Microinjection of Reelin into the mPFC prevents MK-801-induced recognition memory impairment in mice.

Reelin, a large extracellular matrix protein, helps to regulate neuronal plasticity and cognitive function. Several studies have shown that Reelin dysfunction, resulting from factors such as mutations in gene RELN or low Reelin expression, is associated with schizophrenia (SCZ). We previously reported that microinjection of Reelin into cerebral ventricle prevents phencyclidine-induced cognitive and sensory-motor gating deficits. However, it remains unclear whether and how Reelin ameliorates behavioral abnormalities in the animal model of SCZ. In the present study, we evaluated the effect of recombinant Reelin microinjection into the medial prefrontal cortex (mPFC) on abnormal behaviors induced by MK-801, an N-methyl-D-aspartate receptor antagonist. Microinjection of Reelin into the mPFC prevented impairment of recognition memory of MK-801-treated mice in the novel object recognition test (NORT). On the other hand, the same treatment had no effect on deficits in sensory-motor gating and short-term memory in the pre-pulse inhibition and Y-maze tests, respectively. To establish the neural substrates that respond to Reelin, the number of c-Fos-positive cells in the mPFC was determined. A significant increase in c-Fos-positive cells in the mPFC of MK-801-treated mice was observed when compared with saline-treated mice, and this change was suppressed by microinjection of Reelin into the mPFC. A K2360/2467A Reelin that cannot bind to its receptor failed to ameliorate MK-801-induced cognitive deficits in NORT. These results suggest that Reelin prevents MK-801-induced recognition memory impairment by acting on its receptors to suppress neural activity in the mPFC of mice.

Detachment of Chain-Forming Neuroblasts by Fyn-Mediated Control of cell-cell Adhesion in the Postnatal Brain.

In the rodent olfactory system, neuroblasts produced in the ventricular-subventricular zone of the postnatal brain migrate tangentially in chain-like cell aggregates toward the olfactory bulb (OB) through the rostral migratory stream (RMS). After reaching the OB, the chains are dissociated and the neuroblasts migrate individually and radially toward their final destination. The cellular and molecular mechanisms controlling cell-cell adhesion during this detachment remain unclear. Here we report that Fyn, a nonreceptor tyrosine kinase, regulates the detachment of neuroblasts from chains in the male and female mouse OB. By performing chemical screening and in vivo loss-of-function and gain-of-function experiments, we found that Fyn promotes somal disengagement from the chains and is involved in neuronal migration from the RMS into the granule cell layer of the OB. Fyn knockdown or Dab1 (disabled-1) deficiency caused p120-catenin to accumulate and adherens junction-like structures to be sustained at the contact sites between neuroblasts. Moreover, a Fyn and N-cadherin double-knockdown experiment indicated that Fyn regulates the N-cadherin-mediated cell adhesion between neuroblasts. These results suggest that the Fyn-mediated control of cell-cell adhesion is critical for the detachment of chain-forming neuroblasts in the postnatal OB.SIGNIFICANCE STATEMENT In the postnatal brain, newly born neurons (neuroblasts) migrate in chain-like cell aggregates toward their destination, where they are dissociated into individual cells and mature. The cellular and molecular mechanisms controlling the detachment of neuroblasts from chains are not understood. Here we show that Fyn, a nonreceptor tyrosine kinase, promotes the somal detachment of neuroblasts from chains, and that this regulation is critical for the efficient migration of neuroblasts to their destination. We further show that Fyn and Dab1 (disabled-1) decrease the cell-cell adhesion between chain-forming neuroblasts, which involves adherens junction-like structures. Our results suggest that Fyn-mediated regulation of the cell-cell adhesion of neuroblasts is critical for their detachment from chains in the postnatal brain.

Intracellular PAF-Acetylhydrolase Type I.

Platelet-activating factor (PAF) is a phospholipid mediator whose synthesis and degradation depend on specific sets of enzymes. PAF-acetylhydrolase (PAF-AH) hydrolyzes the acetyl moiety of PAF at its sn-2 position and thereby inactivates it. PAF-AH Ib, originally identified in brain, exists in the cytoplasm of many (probably all) types of mammalian cells and tissues. PAF-AH Ib consists of three subunits (α1, α2, and ß), in which the α subunits provide the catalytic activity. The finding that the ß subunit is the product of the causative gene for Miller-Dieker lissencephaly led to extensive analyses of PAF-AH Ib subunits in the field of cell biology and neurobiology. More than 20 molecules are known to bind to PAF-AH Ib subunits, and PAF-AH Ib has been implicated in neuronal development, neuronal functions, Alzheimer's disease, bipolar disorder, cancer, spermatogenesis, and tolerance to hypoxia. However, in almost all of these cases, how the catalytic activity is involved and the identity of the most important substrate of this enzyme are unclear. In this chapter, the structure and functions of PAF-AH Ib and its subunit proteins are summarized and their contributions to human diseases are discussed.

Dab1-mediated colocalization of multi-adaptor protein CIN85 with Reelin receptors, ApoER2 and VLDLR, in neurons.

