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Biomedical subjects

E D Gundelfinger

Publications and source records attributed to E D Gundelfinger.

At least 19 recordsLinked to original sources
PubMed2002

Evidence for different functional properties of the neuronal calcium sensor proteins VILIP-1 and VILIP-3: from subcellular localization to cellular function.

The visinin-like-proteins VILIP-1 and -3 are EF-hand calcium-binding proteins and belong to the family of neuronal calcium sensor (NCS) proteins. Members of this family are involved in the calcium-dependent regulation of signal transduction cascades mainly in the nervous system. VILIP-1 and VILIP-3 are expressed in different populations of neuronal cells. To gain insights into the different functional characteristics of VILIP-1 and -3, we have compared the localization of the proteins in intact cells and the calcium-dependent membrane association in subcellular fractions. Furthermore, we have investigated the different functional properties of the two proteins in activating cGMP signal pathways and have defined different sets of protein interaction partners. Our data indicate that VILIP-3, which is mainly expressed in Purkinje cells, and VILIP-1, which is expressed in granule cells in the cerebellum, show a different calcium-dependent subcellular localization, may activate different cellular signaling pathways, and thus have signaling functions which seem to be cell-type specific.

Animals↗
PubMed2002

The neuron-specific Ca2+-binding protein caldendrin: gene structure, splice isoforms, and expression in the rat central nervous system.

Caldendrin is the founder member of a recently discovered family of calmodulin-like proteins, which are highly abundant in brain. In this study we examined the organization of the murine and human caldendrin gene as well as the expression pattern of transcripts for caldendrin and two novel splice variants. In addition the distribution of caldendrin in rat brain has been assessed by immunohistochemistry. Caldendrin is localized to the somatodendritic compartment of a subpopulation of mainly principal neurons in brain regions with a laminar organization and is present only at a subset of mature excitatory synapses. Caldendrin immunoreactivity (IR) is tightly associated with the cortical cytoskeleton, enriched in the postsynaptic density (PSD) fraction, and associates late during development with the synaptic cytomatrix. The expression is highly heterogenous within cortex, with highest levels of caldendrin IR in layer III of the piriform and layer II/III of the somatosensory cortex. The segregated cortical distribution to areas, which represent the most important primary sensory systems of the rodent brain, may reflect different requirements for dendritic Ca2+-signaling in these neurons. The presence of caldendrin in the PSD of distinct synapses may have important implications for Ca2+-modulated processes of synaptic plasticity.

Alternative Splicing↗
PubMed2002

ProSAP/Shank postsynaptic density proteins interact with insulin receptor tyrosine kinase substrate IRSp53.

The ProSAP/Shank family of multidomain proteins of the postsynaptic density (PSD) can either directly or indirectly interact with NMDA-type and metabotropic glutamate receptors and the actin-based cytoskeleton. In a yeast two hybrid screen utilizing a proline-rich domain that is highly conserved among the ProSAP/Shank family members, we isolated several cDNA clones coding for the insulin receptor substrate IRSp53. The specificity of this interaction was confirmed in transfected COS cells. Co-immunoprecipitation of IRSp53 and ProSAP2 solubilized from rat brain membranes indicates that the interaction occurs in vivo. The C-terminal SH3 domain of IRSp53 is responsible for the interaction with a novel proline-rich consensus sequence of ProSAP/Shank that was characterized by mutational analysis. IRSp53 is a substrate for the insulin receptor in the brain and acts downstream of small GTPases of the Rho family. Binding of Cdc42Hs to IRSp53 induces actin filament assembly, reorganization and filopodia outgrowth in neuronal cell lines. Our data suggest that IRSp53 can be recruited to the PSD via its ProSAP/Shank interaction and may contribute to the morphological reorganization of spines and synapses after insulin receptor and/or Cdc42Hs activation.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Localization of the presynaptic cytomatrix protein Piccolo at ribbon and conventional synapses in the rat retina: comparison with Bassoon.

