Biomedical subjects

R Jahn

Publications and source records attributed to R Jahn.

At least 73 records · Page 4Linked to original sources

PubMed1997

Inhibition of transmitter release correlates with the proteolytic activity of tetanus toxin and botulinus toxin A in individual cultured synapses of Hirudo medicinalis.

We have studied the effects of tetanus toxin and botulinus toxin A on neurotransmitter release in the Retzius-->P-cell synapse of the leech and exploited the unique properties of this system, which allow for combined physiological and biochemical analyses in single-cell pairs. The sequences of Hirudo medicinalis synaptobrevin and synaptosomal-associated protein of 25 kDa (SNAP-25), deduced by cDNA cloning, are 61 and 55% identical, respectively, to their corresponding mammalian homologs. Whereas Hirudo synaptobrevin is proteolyzed by tetanus toxin, its SNAP-25 isoform is resistant to botulinus toxin A cleavage because of amino acid substitutions within and around the putative cleavage site. In close correlation, microinjection of tetanus toxin into the presynaptic neuron produced a block of transmitter release, whereas botulinus toxin A had no effect on synaptic transmission. Subsequent immunoblotting of single-cell pairs demonstrated directly that the tetanus toxin-mediated block of exocytosis is accompanied by cleavage of synaptobrevin in the injected neuron, resulting in the generation of a detectable C-terminal cleavage product. Immunoblotting also confirmed the resistance of SNAP-25 to botulinus toxin A cleavage in vivo. Using recombinant proteins, we show that the N-terminal fragment of synaptobrevin released by tetanus toxin, but not its C-terminal membrane-anchored cleavage product, participates with syntaxin and SNAP-25 in synaptic SNAP receptor (SNARE) ternary complex formation in Hirudo. Our data demonstrate a direct correlation between the inhibition of transmitter release and the ability of the neurotoxin to proteolyze its target protein and support the view that SNARE ternary complex formation is an important step leading to synaptic vesicle exocytosis.

Amino Acid Sequence↗

PubMed1997

A structural change occurs upon binding of syntaxin to SNAP-25.

The highly conserved proteins syntaxin and SNAP-25 are part of a protein complex that is thought to play a key role in exocytosis of synaptic vesicles. Previous work demonstrated that syntaxin and SNAP-25 bind to each other with high affinity and that their binding regions are predicted to form coiled coils. Circular dichroism spectroscopy was used here to study the alpha-helicity of the individual proteins and to gain insight into structural changes associated with complex formation. Syntaxin displayed approximately 43% alpha-helical content. In contrast, the alpha-helical content of SNAP-25 was low under physiological conditions. Formation of the SNAP-25-syntaxin complex was associated with a dramatic increase in alpha-helicity. Interaction of a 90-residue NH2-terminal fragment of SNAP-25 comprising the minimal syntaxin binding domain lead to a similar but less pronounced increase in alpha-helicity. Single amino acid replacements in the putative hydrophobic core of this fragment with hydrophilic amino acids abolished the induced structural change and disrupted the interaction monitored by binding assays. Replacements with hydrophobic residues had no effect. Our findings are consistent with induced coiled coil formation upon binding of syntaxin and SNAP-25.

Amino Acid Sequence↗

PubMed1997

Neurotransmitter release - four years of SNARE complexes.

Exocytosis in neurons requires proteins known as SNAREs, membrane proteins that have now been implicated in many intracellular fusion events. SNAREs assemble into stable ternary complexes that are dissociated by the ATPase NSF (N-ethylmaleimide-sensitive factor), working together with SNAPs (soluble NSF attachment proteins). Recent results have shed new light on the mechanisms underlying SNARE (SNAP receptor) complex assembly and disassembly, and suggest changes in models that relate these reactions to vesicle docking and fusion.

Animals↗

PubMed1997

De novo acquisition of neuronal polarity in retinoic acid-induced embryonal carcinoma cells.

