Carl F. Lagenaur

Integrin-associated Protein Is a Ligand for the P84 Neural Adhesion Molecule

P84 (also known as SHPS-1, BIT, and SIRP) is a heterophilic adhesive membrane protein involved in receptor tyrosine kinase signaling that is found at synapses in the mammalian central nervous system and in non-neural tissues. We have identified a binding partner for P84 using an expression cloning strategy. Here we report that integrin-associated protein (IAP/CD47) is a predominant binding partner of P84. Immunohistochemistry reveals a virtually identical distribution of P84 and IAP in a variety of adult brain regions. Because IAP has been implicated in cell signaling in cells of the immune system, P84 and IAP represent a heterophilic binding pair that is likely to be involved in bi-directional signaling at the synapse and in other tissues.

Molecular cloning of M6: Identification of a PLP/DM20 gene family

M6 is a membrane glycoprotein that is expressed on central neurons and certain polarized epithelia from early developmental stage. Antibodies against M6 interfere with cerebellar neurite outgrowth in vitro. Two closely related cDNAs were obtained by expression cloning, both of which showed high homology with the major CNS myelin protein PLP/DM20. Although M6 and PLP/DM20 share many molecular characteristics, in situ hybridization revealed nonoverlapping distributions of their mRNAs in mouse CNS. The identification of a gene family including neuron-specific M6 and glia-specific PLP/DM20 in CNS suggests a broader functional role for these molecules than myelination.

Central nervous system antigen P84 can serve as a substrate for neurite outgrowth

Neurite outgrowth promoting properties of neural cell surface proteins can be assessed by immobilizing isolated membrane proteins on nitrocellulose-coated petri dishes. Using this method, we have identified a unique cell surface antigen, designated P84, as a new neural cell adhesion molecule. Immunoaffinity purified P84 contains three polypeptides with molecular weights of 167, 85, and 66 kDa. When spotted onto nitrocellulose-coated plates, P84 supports adhesion of mouse cerebellar neurons and neurite outgrowth. Glial cell attachment was also observed. Intact monoclonal antibodies directed against P84 inhibit adhesion and outgrowth on a P84 substrate. This antigen is found on the surfaces of neurons in cultures of cerebellar cells. It is also found on a subclass of unidentified flat cells. P84 is not found on oligodendrocytes or GFAP-positive astrocytes. As early as E9, P84 could be detected in the floor plate region of the spinal cord. This pattern persists throughout embryonic development. Postnatally, widespread expression of P84 is observed in a variety of CNS tissues.

The Surface Immobilization of the Neural Adhesion Molecule L1 on Neural Probes and its Effect on Neuronal Density and Gliosis at the Probe/Tissue Interface

Brain tissue inflammatory responses, including neuronal loss and gliosis at the neural electrode/tissue interface, limit the recording stability and longevity of neural probes. The neural adhesion molecule L1 specifically promotes neurite outgrowth and neuronal survival. In this study, we covalently immobilized L1 on the surface of silicon-based neural probes and compared the tissue response between L1 modified and non-modified probes implanted in the rat cortex after 1, 4, and 8 weeks. The effect of L1 on neuronal health and survival, and glial cell reactions were evaluated with immunohistochemistry and quantitative image analysis. Similar to previous findings, persistent glial activation and significant decreases of neuronal and axonal densities were found at the vicinity of the non-modified probes. In contrast, the immediate area (100 μm) around the L1 modified probe showed no loss of neuronal bodies and a significantly increased axonal density relative to background. In this same region, immunohistochemistry analyses show a significantly lower activation of microglia and reaction of astrocytes around the L1 modified probes when compared to the control probes. These improvements in tissue reaction induced by the L1 coating are likely to lead to improved functionality of the implanted neural electrodes during chronic recordings.

