シンポジウム（Symposium）

Symposium Failure of organelle communications as a key pathomechanism for neurological disorders シンポジウムオルガネラ間の連携破綻からみた神経疾患の発症メカニズム Sponsored by IBRO-APRC Lecturer Exchange Program

7月28日（日）10:50～11:10　第2会場（朱鷺メッセ　2F　メインホールA） 4S02a-1 運動神経変性におけるオルガネラ連携の破綻 Koji Yamanaka（山中宏二）名古屋大環境医学研病態神経 The mitochondria-associated membrane (MAM), contacting site of endoplasmic reticulum (ER) and mitochondria, regulates various functions including calcium (Ca²⁺) transfer from ER to mitochondria. Sigma 1 receptor (Sig1R), a gene product of SIGMAR1, is a chaperone and orphan receptor specifically localized in the MAM. Recessive mutations for SIGMAR1 gene were causative for juvenile amyotrophic lateral sclerosis (ALS), ALS16. On the other hand, mutant SOD1 (Cu/Zn superoxide dismutase) protein, a gene product of ALS causative gene, SOD1, is also known to accumulate in mitochondria and ER. However, the role of the MAM in the pathomechanism for ALS remains elusive. We identified a novel recessive mutation c.283dupC/p.L95fs in SIGMAR1 gene, which is causative for juvenile inherited ALS (ALS16). Mutant Sig1R proteins were unstable, and unable to regulate the intracellular Ca²⁺ flux. The loss of Sig1R accelerated the disease onset of mutant SOD1 mice. Moreover, collapse of the MAM structure was observed both in the motor neurons of Sig1R-deficient mice and mutant SOD1 mice. To extend our findings, we recently established the cultured cells which enable to measure the MAM integrity through luciferase and GFP. By using this system, we found that collapse of the MAM was observed in the cells expressing various ALS-causative genes. Collectively, our findings indicate that collapse of the MAM is a common pathomechanism for ALS, not limited to Sig1R- and SOD1-linked ALS. Furthermore, our result of the selective enrichment of IP₃R3 in motor neurons suggests that integrity of the MAM is crucial for the selective vulnerability in ALS.		7月28日（日）11:10～11:30　第2会場（朱鷺メッセ　2F　メインホールA） 4S02a-2 ミトコンドリア-リソソーム連携の破綻とパーキンソン病 Noriyuki Matsuda（松田憲之）都医学研 Parkinson's disease (PD) is a common movement disorder characterized by dopaminergic neuronal loss. The majority of PD cases are sporadic, however, the discovery of the genes linked to hereditary forms of PD (i.e. hereditary Parkinsonism) has provided important insights into the molecular mechanisms. For example, functional analysis of the recessive familial PD-related genes has revealed that the disease is relevant to mitochondrial quality control. This is consistent with the prior idea that several cases of sporadic PDs and chemical-induced Parkinsonism have been associated with mitochondrial dysfunction. We have focused on PINK1 and PARKIN, responsible genes for hereditary recessive PD. PINK1 and PARKIN encode Ser/Thr kinase and ubiquitin ligase (E3), respectively. We revealed that when the mitochondrial membrane potential decreased, PINK1 phosphorylates ubiquitin at Ser65, and the phosphorylated ubiquitin functions as an activator for E3 function of Parkin (Koyano et al., Nature 2014). We also found that PINK1 phosphorylates ubiquitin-like (Ubl) domain of Parkin at Ser65, and this phosphorylation is important for E3 reaction (ubiquitin-ester transfer) of Parkin (Iguchi et al., JBC 2013). Moreover, phosphorylated poly-ubiquitin chain catalyzed by PINK1 recruits Parkin to damaged mitochondria by functioning as a Parkin receptor (Okatsu et al., JCB 2015). Consequently, trio of PINK1, Parkin, and phospho-ubiquitin rapidly tag outer membrane proteins on depolarized mitochondria with ubiquitin. Several papers reported that ubiquitin chains on depolarized mitochondria are recognized by autophagy adaptors such as OPTN and NDP52. We revealed that the ubiquitin chain is also recognized by RABGEF1, and it directs the downstream Rab proteins, RAB5 and RAB7A, to damaged mitochondria for degradation by lysosome (Yamano, eLife 2018). Impairment of this process predisposes to familial PD. Summary of the latest knowledge for relationship between mitochondrial quality control, lysosome, and Parkinson's disease will be discussed.

