Symposium
Simple is Best. Study of neurological disorders and regeneration using invertebrate models
シンポジウム
シンプルイズベスト。無脊椎動物モデルを利用した神経疾患・再生研究 |
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| 7月25日（木）16:50～17:10 第2会場（朱鷺メッセ 2F メインホールA）
1S02e-1
分子モータータンパク質KIF1Aによるシナプス小胞前駆体の軸索輸送の亢進が神経疾患の原因となる
Shinsuke Niwa（丹羽 伸介）
東北大学際研
The microtubule kinesin motor unc-104/KIF1A is a prominent molecular motor utilized by neurons for the axonal transport of synaptic vesicle precursors (SVPs). The motor domain of kinesin superfamily proteins moves to the plus end of microtubules using the energy of ATP. Mutations in human KIF1A is a cause of a motor neuron disease called Hereditary spastic paraplegia (HSP). As HSP mutations identified so far are mainly in the motor domain of KIF1A, it has been considered that defects in motor activity and axonal transport of SVPs are the molecular mechanism of HSP. Here, we found three independent human KIF1A mutations hyperactivate the motility of KIF1A. Single molecule experiments using TIRF microscopy showed that the disease mutants of KIF1A have higher landing frequency on microtubule than wild-type KIF1A. One mutant showed elevated velocity, Disease models of C.elegans were generated by introducing corresponding point mutations to unc-104 gene by CRISPR/cas9. Previous genetic experiments in C. elegans showed that the axonal transport is tightly regulated by small GTPase ARL-8. In the absence of ARL-8, UNC-104/KIF1A is not properly activated and the axonal transport is significantly reduced. However, disease mutations of UNC-104 allowed the axonal transport of SVPs even in the absence of arl-8, suggesting that KIF1A is misregulated and hyperactivated in HSP neurons. Consistently, we found anterograde axonal transport of SVPs increased in frequency and synaptic vesicles mislaccumulated to the tip of axon in the model worms. Taken together, we suggest that overactivation of molecular motor KIF1A and anterograde axonal transport of synaptic vesicle precursors is a new cause of motor neuron disease. | | 7月25日（木）17:10～17:30 第2会場（朱鷺メッセ 2F メインホールA）
1S02e-2
線虫のテンシンはMet様シグナルを介して軸索再生を制御する
Naoki Hisamoto（久本 直毅），Kazuma Asai（浅井 一真），Tatsuhiro Shimizu（清水 達太），Yoshiki Sakai（酒井 芳樹），Strahil Iv Pastuhov（Pastuhov Iv Strahil），Hiroshi Hanafusa（花房 洋），Kunihiro Matsumoto（松本 邦弘）
名古屋大学大学院理学研究科生命理学専攻
Axon regeneration after nerve injury is a conserved biological process in many animals, including humans. The nematode Caenorhabditis elegans has recently emerged as a genetically tractable model for studying regenerative responses in neurons. It has recently been shown that the C. elegans JNK MAP kinase (MAPK) pathway, consisting of MLK-1 MAPKKK, MEK-1 MAPKK and KGB-1 JNK, plays a critical role in the initiation of axon regeneration. We have previously identified a number of genes affecting the JNK pathway using an RNAi-based screen. Analysis of these genes, called the svh genes, has shed light on the regulation of axon regeneration. We previously demonstrated that a signaling cascade consisting of an HGF-like growth factor SVH-1 and a Met-like receptor tyrosine kinase SVH-2 is required for the activation of the JNK MAPK pathway in axon regeneration. In addition, an integrin signaling pathway, which is activated by external phosphatidylserine, also induces the axon regeneration via activation of the JNK pathway. Here we describe our recent findings about the svh-6 gene, which encodes a homolog of the mammalian Tensin. Similar to mammalian Tensin, SVH-6 has an actin-binding domain, a SH2 domain and a PTB domain. Genetic analysis revealed that the SH2 and PTB domains, but not the actin-binding domain, are essential for the regulation of axon regeneration. Biochemical analysis indicates that SVH-6 interacts with tyrosine-autophosphorylated SVH-2 via the SH2 domain. Furthermore, SVH-6 interacts via its PTB domain with the integrin beta subunit PAT-3, suggesting that SVH-6 plays a positive role in axon regeneration by connecting the SVH-2 and integrin signaling pathways. | | 7月25日（木）17:30～17:50 第2会場（朱鷺メッセ 2F メインホールA）
1S02e-3
ショウジョウバエモデルを活⽤した効率的な神経変性疾患研究
Yoshitaka Nagai（永井 義隆）
⼤阪⼤院医神経難病認知症探索治療学
