Chairs：
Takanori Yokota(Department of Neurology and Neurological Science, Tokyo Medical and Dental University)
Makoto Urushitani(Department of Neurology, Kyoto University Graduate School of Medicine)
≪Objective≫
The last decade is a memorial era for us, in which series of groundbreaking discoveries have struck amyotrophic lateral sclerosis (ALS) . Above all, TAR DNA-binding protein-43kDa (TDP-43) and C9ORF72 have uncovered numerous pathological mysteries, and lead us to the consensus that protein misfolding and RNA errors chiefly underlie ALS pathogenesis. Although C9ORF72 may undoubtedly govern TDP-43 proteinopathy, controversy still exists as to which is more responsible to ALS, dipeptide repeats or RNA repeats;
and the molecular pathomechanism of TDP-43 proteinopathy remains unsolved. Intriguingly, despite that C9ORF72 hexanucleotide expansion is the most popular mutation in sporadic and familial ALS in US and Europe, this mutation is quite rare in Japan, implying the presence of some unidentified machinery corresponding to C9ORF72. This symposium aims to share the latest knowledge on C9ORF72-TDP-43 lineage, focusing on the human pathology, molecular genetics, model animals, and protein degradation mechanisms, presented by distinguished researchers.
Especially, it is our great pleasure that we have Professor Leonard Petrucelli from Mayo Clinic, who is a top runner in this filed. We hope this symposium may provide an opportunity to find some hints to overcome ALS.
HT-13-1
Amyotrophic lateral sclerosis and C9ORF72/TDP-43 pathology
Department of Pathology, Brain Research Institute, Niigata University
○Hitoshi Takahashi
Amyotrophic lateral sclerosis (ALS) is a progressive, generally sporadic, neurological disease of unknown cause affecting adults. In 2006, a nuclear protein, TDP-43, was identified as the major pathological protein in both ALS and frontotemporal lobar degeneration (FTLD). Sporadic ALS is now recognized to be a multisystem TDP-43 proteinopathy widely affecting both neurons and glial cells in the CNS. It is important to note that mutations of the TDP-43 gene can cause an autosomal-dominant disease clinicopathologically indistinguishable from sporadic ALS, strongly suggesting that TDP-43 is related directly to the pathomechanism underlying ALS. In TDP-43 proteinopathy, ALS is much more common than FTLD-TDP, although both diseases are recognized to represent part of a continuous spectrum. On the other hand, in 2011, a GGGGCC hexanucleotide repeat expansion in the non-coding region of C9ORF72 was identified to cause frontotemporal dementia and ALS (c9FTD/ALS). At present, the mutation of C9ORF72 is the major genetic cause of c9FTD/ALS in the Caucasian population, but it is very rare in the Japanese population, possibly because of the difference in genetic background. In the form of c9ALS, the neuropathology of sporadic ALS has been clearly shown to have distinct additional features, including occurrence of TDP-43-negative aggregates of dipeptide repeat (DPR) proteins; of great importance was that the DPR protein pathology was distributed independently of TDP-43 pathology, suggesting that there is no correlation between DPR protein pathology and neuronal loss, or between DPR protein pathology and clinical symptoms. In the present symposium, we will present the neuropathological features observed in a Japanese patient with C9ORF72 repeat expansion (c9ALS), who to our knowledge represents the only autopsy case of this genetic disease to have been reported in the Japanese population so far, and will also review the subsequent development of C9ORF72/TDP pathology.
