Search

The amplitude and duration of Ca2+ signaling is crucial for B-cell development and self-tolerance; however, the mechanisms for terminating Ca2+ signals in B cells have not been determined. In lymphocytes, plasma membrane Ca2+ ATPase (PMCA) isoforms 1 and 4 (PMCA1 and PMCA4, aka ATP2B1 and ATP2B4) are the main candidates for expelling Ca2+ from the cell through the plasma membrane. We report here that Pmca4 (Atp2b4) KO mice had normal B-cell development, while mice with a conditional KO of Pmca1 (Atp2b1) had greatly reduced numbers of B cells, particularly splenic follicular B cells, marginal zone B cells, and peritoneal B-1a cells. Mouse and naive human B cells showed only PMCA1 expression and no PMCA4 by western blot, in contrast to T cells, which did express PMCA4. Calcium handling was normal in Pmca4-/- B cells, but Pmca1 KO B cells had elevated basal levels of Ca2+ , elevated levels in ER stores, and reduced Ca2+ clearance. These findings show that the PMCA1 isoform alone is required to ensure normal B-cell Ca2+ signaling and development, which may have implications for therapeutic targeting of PMCAs and Ca2+ in B cells.

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.

In the brain, the polyamines spermidine (Spd) and spermine (Spm) serve highly specific functions by interacting with various ion channel receptors intimately involved with synaptic signaling. Both, glial cells and neurons contain Spd/Spm, but release and uptake mechanisms could re-distribute polyamines between cell types. The cellular and subcellular localization of polyamine biosynthetic enzymes may therefore offer a more appropriate tool to identify local sources of enhanced Spd/Spm synthesis, which may be related with specific roles in neuronal circuits and synaptic function. A recently characterized antibody against Spd synthase was therefore used to screen the rat brain for compartment-specific peaks in enzyme expression. The resulting labeling pattern indicated a clearly heterogeneous expression predominantly localized to neurons and neuropil. The highest levels of Spd synthase expression were detected in the accumbens nucleus, taenia tecta, cerebellar cortex, cerebral cortical layer I, hippocampus, hypothalamus, mesencephalic raphe nuclei, central and lateral amygdala, and the circumventricular organs. Besides a diffuse labeling of the neuropil in several brain areas, the distinct labeling of mossy fiber terminals in the cerebellar cortex directly indicated a synaptic role for Spd synthesis. Electron microscopy revealed a preferential distribution of the immunosignal in synaptic vesicle containing areas. A pre-synaptic localization was also observed in parallel and climbing fiber terminals. Electrophysiological recordings in acute cerebellar slices revealed a Spd-induced block of evoked extracellular field potentials resulting from mossy fiber stimulation in a dose-dependent manner.

The peptidylarginine deiminase (PAD) family of enzymes are responsible for conversion of protein-bound arginine to citrulline in most tissues of the body and are garnering increased interest for their physiological and pathological roles. Although it has been shown that oligodendrocytes of the CNS express the PAD isoenzyme type 2, nothing is presently known about PAD expression in Schwann cells, the myelinating cells of the PNS. To evaluate PAD expression in the PNS, cultivated rat and human Schwann cells and slices of fetal, juvenile, and normal and regenerated adult sciatic nerves were examined with RT-PCR, Western blot, and immunohistochemical analysis. Samples from cerebellar cultures and skin served as positive controls. One of the principle findings was that cultivated Schwann cells expressed significant levels of mRNA and protein for the PAD isoenzymes 2 and 3. PAD1 and PAD4, however, were not expressed in any types of Schwann cells. Using double immunofluorescence, the majority of PAD2 staining was localized in immature cell stages. Moreover, increased amounts of PAD2, PAD3, and peptidyl-citrulline were also found in human fetal and rat juvenile and regenerated sciatic nerves as compared to similar normal adult specimens. Neuronal and inducible nitric oxide synthases, enzymes that convert free arginine to citrulline, were also expressed in Schwann cells; however, their massive induction by LPS/K+, was not reflected in an enhanced peptidyl-citrulline immunosignal. These data suggest that, similar to the CNS, citrullination of proteins may also exert a specific role in thecourse of PNS development and repair. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2008