In recent years significant progress has been made in the elucidation of the molecular assembly of the postsynaptic density at synapses, whereas little is known as yet about the components of the presynaptic active zone. Piccolo and Bassoon, two structurally related presynaptic cytomatrix proteins, are highly concentrated at the active zones of both excitatory and inhibitory synapses in rat brain. In this study we used immunocytochemistry to examine the cellular and ultrastructural localization of Piccolo at synapses in the rat retina and compared it with that of Bassoon. Both proteins showed strong punctate immunofluorescence in the outer and the inner plexiform layers of the retina. They were found presynaptically at glutamatergic ribbon synapses and at conventional GABAergic and glycinergic synapses. Although the two proteins were coexpressed at all photoreceptor ribbon synapses and at some conventional amacrine cell synapses, at bipolar cell ribbon synapses only Piccolo was present. Our data demonstrate similarities but also differences in the molecular composition of the presynaptic apparatuses of the synapses in the retina, differences that may account for the functional differences observed between the ribbon and the conventional amacrine cell synapses and between the photoreceptor and the bipolar cell ribbon synapses in the retina.

Animals↗
PubMed2001

Synaptic scaffolding proteins in rat brain. Ankyrin repeats of the multidomain Shank protein family interact with the cytoskeletal protein alpha-fodrin.

The postsynaptic density is the ultrastructural entity containing the neurotransmitter reception apparatus of excitatory synapses in the brain. A recently identified family of multidomain proteins termed Src homology 3 domain and ankyrin repeat-containing (Shank), also known as proline-rich synapse-associated protein/somatostatin receptor-interacting protein, plays a central role in organizing the subsynaptic scaffold by interacting with several synaptic proteins including the glutamate receptors. We used the N-terminal ankyrin repeats of Shank1 and -3 to search for interacting proteins by yeast two-hybrid screening and by affinity chromatography. By cDNA sequencing and mass spectrometry the cytoskeletal protein alpha-fodrin was identified as an interacting molecule. The interaction was verified by pull-down assays and by coimmunoprecipitation experiments from transfected cells and brain extracts. Mapping of the interacting domains of alpha-fodrin revealed that the highly conserved spectrin repeat 21 is sufficient to bind to the ankyrin repeats. Both interacting partners are coexpressed widely in the rat brain and are colocalized in synapses of hippocampal cultures. Our data indicate that the Shank1 and -3 family members provide multiple independent connections between synaptic glutamate receptor complexes and the cytoskeleton.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Cholinergic and GABAergic pathways in fly motion vision.

BACKGROUND: The fly visual system is a highly ordered brain structure with well-established physiological and behavioral functions. A large number of interneurons in the posterior part of the third visual neuropil, the lobula plate tangential cells (LPTCs), respond to visual motion stimuli. In these cells the mechanism of motion detection has been studied in great detail. Nevertheless, the cellular computations leading to their directionally selective responses are not yet fully understood. Earlier studies addressed the neuropharmacological basis of the motion response in lobula plate interneurons. In the present study we investigated the distribution of the respective neurotransmitter receptors in the fly visual system, namely nicotinic acetylcholine receptors (nAChRs) and GABA receptors (GABARs) demonstrated by antibody labeling. RESULTS: The medulla shows a laminar distribution of both nAChRs and GABARs. Both receptor types are present in layers that participate in motion processing. The lobula also shows a characteristic layering of immunoreactivity for either receptor in its posterior portion. Furthermore, immunostaining for nAChRs and GABARs can be observed in close vicinity of lobula plate tangential cells. Immunostaining of GABAergic fibers suggests that inhibitory inputs from the medulla are relayed through the lobula to the lobula plate rather than through direct connections between medulla and lobula plate. CONCLUSIONS: The interaction of excitatory and inhibitory pathways is essential for the computation of visual motion responses and discussed in the context of the Reichardt model for motion detection.

Animals↗
PubMed2001

The presynaptic cytomatrix of brain synapses.

Synapses are principal sites for communication between neurons via chemical messengers called neurotransmitters. Neurotransmitters are released from presynaptic nerve terminals at the active zone, a restricted area of the cell membrane situated exactly opposite to the postsynaptic neurotransmitter reception apparatus. At the active zone neurotransmitter-containing synaptic vesicles (SVs) dock, fuse, release their content and are recycled in a strictly regulated manner. The cytoskeletal matrix at the active zone (CAZ) is thought to play an essential role in the organization of this SV cycle. Several multi-domain cytoskeleton-associated proteins, including RIM, Bassoon, Piccolo/Aczonin and Munc-13, have been identified, which are specifically localized at the active zone and thus are putative molecular components of the CAZ. This review will summarize our present knowledge about the structure and function of these CAZ-specific proteins. Moreover, we will review our present view of how the exocytotic and endocytic machineries at the site of neurotransmitter release are linked to and organized by the presynaptic cytoskeleton. Finally, we will summarize recent progress that has been made in understanding how active zones are assembled during nervous system development.

