Aragonite crystalline matrix as an instructive microenvironment for neural development.

The ability to mimic cell-matrix interactions in a way that closely resembles the natural environment is of a great importance for both basic neuroscience and for fabrication of potent scaffolding materials for nervous tissue engineering. Such scaffolding materials should not only facilitate cell attachment but also create a microenvironment that provides essential developmental and survival cues. We previously found that porous aragonite crystalline matrices of marine origin are an adequate and active biomaterial that promotes neural cell growth and tissue development. Here we studied the mechanism underlying these neural cell-material interactions, focusing on the three-dimensional (3D) surface architecture and matrix activity of these scaffolds. We introduced a new cloning technique of the hydrozoan Millepora dichotoma, through which calcein or (45)Ca(2+) were incorporated into the organism's growing skeleton and neuronal cells could then be cultured on the labelled matrices. Herein, we describe the role of matrix 3D architecture on neural cell type composition and survival in culture, and report for the first time on the capacity of neurons and astrocytes to exploit calcium ions from the supporting biomatrix. We found that hippocampal cells growing on the prelabelled aragonite lattice took up aragonite-derived Ca(2+), and even enhanced this uptake when extracellular calcium ions were chelated by EGTA. When the aragonite-derived Ca(2+) uptake was omitted by culturing the cells on coral skeletons coated with gold, cell survival was reduced but not arrested, suggesting a role for matrix architecture in neural survival. In addition, we found that the effects of scaffold architecture and chemistry on cell survival were more profound for neurons than for astrocytes. We submit that translocation of calcium from the biomaterial to the cells activates a variety of membrane-bound signalling molecules and leads to the subsequent cell behaviour. This kind of cell-material interaction possesses great potential for fabricating advanced biomaterials for neural tissue-engineering applications.

Interconnected network of ganglion-like neural cell spheres formed on hydrozoan skeleton.

Identifying scaffolds supporting in vitro reconstruction of active neuronal tissues in their 3-dimensional (3D) conformation is a major challenge in tissue engineering. We have previously shown that aragonite coral exoskeletons support the development of neuronal tissue from hippocampal neurons and astrocytes. Here we show for the first time that the porous aragonite skeleton obtained from bio-fabricated hydrozoan Millepora dichotoma supports the spontaneous organization of dissociated hippocampal cells into highly interconnected 3D ganglion-like tissue formations. The ganglion-like cell spheres expanded hundreds of microns across and included hundreds to thousands of astrocytes and mature neurons, most of them having only cell-cell and no cell-surface interactions. The spheres were linked to the surface directly or through a neck of cells and were interconnected through thick bundles of dendrites, varicosity-bearing axons, and astrocytic processes. Thus, M. dichotoma exoskeleton is a novel scaffold with the unprecedented ability to support a highly ordered organization of neuronal tissue. This unexpected organization opens new opportunities for neuronal tissue regeneration, because the spheres resemble in vivo nervous tissue having high volume of cells associated primarily through cell-cell rather than cell-matrix interactions.

Aragonite crystalline biomatrices support astrocytic tissue formation in vitro and in vivo.

Astrocytes play a pivotal role in the development and function of the central nervous system by regulating synaptic activity and supporting and guiding growing axons. It is therefore a central therapeutic and scientific challenge to develop means to control astrocytic survival and growth. We cultured primary hippocampal astrocytes on a crystalline three-dimensional (3D) aragonite biomatrix prepared from the exoskeleton of the coral Porites lutea. Such culturing led to the formation of astrocytic tissue-like 3D structures in which the cells had a higher survival rate than astrocytes grown in conventional cell culture. Within the pore void areas, multiple layers of astrocytic processes formed concave sheet structures that had no physical contact with the surface. The astrocytes attached to the crystalline perpendicular edges of the crystalline template surface extended processes in 3D and expressed glial fibrillary acidic protein. The astrocytes also expressed gap junctions and developed partly synchronized cytosolic Ca2+ oscillations. Preliminary in vivo models showed that astrocytic networks were also developed when the matrices were implanted into cortical areas of postnatal rat brains. Hence, we suggest that the biomatrix is a biocompatible supportive scaffold for astrocytes and may be exploited in applications for neuronal tissue restoration in injured or diseased central nervous system.

