalization of PRLr, which proceeds through a pathway dependent on clathrin and the assembly polypeptide 2 (AP2) adaptor complexes. Recruitment of AP2 to PRLr is stimulated by PRLr ubiquitination, which also is required for the targeting of already internalized PRLr to the lysosomal compartment. While mass spectrometry analysis revealed that both monoubiquitination and polyubiquitination (via both K48- and K63-linked chains) occur on PRLr, the results of experiments using forced expression of ubiquitin mutants indicate that PRLr polyubiquitination via K63-linked chains is important for efficient interaction of PRLr with AP2 as well as for efficient internalization, postinternalization sorting, and proteolytic turnover of PRLr. We discuss how specific ubiquitination may regulate early and late stages of endocytosis of PRLr and of related receptors to contribute to the negative regulation of the magnitude and duration of downstream signaling.