Biomedical subjects

O Mundigl

Publications and source records attributed to O Mundigl.

17 recordsLinked to original sources

PubMed2001

Digital imaging microscopy of firefly luciferase activity to directly monitor differences in cell transduction efficiencies between AdCMVLuc and Ad5LucRGD vectors having different cell binding properties.

The luciferase reporter gene incorporated into adenoviral vectors is very useful for monitoring viral transduction of different cell types or for comparing the transduction efficiency of different viral constructs of one cell type. Luciferase protein expression can be detected and quantified with very high sensitivity from whole cells or organ extracts. However, its disadvantages become obvious when aiming at evaluation of transduction events at the single cell level. The results obtained from whole cell extracts cannot be directly correlated to single cell events. In this paper direct cellular luciferase imaging using cell permeable luciferin substrates is applied for comparative analysis of cellular transduction events by two adenoviral vectors with different cell binding properties. Using digital imaging microscopy we show a more than ten-fold increase in transduction efficiency by Ad5LucRGD vectors versus AdCMVLuc vectors on human A549 cells.

Adenoviridae↗

PubMed1998

Amphiphysin I antisense oligonucleotides inhibit neurite outgrowth in cultured hippocampal neurons.

Amphiphysin I is an SH3 domain-containing neuronal protein, enriched in axon terminals, which was reported to act as a physiological binding partner for dynamin I in synaptic vesicle endocytosis. Rvs167 and Rvs161, the yeast homologs of amphiphysin I, have been implicated in endocytosis, actin function, and cell polarity. Now we have explored the possibility that amphiphysin I also may have a role in actin dynamics and cell polarity by testing the effect of amphiphysin I suppression on neurite outgrowth. Freshly plated hippocampal neurons were exposed to antisense oligonucleotides via a new delivery system based on a polycationic amphipathic polymer, PS980. Western blot analysis revealed that amphiphysin I levels steadily increased with neuronal differentiation, whereas in antisense-treated cultures amphiphysin I levels were reduced to approximately 10% of control levels at 48 hr. Concomitantly, a collapse of growth cones and a severe inhibition of neurite outgrowth and axon formation were observed. A similar effect was observed previously after dynamin I suppression in the same culture system (). We also have found that amphiphysin I and dynamin I colocalize in developing neurons at all developmental stages and that a pool of both proteins is colocalized with actin patches at the leading edge of growth cones. Our findings suggest a conserved role of the amphiphysin protein family in the dynamics of the cortical cell cytoskeleton and provide new evidence for a close functional link between amphiphysin I and dynamin I.

Actins↗

PubMed1996

Mobility of synaptic vesicles in nerve endings monitored by recovery from photobleaching of synaptic vesicle-associated fluorescence.

In nerve terminals, synaptic vesicles form large clusters anchored to the presynaptic plasmalemma. Recently, FM1-43 photobleaching experiments carried out a frog motor end-plates demonstrated lack of lateral intermixing of synaptic vesicles within clusters, even during sustained nerve terminal stimulation (Henkel and Betz, 1995; Henkel et al., 1996b). We now have investigated the mobility of synaptic vesicle membranes during the endocytic limb of their exo-endocytic cycle. To this aim, we have carried out photobleaching experiments on nerve terminals of hippocampal neurons prelabeled with CY3-conjugated antibodies directed against lumenal epitopes of synaptotagmin I. This conjugate is taken up specifically by synaptic vesicle membranes during endocytosis and then is recovered in newly formed synaptic vesicles. Using this method, we show that synaptic vesicle membranes intermix after endocytosis. Staurosporine, which at hippocampal synapses partially inhibits unloading of FM1-43, but does not block uptake of antibody probes, prevents this intermixing. Our results indicate that synaptic vesicle docking and/or fusion with the plasmalemma correlate with the release of their membranes from a restraining matrix that hinders their lateral mobility. They suggest that membrane intermediates involved in synaptic vesicle reformation interact with a distinct, highly dynamic cytoskeleton and that newly formed synaptic vesicles are recaptured at random within vesicle clusters. Staurosporine, by inhibiting mobility within the terminal, may favor recapture of new vesicles near sites of endocytosis.

