Martin Grumet

The carbonic anhydrase domain of receptor tyrosine phosphatase β is a functional ligand for the axonal cell recognition molecule contactin

Receptor-type protein tyrosine phosphatase beta (RPTP beta) is expressed in the developing nervous system and contains a carbonic anhydrase (CAH) domain as well as a fibronectin type III repeat in its extracellular domain. Fusion proteins containing these domains were used to search for ligands of RPTP beta. The CAH domain bound specifically to a 140 kDa protein expressed on the surface of neuronal cells. Expression cloning in COS7 cells revealed that this protein is contactin, a GPI membrane-anchored neuronal cell recognition molecule. The CAH domain of RPTP beta induced cell adhesion and neurite growth of primary tectal neurons, and differentiation of neuroblastoma cells. These responses were blocked by antibodies against contactin, demonstrating that contactin is a neuronal receptor for RPTP beta. These experiments show that an individual domain of RPTP beta acts as a functional ligand for the neuronal receptor contactin. The interaction between contactin and RPTP beta may generate unidirectional or bidirectional signals during neural development.

Identification of a novel contactin-associated transmembrane receptor with multiple domains implicated in protein-protein interactions

Receptor protein tyrosine phosphatase β (RPTPβ) expressed on the surface of glial cells binds to the glycosylphosphatidylinositol (GPI)‐anchored recognition molecule contactin on neuronal cells leading to neurite outgrowth. We describe the cloning of a novel contactin‐associated transmembrane receptor (p190/Caspr) containing a mosaic of domains implicated in protein–protein interactions. The extracellular domain of Caspr contains a neurophilin/coagulation factor homology domain, a region related to fibrinogen β/γ, epidermal growth factor‐like repeats, neurexin motifs as well as unique PGY repeats found in a molluscan adhesive protein. The cytoplasmic domain of Caspr contains a proline‐rich sequence capable of binding to a subclass of SH3 domains of signaling molecules. Caspr and contactin exist as a complex in rat brain and are bound to each other by means of lateral (cis) interactions in the plasma membrane. We propose that Caspr may function as a signaling component of contactin, enabling recruitment and activation of intracellular signaling pathways in neurons. The binding of RPTPβ to the contactin–Caspr complex could provide a mechanism for cell–cell communication between glial cells and neurons during development.

Vascular Apoptosis and Involution in Gliomas Precede Neovascularization: A Novel Concept for Glioma Growth and Angiogenesis

Vascular changes in gliomas were analyzed by implanting fluorescent-labeled glioma 261 cells in the brains of 28 mice. Seven animals were killed each week for 4 weeks. We investigated the expression of angiopoietin-2 (Ang-2) by in situ hybridization and compared it with the distribution of apoptotic cells identified by DNA strand breaks (using the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling [TUNEL] method) and transmission electron microscopy (TEM). As early as 1 week after implantation, tumor cells accumulated around vessels, which expressed Ang-2 and were TUNEL negative. TEM showed tumor cells adjacent to the vascular cells “lifting up” the normal astrocytic feet processes away from the endothelial cells and disrupting normal pericytic cuffing. After 2 weeks the number of perivascular glioma cells had increased. No increase in the number of blood vessels was detected at this time. Vascular cells remained positive for Ang-2 and rare ones were TUNEL positive. TEM showed closely packed proliferating perivascular tumor cells. After 3 weeks, there was vascular involution with scant zones of tumor necrosis. Ang-2 was still detected in vascular cells, but now numerous vascular cells were TUNEL positive. In addition, TEM showed apoptotic vascular cells. After 4 weeks, there were extensive areas of tumor necrosis with pseudopalisading and adjacent angiogenesis. Ang-2 was detected in vascular cells at the edge of the tumors in the invaded brain and in vessels surrounded by tumor cells. At both 3 and 4 weeks, most of the TUNEL-positive tumor cells lacked morphological features characteristic of apoptosis and displayed features consistent with necrotic cell death as determined by TEM. Only rare tumor cells appeared truly apoptotic. In contrast, the TUNEL-positive endothelial cells and pericytes were round and shrunken, with condensed nuclear chromatin by TEM, suggesting that vascular cells were undergoing an apoptotic cell death. These results suggest that vascular cell apoptosis and involution preceded tumor necrosis and that angiogenesis is a later event in tumor progression in experimental gliomas. Moreover, Ang-2 is detected prior to the onset of apoptosis in vascular cells and could be linked to vascular involution.

