Fumitoshi Irie

Control of hippocampal dendritic spine morphology through ephrin-A3/EphA4 signaling

Communication between glial cells and neurons is emerging as a critical parameter of synaptic function. However, the molecular mechanisms underlying the ability of glial cells to modify synaptic structure and physiology are poorly understood. Here we describe a repulsive interaction that regulates postsynaptic morphology through the EphA4 receptor tyrosine kinase and its ligand ephrin-A3. EphA4 is enriched on dendritic spines of pyramidal neurons in the adult mouse hippocampus, and ephrin-A3 is localized on astrocytic processes that envelop spines. Activation of EphA4 by ephrin-A3 was found to induce spine retraction, whereas inhibiting ephrin/EphA4 interactions distorted spine shape and organization in hippocampal slices. Furthermore, spine irregularities in pyramidal neurons from EphA4 knockout mice and in slices transfected with kinase-inactive EphA4 indicated that ephrin/EphA4 signaling is critical for spine morphology. Thus, our data support a model in which transient interactions between the ephrin-A3 ligand and the EphA4 receptor regulate the structure of excitatory synaptic connections through neuroglial cross-talk.

EphB/Syndecan-2 Signaling in Dendritic Spine Morphogenesis

We previously reported that the cell surface proteoglycan syndecan-2 can induce dendritic spine formation in hippocampal neurons. We demonstrate here that the EphB2 receptor tyrosine kinase phosphorylates syndecan-2 and that this phosphorylation event is crucial for syndecan-2 clustering and spine formation. Syndecan-2 is tyrosine phosphorylated and forms a complex with EphB2 in mouse brain. Dominant-negative inhibition of endogenous EphB receptor activities blocks clustering of endogenous syndecan-2 and normal spine formation in cultured hippocampal neurons. This is the first evidence that Eph receptors play a physiological role in dendritic spine morphogenesis. Our observations suggest that spine morphogenesis is triggered by the activation of Eph receptors, which causes tyrosine phosphorylation of target molecules, such as syndecan-2, in presumptive spines.

EphB receptors regulate dendritic spine development via intersectin, Cdc42 and N-WASP

Dendritic spines show structural plasticity during development and in response to synaptic activity. We previously found that EphB2 receptor tyrosine kinase, a receptor for B-ephrin ligands, is functionally involved in spine morphogenesis, but the downstream signaling mechanisms are not known. Here we show that EphB2 physically associates with the guanine nucleotide exchange factor (GEF) intersectin and activates its GEF activity in cooperation with neural Wiskott-Aldrich syndrome protein (N-WASP), which in turn activates the Rho-family GTPase Cdc42 and spine morphogenesis.

EphrinB-EphB signalling regulates clathrin-mediated endocytosis through tyrosine phosphorylation of synaptojanin 1

Recent studies show that Eph receptors act mainly through the regulation of actin reorganization. Here, we show a novel mode of action for EphB receptors. We identify synaptojanin 1 — a phosphatidylinositol 5′-phosphatase that is involved in clathrin-mediated endocytosis — as a physiological substrate for EphB2. EphB2 causes tyrosine phosphorylation in the proline-rich domain of synaptojanin 1, and inhibits both the interaction with endophilin and the 5′-phosphatase activity of synaptojanin 1. Treatment with the EphB ligand, ephrinB2, elevates the cellular level of phosphatidylinositol 4,5-bisphosphate and promotes transferrin uptake. A kinase inactive mutant of EphB2 and a phosphorylation site mutant of synaptojanin 1 both neutralize the increase of transferrin uptake after ephrinB2 treatment. These mutants also inhibit AMPA glutamate receptor endocytosis in hippocampal neurons. Interestingly, incorporated transferrin does not reach endosomes, suggesting dual effects of EphB signalling on the early and late phases of clathrin-mediated endocytosis. Our results indicate that ephrinB–EphB signalling regulates clathrin-mediated endocytosis in various cellular contexts by influencing protein interactions and phosphoinositide turnover through tyrosine phosphorylation of synaptojanin 1.

