RESEARCH THEMES IN OUR LABORATORY

How does the brain work? Researchers have been struggling with this simple but profound question for a century. Because of its extreme complexity (approximately 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 ‘tabula rasa’ brain and explore how the brain develops.
We are currently studying how the brain, especially the neocortex, develops at both cellular and molecular levels.

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Molecular mechanism of the cortical development: differentiation and migration.

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

We cloned a novel gene (which we call 'FILIP') the mRNAs of which are located in the ventricular zone. FILIP regulates the movement of cortical neurons out of the ventricular zone by inducing the degradation of an actin-binding protein, filamin A (Nagano, T. et al., Nature Cell Biology, 4: 495-501, 2002). When to start migration is essential to the formation of the 6 layers of the neocortex, which are a typical feature of this part of the brain. FILIP is a key molecule controlling this process. The outline of our work is introduced in the highlight section of 'Nature Reviews Molecular Cell Biology, 3:472-473. 2002'. Later, we reported that the amount of filamin A is crucial for the 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 (Yagi et al., Scientific Reports, 4, 6353; DOI:10.1038/srep06353). Concurrently, we also elucidated one of the fundamental mechanisms of directed cell migration and cortical development (Takabayashi et al., Journal of Biological Chemistry, 285(21):16155-16165, 2010; Xie et al., Cerebral Cortex, 23(6):1410-1423, 2013).

In addition to the works mentioned above, we have carried out a couple of collaborative works to address further the mechanisms underlying the cortical development. In conjunction with Horio’s lab at Sapporo Medical University, we elucidated the significance of SIRT1 (Hisahara et al., Proc. Natl. Acad. Sci. (USA). 105 (40):15599-15604, 2008, which was selected as the cover for this issue and was introduced in the “cutting edge” section of Cell) in neuronal differentiation. Working with Hirotsune’s 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: formation of intracortical neural circuits.

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 such as visual, auditory, motor, and association areas, each of which processes information of a certain modality or property. Inter-areal connections underlie these higher functions, comparing and integrating information processed in each area. Moreover, these areas are not interconnected at random. Hierarchical organization of such subdivisions are crucial for the neocortex to exert its higher cognitive functions. It has been revealed that the disorganization of such hierarchy is involved in certain psychiatric diseases and developmental disorders. However, how hierarchical organization is formed and works have 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 a way to manipulate some fundamental connections specifically. With our newly developed technologies, we are currently studying how such connections are formed and work.

(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, and 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 is a largely unanswerd question. We are analyzing the mechanisms of neuronal subtype specification and respective circuit formation by comparing neurons having inter-areal connections with those having inter-hemispheric ones.

Molecular mechanism of cortical development: formation of neural circiuts with subcortical structures.

The neocortex controls many subcortical regions. Especially, what we call the ‘pyramidal tract’ is one of the major subcortical projections and is essential for exerting motor function. During the development of the pyramidal tract, axon collaterals protrude from their main shaft toward nuclei such as the superior colliculi, pontine nuclei, and inferior olivary nuclei. However, the molecular mechanisms by which axon collaterals are induced and project to specific regions 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 target the pons, which is the most important relay structure between the cerebral cortex and the cerebellum. We have developed gene expression/ knockdown techniques including the in utero electroporation method, primary culture of cortical neurons, and organ culture of the brain tissues in collagen gels to examine the pyramidal tract and/or pontine nuclei. Using these techniques we are now trying to identify the molecules involved in the regulation of collateral formation and to disclose the signaling mechanisms that reorganize the cytoskeleton during this axon collateralization.
              Although we are focusing on this pyramidal tract projection, underlying molecular mechanisms are so common that we can apply our knowledge to find a way to rebuild or repair the brain as well as to understand/cure other neurological diseases caused by malformation/malfunction of neural circuits.

Autistic Brain

The incidence of developmental disorders has increased rapidly, and social demands to treat such disorders are growing. They affect close to, or more than, 1 % of all children worldwide. Concurrently, it has been recognized that there exists a wide variation in symptoms in developmental disorders such as autism. Such variations have led to the concept of autism spectrum disorder (ASD). Many responsible mutations in the genome for autism spectrum disorders have been reported, but how they result in similar behavioral patterns has remained an open question. Moreover, the development of curative medicines has not been successful. Recently we made an original mouse model for studying ASD. We are currently tackling this issue with our model

Targeting mechanism of dendritic mRNA

A salient feature of neuronal cells is that they are segmented with spatially and functionally isolated microdomains, such as myriads of synapses and growth cones, that are distant from the cell body, which 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 microenvironment they face. Since these proteins would supposedly be synthesized in the cell body and recruited into each of the targeted microdomains by a protein-transport system, it has become a complicated task to explain the mechanism that enables the delivery of the required proteins to the required domains in response to external stimuli. However, the discovery of localized mRNA molecules in dendrites and growth cones has solved this problem, in part; i.e., on-demand and in situ protein synthesis from these mRNAs transported to the microdomains in advance may allow such delivery. Especially, a group of resident mRNAs in the dendrites (dendritic mRNA), such as αCaMKII, β-actin, and the mRNAs of certain glutamate receptors, have been reported to be translated in the postsynaptic regions. Thus, the neighboring 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 are focusing on αCaMKII mRNA, the product of which 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 3’-untranslated region, including the cis-element. Further studies on the functions of these identified proteins for dendritic targeting are now in progress.

Dendritic distribution of αCaMKII mRNA in the hippocampal neurons (red).

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