Joachim Kappler

University of Bonn, Bonn, North Rhine-Westphalia, Germany

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Publications (39)120.29 Total impact

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    ABSTRACT: The extracellular matrix of the brain is a highly organized hyaluronan-based supramolecular assembly that is involved in neuronal pathfinding, cell migration, synaptogenesis and neuronal plasticity. Here, we analyze the structure of the hyaluronan-rich pericellular matrix of an oligodendroglial precursor cell line using helium ion beam scanning microscopy at a subnanometer resolution. We find that thin nanofibers are the ultimate building elements of this oligodendroglial pericellular matrix. These structures may participate in the regulation of oligodendroglial maturation and motility.
    Matrix biology: journal of the International Society for Matrix Biology 10/2010; 29(8):664-7. · 3.56 Impact Factor
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    ABSTRACT: Hyaluronan and its receptor CD44 are known to contribute to the invasive growth of different tumors of the central nervous system. It is not known, however, if CD44 is sufficient to activate invasive growth into the brain tissue. This study examines how CD44 regulates the motility and invasive growth of B35 neuroblastoma cells into a hyaluronan-rich environment. A comprehensive experimental approach was used encompassing biochemical techniques, single molecule microscopy, correlative confocal and scanning electron microscopy, morphometry of cellular extensions, live-cell imaging and tracking, transplantation onto organotypic brain slices, two-photon imaging and invasion assays. We found that CD44-GFP fusion protein was localized in filopodia and in focal bleb-like protrusions where it provided binding sites for hyaluronan. Transient expression of CD44-GFP was sufficient to increase the length of filopodia, to enhance cell migration and to promote invasive growth into hyaluronan-rich brain tissue. Thus, CD44 controls molecular devices localized in filopodia and bleb-like specializations of the cell surface that enhance cell migration and invasive growth.
    Biochimica et Biophysica Acta 02/2010; 1803(2):261-74. · 4.66 Impact Factor
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    ABSTRACT: Human synovial fluid contains a high concentration of hyaluronan, a high molecular weight glycosaminoglycan that provides viscoelasticity and contributes to joint lubrication. In osteoarthritis synovial fluid, the concentration and molecular weight of hyaluronan decrease, thus impairing shock absorption and lubrication. Consistently, substitution of hyaluronan (viscosupplementation) is a widely used treatment for osteoarthritis. So far, the organization and dynamics of hyaluronan in native human synovial fluid and its action mechanism in viscosupplementation are poorly characterized at the molecular level. Here, we introduce highly sensitive single molecule microscopy to analyze the conformation and interactions of fluorescently labeled hyaluronan molecules in native human synovial fluid. Our findings are consistent with a random coil conformation of hyaluronan in human synovial fluid, and point to specific interactions of hyaluronan molecules with the synovial fluid matrix. Furthermore, single molecule microscopy is capable of detecting the breakdown of the synovial fluid matrix in osteoarthritis. Thus, single molecule microscopy is a useful new method to probe the structure of human synovial fluid and its changes in disease states like osteoarthritis.
    Journal of Biomedical Optics 01/2010; 15(6):060504. · 2.88 Impact Factor
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    ABSTRACT: yes yes
    01/2010;
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    ABSTRACT: Intracellular calcium influx through NMDA receptors triggers a cascade of deleterious signaling events which lead to neuronal death in neurological conditions such as stroke. However, it is not clear as to the molecular mechanism underlying early damage response from axons and dendrites which are important in maintaining a network essential for the survival of neurons. Here, we examined changes of axons treated with glutamate and showed the appearance of betaIII-tubulin positive varicosities on axons before the appearance of neuronal death. Dizocilpine blocked the occurrence of varicosities on axons suggesting that these microstructures were mediated by NMDA receptor activities. Despite early increased expression of pCaMKII and pMAPK after just 10 min of glutamate treatment, only inhibitors to Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and calpain prevented the occurrence of axonal varicosities. In contrast, inhibitors to Rho kinase, mitogen-activated protein kinase and phosphoinositide 3-kinase were not effective, nor were they able to rescue neurons from death, suggesting CaMKII and calpain are important in axon survival. Activated CaMKII directly phosphorylates collapsin response mediator protein (CRMP) 2 which is independent of calpain-mediated cleavage of CRMP2. Over-expression of CRMP2, but not the phosphorylation-resistant mutant CRMP2-T555A, increased axonal resistance to glutamate toxicity with reduced numbers of varicosities. The levels of both pCRMP2 and pCaMKII were also increased robustly within early time points in ischemic brains and which correlated with the appearance of axonal varicosities in the ischemic neurons. Collectively, these studies demonstrated an important role for CaMKII in modulating the integrity of axons through CRMP2 during excitotoxicity-induced neuronal death.
