Kensuke Hayashi

Identification of autotaxin as a neurite retraction-inducing factor of PC12 cells in cerebrospinal fluid and its possible sources

Cerebrospinal fluid (CSF) induced neurite retraction of differentiated PC12 cells; the action was observed in 15 min (a rapid response) and the activity further increased until 6 h (a long‐acting response) during exposure of CSF to the cells. The CSF action was sensitive to monoglyceride lipase and diminished by homologous desensitization with lysophosphatidic acid (LPA) and by pretreatment with an LPA receptor antagonist Ki16425. Although fresh CSF contains LPA to some extent, the LPA content in the medium was increased during culture of PC12 cells with CSF. The rapid response was mimicked by exogenous LPA, and a long‐acting response was duplicated by a recombinant autotaxin, lysophospholipase D (lyso‐PLD). Although the lyso‐PLD substrate lysophosphatidylcholine (LPC) was not detected in CSF, lyso‐PLD activity and an ∼120‐kDa autotaxin protein were detected in CSF. On the other hand, LPC but not lyso‐PLD activity was detected in the conditioned medium of a PC12 cell culture without CSF. Among neural cells examined, leptomeningeal cells expressed the highest lyso‐PLD activity and autotaxin protein. These results suggest that leptomeningeal cells may work as one of the sources for autotaxin, which may play a critical role in LPA production and thereby regulate axonal and neurite morphological change.

Relocalization of a microtubule-anchoring protein, ninein, from the centrosome to dendrites during differentiation of mouse neurons.

Microtubules in typical cells form radial arrays with their plus-ends pointing toward the cell periphery. In contrast, microtubules in dendrites of neurons are free from centrosomes and have a unique arrangement in which about half have a polarity with a minus-end distal orientation. Mechanisms for generation and maintenance of the microtubule arrangement in dendrites are not well understood. Here, we examined dendritic localization of a centrosomal protein, ninein, which has microtubule-anchoring and stabilizing functions. Immunohistochemical analysis of developing mouse cerebral and cerebellar cortices showed that ninein is localized at the centrosome in undifferentiated neural precursors. In contrast, ninein was barely detected in migrating neurons, such as those in the intermediate layer of the cerebral cortex and the internal granular layer of the cerebellar cortex. High expression was observed in thick dendrite-bearing neurons such as pyramidal neurons of the cerebral cortex and Purkinje neurons in the cerebellar cortex. Ninein was not detected at the centrosome of these cells, but was diffusely present in cell soma and dendrites. In cultured cortical neurons, ninein formed granular structures in soma and dendrites, being not associated with γ-tubulin. About 60% of these structures showed resistance to detergent and association with microtubules. Our observations suggest that the minus-ends of microtubules may be anchored and stabilized by centrosomal proteins localized in dendrites.

Visualization of plastid movement in the pollen tube of Arabidopsis thaliana.

Organelle dynamics in the plant male gametophyte has received attention for its importance in pollen tube growth and cytoplasmic inheritance. We recently revealed the dynamic behaviors of plastids in living Arabidopsis pollen grains and tubes, using an inherent promoter-driven FtsZ1–green fluorescent protein (GFP) fusion. Here, we further monitored the movement of pollen tube plastids with an actin1 promoter-driven, stroma-targeted yellow fluorescent protein (YFP). In elongating pollen tubes, most plastids localized to the tube shank, where they displayed either retarded and unsteady motion, or fast, directional, and long-distance movement along the tube polarity. Efficient plastid tracking further revealed a population of tip-forwarding plastids that undergo a fluctuating motion(s) before traveling backward. The behavior of YFP-labeled plastids in pollen basically resembled that of FtsZ1–GFP-labeled plastids, thus validating the use of FtsZ1–GFP for simultaneous visualization of the stroma and the plastid-dividing FtsZ ring.

Loss of γ-tubulin, GCP-WD/NEDD1 and CDK5RAP2 from the Centrosome of Neurons in Developing Mouse Cerebral and Cerebellar Cortex

It has been recently reported that the centrosome of neurons does not have microtubule nucleating activity. Microtubule nucleation requires γ-tubulin as well as its recruiting proteins, GCP-WD/NEDD1 and CDK5RAP2 that anchor γ-tubulin to the centrosome. Change in the localization of these proteins during in vivo development of brain, however, has not been well examined. In this study we investigate the localization of γ-tubulin, GCP-WD and CDK5RAP2 in developing cerebral and cerebellar cortex with immunofluorescence. We found that γ-tubulin and its recruiting proteins were localized at centrosomes of immature neurons, while they were lost at centrosomes in mature neurons. This indicated that the loss of microtubule nucleating activity at the centrosome of neurons is due to the loss of γ-tubulin-recruiting proteins from the centrosome. RT-PCR analysis revealed that these proteins are still expressed after birth, suggesting that they have a role in microtubule generation in cell body and dendrites of mature neurons. Microtubule regrowth experiments on cultured mature neurons showed that microtubules are nucleated not at the centrosome but within dendrites. These data indicated the translocation of microtubule-organizing activity from the centrosome to dendrites during maturation of neurons, which would explain the mixed polarity of microtubules in dendrites.

