Signal mechanisms underlying low-dose endothelial monocyte-activating polypeptide-II-induced opening of the blood-tumor barrier

Our previous studies have demonstrated that both the RhoA/Rho kinase and the protein kinase C (PKC) signaling pathways are involved in the low-dose endothelial monocyte-activating polypeptide-II (EMAP-II)-induced blood-tumor barrier (BTB) opening. In the present study, an in vitro BTB model was used to investigate which isoforms of PKC were involved in this process as well as the interactions between the RhoA/Rho kinase and the PKC signaling pathways. Our results showed that EMAP-II-activated PKC-α, β, and ζ and induced translocations of them from the cytosolic to the membrane fractions of rat brain microvascular endothelial cells. The EMAP-II-induced alterations in BTB permeability and tight junction (TJ) protein expression were partially blocked by G?6976, the inhibitor of PKC-α/β, and PKC-ζ pseudosubstrate inhibitor (PKC-ζ-PI). Meanwhile, we observed that G?6976 partly inhibited the EMAP-II-induced rearrangement of actin cytoskeleton as well as phosphorylation of myosin light chain and cofilin, whereas PKC-ζ-PI had no effect on these above-mentioned changes induced by EMAP-II. Also, our data revealed that inhibition of RhoA or inhibition of Rho kinase significantly diminished the activities and the translocations of PKC-α and PKC-β induced by EMAP-II, whereas PKC-ζ was unaffected. However, inhibition of PKC-α/β or inhibition of PKC-ζ did not cause any changes in the RhoA and Rho kinase activities. The effects of EMAP-II on BTB permeability and TJ proteins expression were completely blocked by inhibition of both RhoA and PKC-ζ, whereas inhibition of both RhoA and PKC-α/β had an effect similar to that of inhibition of RhoA alone. In summary, this study demonstrates for the first time that three PKC isoforms, PKC-α, β, and ζ, are involved in the EMAP-II-induced BTB opening. It is PKC-α/β, but not PKC-ζ, which serves as the downstream target for RhoA and Rho kinase, suggesting that EMAP-II induces BTB opening via the RhoA/Rho kinase/PKC-α/β signaling pathways. However, PKC-ζ is involved in this process by other mechanisms.     

Interleukin-1 receptor accessory protein organizes neuronal synaptogenesis as a cell adhesion molecule

Interleukin-1 receptor accessory protein (IL-1RAcP) is the essential component of receptor complexes mediating immune responses to interleukin-1 family cytokines. IL-1RAcP in the brain exists in two isoforms, IL-1RAcP and IL-1RAcPb, differing only in the C-terminal region. Here, we found robust synaptogenic activities of IL-1RAcP in cultured cortical neurons. Knockdown of IL-1RAcP isoforms in cultured cortical neurons suppressed synapse formation as indicated by decreases of active zone protein Bassoon puncta and dendritic protrusions. IL-1RAcP recovered the accumulation of presynaptic Bassoon puncta, while IL-1RAcPb rescued both Bassoon puncta and dendritic protrusions. Consistently, the expression of IL-1RAcP in cortical neurons enhances the accumulation of Bassoon puncta and that of IL-1RAcPb stimulated both Bassoon puncta accumulation and spinogenesis. IL-1RAcP interacted with protein tyrosine phosphatase (PTP) δ through the extracellular domain. Mini-exon peptides in the Ig-like domains of PTPδ splice variants were critical for their efficient binding to IL-1RAcP. The synaptogenic activities of IL-1RAcP isoforms were diminished in cortical neurons from PTPδ knock-out mice. Correspondingly, PTPδ required IL-1RAcPb to induce postsynaptic differentiation. Thus, IL-1RAcPb bidirectionally regulated synapse formation of cortical neurons. Furthermore, the spine densities of cortical and hippocampal pyramidal neurons were reduced in IL-1RAcP knock-out mice lacking both isoforms. These results suggest that IL-1RAcP isoforms function as trans-synaptic cell adhesion molecules in the brain and organize synapse formation. Thus, IL-1RAcP represents an interesting molecular link between immune systems and synapse formation in the brain.     

