○YukikoMaki,AmiTakahashi,MegumiSeo,NichikaSumitomo, TakaoMitsui
Department of Clinical Research, Tokushima National Hospital, Japan Objective We have found that Parkin is localized in mitochondria and undergoes glycosylation. In particular, it was presumed that Parkin was modified with a sugar chain modification, O-linked N-acetylglucosamine
（O-GlcNAc）. However, It was problematic to detect O-GlcNAc with monoclonal antibodies. In this study, we studied the O-GlcNAc modification of Parkin through various lines of approaches. Methods We investigated the reaction specificity of five monoclonal anti-O-GlcNAc antibodies. The reactivity of antibodies against the nuclear proteins Nup62 and H2B was examined. In addition, the specificity of biotin or TAMRA labeling via Click reaction was tested. After introducing GFP-added protein into cultured cells and labeling O-GlcNAc with TAMRA, the cells were observed by fluorescence resonance energy transfer （FRET）. Furthermore, we attempted to identify the O-GlcNAc addition site of Parkin. Biotin-labeled Parkin was degraded and immunoprecipitated. The precipitated peptide fragments were analyzed by LC-MS / MS. Results GFP / His Parkin and H2B protein were stained with all O-GlcNAc antibodies. The antibodies also reacted with the proteins purified using an E. coli expression system. On the other hand, Click reaction detected only proteins derived from mammalian cells. By the FRET method, GFP Parkin was detected as a wavelength of 580 nm with 488 nm excitation.
Furthermore, a 24-amino acid fragment of Parkin was detected by LC-MS / MS analysis. Conclusions It was strongly suggested that Parkin was modified with O-GlcNAc.
Pe-05-1
Elevationofserumcreatinekinaseattheonset ofsporadicamyotrophiclateralsclerosis○DaisukeIto1,AtsushiHashizume1,YasuhiroHijikata1, ShinichiroYamada1,YoshiyukiKishimoto1,YoheiIguchi1, MadokaIida1,2,AkihiroHirakawa3,MasahisaKatsuno1
1 Department of Neurology, Nagoya University Graduate School of Medicine, Japan, 2 Research Fellow of Japan Society for the Promotion of Science, RPD, 3 Department of Biostatistics and Bioinformatics, Graduate School of Medicine, The University of Tokyo
[Background] The early intervention is a major issue in neurodegenerative diseases, however, pathophysiological change at the timing around clinical disease onset of amyotrophic lateral sclerosis （ALS） is unclear. [Objective] To estimate changes of biomarkers at the onset [Method] Subjects with ALS whose disease durations were less than 2 years and healthy controls（HC） were recruited. Blood test and the appendicular lean soft tissue mass （ALST mass）, a marker of muscle mass, were evaluated every six months. Their values at the onset were estimated by linear mixed models with unstructured correlation and random intercepts. We also analyzed serum creatine kinase （CK）, creatinine （Cr） and the mRNA levels of acetylcholine receptor subunit γ
（Chrng）, a marker of denervation, in the gastrocnemius muscle in superoxide dismutase 1 （SOD1）G93A transgenic mice. [Result] Thirty-nine subjects with sporadic ALS and age-, sex- matched 20 HCs were analyzed. Serum Cr and ALST mass at baseline were decreased and serum CK was elevated in ALS subjects. These three markers decreased longitudinally. The estimated levels of CK at the onset was higher than those of HCs, although the estimated Cr and ALST mass at the onset were equivalent [log CK 2.34, 1.97; Cr （mg/dL） 0.80, 0.75; ALST mass（kg） 19.91, 19.32; respectively]. In SOD1G93A transgenic mice, the levels of CK also increased at the onset with the elevation of Chrng and decreased longitudinally. [Conclusion] The serum CK elevation precedes muscle atrophy in sporadic ALS and it might reflect the initiation of muscle denervation.