Changes in the axo-glial junctions of the optic nerves of cuprizone-treated mice

Demyelination induced by cuprizone in mice has served a useful model system for the study of demyelinating diseases, such as multiple sclerosis. Severity of demyelination by cuprizone, however, varies across different regions of the central nervous system; the corpus callosum is sensitive, while the optic nerves are resistant. Here, we investigated the effects of cuprizone on optic nerves, focusing on the axo-glial junctions. Immunostaining for sodium channels, contactin-associated protein, neurofascins, and potassium channels revealed that there were no massive changes in the density and morphology of the axo-glial junctions in cuprizone-treated optic nerves. However, when we counted the number of incomplete junctional complexes, we observed increased numbers of isolated paranodes. These isolated paranodes were immunopositive for both axonal and glial membrane proteins, indicating that they were the contact sites between axons and glia. These were not associated with sodium channels or potassium channels, suggesting the absence of physiological functions. When teased axons from cuprizone-treated optic nerves were immunostained, the isolated paranodes were found at the internode region of the myelin. From these observations, we conclude that cuprizone induces new contacts between axons and myelins at the internode region.

Cell Shape Change by Drebrin

Drebrin is localized in actin-rich regions of neuronal and non-neuronal cells. In mature neurons, its localization is strictly restricted to the postsynaptic sites. In order to understand the function of drebrin in cells, many studies have been performed to examine the effect of overexpression or knocking down of drebrin in various cell types, including neurons, myoblasts, kidney cells, and intestinal epithelial cells. In most cases alteration of cell shape and impairment or facilitation of actin-based activities of these cells were observed. Interestingly, overexpression of drebrin in matured neurons results in the alteration in dendritic spine morphology. Further studies have shown alteration in the localization of postsynaptic receptors and even changes in synaptic transmission caused by drebrin overexpression or depletion in neurons. These drebrins effects are thought to come from drebrins actin-cross-linking activity or competitive binding to actin against tropomyosin, fascin, and α-actinin. Furthermore, drebrin binds to various molecules, such as homer, EB3, and cell-cell junctional proteins, indicating that drebrin is a multifunctional cytoskeletal regulator.

Involvement of GSK3 in the formation of the leading process and migration of neurons from the embryonic rat medial ganglionic eminence in vitro.

Migrating neurons have leading processes that direct cell movement in response to guidance cues. We investigated the involvement of glycogen synthase kinase 3 (GSK3) in the formation of leading processes and migration of neurons in vitro. We used embryonic rat medial ganglionic eminence (MGE) neurons, which are precursors of inhibitory neurons that migrate into the cerebral cortex. When MGE neurons were placed on an astrocyte layer, they migrated freely with the highest speed among neurons from other parts of the embryonic forebrain. When they were cultured alone, they showed bipolar morphology and extended leading processes within 20 h. Their leading processes had large growth cones, but did not elongate during 3 days in culture, indicating that leading processes are distinct from short axons. Next, we examined the effect of GSK3 inhibitors on leading processes and the migratory behavior of MGE neurons. MGE neurons treated with GSK3 inhibitors showed multipolar morphology and altered process shapes. Moreover, migration of MGE neurons on the astrocyte layer was significantly decreased in the presence of GSK3 inhibitors. These data suggest that GSK3 is involved in the formation of leading processes and in the migration of MGE neurons.

Rear-Side Localization of the Centrosome in Migrating Neuroblastoma Neuro-2a Cells and Its Roles in Process Elongation

Axon elongation is usually performed by the migration of growth cones that leave axons. Axon microtubules are generated by enhanced polymerization of tubulin in the growth cones. Some kinds of neurons like cerebellar granule cells, however, generate axons as a result of migration of the cell body leaving axons at the rear. The mechanism to generate microtubules during such growth cone-independent elongation of axons is not well understood. To establish an experimental model to study this mechanism, we cultured neuroblastoma (Neuro-2a) cells on substrates that facilitate cell migration. When cultured on laminin-treated substrate, cells migrated actively and left processes at the rear. We investigated the role of the centrosome in this process formation. The centrosomes were always located at the base of the processes, i.e., at the rear side of the migrating cell body. Close observation of cytoskeletons revealed microtubules limited around the centrosomes, but concentrated at the periphery of the cells or within the processes. Microtubule regrowth experiments showed the ability of the centrosomes to nucleate microtubules. We thus examined the role of microtubule release from the centrosomes, by knocking down the expression of spastin, a microtubule-severing enzyme. Introducing siRNA for spastin into Neuro-2a cells reduced both the migration speed and the length of the processes. Taken together, Neuro-2a cells on laminin proved useful as a model to study the alternative type of axon elongation in which cell migration leaves axons at the rear. This model provided evidence for the involvement of microtubule release from centrosomes in the mechanisms for this type of process elongation.

Involvement of GSK3 in the formation of leading processes of migratory neurons

Asymmetric elevation of cytosolic Ca2+ concentration across the axonal growth cone, which is triggered upon the reception of extracellular guidance cues, can mediate both attractive and repulsive growth cone turning. Here we show that clathrin-mediated endocytosis acts downstream of Ca2+ signals as driving machinery for growth cone turning. In dorsal root ganglion neurons, the formation of clathrin-coated pits is facilitated asymmetrically across the growth cone by directionally applied chemorepellent, semaphorin 3A, or by Ca2+ signals that mediate repulsive guidance. In contrast, coated pit formation remains symmetric in the presence of attractive Ca2+ signals. Inhibition of clathrin-mediated endocytosis abolishes growth cone repulsion, but not attraction, induced by Ca2+ or extracellular physiological cues. Furthermore, asymmetric perturbation of the balance of endocytosis and exocytosis in the growth cone is sufficient to initiate its turning toward the side with less endocytosis or more exocytosis. With our previous finding that growth cone attraction involves asymmetric exocytosis (Tojima et al., Nat. Neurosci., 10: 58-66. 2007), we propose that the balance between membrane addition and removal dictates bidirectional axon guidance.