Compact self-wiring in cultured neural networks.

We present a novel approach for patterning cultured neural networks in which a particular geometry is achieved via anchoring of cell clusters (tens of cells/each) at specific positions. In addition, compact connections among pairs of clusters occur spontaneously through a single non-adherent straight bundle composed of axons and dendrites. The anchors that stabilize the cell clusters are either poly-D-lysine, a strong adhesive substrate, or carbon nanotubes. Square, triangular and circular structures of connectivity were successfully realized. Monitoring the dynamics of the forming networks in real time revealed that the self-assembly process is mainly driven by the ability of the neuronal cell clusters to move away from each other while continuously stretching a neurite bundle in between. Using the presented technique, we achieved networks with wiring regions which are made exclusively of neuronal processes unbound to the surface. The resulted network patterns are very stable and can be maintained for as long as 11 weeks. The approach can be used to build advanced neuro-chips for bio-sensing applications (e.g. drug and toxin detection) where the structure, stability and reproducibility of the networks are of great relevance.

The Ras/Rac guanine nucleotide exchange factor mammalian Son-of-sevenless interacts with PACSIN 1/syndapin I, a regulator of endocytosis and the actin cytoskeleton.

Mammalian Son-of-sevenless (mSos) functions as a guanine nucleotide exchange factor for Ras and Rac, thus regulating signaling to mitogen-activated protein kinases and actin dynamics. In the current study, we have identified a new mSos-binding protein of 50 kDa (p50) that interacts with the mSos1 proline-rich domain. Mass spectrometry analysis and immunodepletion studies reveal p50 as PACSIN 1/syndapin I, a Src homology 3 domain-containing protein functioning in endocytosis and regulation of actin dynamics. In addition to PACSIN 1, which is neuron-specific, mSos also interacts with PACSIN 2, which is expressed in neuronal and nonneuronal tissues. PACSIN 2 shows enhanced binding to the mSos proline-rich domain in pull-down assays from brain extracts as compared with lung extracts, suggesting a tissue-specific regulation of the interaction. Proline to leucine mutations within the Src homology 3 domains of PACSIN 1 and 2 abolish their binding to mSos, demonstrating the specificity of the interactions. In situ, PACSIN 1 and mSos1 are co-expressed in growth cones and actin-rich filopodia in hippocampal and dorsal root ganglion neurons, and the two proteins co-immunoprecipitate from brain extracts. Moreover, epidermal growth factor treatment of COS-7 cells causes co-localization of PACSIN 1 and mSos1 in actin-rich membrane ruffles, and their interaction is regulated through epidermal growth factor-stimulated mSos1 phosphorylation. These data suggest that PACSINs may function with mSos1 in regulation of actin dynamics.

The endocytic protein intersectin is a major binding partner for the Ras exchange factor mSos1 in rat brain.

We recently identified intersectin, a protein containing two EH and five SH3 domains, as a component of the endocytic machinery. The N-terminal SH3 domain (SH3A), unlike other SH3 domains from intersectin or various endocytic proteins, specifically inhibits intermediate events leading to the formation of clathrin-coated pits. We have now identified a brain-enriched, 170 kDa protein (p170) that interacts specifically with SH3A. Screening of combinatorial peptides reveals the optimal ligand for SH3A as Pp(V/I)PPR, and the 170 kDa mammalian son-of-sevenless (mSos1) protein, a guanine-nucleotide exchange factor for Ras, con- tains two copies of the matching sequence, PPVPPR. Immunodepletion studies confirm that p170 is mSos1. Intersectin and mSos1 are co-enriched in nerve terminals and are co-immunoprecipitated from brain extracts. SH3A competes with the SH3 domains of Grb2 in binding to mSos1, and the intersectin-mSos1 complex can be separated from Grb2 by sucrose gradient centrifugation. Overexpression of the SH3 domains of intersectin blocks epidermal growth factor-mediated Ras activation. These results suggest that intersectin functions in cell signaling in addition to its role in endocytosis and may link these cellular processes.

Splice variants of intersectin are components of the endocytic machinery in neurons and nonneuronal cells.

