Neuroplastin cell recognition molecules have been implicated in synaptic plasticity. Polymorphisms in the regulatory region of the human neuroplastin gene (NPTN) are correlated with cortical thickness and intellectual abilities in adolescents and in individuals with schizophrenia.

Deubiquitinylases (DUBs) are highly specialized enzymes which are responsible for removal of covalently attached ubiquitin(s) from the targeted proteins. DUBs play an important role in maintaining the protein homeodynamics. Recently, DUBs have emerged as novel therapeutic targets in cancer, inflammation, diabetes, and neurodegeneration. Among the different families of DUBs, the metalloprotease group or JAB1/MOV34/MPR1 (JAMMs) proteases are unique in terms of catalytic mechanism. JAMMs exhibit a Zn2+-dependent deubiquitinylase activity. Within the JAMM family, deubiquitinylases Rpn11 and CSN5 are constituents of large bimolecular complexes, namely the 26S proteasome and COP9 signalosome (CSN), respectively. Rpn11 and CSN5 are potential drug targets in cancer and selective inhibitors of both proteins have been reported in the literature. However, the selectivity of JAMM inhibitors (capzimin for RPN11 and CSN5i-3 for CSN5) has not been structurally resolved yet. In the present work, we have explored the binding modes of capzimin and CSN5i-3 and rationalize their selectivity for Rpn11 and CSN5 targets. We found that capzimin interacts with the active site Zn+2 of Rpn11 in a bidentate manner and also interacts with the residues in the distal ubiquitin binding site. MD simulations studies and binding energy analysis revealed that the selective binding of the inhibitors can be only explained by the consideration of larger heterodimeric complexes of Rpn11 (Rpn8-Rpn11) and CSN5 (CSN5-CSN6). Simulation of these protein-protein complexes is necessary to avoid unrealistic large conformational changes. The selective binding of inhibitors is mainly governed by residues in the distal ubiquitin binding site. This study demonstrates that selective inhibitor binding design for Rpn11 and CSN5 JAMM proteases requires consideration of heterodimeric protein-protein target structures.

Cognitive impairments can be devastating for quality of life, and thus, preventing or counteracting them is of great value. To this end, the present study exploits the potential of the plant Rhodiola rosea and identifies the constituent ferulic acid eicosyl ester [icosyl-(2E)-3-(4-hydroxy-3-methoxyphenyl)-prop-2-enoate (FAE-20)] as a memory enhancer. We show that food supplementation with dried root material from R. rosea dose-dependently improves odor-taste reward associative memory scores in larval Drosophila and prevents the age-related decline of this appetitive memory in adult flies. Task-relevant sensorimotor faculties remain unaltered. From a parallel approach, a list of candidate compounds has been derived, including R. rosea-derived FAE-20. Here, we show that both R. rosea-derived FAE-20 and synthetic FAE-20 are effective as memory enhancers in larval Drosophila. Synthetic FAE-20 also partially compensates for age-related memory decline in adult flies, as well as genetically induced early-onset loss of memory function in young flies. Furthermore, it increases excitability in mouse hippocampal CA1 neurons, leads to more stable context-shock aversive associative memory in young adult (3-month-old) mice, and increases memory scores in old (>2-year-old) mice. Given these effects, and given the utility of R. rosea-the plant from which we discovered FAE-20-as a memory enhancer, these results may hold potential for clinical applications.

