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E-mail: makosato@ anat2.med.osaka-u.ac.jp

We are interested in how the brain, especially the neocortex, develops and works. Currently, we are waiting for our new company to tackle on such interesting scientific and medical subjects together.


How does the brain work? Researchers have been struggling with this simple but profound question for a century. Because of its extreme complexity (approximate 86 billion neurons in the human brain!), it is crucial to sculpt essential neural circuits as well as to reveal their key operating principles. Since the architecture, including neural circuits, of the brain is very well organized, one way to address this question is to investigate the etabula rasaf brain and explore how the brain develops.

We are currently studying how the brain, especially the neocortex, develops at celluar and molecular levels.

Molecular mechanisms of the cortical development: differentiation and migration.

              We focus on the differentiation and migration stages of the development. Neurons are born in the ventricular zone (germinal zone, neuroepithelium) and migrate radially and/or horizontally then form the neocortex after migration. We have newly cloned several novel genes that are expressed in the neuroepithelium and elucidated functions of these molecules.

We cloned a novel gene (that we call 'FILIP') of which mRNAs are located in the ventricular zone. FILIP regulates cortical neurons out of the ventricular zone by inducing the degradation of actin-binding protein, filamin A (Nagano, T. et al., Nature Cell Biology, 4: 495-501, 2002). When to start migration is essential to form 6 layers, which are typical feature of the neocortex. FILIP is a key molecule controlling this process. Outline of our work is introduced in the highlight section of 'Nature Reviews Molecular Cell Biology, 3:472-473. 2002'. Later, we reported that an amount of filamin A is crucial for neuronal shape of radially migrating neurons (Nagano, T. et al., Journal of Neuroscience, 24(43):9648-9657, 2004. This paper was selected as a highlight paper). We are now studying FILIP knockout mice and have found that FILIP is involved in neuronal transmission. Concurrently, we also elucidated one fundamental mechanisms of directed cell migration (Takabayashi et al., Journal of Biological Chemistry, 285(21):16155-16165, 2010).

In addition to the works mentioned above, we have carried out a couple of collaborative works and have addressed the mechanisms underlying the cortical development. With Horiofs lab at Sapporo Medical University, we elucidated the significance of SIRT1 (Hisahara et al., Proc. Natl. Acad. Sci. (USA). 105(40):15599-15604, 2008, seleced as a cover for this issue and was introduced in the gcutting edgeh section of Cell) for neuronal differentiation. With Hirotsunefs lab at Osaka City University, we found that modulating calpain activity can be a potential therapeutic approach for lissencephaly due to Lis1 mutation (Yamada et al., Nature Medicine, 15(10): 1202-1207. 2009).

Connections, connections, connections: intracortical neural circuitsf formation.

              The neocortex is well-developed in mammals, especially in primates. It serves as the center for higher cerebral functions such as integration of multi-modal sensory inputs and regulation of voluntary motions. It consists of many functional areas like visual, auditory, motor, and association areas, each of which processes information of a certain modality or property. Inter-areal connections underlie those higher functions comparing and integrating information processed in each area. Moreover, areas are not inter-connected at random. Hierarchical organization of such subdivisions are crucial for exerting its higher cognitive functions. It has been revealed that disorganization of such hierarchy is involved in some psychiatric diseases and developmental disorders. However, how hierarchical organization is formed and works has remained fully open for a century.

(1) Developmental mechanisms of the inter-areal connections in the cerebral neocortex. We are analyzing the developmental mechanisms of these neural circuits using the mouse brain. We have already developed the way to manipulate some fundamental connections specifically. With our newly developed technologies, we are currently studying how such connections are formed and works.

(2) Mechanisms of subtype specific circuit formation of neocortical neurons. The neocortex is made up of many different types of neurons in terms of connections, e.g. inter-areal, local, inter-hemispheric, subcortical connections. These neurons are located in specific layers of the neocortex, often intermingling with each other. How these neurons with different connection patterns are fate-determined and project their axons properly according to their types during development are largely unknown. We are analyzing the mechanisms of neuronal subtype specification and respective circuit formation by comparing neurons with inter-areal connections and those with inter-hemispheric connections.

Molecular mechanisms of the cortical development: formation of neural circuits with subcortical structures.

              The neocortex controls many subcortical regions. Especially, what we call epyramidal tractf is one major subcortical projection, which is essential for exerting motor function. During the development of pyramidal tract, axon collaterals protrude form their main shaft toward the nuclei such as superior colliculi, pontine nuclei, and inferior olivary nuclei. However, molecular mechanisms by which axon collaterals are induced and projected to the specific regiond of the brain are mostly unknown.

              We are currently studying how the pyramidal tract is formed, especially focusing on how collaterals from the main pyramidal tract targets onto the pons, which is the most important relay structure between the cerebral cortex and the cerebellum. We developed the gene expression/ knockdown techniques in the pyramidal tract and/or pontine nuclei by the in utero electroporation method, primary culture of cortical neurons and organ culture of the brain tissues in the collagen gels. Using these techniques we now trying to to identify the molecules, which are involved in the regulation of collateral formation, and to disclose the signaling mechanisms that reorganize the cytoskeleton during the axon collateralization.

              Although we focus on this projection, underlying molecular mechanisms are so common that we can apply our knowledge to find the way to rebuild the repair brain as well as to understand/cure other neurological diseases due to malformation/malfunction of neural circuits.

              In addition to studies on neural network formation, we are also tackling with how neurotransmission works. Especially, we are studying on how dendritic mRNA is targeted to the specific synapse.

Targeting mechanism of dendritic mRNA

              A salient feature of neuronal cells is that they are segmented with spatially and functionally isolated microdomains, like myriads of synapses and growth cones, distant from the cell bodies that is the sole center for gene expression from the genome. Accordingly, such spatial sequestration must make differences in qualitative and quantitative requirements for proteins among the microdomains, depending on which microenvironments they face. Since the proteins were supposed to be synthesized in the cell body and recruited to each of the targeted microdomains by protein transporting system, it has been complicated to explain a mechanism that enables the delivery of the required proteins to the required domains in response to the external stimuli. However, the discovery of localized mRNA in dendrites and growth cones, in part, solved the problem by allowing on-demand and in situ protein synthesis from the mRNAs transported to the microdomains in advance. Especially, a group of resident mRNAs in the dendrites (dendritic mRNA), such as aCaMKII, b-actin, and some of glutamate receptors mRNA, were reported to be translated in the postsynaptic regions. Thus, the neighbouring translation to the regions where the products can work would overcome spatiotemporal complexities that accompany the one-by-one protein transport model. Among dendritic mRNAs, we focus on aCaMKII mRNA, of which the product is a key regulator of synaptic transmission and shape, and have identified cis-element regions for dendritic targeting and a list of proteins that bind to the 3f-untranslated region, including the cis-element. Further studies on the functions of the identified proteins for dendritic targeting are now proceeding.

Functional analysis of RNA-binding proteins (RBPs) and their modulation with arginine di-methylation.

              Some species of RBPs are di-methylated on specific arginine residues, though the biological function remains unknown. To explore the functional significance of this kind of modification, we knock down the responsible enzyme, protein arginine N-methyltransferase, from primary culture neurons and analyze the involvement of arginine methylation of RBPs in the dendritic targeting of mRNA.

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