《Curriculum Vitae》
Education
Niigata University Faculty of Medicine, Niigata, Japan
graduated in 1979; Medical License, 1979; Ph.D. (Medical Science), 1985 Careers
1995 - present
Professor (Department of Pathology) Brain Research Institute, Niigata University 2002 - 2014
Director
Brain Research Institute, Niigata University 2014 - present
Executive Vice President, Niigata University 1981 - 1983
Postdoctoral Fellow (Dr. Kinuko Suzuki)
Department of Pathology, Albert Einstein College of Medicine, NY, USA 2010
-President
The Japanese Society of Neuropathology 2014 - 2018
President
The International Society of Neuropathology
ホットトピックス HT-13：Understanding ALS pathogenesis by Updated
Chairs：
Takanori Yokota(Department of Neurology and Neurological Science, Tokyo Medical and Dental University)
Makoto Urushitani(Department of Neurology, Kyoto University Graduate School of Medicine)
≪Objective≫
The last decade is a memorial era for us, in which series of groundbreaking discoveries have struck amyotrophic lateral sclerosis (ALS) . Above all, TAR DNA-binding protein-43kDa (TDP-43) and C9ORF72 have uncovered numerous pathological mysteries, and lead us to the consensus that protein misfolding and RNA errors chiefly underlie ALS pathogenesis. Although C9ORF72 may undoubtedly govern TDP-43 proteinopathy, controversy still exists as to which is more responsible to ALS, dipeptide repeats or RNA repeats;
and the molecular pathomechanism of TDP-43 proteinopathy remains unsolved. Intriguingly, despite that C9ORF72 hexanucleotide expansion is the most popular mutation in sporadic and familial ALS in US and Europe, this mutation is quite rare in Japan, implying the presence of some unidentified machinery corresponding to C9ORF72. This symposium aims to share the latest knowledge on C9ORF72-TDP-43 lineage, focusing on the human pathology, molecular genetics, model animals, and protein degradation mechanisms, presented by distinguished researchers.
Especially, it is our great pleasure that we have Professor Leonard Petrucelli from Mayo Clinic, who is a top runner in this filed. We hope this symposium may provide an opportunity to find some hints to overcome ALS.
HT-13-1
Amyotrophic lateral sclerosis and C9ORF72/TDP-43 pathology
Department of Pathology, Brain Research Institute, Niigata University
○Hitoshi Takahashi
Amyotrophic lateral sclerosis (ALS) is a progressive, generally sporadic, neurological disease of unknown cause affecting adults. In 2006, a nuclear protein, TDP-43, was identified as the major pathological protein in both ALS and frontotemporal lobar degeneration (FTLD). Sporadic ALS is now recognized to be a multisystem TDP-43 proteinopathy widely affecting both neurons and glial cells in the CNS. It is important to note that mutations of the TDP-43 gene can cause an autosomal-dominant disease clinicopathologically indistinguishable from sporadic ALS, strongly suggesting that TDP-43 is related directly to the pathomechanism underlying ALS. In TDP-43 proteinopathy, ALS is much more common than FTLD-TDP, although both diseases are recognized to represent part of a continuous spectrum. On the other hand, in 2011, a GGGGCC hexanucleotide repeat expansion in the non-coding region of C9ORF72 was identified to cause frontotemporal dementia and ALS (c9FTD/ALS). At present, the mutation of C9ORF72 is the major genetic cause of c9FTD/ALS in the Caucasian population, but it is very rare in the Japanese population, possibly because of the difference in genetic background. In the form of c9ALS, the neuropathology of sporadic ALS has been clearly shown to have distinct additional features, including occurrence of TDP-43-negative aggregates of dipeptide repeat (DPR) proteins; of great importance was that the DPR protein pathology was distributed independently of TDP-43 pathology, suggesting that there is no correlation between DPR protein pathology and neuronal loss, or between DPR protein pathology and clinical symptoms. In the present symposium, we will present the neuropathological features observed in a Japanese patient with C9ORF72 repeat expansion (c9ALS), who to our knowledge represents the only autopsy case of this genetic disease to have been reported in the Japanese population so far, and will also review the subsequent development of C9ORF72/TDP pathology.
