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Nils Brose

Publications and source records attributed to Nils Brose.

At least 19 recordsLinked to original sources
PubMed2005

CAPS1 regulates catecholamine loading of large dense-core vesicles.

CAPS1 is thought to play an essential role in mediating exocytosis from large dense-core vesicles (LDCVs). We generated CAPS1-deficient (KO) mice and studied exocytosis in a model system for Ca2+-dependent LDCV secretion, the adrenal chromaffin cell. Adult heterozygous CAPS1 KO cells display a gene dosage-dependent decrease of CAPS1 expression and a concomitant reduction in the number of docked vesicles and secretion. Embryonic homozygous CAPS1 KO cells show a strong reduction in the frequency of amperometrically detectable release events of transmitter-filled vesicles, while the total number of fusing vesicles, as judged by capacitance recordings or total internal reflection microscopy, remains unchanged. We conclude that CAPS1 is required for an essential step in the uptake or storage of catecholamines in LDCVs.

Animals↗
PubMed2005

Unchanged survival rates of 14-3-3gamma knockout mice after inoculation with pathological prion protein.

The diagnosis of sporadic Creutzfeldt-Jakob disease (CJD) is based on typical clinical findings and is supported by a positive 14-3-3 Western blot of cerebrospinal fluid. However, it is not clear whether 14-3-3 indicates general neuronal damage or is of pathophysiological relevance in CJD. The fact that the 14-3-3 isoform spectrum in cerebrospinal fluid does not correspond to that found in the brain points to a regulated process. To investigate a possible role of 14-3-3 proteins in transmissible spongiform diseases, we generated a 14-3-3gamma-deficient mutant mouse line by using a classical knockout strategy. The anatomy and cage behavior of the mutant mice were normal. Western blot analyses of brain homogenates revealed no changes in the protein expression of other 14-3-3 isoforms (epsilon, beta, zeta, and eta). Proteomic analyses of mouse brains by two-dimensional differential gel electrophoresis showed that several proteins, including growth hormone, 1-Cys peroxiredoxin, CCT-zeta, glucose-6-phosphate isomerase, GRP170 precursor, and alpha-SNAP, were differentially expressed. Mutant and wild-type mice were inoculated either intracerebrally or intraperitoneally with the Rocky Mountain Laboratory strain of scrapie, but no differences were detected in the postinoculation survival rates. These results indicate that 14-3-3gamma is unlikely to play a causal role in CJD and related diseases.

14-3-3 Proteins↗
PubMed2004

Munc13-4 is an effector of rab27a and controls secretion of lysosomes in hematopoietic cells.

Griscelli syndrome type 2 (GS2) is a genetic disorder in which patients exhibit life-threatening defects of cytotoxic T lymphocytes (CTLs) whose lytic granules fail to dock on the plasma membrane and therefore do not release their contents. The disease is caused by the absence of functional rab27a, but how rab27a controls secretion of lytic granule contents remains elusive. Mutations in Munc13-4 cause familial hemophagocytic lymphohistiocytosis subtype 3 (FHL3), a disease phenotypically related to GS2. We show that Munc13-4 is a direct partner of rab27a. The two proteins are highly expressed in CTLs and mast cells where they colocalize on secretory lysosomes. The region comprising the Munc13 homology domains is essential for the localization of Munc13-4 to secretory lysosomes. The GS2 mutant rab27aW73G strongly reduced binding to Munc13-4, whereas the FHL3 mutant Munc13-4Delta608-611 failed to bind rab27a. Overexpression of Munc13-4 enhanced degranulation of secretory lysosomes in mast cells, showing that it has a positive regulatory role in secretory lysosome fusion. We suggest that the secretion defects seen in GS2 and FHL3 have a common origin, and we propose that the rab27a/Munc13-4 complex is an essential regulator of secretory granule fusion with the plasma membrane in hematopoietic cells. Mutations in either of the two genes prevent formation of this complex and abolish secretion.

Animals↗
PubMed2004

Calmodulin and Munc13 form a Ca2+ sensor/effector complex that controls short-term synaptic plasticity.