    Journal of Neurochemistry 10/2009; 111(3):870-81. · 3.97 Impact Factor
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    ABSTRACT: Hyaluronan is an unsulfated linear glycosaminoglycan with the ability to nucleate extracellular matrices by the formation of aggregates with lecticans. These matrices are essential during development of the central nervous system. In the prospective white matter of the developing brain hyaluronan is organized into fiber-like structures according to confocal microscopy of fixed slices which may guide the migration of neural precursor cells [Baier, C., S.L. Baader, J. Jankowski, V. Gieselmann, K. Schilling, U. Rauch, and J. Kappler. 2007. Hyaluronan is organized into fiber-like structures along migratory pathways in the developing mouse cerebellum. Matrix Biol. 26: 348-58]. By using plasmon surface resonance, microinjection into brain slices and fluorescence correlation spectroscopy, we show that the brain-specific lecticans bind to, but also dissociate rather rapidly from hyaluronan. After microinjection into native cerebellar slices a GFP-tagged hyaluronan-binding neurocan fragment was enriched at binding sites in the prospective white matter, which had a directional orientation and formed local stationary concentration gradients in areas where binding sites are abundant. Fluorescence correlation spectroscopy measurements at fixed brain slices revealed that fiber-bound neurocan-GFP was mobile with D(fiber(neurocan-GFP))=4x10(-10)cm(2)/s. Therefore, we propose that hyaluronan-rich fibers in the prospective white matter of the developing mouse cerebellum can guide the diffusion of lecticans. Since lecticans bind a variety of growth and mobility factors, their guided diffusion may contribute to the transport of these polypeptides and to the formation of concentration gradients. This mechanism could serve to encode positional information during development.
    Matrix biology: journal of the International Society for Matrix Biology 08/2009; 28(7):396-405. · 3.56 Impact Factor
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    ABSTRACT: Hyaluronan is an important soluble component of the extracellular matrix of many tissues with well known space-filling, lubricating and signaling functions. As such, hyaluronan can regulate cell adhesion, migration, differentiation and proliferation. Ultrastructural studies showed the existence of fibers and networks of hyaluronan molecules at surfaces, while bulk studies of hyaluronan in solution indicated that the polymer forms random coils. Here, we show that single hyaluronan molecules can be visualized and tracked in three-dimensional samples at room temperature in aqueous buffer. Using a wide-field fluorescence microscope equipped with laser excitation and an sensitive and fast EMCCD camera for fluorescence detection, single FITC-labeled hyaluronan molecules from rooster comb were detected in aqueous solutions. Freely moving hyaluronan-FITC could be tracked over up to 20 images acquired at a frame rate of 98 Hz. Analysis of the trajectories revealed Brownian motion of hyaluronan in tris-buffered saline with an average diffusion coefficient D=3.0+/-0.2 microm(2)/s. These observations confirm the concept that hyaluronan molecules form random coils in solution. The possibility of following the tracks of single hyaluronan molecules in solution facilitates the analysis of processes that lead to the formation of more organized forms of hyaluronan and its interactions with cells with very high spatial and temporal accuracy.