We recently identified and cloned intersectin, a protein containing two Eps15 homology (EH) domains and five Src homology 3 (SH3) domains. Using a newly developed intersectin antibody, we demonstrate that endogenous COS-7 cell intersectin localizes to clathrin-coated pits, and transfection studies suggest that the EH domains may direct this localization. Through alternative splicing in a stop codon, a long form of intersectin is generated with a C-terminal extension containing Dbl homology (DH), pleckstrin homology (PH), and C2 domains. Western blots reveal that the long form of intersectin is expressed specifically in neurons, whereas the short isoform is expressed at lower levels in glia and other nonneuronal cells. Immunofluorescence analysis of cultured hippocampal neurons reveals that intersectin is found at the plasma membrane where it is co-localized with clathrin. Ibp2, a protein identified based on its interactions with the EH domains of intersectin, binds to clathrin through the N terminus of the heavy chain, suggesting a mechanism for the localization of intersectin at clathrin-coated pits. Ibp2 also binds to the clathrin adaptor AP2, and antibodies against intersectin co-immunoprecipitate clathrin, AP2, and dynamin from brain extracts. These data suggest that the long and short forms of intersectin are components of the endocytic machinery in neurons and nonneuronal cells.

Tissue plasminogen activator contributes to the late phase of LTP and to synaptic growth in the hippocampal mossy fiber pathway.

The expression of tissue plasminogen activator (tPA) is increased during activity-dependent forms of synaptic plasticity. We have found that inhibitors of tPA inhibit the late phase of long-term potentiation (L-LTP) induced by either forskolin or tetanic stimulation in the hippocampal mossy fiber and Schaffer collateral pathways. Moreover, application of tPA enhances L-LTP induced by a single tetanus. Exposure of granule cells in culture to forskolin results in secretion of tPA, elongation of mossy fiber axons, and formation of new, active presynaptic varicosities contiguous to dendritic clusters of the glutamate receptor R1. These structural changes are blocked by tPA inhibitors and induced by application of tPA. Thus, tPA may be critically involved in the production of L-LTP and specifically in synaptic growth.

The 3'-untranslated region of CaMKII alpha is a cis-acting signal for the localization and translation of mRNA in dendrites.

Neuronal signaling requires that synaptic proteins be appropriately localized within the cell and regulated there. In mammalian neurons, polyribosomes are found not just in the cell body, but also in dendrites where they are concentrated within or beneath the dendritic spine. The alpha subunit of Ca(2+)-calmodulin-dependent protein kinase II (CaMKII alpha) is one of only five mRNAs known to be present within the dendrites, as well as in the soma of neurons. This targeted subcellular localization of the mRNA for CaMKII alpha provides a possible cell biological mechanism both for controlling the distribution of the cognate protein and for regulating independently the level of protein expression in individual dendritic spines. To characterize the cis-acting elements involved in the localization of dendritic mRNA we have produced two lines of transgenic mice in which the CaMKII alpha promoter is used to drive the expression of a lacZ transcript, which either contains or lacks the 3'-untranslated region of the CaMKII alpha gene. Although both lines of mice show expression in forebrain neurons that parallels the expression of the endogenous CaMKII alpha gene, only the lacZ transcripts bearing the 3'-untranslated region are localized to dendrites. The beta-galactosidase protein shows a variable level of expression along the dendritic shaft and within dendritic spines, which suggests that neurons can control the local biochemistry of the dendrite either through differential localization of the mRNA or variations in the translational efficiency at different sites along the dendrite.

Reconstitution of the hippocampal mossy fiber and associational-commissural pathways in a novel dissociated cell culture system.

Synapses of the hippocampal mossy fiber pathway exhibit several characteristic features, including a unique form of long-term potentiation that does not require activation of the N-methyl-D-aspartate receptor by glutamate, a complex postsynaptic architecture, and sprouting in response to seizures. However, these connections have proven difficult to study in hippocampal slices because of their relative paucity (<0.4%) compared to commissural-collateral synapses. To overcome this problem, we have developed a novel dissociated cell culture system in which we have enriched mossy fiber synapses by increasing the ratio of granule-to-pyramidal cells. As in vivo, mossy fiber connections are composed of large dynorphin A-positive varicosities contacting complex spines (but without a restricted localization). The elementary synaptic connections are glutamatergic, inhibited by dynorphin A, and exhibit N-methyl-D-aspartate-independent long-term potentiation. Thus, the simplicity and experimental accessibility of this enriched in vitro mossy fiber pathway provides a new perspective for studying nonassociative plasticity in the mammalian central nervous system.