The efficacy of synaptic transmission between neurons can be altered transiently during neuronal network activity. This phenomenon of short-term plasticity is a key determinant of network properties; is involved in many physiological processes such as motor control, sound localization, or sensory adaptation; and is critically dependent on cytosolic [Ca2+]. However, the underlying molecular mechanisms and the identity of the Ca2+ sensor/effector complexes involved are unclear. We now identify a conserved calmodulin binding site in UNC-13/Munc13s, which are essential regulators of synaptic vesicle priming and synaptic efficacy. Ca2+ sensor/effector complexes consisting of calmodulin and Munc13s regulate synaptic vesicle priming and synaptic efficacy in response to a residual [Ca2+] signal and thus shape short-term plasticity characteristics during periods of sustained synaptic activity.

Animals↗
PubMed2004

Munc13-1-mediated vesicle priming contributes to secretory amyloid precursor protein processing.

The amyloid precursor protein (APP) gives rise toc beta-amyloid peptides, which are the main constituents of senile plaques in brains of Alzheimer's disease patients. Non-amyloidogenic processing of the APP can be stimulated by phorbol esters (PEs) and by intracellular diacylglycerol (DAG) generation. This led to the hypothesis that classical and novel protein kinase Cs (PKCs), which are activated by DAG/PEs, regulate APP processing. However, in addition to PKCs, there are other DAG/PE receptors present in neurons that may participate in the modulation of APP processing. Munc13-1, a presynaptic protein with an essential role in synaptic vesicle priming, represents such an alternative target of the DAG second messenger pathway. Using Munc13-1 knock-out mice and knock-in mice expressing a Munc13-1(H567K) variant deficient in DAG/PE binding, we determined the relative contributions of PKCs and Munc13-1 to PE-stimulated secretory APP processing. We establish that, in addition to PKC, Munc13-1 significantly contributes to the regulation of secretory APP metabolism.

Amyloid Precursor Protein Secretases↗
PubMed2004

Regulation of releasable vesicle pool sizes by protein kinase A-dependent phosphorylation of SNAP-25.

Protein kinase A (PKA) is a key regulator of neurosecretion, but the molecular targets remain elusive. We combined pharmacological manipulations of kinase and phosphatase activities with mutational studies on the exocytotic machinery driving fusion of catecholamine-containing vesicles from chromaffin cells. We found that constitutive PKA activity was necessary to maintain a large number of vesicles in the release-ready, so-called primed, state, whereas calcineurin (protein phosphatase 2B) activity antagonized this effect. Overexpression of the SNARE protein SNAP-25a mutated in a PKA phosphorylation site (Thr-138) eliminated the effect of PKA inhibitors on the vesicle priming process. Another, unidentified, PKA target regulated the relative size of two different primed vesicle pools that are distinguished by their release kinetics. Overexpression of the SNAP-25b isoform increased the size of both primed vesicle pools by a factor of two, and mutations in the conserved Thr-138 site had similar effects as in the a isoform.

Alanine↗
PubMed2004

Divergent and convergent signaling by the diacylglycerol second messenger pathway in mammals.

Diacylglycerol is an essential second messenger in mammalian cells. The most prominent intracellular targets of diacylglycerol and the functionally analogous phorbol esters belong to the protein kinase C family, but at least five alternative types of high affinity diacylglycerol/phorbol ester receptors are known: protein kinase D, diacylglycerol kinases alpha, beta, and gamma, RasGRPs, chimaerins, and Munc13s. These function independently of protein kinase C isozymes, and form a network of signaling pathways in the diacylglycerol second messenger system that regulates processes as diverse as gene transcription, lipid signaling, cytoskeletal dynamics, intracellular membrane trafficking, or neurotransmitter release.

Animals↗
PubMed2004

Synaptic targeting of neuroligin is independent of neurexin and SAP90/PSD95 binding.

