The neuronal class 2 TSR proteins F-spondin and Mindin: a small family with divergent biological activities

F-spondin and Mindin are members of a subgroup of the thrombospondin type 1 (TSR) class molecules, defined by two domains of homology, the FS1/FS2 and TSR domains. The TSRs of F-spondin proteins are typical of class 2 TSRs. F-spondin and Mindin are evolutionarily conserved proteins. The embryonic expression of the vertebrate genes is enriched in the nervous system, mainly at the floor plate and the hippocampus. Similar to thrombospondin, F-spondin and Mindin are extracellular matrix attached molecules that promote neurite outgrowth and inhibit angiogenesis. Analysis of gain and loss of function experiments reveal that F-spondin is required for accurate pathfinding of embryonic axons. F-spondin plays a dual role in patterning axonal trajectories: it promotes the outgrowth of commissural and inhibits the outgrowth of motor axons. Macrophages of Mindin-deficient mice exhibit defective responses to a broad spectrum of microbial stimuli. This may implicate Mindin and F-spondin in inflammatory processes in the nervous system.     

F-spondin promotes nerve precursor differentiation

Cells in the developing nervous system secrete a large number of proteins that regulate the migration and differentiation of their neighbors. It is shown here that a clonal central nervous system cell line secretes a protein that causes both a rat hippocampal progenitor cell line and primary cortical neural cells to differentiate into cells with the morphological and biochemical features of neurons. This protein was identified as F-spondin. Analysis of F-spondin isoforms secreted from transfected cells shows that the core protein without the thrombospondin type 1 repeats is sufficient to promote neuronal differentiation when adsorbed to a surface. F-spondin can also inhibit neurite outgrowth while allowing the expression of nerve-specific proteins when present in a soluble form at high concentrations. Therefore, F-spondin can alter cell differentiation in multiple ways, depending upon its concentration and distribution between substrate-attached and soluble forms.     

F-spondin/spon1b expression patterns in developing and adult zebrafish

F-spondin, an extracellular matrix protein, is an important player in embryonic morphogenesis and CNS development, but its presence and role later in life remains largely unknown. We generated a transgenic zebrafish in which GFP is expressed under the control of the F-spondin (spon1b) promoter, and used it in combination with complementary techniques to undertake a detailed characterization of the expression patterns of F-spondin in developing and adult brain and periphery. We found that F-spondin is often associated with structures forming long neuronal tracts, including retinal ganglion cells, the olfactory bulb, the habenula, and the nucleus of the medial longitudinal fasciculus (nMLF). F-spondin expression coincides with zones of adult neurogenesis and is abundant in CSF-contacting secretory neurons, especially those in the hypothalamus. Use of this new transgenic model also revealed F-spondin expression patterns in the peripheral CNS, notably in enteric neurons, and in peripheral tissues involved in active patterning or proliferation in adults, including the endoskeleton of zebrafish fins and the continuously regenerating pharyngeal teeth. Moreover, patterning of the regenerating caudal fin following fin amputation in adult zebrafish was associated with F-spondin expression in the blastema, a proliferative region critical for tissue reconstitution. Together, these findings suggest major roles for F-spondin in the CNS and periphery of the developing and adult vertebrate.     

F-spondin: a gene expressed at high levels in the floor plate encodes a secreted protein that promotes neural cell adhesion and neurite extension.

The floor plate is a cell group implicated in the control of neural cell pattern and axonal growth in the developing vertebrate nervous system. To identify molecules that might mediate the functions of the floor plate, we have used subtractive hybridization techniques to isolate floor plate-enriched cDNA clones. One such clone encodes a novel secreted protein, F-spondin, which is expressed at high levels in the floor plate. The C-terminal half of the protein contains six repeats identified previously in thrombospondin and other proteins implicated in cell adhesion. F-spondin is expressed in the floor plate at the time that axons first extend and at lower levels in the peripheral nerve. Recombinant F-spondin promotes the attachment of spinal cord and sensory neuron cells and the outgrowth of neurites in vitro. F-spondin may contribute to the growth and guidance of axons in both the spinal cord and the PNS.     

