Hisato Maruoka

NMDA receptor-dependent synaptic translocation of insulin receptor substrate p53 via protein kinase C signaling.

The activity-dependent remodeling of postsynaptic structure is a fundamental process underlying learning and memory. Insulin receptor substrate p53 (IRSp53), a key player in cytoskeletal dynamics, is enriched in the postsynaptic density (PSD) fraction, but its significance in synaptic functions remains unclear. We report here that IRSp53 is accumulated rapidly at the postsynaptic sites of cultured hippocampal neurons after glutamate or NMDA stimulation in an actin cytoskeleton-dependent manner. Pharmacological profiles showed that a PKC inhibitor, but not other kinase inhibitors, specifically suppressed the synaptic translocation of IRSp53 in response to NMDA, and the selective activation of PKC with phorbol ester markedly induced the synaptic translocation. Reverse transcriptase-PCR and Western blotting showed that IRSp53-S is the major isoform expressed in cultured hippocampal neurons. The synaptic targeting of IRSp53-S was found to be mediated through N-terminal coiled-coil domain and the PDZ (PSD-95/Discs large/zona occludens-1)-binding sequence at its C-terminal end and regulated by the PKC phosphorylation of its N terminus. In electrophysiological experiments, overexpression of IRSp53-S wild type and IRSp53-S mutant that is spontaneously accumulated at the postsynaptic sites enhanced the postsynaptic function as detected by an increased miniature EPSC amplitude. These data suggest that IRSp53 is involved in NMDA receptor-linked synaptic plasticity via PKC signaling.

MALS is a binding partner of IRSp53 at cell–cell contacts

Insulin receptor substrate p53 (IRSp53) is a key player in cytoskeletal dynamics, interacting with the actin modulators WAVE2 and Mena. Here, we identified a PDZ protein, MALS, as an IRSp53‐interacting protein using a yeast two‐hybrid screen. A pull‐down assay showed that IRSp53 and MALS interact through the PDZ domain of MALS and the C‐terminal PDZ‐binding sequence of IRSp53. Their interaction in MDCK cells was also demonstrated by co‐immunoprecipitation. Immunocytochemistry showed the colocalization of IRSp53 and MALS at cell–cell contacts. Cytochalasin D induced the redistribution of both proteins to the cytosol. Thus, MALS is a partner of IRSp53 anchoring the actin‐based membrane cytoskeleton at cell–cell contacts.

The postsynaptic density and dendritic raft localization of PSD-Zip70, which contains an N-myristoylation sequence and leucine-zipper motifs

The postsynaptic site of the excitatory synapse, which is composed of the postsynaptic density (PSD) attached to the postsynaptic membrane, is a center for synaptic plasticity. To reveal the molecular organization and functional regulation of the postsynaptic site, we cloned a 70 kDa protein that is concentrated in PSDs using a monoclonal antibody against the PSD. This protein, named PSD-Zip70, is highly homologous to the human FEZ1/LZTS1 gene product. PSD-Zip70 contains an N-myristoylation consensus sequence, a polybasic cluster in the N-terminal region and four leucine-zipper motifs in the C-terminal region. Light and electron microscopy showed that this protein was localized to the dendritic spines, especially in the PSD and the postsynaptic membrane. Fractionation of the synaptic plasma membrane demonstrated that PSD-Zip70 was localized to the PSD and the dendritic raft. In Madin-Darby canine kidney (MDCK) cells, exogenous PSD-Zip70 was targeted to the apical plasma membrane of microvilli, and its N-myristoylation was necessary for this targeting. In hippocampal neurons, N-myristoylation was also required for the membrane localization and the C-terminal region was critically involved in the synaptic targeting. These results suggest that PSD-Zip70 may be involved in the dynamic properties of the structure and function of the postsynaptic site.

Collaboration of PSD-Zip70 with Its Binding Partner, SPAR, in Dendritic Spine Maturity

Recent studies have reported on the molecular mechanisms underlying dendritic spine (spine) dynamics. Because most of these studies investigated spine dynamics by overexpressing constitutively active or dominant-negative PSD (postsynaptic density) proteins in cultured mature neurons, the results represent the enlargement of mature spines or their return to an immature state. Here, we developed the technique of in utero electroporation to investigate spine dynamics. Using this technique, we demonstrated the suppression of spine maturation by the C-terminal variants of PSD-Zip70 in vitro and in vivo. Transient overexpression of the C terminus of PSD-Zip70 and knock-down of PSD-Zip70 also displayed the destabilization of mature spines. We further found the PSD-Zip70 and SPAR (spine-associated RapGAP) interaction via the short C-terminal region of PSD-Zip70 and the GK-binding domain of SPAR. In association with immature spines induced by overexpression of the PSD-Zip70 C terminus or knock-down of PSD-Zip70, SPAR lost its spine localization. Overexpression of the GK-binding domain of SPAR also induced to form immature spines without affecting the localization of PSD-Zip70 in the small heads of filopodial spines. Our results suggest that PSD-Zip70 in collaboration with SPAR is critically involved in spine maturity, especially in the mature spine formation and the maintenance of spine maturity.