The cell adhesion molecule neuroplastin (Np) is a novel candidate to influence human intelligence. Np-deficient mice display complex cognitive deficits and reduced levels of Plasma Membrane Ca2+ ATPases (PMCAs), an essential regulator of the intracellular Ca2+ concentration ([iCa2+]) and neuronal activity. We show abundant expression and conserved cellular and molecular features of Np in glutamatergic neurons in human hippocampal-cortical pathways as characterized for the rodent brain. In Nptn lox/loxEmx1Cre mice, glutamatergic neuron-selective Np ablation resulted in behavioral deficits indicating hippocampal, striatal, and sensorimotor dysfunction paralleled by highly altered activities in hippocampal CA1 area, sensorimotor cortex layers I-III/IV, and the striatal sensorimotor domain detected by single-photon emission computed tomography. Altered hippocampal and cortical activities correlated with reduction of distinct PMCA paralogs in Nptn lox/loxEmx1Cre mice and increased [iCa2+] in cultured mutant neurons. Human and rodent Np enhanced the post-transcriptional expression of and co-localized with PMCA paralogs in the plasma membrane of transfected cells. Our results indicate Np as essential for PMCA expression in glutamatergic neurons allowing proper [iCa2+] regulation and normal circuit activity. Neuron-type-specific Np ablation empowers the investigation of circuit-coded learning and memory and identification of causal mechanisms leading to cognitive deterioration.

Nuclear factor kappa-B (NF-κB) activates multiple genes with overlapping roles in cell proliferation, inflammation and cancer. Using an unbiased approach we identified human CDK6 as a novel kinase phosphorylating NF-κB p65 at serine 536. Purified and reconstituted CDK6/cyclin complexes phosphorylated p65 in vitro and in transfected cells. The physiological role of CDK6 for basal as well as cytokine-induced p65 phosphorylation or NF-κB activation was revealed upon RNAi-mediated suppression of CDK6. Inhibition of CDK6 catalytic activity by PD332991 suppressed activation of NF-κB and TNF-induced gene expression. In complex with a constitutively active viral cyclin CDK6 stimulated NF-κB p65-mediated transcription in a target gene specific manner and this effect was partially dependent on its ability to phosphorylate p65 at serine 536. Tumor formation in thymi and spleens of v-cyclin transgenic mice correlated with increased levels of p65 Ser536 phosphorylation, increased expression of CDK6 and upregulaton of the NF-κB target cyclin D3. These results suggest that aberrant CDK6 expression or activation that is frequently observed in human tumors can contribute through NF-κB to chronic inflammation and neoplasia.

Infection with the human microbial pathogen Helicobacter pylori is assumed to lead to invasive gastric cancer. We find that H. pylori activates the hepatocyte growth factor/scatter factor receptor c-Met, which is involved in invasive growth of tumor cells. The H. pylori effector protein CagA intracellularly targets the c-Met receptor and promotes cellular processes leading to a forceful motogenic response. CagA could represent a bacterial adaptor protein that associates with phospholipase Cgamma but not Grb2-associated binder 1 or growth factor receptor-bound protein 2. The H. pylori-induced motogenic response is suppressed and blocked by the inhibition of PLCgamma and of MAPK, respectively. Thus, upon translocation, CagA modulates cellular functions by deregulating c-Met receptor signaling. The activation of the motogenic response in H. pylori-infected epithelial cells suggests that CagA could be involved in tumor progression.

In the vertebrate retina, gamma-aminobutyric acid (GABA) mediates inhibitory processes that shape the visual response and is also thought to have neurotrophic functions during retinal development. To investigate the role of GABAergic signaling at the beginning of visual experience, we used immunohistochemistry to compare the distribution of GABA, the two isoforms of glutamic acid decarboxylase GAD65/67, and the GABA receptor types A, B, and C, in neonate versus adult Octodon degus, a native South American rodent with diurnal-crepuscular activity and a high cone-to-rod ratio. In parallel, we used electroretinography to evaluate retinal functionality and to test the contribution of fast GABAergic transmission to light responses at both developmental stages. Neonate O. degus opened their eyes on postnatal day (P)0 and displayed an adult-like retinal morphology at this time. GABA, its biosynthetic sources, and receptors had a similar cellular distribution in neonates and adults, but labeling of the outer plexiform layer and of certain amacrine and ganglion cells was more conspicuous at P0. In neonates, retinal sensitivity was 10 times lower than in adults, responses to ultraviolet light could not be detected, and oscillatory potentials were reduced or absent. Blockade of GABA(A/C) receptors by bicuculline and TPMPA had no noticeable effect in neonates, while it significantly altered the electroretinogram response in adults.