Ectopic expression of the immune adaptor protein CD3zeta in neural stem/progenitor cells disrupts cell-fate specification

Immune signaling and neuroinflammatory mediators have recently emerged as influential variables that regulate neural precursor/stem cell (NPC) behavior and function. In this study, we investigated whether the signaling adaptor protein CD3ζ, a transmembrane protein involved in T cell differentiation and function and recently shown to regulate neuronal development in the central nervous system (CNS), may have a role in NPC differentiation. We analyzed the expression profile of CD3ζ in embryonic rat brain during neurogenic periods and in neurosphere-derived neural cells, and we investigated the action of CD3ζ on cell differentiation. We found that CD3ζ expression coincided with neuronal commitment, but its forced expression in NPCs prevented the production of neurons and oligodendrocytes, but not astroglial cells. This blockade of neuronal differentiation was operated through an ITAM-independent mechanism, but required the Asp36 of the CD3ζ transmembrane domain involved in membrane receptor interaction. Together, our findings show that ectopic CD3ζ expression in NPCs impaired their normal cell-fate specification and suggest that variations of CD3ζ expression in the developing CNS might result in neurodevelopmental anomalies.     

PTP1B regulates neurite extension mediated by cell-cell and cell-matrix adhesion molecules

N-cadherin and beta1-integrin adhesion and signaling play important roles in growth cone adhesion and guidance. Each of these adhesion receptor systems is composed of multiprotein complexes, and both adhesion and downstream signaling events are regulated through the interaction of protein tyrosine kinases and phosphatases with many of the proteins that make up these complex systems. Work from our laboratory reported that the nonreceptor protein tyrosine phosphatase PTP1B is localized to adherens junctions and focal adhesion complexes and regulates both N-cadherin- and beta1-integrin-mediated adhesion. PTP1B appears to modulate integrin-mediated adhesion through regulation of src activation and cadherin-mediated adhesion through dephosphorylation of beta-catenin. We have continued these studies and report that PTP1B is localized to the tips of growing neurites and that introduction of a noncatalytic mutant of PTP1B into PC12 cells results in inhibition of N-cadherin- and beta1-integrin-mediated neurite outgrowth but is without effect on neurite outgrowth on poly-L-lysine. Moreover, suppressing the level of PTP1B in primary embryonic chick neural retina cells using antisense oligonucleotides also inhibits N-cadherin- and beta1-integrin-mediated neurite outgrowth. Neither of these techniques reduces the levels of expression of either adhesion receptor. We conclude that PTP1B is a regulatory component of the molecular complex required for both N-cadherin and beta1-integrin-mediated axon growth.     

Increased PKMζ activity impedes lateral movement of GluA2-containing AMPA receptors

Protein kinase M zeta (PKMζ), a constitutively active, atypical protein kinase C isoform, maintains a high level of expression in the brain after the induction of learning and long-term potentiation (LTP). Further, its overexpression enhances long-term memory and LTP. Thus, multiple lines of evidence suggest a significant role for persistently elevated PKMζ levels in long-term memory. The molecular mechanisms of how synaptic properties are regulated by the increase in PKMζ, however, are still largely unknown. The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR) mediates most of the fast glutamatergic synaptic transmission in the brain and is known to be critical for the expression of synaptic plasticity and memory. Importance of AMPAR trafficking has been implicated in PKMζ-mediated cellular processes, but the detailed mechanisms, particularly in terms of regulation of AMPAR lateral movement, are not well understood. In the current study, using a single-molecule live imaging technique, we report that the overexpression of PKMζ in hippocampal neurons immobilized GluA2-containing AMPARs, highlighting a potential novel mechanism by which PKMζ may regulate memory and synaptic plasticity.     