Animals↗
PubMed2001

Kainate-induced seizures alter protein composition and N-methyl-D-aspartate receptor function of rat forebrain postsynaptic densities.

The postsynaptic density is a highly dynamic structure, which is reorganized in an activity-dependent manner. An animal model for temporal lobe epilepsy, i.e. kainate-induced limbic seizures in rats, was used to study changes in postsynaptic density composition after extensive synaptic activity. Six hours after kainate injection, the protein content of the postsynaptic density fractions from rats that developed strong seizures was increased three-fold compared to saline-treated controls. Immunoblot analysis revealed that the relative amounts of metabotropic glutamate receptor 1alpha, N-ethylmaleimide-sensitive fusion protein, protein kinases C, Fyn and TrkB, as well as the neuronal nitric oxide synthase, were significantly higher in seizure-developing than in control rats. In contrast, the relative contents of the kainate receptor KA2 subunit, beta-actin, alpha-adducin and the membrane-associated guanylate kinase homolog SAP90/PSD-95 were decreased. The relative amounts of additional postsynaptic density proteins, including alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-D-aspartate receptor subunits, calcium/calmodulin-dependent kinase type II, casein kinase 2, tubulin, microtubule-associated protein 2B, the membrane-associated guanylate kinase homolog SAP102, and proline-rich synapse-associated protein 1/cortactin binding protein 1/Shank2 remained essentially unchanged. To assess possible changes in postsynaptic performance, postsynaptic densities were isolated from control and epileptic rats, incorporated into giant liposomes and N-methyl-D-aspartate receptor currents were recorded. A significant reduction in the mean conductance was observed in patches containing postsynaptic densities from animals with high seizure activity. This was due to the presence of reduced conductance levels in each membrane patch compared to control postsynaptic density preparations. From these data, we suggest that intense synaptic activity associated with seizures modifies the composition of postsynaptic densities and has profound consequences on the function of the N-methyl-D-aspartate receptors present in them. This rearrangement may accompany impairment of synaptic plasticity.

Animals↗
PubMed2001

Assembling the presynaptic active zone: a characterization of an active one precursor vesicle.

The active zone is a specialized region of the presynaptic plasma membrane where synaptic vesicles dock and fuse. In this study, we have investigated the cellular mechanism underlying the transport and recruitment of the active zone protein Piccolo into nascent synapses. Our results show that Piccolo is transported to nascent synapses on an approximately 80 nm dense core granulated vesicle together with other constituents of the active zone, including Bassoon, Syntaxin, SNAP-25, and N-cadherin, as well as chromogranin B. Components of synaptic vesicles, such as VAMP 2/synaptobrevin II, synaptophysin, synaptotagmin, or proteins of the perisynaptic plasma membrane such as GABA transporter 1 (GAT1), were not present. These studies demonstrate that the presynaptic active zone is formed in part by the fusion of an active zone precursor vesicle with the presynaptic plasma membrane.

Animals↗
PubMed2001

Increased number of nitric oxide synthase immunoreactive Purkinje cells and dentate nucleus neurons in schizophrenia.

There is growing interest in the cerebellum as a site of neuropathological changes in schizophrenia. Reports showing that schizophrenics have higher nitric oxide synthase (NOS) activity and MAPKinase levels in the vermis, point to possible aberrations in the cerebellar signal transduction of schizophrenics. It has been speculated that Ca(2+)-dependent extracellular to intracellular signal transduction may be disrupted in the cerebellum of schizophrenics. We decided to test this hypothesis by studying the nitrergic system and markers of the Ca(2+)-triggered signal cascade in the cerebellum of schizophrenics, depressives and controls. The cellular distribution of two calcium sensor proteins (VILIP-1 and VILIP-3) and of neuronal NOS immunoreactivity was studied morphometrically in the flocculonodulus, the inferior vermis and the dentate nucleus of 9 schizophrenics, 7 depressive patients and 9 matched controls. In comparison to controls and depressed patients there were fewer Nissl-stained neurons in the dentate nucleus of schizophrenics. The number of NOS-expressing Purkinje neurons was however strongly increased. In the flocculonodulus and the vermis no differences between the groups were found with regard to the density of Nissl-stained Purkinje cells. The number of NOS-expressing Purkinje neurons was increased in schizophrenics, however. No differences between schizophrenics, depressives and controls were found in the number of VILIP-1 immunoreactive dentate nucleus neurons and VILIP-3 immunoreactive vermal and flocculonodular Purkinje cells. Our data provide further histochemical evidence in favor of structural abnormalities in discrete cerebellar regions of schizophrenics. They confirm and extend earlier reports of increased cerebellar NOS immunoreactivity in schizophrenia and point to possible neurodevelopmental disturbances. Our failure to show an altered expression of two calcium sensor proteins possibly points to a less important role of calcium signaling in cerebellar pathology of the disease.