Helicobacter pylori are bacteria that colonize the stomach persistently, which confers risk of serious diseases, including peptic ulceration and gastric neoplasia. Aberrant expression of cell cycle control proteins has been demonstrated in H. pylori infected gastric epithelial cells, suggesting that perturbation of the cell cycle plays a role in the pathogenesis of various H. pylori associated diseases. In this study, we investigate the modulation of the cell cycle control protein p21(WAF1) by H. pylori in the gastric carcinoma cell line NCI-N87 and in primary gastric cells derived from healthy tissue. We observed an up-regulation of p21(WAF1) in both NCI-N87 and primary cells. Chromatin immunoprecipitation analysis revealed that the increased expression of p21(WAF1) induced by H. pylori is associated with the release of HDAC-1 from the p21(WAF1) promoter and hyper-acetylation of histone H4. The elucidation of the epigenetic regulation of p21(WAF1) by H. pylori may help to dissect the pathogenetic mechanisms underlying the development and progression of H. pylori associated diseases.

NMDA (N-methyl-D-aspartate) receptors and calcium can exert multiple and very divergent effects within neuronal cells, thereby impacting opposing occurrences such as synaptic plasticity and neuronal degeneration. The neuronal Ca2+ sensor Caldendrin is a postsynaptic density component with high similarity to calmodulin. Jacob, a recently identified Caldendrin binding partner, is a novel protein abundantly expressed in limbic brain and cerebral cortex. Strictly depending upon activation of NMDA-type glutamate receptors, Jacob is recruited to neuronal nuclei, resulting in a rapid stripping of synaptic contacts and in a drastically altered morphology of the dendritic tree. Jacob's nuclear trafficking from distal dendrites crucially requires the classical Importin pathway. Caldendrin binds to Jacob's nuclear localization signal in a Ca2+-dependent manner, thereby controlling Jacob's extranuclear localization by competing with the binding of Importin-alpha to Jacob's nuclear localization signal. This competition requires sustained synapto-dendritic Ca2+ levels, which presumably cannot be achieved by activation of extrasynaptic NMDA receptors, but are confined to Ca2+ microdomains such as postsynaptic spines. Extrasynaptic NMDA receptors, as opposed to their synaptic counterparts, trigger the cAMP response element-binding protein (CREB) shut-off pathway, and cell death. We found that nuclear knockdown of Jacob prevents CREB shut-off after extrasynaptic NMDA receptor activation, whereas its nuclear overexpression induces CREB shut-off without NMDA receptor stimulation. Importantly, nuclear knockdown of Jacob attenuates NMDA-induced loss of synaptic contacts, and neuronal degeneration. This defines a novel mechanism of synapse-to-nucleus communication via a synaptic Ca2+-sensor protein, which links the activity of NMDA receptors to nuclear signalling events involved in modelling synapto-dendritic input and NMDA receptor-induced cellular degeneration.

Mechanisms regulating the turnover of synaptic vesicle (SV) proteins are not well understood. They are thought to require poly-ubiquitination and degradation through proteasome, endo-lysosomal or autophagy-related pathways. Bassoon was shown to negatively regulate presynaptic autophagy in part by scaffolding Atg5. Here, we show that increased autophagy in Bassoon knockout neurons depends on poly-ubiquitination and that the loss of Bassoon leads to elevated levels of ubiquitinated synaptic proteins per se. Our data show that Bassoon knockout neurons have a smaller SV pool size and a higher turnover rate as indicated by a younger pool of SV2. The E3 ligase Parkin is required for increased autophagy in Bassoon-deficient neurons as the knockdown of Parkin normalized autophagy and SV protein levels and rescued impaired SV recycling. These data indicate that Bassoon is a key regulator of SV proteostasis and that Parkin is a key E3 ligase in the autophagy-mediated clearance of SV proteins.