Neurodevelopmental and neuropsychiatric behaviour defects arise from 14-3-3ζ deficiency

Complex neuropsychiatric disorders are believed to arise from multiple synergistic deficiencies within connected biological networks controlling neuronal migration, axonal pathfinding and synapse formation. Here, we show that deletion of 14-3-3ζ causes neurodevelopmental anomalies similar to those seen in neuropsychiatric disorders such as schizophrenia, autism spectrum disorder and bipolar disorder. 14-3-3ζ-deficient mice displayed striking behavioural and cognitive deficiencies including a reduced capacity to learn and remember, hyperactivity and disrupted sensorimotor gating. These deficits are accompanied by subtle developmental abnormalities of the hippocampus that are underpinned by aberrant neuronal migration. Significantly, 14-3-3ζ-deficient mice exhibited abnormal mossy fibre navigation and glutamatergic synapse formation. The molecular basis of these defects involves the schizophrenia risk factor, DISC1, which interacts isoform specifically with 14-3-3ζ. Our data provide the first evidence of a direct role for 14-3-3ζ deficiency in the aetiology of neurodevelopmental disorders and identifies 14-3-3ζ as a central risk factor in the schizophrenia protein interaction network.     

Expression of PTPRO in the interneurons of adult mouse olfactory bulb

PTPRO is a receptor‐type protein tyrosine phosphatase (PTP) with a single catalytic domain in its cytoplasmic region and multiple fibronectin type III‐like domains in its extracellular region. In the chick, PTPRO mRNA has been shown to be particularly abundant in embryonic brain, and PTPRO is implicated in axon growth and guidance during embryonic development. However, the temporal and spatial expression of PTPRO protein in the mammalian CNS, particularly in the juvenile and adult mammalian brain, has not been evaluated in any detail. By immunohistofluorescence analysis with a monoclonal antibody to PTPRO, we show that PTPRO is widely expressed throughout the mouse brain from embryonic day 16 to postnatal day 1, while expression is largely confined to the olfactory bulb (OB) and olfactory tubercle in the adult brain. In the OB, PTPRO protein is expressed predominantly in the external plexiform layer, the granule cell layer, and the glomerular layer (GL). In these regions, expression of PTPRO is predominant in interneurons such as γ‐aminobutyric acid (GABA)‐ergic or calretinin (CR)‐positive granule cells. In addition, PTPRO is expressed in GABAergic, CR‐positive, tyrosine hydroxylase‐positive, or neurocalcin‐positive periglomerular cells in the GL. Costaining of PTPRO with other neuronal markers suggests that PTPRO is likely to be localized to the dendrites or dendritic spines of these olfactory interneurons. Thus, PTPRO might participate in regulation of dendritic morphology or synapse formation of interneurons in the adult mouse OB. J. Comp. Neurol. 518:119–136, 2010. © 2009 Wiley‐Liss, Inc.     

Protein kinase C-α and -ε are enriched in growth cones of differentiating SH-SY5Y human neuroblastoma cells

SH-SY5Y cells differentiate into neuronal-like cells and express marker proteins like growth-associated protein (GAP-43) and neuropeptide tyrosine when treated with a low concentration (16 nM) of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of growth factors or serum. Both control and differentiated cells expressed protein kinase C-α (PKC-α), PKC-ε, and PKC-ζ as revealed by Western blot analyses, but the subcellular distribution of the three isoforms was not uniform, indicating specific localized functions of the enzymes. In growth cones prepared from differentiating cells PKC-α and PKC-ε were enriched. In contrast, PKC-ζ was more evenly distributed within the differentiating cell. Cells treated with a high concentration of TPA (1.6 μM) differentiate poorly and continue to proliferate. In those cells, PKC-α and PKC-ε were found to be down-regulated while PKC-ζ remained present. Thus, down-regulation of PKC-α and PKC-ζ appears to be incompatible with neuronal differentiation of SH-SY5Y cells. These cells also differentiate when treated with a combination of basic fibroblast growth factor and insulin-like growth factor I. Growth cones isolated from such cells are also enriched in PKC-α and PKC-ε, but not in PKC-ζ. Based on the subcellular distribution of PKC-α and ε, and that PKC substrates like GAP-43 and pp60^c-src are enriched in SH-SY5Y growth cones, a role during neurite growth is suggested. © 1995 Wiley-Liss, Inc.     