    Glycoconjugate Journal 09/2008; 25(6):555-60. · 1.88 Impact Factor
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    ABSTRACT: Lysosulfatide is a derivative of the glycosphingolipid sulfatide. It is a major component of high density lipoproteins and was detected in the human brain. Here, we show that lysosulfatide acts as an extracellular signal regulating the migration of a neural precursor cell line (B35 neuroblastoma cells) by rapidly promoting process retraction and cell rounding. These cells express the lysosulfatide receptor S1P3 according to RT-PCR, western blotting and immunocytochemistry, but S1P3 does not mediate the effect since preincubation with three different compounds known to inhibit S1P3 did not block lysosulfatide-induced cell rounding. The signal transduction after stimulation with 3 microM lysosulfatide involves a rapid increase of [Ca2+]i which causes process retraction. This mechanism may be relevant under conditions where neural cells encounter elevated lysosulfatide levels as for example under pathological conditions after breakdown of the blood brain barrier or possibly in the lysosomal sulfatide storage disorder metachromatic leukodystrophy.
    Neurochemical Research 09/2008; 34(3):508-17. · 2.13 Impact Factor
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    ABSTRACT: In long-term time-lapse studies of cell migration, it is often important to distinguish active movement of individual cells from global tissue motion caused, for instance, by morphogenetic changes, or due to artefacts. We have developed a method to define and correct global movements. This is realized by the sequential morphing of image sequences to the initial image based on the position of immobile reference objects. Technically, the approach is implemented in ImageJ, using the plugin UnwarpJ. We describe an efficient way to select parameter settings such as to optimize image correction. To this end, we implemented a strict statistical control that allows to quantify image registration quality. We document this approach using a time-lapse sequence of migrating interneurons in slice cultures of the developing cerebellum.
    Journal of Neuroscience Methods 08/2008; 172(1):67-73. · 2.11 Impact Factor
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    ABSTRACT: Cerebroside sulfotransferase (CST) catalyzes the 3'-sulfation of galactose residues in several glycolipids. Its major product in the mammalian brain is sulfatide, which is an essential myelin component. Using epitope-tagged variants, murine CST was found to localize to the Golgi apparatus, but in contrast to previous assumptions, not to the trans-Golgi network. An examination of enhanced green fluorescent protein (EGFP)-tagged CST suggests that CST forms homodimers and that dimerization is mediated by the lumenal domain of the enzyme, as shown by immunoprecipitation and density gradient centrifugation. In order to verify that dimerization of CST observed by biochemical methods reflects the behavior of the native protein within living cells, the mobility of CST-EGFP was examined using fluorescence correlation spectroscopy. These experiments confirmed the homodimerization of CST-EGFP fusion proteins in vivo. In contrast to full-length CST, a fusion protein of the amino-terminal 36 amino acids of CST fused to EGFP was exclusively found as a monomer but nevertheless showed Golgi localization.
    Biochemistry 09/2007; 46(32):9260-9. · 3.38 Impact Factor
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    ABSTRACT: Collapsin response mediator proteins (CRMPs) are important brain-specific proteins with distinct functions in modulating growth cone collapse and axonal guidance during brain development. Our previous studies have shown that calpain cleaves CRMP3 in the adult mouse brain during cerebral ischemia [S.T. Hou et al. (2006) J. Neurosci., 26, 2241-2249]. Here, the expression of all CRMP family members (1-5) was examined in mouse brains that were subjected to middle cerebral artery occlusion. Among the five CRMPs, the expressions of CRMP1, CRMP3 and CRMP5 were the most abundant in the cerebral cortex and all CRMPs were targeted for cleavage by ischemia-activated calpain. Sub-cellular fractionation analysis showed that cleavage of CRMPs by calpain occurred not only in the cytoplasm but also in the synaptosomes isolated from ischemic brains. Moreover, synaptosomal CRMPs appeared to be at least one-fold more sensitive to cleavage compared with those isolated from the cytosolic fraction in an in-vitro experiment, suggesting that synaptosomal CRMPs are critical targets during cerebral ischemia-induced neuronal injury. Finally, the expression of all CRMPs was colocalized with TUNEL-positive neurons in the ischemic mouse brain, which further supports the notion that CRMPs may play an important role in neuronal death following cerebral ischemia. Collectively, these studies demonstrated that CRMPs are targets of calpains during cerebral ischemia and they also highlighted an important potential role that CRMPs may play in modulating ischemic neuronal death.
