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Abstract Background To investigate the capacity of 99mTc-labeled 1-thio-β-D-glucose (1-TG) and 5-thio-D-glucose (5-TG) to act as a marker for glucose consumption in tumor cells in vivo as well as to evaluate the biodistribution of 1-TG and 5-TG. We investigated the biodistribution, including tumor uptake, of 1-TG and 5-TG at various time points after injection (0.5, 2 and 4 h) in human colorectal carcinoma (HCT-116) and human lung adenocarcinoma (A549) xenograft bearing nude mice (N = 4 per tracer and time point). Results Ex vivo biodistribution studies revealed a moderate uptake with a maximum tumor-to-muscle ratio of 4.22 ± 2.7 and 2.2 ± 1.3 (HCT-116) and of 3.2 ± 1.1 and 4.1 ± 1.3 (A549) for 1-TG and 5-TG, respectively, with a peak at 4 h for 1-TG and 5-TG. Biodistribution revealed a significantly higher uptake compared to blood in kidneys (12.18 ± 8.77 and 12.69 ± 8.93%ID/g at 30 min) and liver (2.6 ± 2.8%ID/g) for 1-TG and in the lung (7.24 ± 4.1%ID/g), liver (6.38 ± 2.94%ID/g), and kidneys (4.71 ± 1.97 and 4.81 ± 1.91%ID/g) for 5-TG. Conclusions 1-TG and 5-TG showed an insufficient tumor uptake with a moderate tumor-to-muscle ratio, not reaching the levels of commonly used tracer, for diagnostic use in human colorectal carcinoma and human lung adenocarcinoma xenograft model.

Abstract Cell death by glutamate excitotoxicity, mediated by N-methyl-d-aspartate (NMDA) receptors, negatively impacts brain function, including but not limited to hippocampal neurons. The NF-κB transcription factor (composed mainly of p65/p50 subunits) contributes to neuronal death in excitotoxicity, while its inhibition should improve cell survival. Using the biotin switch method, subcellular fractionation, immunofluorescence, and luciferase reporter assays, we found that NMDA-stimulated NF-κB activity selectively in hippocampal neurons, while endothelial nitric oxide synthase (eNOS), an enzyme expressed in neurons, is involved in the S-nitrosylation of p65 and consequent NF-κB inhibition in cerebrocortical, i.e., resistant neurons. The S-nitro proteomes of cortical and hippocampal neurons revealed that different biological processes are regulated by S-nitrosylation in susceptible and resistant neurons, bringing to light that protein S-nitrosylation is a ubiquitous post-translational modification, able to influence a variety of biological processes including the homeostatic inhibition of the NF-κB transcriptional activity in cortical neurons exposed to NMDA receptor overstimulation.

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.

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.

Abstract 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.

Abstract The outcome of T cell activation is determined by mechanisms that balance Ca2+ influx and clearance. Here we report that murine CD4 T cells lacking Neuroplastin (Nptn −/−), an immunoglobulin superfamily protein, display elevated cytosolic Ca2+ and impaired post-stimulation Ca2+ clearance, along with increased nuclear levels of NFAT transcription factor and enhanced T cell receptor-induced cytokine production. On the molecular level, we identified plasma membrane Ca2+ ATPases (PMCAs) as the main interaction partners of Neuroplastin. PMCA levels were reduced by over 70% in Nptn −/− T cells, suggesting an explanation for altered Ca2+ handling. Supporting this, Ca2+ extrusion was impaired while Ca2+ levels in internal stores were increased. T cells heterozygous for PMCA1 mimicked the phenotype of Nptn −/− T cells. Consistent with sustained Ca2+ levels, differentiation of Nptn −/− T helper cells was biased towards the Th1 versus Th2 subset. Our study thus establishes Neuroplastin-PMCA modules as important regulators of T cell activation.

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.

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.

Neuroplastin (np) 55 and 65 are immunoglobulin superfamily members that arise by alternative splicing of the same gene and have been implicated in long-term activity-dependent synaptic plasticity. Both biochemical and immunocytochemical data suggest that np55 is the predominant isoform (>95% of total neuroplastin) in cerebellum. Neuroplastin immunoreactivity is concentrated in the molecular layer and synaptic glomeruli in the granule cell layer. Expression in the molecular layer appears to be postsynaptic. First, neuroplastin is associated with Purkinje cell dendrites in two mouse granuloprival cerebellar mutants, disabled and cerebellar deficient folia. Second, in an acid sphingomyelinase knockout mouse with widespread protein trafficking defects, neuroplastin accumulates in the Purkinje cell somata. Finally, primary cerebellar cultures show neuroplastin expression in Purkinje cell dendrites and somata lacking normal histotypic organization and synaptic connections, and high-magnification views indicate a preferential association with dendritic spines. In the molecular layer, differences in neuroplastin expression levels present as a parasagittal array of stripes that alternates with that revealed by the expression of another compartmentation antigen, zebrin II/aldolase c. Neuroplastin immunoreactivity is first detected weakly at postnatal day 3 (P3) in the anterior lobe vermis. By P5, parasagittal stripes are already apparent in the immature molecular layer. At this stage, punctate deposits are also localised at the perimeter of the Purkinje cell perikarya; these are no longer detected by P15. The data suggest a role for neuroplastins in the development and maintenance of normal synaptic connections in the cerebellum. J. Comp. Neurol. 462:286–301, 2003. © 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract Background Chronic infection with the neurotropic parasite Toxoplasma gondii has been implicated in the risk for several neuropsychiatric disorders. The mechanisms, by which the parasite may alter neural function and behavior of the host, are not yet understood completely. Methods Here, a novel proteomic approach using mass spectrometry was employed to investigate the alterations in synaptic protein composition in a murine model of chronic toxoplasmosis. In a candidate-based strategy, immunoblot analysis and immunohistochemistry were applied to investigate the expression levels of key synaptic proteins in glutamatergic signaling. Results A comparison of the synaptosomal protein composition revealed distinct changes upon infection, with multiple proteins such as EAAT2, Shank3, AMPA receptor, and NMDA receptor subunits being downregulated, whereas inflammation-related proteins showed an upregulation. Treatment with the antiparasitic agent sulfadiazine strongly reduced tachyzoite levels and diminished neuroinflammatory mediators. However, in both conditions, a significant number of latent cysts persisted in the brain. Conversely, infection-related alterations of key synaptic protein levels could be partly reversed by the treatment. Conclusion These results provide evidence for profound changes especially in synaptic protein composition in T. gondii-infected mice with a downregulation of pivotal components of glutamatergic neurotransmission. Our results suggest that the detected synaptic alterations are a consequence of the distinct neuroinflammatory milieu caused by the neurotropic parasite.