In situ expression of angiopoietins in astrocytomas identifies angiopoietin-2 as an early marker of tumor angiogenesis.

Angiopoietin-1 (Ang-1) and its naturally occurring antagonist angiopoietin-2 (Ang-2) are novel ligands that regulate tyrosine phosphorylation of the Tie2/Tek receptor on endothelial cells. Proper regulation of Tie2/Tek is absolutely required for normal vascular development, seemingly by regulating vascular remodeling and endothelial cell interactions with supporting pericytes/smooth muscle cells. We investigated the expression of Ang-1 and Ang-2 in human astrocytomas by in situ hybridization and compared them to the distribution of pericytes/smooth muscle cells by immunohistochemistry for alpha-smooth muscle actin (SMA). Ang-1 mRNA was localized in tumor cells and Ang-2 mRNA was detected in endothelial cells of hyperplastic and nonhyperplastic tumor vessels. Ang-2 was also expressed in partially sclerotic vessels and in vascular channels surrounded by tumor cells in brain adjacent to the tumor. Neither Ang-1 nor Ang-2 was detected in normal brain. Dynamic changes in SMA expression during glioma tumorigenesis appear to progress from fragmentation in early vascular hyperplasia to subsequent reassociation and enhanced expression in later stages of vascular proliferation in hyperplastic complexes in high-grade gliomas. All these vessels displaying dynamic changes in SMA immunoreactivity also expressed Ang-2 mRNA. Moreover, SMA immunoreactive intratumoral vascular channels lacking morphological evidence of hyperplasia also showed upregulation of Ang-2. These results suggest that angiopoietins are involved in the early stage of vascular activation and in advanced angiogenesis, and they identify Ang-2 as an early marker of glioma-induced neovascularization. The association between Ang-2 expression and alterations in SMA immunoreactivity suggests a role for Ang-2 in tumor-associated activation of pericytes/smooth muscle cells.

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.

Nodes of Ranvier and axon initial segments are ankyrin G–dependent domains that assemble by distinct mechanisms

Axon initial segments (AISs) and nodes of Ranvier are sites of action potential generation and propagation, respectively. Both domains are enriched in sodium channels complexed with adhesion molecules (neurofascin [NF] 186 and NrCAM) and cytoskeletal proteins (ankyrin G and βIV spectrin). We show that the AIS and peripheral nervous system (PNS) nodes both require ankyrin G but assemble by distinct mechanisms. The AIS is intrinsically specified; it forms independent of NF186, which is targeted to this site via intracellular interactions that require ankyrin G. In contrast, NF186 is targeted to the node, and independently cleared from the internode, by interactions of its ectodomain with myelinating Schwann cells. NF186 is critical for and initiates PNS node assembly by recruiting ankyrin G, which is required for the localization of sodium channels and the entire nodal complex. Thus, initial segments assemble from the inside out driven by the intrinsic accumulation of ankyrin G, whereas PNS nodes assemble from the outside in, specified by Schwann cells, which direct the NF186-dependent recruitment of ankyrin G.

A Glial Signal Consisting of Gliomedin and NrCAM Clusters Axonal Na+ Channels during the Formation of Nodes of Ranvier

Saltatory conduction requires high-density accumulation of Na(+) channels at the nodes of Ranvier. Nodal Na(+) channel clustering in the peripheral nervous system is regulated by myelinating Schwann cells through unknown mechanisms. During development, Na(+) channels are first clustered at heminodes that border each myelin segment, and later in the mature nodes that are formed by the fusion of two heminodes. Here, we show that initial clustering of Na(+) channels at heminodes requires glial NrCAM and gliomedin, as well as their axonal receptor neurofascin 186 (NF186). We further demonstrate that heminodal clustering coincides with a second, paranodal junction (PNJ)-dependent mechanism that allows Na(+) channels to accumulate at mature nodes by restricting their distribution between two growing myelin internodes. We propose that Schwann cells assemble the nodes of Ranvier by capturing Na(+) channels at heminodes and by constraining their distribution to the nodal gap. Together, these two cooperating mechanisms ensure fast and efficient conduction in myelinated nerves.