    European Journal of Neuroscience 09/2007; 26(4):801-9. · 3.75 Impact Factor
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    ABSTRACT: Hyaluronan is a free glycosaminoglycan which is abundant in the extracellular matrix of the developing brain. Although not covalently linked to any protein it can act as a backbone molecule forming aggregates with chondroitin sulfate proteoglycans of the lectican family and link proteins. Using neurocan-GFP as a direct histochemical probe we analyzed the distribution and organization of hyaluronan in the developing mouse cerebellum, and related its fine structure to cell types of specified developmental stages. We observed a high affinity of this probe to fiber-like structures in the prospective white matter which are preferentially oriented parallel to the cerebellar cortex during postnatal development suggesting a specially organized form of hyaluronan. In other layers of the cerebellar cortex, the hyaluronan organization seemed to be more diffuse. During the second postnatal week, the overall staining intensity of hyaluronan in the white matter declined but fiber-like structures were still present at the adult stage. This type of hyaluronan organization is different from perineuronal nets e.g. found in deep cerebellar nuclei. Double staining experiments with cell type specific markers indicated that these fiber-like structures are predominantly situated in regions where motile cells such as Pax2-positive inhibitory interneuron precursors and MBP-positive oligodendroglial cells are located. In contrast, more stationary cells such as mature granule cells and Purkinje cells are associated with lower levels of hyaluronan in their environment. Thus, hyaluronan-rich fibers are concentrated at sites where specific neural precursor cell types migrate, and the anisotropic orientation of these fibers suggests that they may support guided neural migration during brain development.
    Matrix Biology 07/2007; 26(5):348-58. · 3.19 Impact Factor
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    ABSTRACT: Cell death after stroke involves apoptotic, autophagocytic and necrotic mechanisms which may cause the release of cytosolic proteins to the extracellular space. Aldolase C (AldC) is the brain specific isoform of the glycolytic enzyme fructose-1,6-bisphosphate aldolase. According to its characteristic striped expression pattern in the adult cerebellum AldC is also termed zebrin II. Here, we demonstrate release of AldC into the cerebrospinal fluid (CSF) after stroke in vivo. Studies with cell cultures confirmed that AldC is released to the extracellular space after hypoxia. Moreover, addition of purified recombinant AldC to networks of cortical neurons plated on multielectrode arrays reversibly inhibited the spontaneous generation of action potentials at AldC concentrations which can be expected to occur after lesions of the human cerebral cortex. This mechanism could be relevant in the pathogenesis of the electrophysiological changes in the penumbra region after stroke.
    Neurochemical Research 12/2006; 31(11):1297-303. · 2.13 Impact Factor
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    ABSTRACT: Collapsin response mediator proteins (CRMPs) mediate growth cone collapse during development, but their roles in adult brains are not clear. Here we report the findings that the full-length CRMP-3 (p63) is a direct target of calpain that cleaves CRMP-3 at the N terminus (+76 amino acid). Interestingly, activated calpain in response to excitotoxicity in vitro and cerebral ischemia in vivo also cleaved CRMP-3, and the cleavage product of CRMP-3 (p54) underwent nuclear translocation during neuronal death. The expression of p54 was colocalized with the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive nuclei in glutamate-treated cerebellar granule neurons (CGNs) and in ischemic neurons located in the infarct core after focal cerebral ischemia, suggesting that p54 might be involved in neuronal death. Overexpression studies showed that p54, but not p63, caused death of human embryonic kidney cells and CGNs, whereas knock-down CRMP-3 expression by selective small interfering RNA protected neurons against glutamate toxicity. Collectively, these results reveal a novel role of CRMP-3 in that calpain cleavage of CRMP-3 and the subsequent nuclear translocation of the truncated CRMP-3 evokes neuronal death in response to excitotoxicity and cerebral ischemia. Our findings also establish a novel route of how calpain signals neuron death.
