Rumi Kurokawa

UNFOLDED PROTEIN RESPONSE AND AGGRESOME FORMATION IN HEREDITARY REDUCING-BODY MYOPATHY

Reducing‐body myopathy (RBM) is a rare myopathy characterized by the presence of unique sarcoplasmic inclusions called reducing bodies (RBs). We characterized the aggresomal features of RBs that contained γ–tubulin, ubiquitin, and endoplasmic reticulum (ER) chaperones, together with a set of membrane proteins, in a family with hereditary RBM. Increased messenger ribonucleic acid and protein levels of a molecular chaperone, glucose‐related protein 78, were also observed. These results suggest that the unfolded protein response caused by the accumulation of misfolded proteins in the endoplasmic reticulum plays an important role in the formation of RBs. Muscle Nerve, 2006

Characterization of lobulated fibers in limb girdle muscular dystrophy type 2A by gene expression profiling.

Limb girdle muscular dystrophy type 2A (LGMD2A) is caused by mutations in CAPN3, which encodes an intracellular cysteine protease. To elucidate the fundamental molecular changes that may be responsible for the pathological features of LGMD2A, we employed cDNA microarray analysis. We divided LGMD2A muscles into two groups according to specific pathological features: an early-stage group characterized by the presence of active necrosis and a regeneration process and a later-stage group characterized by the presence of lobulated fibers. After comparing the gene expression profiles of the two groups of LGMD2A muscles with control muscles, we identified 29 genes whose mRNA expression profiles were specifically altered in muscles with lobulated fibers. Interestingly, this group included genes that encode actin filament binding and regulatory proteins, such as gelsolin, PDZ and LIM domain 3 (PDLIM3) and troponin I1. Western blot analysis confirmed the upregulation of these proteins. From these results, we propose that abnormal increased expression of actin filament binding proteins may contribute to the changes of the intra-myofiber structures, observed in lobulated fibers in LGMD2A.

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.

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.

Micro-periodic functional organization in layer V composed of subcerebral projection neurons

Visual signal information reaches the primary visual cortex via axons of the dorsal lateral geniculate nucleus (dLGN) terminating chiefly in layer 4 and 6. Since these layers are targeted by different sources of dLGN in cat area 18, for example, C lamina of dLGN mainly innervates layer 4, intracortical projections can differ from each other and contribute to functional architectures in a different way. Firstly, we examined vesicular glutamate transporter 2 immunohistochemistry and revealed putative thalamic terminals were very dense in layer 4, and sparse in layer 6. Within individual layers, the terminals were found almost homogeneously. We reconstructed single cells (N = 46) and analyzed their bouton distribution on orientation and ocular dominance maps. Layer 4 cells are composed of spiny stellate and star pyramidal cells, whereas layer 6 cells can be divided into at least three morphological types. With respect to orientation preference, boutons of layer 6 cells were better tuned to iso-orientation than those of layer 4 cells, but less tuned than those of superficial layer cells. For ocular dominance, boutons of layer 6 cells were found more often in areas representing the same ocular dominance as the parent soma than other layer cells. Layer 4 cell boutons were composed of clusters revealed by the mean shift algorithm. We found individual clusters of layer 4 cell boutons were highly “tuned” into a given orientation. It suggests that each bouton cluster can be functionally independent. In summary, intracortical connections by layer 6 cells are more selective for both orientation and ocular dominance than those of layer 4 cells, nonetheless both two tiers receive thalamo-cortical inputs. It suggests that separate pathways can contribute visual signal integration in the cat primary visual cortex.

Micro cortical unit: A functional unit of neurons periodically repeated in neocortical layer V

Understanding how sensory representations are transformed at each brain region is a fundamental step towards obtaining the entire picture of sensory processing. To accomplish this, one would like to examine the relationship between stimulus evoked spike trains of input and output neurons in vivo. To further understand the mechanisms underlying this transformation, one would like to investigate the properties of synapses as well as the pattern of connectivity between populations of input and output neurons. Here we used the Drosophila antennal lobe, an analogue of the vertebrate olfactory bulb, as a model system to achieve these goals. We characterized the synapse between olfactory receptor neurons (ORNs) and projection neurons (PNs) and found that this synapse is unusually strong, reliable and exhibits prominent shortterm depression. These synaptic properties help explain how odor-evoked spikes are transformed in the antennal lobe in vivo. Further, we investigated the pattern of connectivity between ORNs and PNs. In flies, as in vertebrates, a group of ORNs expressing the same olfactory receptor converge to the same compartment called glomerulus. Whether each second order neuron receives input from all or a subset of ORNs was unknown in any species. Using dual recordings from PNs in the same glomerulus (homotypic PNs), we found that every PN listens to all the ORNs with in the glomerulus. Because ORNs function independently, pooling of ORN inputs will increase the signal to noise ratio of the total input to PNs. Thus, convergence of ORNs helps achieve the high sensitivity of PNs to odors. Moreover, this all-to-all connectivity produces correlated spikes between homotypic PNs, which may have implications on the downstream processing. In sum, these advances demonstrate that in vivo physiology is feasible in the Drosophila brain, and should permit a new integration of genetic, synaptic, cellular and systems approaches to investigating neural processing.

Periodic microcolumns in neocortical layer V

Visual motion can be sensed by low-level (energy-based) and/or high-level (featurebased) mechanisms. To understand the neural mechanism sensing visual motion in the MST area, single unit activities were recorded from two monkeys while they were exposed to motion of square wave gratings lacking the fundamental (“missing fundamental (mf) stimulus”). Motion consisted of successive 1/4 wavelength steps, so the overall pattern (feature) shifted in the direction of the steps, whereas the major Fourier component (the 3rd harmonic) shifted in the reverse direction because of spatial aliasing. The direction-selective MST neurons, whose preferred directions were determined by responses to a moving random-dot pattern, generally showed larger responses when the mf stimulus shifted in the opposite to their preferred directions than shifted in their preferred directions. The result suggests that the MST neurons are sensitive to the motion of the major Fourier component rather than the feature, consistent with the low-level, energy-based mechanism.