Kazutada Watanabe

F3/Contactin Acts as a Functional Ligand for Notch during Oligodendrocyte Maturation

Axon-derived molecules are temporally and spatially required as positive or negative signals to coordinate oligodendrocyte differentiation. Increasing evidence suggests that, in addition to the inhibitory Jagged1/Notch1 signaling cascade, other pathways act via Notch to mediate oligodendrocyte differentiation. The GPI-linked neural cell recognition molecule F3/contactin is clustered during development at the paranodal region, a vital site for axoglial interaction. Here, we show that F3/contactin acts as a functional ligand of Notch. This trans-extracellular interaction triggers gamma-secretase-dependent nuclear translocation of the Notch intracellular domain. F3/Notch signaling promotes oligodendrocyte precursor cell differentiation and upregulates the myelin-related protein MAG in OLN-93 cells. This can be blocked by dominant negative Notch1, Notch2, and two Deltex1 mutants lacking the RING-H2 finger motif, but not by dominant-negative RBP-J or Hes1 antisense oligonucleotides. Expression of constitutively active Notch1 or Notch2 does not upregulate MAG. Thus, F3/contactin specifically initiates a Notch/Deltex1 signaling pathway that promotes oligodendrocyte maturation and myelination.

Association of TAG-1 with Caspr2 is essential for the molecular organization of juxtaparanodal regions of myelinated fibers.

Myelination results in a highly segregated distribution of axonal membrane proteins at nodes of Ranvier. Here, we show the role in this process of TAG-1, a glycosyl-phosphatidyl-inositol–anchored cell adhesion molecule. In the absence of TAG-1, axonal Caspr2 did not accumulate at juxtaparanodes, and the normal enrichment of shaker-type K+ channels in these regions was severely disrupted, in the central and peripheral nervous systems. In contrast, the localization of protein 4.1B, an axoplasmic partner of Caspr2, was only moderately altered. TAG-1, which is expressed in both neurons and glia, was able to associate in cis with Caspr2 and in trans with itself. Thus, a tripartite intercellular protein complex, comprised of these two proteins, appears critical for axo–glial contacts at juxtaparanodes. This complex is analogous to that described previously at paranodes, suggesting that similar molecules are crucial for different types of axo–glial interactions.

A TAG1-APP signalling pathway through Fe65 negatively modulates neurogenesis

The release of amyloid precursor protein (APP) intracellular domain (AICD) may be triggered by extracellular cues through γ-secretase-dependent cleavage. AICD binds to Fe65, which may have a role in AICD-dependent signalling; however, the functional ligand has not been characterized. In this study, we have identified TAG1 as a functional ligand of APP. We found that, through an extracellular interaction with APP, TAG1 increased AICD release and triggered Fe65-dependent activity in a γ-secretase-dependent manner. TAG1, APP and Fe65 colocalized in the neural stem cell niche of the fetal ventricular zone. Neural precursor cells from TAG1−/−, APP−/− and TAG1−/−;APP−/− mice had aberrantly enhanced neurogenesis, which was significantly reversed in TAG1−/− mice by TAG1 or AICD but not by AICD mutated at the Fe65 binding site. Notably, TAG1 reduced normal neurogenesis in Fe65+/+ mice. Abnormally enhanced neurogenesis also occurred in Fe65−/− mice but could not be reversed by TAG1. These results describe a TAG1–APP signalling pathway that negatively modulates neurogenesis through Fe65.

Contactins: emerging key roles in the development and function of the nervous system.

Contactins are a subgroup of molecules belonging to the immunoglobulin superfamily that are expressed exclusively in the nervous system. The subgroup consists of six members: contactin, TAG-1, BIG-1, BIG-2, NB-2 and NB-3. Since their identification in the late 1980s, contactin and TAG-1 have been studied extensively. Axonal expression and the neurite extension activity of contactin and TAG-1 attracted researchers to study the function of these molecules in axon guidance during development. After the exciting discovery of the molecular function of contactin and TAG-1 in myelination earlier this decade, these two molecules have come to be known as the principal molecules in the function and maintenance of myelinated neurons. In contrast, the function of the other four members of this subgroup remained unknown until recently. Here, we will give an overview of contactin function, including recent progress on BIG-2, NB-2 and NB-3.