The action of nitric oxide (NO) synthesized by NO synthases (NOS) is spatially restricted. Hence, the intracellular location of NOS might play an important role for the functional interactions of NO with its target molecules. In the skeletal muscle the neuronal NOS (nNOS) is considered to be the predominant isoform expressed as a muscle specific elongated splice variant. There are only a few and highly discrepant reports of the subcellular distribution of nNOS, which prompted us to re-examine the distribution of nNOS in the skeletal muscle of rat and mouse applying immunocytochemistry and NADPH-diaphorase (NADPH-d) histochemistry. Light microscopically, the sarcolemma, areas beneath the sarcolemma, areas around the nuclei, and the cross striation were labeled by antibodies and by the NADPH-d reaction as well. Ultrastructurally, nNOS visualized immunocytochemically or by the histochemical BSPT-reaction, was associated discretely with extrajunctional portions of the sarcolemma. Both reaction products were additionally observed in the vicinity of endoplasmic reticulum and mitochondria, or associated with their outer membranes. In the neuromuscular junction (NMJ)-region NOS was localized to the cytoplasm of nerve terminals and terminal Schwann cells. In contrast to the commonly accepted assumption, the enzyme was found in association with the presynaptic, and not with the postsynaptic membrane. Cytosolic NADPH-d was exhibited especially between mitochondria accumulated in the postsynaptic region of the NMJ. Surprisingly, in nNOS −/− -mice the skeletal muscle showed patterns of significant nNOS-immunoreactivity and NADPH-d activity possibly due to alternative nNOS-splice isoforms, which might be up-regulated to compensate for decreased NO formation.

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.

Using antibodies against synaptic protein preparations, we cloned the cDNA of a new Ca2+-binding protein. Its C-terminal portion displays significant similarity with calmodulin and contains two EF-hand motifs. The corresponding mRNA is highly expressed in rat brain, primarily in cerebral cortex, hippocampus, and cerebellum; its expression appears to be restricted to neurons. Transcript levels increase during postnatal development. A recombinant C-terminal protein fragment binds Ca2+ as indicated by a Ca2+-induced mobility shift in SDS-polyacrylamide gel electrophoresis. Antisera generated against the bacterial fusion protein recognize a brain-specific protein doublet with apparent molecular masses of 33 and 36 kDa. These data are confirmed by in vitro translation, which generates a single 36-kDa polypeptide, and by the heterologous expression in 293 cells, which yields a 33/36-kDa doublet comparable to that found in brain. On two-dimensional gels, the 33-kDa band separates into a chain of spots plausibly due to differential phosphorylation. This view is supported by in situ phosphorylation studies in hippocampal slices. Most of the immunoreactivity is detectable in cytoskeletal preparations with a further enrichment in the synapse-associated cytomatrix. These biochemical data, together with the ultra-structural localization in dendrites and the postsynaptic density, strongly suggest an association with the somato-dendritic cytoskeleton. Therefore, this novel Ca2+-binding protein was named caldendrin.

Gp65 and gp55 are immunoglobulin superfamily members produced by alternative splicing of the same gene transcript, and originally identified as components of synaptic membranes. A monoclonal antibody specific for gp65 and gp55 has been used to detect immunoreactive species in a wide range of tissues. All immunoreactive species bind to concanavalin A and deglycosylation studies show that in all tissues tested other than brain the immunoreactive species are derived from gp55. HEK cells transfected with gp65 or gp55 express different glycoforms from brain showing that the pattern of glycosylation of these molecules is dependent upon the cell type in which they are expressed.