In the research on neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, the polyglutamine (polyQ) diseases, various genetic animal models have been developed, and they have significantly contributed to elucidation of their pathomechanisms and development of therapeutic candidates for these diseases. Among various animal models, mouse models have been most widely used, but enormous labor and time are required for their analysis. Therefore, development of simpler animal models suitable for rapid and efficient analyses have been anticipated. Drosophila melanogaster has been employed as a powerful organism for modeling human neurodegenerative diseases. Most striking advantages of Drosophila are their rapid generation cycle (10-14 days) and short life span (50-60 days), which enable us to perform rapid and efficient analyses. Approximately 70% of Drosophila genes have human homologous genes, and 75% of human disease genes have Drosophila homologues. Drosophila has been used as model organism for genetics for many years, and thus a huge number of genetic mutants and genetically-engineered strains has been established and is maintained in public stock centers. Furthermore, Drosophila can be easily maintained and analyzed in a small space at low cost, which enable us to efficiently perform genetic screening and drug screening in a short period. Taking advantages of these features, we employ Drosophila models of neurodegenerative diseases as high-throughput animal models, and have been working with them for more than 15 years. In this symposium, I will introduce our research on neurodegenerative diseases using Drosophila models together with nationwide collaborations with Drosophila models. | | 7月25日（木）17:50～18:10 第2会場（朱鷺メッセ 2F メインホールA）
1S02e-4
老化と神経変性疾患に関わるタンパク質の恒常性維持機構
Mari Suzuki（鈴木 マリ）
東京都医学総合研 糖尿病性神経障害プロジェクト
Aging is known to be a significant risk factor for neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and polyglutamine (polyQ) diseases. These diseases are thought to share a common molecular pathogenesis involving accumulation of misfolded proteins. Not only in these disease conditions, physiologically aged cells also accumulate damaged and misfolded proteins, suggesting a functional decline in their protein homeostasis (proteostasis) ability, which regulates biogenesis, folding, trafficking and degradation of proteins. Therefore, proteostasis is thought to be key processes linking neurodegenerative diseases and aging, but it remains unclear by which mechanisms the interventions that delays aging affect misfolded protein-induced neurodegeneration. Since dietary restriction (DR) delays age-associated changes and extends lifespan across multiple species, we explored whether DR improves misfolding protein-induced neurodegeneration and its molecular mechanisms by using Drosophila models. We found that the DR suppresses neurodegeneration through affecting an accumulation of the misfolded proteins, and suggest the role of metabolic and immune signaling in the effect of the DR. In addition to DR, several conserved longevity pathways have been identified: mild reduction of insulin/IGF-1 signaling, germline removal, reduced mitochondrial respiration, and reduced TOR signaling. Importantly, all of these interventions activate autophagy and extend animal lifespan in a manner that depends on the autophagic pathways, suggesting that autophagy is one of the convergent mechanisms of many longevity pathways. However, our knowledge of the molecular mechanism by which autophagic activity declines with age is still limited. We focused on one of few negative regulator of autophagy, Rubicon (Run domain Beclin-1 interacting and cysteine-rich containing protein), and found that the expression of Rubicon increased in aged animals and knockdown of Rubicon extends lifespan in flies and worms. Furthermore, knockdown of Rubicon exerts beneficial effects on polyQ-induced toxicity in the fly model. Our results suggest that suppression of autophagic activity by Rubicon is one of signatures of aging, and our study also provide basic insights into the possibility of Rubicon as a molecular target for treatment of misfolded protein-associated neurodegenerative diseases. | | 7月25日（木）18:10～18:30 第2会場（朱鷺メッセ 2F メインホールA）
1S02e-5
ショウジョウバエ脳における細胞内小器官および微細構造の三次元電子顕微鏡法による可視化
