Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function. Molecular & Cellular Proteomics 9:1324 -1338, 2010.

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N-methyl-D-aspartate (NMDA) receptors have been implicated in many neurological disorders. Although NMDA receptors are best known for their high calcium permeability, the recently discovered NR3 subunits, NR3A and NR3B, have been shown to reduce the calcium permeability of the NMDA receptor. Thus, NR3 subunits may be important players in modulating synaptic plasticity in neurons. Although NR3B expression in the rodent and human brain has been studied, little is known about its distribution in different cell types. Here we used immunolabeling with a specific NR3B antibody together with antibodies against established neurochemical markers to determine the cellular and subcellular localization of NR3B. The nucleus was concurrently stained with NR3B immunolabeling to show that NR3B is widely expressed by many cells in each brain region. Our findings indicate that NR3B is widely expressed in the structures examined in the rat forebrain (hippocampus, cerebral cortex, caudoputamen, and nucleus accumbens), cerebellum, and lumbar sections of the spinal cord. Within these regions NR3B was found to be expressed in all the substructures of the hippocampus (CA1, CA3, dentate gyrus), the various layers of the cerebral cortex, projection neurons and interneurons of the striatum, different cell types of the cerebellum, and motor neurons of the spinal cord. Furthermore, when stained with NR1-the obligatory subunit responsible for forming functional NMDA receptors-the distribution of NR3B appears to be as ubiquitous as NR1. Taken together, our data suggest that there may be a population of NR3B-containing NMDA receptors conferring new functional roles in the mammalian central nervous system.

Significant reduction in glutamate receptor 1 (GluR1)- and GluR2/3-immunopositive neurons was demonstrated in the hilus of the dentate gyrus in mice killed on days 1, 7 and 60 after pilocarpine-induced status epilepticus (PISE). In addition, GluR1 and GluR2/3 immunostaining in the strata oriens, radiatum and lacunosum moleculare of areas CA1-3 decreased drastically on days 7 and 60 after PISE. Neuronal loss observed in the above regions may account, at least in part, for a decrease in GluR immunoreactivity. By contrast, many GluR1-immunopositive neurons were observed in the gliotic area of CA1. Of these, about 42.8% were immunopositive for markers for hippocampal interneurons, namely calretinin (7.6%), calbindin (12.8%) and parvalbumin (22.4%). GluR1 or GluR2/3 and BrdU double-labelling showed that the GluR1- and GluR2/3-immunopositive neurons at 60 days after PISE were neurons that had survived rather than newly generated neurons. Furthermore, anterograde tracer and double-labelling studies performed on animals at 60 days after PISE indicated a projection from the hilus of the dentate gyrus to gliotic areas in both CA3 and CA1, where the projecting fibres apparently established connections with GluR1-immunopositive neurons. The projection to CA1 was unexpected. These novel findings suggest that the intrinsic hippocampal neuronal network is altered after PISE. We speculate that GluR1-immunopositive neurons in gliotic CA1 act as a bridge between dentate gyrus and subiculum contributing towards epileptogenesis.