Receptor-like protein tyrosine phosphatase zeta/RPTP beta is expressed on tangentially aligned neurons in early mouse neocortex

Protein tyrosine phosphatase zeta (PTPzeta)/RPTPbeta is a chondroitin sulfate proteoglycan predominantly expressed in the brain. In this study, we examined immunohistochemical localisation of PTPzeta in the mouse telencephalon from embryonic day 9.5 (E9.5) to E15.5. During E10.5-E12.5, immunoreactivities for PTPzeta are specifically observed on the tangentially aligned neurons at the preplate (PP) of the neocortex, as well as on the neurons at the mantle layer (ML) of the ganglionic eminences (GEs). Likewise, neurons immunoreactive for CR50, a marker for Cajal-Retzius neurons, are aligned from the ML of the ganglionic eminences to the PP of the neocortex and co-express PTPzeta. During E13.5-E15.5, PTPzeta-positive neurons are present at the subplate (SP) as well as at the marginal zone (MZ) of the neocortex. These results indicate that PTPzeta is a useful marker for early-generated neocortical neurons in mice: Cajal-Retzius neurons as well as the subplate neurons.     

Protein kinase C-ε is a developmentally regulated,neuronal isoform in the chick embryo central nervous system

Protein kinase C (PKC) is expressed as many isoforms and in high quantities in the central nervous system (CNS), which suggests an important role for this enzyme in neuronal development and function. We used specific antibodies to investigate the expression of the known PKC isoforms in extracts from chick major CNS areas during embryogenesis, from day 3 (E3) of incubation to day 1 post-hatching (P1). PKC-ε was the predominant isoform and was expressed from E6 onward in all brain regions, except retina (E12 and on). PKC-α/β and -ζ isoforms were expressed at lower levels prior to PKC-ε expression and throughout embryogenesis. No other isoforms were detected in neural tissue preparations. We then used neural culture systems derived from the chick CNS to study the expression of PKC isoforms in neuroblasts, cortical neurons, and cortical glial cells. Western blotting and immunostaining of neuroblastenriched cultures, derived from E3 CNS, showed only the Ca²⁺-dependent PKC-α/β to be present. Studies on neuronal cultures derived from E6 cerebral hemispheres revealed only the Ca²⁺-independent PKC-ε to be expressed in neurons, as predicted by the developmental studies on tissue homogenates. PKC-ε immunoreactivity was seen intracellularly in differentiating neurons, regardless of their neurotransmitter phenotypes, and it correlated well with the level of neuronal activity. Furthermore, PKC-α/β immunoreactivity was verified on glia cells, as the glial lineage emerges in E15 cortical cultures. These data suggest that PKC-ε expression is associated with the final neuroblast division in neurons, and the correlation of PKC isoform expression and neural cell lineage is discussed. © 1993 Wiley-Liss, Inc.     

Protein tyrosine phosphatase zeta/RPTPbeta interacts with PSD-95/SAP90 family.

PTPzeta/RPTPbeta is a proteoglycan-type receptor-like protein tyrosine phosphatase specifically expressed in the brain. Although several ligands of PTPzeta have been identified, proteins interacting with the intracellular region of PTPzeta are still unknown. We performed yeast two-hybrid screening using the intracellular region of PTPzeta as a bait, and found that the C-terminal sequence of PTPzeta binds to the PSD-95/SAP90 family through the second PDZ domain. Immunohistochemical analysis revealed that PTPzeta and PSD-95/SAP90 are similarly distributed in the dendrites of pyramidal neurons of the hippocampus and neocortex. Furthermore, subcellular fractionation experiments indicated that PTPzeta is concentrated in the postsynaptic density fraction. These results suggested that PTPzeta is involved in the regulation of synaptic function as postsynaptic macromolecular complexes with PSD-95/SAP90.     