Adenoviral gene transfer of GDNF, BDNF and TGFβ2, but not CNTF, cardiotrophin-1 or IGF1, protects injured adult motoneurons after facial nerve avulsion

We examined neuroprotective effects of recombinant adenoviral vectors encoding glial cell line‐derived neurotrophic factor (GDNF), brain‐derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), cardiotrophin‐1 (CT1), insulin‐like growth factor‐1 (IGF1), and transforming growth factor‐β2 (TGFβ2) on lesioned adult rat facial motoneurons. The right facial nerves of adult Fischer 344 male rats were avulsed and removed from the stylomastoid foramen, and adenoviral vectors were injected into the facial canal. Animals avulsed and treated with adenovirus encoding GDNF, BDNF, CNTF, CT1, IGF1 and TGFβ2 showed intense immunolabeling for these factors in lesioned facial motoneurons, respectively, indicating adenoviral induction of the neurotrophic factors in these neurons. The treatment with adenovirus encoding GDNF, BDNF, or TGFβ2 after avulsion significantly prevented the loss of lesioned facial motoneurons, improved choline acetyltransferase immunoreactivity and prevented the induction of nitric oxide synthase activity in these neurons. The treatment with adenovirus encoding CNTF, CT1 or IGF1, however, failed to protect these neurons after avulsion. These results indicate that the gene transfer of GDNF and BDNF and TGFβ2 but not CNTF, CT1 or IGF1 may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.

Animal model of dementia induced by entorhinal synaptic damage and partial restoration of cognitive deficits by BDNF and carnitine.

A rat dementia model with cognitive deficits was generated by synapse‐specific lesions using botulinum neurotoxin (BoNTx) type B in the entorhinal cortex. To detect cognitive deficits, different tasks were needed depending upon the age of the model animals. Impaired learning and memory with lesions were observed in adult rats using the Hebb‐Williams maze, AKON‐1 maze and a continuous alternation task in T‐maze. Cognitive deficits in lesioned aged rats were detected by a continuous alternation and delayed non‐matching‐to‐sample tasks in T‐maze. Adenovirus‐mediated BDNF gene expression enhanced neuronal plasticity, as revealed by behavioral tests and LTP formation. Chronic administration of carnitine over time pre‐ and post‐lesions seemed to partially ameliorate the cognitive deficits caused by the synaptic lesion. The carnitine‐accelerated recovery from synaptic damage was observed by electron microscopy. These results demonstrate that the BoNTx‐lesioned rat can be used as a model for dementia and that cognitive deficits can be alleviated in part by BDNF gene transfer or carnitine administration.

Plasticity of neurohypophysial terminals with increased hormonal release during dehydration: Ultrastructural and biochemical analyses

Arginine vasopressin‐ (AVP) and oxytocin‐ (OXT) secreting magnocellular neurons undergo gross structural changes with chronic physiological stimulation. Here, we investigated subcellular aspects of plasticity in rat neurohypophysial terminals during dehydration. Ultrastructural analyses demonstrated that chronic dehydration by 2% NaCl drinking for 7 days significantly decreased the numbers of neurosecretory granules and microvesicles but not the numbers of mitochondria. Moreover, in dehydrated rats, terminals making neurovascular contacts enlarged, whereas terminals in apposition to astrocytes, i.e., neuroglial contacts, became smaller. Western blot analyses demonstrated significant decreases in the levels of F3 and Thy‐1 together with those of AVP‐ and OXT‐neurophysin, but the levels of synaptophysin, SNAP‐25, and GAP‐43 were unchanged. Both F3 and Thy‐1 were recovered in the buffer‐insoluble pellet, and phosphatidyl inositol‐specific phospholipase C treatment released both molecules from the crude membrane fraction, indicating that they are attached to terminal membranes by glycosylphosphatidyl inositol anchors. Confocal microscopic observations demonstrated that F3 colocalized with Thy‐1 in the same terminals of magnocellular neurons. In contrast, the level of calretinin, a Ca2+ binding protein was significantly increased with chronic dehydration. Thus, the present results suggest that enhancement of neurovascular contacts results from rearrangement of terminal‐astrocyte and terminal‐vessel contacts rather than enlargement or sprouting of magnocellular terminals themselves. The down‐regulation of F3 and Thy‐1 may contribute to enhancement of neurovascular contacts that accompany increased peptide release during dehydration. J. Comp. Neurol. 434:413–427, 2001.