The mouse embryonal carcinoma cell line PCC7-Mz1 represents an advantageous model to study acquisition of polarity by neurons. During the first two days after differentiation is induced by the addition of retinoic acid, the neuronal derivatives develop extensions which for at least four more days do not differ from each other in growth characteristics, morphology, and marker expression. Beginning around differentiation day 6 and following the relocation of the nucleus from a central to a polar position in the cell soma, the morphology and marker expression changes dramatically: expression of MAP2 diminishes and eventually disappears in the thinner neurite (future axon), which originates at the nucleated pole, but remains strong in the branched, broad based neurite(s). The opposite changes in expression are observed for synaptophysin, together with a clustering of the vesicle protein in varicosity-like areas. Complete segregation of expression of the two markers is achieved around day 12, shortly followed by dendrite-specific location of MAP2 mRNA and the ability to generate and conduct action potentials. Our studies add several aspects to the process of neuronal polarity acquisition, as it was previously studied in primary cultures of embryonic neurons: (i) we monitored neuronal differentiation from the birth of neurons, rather than from later and less defined maturation stages, (ii) cell nucleus relocation may be associated with the induction of neuronal polarity, and (iii) functional competence of neurons is closely associated with previous acquisition of polarity. Acquisition of polarity by PCC7-Mz1 neuronal derivatives probably refers to de novo acquisition rather than to reestablishment of polarity.

Animals↗

PubMed1996

Alzheimer amyloid protein precursor is localized in nerve terminal preparations to Rab5-containing vesicular organelles distinct from those implicated in the synaptic vesicle pathway.

In order to localize amyloid protein precursor (APP) in nerve terminals, we have immunoisolated vesicular organelles from nerve terminal preparations using antibodies to Rab5 and synaptophysin. These immunoisolates were then analyzed by electron microscopy and by immunoblotting. The synaptophysin immunoisolates represented a nearly homogeneous population of small synaptic vesicles, with less than 10% contamination by other organelles, and very little APP. In contrast, Rab5 immunoisolates contained, in addition to small synaptic vesicles, substantial numbers of large uni- and bilamellar vesicles and high levels of APP. Thus, it appears that nerve terminal APP is contained predominantly in large vesicular organelles, distinct from synaptic vesicles and from the synaptic vesicle recycling pathway.

Alzheimer Disease↗

PubMed1996

16-BAC/SDS-PAGE: a two-dimensional gel electrophoresis system suitable for the separation of integral membrane proteins.

Integral membrane proteins, particularly those with more than one transmembrane domain, have traditionally been difficult to separate by two-dimensional electrophoretic methods. Here we report the adaptation of a previously published procedure [D. E. Macfarlane (1989) Anal. Biochem. 176, 457-463] for the analytical and semipreparative separation of membrane proteins. The first dimension involves discontinuous gel electrophoresis in an acidic buffer system using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC). The second dimension consists of discontinuous SDS-PAGE. Using carbonate-washed membranes of synaptic vesicles and clathrin-coated vesicles as examples, we demonstrate that complex membrane protein mixtures can be resolved with a resolution at least fivefold higher than that of one-dimensional SDS-PAGE. Protein patterns are highly reproducible and proteins with single or multiple transmembrane domains are resolved as clearly distinct spots. Smearing of bands or losses of protein are minimal. Several spots were identified by immunoblotting and internal sequencing. Thus, this method is suitable for the analytical and semipreparative characterization of membrane proteins derived from complex biological samples.

Animals↗

PubMed1996

Fatty acylation of synaptotagmin in PC12 cells and synaptosomes.