    Journal of Neuroscience 03/2006; 26(8):2241-9. · 6.91 Impact Factor
  • Uwe Rauch, Joachim Kappler
    Advances in pharmacology (San Diego, Calif.) 02/2006; 53:337-56.
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    ABSTRACT: We describe a simple and widely applicable method to measure cell migration in time-lapse sequences of fluorescently labeled cells in culture. Briefly, binarized cell images obtained after thresholding were cumulatively projected, and the covered areas were measured. This procedure determines the time course of the track area successively covered by the cell population. Under conditions where cell growth is negligible, a robust index of cell motility is derived from normalized plots for the displacement of cells over time. We applied this method to quantitatively examine the migration of B35 neuroblastoma cells transiently expressing GFP and to C6 glioma cells after staining with Hoechst 33258. This sensitive assay detected the influence of agents which inhibit actin polymerization (cytochalasin B) or interfere with the maintenance of cell polarity (methyl-beta-cyclodextrin) on cell migration. Thus, this assay is a versatile tool to measure quickly the migration of different cell types using different labeling strategies.
    Experimental Cell Research 01/2006; 311(2):272-80. · 3.56 Impact Factor
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    ABSTRACT: Collapsin response mediator proteins (CRMPs) form a family of cytosolic phosphoproteins which are involved in the signal transduction of semaphorin 3A leading to growth cone collapse. These proteins interact with a variety of cytosolic proteins including tubulin heterodimers. Here, we show that CRMP-4 co-localizes with F-actin in regular rib-like structures within lamellipodia of B35 neuroblastoma cells. Furthermore, depolymerization of actin fibers changed the distribution of GFP-CRMP-4 in vivo. In vitro, recombinant CRMP-4 formed homo-oligomers, bound to F-actin and organized F-actin into tight bundles. Both oligomerization and F-actin bundling depended on the C-terminal part of CRMP-4. The stoichiometry of actin and CRMP-4 in bundles was approximately 1:1 and the apparent equilibrium constant of the microfilament-CRMP-4 interaction was estimated from bundling assays as K(app) = 730 mM(-1). CRMP-4 was abundant in the cytosol of B35 neuroblastoma cells and its concentration was measured as approximately 1.7 microM. Overexpression of CRMP-4 inhibited the migration of B35 neuroblastoma cells, while knockdown of CRMP-4 enhanced cell migration and disturbed rib-like actin-structures in lamellipodia. Taken together, our data indicate that CRMP-4 promotes bundling of F-actin in vitro, that it is an important component of rib-like actin bundles in lamellipodia in vivo and that it functionally regulates the actin cytoskeleton in motile cells. These findings suggest a specific regulatory role of CRMP-4 towards the actin cytoskeleton which may by be relevant for growth cone collapse.
    Experimental Cell Research 12/2005; 310(2):434-44. · 3.56 Impact Factor
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    ABSTRACT: NeuN (neuronal nuclei) is an antigen used widely in research and diagnostics to identify postmitotic neurons. The present study aims at an initial understanding of the molecular nature and functional significance of this as yet ill-defined antigen. Using isoelectric focusing, both the 46- and 48-kDa isoforms of NeuN can be separated in multiple spots spanning a pH range of 8-10.5, suggesting that they might be phosphorylated. Enzymatic dephosphorylation abolishes NeuN immunoreactivity, confirming that NeuN is indeed a phosphoprotein, and establishing that binding of the defining antibody depends on its state of phosphorylation. Combined biochemical and immunohistochemical analysis show that both the 46- and the 48-kDa NeuN isoforms can be localized to the cell nucleus as well as in the neuronal cytoplasm. Their relative concentration in these compartments is distinct, however, with the 48-kDa isoform being the predominant isoform in the cytoplasm. Within the nucleus, NeuN is found preferentially in areas of low chromatin density and virtually excluded from areas containing densely packed DNA. The present identification of multiple differentially phosphorylated isoforms of NeuN, together with recent reports on the dependence of NeuN immunoreactivity levels on a variety of physiologic or pathologic signals, suggests a previously unappreciated level of complexity in the regulation of this enigmatic, neuron-specific antigen.
