Biomedical subjects

Andreas W Püschel

Publications and source records attributed to Andreas W Püschel.

14 recordsLinked to original sources

PubMed2006

GTPases and the control of neuronal polarity.

Neurons are probably the most highly polarized cell type and typically develop a single axon and several dendrites. The establishment of a polarized morphology and the functional specialization of axonal and dendritic compartments are essential steps in the differentiation of neurons. Primary cultures of dissociated hippocampal neurons are a widely used system to study the development of neuronal differentiation. In this article, we will describe gain-of-function and loss-of-function approaches that allow us to analyze the role of GTPases in neuronal differentiation.

Animals↗

PubMed2005

Dual functional activity of semaphorin 3B is required for positioning the anterior commissure.

Chemorepulsion by semaphorins plays a critical role during the development of neuronal projections. Although semaphorin-induced chemoattraction has been reported in vitro, the contribution of this activity to axon pathfinding is still unclear. Using genetic and culture models, we provide evidence that both attraction and repulsion by Sema3B, a secreted semaphorin, are critical for the positioning of a major brain commissural projection, the anterior commissure (AC). NrCAM, an immunoglobulin superfamily adhesion molecule of the L1 subfamily, associates with neuropilin-2 and is a component of a receptor complex for Sema3B and Sema3F. Finally, we show that activation of the FAK/Src signaling cascade distinguishes Sema3B-mediated attractive from repulsive axonal responses of neurons forming the AC, revealing a mechanism underlying the dual activity of this guidance cue.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Semaphorin 4B interacts with the post-synaptic density protein PSD-95/SAP90 and is recruited to synapses through a C-terminal PDZ-binding motif.

The semaphorins are a large family of proteins that act as guidance signals for axons and dendrites. The class 4 semaphorins are integral membrane proteins that are widely expressed throughout the nervous system. Here, we show that a subclass of these semaphorins is characterized by a PDZ-binding motif at their carboxy-terminus. This sequence mediates the interaction with the post-synaptic density protein PSD-95/SAP90. Co-expression of Sema4B with PSD-95 in COS 7 cells results in the clustering of Sema4B. Sema4B co-localizes with PSD-95 at synaptic contacts between cultured hippocampal neurons. This synaptic localization depends on the presence of the PDZ-binding motif.

Amino Acid Motifs↗

PubMed2005

Inactivation of the Sema5a gene results in embryonic lethality and defective remodeling of the cranial vascular system.

The semaphorins are a large family of proteins involved in the patterning of both the vascular and the nervous systems. In order to analyze the function of the membrane-bound semaphorin 5A (Sema5A), we generated mice homozygous for a null mutation in the Sema5a gene. Homozygous null mutants die between embryonic development days 11.5 (E11.5) and E12.5, indicating an essential role of Sema5A during embryonic development. Mutant embryos did not show any morphological defects that could account for the lethality of the mutation. A detailed analysis of the vascular system uncovered a role of Sema5A in the remodeling of the cranial blood vessels. In Sema5A null mutants, the complexity of the hierarchically organized branches of the cranial cardinal veins was decreased. Our results represent the first genetic analysis of the function of a class 5 semaphorin during embryonic development and identify a role of Sema5A in the regional patterning of the vasculature.

Animals↗

PubMed2004

The sequential activity of the GTPases Rap1B and Cdc42 determines neuronal polarity.

The establishment of a polarized morphology is an essential step in the differentiation of neurons with a single axon and multiple dendrites. In cultured rat hippocampal neurons, one of several initially indistinguishable neurites is selected to become the axon. Both phosphatidylinositol 3,4,5-trisphosphate and the evolutionarily conserved Par complex (comprising Par3, Par6 and an atypical PKC (aPKC) such as PKClambda or PKCzeta) are involved in axon specification. However, the initial signals that establish cellular asymmetry and the pathways that subsequently translate it into structural changes remain to be elucidated. Here we show that localization of the GTPase Rap1B to the tip of a single neurite is a decisive step in determining which neurite becomes the axon. Using GTPase mutants and RNA interference, we found that Rap1B is necessary and sufficient to initiate the development of axons upstream of Cdc42 and the Par complex.

Animals↗

PubMed2004

Semaphorin 3A stimulates neurite extension and regulates gene expression in PC12 cells.

The secreted semaphorin 3A (Sema3A) is a member of a large family of proteins that act as guidance signals for axons and dendrites. While the receptors and signaling pathways that mediate the repulsive effects of semaphorins are beginning to be understood in some detail, the mechanisms that are responsible for the ability of Sema3A to stimulate the extension of dendrites remain to be elucidated. Here we show that PC12 cells, a model widely used to study neuronal differentiation, can be used to dissect this pathway. Sema3A is as effective as nerve growth factor in stimulating the extension of neurites from PC12 cells. We show that Sema3A is able to regulate gene expression and identify mitochondria as a novel target of Sema3A signaling. Pharmacological block of mitochondrial reactive oxygen species production abolishes the extension of neurites in response to Sema3A. These results show that the characterization of transcripts that are regulated by axon guidance signals may help to identify novel components of their signaling pathways.