Protein tyrosine phosphatases in glioma biology

Gliomas are a diverse group of brain tumors of glial origin. Most are characterized by diffuse infiltrative growth in the surrounding brain. In combination with their refractive nature to chemotherapy this makes it almost impossible to cure patients using combinations of conventional therapeutic strategies. The drastically increased knowledge about the molecular underpinnings of gliomas during the last decade has elicited high expectations for a more rational and effective therapy for these tumors. Most studies on the molecular pathways involved in glioma biology thus far had a strong focus on growth factor receptor protein tyrosine kinase (PTK) and phosphatidylinositol phosphatase signaling pathways. Except for the tumor suppressor PTEN, much less attention has been paid to the PTK counterparts, the protein tyrosine phosphatase (PTP) superfamily, in gliomas. PTPs are instrumental in the reversible phosphorylation of tyrosine residues and have emerged as important regulators of signaling pathways that are linked to various developmental and disease-related processes. Here, we provide an overview of the current knowledge on PTP involvement in gliomagenesis. So far, the data point to the potential implication of receptor-type (RPTPδ, DEP1, RPTPμ, RPTPζ) and intracellular (PTP1B, TCPTP, SHP2, PTPN13) classical PTPs, dual-specific PTPs (MKP-1, VHP, PRL-3, KAP, PTEN) and the CDC25B and CDC25C PTPs in glioma biology. Like PTKs, these PTPs may represent promising targets for the development of novel diagnostic and therapeutic strategies in the treatment of high-grade gliomas.     

Expression and immunohistochemical localization of heparan sulphate proteoglycan N-syndecan in the migratory pathway from the rat olfactory placode

N-syndecan, a membrane-bound heparan sulphate proteoglycan, is abundantly present in the developing nervous system and thought to play important roles in the neurite outgrowth. In the present study, we examined the distribution of N-syndecan in the migratory route from the rat olfactory placode using immunohistochemistry and in situ hybridization. At embryonic day 15, both heparan sulphate and N-syndecan immunoreactivities were localized in and around the migrating cell clusters, which contained luteinizing hormone-releasing hormone (LHRH) and calbindin D-28k. Immunoreactivity for other glycosaminoglycan chains, such as chondroitin and keratan sulphate, and core proteins of the chondroitin sulphate proteoglycan, neurocan and phosphacan, were barely detected in the migratory pathway from the olfactory placode. By in situ hybridization histochemistry, N-syndecan mRNA was localized in virtually all of migrating neurons as well as in cells of the olfactory epithelium and the vomeronasal organ. N-syndecan immunoreactivity surrounded cells migrating along the vomeronasal nerves that were immunoreactive for neural cell adhesion molecules, NCAM, L1 and TAG-1. Considering that NCAM is implicated in the migratory process of LHRH neurons and specifically binds to heparan sulphate, it is likely that a heterophilic interaction between NCAM and N-syndecan participates in the neuronal migration from the rat olfactory placode.     

Initiation and maintenance of CNTF-Jak/STAT signaling in neurons is blocked by protein tyrosine phosphatase inhibitors

Cytokines, including interferon-gamma and ciliary neurotrophic factor (CNTF), act in common through tyrosine kinase-based Jak/STAT signaling pathways. We found that activation of the Jak/STAT pathway by both interferon-gamma and CNTF in nerve cells was rapidly terminated by tyrosine phosphatase inhibitors. Exposure of human neuroblastoma cells, BE(2)-C, first to tyrosine phosphatase inhibitors (either phenylarsine oxide or PTP inhibitor-2) prevented Jak1, STAT1 and STAT3 activation elicited subsequently by either CNTF or interferon-gamma. In contrast, exposure of these cells to phosphatase inhibitors after initial stimulation by CNTF or interferon-gamma prevented the normal time-dependent decrease of total cellular phosphotyrosine-STAT levels as expected, while excluding already formed phosphotyrosine-STAT from the nucleus. Thus, treatment of nerve cells with a tyrosine phosphatase inhibitor blocked nuclear signal transduction. A similar inhibition of CNTF-Jak/STAT signaling was observed following tyrosine phosphatase inhibition in SH-SY5Y human neuroblastoma cells, HMN-1 mouse motor neuron-neuroblastoma hybrid cells, HepG2 human hepatoma cells and embryonic chick ciliary ganglion and retinal neurons. Expression of dominant-negative forms of the tyrosine phosphatases, SHP-1 and/or SHP-2, in BE(2)-C cells had no effect on CNTF activation of STAT or on the ability of phosphatase inhibitors to block signaling. Further, results from H-35 cells expressing gp130 receptor subunits lacking functional SHP-2 binding sites revealed normal cytokine activation of Jak and STAT that was inhibited by phosphatase inhibitors. These findings suggest a critical control for regulating the initiation of Jak/STAT signaling requiring tyrosine phosphatase activity.     