Pe-05-2
ClinicalandgeneticcharacteristicsinJapanese familieswithVCP-relatedmultisystemproteinopathy○TakashiAndo1,RyoichiNakamura1,SatoshiKuru2,DaichiYokoi1,3,NaokiAtsuta1, HarukiKoike1,MasashiSuzuki1,KazuhiroHara1,YoheiIguchi1,AyukaMurakami1,2,
SeiyaNoda1,2,JunSone1,2,TomohikoNakamura1,YojiGoto4,KazuoMano4, HisashiOkada5,IchizoNishino6,TomooOgi7,GenSobue8,MasahisaKatsuno1
1 Department of Neurology, Nagoya University Graduate School of Medicine, Japan, 2 Department of Neurology, National Hospital Organization Suzuka National Hospital, 3 Department of Neurology, Kakeyu-Misayama Rehabilitation Center Kakeyu Hospital, 4 Department of Neurology, Japanese Red Cross Nagoya Daiichi Hospital, 5 Department of Neurology, National Hospital Organization Nagoya Medical Center, 6 Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 7 Department of Genetics, Research Institute of Environmental Medicine （RIeM）, Nagoya University, 8 Brain and Mind Research Center, Nagoya University
[Objective] Valosin-containing protein （VCP） gene mutations are known to cause various neurodegenerative disorders, such as inclusion body myopathy （IBM） associated with frontotemporal dementia （FTD）, amyotrophic lateral sclerosis （ALS）, and axonal neuropathy. In this study, we investigated and characterized the VCP gene mutations in Japanese patients. [Methods] We report eight patients [six males, two females;
median onset age: 49.5 years] from five unrelated families with VCP missense mutations. Clinical, physiological, pathological, and genetic assessments were performed for each patient. [Results] Although 7 of 8 patients had either IBM or ALS, one patient had demyelinating polyneuropathy without myopathy or motor neuron disease. Three known pathogenic VCP mutations （p.Arg191Gln, p.Arg155Cys, and p.Ile126Phe）
were detected. Moreover, a novel mutation, c.293A>T （p.Asp98Val）, was identified in a patient with ALS and FTD. This mutation was predicted to be 'Deleterious' or 'Disease causing' using in silico mutation analyses. [Conclusions] The p.Asp98Val mutation was found to be a novel pathogenic mutation of VCP-related multisystem proteinopathy. In addition, demyelinating polyneuropathy may be a novel phenotype of VCP-related multisystem proteinopathy. Our cases represented a wide clinical spectrum with VCP mutations.
Pe-04-10
withdrawninpatientswithamyotrophiclateralsclerosis
○AkihiroNishida1,JunTsugawa2,ShinsukeFujioka1,YoshioTsuboi1
1 Department of Neurology, Fukuoka University, Japan, 2 Stroke Center, Fukuoka University Chikushi Hospital
[Objective]Fasciculation is an important clinical feature for the diagnosis of Amyotrophic Lateral Sclerosis （ALS）. Recently, muscle ultrasound （MUS） has been widely used as a supportive diagnostic tool for detecting fasciculations in ALS. However, few studies have focused on the quantitative assessment of fasciculations by MUS in ALS. To investigate the impact of quantitative assessment of fasciculations in patients with ALS. [Methods]Twenty-four ALS patients were enrolled. Patients underwent MUS to detect muscle fasciculations quantitatively in 25 regions in each subject, including the tongue, trunk, and all 4 limbs. The sum was calculated as the MUS fasciculation score. We analyzed the correlation between this score and clinical parameters （age, sex, disease duration, ALS functional rating scale-revised, Δfs, SAO2, median compound action potential, tongue pressure） in ALS. [Results] Overall, the fasciculation positive rate on MUS in each muscle was 59.3±15.6%. Of those, the flexor carpi ulnaris muscle had the highest MUS positive rate （79.2%）, while the abdominal muscle had the lowest （25%）. Disease duration negatively correlated with overall MUS fasciculation score （r=-0.557, p=0.005）. Respiratory function （SaO2）
positively correlated with the MUS fasciculation score （r=0.502, p=0.12）. There is no association between other parameters. [Conclusions]We confirmed that the MUS fasciculation score was higher in the early stage in ALS patients.
Quantitative assessment of fasciculations by MUS may be correlated with the stage of ALS and may predict respiratory outcome.