A presynaptic locus for long-term potentiation of elementary synaptic transmission at mossy fiber synapses in culture.

The complex circuitry of the CA3 region and the abundance of collateral connections has made it difficult to study the mossy fiber pathway in hippocampal slices and therefore to establish the site of expression of long-term potentiation at these synapses. Using a novel cell culture system, we have produced long-term potentiation of the elementary synaptic connections on single CA3 pyramidal neurons following tetanic stimulation of individual dentate gyrus granule cells. As is the case for the hippocampal slice, this potentiation was independent of N-methyl-D-aspartate receptor activation, was simulated by application of forskolin, and its induction did not require any modulatory input. The increase in synaptic strength was accompanied by a reduction in the number of failures of transmission and by an increase in the coefficient of variation of the responses and was prevented by presynaptic injection of an inhibitor of protein kinase A. These findings show that mossy fiber long-term potentiation has a presynaptic locus and that its expression is dependent on protein kinase A.

Serum modulates mast cell responses to IgE antigen stimulation.

Serum induces the expression of the fos and jun gene families, which encode the transcription factor AP-1. Since we previously found that activation of mast cells by IgE-antigen (Ag) induces the mRNA accumulation of c-fos, c-jun, junB and junD proto-oncogenes, we were prompted to investigate whether serum could affect such accumulation in these cells. In addition, we investigated whether serum could modulate inhibition of DNA synthesis in immunologically stimulated mast cells. Mast cells, which were cultured in the presence of fetal calf serum (FCS), were characterized by a high proliferation rate and high accumulation of the mRNA of c-fos, junB and junD proto-oncogenes. After sustained FCS deprivation both DNA synthesis and the level of c-fos mRNA were significantly decreased, as expected, whereas the level of c-jun, junB and junD mRNA were not affected. As opposed to mast cells which were cultured in the presence of FCS, immunological stimulation of FCS-deprived cells resulted in DNA synthesis inhibition and an increase in c-fos expression. The results also show that the level of c-fos mRNA was increased by either IgE-Ag or FCS up to a similar level, while these two triggers could not act synergistically to enhance this expression further. Thus, changes in DNA synthesis, induced by FCS, block the ability of the immunological challenge to inhibit mast cell growth and to enhance c-fos mRNA accumulation.

Protein kinase C regulates proliferation of mast cells and the expression of the mRNAs of fos and jun proto-oncogenes during activation by IgE-Ag or calcium ionophore A23187.

Short-term stimulation (up to 16 hours) of interleukin-3 (IL-3)-dependent mouse bone marrow-derived mast cells, Abelson transformed mouse liver-derived mast cells, or rat basophilic leukemia cells by either IgE-Ag or calcium ionophore A23187 resulted in inhibition of their proliferation as measured by 3H-thymidine incorporation and MTT (tetrazolium) assays, and in accumulation of the mRNAs of c-fos, c-jun, junB and slightly of junD proto-oncogenes. The involvement of protein kinase C (PKC) in these responses was investigated by using several approaches of enzyme activity regulation. Direct activation of the PKC was achieved by short-term exposure of the cells to the PKC-specific activator phorbol 12-myristate-13-acetate (PMA). Inhibition of PKC activity was obtained by either prolonged treatment of the cells with PMA or by exposure of the cells to the PKC inhibitors H-7 and staurosporine. The results showed the following: (1) Short-term exposure of mast cells to PMA was sufficient to induce inhibition of proliferation. (2) An increase in PKC activity was associated with a decrease in the proliferation of IgE-dinitrophenol (DNP) or calcium ionophore A23187-stimulated cells. (3) A direct correlation was found between the increase in PKC activity and the increase in the level of the mRNAs of the jun proto-oncogenes in cells activated by both stimuli mentioned. (4) While an increase in PKC activity was associated with the upregulation of the level of c-fos mRNA during calcium ionophore A23187 stimulation, it showed the opposite effect on the expression of the mRNA of this proto-oncogene when the cells were triggered by IgE-DNP. Therefore, we concluded that PKC plays various roles in the expression of the mRNA of c-fos in activated mast cells depending on the stimulus involved. In addition, the expression of the mRNA of c-jun and junB proto-onogenes is not coordinately regulated with that of c-fos during immunologic stimulation. This discordancy, which is associated with the increase in PKC activity in mast cells, may play a role in the regulation of the transcription of AP-1-responsive genes, and therefore could be associated with the regulation of proliferation of these cells.