Plasmin-mediated release of the guidance molecule F-spondin from the extracellular matrix

Serine proteases are implicated in a variety of processes during neurogenesis, including cell migration, axon outgrowth, and synapse elimination. Tissue-type plasminogen activator and urokinase-type activator are expressed in the floor plate during embryonic development. F-spondin, a gene also expressed in the floor plate, encodes a secreted, extracellular matrix-attached protein that promotes outgrowth of commissural axons and inhibits outgrowth of motor axons. F-spondin is processed in vivo to yield an amino half protein that contains regions of homology to reelin and mindin, and a carboxyl half protein that contains either six or four thrombospondin type I repeats (TSRs). We have tested F-spondin to see whether it is subjected to processing by plasmin and to determine whether the processing modulates its biological activity. Plasmin cleaves F-spondin at its carboxyl terminus. By using nested deletion proteins and mutating potential plasmin cleavage sites, we have identified two cleavage sites, the first between the fifth and sixth TSRs, and the second at the fifth TSR. Analysis of the extracellular matrix (ECM) attachment properties of the TSRs revealed that the fifth and sixth TSRs bind to the ECM, but repeats 1-4 do not. Structural functional experiments revealed that two basic motives are required to elicit binding of TSR module to the ECM. We demonstrate further that plasmin releases the ECM-bound F-spondin protein.     

Control of neuronal morphology and connectivity: Emerging developmental roles for gap junctional proteins

Recent evidence indicates that gap junction (GJ) proteins can play a critical role in controlling neuronal connectivity as well as cell morphology in the developing nervous system. GJ proteins may function analogously to cell adhesion molecules, mediating cellular recognition and selective neurite adhesion. Moreover, during synaptogenesis electrical synapses often herald the later establishment of chemical synapses, and thus may help facilitate activity-dependent sculpting of synaptic terminals. Recent findings suggest that the morphology and connectivity of embryonic leech neurons are fundamentally organized by the type and perhaps location of the GJ proteins they express. For example, ectopic expression in embryonic leech neurons of certain innexins that define small GJ-linked networks of cells leads to the novel coupling of the expressing cell into that network. Moreover, gap junctions appear to mediate interactions among homologous neurons that modulate process outgrowth and stability. We propose that the selective formation of GJs between developing neurons and perhaps glial cells in the CNS helps orchestrate not only cellular synaptic connectivity but also can have a pronounced effect on the arborization and morphology of those cells involved.     

Developmental Expression of GABAA Receptor Subunit mRNAs in Individual Hippocampal Neurons In Vitro and In Vivo

Abstract: The GABA_A receptor is a heterooligomeric protein complex composed of multiple receptor subunits. Developmental changes in the pattern of expression of 11 GABA_A receptor subunits in individual rat embryonic hippocampal neurons on days 1–21 in culture and acutely dissociated hippocampal neurons from postnatal day (PND) 5 rat pups were investigated using the technique of single-cell mRNA amplification. We demonstrate that multiple GABA_A receptor subunits are expressed within individual hippocampal neurons, with most cells simultaneously expressing α1, α2, α5, β1, and γ2 mRNAs. Further, relative expression of several GABA_A receptor subunit mRNAs changes significantly in embryonic hippocampal neurons during in vitro development, with the relative abundance (compared with β-actin) of α1, α5, and γ2 mRNAs increasing 2.3-, 2.7-, and 3.8-fold, respectively, from days 1 to 14, and β1 increasing 5-fold from days 1 to 21. In situ hybridization with antisense digoxigenin-labeled α1, β1, and γ2 RNA probes demonstrates a similar increase in expression of subunit mRNAs as embryonic hippocampal neurons mature in vitro. Relative abundances of α1, β1, and γ2 subunit mRNAs in acutely dissociated PND 5 hippocampal neurons are also significantly greater than in embryonic day 17 neurons on day 1 in vitro and exceed the peak values seen in cultured neurons on days 14–21, suggesting that GABA_A receptor subunit mRNA expression within individual hippocampal neurons follows a similar, if somewhat delayed, developmental pattern in vitro compared with in vivo. These findings suggest that embryonic hippocampal neuronal culture provides a useful model in which to study the developmental regulation of GABA_A receptor expression and that developmental changes in GABA_A receptor subunit expression may underlie some of the differences in functional properties of GABA_A receptors in neonatal and mature hippocampal neurons.     