Synaptotagmin I is localized to synaptic vesicles where it functions in the calcium-triggered release of neurotransmitters. Here we demonstrate that synaptotagmin I covalently incorporated [3H]palmitate after metabolic labelling of PC-12 cells and rat brain synaptosomes. Labeling was localized to a tryptic fragment that contains a cluster of cysteine residues adjacent to the molecule's single transmembrane anchor. Neutral hydroxylamine released the [3H]palmitate from this fragment and increased its electrophoretic mobility, demonstrating that acylation occurs at the membrane-proximal cysteine cluster. In addition, hydroxylamine-induced mobility shifts were also apparent for synaptotagmins II and III, suggesting that posttranslational palmitoylation via thioester bonds may be a general modification of all synaptotagmins.

Acylation↗

PubMed1996

Rab3 reversibly recruits rabphilin to synaptic vesicles by a mechanism analogous to raf recruitment by ras.

GTP activates the interaction between the synaptic vesicle proteins rabphilin and rab3. This raises the question of whether rabphilin is a resident vesicle protein that recruits rab3 in a stage-dependent fashion, or if it is instead an effector protein recruited by rab3. We now show that rabphilin, like rab3, dissociates from synaptic vesicles after exocytosis in a manner requiring both Ca2+ and membrane fusion. Rabphilin interacts with GTP-rab3 via a N-terminal domain comprising a novel Zn2+(-)finger motif, and this interaction is essential for rabphilin binding to synaptic vesicles. Thus, in the same way that ras recruits raf to the plasma membrane, rab3 reversibly recruits rabphilin to synaptic vesicles in a stage-dependent manner. These results reveal an unexpected similarity between the molecular mechanisms by which small G protein function in recruiting effector proteins to membranes during membrane traffic and signal transduction.

Adaptor Proteins, Signal Transducing↗

PubMed1996

A novel function for the second C2 domain of synaptotagmin. Ca2+-triggered dimerization.

Synaptotagmin serves as the major Ca2+ sensor for regulated exocytosis from neurons. While the mechanism by which synaptotagmin regulates membrane fusion remains unknown, studies using Drosophila indicate that the molecule functions as a multimeric complex and that its second C2 domain is essential for efficient excitation-secretion coupling. Here we describe biochemical data that may account for these phenomena. We report that Ca2+ causes synaptotagmin to oligomerize, primarily forming dimers, via its second C2 domain. This effect is specific for divalent cations that can stimulate exocytosis of synaptic vesicles (Ca2+ >> Ba2+, Sr2+ >> Mg2+) and occurs with an EC50 value of 3-10 microM Ca2+. In contrast, a separate Ca2+-dependent interaction between synaptotagmin and syntaxin, a component of the fusion apparatus, occurs with an EC50 value of approximately 100 microM Ca2+ and involves the synergistic action of both C2 domains of synaptotagmin. We propose that Ca2+ triggers two consecutive protein-protein interactions: the formation of synaptotagmin dimers at low Ca2+ concentrations followed by the association of synaptotagmin dimers with syntaxin at higher Ca2+-concentrations. Our findings, in conjunction with physiological studies, indicate that the Ca2+-induced dimerization of synaptotagmin is important for the efficient regulation of exocytosis by Ca2+.

Animals↗

PubMed1996

Distribution of Rab3a in rat nervous system: comparison with other synaptic vesicle proteins and neuropeptides.

In the present study we have investigated the distribution of Rab3a in rat peripheral nervous system and compared it with the distribution of other synaptic vesicle proteins (synaptophysin, synapsin I), neuropeptides (CGRP, SP, NPY) and tyrosine hydroxylase (TH). Rab3a immunoreactivity (-IR) was always colocalized with synaptophysin-IR and synapsin I-IR in nerve terminals of the spinal cord and peripheral nerve endings. In many cases, Rab3a-IR was also present in the same axons and terminals as peptides. In crushed sciatic nerve axons, Rab3a was colocalized, proximal to the crush, with synaptophysin-IR, synapsin I-IR, CGRP-IR, and TH-IR, but only partially co-localized with NPY-IR and SP-IR. In the area distal to the crush, Rab3a-IR was very weakly positive in a few thin axons, while larger amount of synaptophysin, CGRP, NPY and SP immunoreactivities were detected. The subcellular distribution of peptides and Rab3a differed in that peptides were observed mainly in large granular structures, while Rab3a-IR was observed mainly as diffuse, finely granular immunoreactivity, in addition to a few exceptional large granules present in some axons. The results demonstrate that Rab3a is widely distributed in different types of neurons, i.e. motor, sensory, autonomic adrenergic and cholinergic neurons, and colocalized with other synaptic vesicle proteins, suggesting that Rab3a may play an essential role in neuronal function. Furthermore, Rab3a is present in many peptide containing axons and terminals, but with an apparently different subcellular distribution, being affiliated mostly with small synaptic vesicles and only occasionally with large vesicles, that may represent peptide contained vesicles.