Animals↗

PubMed2004

Plexin-B family members demonstrate non-redundant expression patterns in the developing mouse nervous system: an anatomical basis for morphogenetic effects of Sema4D during development.

Semaphorins and their receptors play important roles in patterning the connectivity of the developing nervous system and recent data suggest that members of the plexin-B family of semaphorin receptors may be involved in axonal guidance. Here we show that the mRNAs of the three plexin-B genes, plxnb1, plxnb2 and plxnb3 (plexin-B1, plexin-B2 and plexin-B3), respectively, are expressed in highly specific and non-redundant patterns in peripheral and central components of the nervous system over defined periods during murine development. Whereas plexin-B1 and plexin-B2 are strongly expressed in the neuroepithelium and developing neurons, plexin-B3 mRNA is selectively localized to the white matter. Moreover, plexin-B1 and its ligand Sema4D are expressed in complementary patterns in several regions such as the developing neopallial cortex, the dorsal root ganglia and the spinal cord over embryonic stages. The Sema4d gene demonstrates a dramatic switch from prenatal expression in neuronal populations to a postnatal expression in oligodendrocytes. In contrast to its collapsing activity on growth cones of embryonic retinal ganglion cells and hippocampal neurons, soluble Sema4D enhances axonal outgrowth in embryonic cortical explants cultured in collagen matrices. Thus, plexin-B family members and Sema4D are likely to play complex and non-redundant roles during the development of the nervous system.

Animals↗

PubMed2004

Semaphorin 3A-mediated axon guidance regulates convergence and targeting of P2 odorant receptor axons.

Semaphorins are known to play an important role in axon guidance of vertebrate olfactory sensory neurons to their targets in specific glomeruli of the olfactory bulb (OB). However, it is not clear how semaphorin-mediated guidance contributes to a systematic hierarchy of cues that govern the organization of this system. Because of the putative role that odorant receptor molecules such as P2 could play in establishing appropriate glomerular destinations for growing olfactory axons, we have also determined the spatial organization of P2 glomeruli in semaphorin 3A (Sema3A) mutant mice. First, in the postnatal OB of control and Sema3A(-/-) mice, we analysed the trajectories of olfactory axons that express the Sema3A receptor, neuropilin-1 (npn-1) and the positions of npn-1(+) glomeruli. Sema3A at the ventral OB midline guides npn-1(+) axons to targets in the lateral and medial OB. Absence of Sema3A permits many npn-1 axons to terminate aberrantly in the rostral and ventral OB. Second, in Sema3A(-/-) mice, many P2 axons are abnormally distributed throughout the ventral OB nerve layer and converge in atypical locations compared with littermate controls where P2 axons converge on stereotypically located lateral and medial glomeruli. In addition to their radically altered spatial distribution, P2 glomeruli in Sema3A(-/-) mice are significantly smaller and more numerous than in heterozygote littermates. These data show that Sema3A is an important repulsive olfactory guidance cue that establishes restricted npn-1(+) subcompartments in the olfactory bulb. Furthermore, Sema3A plays a key role in the convergence of axons expressing the odorant receptor P2 onto their appropriate targets.

Animals↗

PubMed2003

Structure of the semaphorin-3A receptor binding module.

The semaphorins are a large group of extracellular proteins involved in a variety of processes during development, including neuronal migration and axon guidance. Their distinctive feature is a conserved 500 amino acid semaphorin domain, a ligand-receptor interaction module also present in plexins and scatter-factor receptors. We report the crystal structure of a secreted 65 kDa form of Semaphorin-3A (Sema3A), containing the full semaphorin domain. Unexpectedly, the semaphorin fold is a variation of the beta propeller topology. Analysis of the Sema3A structure and structure-based mutagenesis data identify the neuropilin binding site and suggest a potential plexin interaction site. Based on the structure, we present a model for the initiation of semaphorin signaling and discuss potential similarities with the signaling mechanisms of other beta propeller cell surface receptors, such as integrins and the LDL receptor.

Amino Acid Sequence↗

PubMed2003

Class 3 semaphorins control vascular morphogenesis by inhibiting integrin function.

The motility and morphogenesis of endothelial cells is controlled by spatio-temporally regulated activation of integrin adhesion receptors, and integrin activation is stimulated by major determinants of vascular remodelling. In order for endothelial cells to be responsive to changes in activator gradients, the adhesiveness of these cells to the extracellular matrix must be dynamic, and negative regulators of integrins could be required. Here we show that during vascular development and experimental angiogenesis, endothelial cells generate autocrine chemorepulsive signals of class 3 semaphorins (SEMA3 proteins) that localize at nascent adhesive sites in spreading endothelial cells. Disrupting endogenous SEMA3 function in endothelial cells stimulates integrin-mediated adhesion and migration to extracellular matrices, whereas exogenous SEMA3 proteins antagonize integrin activation. Misexpression of dominant negative SEMA3 receptors in chick embryo endothelial cells locks integrins in an active conformation, and severely impairs vascular remodelling. Sema3a null mice show vascular defects as well. Thus during angiogenesis endothelial SEMA3 proteins endow the vascular system with the plasticity required for its reshaping by controlling integrin function.