《Curriculum Vitae》
Education
Niigata University Faculty of Medicine, Niigata, Japan
graduated in 1979; Medical License, 1979; Ph.D. (Medical Science), 1985 Careers
1995 - present
Professor (Department of Pathology) Brain Research Institute, Niigata University 2002 - 2014
Director
Brain Research Institute, Niigata University 2014 - present
Executive Vice President, Niigata University 1981 - 1983
Postdoctoral Fellow (Dr. Kinuko Suzuki)
Department of Pathology, Albert Einstein College of Medicine, NY, USA 2010
-President
The Japanese Society of Neuropathology 2014 - 2018
President
The International Society of Neuropathology
ホットトピックス HT-13：Understanding ALS pathogenesis by Updated progress in C9ORF72-TDP43 lineage
5月20日（金） 8：00～10：00 第13会場(神戸国際会議場4F Room 401＋402 )
216 -ホッ
トト ピッ クス
HT-13-2
Deciphering the expression and function of C9ORF72 using mouse model
1Department of Neurology, Tohoku University，
2Department of Stem Cell and Regenerative Biology, Harvard University, USA
○Naoki Suzuki1,2
Recently, a hexanucleotide repeat expansion at C9ORF72 is found in a significant fraction of patients suffering from amyotrophic lateral sclerosis (ALS) and frototemporal dementia in Western countries. However, it remains unclear to what extent this mutation acts through a gain of function or loss of function mechanism to contribute to disease initiation. The expanded GGGGCC repeat in C9ORF72 has been proposed to mediate its effects through several potential mechanisms. However, it is still unclear which of these is responsible for neural degeneration in vivo. Individuals harboring the expanded repeat exhibit significantly reduced abundance of two isoforms of the C9ORF72 transcript and it has been suggested that the GC rich expansion may silence the mutant allele through a mechanism similar to that observed in Fragile X Syndrome. It has also been observed that the repeat expansion itself is expressed in at least one transcriptional isoform and that the resulting RNA accumulates in nuclear foci. These foci can sequester RNA binding proteins, potentially unbalancing RNA metabolism and promoting nucleolar stress within mutant cells. Once transcribed, reports suggest the repeat expansion is also subject to a form of non-canonical repeat associated non-ATG translation initiation, leading to accumulation of potentially toxic and aggregation prone di-peptide repeat proteins. It is possible that only one of the immediate effects of the repeat expansion could be sufficient to cause ALS.
Alternatively, all three known consequences of the expansion could collaborate in the development of motor neuron disease. Resolution of the relevant pathological mechanism, or mechanisms, will be essential for devising effective and safe therapeutics. Furthermore, relatively little has been learned concerning the physiological function of C9ORF72. In this talk, we present our data about C9ORF72-ortholog mutant mice and provide an overview of current literatures in this field.
《Curriculum Vitae》
2001 M.D. Tohoku University School of Medicine
2001-2002 Department of Neurology, Tohoku University Hospital 2002-2003 Iwaki Kyoritsu Hospital & Tohoku Kosei Nenkin Hospital 2003-2007 Department of Neurology, Tohoku University School of Medicine 2004-2007 Graduate study at Department of Molecular Therapy, National
Institute of Neuroscience
2007-2011 Clinical Fellow at Department of Neurology, Tohoku University Hospital
2011 Assistant Professor at Department of Neurology, Tohoku University Hospital
2011-2014 Postdoctoral Fellow, Eggan Lab, Department of Stem Cell and Regenerative Biology, HSCI, Harvard University
2014-Present Assistant Professor at Department of Neurology, Tohoku University Hospital
Awards:
2006 The WMS-Elsevier Membership Award, World Muscle Society 2008 Best Paper Award, National Institute of Neuroscience
2011 Lilly Scientific Fellowship Program selected by Japanese Society of Neurology
HT-13-3 withdrawn
ホットトピックス HT-13：Understanding ALS pathogenesis by Updated progress in C9ORF72-TDP43 lineage
5月20日（金） 8：00～10：00 第13会場(神戸国際会議場4F Room 401＋402 )
217
-ホッ トト ピッ クス
HT-13-4 一般演題から採用 C9orf72-Rab mediates defective vesicle trafficking in C9ALS/FTD and rescue by antisense intervention
1Department of Molecular Therapy, National Institute
of Neuroscience, National Center of Neurology and Psychiatry，
2Department of Physiology, Anatomy and Genetics, University of Oxford，3Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University，4Nuffield Department of Clinical Neurosciences, University of Oxford
○Yoshitsugu Aoki1，Raquel Manzano2， Mitsunori Fukuda3，Kevin Talbot4， Shin’ichi Takeda1，Matthew Wood2 [Background]
A hexanucleotide repeat expansion mutation in the C9orf72 locus is the commonest genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD) . Currently, the biological function of C9orf72 remains unknown. However, bioinformatic analysis has revealed that the C9orf72 gene product has a differentially expressed in normal and neoplasia (DENN)-like domain and might regulate membrane trafficking as a Rab-GTPase GDP/GTP exchange factor.