Enzymatic removal of chondroitin sulphates abolishes the age-related axon order in the optic tract of mouse embryos

Retinal axons undergo an age-related reorganization at the junction of the chiasm and the optic tract. We have investigated the effects of removal of chondroitin sulphate on this order change in mouse embryos aged embryonic day 14, when most axons are growing in the optic tract. Enzymatic removal of chondroitin sulphate but not keratan sulphate in brain slice preparations of the retinofugal pathway abolished the accumulation of phalloidin-positive growth cones in the subpial region of the optic tract. The loss of chronotopicity was further demonstrated by anterograde filling of single retinal axons, which showed a dispersion of growth cones from subpial to the whole depth of the tract. The enzyme treatment neither produced detectable changes in growth cone morphology and growth dynamic of retinal neurites nor affected the radial glial processes in the tract, indicating a specific effect of removal of chondroitin sulphate from the pathway to the axon order in the tract. Although chondroitin sulphate was also found at the midline of the chiasm, growth cone distribution across the depth of fibre layer at the midline was not affected by the enzyme treatment. These results suggest a mechanism in which retinal axons undergo changes in response to chondroitin sulphate at the chiasm-tract junction, but not at the midline, that produce a chronotopic fibre rearrangement in the mouse retinofugal pathway.     

Early memory formation disrupted by atypical PKC inhibitor ZIP in the medial prefrontal cortex but not hippocampus

Atypical isoforms of protein kinase C (aPKCs; particularly protein kinase M zeta: PKMζ) have been hypothesized to be necessary and sufficient for the maintenance of long‐term potentiation (LTP) and long term memory by maintaining postsynaptic AMPA receptors via the GluA2 subunit. A myristoylated PKMζ pseudosubstrate peptide (ZIP) blocks PKMζ activity. We examined the actions of ZIP in medial prefrontal cortex (mPFC) and hippocampus in associative recognition memory in rats during early memory formation and memory maintenance. ZIP infusion in either hippocampus or mPFC impaired memory maintenance. However, early memory formation was impaired by ZIP in mPFC but not hippocampus; and blocking GluA2‐dependent removal of AMPA receptors did not affect this impairment caused by ZIP in the mPFC. The findings indicate: (i) a difference in the actions of ZIP in hippocampus and medial prefrontal cortex, and (ii) a GluA2‐independent target of ZIP (possibly PKCλ) in the mPFC during early memory formation. © 2014 Wiley Periodicals, Inc.     

Receptor tyrosine phosphatase PTPγ is a regulator of spinal cord neurogenesis

During spinal cord development the proliferation, migration and survival of neural progenitors and precursors is tightly controlled, generating the fine spatial organisation of the cord. In order to understand better the control of these processes, we have examined the function of an orphan receptor protein tyrosine phosphatase (RPTP) PTPγ, in the developing chick spinal cord. Widespread expression of PTPγ occurs post-embryonic day 3 in the early cord and is consistent with a potential role in either neurogenesis or neuronal maturation. Using gain-of-function and loss-of-function approaches in ovo, we show that PTPγ perturbation significantly reduces progenitor proliferation rates and neuronal precursor numbers, resulting in hypoplasia of the neuroepithelium. PTPγ gain-of-function causes widespread suppression of Wnt/β-catenin-driven TCF signalling. One potential target of PTPγ may therefore be β-catenin itself, since PTPγ can dephosphorylate it in vitro, but alternative targets are also likely. PTPγ loss-of-function is not sufficient to alter TCF signalling. Instead, loss-of-function leads to increased apoptosis and defective cell-cell adhesion in progenitors and precursors. Furthermore, motor neuron precursor migration is specifically defective. PTPγ therefore regulates neurogenesis during a window of spinal cord development, with molecular targets most likely related to Wnt/β-catenin signalling, cell survival and cell adhesion.     