Autism-like socio-communicative deficits and stereotypies in mice lacking heparan sulfate

Heparan sulfate regulates diverse cell-surface signaling events, and its roles in the development of the nervous system recently have been increasingly uncovered by studies using genetic models carrying mutations of genes encoding enzymes for its synthesis. On the other hand, the role of heparan sulfate in the physiological function of the adult brain has been poorly characterized, despite several pieces of evidence suggesting its role in the regulation of synaptic function. To address this issue, we eliminated heparan sulfate from postnatal neurons by conditionally inactivating Ext1, the gene encoding an enzyme essential for heparan sulfate synthesis. Resultant conditional mutant mice show no detectable morphological defects in the cytoarchitecture of the brain. Remarkably, these mutant mice recapitulate almost the full range of autistic symptoms, including impairments in social interaction, expression of stereotyped, repetitive behavior, and impairments in ultrasonic vocalization, as well as some associated features. Mapping of neuronal activation by c-Fos immunohistochemistry demonstrates that neuronal activation in response to social stimulation is attenuated in the amygdala in these mice. Electrophysiology in amygdala pyramidal neurons shows an attenuation of excitatory synaptic transmission, presumably because of the reduction in the level of synaptically localized AMPA-type glutamate receptors. Our results demonstrate that heparan sulfate is critical for normal functioning of glutamatergic synapses and that its deficiency mediates socio-communicative deficits and stereotypies characteristic for autism.

Two species of antiganglioside antibodies in a patient with a pharyngeal-cervical-brachial variant of Guillain-Barré syndrome.

A patient with a pharyngeal-cervical-brachial variant of Guillain-Barré syndrome had anti-GT1a and anti-GD1a antibodies (IgG) in the serum. The activities of anti-GT1a antibodies were stronger than anti-GD1a antibodies and their activities declined later in the clinical course. These two different antibodies bound independently to each ganglioside in an absorption study with polystyrene beads coated with GT1a or GD1a.

A mouse model of chondrocyte-specific somatic mutation reveals a role for Ext1 loss of heterozygosity in multiple hereditary exostoses

Multiple hereditary exostoses (MHE) is one of the most common skeletal dysplasias, exhibiting the formation of multiple cartilage-capped bony protrusions (osteochondroma) and characteristic bone deformities. Individuals with MHE carry heterozygous loss-of-function mutations in Ext1 or Ext2, genes which together encode an enzyme essential for heparan sulfate synthesis. Despite the identification of causative genes, the pathogenesis of MHE remains unclear, especially with regard to whether osteochondroma results from loss of heterozygosity of the Ext genes. Hampering elucidation of the pathogenic mechanism of MHE, both Ext1+/− and Ext2+/− heterozygous mutant mice, which mimic the genetic status of human MHE, are highly resistant to osteochondroma formation, especially in long bones. To address these issues, we created a mouse model in which Ext1 is stochastically inactivated in a chondrocyte-specific manner. We show that these mice develop multiple osteochondromas and characteristic bone deformities in a pattern and a frequency that are almost identical to those of human MHE, suggesting a role for Ext1 LOH in MHE. Surprisingly, however, genotyping and fate mapping analyses reveal that chondrocytes constituting osteochondromas are mixtures of mutant and wild-type cells. Moreover, osteochondromas do not possess many typical neoplastic properties. Together, our results suggest that inactivation of Ext1 in a small fraction of chondrocytes is sufficient for the development of osteochondromas and other skeletal defects associated with MHE. Because the observed osteochondromas in our mouse model do not arise from clonal growth of chondrocytes, they cannot be considered true neoplasms.