[Objective]
To clarify the molecular mechanism of C9orf72, how this could relate to C9ALS/FTD and whether antisense oligonucleotide-based therapy is applicable to the disease.
[Method and Results]
Here, using comprehensive GST-Rab pulldown analysis, we show that C9orf72 physically interacts with the Rab29 GTPase (also known as Rab7L1), suggesting that C9orf72 is a Rab29 effector. Further, we show that loss of the C9orf72/Rab29 interaction leads to dramatically reduced trafficking of extracellular vesicles (EVs), impaired intracellular trans-Golgi trafficking, and a consequent deficiency of autophagy in fibroblasts and iPSC-derived neurons from patients with C9ALS/FTD. Both overexpression of C9orf72 and antisense oligonucleotides targeting repeat-containing C9orf72 transcripts to upregulate normal variant 1 transcript levels are able independently to rescue the defective EV, trans-Golgi vesicle trafficking and basal autophagy phenotype.
[Conclusions]
Our findings identify C9orf72 haploinsufficiency as a major contributing factor in ALS/FTD neurodegenerative disorders and directly link ALS/FTD to EV biogenesis.
《Curriculum Vitae》
Yoshitsugu AOKI, M.D., Ph.D. graduated from Tohoku University, School of Medicine and received an M.D. in 2001. He received specialist training as a neurologist from 2004 to 2007 at the National Center Hospital, National Center of Neurology and Psychiatry (NCNP) . In 2011, he graduated from Tokyo Medical and Dental University Graduate School of Medicine with a Ph.D. (Mentor: Dr.
Shin’ichi Takeda). Then he worked as a Uehara postdoctoral fellow and then a team leader from 2012 to 2015 for Prof. Wood at the Department of Physiology, Anatomy and Genetics, Oxford. He is currently a Section Chief at National Institute of Neuroscience, NCNP and a Visiting Scholar at University of Oxford.
HT-13-5
Impaired VHL/CUL2-mediated clearance of misfolded TDP-43
underlies oligodendrocyte inclusions in ALS
Department of Neurology, Kyoto Graduate School of Medicine
○Makoto Urushitani
Cytosolic accumulation of misfolded and fragmented TDP-43 is a pathological signature for ALS, whose pathomechanisms remain elusive. Here we identified a cullin-2 (CUL2) RING complex as a novel ubiquitin ligase for fragmented forms of TDP-43. The von Hippel-Lindau protein (VHL), a substrate binding component of the complex, preferentially recognized misfolded TDP-43 at Glu246 in RNA-recognition motif 2, a previously reported epitope for mislocalized/misfolded TDP-43. Interestingly, CUL2 predominantly ubiquitinates and degraded carboxyl fragments of TDP-43 rather than the full-length by the 24h chase study. Moreover, Recombinant full-length TDP-43 was structurally fragile and readily cleaved, suggesting that misfolded TDP-43 is cleared by VHL/CUL2 in a step-wise manner via fragmentation. Conversely, however, the excess amount of VHL and TDP-43 stabilized each other and promoted their accumulation at juxtanuclear protein quality control center (JUNQ). TDP-43 knockdown induced VHL expression in culture cells, suggesting the linkage between VHL and mislocalized TDP-43 in TDP-43proteinopathy in ALS. Finally, cytoplasmic inclusions, especially in oligodendrocytes in ALS spinal cords, were immunoreactive to both phosphorylated TDP-43 and VHL. Notably, VHL also recognized ALS-relevant SOD1 mutants and promoted the inclusion formation. These results indicate that double-edged action of VHL may underlie oligodendrocyte dysfunction in ALS, and the regulation of VHL/CUL2 balance has a huge potential to understand and find a new cure for ALS.