Adult↗
PubMed2001

Semaphorin4F interacts with the synapse-associated protein SAP90/PSD-95.

Semaphorins are a family of secreted and membrane-associated proteins involved in growth cone guidance during development. Here, we describe the interaction of Semaphorin4F (Sema4F) with the post-synaptic density protein SAP90/PSD-95. Using the yeast two-hybrid system and coprecipitation assays we were able to show an interaction between the extreme C-terminus of Sema4F and the PDZ domains of SAP90/PSD-95. Heterologous coexpression of a chimeric EphrinB1/Semaphorin4F protein with SAP90/PSD-95 in COS cells leads to translocation of SAP90/PSD-95 from the cytosol to the membrane. Deletion analysis shows that this translocation activity of Sema4F is completely dependent on the presence of the last three C-terminal amino acids. In addition, Sema4F immunoreactivity is present in synaptosome fractions and enriched in post-synaptic density fractions. Consistently, in cultured hippocampal neurons, we demonstrate punctate colocalization of Sema4F and SAP90/PSD-95 in dendrites, furthermore we found colocalization of Sema4F with synapsin1 suggesting a synaptic localization. Our data implicate a new functional context for semaphorins at glutamatergic synapses.

Amino Acid Sequence↗
PubMed2001

Intracellular neuronal calcium sensor (NCS) protein VILIP-1 modulates cGMP signalling pathways in transfected neural cells and cerebellar granule neurones.

The family of intracellular neuronal calcium-sensors (NCS) belongs to the superfamily of EF-hand proteins. Family members have been shown by in vitro assays to regulate signal cascades in retinal photoreceptor cells. To study the functions of NCS proteins not expressed in photoreceptor cells we examined Visinin-like protein-1 (VILIP-1) effects on signalling pathways in living neural cells. Visinin-like protein-1 expression increased cGMP levels in transfected C6 and PC12 cells. Interestingly, in transfected PC12 cells stimulation was dependent on the subcellular localization of VILIP-1. In cells transfected with membrane-associated wild-type VILIP-1 particulate guanylyl cyclase (GC) was stimulated more strongly than soluble GC. In contrast, deletion of the N-terminal myristoylation site resulted in cytosolic localization of VILIP-1 and enhanced stimulation of soluble GC. To study the molecular mechanisms underlying GC stimulation VILIP-1 was examined to see if it can physically interact with GCs. A direct physical interaction of VILIP-1 with the recombinant catalytic domain of particulate GCs-A, B and with native GCs enriched from rat brain was observed in GST pull-down as well as in surface plasmon resonance interaction studies. Furthermore, following trituration of recombinant VILIP-1 protein into cerebellar granule cells the protein influenced only signalling by GC-B. Together with the observed colocalization of GC-B, but not GC-A, with VILIP-1 in cerebellar granule cells, these results suggest that VILIP-1 may be a physiological regulator of GC-B.

Animals↗
PubMed2001

Neurocan is dispensable for brain development.

Neurocan is a component of the extracellular matrix in brain. Due to its inhibition of neuronal adhesion and outgrowth in vitro and its expression pattern in vivo it was suggested to play an important role in axon guidance and neurite growth. To study the role of neurocan in brain development we generated neurocan-deficient mice by targeted disruption of the neurocan gene. These mice are viable and fertile and have no obvious deficits in reproduction and general performance. Brain anatomy, morphology, and ultrastructure are similar to those of wild-type mice. Perineuronal nets surrounding neurons appear largely normal. Mild deficits in synaptic plasticity may exist, as maintenance of late-phase hippocampal long-term potentiation is reduced. These data indicate that neurocan has either a redundant or a more subtle function in the development of the brain.