Netrin-1/DCC Signaling in Commissural Axon Guidance Requires Cell-Autonomous Expression of Heparan Sulfate

There is increasing evidence that heparan sulfate (HS) plays an essential role in various axon guidance processes. These observations, however, have not addressed whether HS is required cell autonomously as an axonal coreceptor or as an environmental factor that modulates the localization of guidance molecules in the terrain in which growing axons navigate. Here we demonstrate that netrin-1-mediated commissural axon guidance requires cell-autonomous expression of HS in commissural neurons in vivo. We used the Wnt1 –Cre transgene to drive region-specific ablation of Ext1, which encodes an enzyme essential for HS synthesis, in the dorsal part of the spinal cord. Remarkably, Wnt1–Cre-mediated ablation of Ext1 causes commissural axon pathfinding defects that share similarities with those of Netrin-1-deficient and DCC (deleted in colorectal cancer)-deficient mice. Neither Ext1-deficient dorsal spinal cord explants nor wild-type explants in which HS expression was ablated could extend axons in response to netrin-1. Intracellular signaling downstream of netrin-1 and DCC was defective in Ext1-deficient commissural neurons and in DCC-transfected HEK293T cells from which HS was removed. These results demonstrate that the expression of HS by commissural neurons is essential for these neurons to transduce netrin-1 signals, thus providing evidence for a cell-autonomous role of HS in netrin-1/DCC-mediated axon guidance.

Application of exogenous ceramide to cultured rat spinal motoneurons promotes survival or death by regulation of apoptosis depending on its concentrations

The membrane lipid ceramide (Cer) has been shown to be involved in the survival and dendritic growth of cerebellar Purkinje cells and hippocampal neurons. We examined the effects of Cer on isolated rat spinal motoneurons. Basal neuronal cell death due to apoptosis occurs under these culture conditions. This cell death was prevented by treatment with 2.5 μM of D‐erythro‐N‐hexsanoylsphingosine (C6‐Cer), a cell‐permeable analogue, and the surviving cell number was increased approximately 1.6‐fold compared with the control cell number on 5 days in vitro (DIV). Application of the same amount of C6‐Cer improved axonal elongation. Conversely, addition of 10 μM of C6‐Cer led all motoneurons to apoptotic cell death by 2DIV. A stereo isomer, threo‐C6‐Cer, which is not metabolized to C6‐glucosylceramide, also promoted survival, death, and axonal growth in the same manners as C6‐Cer. However, C6‐dihydro‐Cer, a biologically inactive analogue, had no effects on survival or death, indicating that the presence of a double bond in the sphingosine base is essential for its activity. In addition, treatment with bacterial sphingomyelinase, which generates endogenous Cer, increases motoneuron survival and axonal growth. These observations suggest that Cer, but not its metabolites, regulates survival and development of spinal motoneurons, depending on its intracellular concentration. J. Neurosci. Res. 54:475–485, 1998.

Heparan sulfate regulates ephrin-A3/EphA receptor signaling.

Increasing evidence indicates that many signaling pathways involve not only ligands and receptors but also various types of coreceptors and matrix components as additional layers of regulation. Signaling by Eph receptors and their ephrin ligands plays a key role in a variety of biological processes, such as axon guidance and topographic map formation, synaptic plasticity, angiogenesis, and cancer. Little is known about whether the ephrin-Eph receptor signaling system is subject to such additional layers of regulation. Here, we show that ephrin-A3 binds to heparan sulfate, and that the presence of cell surface heparan sulfate is required for the full biological activity of ephrin-A3. Among the ephrins tested, including ephrin-A1, -A2, -A5, -B1, and -B2, only ephrin-A3 binds heparin or heparan sulfate. Ephrin-A3-dependent EphA receptor activation is reduced in mutant cells that are defective in heparan sulfate synthesis, in wild-type cells from which cell surface heparan sulfate has been removed, and in the hippocampus of conditional knockout mice defective in heparan sulfate synthesis. Ephrin-A3-dependent cell rounding is impaired in CHO cells lacking heparan sulfate, and cortical neurons lacking heparan sulfate exhibit impaired growth cone collapse. In contrast, cell rounding and growth cone collapse in response to ephrin-A5, which does not bind heparan sulfate, are not affected by the absence of heparan sulfate. These results show that heparan sulfate modulates ephrin/Eph signaling and suggest a physiological role for heparan sulfate proteoglycans in the regulation of ephrin-A3-dependent biological processes.