7月28日（日）11:30～11:50　第2会場（朱鷺メッセ　2F　メインホールA） 4S02a-3 メンブレントラフィックが切り開くパーキンソン病の新展開 Takafumi Hasegawa（長谷川隆文）東北大学大学院医学系研究科神経内科学分野 Membrane trafficking is a type of cellular logistics by which proteins and other macromolecules are able to reach their appropriate destinations without crossing a membrane, thereby plays crucial roles in not only in maintaining cellular homeostasis but also in fulfilling specific demands during development, differentiation, signal perception and transduction. Intriguingly, defects in membrane trafficking are hallmarks in a variety of neurodegenerative diseases. Such defects result in the accumulation of misfolded proteins due to aberrant endosomal sorting, lysosomal degradation, or autophagy. The genetic as well environmental causes of a specific disease may directly or indirectly affect these membrane traffic machinery. Likewise, changes in intracellular sorting and degradation profoundly influence on the cellular trafficking of misfolded proteins, thereby facilitating the cell-to-cell spreading of proteinaceous aggregates. Here, I will review our current knowledge about the functional roles of membrane trafficking in neurodegenerating process. In particular, I will provide several examples of how alterations of membrane trafficking contribute to the pathogenesis of neurodegenerative disorders such as Parkinson's disease. Defining the precise mode of intercellular trafficking of toxic protein aggregates will shed light on the pathogenic mechanisms involved, and will open up new possibilities for novel therapeutic interventions of devastating neurodegenerative diseases. [Selected publications] 1. Kobayashi J, Hasegawa T, et al, FASEB J, in press. 2. Hasegawa T, Yoshida S, et al, Front Neurosci 2018 3. Yoshida S, Hasegawa T, et al., Hum Mol Genet 2018 4. Hasegawa T, Sugeno N, et al, Tohoku J Exp Med 2017 5. Oshima R, Hasegawa T, et al., Sci Rep 2016 6. Miura E, Hasegawa T, et al., Neurobiol Dis 2014 7. Sugeno N, Hasegawa T, et al., J Biol Chem 2014 8. Konno M, Hasegawa T, et al. Mol Neurodegener 2012 9. Hasegawa T, Konno M, et al., PLoS One 2011		7月28日（日）11:50～12:10　第2会場（朱鷺メッセ　2F　メインホールA） 4S02a-4 Traffic Jam仮説：エンドサイトーシス障害とアルツハイマー病態 Nobuyuki Kimura（木村展之）国立長寿医療セ研究所アルツハイマー Accumulating evidence suggests that β-amyloid protein (Aβ) would be the key factor for Alzheimer's disease (AD) pathogenesis, however, it remains unclear how aging induces AD pathology. It is widely accepted that the cleavage of Aβ from its precursor protein, β-amyloid precursor protein (APP), occurs through the membrane traffic in endocytic pathway, so-called endocytosis. In early stage of AD patient neurons, endocytic pathology, the intracellular accumulation of abnormally enlarged endosomes, is frequently observed, indicating that something wrong happens in endocytic membrane traffic pathway. We previously found that aging attenuates the interaction of dynein with dynactin, the functional protein complex required for retrograde axonal transport, in aged nonhuman primate brain. We also demonstrated that dynein dysfunction disturbs several endosome trafficking pathways concomitantly, and such endocytic disturbance alters Aβ metabolism to induce intracellular accumulation of Aβ. Besides Aβ pathology, we also observed that endocytic disturbance disrupts synaptic vesicle transport and exocytosis, indicating that endocytic disturbance can alter neuronal activity by itself. These findings suggest that endocytic disturbance is involved in AD pathology, and genetic studies also support the idea, since genome-wide association studies identified several endocytosis-related genes as risk factors for AD onset. Moreover, our recent studies showed that even type 2 diabetes mellitus, one of the acquired risk factors for AD, alters cholesterol metabolism in brain to enhance endocytic disturbance, resulting in much severe Aβ pathology. Thus the "traffic jam" in endocytic pathway may represent a brand new target for the development of new therapeutics against AD.