Efferent Projections to the Host Brain from Intrastriatal Striatal Mouse-to-rat Grafts: Time Course and Tissue-type Specificity as Revealed by a Mouse Specific Neuronal Marker

The developmental time‐course and growth characteristics of efferent graft‐to‐host projections were studied from mouse fetal striatal grafts (E13–14) implanted as a cell suspension into the ibotenate‐lesioned striatum of immunosuppressed adult rats. A cell surface monoclonal antibody specific for mouse neurons (M6) was used to identify the donor cells and their projections into the host brain. At 3–5 days after implantation, sparse fascicles of M6‐positive graft‐derived fibres extended for ∼0.3–0.4 mm across the graft‐host border into the surrounding host striatum. From the beginning they were selectively orientated in one direction, i.e. caudally along the myelinated fibre bundles of the internal capsule. At 8 days, the graft‐derived fibres were more numerous and more densely labelled. They ran in dense fascicles inside the myelinated bundles of the host internal capsule and reached the rostral host globus pallidus, a distance of ∼ 1.2 mm from the caudal tip of the graft. Two weeks after grafting, the M6‐positive fibre fascicles were clearly seen to branch within the globus pallidus to form terminal‐like networks. From this time onwards, the immunoreactivity of the outgrowing fibre fascicles gradually diminished, although small but dense terminal‐like networks could be found in the host globus pallidus in most, but not all, of the rats at longer survival times (3–15 weeks). This is consistent with previous work showing that outgrowing axons lose their M6 immunoreactivity as they mature and become myelinated. Control grafts of fetal neocortical and fetal cerebellar tissue were used to assess the tissue‐type specificity of the efferent fibre growth. The neocortical implants projected densely up to about 3 mm into the host brain, along the internal capsule and the corpus callosum and into the overlying cortex. By contrast, although the cerebellar grafts survived well, they showed very little efferent fibre growth. Double immunostaining for DARPP‐32 and M6 revealed that all M6‐positive fibre fascicles extending from the striatal (but not neocortical) grafts also showed DARPP‐32 positivity, and thus that it was the DARPP‐32‐positive regions of the striatal grafts that projected to the host brain. It is concluded that graft‐to‐host projections, running along and inside host myelinated bundles, are formed from intrastriatal striatal grafts within 1–2 weeks of implantation. Grafts of neocortical tissue grew well along the same trajectory, whereas neurons of a type not normally projecting along the internal capsule, i.e. cerebellum, failed to extend axons over any significant distance along this trajectory.

Expression of a synapse-associated membrane protein, P84/SHPS-1, and its ligand, IAP/CD47, in mouse retina

P84 and integrin associated protein (IAP) are heterophilic binding partners that are expressed in the central nervous system in addition to a variety of other tissues. Both molecules are known to be involved in cell signaling in nonneural tissues. In the retina, both molecules are expressed prominently in plexiform layers, suggesting a possible association with synapses. Here, we examined the cellular expression and ultrastructural localization of the two molecules in the developing mouse retina. Both appeared to be expressed at one or both sides of synaptic sites, although the expression of IAP in the retina precedes that of P84. Examination of transgenic IAP‐null retinae revealed a failure of P84 to become associated with synaptic sites, suggesting the interaction of P84 with IAP was necessary for P84s synaptic localization. These findings suggest that the signaling activities of P84 and IAP are localized to sites of synaptic contact in the retina. Thus this pair of synapse‐associated molecules represents a bidirectional signaling system that could function to modify synaptic activity or possibly trophic interactions between central neurons. J. Comp. Neurol. 416:335–344, 2000.

Surface immobilization of neural adhesion molecule L1 for improving the biocompatibility of chronic neural probes: In vitro characterization

Silicon-based implantable neural electrode arrays are known to experience failure during long-term recording, partially due to host tissue responses. Surface modification and immobilization of biomolecules may provide a means to improve their biocompatibility and integration within the host brain tissue. Previously, the laminin biomolecule or laminin fragments have been used to modify the neural probes silicon surface to promote neuronal attachment and growth. Here we report the successful immobilization of the L1 biomolecule on a silicon surface. L1 is a neuronal adhesion molecule that can specifically promote neurite outgrowth and neuronal survival. Silane chemistry and the heterobifunctional coupling agent 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS) were used to covalently bind these two biomolecules onto the surface of silicon dioxide wafers, which mimic the surface of silicon-based implantable neural probes. After covalent binding of the biomolecules, polyethylene glycol (PEG)-NH(2) was used to cap the unreacted GMBS groups. Surface immobilization was verified by goniometry, dual polarization interferometry, and immunostaining techniques. Primary murine neurons or astrocytes were used to evaluate the modified silicon surfaces. Both L1- and laminin-modified surfaces promoted neuronal attachment, while the L1-modified surface demonstrated significantly enhanced levels of neurite outgrowth (p<0.05). In addition, the laminin-modified surface promoted astrocyte attachment, while the L1-modified surface showed significantly reduced levels of astrocyte attachment relative to the laminin-modified surface and other controls (p<0.05). These results demonstrate the ability of the L1-immobilized surface to specifically promote neuronal growth and neurite extension, while inhibiting the attachment of astrocytes, one of the main cellular components of the glial sheath. Such unique properties present vast potentials to improve the biocompatibility and chronic recording performance of neural probes.