Animals↗

PubMed2003

The function of semaphorins during nervous system development.

The wiring of the nervous system is established through a progressive refinement of the choices made by a growing axon. The growth cone is a highly motile structure at the tip of the axon that integrates the multitude of signals present in its environment and translates these signals into structural changes of the cytoskeleton that determine the rate and direction of extension. Four families of guidance cues were identified that provide directional information to growing axons: the ephrins, the netrins, the slit proteins, and the semaphorins. The semaphorins represent the largest family of guidance cues identified so far that can be divided into 8 classes based on the degree of sequence similarity between their semaphorin domains and the presence of class specific carboxy-terminal domains (CTD). Collectively, the in vitro data indicate that semaphorins function mainly as chemorepellents that direct axons away from tissues marked by their expression, but can also act as chemoattractants in some cases. Genetic analysis of the semaphorins and their receptor showed that these proteins play an important role in the development and function of different tissues other than the nervous system. The effects of the different semaphorins are mediated by a variety of receptor complexes that include members of the neuropilin and plexin protein families as well as other membrane proteins belonging to the immunoglobulin superfamily. Plexins directly and indirectly interact with members of the Rho-like GTPases, the kinases Fes, Fyn, and Cdk5, the oxidoreductase MICAL, lipoxygenase, and the CRMP proteins. The signal transduction cascades that include these signalling proteins and link semaphorin receptors to the cytoskeleton are still incompletely understood.

Animals↗

PubMed2002

Antagonistic effects of Rnd1 and RhoD GTPases regulate receptor activity in Semaphorin 3A-induced cytoskeletal collapse.

The semaphorins are a large protein family that is involved in the patterning of neuronal connections in the developing nervous system of both vertebrates and invertebrates. The chemorepulsive axon guidance signal Semaphorin 3A (Sema3A) induces the depolymerization of actin filaments and the collapse of sensory growth cones by activating a receptor complex that contains a plexin as the signal-transducing subunit. Here we show that, of a large number of GTPases tested, only Rnd1 and RhoD bind the cytoplasmic domain of Plexin-A1. Recruitment of active Rnd1 is sufficient to trigger signaling by Plexin-A1, even in the absence of Sema3A, and initiates cytoskeletal collapse by activating its cytoplasmic domain. RhoD, in contrast, blocks Plexin-A1 activation by Rnd1 and repulsion of sympathetic axons by Sema3A. Thus, the antagonism of two GTPases regulates the activity of the Sema3A receptor, and activation by Rnd1 appears to be an essential step in signaling by Plexin-A1.

Animals↗

PubMed2002

The function of neuropilin/plexin complexes.

Neuropilins bind the secreted class 3 semaphorins with high affinity but require a member of the plexin family to form receptors that are able to activate downstream signal transduction cascades. In this receptor complex neuropilins act as the ligand-binding subunit while plexins function as the signal-transducing subunit in the induction of cytoskeletal collapse by semaphorins. The cytoplasmic domain is highly conserved within the plexin family and interacts with Rho-like GTPases.

Animals↗

PubMed2002

Cell type-specific expression of neuropilins in an MCA-occlusion model in mice suggests a potential role in post-ischemic brain remodeling.

Neuropilin-1 and -2 (NP-1/NP-2) are transmembrane receptors that play a role in axonal guidance by binding of class III semaphorins, and in angiogenesis by binding of the vascular endothelial growth factor isoform VEGF165 and placenta growth factor (PLGF). We investigated the expression pattern of NP-1/NP-2, their co-receptors, vascular endothelial growth factor receptor-1 and -2 (VEGFR-1, VEGFR-2), and their ligands, class III semaphorins, VEGF and PLGF, following experimental cerebral ischemia in mice. By means of in situ hybridization and immunohistochemistry we observed loss of expression of class III semaphorins in neurons in the infarct/peri-infarct area. In contrast, we observed high expression of NP-1 in vessels, neurons, and astrocytes surrounding the infarct. VEGF and PLGF were upregulated in different cell types following stroke. Our results suggest a shift in the balance between semaphorins and VEGF/PLGF, which compete for NP-binding. Possibly, the loss of semaphorins facilitates binding of the competing ligands (VEGF/PLGF), thus inducing angiogenesis. In addition, the observed expression patterns further suggest a neurotrophic/neuroprotective role of VEGF/PLGF.

Animals↗