Animals↗

PubMed1996

Polarized expression of GABA transporters in Madin-Darby canine kidney cells and cultured hippocampal neurons.

At least three high affinity Na+- and Cl--dependent gamma-aminobutyric acid (GABA) transporters are known to exist in the rat and mouse brain. These transporters share 50-65% amino acid sequence identity with the kidney betaine transporter which also transports GABA but with lower affinity. The betaine transporter (BGT) is expressed on the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. Recent evidence suggests that the signals and mechanisms involved in membrane protein sorting share many functional characteristics in polarized neurons and epithelial cells. It was previously shown that the rat GABA transporter GAT-1 is located in the presynaptic membrane of axons where it plays a role in terminating GABAergic neurotransmission. When expressed in MDCK cells by transfection, GAT-1 was sorted to the apical membrane. In this report, we have localized the other two GABA transporters, GAT-2 and GAT-3, in transfected MDCK cells by GABA uptake, immunofluorescence, and cell surface biotinylation. GAT-3, like GAT-1, localized to the apical membrane of MDCK cells while GAT-2, like BGT, localized to the basolateral membrane. We have also expressed BGT in low density cultures of hippocampal neurons by microinjection and immunolocalized it to the dendrites. The distribution of GAT-3 in these neurons after transfection was axonal as well as somatodendritic. These results indicate that highly homologous subtypes of GABA transporters are sorted differently when expressed in epithelial cells or neurons and suggest that these two cell types share the capacity to distinguish among these isoforms and target them to distinct destinations.

Amino Acid Sequence↗

PubMed1996

A role of amphiphysin in synaptic vesicle endocytosis suggested by its binding to dynamin in nerve terminals.

Amphiphysin, a major autoantigen in paraneoplastic Stiff-Man syndrome, is an SH3 domain-containing neuronal protein, concentrated in nerve terminals. Here, we demonstrate a specific, SH3 domain-mediated, interaction between amphiphysin and dynamin by gel overlay and affinity chromatography. In addition, we show that the two proteins are colocalized in nerve terminals and are coprecipitated from brain extracts consistent with their interactions in situ. We also report that a region of amphiphysin distinct from its SH3 domain mediates its binding to the alpha c subunit of AP2 adaptin, which is also concentrated in nerve terminals. These findings support a role of amphiphysin in synaptic vesicle endocytosis.

Adaptor Protein Complex 2↗

PubMed1996

The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin.

Strong evidence implicates clathrin-coated vesicles and endosome-like vacuoles in the reformation of synaptic vesicles after exocytosis, and it is generally assumed that these vacuoles represent a traffic station downstream from clathrin-coated vesicles. To gain insight into the mechanisms of synaptic vesicle budding from endosome-like intermediates, lysed nerve terminals and nerve terminal membrane subfractions were examined by EM after incubations with GTP gamma S. Numerous clathrin-coated budding intermediates that were positive for AP2 and AP180 immunoreactivity and often collared by a dynamin ring were seen. These were present not only on the plasma membrane (Takei, K., P.S. McPherson, S.L.Schmid, and P. De Camilli. 1995. Nature (Lond.). 374:186-190), but also on internal vacuoles. The lumen of these vacuoles retained extracellular tracers and was therefore functionally segregated from the extracellular medium, although narrow connections between their membranes and the plasmalemma were sometimes visible by serial sectioning. Similar observations were made in intact cultured hippocampal neurons exposed to high K+ stimulation. Coated vesicle buds were generally in the same size range of synaptic vesicles and positive for the synaptic vesicle protein synaptotagmin. Based on these results, we suggest that endosome-like intermediates of nerve terminals originate by bulk uptake of the plasma membrane and that clathrin- and dynamin-mediated budding takes place in parallel from the plasmalemma and from these internal membranes. We propose a synaptic vesicle recycling model that involves a single vesicle budding step mediated by clathrin and dynamin.