Developmental distribution of platelet-derived growth factor in the mouse central nervous system.

Immunoblotting and immunohistochemical techniques have been used to characterize the developmental changes in the distribution and relative quantity of platelet-derived growth factor (PDGF), an important mitogen and growth regulator for glial (and possibly neuronal) cells. PDGF exists as a dimer of two chains, A and B, and antibodies which are relatively specific for one chain or the other can be used to localize PDGF isoforms during development. We have also studied the distribution of PDGF receptor beta subunit (PDGF-R beta)-like immunoreactivity using an antibody probe. All 3 isoforms of PDGF are found in neural structures during development, beginning at about the midpoint of embryogenesis. Immunoblotting studies confirm the presence of PDGF isoforms in brain during embryonic and postnatal development, with the distribution and relative abundance of each isoform appearing to be independently regulated. Similarly, immunoblotting studies have verified the relative abundance and specificity of PDGF receptor beta subunit. The immunohistochemical findings confirm and extend these biochemical observations. Each PDGF chain (A and B) has a discrete localization during nervous system development, and the immunohistochemical distribution of PDGF-R beta is distinct from each of the PDGF isoforms. PDGF A-chain (localized with an antibody to PDGF(AA) dimers) appears to be found in growth cones of developing neurons in mid-embryonic brain development. By 11.5 days post-conception (embryonic day 11.5, E11.5) to E12, PDGF isoforms are found in apparent neurons in the basal plate (future ventral horn) of spinal cord. PDGF-R beta-like immunoreactivity is localized to the boundary cap region of the developing spinal cord at the same age. Similarly, at E13.5, all 3 PDGF isoforms are found, to varying extents, within cells of the dorsal root ganglia and trigeminal ganglia. At the same developmental stage, PDGF receptor protein is most prevalent in the nerves accompanying these structures. By E15, both PDGF isoform and PDGF receptor beta subunit immunoreactivity have declined to near-background levels in the sensory ganglia, while in the spinal cord and developing forebrain, levels of all PDGF-related proteins remain high.     

Brain-derived neurotrophic factor enhances association of protein tyrosine phosphatase PTP1D with the NMDA receptor subunit NR2B in the cortical postsynaptic density.

Our recent studies revealed that brain-derived neurotrophic factor (BDNF) rapidly enhances tyrosine phosphorylation and dephosphorylation of the NMDA receptor subunit, NR2B, in the postsynaptic density (PSD), potentially regulating synaptic plasticity. To explore the molecular mechanisms underlying synaptic NR2B signaling, we examined the protein tyrosine phosphatase, PTP1D; BDNF reportedly increases association of PTP1D with tyrosine phosphorylated proteins in cortical neurons and PC 12 cells. We now report that PTP1D is an intrinsic component of the rat cerebrocortical PSD, based on Western blot analysis using specific anti-PTP1D antibodies. In addition, NR2B was co-immunoprecipitated with PTP1D using anti-NR2B antibodies or anti-PTP1D antibodies, indicating physical association of the subunit with PTP1D. Moreover, treatment of the purified PSD with BDNF for 5 min elicited a two-fold increase in the association of NR2B with PTP1D. The BDNF action appeared to be specific, since nerve growth factor, another member of the neurotrophin gene family, did not alter the association. Finally, an overlay assay revealed that BDNF caused a two-fold increase in binding of blotted PSD NR2B proteins to PTP1D-SH2 domains, revealing molecular mechanisms mediating the PTP1D-NR2B binding. Taken together, our results raise the possibility that PTP1D participates in BDNF-mediated NR2B signaling cascades at the postsynaptic site, thereby regulating synaptic plasticity.