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 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, seleced as a cover for this
issue and was introduced in the “cutting edge” section of Cell) for neuronal
differentiation. 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: intracortical neural circuits’ 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 ‘pyramidal tract’ 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 3’-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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