Reelin-Dab1 signaling is indispensable for proper positioning of neurons in mammalian brain. Reelin is a glycoprotein secreted from Cajal-Reztuis cells in marginal zone of cerebral cortex, and its receptors are Apolipoprotein E receptor 2 (ApoER2) or very low density lipoprotein receptor (VLDLR) expressed on migrating neurons. When Reelin binds to ApoER2 or VLDLR, an adaptor protein Dab1 bound to the receptors undergoes Tyr phosphorylation that is essential for Reelin signaling. We reported previously that Cdk5-p35 phosphorylates Dab1 at Ser400 and Ser491 and the phosphorylation regulates its binding to CIN85, which is an SH3-containing multiadaptor protein involved in endocytic downregulation of receptor-tyrosine kinases. However, the interaction of CIN85 with Dab1 has not been addressed in neurons. We examined here a possibility that CIN85 has a role in Reelin signaling. We found nonpho-sphorylated Dab1-mediated colocalization of CIN85 with ApoER2. The colocalization of CIN85 with ApoER2 was increased in neurons stimulated with Reelin repeats 3-6, an active Reelin fragment. The stimulation recruited CIN85 to domains in plasma membrane where it colocalized with ApoER2 and Dab1 and then to EEA1-labeled early endosomes in the cytoplasm. In addition, Tyr phosphorylation of Dab1 strengthened the binding to CIN85. These results suggest that CIN85 participates in Reelin signaling through the binding to Dab1.

Reelin controls neuronal positioning by promoting cell-matrix adhesion via inside-out activation of integrin α5ß1.

Birthdate-dependent neuronal layering is fundamental to neocortical functions. The extracellular protein Reelin is essential for the establishment of the eventual neuronal alignments. Although this Reelin-dependent neuronal layering is mainly established by the final neuronal migration step called "terminal translocation" beneath the marginal zone (MZ), the molecular mechanism underlying the control by Reelin of terminal translocation and layer formation is largely unknown. Here, we show that after Reelin binds to its receptors, it activates integrin α5ß1 through the intracellular Dab1-Crk/CrkL-C3G-Rap1 pathway. This intracellular pathway is required for terminal translocation and the activation of Reelin signaling promotes neuronal adhesion to fibronectin through integrin α5ß1. Since fibronectin is localized in the MZ, the activated integrin α5ß1 then controls terminal translocation, which mediates proper neuronal alignments in the mature cortex. These data indicate that Reelin-dependent activation of neuronal adhesion to the extracellular matrix is crucial for the eventual birth-date-dependent layering of the neocortex.

Functional importance of covalent homodimer of reelin protein linked via its central region.

Reelin is a 3461-residue secreted glycoprotein that plays a critical role in brain development through its action on target neurons. Although it is known that functional reelin protein exists as multimer formed by interchain disulfide bond(s) as well as through non-covalent interactions, the chemical nature of the multimer assembly has been elusive. In the present study, we identified, among 122 cysteines present in full-length reelin, the single critical cysteine residue (Cys(2101)) responsible for the covalent multimerization. C2101A mutant reelin failed to assemble into disulfide-bonded multimers, whereas it still exhibited non-covalently associated high molecular weight oligomeric states in solution. Detailed analysis of tryptic fragments produced from the purified reelin proteins revealed that the minimum unit of the multimer is a homodimeric reelin linked via Cys(2101) present in the central region and that this cysteine does not connect to the N-terminal region of reelin, which had been postulated as the primary oligomerization domain. A surface plasmon resonance binding assay confirmed that C2101A mutant reelin retained binding capability toward two neuronal receptors apolipoprotein E receptor 2 and very low density lipoprotein receptor. However, it failed to show signaling activity in the assay using the cultured neurons. These results indicate that an intact higher order architecture of reelin multimer maintained by both Cys(2101)-mediated homodimerization and other non-covalent association present elsewhere in the reelin primary structure are essential for exerting its full biological activity.

Fyn tyrosine kinase regulates the surface expression of glycosylphosphatidylinositol-linked ephrin via the modulation of sphingomyelin metabolism.

Glycosylphosphatidylinositol-linked ephrin-As play important roles in various biological events, such as neuronal development and immune responses. Because the surface amount of ephrin-As is critical in these events, the trafficking of ephrin-As must be regulated by intracellular machinery. In particular, Src family protein-tyrosine kinases regulate the intracellular trafficking of several membrane molecules and act downstream of ephrin-As; whether they affect the trafficking of ephrin-As, however, has remained unexplored. Here, we report that the activity of Src family protein-tyrosine kinases, particularly Fyn, negatively regulates the cell-surface amount of ephrin-As. The expression of constitutively active Fyn decreases the surface amount of ephrin-As. Conversely, the expression of dominant-negative Fyn or the application of a Src-family inhibitor increases the surface amount of ephrin-A2. The total cellular amount of ephrin-A is inversely correlated with its amount on the surface, suggesting that ephrin-As are more stable in the intracellular compartment. The expression of constitutively active Fyn increases the amount of sphingomyelin clusters on the plasma membrane, whereas inhibiting Fyn decreases it. Moreover, the inhibition of sphingomyelin synthesis greatly increases the surface amount of ephrin-As. Altogether, these results suggest that Fyn regulates the surface amount of ephrin-As by modulating the metabolism of sphingomyelin, which presumably inhibits the trafficking of ephrin-As from endosomes to the plasma membrane. The signaling cascade described here may function as part of the negative feedback loop of ephrin-A function.