Multi-ligand interactions with receptor-like protein tyrosine phosphatase β: implications for intercellular signaling

Receptor-like protein tyrosine phosphatase beta (RPTP beta) shows structural and functional similarity to cell adhesion molecules (CAMs). It binds to several neuronal CAMs and extracellular matrix (ECM) proteins that combine to form cell-recognition complexes. Here, the authors discuss the implications of such complexes for intercellular signaling, and the regulation of RPTP activity by cell-cell and cell-ECM contact.

Molecular events implicated in brain tumor angiogenesis and invasion

We have conducted studies designed to help elucidate the molecular mechanisms involved in brain tumor invasion and angiogenesis, which are critical in the growth of malignant tumors of the central nervous system. A variety of molecular factors have been implicated in these processes. Here we focus on three that are of particular importance in the progression of brain tumors. Angiopoietins are involved in the regulation of vascular development. Hypoxia inducible factor-1 is a transcription factor that up-regulates genes, including genes encoding vascular endothelial growth factor under hypoxic conditions. Focal adhesion kinase is associated with infiltration of tumor cells and angiogenesis.

Soluble Cell Adhesion Molecule L1-Fc Promotes Locomotor Recovery in Rats after Spinal Cord Injury

Previous studies suggest that the cell adhesion molecule L1 promotes neurite growth by neutralizing white matter associated inhibitors of axonal growth. We made a soluble chimeric dimer by linking mouse L1 to human Fc. This L1-Fc construct (40 microg/mL) markedly facilitated neurite outgrowth, as well as neuronal adhesion to white matter on frozen sections of spinal cord. We applied L1-Fc intrathecally (200 microg/mL at 0.5 microL/h) to rat spinal cords for 2 weeks after a 25-mm weight drop contusion of the T13 spinal cord. Initial experiments indicated that L1-Fc is present in the spinal cord after 2 weeks of intrathecal infusion and significantly improved locomotor recovery by 6-12 weeks after injury. We then randomized 45 rats to intrathecal infusion of L1-Fc (L1), phosphate-buffered saline controls (PBS), and a mouse monoclonal IgM antibody (M1). By 12 weeks after injury, L1-treated rats recovered significantly (p < 0.005) better locomotor function (BBB score 10.57 +/- 0.25, n = 14) than PBS-treated rats (BBB score 9.00 +/- 0.33, n = 14) or M1-treated (BBB score 8.71 +/- 0.16, n = 14). Only two rats of 22 treated with saline recovered weight-supported ambulation. Of 20 L1-Fc-treated rats, however, 18 recovered weight-supported walking by 12 weeks. The L1-Fc-treated rats also showed more consistent hindlimb contact placing than saline controls. We injected biotinylated dextran amine (BDA) into the motor cortices of 14 rats treated with L1-Fc to label corticospinal axons, comparing these with 13 rats treated with saline. In saline-treated rats, BDA-labeled corticospinal axons often grew up to the impact edge and occasionally into the impact site. L1-treated rats showed longer corticospinal tract growth at the injury site. Three rats had BDA-labeled axons that extended beyond the impact center. One L1-Fc-treated rat showed axonal extension and synapse formation in cord distal to the injury. These results indicate that soluble L1-Fc promotes axonal growth and functional recovery after spinal cord injury. However, the limited corticospinal tract growth across the injury site cannot account for the observed locomotor recovery. Thus, L1 may be stimulating growth of other motor tracts or protecting axons and neurons. More studies are required to elucidate the mechanisms of L1-Fc-induced locomotor recovery.