Expression of members of the proteolipid protein gene family in the developing murine central nervous system

Two homologous cDNAs were previously isolated by expression cloning with a monoclonal antibody that recognized a CNS neuronal membrane protein. Both cDNAs, M6a and M6b, bore significant homology with the major myelin proteolipid protein, PLP/DM20. Our initial studies of M6 gene expression in the adult mouse brain showed that M6a was present in neurons, PLP/DM20 in oligodendrocytes, and M6b in both neurons and glia. This led to the recognition of a novel gene family that included the oligodendrocyte‐specific PLP/DM20 gene and the neuronal M6 genes. These observations supported the idea that PLP/DM20 may have functions other than myelination.

Alternate strategies in lesion-induced reactive synaptogenesis: differetial expression of L1 in two populations of sprouting axons

In the CNS the cell adhesion molecule L1 plays a role in axonal growth and fasciculation. Since its roles in synapse formation and CNS regeneration are unknown, we followed the staining of L1 through the sequence of degeneration and reactive axon sprouting in the denervated outer molecular layer (ML) of the hippocampal dentate gyrus following ipsilateral entorhinal cortex (ERC) lesion. We compared immunohistological and ultrastructural localization of L1 and employed image analysis to evaluate lamina-specific changes over time. L1 staining was uniformly distributed over the ML in unlesioned animals. Following ERC lesion, L1 staining markedly declined in the outer ML; L1 staining in the inner ML remained constant. Over 30 days postlesion, commissural and associational (C/A) afferents from inner ML sprouted partway into the denervated zone, and L1 was expressed on these sprouting afferents. L1 staining exactly corresponded to fiber outgrowth as assessed by Holmes fiber stain. As the L1-bearing axons of the C/A projection expanded, staining for embryonic N-CAM (reexpressed on the dendrites of the denervated zone) appeared to recede. There was never overlap of L1 and embryonic N-CAM staining; the difference always marked the boundary between inner and outer ML. Ultrastructural analysis confirmed localization of L1 staining to axonal profiles, indicating that the new pattern of L1 staining reflected distinct types of axonal growth. These changes in cell adhesion molecule expression closely paralleled the known sequence of reactive synaptogenesis and axonal sprouting and demonstrate a link between cell adhesion molecule expression and axonal sprouting during self-repair by the CNS.

Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Substrate 1 Regulates the Migration of Langerhans Cells from the Epidermis to Draining Lymph Nodes

Src homology 2 domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1) is a member of the signal regulatory protein family in which the extracellular region interacts with its ligand, CD47. Recent studies have demonstrated that SHPS-1 plays an important role in cell migration and cell adhesion. We demonstrate in this study, using immunohistochemical and flow cytometric analyses, that murine Langerhans cells (LCs) express SHPS-1. Treatment of mice ears with 2,4-dinitro-1-fluorobenzene significantly reduced the number of epidermal LCs, and that reduction could be reversed by pretreatment with mAb to SHPS-1 or the CD47-Fc fusion protein. Treatment with the SHPS-1 mAb in vivo reduced the number of FITC-bearing cells in the lesional lymph nodes after the application of FITC to the skin. The SHPS-1 mAb inhibited the in vivo TNF-α-induced migration of LCs. The emigration of dendritic cells expressing I-Ab+ from skin explants to the medium was also reduced by the SHPS-1 mAb. We further demonstrate that the chemotaxis of a murine dendritic cell line, XS52, by macrophage inflammatory protein-3β was significantly inhibited by treatment with the SHPS-1 mAb or CD47-Fc recombinant protein. Finally, we show that migration of LCs was attenuated in mutant mice that lack the intracellular domain of SHPS-1. These observations show that the ligation of SHPS-1 with the SHPS-1 mAb or with CD47-Fc abrogates the migration of LCs in vivo and in vitro, which suggests that the SHPS-1-CD47 interaction may negatively regulate LC migration.