Mechanisms and principles of branch pattern formation of dendrites

Dendrite morphologies of CNS neurons are highly diverse, depending on cell type and function. The architecture of dendritic arbors critically affects the integration of neuronal inputs and propagation of chemical signals, and hence determines the connectivity of neurons. The question of how neurons acquire their appropriate morphology is a major issue in the study of neuronal development. In spite of the increasing number of molecular signals that have been identified as regulators of dendritic arborization patterns, the precise function of each molecule in the specific steps of branch dynamics largely remains elusive. Cerebellar Purkinje cells develop intricate dendritic arbors with minimal branch overlap. We developed a method of long-term time-lapse observation of dendritic branch dynamics in growing Purkinje cells in culture. Using a combinatorial approach with quantitative image analyses and computer-aided simulation, we identified the fundamental rules of growth dynamics that govern the construction of the characteristic dendritic patterns in Purkinje cells(Fujishima et al. 2012). We also demonstrated that a small change in actin dynamics led to a siginifant difference in dendrite growth dynamics and the untimate branch pattern in a mature neuron (Kawabata Galbraith et al., 2018).

Time-lapse observation and computer-assisted simulation of dendrite formation in cultured Purkinje cells

培養プルキンエ細胞の樹状突起形成ダイナミクスとシミュレーション

長期タイムラプス観察により樹状突起形成過程を解析し、必要最小限のパラメータを抽出して モデル細胞を再構築した。

We have successfully visualized the dynamic motility of organelles, including the nucleus, centrosomes, mitochondria and Golgi apparatus, in developing neurons. We recently found that developing neurons actively transport mitochondria into growing dendrites to fuel ATP energy necessary for arbor formation. We also found that dendrites sense local ATP levels and tune their growth rates by slowing actin turnover to avoid overconsumption of the ATP necessary for cellular metabolism.

Super-resolution images of mitochondria dynamics in cultured hippocampus neuron

培養した海馬ニューロンのミトコンドリア動態の超解像イメージ

培養した海馬ニューロンの細胞質(緑)とミトコンドリア(マゼンダ、白)の動態を超解像顕微鏡(Zeiss Airy scan)を用いて観察した。

Neuronal dendrites tend to extend radially within the brain to form perpendicular contacts with the afferent axon fibers, which run horizontally. Such organization has been shown to maximize the number of potential anatomical connections, yet the mechanism of how neurons orient dendrites perpendicular to afferent axons is unknown. Using electrospun carbon nanofibers as an artificial scaffold, we cultivated cerebellar neurons and reproduced the perpendicular contact observed between Purkinje cell dendrites and the aligned granule cell axons in culture dishes. Utilizing this system, we seek to identify the molecular and mechanical bases underlying axon-dendrite wiring topology.

樹状突起パターン形成のダイナミクスと原理の解明

中枢神経系ニューロン樹状突起の分岐パターンは、シナプス結合する入力線維の種類と数を決め、また膜電位と細胞内シグナルの拡散に影響してニューロンの情報処理特性を規定します。このためニューロンは機能に応じて個性的な分岐パターンを獲得します。同一細胞種で共通の樹状形態が獲得されることから、樹状突起形成のダイナミクスは細胞種に共通の原理が作動すると考えられます。しかし発達中の脳で数日から数週間かけて完成する樹状突起のパターン形成過程を長期間観察するのは技術的に困難で、ダイナミクス(伸長・分岐・退縮)の組合せにより細胞種毎に異なる分岐パターンが獲得される機構は明らかでありません。  我々は中枢神経系ニューロンの中でも際立って緻密な樹状突起を形成する小脳プルキンエ細胞を用い、分散培養下で樹状突起発達過程を1週間以上連続観察する系を確立し、樹状突起ダイナミクスの定量的解析と数理解析を用いてその形成原理を明らかにしました(Fujishima et al. 2012)。また、分化中の細胞骨格アクチン動態のわずかなズレが樹状突起ダイナミクスに影響し、成熟したニューロンの分岐の形を大きく変えてしまうことを証明しました(Kawabata Galbraith et al. 2018)。

References

Kawabata-Galbraith, K., Fujishima, K., Mizuno, H., Lee, S.J., Uemura, T., Sakimura, K., Mishina, M., Watanabe, N.  and Kengaku, M. (2018) MTSS1 regulation of actin-nucleating formin DAAM1 in dendritic filopodia determines final dendritic configuration of Purkinje cells. Cell Rep. 24(1):95-106.

Hatsukano, T., Kurisu, J., Fukumitsu, K., Fujishima, K. and Kengaku, M. (2017) Thyroid hormone induces PGC-1 α during dendritic outgrowth in mouse cerebellar Purkinje cells. Front Cell Neurosci. 11:133

Fukumitsu, K., Hatsukano, T., Yoshimura, A., Heuser, J., Fujishima, K. and Kengaku, M. (2016) Mitochondrial fission protein Drp1 regulates mitochondrial transport and dendritic arborization in cerebellar Purkinje cells. Mol Cell Neurosci.71:56-65

Fukumitsu, K., Fujishima, K., Yoshimura, A., Wu, Y.K., Heuser, J. and Kengaku, M. (2015) Synergistic action of dendritic mitochondria and creatine kinase maintains ATP homeostasis and actin dynamics in growing neuronal dendrites. J. Neurosci. 35(14):5707- 5723.

Wu, Y.K., Fujishima, K. and Kengaku, M. (2015) Differentiation of Apical and Basal Dendrites in Pyramidal Cells and Granule Cells in Dissociated Hippocampal Cultures. PLoS ONE 10(2) e0118482.

Fujishima, K., Horie, R., Mochizuki, A. and Kengaku M. (2012) Principles of branch dynamics governing shape characteristics of cerebellar Purkinje cell dendrites. Development 139: 3442-3455.

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