Novel affinity tag system using structurally defined antibody-tag interaction: application to single-step protein purification.

Biologically important human proteins often require mammalian cell expression for structural studies, presenting technical and economical problems in the production/purification processes. We introduce a novel affinity peptide tagging system that uses a low affinity anti-peptide monoclonal antibody. Concatenation of the short recognition sequence enabled the successful engineering of an 18-residue affinity tag with ideal solution binding kinetics, providing a low-cost purification means when combined with nondenaturing elution by water-miscible organic solvents. Three-dimensional information provides a firm structural basis for the antibody-peptide interaction, opening opportunities for further improvements/modifications.

Molecular cloning of mouse type 2 and type 3 inositol 1,4,5-trisphosphate receptors and identification of a novel type 2 receptor splice variant.

We isolated cDNAs encoding type 2 and type 3 inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R2 and IP(3)R3, respectively) from mouse lung and found a novel alternative splicing segment, SI(m2), at 176-208 of IP(3)R2. The long form (IP(3)R2 SI(m2)(+)) was dominant, but the short form (IP(3)R2 SI(m2)(-)) was detected in all tissues examined. IP(3)R2 SI(m2)(-) has neither IP(3) binding activity nor Ca(2+) releasing activity. In addition to its reticular distribution, IP(3)R2 SI(m2)(+) is present in the form of clusters in the endoplasmic reticulum of resting COS-7 cells, and after ATP or Ca(2+) ionophore stimulation, most of the IP(3)R2 SI(m2)(+) is in clusters. IP(3)R3 is localized uniformly on the endoplasmic reticulum of resting cells and forms clusters after ATP or Ca(2+) ionophore stimulation. IP(3)R2 SI(m2)(-) does not form clusters in either resting or stimulated cells. IP(3) binding-deficient site-directed mutants of IP(3)R2 SI(m2)(+) and IP(3)R3 fail to form clusters, indicating that IP(3) binding is involved in the cluster formation by these isoforms. Coexpression of IP(3)R2 SI(m2)(-) prevents stimulus-induced IP(3)R clustering, suggesting that IP(3)R2 SI(m2)(-) functions as a negative coordinator of stimulus-induced IP(3)R clustering. Expression of IP(3)R2 SI(m2)(-) in CHO-K1 cells significantly reduced ATP-induced Ca(2+) entry, but not Ca(2+) release, suggesting that the novel splice variant of IP(3)R2 specifically influences the dynamics of the sustained phase of Ca(2+) signals.

Subtype-specific and ER lumenal environment-dependent regulation of inositol 1,4,5-trisphosphate receptor type 1 by ERp44.

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are intracellular channel proteins that mediate Ca(2+) release from the endoplasmic reticulum (ER) and are involved in many biological processes and diseases. IP(3)Rs are differentially regulated by a variety of cytosolic proteins, but their regulation by ER lumenal protein(s) remains largely unexplored. In this study, we found that ERp44, an ER lumenal protein of the thioredoxin family, directly interacts with the third lumenal loop of IP(3)R type 1 (IP(3)R1) and that the interaction is dependent on pH, Ca(2+) concentration, and redox state: the presence of free cysteine residues in the loop is required. Ca(2+)-imaging experiments and single-channel recording of IP(3)R1 activity with a planar lipid bilayer system demonstrated that IP(3)R1 is directly inhibited by ERp44. Thus, ERp44 senses the environment in the ER lumen and modulates IP(3)R1 activity accordingly, which should in turn contribute to regulating both intralumenal conditions and the complex patterns of cytosolic Ca(2+) concentrations.

Sustained activation of N-WASP through phosphorylation is essential for neurite extension.

Neurite extension is a key process for constructing neuronal circuits during development and remodeling of the nervous system. Here we show that Src family tyrosine kinases and proteasome degradation signals synergistically regulate N-WASP in neurite extension. Src family kinases activate N-WASP through tyrosine phosphorylation, which induces Arp2/3 complex-mediated actin polymerization. Tyrosine phosphorylation of N-WASP also initiates its degradation through ubiquitination. When neurite growth is stimulated in culture, degradation of N-WASP is markedly inhibited, leading to accumulation of the phosphorylated N-WASP. On the other hand, under culture conditions that inhibit neurite extension, but favor proliferation, the phosphorylated N-WASP is degraded rapidly. Collectively, neurite extension is regulated by the balance of N-WASP phosphorylation (activation) and degradation (inactivation), which are induced by tyrosine phosphorylation.