F-spondin interaction with the apolipoprotein E receptor ApoEr2 affects processing of amyloid precursor protein

A recent study showed that F-spondin, a protein associated with the extracellular matrix, interacted with amyloid precursor protein (APP) and inhibited beta-secretase cleavage. F-spondin contains a thrombospondin domain that we hypothesized could interact with the family of receptors for apolipoprotein E (apoE). Through coimmunoprecipitation experiments, we demonstrated that F-spondin interacts with an apoE receptor (apoE receptor 2 [ApoEr2]) through the thrombospondin domain of F-spondin and the ligand binding domain of ApoEr2. Full-length F-spondin increased coimmunoprecipitation of ApoEr2 and APP in transfected cells and primary neurons and increased surface expression of APP and ApoEr2. Full-length F-spondin, but none of the individual F-spondin domains, increased cleavage of APP and ApoEr2, resulting in more secreted forms of APP and ApoEr2 and more C-terminal fragments (CTF) of these proteins. In addition, full-length F-spondin, but not the individual domains, decreased production of the beta-CTF of APP and Abeta in transfected cells and primary neurons. The reduction in APP beta-CTF was blocked by receptor-associated protein (RAP), an inhibitor of lipoprotein receptors, implicating ApoEr2 in the altered proteolysis of APP. ApoEr2 coprecipitated with APP alpha- and beta-CTF, and F-spondin reduced the levels of APP intracellular domain signaling, suggesting that there are also intracellular interactions between APP and ApoEr2, perhaps involving adaptor proteins. These studies suggest that the extracellular matrix molecule F-spondin can cluster APP and ApoEr2 together on the cell surface and affect the processing of each, resulting in decreased production of Abeta.     

Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons

The identification of surface proteins restricted to subsets of embryonic axons and growth cones may provide information on the mechanisms underlying axon fasciculation and pathway selection in the vertebrate nervous system. We describe here the characterization of a 135 kd cell surface glycoprotein, TAG-1, that is expressed transiently on subsets of embryonic spinal cord axons and growth cones. TAG-1 is immunochemically distinct from the cell adhesion molecules N-CAM and L1 (NILE) and is expressed on commissural and motor neurons over the period of initial axon extension. Moreover, TAG-1 and L1 appear to be segregated on different segments of the same embryonic spinal axons. These observations provide evidence that axonal guidance and pathway selection in vertebrates may be regulated in part by the transient and selective expression of distinct surface glycoproteins on subsets of developing neurons.     

Characterization of two novel nuclear BTB/POZ domain zinc finger isoforms. Association with differentiation of hippocampal neurons, cerebellar granule cells, and macroglia.

BTB/POZ (broad complex tramtrack bric-a-brac/poxvirus and zinc finger) zinc finger factors are a class of nuclear DNA-binding proteins involved in development, chromatin remodeling, and cancer. However, BTB/POZ domain zinc finger factors linked to development of the mammalian cerebral cortex, cerebellum, and macroglia have not been described previously. We report here the isolation and characterization of two novel nuclear BTB/POZ domain zinc finger isoforms, designated HOF(L) and HOF(S), that are specifically expressed in early hippocampal neurons, cerebellar granule cells, and gliogenic progenitors as well as in differentiated glia. During embryonic development of the murine cerebral cortex, HOF expression is restricted to the hippocampal subdivision. Expression coincides with early differentiation of presumptive CA1 and CA3 pyramidal neurons and dentate gyrus granule cells, with a sharp decline in expression at the CA1/subicular border. By using bromodeoxyuridine labeling and immunohistochemistry, we show that HOF expression coincides with immature non-dividing cells and is down-regulated in differentiated cells, suggesting a role for HOF in hippocampal neurogenesis. Consistent with the postulated role of the POZ domain as a site for protein-protein interactions, both HOF isoforms are able to dimerize. The HOF zinc fingers bind specifically to the binding site for the related promyelocytic leukemia zinc finger protein as well as to a newly identified DNA sequence.     

A developmentally regulated switch in neuronal responsiveness to NCAM and N-cadherin in the rat hippocampus.