Pe-05-4
withdrawn一 般 演 題
ポ ス タ ー （ 英 語 ）
Pe-05-3
ImpactofquantitativeassessmentoffasciculationsPe-06-1
Increasedβ-tubulinglutamylationintheataxia andmalesterilitymousehindersneuralmaturation○YoshieIto1,SheikhAbdullah2,ShozoYano2,TabassumShatera2, KojiOmura3,AsukaAraki3,SatoshiInagaki1,SatoshiAbe1, ShingoMitaki1,AtsushiNagai1
1 Department of Neurology, Shimane University Faculty of Medicine, Japan,
2 Department of Laboratory Medicine, Shimane University Faculty of Medicine, 3 Department of Organ Pathology, Shimane University Faculty of Medicine
【Background】 Ataxia and male sterility （AMS） is a mutant mouse strain, which contains a missense mutation in the coding region of NNA1 gene. AMS demonstrates cerebellar Purkinje cell （PC） degeneration at very early age, and ataxic phenotype is expressed in an autosomal recessive manner. 【Objective】 The objective of this study is to understand the molecular mechanism that is affected by this mutation during PC degeneration. 【Methods】 We have generated neuronal stem cell （NSC） lines from wild type （NMW7）, NNA1 mutation heterozygous （NME） and NNA1 mutation homozygous
（NMO1） mice brains. We also analyzed time-dependent changes in AMS cerebellum at pathological and molecular levels. 【Results】 The growth rate of NMO1 was faster than other NSC lines. Although NMO1 could make neurospheres, some of them were attached to the plate after 3 days' culture. Immunostaining revealed that SOX2 and nestin levels were decreased in NMO1-derived neurospheres. Conversely, β-tubulin level was high in NMO1 and NME. Neuron differentiation potentials of NMO1 were less than NME or NMW7. Further, β-tubulin was high in NMO1 after neuron differentiation. Although total β-tubulin was not changed, glutamylated tubulin level was increased in AMS mice cerebellum both at 15 and 30 days. Similarly, glutamylated tubulin level was increased in NMO1-derived neurospheres without changing β-tubulin transcription. 【Conclusion】
Thus, our results suggest that glutamylated β-tubulin level is increased early in the cerebellum, which disturbs neuron maturation and induce PC death in AMS mice.
Pe-06-2
Establishmentofspinocerebellarataxiatype 36modelfly○TomoyaTaminato1,MorioUeyama1,ToruYamashita3, YoshioIkeda2,KojiAbe3,YoshitakaNagai1
1 Dept Neurotherapeutics, Osaka Univ Grad Sch of Med, Osaka, Japan, 2 Dept Neurology, Gunma Univ Grad Sch of Med, Gunma, Japan, 3 Dept Neurology, Okayama Univ Grad Sch of Med, Okayama, Japan
OBJECTIVE:Spinocerebellar Ataxia type 36 （SCA36） is one of the non-coding repeat expansion disorders （NRDs） which is caused by an GGCCTG repeat expansion （>25） in the first intron of the NOP56 gene. Remarkably, SCA36 patients develop not only ataxia but also motor neuron symptoms similar to ALS. A pathological hallmark of NRDs, such as C9orf72-linked ALS/FTD, is the accumulation of expanded repeat RNA as RNA foci. Furthermore, it is known that the expanded repeat RNA is translated into proteins by unconventional repeat associated non-ATG （RAN） translation. METHODS:To elucidate the molecular mechanisms of SCA36, we established novel Drosophila models expressing GGCCTG repeat RNA. Drosophila is a powerful genetic system for identification of genetic modifier or drug candidates because of their short lifespan, large numbers of embryos and accumulated knowledge about their genetics. In order to obtain a long repeat sequence with flanking region of NOP56 gene, we amplified subcloned （GGCCTG）43 sequences from the SCA36 patient by PCR and ligated these amplicons. Then transgenic flies were made using these constructs.RESULTS:When we expressed GGCCTG repeats in the compound eyes, we observed obvious eye degeneration in （GGCCTG）118 expressing flies, while
（GGCCTG）7 expressing flies showed almost normal eye phenotype. Consistent with SCA36 patients, intranuclear RNA foci were detected by fluorescent in situ hybridization. Furthermore, we found the expression of poly （Gly-Pro）, which is one of the RAN proteins, in eye imaginal discs by immunohistochemistry.