Adaptor Protein Complex 2↗

PubMed1995

v- and t-SNAREs in neuronal exocytosis: a need for additional components to define sites of release.

Synaptic vesicle recycling is a specialized form of membrane recycling which takes place in all cells between early endosomes and the plasmalemma. Synaptic vesicles exocytosis is highly regulated and occurs only at presynaptic active zones. In contrast, exocytosis of endosome-derived vesicles of the housekeeping recycling pathway takes place constitutively and throughout the cell surface. Since v- and t-SNAREs play a key role in membrane interactions leading to fusion, unique v- and t-SNAREs may be implicated in synaptic vesicle exocytosis. It was found, however, that the same v-SNAREs of the synaptobrevin family are found both on synaptic vesicles and on endosome-derived vesicles which undergo constitutive fusion. Likewise, t-SNAREs which act as plasmalemmal receptors for synaptic vesicles are not restricted to synaptic active zones. Thus, v- and t-SNAREs interactions may define which organelles can fuse with the plasmalemma, but require additional components to define properties of the exocytotic reaction which are specific for distinct classes of secretory organelles.

Animals↗

PubMed1995

Mechanisms of synaptogenesis in hippocampal neurons in primary culture.

To improve our understanding of the mechanisms which regulate the formation and the functional maturation of synaptic contacts between neurons, we used hippocampal neurons maintained in primary cultures as experimental system. In this model, which offers several advantages for the study of neuronal development and synaptogenesis, we investigated some of the cellular mechanisms underlying the formation of presynaptic and postsynaptic compartments.

Animals↗

PubMed1995

Cellubrevin and synaptobrevins: similar subcellular localization and biochemical properties in PC12 cells.

There is strong evidence to indicate that proteins of the synaptobrevin family play a key role in exocytosis. Synaptobrevin 1 and 2 are expressed at high concentration in brain where they are localized on synaptic vesicles. Cellubrevin, a very similar protein, has a widespread tissue distribution and in fibroblasts is localized on endosome-derived, transferin receptor-positive vesicles. Since brain cellubrevin is not detectable in synaptic vesicles, we investigated whether cellubrevin and the synaptobrevins are differentially targeted when co-expressed in the same cell. We report that in the nervous system cellubrevin is expressed at significant levels only by glia and vascular cells. However, cellubrevin is coexpressed with the two synaptobrevins in PC12 cells, a neuroendocrine cell line which contains synaptic vesicle-like microvesicles. In PC12 cells, cellubrevin has a distribution very similar to that of synaptobrevin 1 and 2. The three proteins are targeted to neurites which exclude the transferrin receptor and are enriched in synaptic-like microvesicles and dense-core granules. They are recovered in the synaptic-like microvesicle peak of glycerol velocity gradients, have a similar distribution in isopycnic fractionation and are coprecipitated by anti-synaptobrevin 2 immunobeads. Finally, cellubrevin, like the synaptobrevins, interact with the neuronal t-SNAREs syntaxin 1 and SNAP-25. These results suggest that cellubrevin and the synaptobrevins have similar function and do not play a specialized role in constitutive and regulated exocytosis, respectively.

Amino Acid Sequence↗

PubMed1995

Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with CY3-conjugated antibodies directed against the lumenal domain of synaptotagmin.