Genetic disruption of synaptic proteins results in a whole variety of human neuropsychiatric disorders including intellectual disability, schizophrenia or autism spectrum disorder (ASD). In a wide range of these so-called synaptopathies a sex bias in prevalence and clinical course has been reported. Using an unbiased proteomic approach, we analyzed the proteome at the interaction site of the pre- and postsynaptic compartment, in the prefrontal cortex, hippocampus, striatum and cerebellum of male and female adult C57BL/6J mice. We were able to reveal a specific repertoire of synaptic proteins in different brain areas as it has been implied before. Additionally, we found a region-specific set of novel synaptic proteins differentially expressed between male and female individuals including the strong ASD candidates DDX3X, KMT2C, MYH10 and SET. Being the first comprehensive analysis of brain region-specific synaptic proteomes from male and female mice, our study provides crucial information on sex-specific differences in the molecular anatomy of the synapse. Our efforts should serve as a neurobiological framework to better understand the influence of sex on synapse biology in both health and disease.

Influenza A virus (IAV) causes respiratory tract disease and is responsible for seasonal and reoccurring epidemics affecting all age groups. Next to typical disease symptoms, such as fever and fatigue, IAV infection has been associated with behavioral alterations presumably contributing to the development of major depression. Previous experiments using IAV/H1N1 infection models have shown impaired hippocampal neuronal morphology and cognitive abilities, but the underlying pathways have not been fully described. In this study, we demonstrate that infection with a low-dose non-neurotrophic H1N1 strain of IAV causes ample peripheral immune response followed by a temporary blood-brain barrier disturbance. Although histological examination did not reveal obvious pathological processes in the brains of IAV-infected mice, detailed multidimensional flow cytometric characterization of immune cells uncovered subtle alterations in the activation status of microglial cells. More specifically, we detected an altered expression pattern of major histocompatibility complex classes I and II, CD80, and F4/80 accompanied by elevated mRNA levels of CD36, CD68, C1QA, and C3, suggesting evolved synaptic pruning. To closer evaluate how these profound changes affect synaptic balance, we established a highly sensitive multiplex flow cytometry-based approach called flow synaptometry. The introduction of this novel technique enabled us to simultaneously quantify the abundance of pre- and postsynapses from distinct brain regions. Our data reveal a significant reduction of VGLUT1 in excitatory presynaptic terminals in the cortex and hippocampus, identifying a subtle dysbalance in glutamatergic synapse transmission upon H1N1 infection in mice. In conclusion, our results highlight the consequences of systemic IAV-triggered inflammation on the central nervous system and the induction and progression of neuronal alterations. IMPORTANCE Influenza A virus (IAV) causes mainly respiratory tract disease with fever and fatigue but is also associated with behavioral alterations in humans. Here, we demonstrate that infection with a low-dose non-neurotrophic H1N1 strain of IAV causes peripheral immune response followed by a temporary blood-brain barrier disturbance. Characterization of immune cells uncovered subtle alterations in the activation status of microglia cells that might reshape neuronal synapses. We established a highly sensitive multiplex flow cytometry-based approach called flow synaptometry to more closely study the synapses. Thus, we detected a specific dysbalance in glutamatergic synapse transmission upon H1N1 infection in mice. In conclusion, our results highlight the consequences of systemic IAV-triggered inflammation on the central nervous system and the induction and progression of neuronal alterations.

The second beta-like subunit (SBD) is a putative structural subunit of Drosophila melanogaster nicotinic acetylcholine receptors (nAChRs). Here we have produced specific antibodies against SBD to study, which other nAChR subunits can co-assemble with SBD in receptor complexes of the Drosophila nervous system. Immunohistochemical studies in the adult optic lobe revealed that SBD has a distribution similar to that of the alpha-subunit ALS in the synaptic neuropil. The subunits ALS, D(alpha)2 and SBD can be co-purified by alpha-bungarotoxin affinity chromatography. Moreover, anti-SBD antibodies co-precipitate ALS and D(alpha)2 and, vice versa, ALS and D(alpha)2 antibodies co-immunoprecipitate SBD protein. Two-step immunoaffinity chromatography with immobilized antibodies against ALS and D(alpha)2 revealed the existence of nAChR complexes that include ALS, D(alpha)2 and SBD as integral components. Interestingly, the genes encoding these three subunits appear to be directly linked in the Drosophila genome at region 96 A of the third chromosome. In addition, SBD appears to be a component of a different receptor complex, which includes the ARD protein as an additional beta-subunit, but neither ALS nor D(alpha)2 nor the third alpha-subunit D(alpha)3. These findings suggest a considerable complexity of the Drosophila nicotinic receptor system.

Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.

Graft-vs.-host disease (GvHD) is a major complication of allogenic hematopoietic stem-cell(HSC) transplantation. GvHD is associated with loss of endothelial thrombomodulin, but the relevance of this for the adaptive immune response to transplanted HSCs remains unknown. Here we show that the protease-activated protein C (aPC), which is generated by thrombomodulin, ameliorates GvHD aPC restricts allogenic T-cell activation via the protease activated receptor (PAR)2/PAR3 heterodimer on regulatory T-cells (Tregs, CD4+FOXP3+). Preincubation of pan T-cells with aPC prior to transplantation increases the frequency of Tregs and protects from GvHD. Preincubation of human T-cells (HLA-DR4-CD4+) with aPC prior to transplantation into humanized (NSG-AB°DR4) mice ameliorates graft-vs.-host disease. The protective effect of aPC on GvHD does not compromise the graft vs. leukaemia effect in two independent tumor cell models. Ex vivo preincubation of T-cells with aPC, aPC-based therapies, or targeting PAR2/PAR3 on T-cells may provide a safe and effective approach to mitigate GvHD.Graft-vs.-host disease is a complication of allogenic hematopoietic stem cell transplantation, and is associated with endothelial dysfunction. Here the authors show that activated protein C signals via PAR2/PAR3 to expand Treg cells, mitigating the disease in mice.

Transforming growth factor-β (TGFβ)-activated kinase 1 (TAK1) plays a central role in controlling the cellular pro-inflammatory response via the activation of the nuclear factor κB (NF-κB)- and mitogen-activated protein (MAP) kinases-dependent transcriptional programs. Here, we show that depletion of TAK1 and the TAK1-binding proteins TAB1 and TAB2 affects NF-κB, JNK and p38 phosphorylation and suppresses NF-κB activity in AGS cells infected with Helicobacter pylori or stimulated with the cytokines TNF and IL-1β. To increase our understanding of TAK1 regulation and function, we performed mass spectrometry (MS)-based TAK1 interactomics. In addition to the identification of known and novel TAK1 interacting proteins, including TRIM28, CDC37 and STOML2, analysis of the MS data revealed various post-translational modifications within the TAK1/TAB complex. By applying siRNAs, TRIM28 and CDC37 were found to regulate phosphorylations of TAK1, IκB kinases IKKα/IKKβ and MAP kinases, NF-κB transactivation activity and IL-8 expression in the infected epithelial cells.

Every year, gastric cancer causes around 819,000 deaths worldwide. The incidence of gastric cancer in the western world is slowly declining, but the prognosis is unpromising. In Germany, the 5-year-survival rate is around 32%, and the average life span after diagnosis is 6 to 9 months. Therapy of gastric cancer patients comprises a gastrectomy and perioperative or adjuvant chemotherapy. However, resistance of gastric cancer cells to these agents is widespread; thus, improved chemotherapeutic approaches are required. Nuclear factor kappa B (NF-κB) transcription factors are associated with anti-apoptosis, carcinogenesis, and chemoresistance, and thus, constitute attractive targets for therapeutic intervention. In immunoblots, we show that ubiquitin specific protease 47 (USP47) promotes β-transducin repeat-containing protein (βTrCP) stability and phosphorylation of RelA. Furthermore, after knockdown of USP47 by RNA interference, we analyzed in gastric cancer cell lines metabolic activity/viability in an MTT assay, and apoptotic cell death by Annexin V staining and poly(ADP-Ribose) polymerase (PARP)-1, caspase 3, and caspase 8 cleavage, respectively. We found that USP47 contributes to cell viability and chemoresistance in NCI-N87 gastric carcinoma cells treated with etoposide and camptothecin. Inhibition of USP47 might be a suitable strategy to downregulate NF-κB activity, and to overcome chemoresistance in gastric cancer.

