[Clinical pathway (workflow) for diagnostic, therapy and follow-up in patients with rectal cancer].
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J Roder
Publications and source records attributed to J Roder.
Activity-dependent regulation of the subcellular localization of neuronal calcium sensor-1 in the avian cochlear nucleus.
Neurons in the avian cochlear nucleus, nucleus magnocellularis (NM), are highly sensitive to manipulations of afferent input, and removal of afferent activity through cochlear ablation results in the death of approximately 20-40% of ipsilateral NM neurons. The intracellular cascades that determine whether an individual NM neuron will die or survive are not fully understood. One early event observed in NM following deafferentation is a rapid rise in intracellular calcium concentration. In most cellular systems, the activity of calcium-binding proteins is believed to accommodate calcium influx. The calcium-binding protein, neuronal calcium sensor-1 (NCS-1), is an intracellular neuronal calcium sensor belonging to the EF-hand superfamily. NCS-1 has been implicated in calcium-dependent regulation of signaling cascades. To evaluate NCS-1 action in NM neurons, the localization of NCS-1 protein was examined. Double-label immunofluorescence experiments revealed that NCS-1 expression is evident in both the presynaptic nerve terminal and postsynaptic NM neuron. The postsynaptic expression of NCS-1 typically appears to be closely associated with the cell membrane. This close proximity of NCS-1 to the postsynaptic membrane could allow NCS-1 to function as a modulator of postsynaptic signaling events. Following deafferentation, NM neurons were more likely to show diffuse cytoplasmic NCS-1 labeling. This increase in the number of cells showing diffuse cytoplasmic labeling was observed 12 and 24 h following cochlea ablation, but was not observed 4 days following surgery. This activity-dependent regulation of NCS-1 subcellular localization suggests it may be associated with, or influenced by, processes important for the survival of NM neurons.
Action Potentials↗
Immunocytochemical localization of neuronal calcium sensor-1 in the hippocampus and cerebellum of the mouse, with special reference to presynaptic terminals.
Neuronal calcium sensor-1 (NCS-1) is a member of the EF-hand calcium-binding protein superfamily, which is considered to modulate synaptic transmission and plasticity. In this work, we first examined the distribution patterns of NCS-1 in the hippocampus and cerebellum. The intense NCS-1-immunoreactive (IR) elements in the hippocampus were restricted to dendritic layers, while those in the cerebellum occurred in both dendritic and cellular layers. Then, we examined the exact localization of NCS-1 using immunofluorescent double labeling for NCS-1 and synaptophysin, a marker of presynaptic terminals. In the hippocampus, the mossy fiber systems (terminals and bundles) exhibited intense NCS-1 immunoreactivity. On the other hand, the presumed principal cell dendrites were also NCS-1-IR in the stratum lacunosum-moleculare of Ammon's horn and molecular layer of the dentate gyrus, where NCS-1-IR elements and synaptophysin-IR presynaptic terminals showed characteristic complementary distribution patterns. In the cerebellum, some of the basket cell axon terminals surrounding the somata of Purkinje cells exhibited NCS-1 immunoreactivity, while the pinceau showed consistent labeling for NCS-1. Higher magnification observations revealed that the NCS-1-IR presumed granule cell dendrites and synaptophysin-IR mossy fiber terminals in the glomeruli of the cerebellum showed characteristic complementary distribution patterns. Furthermore, we estimated quantitatively the relative amount of NCS-1 in the presynaptic terminals in individual layers, and confirmed that the mossy fiber terminals in the hippocampus contained comparatively high amounts of NCS-1. These results showed the diverse localization of NCS-1 in pre- and/or postsynaptic elements of the hippocampus and cerebellum, and suggest potential roles in specific synaptic transmission.
Animals↗
Intestinal inflammation modulates expression of the synaptic vesicle protein neuronal calcium sensor-1.