Periodic Organization of a Major Subtype of Pyramidal Neurons in Neocortical Layer V

A major question in neocortical research is the extent to which neuronal organization is stereotyped. Previous studies have revealed functional clustering and neuronal interactions among cortical neurons located within tens of micrometers in the tangential orientation (orientation parallel to the pial surface). In the tangential orientation at this scale, however, it is unknown whether the distribution of neuronal subtypes is random or has any stereotypy. We found that the tangential arrangement of subcerebral projection neurons, which are a major pyramidal neuron subtype in mouse layer V, was not random but significantly periodic. This periodicity, which was observed in multiple cortical areas, had a typical wavelength of 30 μm. Under specific visual stimulation, neurons in single repeating units exhibited strongly correlated c-Fos expression. Therefore, subcerebral projection neurons have a periodic arrangement, and neuronal activity leading to c-Fos expression is similar among neurons in the same repeating units. These results suggest that the neocortex has a periodic functional micro-organization composed of a major neuronal subtype in layer V.

Lattice system of functionally distinct cell types in the neocortex

The basic modules of the neocortex The fundamental organization of excitatory and inhibitory neurons in the neocortex is still poorly understood. Subcerebral projection neurons, a major excitatory cell type in neocortical layer 5, form small cell clusters called microcolumns. Maruoka et al. examined large regions of mouse brain layer 5 and observed that thousands of these microcolumns make up a hexagonal lattice with a regular gridlike spacing. The other major layer 5 excitatory cell class, cortical projection neurons, also form microcolumns that interdigitate with those of the subcerebral projection neurons. Microcolumns received common presynaptic inputs and showed synchronized activity in many cortical areas. These microcolumns developed from nonsister neurons coupled by cell type–specific gap junctions, suggesting that their development is lineage-independent but guided by local electrical transmission. Science, this issue p. 610 Neocortical layer 5 is composed of microcolumns containing specific cell types that form a brainwide hexagonal lattice system. The mammalian neocortex contains many cell types, but whether they organize into repeated structures has been unclear. We discovered that major cell types in neocortical layer 5 form a lattice structure in many brain areas. Large-scale three-dimensional imaging revealed that distinct types of excitatory and inhibitory neurons form cell type–specific radial clusters termed microcolumns. Thousands of microcolumns, in turn, are patterned into a hexagonal mosaic tessellating diverse regions of the neocortex. Microcolumn neurons demonstrate synchronized in vivo activity and visual responses with similar orientation preference and ocular dominance. In early postnatal development, microcolumns are coupled by cell type–specific gap junctions and later serve as hubs for convergent synaptic inputs. Thus, layer 5 neurons organize into a brainwide modular system, providing a template for cortical processing.

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Single-cell level multi-layered substructures of neocortical layer V

ber of extracellular axon guidance factors, their receptors and intracellular signaling molecules involved in controlling axon morphology, the signaling pathways in axon development remain to be elucidated. We have previously reported that R-Ras, a member of Ras family GTPases, plays an important role in axon specification and guidance. However, direct effectors of R-Ras in neurons have not been identified except PI3K. In this study, we report that lAfadin, an actin filament-binding protein having Ras association (RA) domain and PDZ domain, functions as an effector of R-Ras and regulates axon branching downstream of R-Ras in cultured hippocampal neurons. Pull-down and immunoprecipitaion assays showed that l-Afadin bound to active R-Ras. In Neuro-2a cells, constitutively active R-Ras recruited l-Afadin to the plasma membrane in a RA domain-dependent manner. In cultured neurons, overexpression of l-Afadin promoted axon branching, while knockdown of l-Afadin suppressed the branching activity. Overexpression of constitutively active R-Ras increased axon arborization and it was partially repressed by knockdown of l-Afadin. These results suggest that activated R-Ras induces axon branching in part by recruiting l-Afadin to the plasma membrane.

Precise three-dimensional functional micro-organization in neocortical layer V

Zic2 is a causal gene of holoprosencephaly (a dysgenesis of medial forebrain). Although it is broadly expressed in CNS, there was a difficulty to fully show its role due to its critical role in early embryogenesis. Here we developed a conditionally targeted Zic2 mutant mice and clarified its role in the development of dorsal cochlear nucleus (DCoN) and in the auditory function of mature mice. Soon after the neural tube closure, Zic2 and its close relatives (Zic1 and Zic3) are differentially expressed in hindbrain region along the rostrocaudal axis. Zic2 expression was dominant in the DCoN forming region. In Zic2 hypomorphic mutants (60% of wild type level), slight reduction of DCoN size was observed whereas the DCoN size reduction was severe in the midbrainhindbrain restricted Zic2 conditional knockout (CKO). Both granule cells and unipolar brush cells were decreased in DCoN. We observed the increased acoustic startle response and the altered auditory brain stem responses in both Zic2 hypomorphic and Zic2 CKO animals. Furthermore, we measured the activities of the primary auditory cortices during various sound stimuli application by means of autofluorescence imaging. These results indicated that optimal Zic2 gene dosage is a critical parameter for the auditory neural circuit formation and the auditory function. Further analyses using the Zic2 mutants would be beneficial for understanding physiological regulation of auditory information processing in mammalian. Research fund: RIKEN BSI funds.

Analysis of the formation of single-cell level micro-organization in neocortical layer V

We have previously found that layer V projection neurons that have different axonal projection or gene expression make precise three-dimensional organization in the radial orientation, projection neurons form thin sublayers with single-cell precision, and multiple sublayers are stacked in layer V. As a first step to investigate mechanisms of the multi-layer formation, we analyzed correlations between axonal projections and gene expression. The result showed that the sublayers defined by gene expression precisely correlate with the sublayers defined by axonal projection targets. Our analysis suggest that layer V is precisely subdivided into multiple sublayers of functionally different neuronal types and that microcircuits involving layer V projection neurons may be strictly specified by molecular mechanisms. Analysis of over-expression experiments will be discussed.