Animals↗
PubMed2001

The cortactin-binding postsynaptic density protein proSAP1 in non-neuronal cells.

Proline-rich synapse-associated protein-1 (ProSAP1) is a neuronal PDZ domain-containing protein that has recently been identified as an essential element of the postsynaptic density. Via its interaction with the actin-binding protein cortactin and its integrative function in the organization of neurotransmitter receptors, ProSAP1 is believed to be involved in the linkage of the postsynaptic signaling machinery to the actin-based cytoskeleton, and may play a role in the cytoskeletal rearrangements that underlie synaptic plasticity. As a result of our ongoing studies on the distribution and function of this novel PDZ domain protein, we now report that the expression of ProSAP1 is restricted neither to neurons and interneuronal junctions nor to the nervous system. Using immunohistochemical techniques in conjunction with specific antibodies, we found that, in the CNS, ProSAP1 can be detected in certain glial cells, such as ependymal cells, tanycytes, subpial/radial astrocytes, and in the choroid plexus epithelium. Moreover, our immunohistochemical analyses revealed the presence of ProSAP1 in endocrine cells of the adenohypophysis and of the pancreas, as well as in non-neuronal cell types of other organs. In the pancreas, ProSAP1 immunoreactivity was also localized in the duct system of the exocrine parenchyma. Our findings demonstrate that, in addition to neurons, ProSAP1 is present in various non-neuronal cells, in which it may play a crucial role in the dynamics of the actin-based cytoskeleton. (J Histochem Cytochem 49:639-648, 2001)

Animals↗
PubMed2001

Distribution of transcript and protein isoforms of the synaptic glycoprotein neuroplastin in rat retina.

PURPOSE: To examine the expression and localization of the neuroplastins (np), two synapse-enriched members of the immunoglobulin (Ig) superfamily of cell-adhesion molecules, in the developing and adult retina and optic nerve. METHODS: Expressions of the two isoforms np55 and np65 and carboxyl-terminal splice variants were investigated by immunocytochemistry, Western blot analysis, RT-PCR, and in situ hybridization. RESULTS: Immunoreactivity for both neuroplastins was confined to the two synaptic layers of the retina: the inner (IPL) and outer plexiform layer (OPL). Significant overlap was found in staining at synaptic structures with synaptophysin. A large proportion of immunoreactivity for both isoforms, however, was of perisynaptic origin. In situ hybridization studies were suggestive of a pre- and postsynaptic localization of np65 in the OPL. Transcripts for np55 were already present at birth in the inner retina, but the hybridization signals increased during postnatal development. Np65 transcripts and immunosignals appeared at later developmental ages, concomitant with synapse formation in the OPL. Several C-terminal neuroplastin cDNA clones harbor an insert of 12 bp, coding for four amino acids (DDEP) in the intracellular domain of neuroplastins. Splice isoforms containing the insert exhibited a developmental expression pattern similar to that of np55; however, both neuroplastins could harbor the C-terminal insert. Neuroplastins were also detected in optic nerve homogenates. RT-PCR and blockade of axonal transport by nerve crush confirmed transcript and protein expression in optic nerve tissue. CONCLUSIONS: The findings suggest a role for neuroplastins in cell adhesion in the plexiform layers during histogenesis, as well as in maintenance of connections between specific cellular structures.

Animals↗
PubMed2000

Intramolecular interactions regulate SAP97 binding to GKAP.

Membrane-associated guanylate kinase homologs (MAGUKs) are multidomain proteins found to be central organizers of cellular junctions. In this study, we examined the molecular mechanisms that regulate the interaction of the MAGUK SAP97 with its GUK domain binding partner GKAP (GUK-associated protein). The GKAP-GUK interaction is regulated by a series of intramolecular interactions. Specifically, the association of the Src homology 3 (SH3) domain and sequences situated between the SH3 and GUK domains with the GUK domain was found to interfere with GKAP binding. In contrast, N-terminal sequences that precede the first PDZ domain in SAP97, facilitated GKAP binding via its association with the SH3 domain. Utilizing crystal structure data available for PDZ, SH3 and GUK domains, molecular models of SAP97 were generated. These models revealed that SAP97 can exist in a compact U-shaped conformation in which the N-terminal domain folds back and interacts with the SH3 and GUK domains. These models support the biochemical data and provide new insights into how intramolecular interactions may regulate the association of SAP97 with its binding partners.

Adaptor Proteins, Signal Transducing↗