《Curriculum Vitae》
Education, Professional Training and Employment:
1985-1991 Student, Faculty of Medicine, Kyoto University 1991-1992 Resident, Dept. of Neurology, Kyoto University Hospital 1992-1996 Staff Doctor, Dept. of Neurology, Sumitomo Hospital
1996-2000 Ph.D. Student. Department of Neurology,Kyoto University Graduate School of Medicine
2000-2003 Staff Scientist (Postdoctoral fellow). Brain Science Institute, RIKEN 2003-2006 Postdoctoral fellow of Laval University. c.o. Jean-Pierre Julien 2006-2009 Assistant Professor, Shiga-University of Medical Science (SUMS) 2009-2013 Associate Professor and Principal Investigator, SUMS
2013-present Associate Professor and Laboratory Head, Department of NeurologyKyoto University Graduate School of Medicine
Awards:
Fellowship of Canadian Institute of Health and Research (CIHR) (2005-2006) Brain Star Award (2006); Canadian Institute of Health Science
Research interests
1. Structural Analysis of Pathogenic protein of ALS
2. Development of antibody-mediated molecular targeting therapy for ALS-causal proteins
ホットトピックス HT-13：Understanding ALS pathogenesis by Updated progress in C9ORF72-TDP43 lineage
5月20日（金） 8：00～10：00 第13会場(神戸国際会議場4F Room 401＋402 )
ホッ トト ピッ クス
HT-13-4 一般演題から採用 C9orf72-Rab mediates defective vesicle trafficking in C9ALS/FTD and rescue by antisense intervention
1Department of Molecular Therapy, National Institute
of Neuroscience, National Center of Neurology and Psychiatry，
2Department of Physiology, Anatomy and Genetics, University of Oxford，3Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University，4Nuffield Department of Clinical Neurosciences, University of Oxford
○Yoshitsugu Aoki1，Raquel Manzano2， Mitsunori Fukuda3，Kevin Talbot4， Shin’ichi Takeda1，Matthew Wood2 [Background]
A hexanucleotide repeat expansion mutation in the C9orf72 locus is the commonest genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD) . Currently, the biological function of C9orf72 remains unknown. However, bioinformatic analysis has revealed that the C9orf72 gene product has a differentially expressed in normal and neoplasia (DENN)-like domain and might regulate membrane trafficking as a Rab-GTPase GDP/GTP exchange factor.
[Objective]
To clarify the molecular mechanism of C9orf72, how this could relate to C9ALS/FTD and whether antisense oligonucleotide-based therapy is applicable to the disease.
[Method and Results]
Here, using comprehensive GST-Rab pulldown analysis, we show that C9orf72 physically interacts with the Rab29 GTPase (also known as Rab7L1), suggesting that C9orf72 is a Rab29 effector. Further, we show that loss of the C9orf72/Rab29 interaction leads to dramatically reduced trafficking of extracellular vesicles (EVs), impaired intracellular trans-Golgi trafficking, and a consequent deficiency of autophagy in fibroblasts and iPSC-derived neurons from patients with C9ALS/FTD. Both overexpression of C9orf72 and antisense oligonucleotides targeting repeat-containing C9orf72 transcripts to upregulate normal variant 1 transcript levels are able independently to rescue the defective EV, trans-Golgi vesicle trafficking and basal autophagy phenotype.
[Conclusions]
Our findings identify C9orf72 haploinsufficiency as a major contributing factor in ALS/FTD neurodegenerative disorders and directly link ALS/FTD to EV biogenesis.