Antibodies directed against the lumenal domain of synaptotagmin I conjugated to CY3 (CY3-Syt1-Abs) and video microscopy were used to study the dynamics of synaptic vesicles in cultured hippocampal neurons. When applied to cultures after synapse formation, CY3-Syt1-Abs produced a strong labeling of presynaptic vesicle clusters which was markedly increased by membrane depolarization. The increase of the rate of CY3-Syt1-Ab uptake in a high K+ medium was maximal during the first few minutes but persisted for as long as 60 min. In axons developing in isolation, CY3-Syt1-Abs, in combination with electron microscopy immunocytochemistry, revealed the presence of synaptic vesicle clusters which move in bulk in anterograde and retrograde direction. Clusters are present both in the axon shaft and in filopodia but not in the filopodia of the growth cone. Both presynaptic vesicle clusters and clusters present in isolated axons were disrupted by okadaic acid as previously shown for synaptic vesicle clusters at the frog neuromuscular junction. These findings indicate that synaptic vesicle aggregation may occur independently of cell-cell interaction, but that, in the absence of a synaptic contact, vesicle clusters are not stably anchored to a given region of the cell surface. Labeling of synaptic vesicles in immature isolated neurons was found to be depolarization and Ca2+ dependent, demonstrating that Ca(2+)-regulated exocytosis is an intrinsic characteristic of synaptic vesicles irrespective of their localization at a synapse.

Animals↗

PubMed1995

A radioimmunoassay to monitor synaptic activity in hippocampal neurons in vitro.

Exocytosis of synaptic vesicles (SV) results in the surface exposure of lumenal epitopes of SV proteins. We have recently described the use of antibodies directed against the lumenal N-terminus of synaptotagmin I (Sytlum-Abs) to morphologically monitor exo-endocytic recycling of SVs. We report here that a radioimmunoassay based on these antibodies can be used to quantify levels of synaptic activity in primary neuronal cultures. High density cultures of hippocampal neurons grown in the absence of glia were used for these studies. A significant cell surface pool of synaptotagmin I immunoreactivity was detectable by Sytlum-Abs at steady state. The increase in the amount of Sytlum-Abs which became cell bound during a 3 min incubation at 37 degrees C over the Ab bound to this cell surface pool, was substantially higher in depolarizing media containing extracellular Ca2+ than in Ca(2+)-free media. Incubation of the cultures with Sytlum-Abs for longer time periods indicated a sustained increase in the rate of SV exocytosis in depolarizing media which lasted for at least 1 h. This increase was completely abolished by pretreating the neurons with tetanus toxin and this block correlated with a disappearance of synaptobrevin immunoreactivity. This radioimmunoassay therefore offers a new way to monitor SV exocytosis of neuronal populations in vitro irrespective of the type of neurotransmitter secreted and of postsynaptic effects.

Animals↗

PubMed1994

Formation of synaptic vesicles.

Synaptic vesicles (SVs) are specialized secretory organelles used for the fast and focal signaling between nerve cells. They are small and homogeneous in size (50 nm), and contain non-peptide neurotransmitters such as glutamate, gamma-aminobutyric acid (GABA) and acetylcholine. The exocytosis of SVs occurs at low rates in resting nerve terminals and is greatly stimulated by depolarization-induced Ca2+ influx. Following exocytosis, SV membranes are rapidly retrieved, refilled locally with neurotransmitters and reused for the assembly of new SVs. Over the past few years, significant progress has been made in characterizing the molecular composition of SVs. From these studies, we know that SVs share a conserved set of membrane proteins with transport vesicles involved in other pathways. Furthermore, these findings have provided us with a new understanding about the evolutionary origin of SVs from recycling vesicles present in all cells.

Animals↗

PubMed1994

Tetanus toxin-mediated cleavage of cellubrevin impairs exocytosis of transferrin receptor-containing vesicles in CHO cells.