Animals↗

PubMed1996

Synaptotagmin, a synaptic vesicle protein, is present in human cerebrospinal fluid: a new biochemical marker for synaptic pathology in Alzheimer disease?

Using a novel approach, including affinity chromatography, reversed-phase chromatography, and chemiluminescence immunoblotting, we have for the first time been able to demonstrate one of the small synaptic vesicle proteins, synaptotagmin I, in cerebrospinal fluid (CSF). Two other small synaptic vesicle proteins, rab3a and synaptophysin, were not detectable. The approximate molecular weight of CSF-synaptotagmin was 65 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Further characterization of CSF synaptotagmin by high-performance capillary electrophoresis (HPCE) showed a single peak. These findings support that the whole synaptotagmin molecule is present in CSF, without significant proteolytic degradation. After high-speed centrifugation of CSF, synaptotagmin was exclusively found in the supernatant, suggesting that synaptotagmin is present in CSF as a free protein, and not as a constituent of synaptic vesicles. In a preliminary study, we found a marked reduction of CSF synaptotagmin in patients with early onset Alzheimer disease (EAD) as compared with age-matched healthy individuals. To elucidate the biological relevance of this finding, we also quantified synaptotagmin in brain tissue. A marked reduction in synaptotagmin was found both in the hippocampus and frontal cortex of EAD, suggesting that a decrease in synaptotagmin in the brain is followed by a concomitant decrease in the CSF. Analysis of CSF synaptotagmin might provide a tool to study synaptic function and pathology in the human brain.

Aged↗

PubMed1996

Axonal transport and distribution of synaptobrevin I and II in the rat peripheral nervous system.

Synaptobrevin, a membrane protein of synaptic vesicles that plays a key role in exocytosis, occurs in two closely related isoforms, synaptobrevin I and II. We have analyzed the axonal transport of both isoforms in sciatic nerve and spinal roots. When fast axonal transport was interrupted by crushing, the proteins accumulated continuously proximal to the crush. Accumulation also was observed distal to the crush, but to a lesser extent (47 and 63% of the proximal accumulation for synaptobrevin I and II, respectively). Immunoelectron microscopy revealed that, proximal to the crush, synaptobrevin I and II were associated with small clear vesicles reminiscent of typical synaptic vesicles. Distal to the crush, membranes positive for synaptobrevin I or II were more heterogeneous, including larger membrane profiles that may represent endosomes. In spinal cord, synaptobrevin I and II were colocalized in many terminals. However, labeling for synaptobrevin I was more intense whereas labeling for synaptobrevin II was stronger in dorsal than in ventral horn terminals. Motor endplates contained only synaptobrevin I. In the sciatic nerve, synaptobrevin I was present predominantly in large, myelinated axons, whereas synoptobrevin II was virtually absent but abundant in small- and medium-sized axons. Lumbar sympathectomy, ventral rhizotomy, and double-labeling studies confirmed that synaptobrevin I is present predominantly in motor neurons whereas synaptobrevin II is present in adrenergic and sensory neurons. We conclude that synaptobrevin I and II are transported bidirectionally by fast axonal transport and are expressed heterogeneously in different neurons in the peripheral nervous system of the adult rat, suggesting that these isoforms have special functional roles in different sets of neurons.