《Curriculum Vitae》
Yoshitsugu AOKI, M.D., Ph.D. graduated from Tohoku University, School of Medicine and received an M.D. in 2001. He received specialist training as a neurologist from 2004 to 2007 at the National Center Hospital, National Center of Neurology and Psychiatry (NCNP) . In 2011, he graduated from Tokyo Medical and Dental University Graduate School of Medicine with a Ph.D. (Mentor: Dr.
Shin’ichi Takeda). Then he worked as a Uehara postdoctoral fellow and then a team leader from 2012 to 2015 for Prof. Wood at the Department of Physiology, Anatomy and Genetics, Oxford. He is currently a Section Chief at National Institute of Neuroscience, NCNP and a Visiting Scholar at University of Oxford.
HT-13-5
Impaired VHL/CUL2-mediated clearance of misfolded TDP-43
underlies oligodendrocyte inclusions in ALS
Department of Neurology, Kyoto Graduate School of Medicine
○Makoto Urushitani
Cytosolic accumulation of misfolded and fragmented TDP-43 is a pathological signature for ALS, whose pathomechanisms remain elusive. Here we identified a cullin-2 (CUL2) RING complex as a novel ubiquitin ligase for fragmented forms of TDP-43. The von Hippel-Lindau protein (VHL), a substrate binding component of the complex, preferentially recognized misfolded TDP-43 at Glu246 in RNA-recognition motif 2, a previously reported epitope for mislocalized/misfolded TDP-43. Interestingly, CUL2 predominantly ubiquitinates and degraded carboxyl fragments of TDP-43 rather than the full-length by the 24h chase study. Moreover, Recombinant full-length TDP-43 was structurally fragile and readily cleaved, suggesting that misfolded TDP-43 is cleared by VHL/CUL2 in a step-wise manner via fragmentation. Conversely, however, the excess amount of VHL and TDP-43 stabilized each other and promoted their accumulation at juxtanuclear protein quality control center (JUNQ). TDP-43 knockdown induced VHL expression in culture cells, suggesting the linkage between VHL and mislocalized TDP-43 in TDP-43proteinopathy in ALS. Finally, cytoplasmic inclusions, especially in oligodendrocytes in ALS spinal cords, were immunoreactive to both phosphorylated TDP-43 and VHL. Notably, VHL also recognized ALS-relevant SOD1 mutants and promoted the inclusion formation. These results indicate that double-edged action of VHL may underlie oligodendrocyte dysfunction in ALS, and the regulation of VHL/CUL2 balance has a huge potential to understand and find a new cure for ALS.
《Curriculum Vitae》
Education, Professional Training and Employment:
1985-1991 Student, Faculty of Medicine, Kyoto University 1991-1992 Resident, Dept. of Neurology, Kyoto University Hospital 1992-1996 Staff Doctor, Dept. of Neurology, Sumitomo Hospital
1996-2000 Ph.D. Student. Department of Neurology,Kyoto University Graduate School of Medicine
2000-2003 Staff Scientist (Postdoctoral fellow). Brain Science Institute, RIKEN 2003-2006 Postdoctoral fellow of Laval University. c.o. Jean-Pierre Julien 2006-2009 Assistant Professor, Shiga-University of Medical Science (SUMS) 2009-2013 Associate Professor and Principal Investigator, SUMS
2013-present Associate Professor and Laboratory Head, Department of NeurologyKyoto University Graduate School of Medicine
Awards:
Fellowship of Canadian Institute of Health and Research (CIHR) (2005-2006) Brain Star Award (2006); Canadian Institute of Health Science
Research interests
1. Structural Analysis of Pathogenic protein of ALS
2. Development of antibody-mediated molecular targeting therapy for ALS-causal proteins
ホットトピックス HT-13：Understanding ALS pathogenesis by Updated progress in C9ORF72-TDP43 lineage
5月20日（金） 8：00～10：00 第13会場(神戸国際会議場4F Room 401＋402 )
218