Neural recognition molecules CHL1 and NB-3 regulate apical dendrite orientation in the neocortex via PTPα

Apical dendrites of pyramidal neurons in the neocortex have a stereotypic orientation that is important for neuronal function. Neural recognition molecule Close Homolog of L1 (CHL1) has been shown to regulate oriented growth of apical dendrites in the mouse caudal cortex. Here we show that CHL1 directly associates with NB‐3, a member of the F3/contactin family of neural recognition molecules, and enhances its cell surface expression. Similar to CHL1, NB‐3 exhibits high‐caudal to low‐rostral expression in the deep layer neurons of the neocortex. NB‐3‐deficient mice show abnormal apical dendrite projections of deep layer pyramidal neurons in the visual cortex. Both CHL1 and NB‐3 interact with protein tyrosine phosphatase α (PTPα) and regulate its activity. Moreover, deep layer pyramidal neurons of PTPα‐deficient mice develop misoriented, even inverted, apical dendrites. We propose a signaling complex in which PTPα mediates CHL1 and NB‐3‐regulated apical dendrite projection in the developing caudal cortex.

Nogo‐A at CNS paranodes is a ligand of Caspr: possible regulation of K+ channel localization

We report Nogo‐A as an oligodendroglial component congregating and interacting with the Caspr–F3 complex at paranodes. However, its receptor Nogo‐66 receptor (NgR) does not segregate to specific axonal domains. CHO cells cotransfected with Caspr and F3, but not with F3 alone, bound specifically to substrates coated with Nogo‐66 peptide and GST–Nogo‐66. Binding persisted even after phosphatidylinositol‐ specific phospholipase C (PI‐PLC) removal of GPI‐linked F3 from the cell surface, suggesting a direct interaction between Nogo‐66 and Caspr. Both Nogo‐A and Caspr co‐immunoprecipitated with Kv1.1 and Kv1.2, and the developmental expression pattern of both paralleled compared with Kv1.1, implicating a transient interaction between Nogo‐A–Caspr and K+ channels at early stages of myelination. In pathological models that display paranodal junctional defects (EAE rats, and Shiverer and CGT−/− mice), distances between the paired labeling of K+ channels were shortened significantly and their localization shifted toward paranodes, while paranodal Nogo‐A congregation was markedly reduced. Our results demonstrate that Nogo‐A interacts in trans with axonal Caspr at CNS paranodes, an interaction that may have a role in modulating axon–glial junction architecture and possibly K+‐channel localization during development.

Novel neural adhesion molecules in the Contactin/F3 subgroup of the immunoglobulin superfamily: isolation and characterization of cDNAs from rat brain

We cloned two kinds of cDNAs encoding novel Contactin/F3-subgroup adhesion molecules, which we termed NB-2 and NB-3. Nucleotide sequence analyses have shown that NB-2 and NB-3 are comprised of 1099 and 1028 amino acid residues, respectively. There was 51% similarity in the amino acid sequence of NB-2 and NB-3. NB-2 shared 46, 43, 55 and 55% identities with Contactin/F3, Tag-1, Big-1 and Big-2, respectively, at the amino acid sequence level. Likewise, the amino acid sequence of NB-3 exhibited 42, 44, 58 and 60% identities with Contactin/F3, Tag-1, Big-1 and Big-2, respectively. Expression of NB-2 mRNA was restricted to cerebrum, cerebellum and was hardly detectable, if any, in spinal cord. On the other hand, high expression of NB-3 mRNA was observed in spinal cord, as well as in cerebrum and cerebellum. In the other tissues, no expression of NB-2 and NB-3 mRNAs was detected.