Spatial and temporal modulation of intracellular Ca2+ fluxes controls the cellular response of B lymphocytes to antigen stimulation. Herein, we identify the hematopoietic adaptor protein Dok-3 (downstream of kinase-3) as a key component of negative feedback regulation in Ca2+ signaling from the B-cell antigen receptor. Dok-3 localizes at the inner leaflet of the plasma membrane and is a major substrate for activated Src family kinase Lyn. Phosphorylated Dok-3 inhibits antigen receptor-induced Ca2+ elevation by recruiting cytosolic Grb2, which acts at this location as a negative regulator of Bruton's tyrosine kinase. This leads to diminished activation of phospholipase C-gamma2 and reduced production of soluble inositol trisphosphate. Hence, the Dok-3/Grb2 module is a membrane-associated signaling organizer, which orchestrates the interaction efficiency of Ca2+-mobilizing enzymes.

By changing the relative abundance of generated antigenic peptides through alterations in the proteolytic activity, interferon (IFN)-γ-induced immunoproteasomes influence the outcome of CD8+ cytotoxic T lymphocyte responses. In the present study, we investigated the effects of hepatitis C virus (HCV) infection on IFN-γ-induced immunoproteasome expression using a HCV infection cell culture system. We found that, although IFN-γ induced the transcriptional expression of mRNAs encoding the β1i/LMP2, β2i/MECL-1 and β5i/LMP7 immunoproteasome subunits, the formation of immunoproteasomes was significantly suppressed in HCV-infected cells. This finding indicated that immunoproteasome induction was impaired at the translational or posttranslational level by HCV infection. Gene silencing studies showed that the suppression of immunoproteasome induction is essentially dependent on protein kinase R (PKR). Indeed, the generation of a strictly immunoproteasome-dependent cytotoxic T lymphocyte epitope was impaired in in vitro processing experiments using isolated 20S proteasomes from HCV-infected cells and was restored by the silencing of PKR expression. In conclusion, our data point to a novel mechanism of immune regulation by HCV that affects the antigen-processing machinery through the PKR-mediated suppression of immunoproteasome induction in infected cells.

The ubiquitin-modifying enzyme A20, an important negative feedback regulator of NF-κB, impairs the expansion of tumor-specific CD8+ T cells but augments the proliferation of autoimmune CD4+ T cells. To study the T cell-specific function of A20 in bacterial infection, we infected T cell-specific A20 knockout (CD4-Cre A20fl/fl) and control mice with Listeria monocytogenes. A20-deficient pathogen-specific CD8+ T cells expanded stronger resulting in improved pathogen control at day 7 p.i. Imaging flow cytometry revealed that A20-deficient Listeria-specific CD8+ T cells underwent increased apoptosis and necroptosis resulting in reduced numbers of memory CD8+ T cells. In contrast, the primary CD4+ T cell response was A20-independent. Upon secondary infection, the increase and function of pathogen-specific CD8+ T cells, as well as pathogen control were significantly impaired in CD4-Cre A20fl/fl mice. In vitro, apoptosis and necroptosis of Listeria-specific A20-deficient CD8+ T cells were strongly induced as demonstrated by increased caspase-3/7 activity, RIPK1/RIPK3 complex formation and more morphologically apoptotic and necroptotic CD8+ T cells. In vitro, A20 limited CD95L and TNF-induced caspase3/7 activation. In conclusion, T cell-specific A20 limited the expansion but reduced apoptosis and necroptosis of Listeria-specific CD8+ T cells, resulting in an impaired pathogen control in primary but improved clearance in secondary infection.