7月28日（日）12:10～12:30　第2会場（朱鷺メッセ　2F　メインホールA） 4S02a-5 小胞体と細胞膜のクロストーク Yasunori Saheki（佐伯恭範）^1,2 ¹シンガポール南洋理工大学・医学部 ²熊本大生命資源研究・支援センター Lipid homeostasis plays a central role in membrane dynamics and integrity, as well as in cell viability in all eukaryotic cells, including neurons. The endoplasmic reticulum (ER), where the majority of membrane lipids are synthesized, extends throughout the cells and forms close appositions with virtually all other membranous organelles and with the plasma membrane (PM). A well-established function of ER-PM contacts is the regulation of Ca²⁺-dynamics. Growing evidence, however, suggests that these contacts play more general roles in cell physiology, including signaling and lipid exchange between bilayers independent of membrane traffic. In my presentation, I will describe the characterization of evolutionary conserved ER-PM tethering proteins, focusing on the extended synaptotagmins (E-Syt1/2/3; tricalbins in yeast). Structural and biochemical analysis of lipid-harboring modules present in E-Syts and their ability to transport glycerolipids between membranes will be discussed. Finally, I will describe our recent characterization of the role of Ca²⁺ in coupling E-Syt1-mediated tethering and lipid transport. Our analysis of genome-edited cells lacking all the three E-Syts suggests their roles in the control of PM lipid homeostasis in response to acute changes in the lipids in this membrane. Collectively, our studies suggest that E-Syts may cooperate with other lipid transfer proteins at ER-PM contact sites and regulate homeostatic responses that compensate acute metabolic changes of PM lipids during signaling reactions.		7月28日（日）12:30～12:50　第2会場（朱鷺メッセ　2F　メインホールA） 4S02a-6 ERAD遮断によるリソソーム機能回復とムコ多糖症治療 Atsushi Saito（齋藤敦）¹，Yosuke Osaki（尾﨑陽介）^2,3，Kazunori Imaizumi（今泉和則）² ¹広島大院医歯薬ストレス分子 ²広島大院医歯薬分子細胞 ³広島大病院腎臓内科 Endoplasmic reticulum (ER) is a central organelle responsible for the synthesis, folding and posttranslational modification of various proteins. Multiple pathophysiological conditions such as expression of mutated proteins, oxidative stress and calcium depletion in the ER lumen interfere with the correct folding of proteins. Those misfolded proteins are known to accumulate in the ER and are degraded by the ubiquitin-proteasome pathway, known as ER-associated degradation (ERAD). The appropriate communication between ER and proteasome maintains the qualities and functions of proteins synthesized in the ER. Mutant proteins linked to several diseases are degraded by ERAD. The excessive degradation of the mutant proteins may trigger the reduction of intracellular functions of these proteins. Mucopolysaccharidosis type II (MPS II) is a devastating progressive disease caused by mutations in iduronate 2-sulfatase (IDS) gene. IDS is one of sulfatase enzymes required for lysosomal degradation of glycosaminoglycans (GAGs). MPS II is characterized by the progressive lysosomal accumulation of GAGs because of loss of the IDS function. Patients with MPS II exhibit systemic manifestations including mental retardation. Mutant proteins linked to diseases are prone to be misfolded. Decreased enzyme activities of IDS mutants may be due to accelerated degradation of those mutant proteins by ERAD. However, intracellular dynamics including degradation of IDS mutants is unexplored. We examined biochemical and biological characteristics of wild-type (WT) IDS and IDS mutants using Hela cells expressing WT IDS and several types of IDS mutants. IDS was subjected to proteolytic cleavage to generate mature forms at Golgi apparatus. The mature forms of IDS were translocated to lysosome. In contrast, all of IDS mutants we examined were severely accumulated in the ER and had properties of difficulty to be translocated to lysosome. Accumulated IDS mutants in the ER were ubiquitinated by ERAD-related ubiquitin E3 ligase HRD1, followed by being degraded via ERAD. Suppressed degradation of "attenuated" mutant A85T IDS (the late-onset form of MPS II) by inhibiting ERAD components improved the translocation to lysosome and its enzyme activities. Our novel findings indicate alternative target to current principal therapies for MPS II. These perspectives provide a potential to achieve development of fundamental therapeutic strategies and agents.