Monolayers of control 3T3 fibroblasts and 3T3 cells expressing transfected NCAM or N-cadherin have been used as a culture substratum for rat hippocampal neurons. Both NCAM and N-cadherin are expressed in the hippocampus through embryonic day 17 (E17) to postnatal day 4 (PND4); however, whereas E17 neurons responded to transfected NCAM by extending considerably longer neurites, PND4 neurons responded very poorly. The converse was true for responsiveness to N-cadherin. These data demonstrate a switch in neuronal responsiveness to NCAM and N-cadherin in the developing hippocampus. NCAM-dependent neurite outgrowth from E17 neurons was largely dependent on the presence of alpha 2-8-linked polysialic acid (PSA) on neuronal NCAM. NCAM-dependent neurite outgrowth could be fully inhibited by pertussis toxin or a combination of L- and N-type calcium channel antagonists thus providing direct evidence concerning the nature of the second messenger pathway activated in primary neurons by cell adhesion molecules (CAMs).     

The role of selective transport in neuronal protein sorting

To assess whether selective microtubule-based vesicle transport underlies the polarized distribution of neuronal proteins, we expressed green fluorescent protein- (GFP-) tagged chimeras of representative axonal and dendritic membrane proteins in cultured hippocampal neurons and visualized the transport of carrier vesicles containing these proteins in living cells. Vesicles containing a dendritic protein, transferrin receptor (TfR), were preferentially transported into dendrites and excluded from axons. In contrast, vesicles containing the axonal protein NgCAM (neuron-glia cell adhesion molecule) were transported into both dendrites and axons. These data demonstrate that neurons utilize two distinct mechanisms for the targeting of polarized membrane proteins, one (for dendritic proteins) based on selective transport, the other (for axonal proteins) based on a selectivity "filter" that occurs downstream of transport.     

Characterization of an RNA granule from developing brain

In brain, mRNAs are transported from the cell body to the processes, allowing for local protein translation at sites distant from the nucleus. Using subcellular fractionation, we isolated a fraction from rat embryonic day 18 brains enriched for structures that resemble amorphous collections of ribosomes. This fraction was enriched for the mRNA encoding beta-actin, an mRNA that is transported in dendrites and axons of developing neurons. Abundant protein components of this fraction, determined by tandem mass spectrometry, include ribosomal proteins, RNA-binding proteins, microtubule-associated proteins (including the motor protein dynein), and several proteins described only as potential open reading frames. The conjunction of RNA-binding proteins, transported mRNA, ribosomal machinery, and transporting motor proteins defines these structures as RNA granules. Expression of a subset of the identified proteins in cultured hippocampal neurons confirmed that proteins identified in the proteomics were present in neurites associated with ribosomes and mRNAs. Moreover many of the expressed proteins co-localized together. Time lapse video microscopy indicated that complexes containing one of these proteins, the DEAD box 3 helicase, migrated in dendrites of hippocampal neurons at the same speed as that reported for RNA granules. Although the speed of the granules was unchanged by activity or the neurotrophin brain-derived neurotrophic factor, brain-derived neurotrophic factor, but not activity, increased the proportion of moving granules. These studies define the isolation and composition of RNA granules expressed in developing brain.     

Carbohydrate-protein interactions between HNK-1-reactive sulfoglucuronyl glycolipids and the proteoglycan lectin domain mediate neuronal cell adhesion and neurite outgrowth

Lecticans, a family of chondroitin sulfate proteoglycans, represent the largest group of proteoglycans expressed in the nervous system. We previously showed that the C-type lectin domains of lecticans bind two classes of sulfated cell surface glycolipids, sulfatides and HNK-1-reactive sulfoglucuronylglycolipids (SGGLs). In this paper, we demonstrate that the interaction between the lectin domain of brevican, a nervous system-specific lectican, and cell surface SGGLs acts as a novel cell recognition system that promotes neuronal adhesion and neurite outgrowth. The Ig chimera of the brevican lectin domain bind to the surface of SGGL-expressing rat hippocampal neurons. The substrate of the brevican chimera promotes adhesion and neurite outgrowth of hippocampal neurons. The authentic, full-length brevican also promotes neuronal cell adhesion and neurite outgrowth. These activities of brevican substrates are neutralized by preincubation of cells with HNK-1 monoclonal antibodies and by pretreatment of the brevican substrates with purified SGGLs. Brevican and HNK-1 carbohydrates are coexpressed in specific layers of the developing hippocampus where axons from entorhinal neurons elongate. Our observations suggest that cell surface SGGLs and extracellular lecticans comprise a novel cell-substrate recognition system operating in the developing nervous system.