Kazunori Shinomiya（四宮 和範），Patricia K Rivlin（Rivlin K Patricia），Stephen M Plaza（Plaza M Stephen）
HHMI Janelia Research Campus, Ashburn, VA, USA
Studying properties of the wild-type tissue is the first step of disease researches. Even a slight change of structure or distribution of organelles could alter the functions of a normal cell and cause neurological disorders. We have been using the brain of the fruit fly, Drosophila melanogaster, as a model system to identify neuronal circuits and visualize organelles of the wild-type animal. We use a three-dimensional electron microscopy (3D-EM) method called the focused ion beam-aided scanning EM (FIB-SEM) to image the brain. FIB-SEM combines a scanning EM and a focused gallium ion beam instead of a diamond knife to mill the surface of the sample block, producing 3D images with an isotropic resolution of 4 to 8 nanometers per voxel. We imaged multiple brain regions in a single dataset so that neurons in different areas can be compared under the same condition. We visualized organelles and ultrastructures such as pre- and postsynaptic motifs, mitochondria, endoplasmic reticulum, and cytoplasm in different parts of the brain, and categorized them by subtypes. We revealed that the appearance of some ultrastructure differs significantly in different types of neurons. For example, regular neurons have T-shaped presynaptic densities (thus commonly known as the "T-bars") with a pedestal and a platform, while some neuron types turned out to have elongated presynaptic motifs which lack the platform entirely. We also have identified that there are variations in size, contrast, and distribution of mitochondria and ribosomes in synaptic terminals, potentially reflecting the activity level of the neurons. Using automated motif detection algorithms, we could identify synaptic sites and mitochondria efficiently in large volumetric datasets. The method we have developed can be further applied to mutant analysis, and possibly to diagnosis of diseases caused by alteration of ultrastructures as well. | | 7月25日（木）18:30～18:50 第2会場（朱鷺メッセ 2F メインホールA）
1S02e-6
ショウジョウバエモデルを利用した、細胞間コミュニケーション破綻による進行性神経変性の機序解明
Atsushi Sugie（杉江 淳）1，Melisande Richard（Richard Melisande）2，Yohei Nitta（新田 陽平）1，Gaia Tavosanis（Tavosanis Gaia）2，Takashi Suzuki（鈴木 崇之）3
1新潟大学研究推進機構超域学術院
2German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany
3東工大院生命理工
Individual neurons grow and migrate during development, locating their appropriate target and forming functional neural circuits. Although they cannot be regenerated, the neurons in a circuit could be maintained for a lifetime, implying that specific mechanisms exist to maintain the long-term health and integrity of the nervous system. This is in contrast to somatic cells, which are constantly replaced. If the inter-neuronal communication mechanism for maintaining cell health gets disrupted, it could cause irreversible functional deterioration of neural circuits, leading to aging, or neurodegenerative diseases and mental disorders. However, the molecular mechanism underlying the intercellular communication keeping neuronal circuits healthy is still barely understood.
One way to address this question would be to induce a degeneration in a specific neural circuit and to then quantify the strength of synaptic connection and the level of neurodegeneration in specific cells in the circuit. This study, using the Drosophila visual system, reports a new, innovative experimental model capable of examining these factors. we investigated how specific pre- and postsynapses interact during degeneration and we identified that the neuronal activity and the divergent canonical WNT pathway are responsible for intercellular communication to keep neuron healthy.
Results of this study indicate the existence of a new intercellular communication mechanism responsible for maintaining the long-term health of neural circuits. The novel intercellular communication mechanism described also exhibits potential as an inherent protective mechanism in neurodegeneration which, in the future, could be expected to underpin the development of neuroprotective therapies using endogenous factors. | |
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