Synaptic cell adhesion and synaptogenesis are thought to involve the interaction of neuroligin, a postsynaptic transmembrane protein, with its presynaptic ligand neurexin. Neuroligin also interacts with SAP90/PSD95, a multidomain scaffolding protein thought to recruit proteins to postsynaptic sites. Using expression of GFP-tagged versions of neuroligin in cultured hippocampal neurons, we find that neuroligin is targeted to synapses via intracellular sequences distinct from its SAP90/PSD95 binding site. A neuroligin mutant lacking the intracellular domain fails to target to synapses. These data indicate that postsynaptic targeting of neuroligin does not rely on the scaffolding action of SAP90/PSD95 and is not induced by binding to presynaptic neurexin. Neuroligin is rather targeted to synapses via a postsynaptic mechanism, which may precede and be necessary for subsequent recruitment of neurexin and other neuroligin interactors such as SAP90/PSD95, suggesting a pivotal position for neuroligin in a putative hierarchy of interactions assembling or stabilizing synapses.

Animals↗
PubMed2004

The complexity of PDZ domain-mediated interactions at glutamatergic synapses: a case study on neuroligin.

The postsynaptic specialisation at glutamatergic synapses is composed of a network of proteins located within the membrane and the underlying postsynaptic density. The strong interconnectivity between the protein components is mediated by a limited number of interaction modes. Particularly abundant are PDZ domain-mediated interactions. An obstacle in understanding the fidelity of postsynaptic processes involving PDZ domains is the high degree of overlap with respect to their binding specificities. Focussing on transsynaptic adhesion molecules, we used the yeast two-hybrid system to obtain an overview of the binding specificities of selected C-terminal PDZ binding motifs. Neuroligin, a postsynaptic cell surface protein that spans the synaptic cleft and interacts with beta-neurexin, served as a starting point. Neuroligin binds to the PDZ domain-containing proteins PSD95, SAP102, Chapsyn110, S-SCAM, Magi1 and 3, Shank1 and 3, Pick1, GOPC, SPAR, Semcap3 and PDZ-RGS3. Next, we examined the relationship between neuroligin and synaptic cell adhesion molecules or glutamate receptor subunits with respect to PDZ-mediated interactions. We found a limited overlap in the PDZ-domain binding specificities of neuroligin with those of Sidekick2 and Ephrin-B2. In contrast, Syndecan2 and IgSF4 show no overlap with the PDZ-domain specificity of neuroligin, instead, they bind to GRIP and syntenin. The AMPA receptor subunit GluR2 interacts with Semcap3 and PDZ-RGS3, whereas the kainate receptor subunits GluR5 and GluR6 show weak interactions with PSD95. In summary, we can sketch a complex pattern of overlap in the binding specificities of synaptic cell surface proteins towards PDZ-domain proteins.

Animals↗
PubMed2004

Neuroligin 2 is exclusively localized to inhibitory synapses.

Neuroligins are cell adhesion proteins that are thought to instruct the formation and alignment of synaptic specializations. The three known rodent neuroligin isoforms share homologous extracellular acetylcholinesterase-like domains that bridge the synaptic cleft and bind beta-neurexins. All neuroligins have identical intracellular C-terminal motifs that bind to PDZ domains of various target proteins. Neuroligin 1 is specifically localized to glutamatergic postsynaptic specializations. We show here that neuroligin 2 is exclusively localized to inhibitory synapses in rat brain and dissociated neurons. In immature neurons, neuroligin 2 is found at synapses and also at GABAA receptor aggregates that are not facing presynaptic termini, indicating that postsynaptic mechanisms lead to synaptic recruitment of neuroligin 2. Our findings identify neuroligin 2 as a new cell adhesion protein specific for inhibitory synapses and open new avenues for identifiying the constituents of this unique type of postsynaptic specialization.

Animals↗
PubMed2003

A family of Ca2+-dependent activator proteins for secretion: comparative analysis of structure, expression, localization, and function.