The calcium-binding protein neuronal calcium sensor 1 (NCS-1) is involved in modulation of neurotransmitter release in the peripheral and central nervous systems. Since intestinal inflammation impairs neurotransmitter release, we evaluated the expression of NCS-1 in the normal rat colon and in dinitrobenzene sulfonic acid (DNBS)-induced colitis. Immunocytochemistry and Western blots showed high levels of NCS-1 in the myenteric plexus and in axons in the smooth muscle layers; 23 +/- 2% of myenteric neurons were NCS-1 positive, with staining restricted to the largest neurons. NCS-1-positive axons decreased to 13.3 +/- 0.4% of total axons by day 2 and dropped further to 7.0 +/- 0.1% by day 4, returning to control levels by day 16. Dual-label Western blot analysis showed that the expression of NCS-1 relative to PGP 9.5 decreased by 50% on day 4 but returned to control by day 16. The selective loss of NCS-1 during colitis may underlie the altered neural function seen in the inflamed intestine.
Animals↗
Over-expression of NCS-1 in AtT-20 cells affects ACTH secretion and storage.
The effect of over-expressing neuronal calcium sensor 1 (NCS-1) upon stimulated adrenocorticotrophin (ACTH) secretion was studied in AtT-20 cells. Stably-transfected AtT-20 cell lines over-expressing NCS-1 were obtained and compared to wild type AtT-20 cells. Corticotrophin releasing factor (CRF-41)-stimulated ACTH secretion from NCS-1 over-expressing cells was significantly reduced from that obtained in wild type AtT-20 cells. The effects of other stimulants of ACTH secretion from wild type AtT-20 cells were not attenuated in NCS-1 over-expressing cells. Calcium, guanosine 5'-O-(3'-thiotriphosphate) (GTP-gamma-S) and mastoparan stimulated ACTH secretion from permeabilised wild type AtT-20 and NCS-1 over-expressing AtT-20 cells with significantly greater ACTH secretion obtained in NCS-1 over-expressing cells. This study shows that in intact cells over-expression of NCS-1 reduces exocytotic ACTH release, while in permeabilised cells increases ACTH release. NCS-1 has multiple cellular targets and that directly and indirectly via these targets acts to increase the releasable ACTH pool while inhibiting CRF-41 stimulus-secretion coupling.
Adrenocorticotropic Hormone↗
Interaction of neuronal calcium sensor-1 (NCS-1) with phosphatidylinositol 4-kinase beta stimulates lipid kinase activity and affects membrane trafficking in COS-7 cells.
Phosphatidylinositol 4-kinases (PI4K) catalyze the first step in the synthesis of phosphatidylinositol 4,5-bisphosphate, an important lipid regulator of several cellular functions. Here we show that the Ca(2+)-binding protein, neuronal calcium sensor-1 (NCS-1), can physically associate with the type III PI4Kbeta with functional consequences affecting the kinase. Recombinant PI4Kbeta, but not its glutathione S-transferase-fused form, showed enhanced PI kinase activity when incubated with recombinant NCS-1, but only if the latter was myristoylated. Similarly, in vitro translated NCS-1, but not its myristoylation-defective mutant, was found associated with recombinant- or in vitro translated PI4Kbeta in PI4Kbeta-immunoprecipitates. When expressed in COS-7 cells, PI4Kbeta and NCS-1 formed a complex that could be immunoprecipitated with antibodies against either proteins, and PI 4-kinase activity was present in anti-NCS-1 immunoprecipitates. Expressed NCS-1-YFP showed co-localization with endogenous PI4Kbeta primarily in the Golgi, but it was also present in the walls of numerous large perinuclear vesicles. Co-expression of a catalytically inactive PI4Kbeta inhibited the development of this vesicular phenotype. Transfection of PI4Kbeta and NCS-1 had no effect on basal PIP synthesis in permeabilized COS-7 cells, but it increased the wortmannin-sensitive [(32)P]phosphate incorporation into phosphatidylinositol 4-phosphate during Ca(2+)-induced phospholipase C activation. These results together indicate that NCS-1 is able to interact with PI4Kbeta also in mammalian cells and may play a role in the regulation of this enzyme in specific cellular compartments affecting vesicular trafficking.