Pe-06-3
Geneticscreeningforpotassiumchannelmutations inautosomaldominantspinocerebellarataxia○HiroyukiMorino1,YuiTada1,2,KodaiKume1,YukikoMatsuda1, TakashiKurashige3,YuheiKanaya1,HayatoTabu4,SatoshiKaneko5, ToshihikoSuenaga6,AkiraKakizuka2,HideshiKawakami1
1 Department of Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan, 2 Laboratory of Functional Biology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan,
3 Department of Neurology, National Hospital Organization Kure Medical Center and Chugoku Cancer Center, Kure, Japan, 4 Department of Neurology, Kitano Hospital, Osaka, Japan, 5 Department of Neurology, Kansai Medical University, Osaka, Japan, 6 Department of Neurology, Tenri Hospital, Tenri, Japan
Objective: Spinocerebellar ataxia （SCA） is a genetically heterogeneous disease characterized by cerebellar ataxia, and many causative genes have been identified so far. The most common etiology is the abnormal expansion of repeat sequences, and mutations of ion channel genes also play an important role in the development of SCA. However, there are still few reports on the mutations of the potassium channel genes.
Methods: We screened 192 Japanese individuals with dominant inherited SCA, who had no abnormal repeat expansions of causative genes, for potassium channel mutations （KCNC3 for SCA13 and KCND3 for SCA19/
SCA22） by target sequencing. Results: Two variants were identified from two patients: c.1973G>A,p.R658Q and c.1018G>A,p.V340M in KCNC3, and no pathogenic variant was identified in KCND3. The newly identified p.V340M exists in the extracellular domain, and p.R658Q exists in the intracellular domain on the C-terminal side, although most of the reported KCNC3 mutations are present at the transmembrane site. Discussion: Adult-onset and slowly progressive cerebellar ataxia are the main clinical features of SCA13 and SCA19 caused by potassium channel mutations, which was similar in our cases. SCA13 caused by KCNC3 mutations may present with deep sensory loss and cognitive impairment in addition to cerebellar ataxia. In this study, mild deep sensory loss was observed in one case. SCA caused by potassium channel gene mutations is extremely rare, and more cases should be accumulated in the future to elucidate its pathogenesis due to channel dysfunction.
Pe-06-4
EffectsoftheoverexpressionofTFEBincellular andmousemodelsofneurodegenerativediseases○HiroakiAdachi1,ZheHuang2,KazumasaOkada1,KeikoOhnari1, TomoyoHashimoto1,TomokoToyota1,YukioIwanaka1
1 Department of Neurology, University of Occupational and Environmental Health School of Medicine, Japan, 2 Department of Community Healthcare and Geriatrics, Nagoya University Graduate School of Medicine
[Background] Macroautophagy is a set of bulk degradation processesin which cells form double-membrane vesicles, called autophagosomes,around a portion of the cytoplasm. The transcription factor EB （TFEB） has been reported to regulate autophagy by upregulating genes that belong to the coordinated lysosomal expression and regulation （CLEAR） network, thereby controlling lysosomal biogenesis. Spinal and bulbar muscular atrophy （SBMA） is an inherited motor neuron disease caused by the expansion of a polyglutamine （polyQ） tract within the androgen receptor （AR）. We examined the effects of the overexpression of TFEB in cultured cell models of neurodegenerative diseases and in a transgenic mouse model that overexpressed the mutant AR （SBMA mice）. [Materials and Methods] Cells were transfected with plasmids encoding mutant AR, huntingtin, ataxin-1, ataxin-3 and TFEB. We generated transgenic mice overexpressing full-length human TFEB under the control of prion protein promoter, and crossed the SBMA mice with mice overexpressing human TFEB. [Results] The overexpression of TFEB decreased the expression of each causative protein in the neuronal cell models （p< 0.01）. The expression of the autophagic marker LC-3 II was significantly elevated in the cells expressing TFEB （p< 0.01）. TFEB overexpression improved SBMA mouse phenotypes by inducing autophagy.
[Conclusions] These findings demonstrated that the high expression of TFEB induced autophagy and enhanced the preferential degradation of the disease-causative proteins, and promoted therapeutic effects in an SBMA model mice.
Pe-06-5
Biochemicalanalysisofmiddle-age-onsetSCAR causedbyabiallelicmutationofHSD17B4○YukikoMatsuda1,HiroyukiMorino1,TakashiKurashige2, RyosukeMiyamoto3,HirofumiMaruyama4,HideshiKwakami1
1 Dept. Epidemiology, RIRBM, Hiroshima Univ., Japan, 2 Dept. Neurology, Natl. Hosp. Org. Kure Med. Ctr. and Chugoku Cancer Ctr., 3 Dept. Clinical Neuroscience., Inst. Biomed. Sci., Tokushima Univ., 4 Dept. Clinical Neuroscience and Therapeutics, Hiroshima Univ.