Ca2+-dependent activator protein for secretion (CAPS) 1 is an essential cytosolic component of the protein machinery involved in large dense-core vesicle (LDCV) exocytosis and in the secretion of a subset of neurotransmitters. In the present study, we report the identification, cloning, and comparative characterization of a second mammalian CAPS isoform, CAPS2. The structure of CAPS2 and its function in LDCV exocytosis from PC12 cells are very similar to those of CAPS1. Both isoforms are strongly expressed in neuroendocrine cells and in the brain. In subcellular fractions of the brain, both CAPS isoforms are enriched in synaptic cytosol fractions and also present on vesicular fractions. In contrast to CAPS1, which is expressed almost exclusively in brain and neuroendocrine tissues, CAPS2 is also expressed in lung, liver, and testis. Within the brain, CAPS2 expression seems to be restricted to certain brain regions and cell populations, whereas CAPS1 expression is strong in all neurons. During development, CAPS2 expression is constant between embryonic day 10 and postnatal day 60, whereas CAPS1 expression is very low before birth and increases after postnatal day 0 to reach a plateau at postnatal day 21. Light microscopic data indicate that both CAPS isoforms are specifically enriched in synaptic terminals. Ultrastructural analyses show that CAPS1 is specifically localized to glutamatergic nerve terminals. We conclude that at the functional level, CAPS2 is largely redundant with CAPS1. Differences in the spatial and temporal expression patterns of the two CAPS isoforms most likely reflect as yet unidentified subtle functional differences required in particular cell types or during a particular developmental period. The abundance of CAPS proteins in synaptic terminals indicates that they may also be important for neuronal functions that are not exclusively related to LDCV exocytosis.

Amino Acid Sequence↗
PubMed2003

A brain-specific isoform of small glutamine-rich tetratricopeptide repeat-containing protein binds to Hsc70 and the cysteine string protein.

Small glutamine-rich tetratricopeptide repeat-containing protein (SGT) is a ubiquitously expressed cochaperone of heat shock cognate protein of 70 kDa (Hsc70). SGT binds to the C terminus of Hsc70, a site used by several tetratricopeptide repeat-containing binding partners to recruit Hsc70 into complexes of diverse function. We describe here an isoform of SGT with 60% amino acid sequence identity that we name betaSGT. In contrast to the previously published alphaSGT, betaSGT is almost exclusively expressed in brain. Both isoforms of SGT possess similar binding properties toward Hsc70 and cysteine string protein, a synaptic vesicle-associated J-domain-containing protein. In addition, SGTs oligomerize without preferences among isoforms. The distribution of protein binding motifs on SGTs reveals a modular structure. The N-terminal domains mediate oligomerization. Binding to Hsc70 is impaired by mutations of basic residues within the central tetratricopeptide repeat domain of betaSGT, indicating a two-carboxylate clamp as the binding mode. The tetratricopeptide repeats are also necessary for binding to the cysteine string protein. However, this binding mode is distinct from the two-carboxylate clamp that is involved in Hsc70 binding. The C-terminal regions of SGTs might constitute independent protein interaction domains. We conclude that betaSGT is likely to cooperate with alphaSGT as co-chaperone of Hsc70 in the brain. The modular structure of SGTs allows them to recruit client proteins to Hsc70 and to direct the resulting complex toward downstream proteins that take over the respective client proteins.

Amino Acid Motifs↗
PubMed2003

Differential control of the releasable vesicle pools by SNAP-25 splice variants and SNAP-23.

The SNARE complex, consisting of synaptobrevin, syntaxin, and SNAP-25, is essential for calcium-triggered exocytosis in neurosecretory cells. Little is known, however, about how developmentally regulated isoforms and other cognate SNARE components regulate vesicular fusion. To address this question, we examined neuroexocytosis from chromaffin cells of Snap25 null mice rescued by the two splice variants SNAP-25a and SNAP-25b and the ubiquitously expressed homolog SNAP-23. In the absence of SNAP-25, vesicle docking persisted, but primed vesicle pools were empty and fast calcium-triggered release abolished. Single vesicular fusion events showed normal characteristics, except for a shorter duration of the fusion pore. Overexpression of SNAP-25a, SNAP-25b, and SNAP-23 resulted in three distinct phenotypes; SNAP-25b induced larger primed vesicle pools than SNAP-25a, whereas SNAP-23 did not support a standing pool of primed vesicles. We conclude that three alternative SNARE components support exocytosis, but they differ in their ability to stabilize vesicles in the primed state.