1-Phosphatidylinositol 4-Kinase↗
Overexpression of rat neuronal calcium sensor-1 in rodent NG108-15 cells enhances synapse formation and transmission.
The role of rat neuronal calcium sensor-1 (NCS-1), a Ca2+-binding protein, in synapse formation and transmitter release was examined in mouse neuroblastoma x rat glioma hybrid NG108-15 cells in culture. Wild-type NG108-15 cells expressed rodent NCS-1. Endogenous NCS-1 was partially co-localized with the synaptic protein SNAP-25 at the plasma membrane in both cell bodies and processes, but not with the Golgi marker [beta]-COP, an individual coat subunit of the coatomer complex present on Golgi-derived vesicles. In NG108-15 cells co-cultured with rat myotubes, partial co-localization of SNAP-25 and NCS-1 was observed at the plasma membrane of neurites and growth cones, some of which had synaptic contacts to muscle cells. Transient co-transfection of the rat NCS-1 cDNA and green fluorescent protein (GFP) resulted in NCS-1 overexpression in about 30 % of the cells as determined by fluorescence microscopy. The rate of functional synapse formation with co-cultured rat myotubes increased 2-fold as determined by the presence of miniature endplate potentials (MEPPs) in NCS-1-overexpressing NG108-15 cells compared to non- and mock-transfected cells. The number of neurites per cell, branches per neurite and length of neurites was slightly less in cells that were either transiently transfected (GFP-NCS-1-fluorescence positive) or stably transformed with NCS-1 compared to GFP-NCS-1-negative, non-transfected or mock-transfected NG108-15 cells. The number of action potentials that elicited endplate potentials increased in NG108-15 cells stably transformed with rat NCS-1. The mean number of quanta per impulse (m) increased 5-fold. These results show that NCS-1 functions to facilitate synapse formation, probably because of the increased quantal content of evoked acetylcholine release.
Acetylcholine↗
Gamma-hydroxybutyric acid-induced absence seizures in GluR2 null mutant mice.
In this electrophysiological study, we examined the susceptibility of GluR2 mutant null mice to absence seizures in comparison with wild-type controls. The prodrug of (GHB), gamma-butyrolactone (GBL) was given systemically to induce the absence seizures. We also tested the severity and duration of the seizure activity in this model. The results showed that the latency from GBL administration to onset of seizure was significantly prolonged in GluR2(-/-) mice when compared to GluR2(+/+) mice. The duration of spike-and-wave discharges (SWD) was also significantly decreased in the GluR2(-/-) mice. Ninety minutes following GBL administration, wild-type animals continued to exhibit intermittent SWD bursts while GluR2(-/-) mice had returned to baseline. These data suggest that the GluR2 subunit may be involved in the initiation and maintenance of absence seizures induced by GBL.
Animals↗
Learning and memory in S100-beta transgenic mice: an analysis of impaired and preserved function.
S100-beta, a calcium-binding astrocytic protein from chromosome 21, has been implicated in CNS function generally and the hippocampus in particular. Elevated levels of S100-beta have been observed reliably in the brains of patients with Alzheimer's Disease and Down Syndrome. Groups of transgenic mice, carrying multiple S100-beta gene copies, and nontransgenic controls were administered a series of behavioral tests (delayed spatial and nonspatial non-matching-to-sample, radial arm maze, socially acquired food preference) that assessed a wide range of cognitive functions. Consistent with the widespread presence of S100-beta throughout the brain, transgenic mice exhibited learning or memory impairment on all tasks. The dementia-like cognitive profile of S100-beta mice represents a promising model for studying comparable cognitive deficits associated with neurodegenerative diseases.