The molecular architecture of the cytomatrix of presynaptic nerve terminals is poorly understood. Here we show that Bassoon, a novel protein of >400,000 Mr, is a new component of the presynaptic cytoskeleton. The murine bassoon gene maps to chromosome 9F. A comparison with the corresponding rat cDNA identified 10 exons within its protein-coding region. The Bassoon protein is predicted to contain two double-zinc fingers, several coiled-coil domains, and a stretch of polyglutamines (24 and 11 residues in rat and mouse, respectively). In some human proteins, e.g., Huntingtin, abnormal amplification of such poly-glutamine regions causes late-onset neurodegeneration. Bassoon is highly enriched in synaptic protein preparations. In cultured hippocampal neurons, Bassoon colocalizes with the synaptic vesicle protein synaptophysin and Piccolo, a presynaptic cytomatrix component. At the ultrastructural level, Bassoon is detected in axon terminals of hippocampal neurons where it is highly concentrated in the vicinity of the active zone. Immunogold labeling of synaptosomes revealed that Bassoon is associated with material interspersed between clear synaptic vesicles, and biochemical studies suggest a tight association with cytoskeletal structures. These data indicate that Bassoon is a strong candidate to be involved in cytomatrix organization at the site of neurotransmitter release.

We analyzed the long-term consequences of asphyxial cardiac arrest for hippocampal cell proliferation in rats to evaluate if the ischaemia-induced degenerated CA1 region may be repopulated by endogenous (stem) cells. Studies were performed in an asphyxial cardiac arrest model with 5 minutes of asphyxiation and three different survival times: 7, 21, and 90 days. Sham-operated non-asphyxiated rats served as control. Cell proliferation was studied by labeling dividing cells with 5-bromo-2′-deoxy-uridine (BrdU). The neurodegenerative/regenerative pattern at single cell levels was monitored by immunohistochemistry. Alterations of gene expression were analyzed by real-time quantitative RT-PCR. Analysis of BrdU-incorporation demonstrated an increase at 7, 21 as well as 90 days after global ischaemia in the hippocampal CA1 pyramidal cell layer. Similar results were found in the dentate gyrus. Differentiation of BrdU-positive cells, investigated by cell phenotype-specific double fluorescent labeling, showed increased neurogenesis only in the dentate gyrus of animals surviving the cardiac arrest for 7 days. The majority of newcomers, especially in the damaged CA1 region, consisted of glial cells. Moreover, asphyxia seemed to be able to induce the migration of microglia and astroglia from adjacent areas into the damaged area and/or the activation of resident cells. In addition, we show microglia proliferation/activation even 90 days after cardiac arrest. This morphological finding was confirmed by PCR analysis. The results indicate that asphyxia triggers cell proliferation in general and gliogenesis in particular — a possible pro-reparative event. Furthermore, from the finding of microglia proliferation up to 90 days after insult we conclude that delayed cell death processes take place which should be considered for further therapy strategies.

Bassoon is a 420-kDa presynaptic cytomatrix protein potentially involved in the structural organization of neurotransmitter release sites. In this study, we have investigated a possible role for Bassoon in synaptogenesis and in defining synaptic vesicle recycling sites. We find that it is expressed at early stages of neuronal differentiation in which it is selectively sorted into axons. As synaptogenesis begins, Bassoon clusters appear along dendritic profiles simultaneously with synaptotagmin I, sites of synaptic vesicle recycling, and the acquisition of functional excitatory and inhibitory synapses. A role for Bassoon in the assembly of excitatory and inhibitory synapses is supported by the colocalization of Bassoon clusters with clusters of GKAP and AMPA receptors as well as GABA(A) receptors. These data indicate that the recruitment of Bassoon is an early step in the formation of synaptic junctions.