Animals↗

PubMed1996

Axonal transport and targeting of the t-SNAREs SNAP-25 and syntaxin 1 in the peripheral nervous system.

Axonal transport and targeting of the t-SNAREs SNAP-25 and syntaxin 1 were investigated in the rat peripheral nervous system using a stop-flow (crush) technique. In crush-operated sciatic nerves, accumulations of SNAP-25 and syntaxin 1 immunoreactivities were detected as early as 1 h after operation, indicating fast axonal transport. The amounts increased on the proximal side of the crush with time after crushing. Distal accumulations of SNAP-25, representing recycling to the cell body, were less than 10% of the proximal accumulations, but 40% for syntaxin 1, 50% for synaptobrevin II and 70% for synaptophysin. Immunoelectron microscopic studies demonstrated that SNAP-25 and syntaxin 1 are present on pleiotropic membranes within a diameter of 50 to 100 nm in axons proximal to a crush. Distal to the crush, labeling for syntaxin 1 and SNAP-25 were sparse and barely detectable, respectively. In addition, the two proteins were found in the axolemma. In nerve terminals of the spinal cord, both proteins were concentrated around small synaptic vesicles (about 50 nm in diameter), whereas only very few gold particles were observed near the presynaptic membrane or the active zones.

Animals↗

PubMed1995

Expression of synaptobrevin II, cellubrevin and syntaxin but not SNAP-25 in cultured astrocytes.

Astrocytes, a sub-type of glial cell in the central nervous system, can release the excitatory transmitters glutamate and aspartate in response to elevated levels of internal calcium. To investigate potential release mechanisms that may be present in these cells we have determined whether protein components of the neuronal secretory apparatus are expressed in astrocytes. Western blots, immunocytochemistry and RT PCR demonstrate that astrocytes express cellubrevin, synaptobrevin II and syntaxin, proteins known to form a macromolecular fusion complex. However, SNAP-25 which is another neuronal protein of the fusion complex, was not detected. Astrocyte cellubrevin and synaptobrevin II were also shown to be sensitive to the proteolytic activity of tetanus toxin. Together these data indicate that astrocytes express some proteins that are known to form a fusion complex indicating that regulated exocytosis might mediate calcium-regulated transmitter release from these cells.

Animals↗

PubMed1995

Ca2+ regulates the interaction between synaptotagmin and syntaxin 1.

While there is compelling evidence that the synaptic vesicle protein synaptotagmin serves as the major Ca2+ sensor for regulated exocytosis, it is not known how Ca2+ binding initiates membrane fusion. Here we report that Ca2+ increases the affinity, by approximately 2 orders of magnitude, between synaptotagmin and syntaxin 1, a component of the synaptic fusion apparatus. This effect is specific for divalent cations which can stimulate exocytosis of synaptic vesicles (Ca2+ > Ba2+, Sr2+ >> Mg2+). The Ca(2+)-dependence of the interaction was composed of two components with EC50 values of 0.7 and 180 microM Ca2+. The interaction is mediated by the carboxyl-terminal region of syntaxin 1 (residues 194-288) and is regulated by a novel Ca(2+)-binding site(s) which does not require phospholipids and is not disrupted by mutations that abolish Ca(2+)-dependent phospholipid binding to synaptotagmin. We propose that this interaction constitutes an essential step in excitation-secretion coupling.

Amino Acid Sequence↗

PubMed1995

Real-time measurement of transmitter release from single synaptic vesicles.