Animals↗
Gene targeting reveals a role for the glutamate receptors mGluR5 and GluR2 in learning and memory.
This work suggests that class I mGluRs are involved in long-term potentiation (LTP) at CA1 synapses within the hippocampus. Our data support a pathway linking class I-mGluRs with PKC and src to enhance the open probability of the NMDAR channel. This leads to LTP of the NMDAR, but not the AMPAR. We are currently analyzing double mGluR1 X mGluR5 knockouts with Collingridge for a loss of the LTP induction switch [Nature 368 (1994) 740.]. This induction of LTP of the NMDAR is necessary for "spatial" learning and memory to occur, since mice lacking the mGluR5 are deficient in the Morris water maze and context-dependent fear conditioning. We postulate that AMPARs may provide negative feedback inhibition to the NMDAR. Hence, in null mutants lacking the AMPAR subtype, GluR2, LTP in the CA1 region of hippocampal slices was markedly enhanced (twofold) and non-saturating, whereas neuronal excitability and paired-pulse facilitation were normal. The ninefold increase in Ca(2+) permeability, in response to kainate application, suggests one possible mechanism for enhanced LTP. Enhanced LTP could result from enhanced AMPAR channel conductance or increased recruiting of previously silent synapses. Since the GluR2 null mutants showed reduced exploration and impaired motor coordination, we could make no conclusion about its role in learning and memory. Future work will be directed to inducible deletion of GluR2 only in CA1 after development is complete. These results support the correlation between LTP and learning and memory.
Animals↗
Regulated expression of the neuronal calcium sensor-1 gene during long-term potentiation in the dentate gyrus in vivo.
Neuronal calcium sensor-1 (NCS-1), the mammalian homologue of frequenin, is a member of a highly conserved family of neuron-specific calcium-binding proteins which has been implicated in exocytosis and in multiple calcium-signalling pathways, suggesting a potential involvement in mechanisms of neuronal plasticity. Here, using in situ hybridization, we report an increased induction of the mRNA encoding NCS-1 in dentate granule cells following the induction of long-term potentiation in the awake rat. We show that NCS-1 mRNA levels are increased 1 and 3 h after long-term potentiation in an N-methyl-D-aspartate receptor-dependent manner, returning to baseline expression levels by 6 h. Electroconvulsive stimulation also induced NCS-1 mRNA transcription in the dentate gyrus, but at the different time of 6 h post-seizure, returning to baseline by 12 h. These results show that regulated expression of the NCS-1 gene is part of the transcriptional response associated with activity-dependent neuronal plasticity in vivo and suggest a molecular mechanism capable of mediating a functional change in synapse sensitivity to calcium and calcium-signalling pathways after long-term potentiation.
Animals↗
PLC-beta1, activated via mGluRs, mediates activity-dependent differentiation in cerebral cortex.
During development of the cerebral cortex, the invasion of thalamic axons and subsequent differentiation of cortical neurons are tightly coordinated. Here we provide evidence that glutamate neurotransmission triggers a critical signaling mechanism involving the activation of phospholipase C-beta1 (PLC-beta1) by metabotropic glutamate receptors (mGluRs). Homozygous null mutation of either PLC-beta1 or mGluR5 dramatically disrupts the cytoarchitectural differentiation of 'barrels' in the mouse somatosensory cortex, despite segregation in the pattern of thalamic innervation. Furthermore, group 1 mGluR-stimulated phosphoinositide hydrolysis is dramatically reduced in PLC-beta1-/- mice during barrel development. Our data indicate that PLC-beta1 activation via mGluR5 is critical for the coordinated development of the neocortex, and that presynaptic and postsynaptic components of cortical differentiation can be genetically dissociated.
Animals↗
PtdIns 4-kinasebeta and neuronal calcium sensor-1 co-localize but may not directly associate in mammalian neurons.