Thrombin-induced calcium-independent degranulation of human neutrophils.

Thrombin, a highly specific coagulation factor, can rapidly trigger lysozyme release from human neutrophils without concomitant activation of the 5-lipoxygenase pathway. This activation was not dependent on the presence of extracellular calcium. Since thrombin also induces the release of hemostatic and inflammatory metabolites from platelets and mast cells, it is proposed that it plays a significant role in amplification of the inflammatory response.

Thrombin and IgE antigen induce formation of inositol phosphates by mouse E-mast cells.

Stimulation of murine chondroitin sulfate E containing mast cells (E-MC) in vitro either by thrombin or immunologically resulted in a rapid formation of inositol phosphates (IPs). Increase in all of the three IPs (IP1, IP2 and IP3) could be detected 20 s after stimulation. The depletion of Ca2+ from the medium resulted in more than 80% reduction in beta-hexosaminidase release from either thrombin or IgE antigen stimulated cells. However, both thrombin and IgE antigen increased the formation of IP3 under these conditions independent of the presence of extracellular Ca2+.

Regulation of thrombin-induced mast cell degranulation by zinc and manganese.

This study was undertaken to determine whether zinc, manganese and copper could regulate the thrombin-induced secretion of the granule-associated mediator, beta-hexosaminidase, from mast cells derived from mouse bone marrow. Exposure of thrombin to copper (2-100 microM) does not affect the enzyme-induced release of beta-hexosaminidase from the mast cells. Zinc at 50 microM reduced the degranulation of calcium ionophore A23187 activated cells by 75% and that of immunological challenge or thrombin by 30% each. Exposure of the thrombin to incremental concentrations of manganese (2-100 microM) prevents its degranulation activity in a dose-related fashion. 75% inhibition of the enzyme activity was achieved at 100 microM manganese. However, exposure of IgE sensitized or unsensitized cells to incremental concentrations of manganese (2-400 microM) prior to antigen or calcium ionophore A23187 stimulation, does not significantly affect the exocytosis of beta-hexosaminidase. The binding of purified human FITC-thrombin to E-mast cells was analyzed by fluorescence flow cytometry. All cells bound specifically the labelled thrombin. Pretreatment of the FITC-thrombin with 100 micron zinc or manganese had no effect on the binding of the labelled thrombin to the cells. It was assumed that manganese modulates either directly the thrombin activity or the substrate for the enzyme on the cell surface.

Thrombin-mast cell interactions. Binding and cell activation.

Activation of mouse bone marrow-derived mast cells (BMMC) by thrombin (0.05-0.5 U/million cells) resulted in a concentration-dependent release of histamine, which levelled off by 0.1 U thrombin. Rat peritoneal mast cells (RMC) were not stimulated by thrombin, though in control experiments, both types of mast cells degranulated upon exposure to IgE-antigen. Pretreatment of thrombin with 0.2 mM diisopropylfluorophosphate (DFP), a specific serine protease inhibitor, resulted in 90% loss of thrombin degranulation and coagulant activity. Fluorescently labelled thrombin (FITC-thrombin) specifically bound to the BMMC surface, as measured by fluorescence cytometry. Pre-exposure of the BMMC to 20-fold excess of unlabelled thrombin prior to incubation with FITC-thrombin, prevented the binding of the labelled-thrombin to the cells. Incubation of thrombin with DFP or with antithrombin III (AT-III) resulted in losses of procoagulant and of BMMC degranulatory activities. DFP treatment of FITC-thrombin had no effect on the binding of the labelled enzyme to the cell surface. However, preincubation of the FITC-thrombin with AT-III prevented thrombin binding to the BMMC. Thus, the binding and the catalytic regions of the thrombin molecule are operationally distinct from one another. Kinetic analysis of the BMMC exposed to 0.5 U thrombin revealed a transient rise in intracellular cAMP, which peaked by 15 sec and was not measurable after 1 min. This suggests that differential activation of mast cells can occur at sites of tissue injury.