Neurotransmitter release is mediated by Ca2+ dependent exocytosis of synaptic vesicles. Neither the amount of transmitter released from individual synaptic vesicles nor the kinetics of this process have yet been directly determined. Using carbon fibres as electrochemical detectors, we have measured release of the neurotransmitter serotonin from cultured neurons of the leech. This technique allowed us to monitor transmitter discharge from single synaptic vesicles as spike-like oxidation currents at high time resolution, providing new insight into the mechanism of neuronal exocytosis. Two types of signals were characterized, corresponding to exocytosis of small clear and large dense core vesicles present in these cells. A small vesicle discharges about 4,700 transmitter molecules with a time constant in the region of 260 microseconds, whereas large vesicles release their content of approximately 80,000 molecules with a time constant of about 1.3 ms. Release from both vesicle types is initiated rapidly, with a rise time of less than 60 microseconds, suggesting an abrupt opening of a preassembled fusion pore.

Action Potentials↗

PubMed1995

Poisoning by botulinum neurotoxin A does not inhibit formation or disassembly of the synaptosomal fusion complex.

We investigated the effect of poisoning rat brain synaptosomes with botulinum neurotoxin A on the NSF-mediated disassembly of a complex consisting of syntaxin, SNAP-25 and synaptobrevin (fusion complex). Botulinum neurotoxin A specifically removes 9 amino acids from the C-terminus of SNAP-25 and efficiently blocks KCl-evoked glutamate release from synaptosomes. We report that truncated SNAP-25 is incorporated into the fusion complex of poisoned synaptosomes. The presence of truncated SNAP-25 does not interfere with the NSF-induced disassembly of the fusion complex. Also, the release of truncated SNAP-25 from the fusion complex is similar to that of the native SNAP-25. Since neither the formation of the complex nor its disassembly seems to be affected by the SNAP-25 fragment, this fragment is likely to block exocytosis by disrupting events between disassembly of the synaptosomal fusion complex and membrane fusion itself.

Animals↗

PubMed1995

Stimulation of inositol 1,4,5-trisphosphate production by peptides corresponding to the effector domain of different Rab3 isoforms and cross-linking of an effector domain peptide target.

Rab3 proteins are localized on secretory vesicles and appear to be involved in regulated exocytosis. We have previously shown that a modified peptide corresponding to the effector domain of the small molecular mass GTP-binding protein Rab3A, Rab3AAL, stimulates inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production and amylase release in digitonin-permeabilized pancreatic acini. Experiments using monoclonal antibodies reveal that the Rab3-like protein present in pancreatic acini is not the Rab3A isoform. However, since the putative effector domains of the four as yet known Rab3 proteins (A, B, C and D) differ only in the C-terminal four amino acid residues, Rab3A effector domain peptide could mimic the action of the pancreas-specific Rab3 isoform. In the present study we report that peptides corresponding to the different Rab3 isoforms stimulate both Ins(1,4,5)P3 production and amylase secretion with an order of potency Rab3B/D > Rab3AAL > Rab3A = Rab3C. For Rab3A, B/D and C effector domain peptides the concentrations causing half-maximal response (EC50) were 3, 0.2 and 3 nM for Ins(1,4,5)P3 accumulation and 0.3, 0.02 and 0.3 nM for amylase release, respectively. A Rab1A effector domain peptide, Rab1AAL, and a scrambled peptide of Rab3AAL were less potent by several orders of magnitude in eliciting these responses compared with native Rab3 effector domain peptides. None of the peptides influenced Ins(1,4,5)P3 production and amylase release in intact acini. Cross-linking of 125I-Rab3B/D peptide to pancreatic acinar membranes showed a band at 70 to 75 kDa with maximum intensity at 75 kDa. Radiolabelling of the substrates could be displaced by unlabelled Rab3B/D peptide, and to a lesser extend by Rab3A peptide, whereas the scrambled peptide of Rab3AAL had no effect. These data suggest that phospholipase C and exocytosis might be regulated by Rab3B-or Rab3D-like proteins in pancreatic acinar cells. A 75 kDa protein that preferentially cross-linked to 125I-Rab3B/D effector domain peptide is a potential candidate as an effector protein of Rab3 effector domain peptides.

Amino Acid Sequence↗