Alternative Splicing↗
PubMed2003

Regulation of insulin exocytosis by Munc13-1.

The slower kinetics of insulin release from pancreatic islet beta cells, as compared with other regulated secretory processes such as chromaffin granule secretion, can in part be explained by the small number of the insulin granules that are docked to the plasma membrane and readily releasable. In type-2 diabetes, the kinetics of insulin secretion become grossly distorted, and, to therapeutically correct this, it is imperative to elucidate the mechanisms that regulate priming and secretion of insulin secretory granules. Munc13-1, a synaptic protein that regulates SNARE complex assembly, is the major protein determining the priming of synaptic vesicles. Here, we demonstrate the presence of Munc13-1 in human, rat, and mouse pancreatic islet beta cells. Expression of Munc13-1, along with its cognate partners, syntaxin 1a and Munc18a, is reduced in the pancreatic islets of type-2 diabetes non-obese Goto-Kakizaki and obese Zucker fa/fa rats. In insulinoma cells, overexpressed Munc13-1-enhanced green fluorescent protein is translocated to the plasma membrane in a temperature-dependent manner. This, in turn, greatly amplifies insulin exocytosis as determined by patch clamp capacitance measurements and radioimmunoassay of the insulin released. The potentiation of exocytosis by Munc13-1 is dependent on endogenously produced diacylglycerol acting on the overexpressed Munc13-1 because it is blocked by a phospholipase C inhibitor (U73122) and abrogated when the diacylglycerol binding-deficient Munc13-1H567K mutant is expressed instead of the wild type protein. Our data demonstrate that Munc13-mediated vesicle priming is not restricted to neurotransmitter release but is also functional in insulin secretion, where it is subject to regulation by the diacylglycerol second messenger pathway. In view of our findings, Munc13-1 is a potential drug target for therapeutic optimization of insulin secretion in diabetes.

Animals↗
PubMed2003

Molecular mechanisms of active zone function.

In response to incoming action potentials the presynapse exocytoses neurotransmitter filled vesicles. Proper information encoding requires that the release occurs exactly at the junction to the postsynapse, that it is temporally tightly coupled to the electrical signal and that it proceeds efficiently. This review discusses the structure and function of the active zone, which is defined as the region of the presynapse that is specialized for vesicle release. Particular emphasis is put on the molecular players that control spatial restriction, efficiency and timing of exocytosis in the mammalian nervous system.

Action Potentials↗
PubMed2002

Move over protein kinase C, you've got company: alternative cellular effectors of diacylglycerol and phorbol esters.

Diacylglycerol is an essential second messenger in mammalian cells. The most prominent intracellular targets of diacylglycerol and of the functionally analogous phorbol esters belong to the protein kinase C (PKC) family. However, at least five alternative types of high-affinity diacylglycerol/phorbol-ester receptor are known: chimaerins, protein kinase D, RasGRPs, Munc13s and DAG kinase gamma. Recent evidence indicates that these have functional roles in diacylglycerol second messenger signalling in vivo and that several cellular processes depend on these targets rather than protein kinase C isozymes. These findings contradict the still prevalent view according to which all diacylglycerol/phorbol-ester effects are caused by the activation of protein kinase C isozymes. RasGRP1 (in Ras/Raf/MEK/ERK signalling) and Munc13-1 (in neurotransmitter secretion) are examples of non-PKC diacylglycerol/phorbol-ester receptors that mediate diacylglycerol and phorbol-ester effects originally thought to be caused by PKC isozymes. In the future, pharmacological studies on PKC must be complemented with alternative experimental approaches to allow the separation of PKC-mediated effects from those caused by alternative targets of the diacylglycerol second messenger pathway. The examples of RasGRP1 and Munc13-1 show that detailed genetic analyses of C(1)-domain-containing non-PKC diacylglycerol/phorbol-ester receptors in mammals are ideally suited to achieve this goal.

Animals↗