Cellubrevin is a member of the synaptobrevin/VAMP family of SNAREs, which has a broad tissue distribution. In fibroblastic cells it is concentrated in the vesicles which recycle transferrin receptors but its role in membrane trafficking and fusion remains to be demonstrated. Cellubrevin, like the synaptic vesicle proteins synaptobrevins I and II, can be cleaved by tetanus toxin, a metallo-endoprotease which blocks neurotransmitter release. However, nonneuronal cells are unaffected by the toxin due to lack of cell surface receptors for its heavy chain. To determine whether cellubrevin cleavage impairs exocytosis of recycling vesicles, we tested the effect of tetanus toxin light chain on the release of preinternalized transferrin from streptolysin-O-perforated CHO cells. The release was found to be temperature and ATP dependent as well as NEM sensitive. Addition of tetanus toxin light chain, but not of a proteolytically inactive form of the toxin, resulted in a partial inhibition of transferrin release which correlated with the toxin-mediated cleavage of cellubrevin. The residual release of transferrin occurring after complete cellubrevin degradation was still ATP dependent. Our results indicate that cellubrevin plays an important role in the constitutive exocytosis of vesicles which recycle plasmalemma receptors. The incomplete inhibition of transferrin release produced by the toxin suggests the existence of a cellubrevin-independent exocytotic mechanism, which may involve tetanus toxin-insensitive proteins of the synaptobrevin/VAMP family.

Animals↗

PubMed1994

A signal located within amino acids 1-27 of GAD65 is required for its targeting to the Golgi complex region.

The mechanisms involved in the targeting of proteins to different cytosolic compartments are still largely unknown. In this study we have investigated the targeting signal of the 65-kD isoform of glutamic acid decarboxylase (GAD65), a major autoantigen in two autoimmune diseases: Stiff-Man syndrome and insulin-dependent diabetes mellitus. GAD65 is expressed in neurons and in pancreatic beta-cells, where it is concentrated in the Golgi complex region and in proximity to GABA-containing vesicles. GAD65, but not the similar isoform GAD67 which has a more diffuse cytosolic distribution, is palmitoylated within its first 100 amino acids (a.a.). We have previously demonstrated that the domain corresponding to a.a. 1-83 of GAD65 is required for the targeting of GAD65 to the Golgi complex region. Here we show that this domain is sufficient to target an unrelated protein, beta-galactosidase, to the same region. Site-directed mutagenesis of all the putative acceptor sites for thiopalmitoylation within this domain did not abolish targeting of GAD65 to the Golgi complex region. The replacement of a.a. 1-29 of GAD67 with the corresponding a.a. 1-27 of GAD65 was sufficient to target the otherwise soluble GAD67 to the Golgi complex region. Conversely, the replacement of a.a. 1-27 of GAD65 with a.a. 1-29 of GAD67 resulted in a GAD65 protein that had a diffuse cytosolic distribution and was primarily hydrophilic, suggesting that targeting to the Golgi complex region is required for palmitoylation of GAD65. We propose that the domain corresponding to a.a. 1-27 of GAD65, contains a signal required for the targeting of GAD65 to the Golgi complex region.

Amino Acid Sequence↗

PubMed1994

Morphologic and biochemical analysis of the intracellular trafficking of the Alzheimer beta/A4 amyloid precursor protein.

Abnormal metabolic processing of the beta/A4 amyloid precursor protein (APP) has been implicated in the pathogenesis of Alzheimer disease. Several aspects of normal APP processing have been elucidated, but the precise cellular trafficking of APP remains unclear. To investigate APP trafficking pathways further, we have examined the subcellular distribution of APP in rat brain tissue and a variety of cultured cell types, and correlated this distribution with the biochemical processing of APP. In immunofluorescence microscopy of rat brain sections, APP immunoreactivity was concentrated in the Golgi complex and in proximal axon segments. In addition, a lower level of punctate fluorescence was visible throughout the neuropil. By immunoelectron microscopy of rat brain tissue fragments, APP was found associated with Golgi elements and with medium-sized, invaginated vesicles in both axons and dendrites. Prominent localization of APP to the Golgi complex was also found in primary cultures of rat hippocampal neurons and in non-neuronal cell lines. When cultured cells were treated with brefeldin A (BFA), APP immunoreactivity changed from a Golgi-like to an endoplasmic reticulum-like distribution. No APP was detected in the BFA-induced reticulum identified by the transferrin receptor, indicating that concentration of APP in the Golgi does not reflect recycling between the trans-Golgi network and early endosomal system. In immunoblots of BFA-treated cells, there was an accumulation of full-length APP and inhibition of APP secretory processing. Treatment with phorbol ester resulted in a marked elevation of APP secretion, but no obvious redistribution of APP immunoreactivity was apparent at the light microscope level. The lysosomotropic drug chloroquine induced accumulation of APP in cell lysates, as seen by immunoblotting. Immunofluorescence microscopy of chloroquine-treated cells demonstrated a colocalization of APP with the lysosomal marker Igp 120, whereas no colocalization was seen in untreated cells. Taken together, these results support a scheme in which APP is concentrated in the Golgi complex as it travels through the central vacuolar system en route to the plasma membrane for secretion of its amino-terminal domain and/or to lysosomes for degradation.