It was recently demonstrated that the yeast homologue of phosphatidylinositol 4-kinasebeta PIK1 is directly associated with frq1, the yeast homologue of mammalian neuronal calcium sensor-1 (NCS-1) (Hendricks et al., [1999] Nat. Cell Biol. 1:234- 241). This was a novel finding and suggests that a calcium binding protein activates and regulates PtdIns 4-kinasebeta. This finding had not been shown in mammalian cells and both PtdIns 4-kinasebeta and NCS-1 have been shown to have important roles in the regulation of exocytotic release associated with neurotransmission. The aims of this study were to determine if PtdIns 4-kinasebeta and NCS-1 directly associate in mammalian neural tissues. We show that the immunostaining pattern for PtdIns 4-kinasebeta and NCS-1 is co-localized throughout the neurites of newborn cultured dorsal root ganglia (DRG) neurons but not in E13 DRG neurons. We then provide biochemical evidence that PtdIns 4-kinasebeta may not be in physical association with NCS-1 in mammalian nervous tissue unlike that previously reported in yeast.
1-Phosphatidylinositol 4-Kinase↗
Overexpression of frequenin, a modulator of phosphatidylinositol 4-kinase, inhibits biosynthetic delivery of an apical protein in polarized madin-darby canine kidney cells.
Polyphosphoinositides regulate numerous steps in membrane transport. The levels of individual phosphatidylinositols are controlled by specific lipid kinases, whose activities and localization are in turn regulated by a variety of effectors. Here we have examined the effect of overexpression of frequenin, a modulator of phosphatidylinositol 4-kinase activity, on biosynthetic and postendocytic traffic in polarized Madin-Darby canine kidney cells. Endogenous frequenin was identified in these cells by polymerase chain reaction, Western blotting, and indirect immunofluorescence. Adenoviral-mediated overexpression of frequenin had no effect on early Golgi transport of membrane proteins, as assessed by acquisition of resistance to endoglycosidase H. However, delivery of newly synthesized influenza hemagglutinin from the trans-Golgi network to the apical cell surface was severely inhibited in cells overexpressing frequenin, whereas basolateral delivery of the polymeric immunoglobulin receptor was unaffected. Overexpression of frequenin did not affect postendocytic trafficking steps including apical and basolateral recycling and basal-to-apical transcytosis. We conclude that frequenin, and by inference, phosphatidylinositol 4-kinase, plays an important and selective role in apical delivery in polarized cells.
1-Phosphatidylinositol 4-Kinase↗
Enhanced calcium transients in glial cells in neonatal cerebellar cultures derived from S100B null mice.
S100B is the major low-affinity Ca(2+)-binding protein in astrocytes. In order to study the role of S100B in the maintenance of Ca(2+) homeostasis, we generated S100B null mice by a targeted inactivation of the S100B gene. Absence of S100B expression was demonstrated by Northern and Western blotting for S100B mRNA and protein, respectively, and immunoperoxidase staining of sections of various brain regions. S100B null mice were viable, fertile, and exhibited no overt behavioral abnormalities up to 12 months of age. On the basis of light microscopy and immunohistochemical staining, there were no discernable alterations in the distribution and morphology of astrocytes or neurons in sections of adult brains of these mice. Astrocytes in cerebellar cultures derived from 6-day-old S100B null mice exhibited enhanced Ca(2+) transients in response to treatment with KCl or caffeine. On the other hand, granule neurons, in the same cultures, exhibited normal Ca(2+) transients in response to treatment with KCl, caffeine, or N-methyl-d-aspartate. These results demonstrate a specific decrease in Ca(2+)-handling capacity in astrocytes derived from S100B null mice and suggest that S100B plays a role in the maintenance of Ca(2+) homeostasis in astrocytes.
Animals↗
Expression of frequenin at the frog (Rana) neuromuscular junction, muscle spindle and nerve.