The postsynaptic density (PSD) is crucially involved in the structural and functional organization of the postsynaptic neurotransmitter reception apparatus. Using antisera against rat brain synaptic junctional protein preparations, we isolated cDNAs coding for proline-rich synapse-associated protein-1 (ProSAP1), a PDZ-domain protein. This protein was found to be identical to the recently described cortactin-binding protein-1 (CortBP1). Homology screening identified a related protein, ProSAP2. Specific antisera raised against a C-terminal fusion construct and a central part of ProSAP1 detect a cluster of immunoreactive bands of 180 kDa in the particulate fraction of rat brain homogenates that copurify with the PSD fraction. Transcripts and immunoreactivity are widely distributed in the brain and are upregulated during the period of synapse formation in the brain. In addition, two short N-terminal insertions are detected; they are differentially regulated during brain development. Confocal microscopy of hippocampal neurons showed that ProSAP1 is predominantly localized in synapses, and immunoelectron microscopyin siturevealed a strong association with PSDs of hippocampal excitatory synapses. The accumulation of ProSAP1 at synaptic structures was analyzed in the developing cerebral cortex. During early postnatal development, strong immunoreactivity is detectable in neurites and somata, whereas from postnatal day 10 (P10) onward a punctate staining is observed. At the ultrastructural level, the immunoreactivity accumulates at developing PSDs starting from P8. Both interaction with the actin-binding protein cortactin and early appearance at postsynaptic sites suggest that ProSAP1/CortBP1 may be involved in the assembly of the PSD during neuronal differentiation.

Glycoproteins gp65 and gp55 are major components of synaptic membranes prepared from rat forebrain. Both are recognized by the monoclonal antibody SMgp65. We have used SMgp65 to screen a rat brain cDNA expression library. Two sets of overlapping cDNAs that contain open reading frames of 397 and 281 amino acids were isolated. The deduced proteins are members of the immunoglobulin (Ig) superfamily containing three and two Ig domains, respectively. The common part has approximately 40% sequence identity with neurothelin/basigin. The identity of the proteins as gp65 and gp55 was confirmed by production of new antisera against a common recombinant protein fragment. These antisera immunoprecipitate gp65 and gp55. Furthermore, expression of gp65 and gp55 cDNAs in human 293 cells treated with tunicamycin results in the production of unglycosylated core proteins of identical size to deglycosylated gp65 and gp55. Northern analysis revealed that gp65 transcripts are brain-specific, whereas gp55 is expressed in most tissues and cell lines examined. The tissue distribution was confirmed at the protein level though the pattern of glycosylation of gp55 varies between tissues. In situ hybridization experiments with a common and a gp65-specific probe suggest differential expression of gp65 and gp55 transcripts in the rat brain.

Antisera against a rat brain synaptic protein preparation, the postsynaptic density (PSD) fraction, were used to isolate cDNA clones by expression screening of a rat brain cDNA library. About one fifth of more than 200 analyzed cDNAs encoding potential synapse-associated proteins were previously unknown. Identifiable proteins include, among others, components of the pre- and postsynaptic cytoskeleton, synaptic vesicle proteins and several protein kinases and kinase substrates. This demonstrates that both pre- and postsynaptic elements purify with the PSD fraction.

To investigate the expression of isoforms of the cyclic AMP (cAMP)-dependent protein kinase (cAK) in the transition zone of the human prostate and the functional significance of the enzyme in the control of prostate smooth muscle.Using Western blot analysis and immunohistochemistry, the expression and distribution in the prostate of cAKIalpha, cAKIbeta, cAKIIalpha, and cAKIIbeta in relation to alpha-actin and the phosphodiesterase PDE4 (types A and B) were investigated. The effects of the cAK inhibitor Rp-8-CPT-cAMPS on the reversion of the adrenergic tension of isolated prostate tissue induced by forskolin, rolipram, sodium nitroprusside (SNP), and tadalafil were examined by means of the organ bath technique.Immunosignals specific for cAKIalpha, cAKIIalpha, and cAKIIbeta were observed in the smooth musculature and glandular structures of the prostate. Double stainings revealed the colocalization of alpha-actin and PDE4 with the cAK isoforms. The expression of the cAK isoforms was confirmed by Western blot analysis. The relaxation of the tension induced by norepinephrine brought about by forskolin, rolipram, SNP, and tadalafil was significantly attenuated by Rp-8-CPT-cAMPS.The colocalization of smooth muscle alpha-actin and PDE4 with cAK, as well as the results from the organ bath experiments, provide further evidence for a pivotal role of the cAMP-dependent signaling in the regulation of prostate smooth muscle contractility. Compounds interacting with the cAMP/cAK pathway might represent a new therapeutic avenue to treat symptoms of benign prostatic hyperplasia and lower urinary tract symptomatology.