Alzheimer Disease↗

PubMed1993

Synaptic vesicle proteins and early endosomes in cultured hippocampal neurons: differential effects of Brefeldin A in axon and dendrites.

The pathways of synaptic vesicle (SV) biogenesis and recycling are still poorly understood. We have studied the effects of Brefeldin A (BFA) on the distribution of several SV membrane proteins (synaptophysin, synaptotagmin, synaptobrevin, p29, SV2 and rab3A) and on endosomal markers to investigate the relationship between SVs and the membranes with which they interact in cultured hippocampal neurons developing in isolation. In these neurons, SV proteins are detected as punctate immunoreactivity that is concentrated in axons but is also present in perikarya and dendrites. In the same neurons, the transferrin receptor, a well established marker of early endosomes, is selectively concentrated in perikarya and dendrites. In the perikaryal-dendritic region, BFA induced a dramatic tubulation of transferrin receptors as well as a cotubulation of the bulk of synaptophysin. Synaptotagmin, synaptobrevin, p29 and SV2 immunoreactivities retained a primarily punctate distribution. No tubulation of rab3A was observed. In axons, BFA did not produce any obvious alteration of the distribution of SV proteins, nor of peroxidase- or Lucifer yellow-labeled early endosomes. The selective effect of BFA on dendritic membranes suggests the existence of functional differences between the endocytic systems in dendrites and axons. Cotubulation of transferrin receptors and synaptophysin in the perikaryal-dendritic region is consistent with a functional interconnection between the traffic of SV proteins and early endosomes. The heterogeneous effects of BFA on SV proteins in this cell region indicates that SV proteins are differentially sorted upon exit from the TGN and are coassembled into SVs at the cell periphery.

Amino Acid Sequence↗

PubMed1993

Traffic of synaptic vesicle proteins in polarized and nonpolarized cells.

Neurons have at least two pathways of regulated secretion, which involve two classes of secretory organelles: typical synaptic vesicles (SVs) and large dense-core vesicles. Large dense-core vesicles store and secrete peptide neurotransmitters and amines, and may be seen as the neuronal counterpart of secretory granules of endocrine cells. SVs are highly specialized secretory organelles, which store and secrete non-peptide hormones and play a dominant role in the fast, point-to-point signalling typical of the nervous system. Microvesicles that share a variety of biochemical and functional similarities with SVs (synaptic-like microvesicles) have recently been described in endocrine cells. SVs and synaptic-like microvesicles are closely related to vesicular carriers of the receptor-mediated recycling pathway. They undergo repeated cycles of exo-endocytosis, which are thought to involve endosomal intermediates. In mature neurons, SVs are concentrated in axon endings. To gain insight into the mechanisms responsible for SV targeting, we have studied the traffic of SV proteins in both endocrine cells and developing hippocampal neurons in primary culture at different stages of differentiation. Additionally, the distribution of the SV protein synaptophysin, when expressed by transfection in fibroblastic cells or in polarized epithelial cells (MDCK cells), was investigated. SV proteins are already present in developing neurons at stages preceding the establishment of neuronal polarity. As axons and dendrites form, SV proteins are found in both types of processes, although they become progressively more concentrated in the axon. Throughout these developmental stages SVs undergo active exo-endocytotic recycling.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