    Journal of Neuroscience Research 03/2005; 79(3):295-302. · 2.97 Impact Factor
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    ABSTRACT: Although the syndrome of mesial temporal lobe epilepsy (MTLE) associated with hippocampal sclerosis has been elaborated in recent years, pathogenesis and pathomechanisms are still elusive. Performing protein hunting in hippocampus of patients with MTLE we detected derangement of collapsin response mediated protein-2 (CRMP-2). Hippocampal tissue from controls and MTLEs was taken and two-dimensional gel electrophoresis with subsequent MALDI-MS-characterisation was applied. The proteomic approach identified 13 spots unambiguously assigned to CRMP-2. Three spots at molecular weight 55 kDa showed a significant decrease in MTLE and other 3 spots at 65 kDa showed deranged in MTLE. Immunoblotting revealed two bands at 65 and 55 kDa in the control group whereas the 55 kDa band was extremely low expressed in MTLE. CRMP-2 is required to induce axonal outgrowth and maintaining neuronal polarity in hippocampal neurons and the significant decrease of this protein may represent or underlie impaired neuronal plasticity, neurodegeneration, wiring of the brain in MTLE and may explain abnormal migration. Therefore, the decrease of CRMP-2 may well contribute to the understanding of the still unclear pathomechanisms involved in MTLE.
    Neurochemical Research 01/2005; 29(12):2189-96. · 2.13 Impact Factor
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    ABSTRACT: Hyaluronan is an unsulfated glycosaminoglycan (GAG) that is ubiquitously expressed in the extracellular matrix (ECM) of all vertebrates, where hyaluronan rich matrices constitute a particular permissive environment for the development of complex biological structures and also for tumor progression. Because of its conserved structure and ubiquitous expression, antibodies for its histochemical detection cannot be produced. We have engineered a fusion protein, neurocan-GFP, and expressed it as a secreted molecule in mammalian cells. Neurocan-GFP fusion protein specifically binds to hyaluronan and directly visualizes hyaluronan on tissue sections, revealing a very detailed picture of hyaluronan distribution. The fluorescent fusion protein can be used in combination with antibodies and nuclear markers for double or triple staining. In addition, it is suitable to visualize hyaluronan on living cells by time-lapse video microscopy. The successful production and application of the neurocan-GFP fusion protein opens up new perspectives for using GFP fusion proteins as detection tools in histological and cytological studies complementing conventional antibody and biotin/avidin techniques.
    Journal of Histochemistry and Cytochemistry 08/2004; 52(7):915-22. · 2.26 Impact Factor

Publication Stats

960 Citations
120.29 Total Impact Points

Institutions

  • 2000–2010
    • University of Bonn
      • • Institute of Biochemistry and Molecular Biology
      • • Institut für Physiologie I
      • • Physics Institute
      Bonn, North Rhine-Westphalia, Germany
  • 2006
    • National Research Council Canada
      • Institute for Biological Sciences (IBS)
      Ottawa, Ontario, Canada
    • Lund University
      • Department of Experimental Medical Science
      Lund, Skåne, Sweden
  • 1995–2002
    • Heinrich-Heine-Universität Düsseldorf
      • Neurologische Klinik
      Düsseldorf, North Rhine-Westphalia, Germany