Frequenin is a calcium binding protein previously implicated in the regulation of neurotransmission in Drosophila and Xenopus. We have used the frog (Rana pipiens) to study the localization and regulation of expression of frequenin-like molecules in the vertebrate peripheral nervous system. Affinity purified antibodies to frequenin recognize molecules in the neuromuscular junction, axons in the peripheral nerve, and neuronal processes in muscle spindles. Western blots of endplate regions, peripheral nerve, and brain, resulted in the labelling of a single 24 kDa band, which is the expected size for frequenin. These results suggest that frequenin expression is high in the frog peripheral nervous system, and may reflect a function for frequenin in synaptic transmission in vertebrates.
Animals↗
Endogenous Zn(2+) is required for the induction of long-term potentiation at rat hippocampal mossy fiber-CA3 synapses.
The functional role of the abundant Zn(2+) found in some hippocampal synapses has been an enigma. We show here, using N-[6-methoxy-8-quinolyl]-P-toluenesulfonamide (TSQ) staining, that chelatable-Zn(2+) can be removed from hippocampal synaptic boutons using dietary depletion or with Zn(2+) chelators. A chronic dietary deficiency of bouton Zn(2+) resulted in the impairment of long-term potentiation (LTP) at mossy fiber-CA3 synapses. The averaged normalized fEPSP slope 30 min after tetanus was 209 +/- 28% of baseline value in control (mean +/- SEM, n = 10), and 118 +/- 12% in Zn(2+)-deficient rats (mean +/- SEM, n = 12, P < 0.01). In the deficient rats with Zn(2+) supplements, mossy fiber LTP returned to normal levels. The acute depletion of bouton Zn(2+) in the hippocampal slice with membrane-permeable Zn(2+) chelators, dithizone, or diethyldithiocarbamic acid (DEDTC) blocked the induction of mossy fiber LTP. The mean amplitudes of EPSCs after tetanus were 194 +/- 22% of baseline value in control (n = 5), compared to 108 +/- 14% in dithizone (n = 6) and 101 +/- 12% in DEDTC (n = 5). The averaged value of LTP, at the associational commisural fiber-CA3 synapses, was 193 +/- 20% in the control (n = 6), compared to 182 +/- 21% (n = 6, P > 0.1) in the presence of dithizone. The blockade of mossy fiber LTP by dithizone was reversible after washout. In addition, normal LTP could be induced by tetanus if exogenous Zn(2+) was applied immediately following dithizone. Our results indicate that the endogenous Zn(2+) is specifically required for LTP induction at the mossy fiber input into CA3 neurons.
Animals↗
Purification of myristoylated and nonmyristoylated neuronal calcium sensor-1 using single-step hydrophobic interaction chromatography.
Neuronal calcium sensors (NCSs) belong to a family of Ca(2+)-binding proteins, which serve important functions in neurotransmission, and are highly conserved from yeast to humans. Overexpression of the neuronal calcium sensor-1, called frequenin in the fruit fly and in frog, increases the release of neurotransmitters. Studying the functional role of frequenin in mammals and understanding its structural dynamics is critically dependent on the availability of active purified protein. Neuronal calcium sensors like other members of the family share common structural features: they contain four EF-hands as potential binding sites for Ca(2+) and an N-terminal consensus sequence for myristoylation. Previously, recoverin, distantly related to NCSs, has been expressed and purified from Escherichia coli, involving a combination of different chromatographic steps. NCS-1 has earlier been purified adopting a two-step procedure used for recoverin purification. We have overexpressed NCS-1 from rat in its myristoylated and nonmyristoylated form in E. coli and purified it from crude lysates using a single-step hydrophobic interaction chromatography. The purified protein was identified by Western blotting and mass spectrometry and assayed for its ability to bind Ca(2+) using a Ca(2+) shift assay, terbium fluorescence, and Stains-all binding. The present protocol provides a rapid, more efficient and simplified, single-step method for purifying NCS-1 for structural and functional studies. This method can also be applied to purify related proteins of the superfamily.
Animals↗