[Objective] We identified a homozygous mutation in the dehydrogenase domain of HSD17B4 gene as a cause for slowly progressive spinocerebellar ataxia, autosomal recessive （SCAR） using exome sequencing. In order to determine the biochemical underpinnings, we conducted the functional analysis of D-bifunctional protein （DBP） encoded by HSD17B4. [Methods] We assessed the expression level of DBP and the amount of dimerized DBP by immunoblotting and quantitative reverse transcription polymerase chain reaction using patient-derived fibroblasts and HEK293T cells overexpressing mutant DBP. We also performed morphological evaluation using immunocytochemistry. [Results]
mRNA expression levels of DBP were normal. However, protein levels were diminished in the patient-derived fibroblasts, and relative amount of DBP in peroxisome was reduced. A decrease in DBP expression was observed in HEK293T cells overexpressing mutant DBP. Moreover, the dimer which is a functional form of DBP was also decreased. [Conclusions] DBP mutations that cause severe functional impairment lead to severe infant-onset abnormalities.
The DBP mutation causing mild dysfunction analyzed in this study showed mild clinical symptoms and a phenotype of middle-age-onset slowly progressive SCAR. This study broadens the scope of DBP deficiency phenotypes and indicates that residual functional DBP may be used to estimate the severity of DBP deficiencies.
Pe-06-6
AssociationofFMR1intermediatealleleswith multiplesystematrophy○MAsemAlmansour1,HiroyukiIshiura1,JunMitsui2,ShojiTsuji2,3, TatsushiToda1
1 Department of Neurology, Graduate School of Medicine, The University of Tokyo, Japan, 2 Department of Molecular Neurology, Graduate School of Medicine, The University of Tokyo, 3 International University of Health and Welfare
Objective: To investigate the correlation between intermediate CGG expansion in the FMR1 gene and patients with multiple system atrophy（MSA） Method:
391 Japanese patients with MSA （195 males/ 167 females, 29 not identified）and 105healthy Japanese male subjects as controls were included. 225 patients were MSA-C（133males/92females）,and 137 patients were MSA-P （62males/75females）. All subjects were genetically assessed forFMR1 by repeat-primed PCR（RP-PCR） and the expansion alleles were confirmed by fragment analysis and/or Sanger sequencing. The sizes of CGG repeats of normal, intermediate, permutation, and full mutation alleles are defined as <45,45-60,60-200, and>200 CGG repeats, respectively. Results: Of the 195 MSA male patients, we identified three patients with FMR1 intermediate, giving an estimation of 1.6% among the male patients with MSA and4.9% of62 male patients with MSA-P. FMR1intermediate carriers harbor 45, 46, and 60 CGG repeats. No intermediate was detected in control group. Premutation and fullmutation were found in MSA or control cohorts. Our study showed FMR1intermediate is overrepresented in patients with MSA-P. Intermediate alleles were not observed in females with MSA in Japan. Conclusion: We revealed that FMR1intermediate alleles are observed at a considerably increased frequency in MSA-P（4.9% in MSA-P versus null in control）.
Previous studies have also suggested an increased risk in patients with Parkinson disease carrying FMR1 intermediate. Further studies of a larger group of patients with MSA-Parkinsonism and those with Parkinson disease will be needed.
一 般 演 題
ポ ス タ ー （ 英 語 ）
Pe-06-7
GeneticsandiPSCanalysisofthepatientswith thevariantsofvacuolarproteinsorting13Cgene○ArisaHayashida1,YuanzheLi1,HiroyoYoshino2,Kei-ichiIshikawa1,3, RisaNonaka3,4,YukinoriOkada5,WadoAkamatsu3,
ManabuFunayama1,2,KenyaNishioka1,NobutakaHattori1,2
1 Department of Neurology, Juntendo University School of Medicine, Tokyo, Japan, 2 Research Institute for Diseases of Old Age, Graduate School of Medicine, Juntendo University, Tokyo, Japan, 3 Center for Genomic and Regeneration Medicine, Juntendo University, Tokyo, Japan, 4 Department of Diagnosis, Prevention and Treatment Dementia, Graduate School of Medicine, Juntendo University, Tokyo, Japan, 5 Department of Statistical Genetics, Osaka University Graduate School of Medicine, Osaka, Japan [Objective] Vacuolar protein sorting 13C （VPS13C） is recently reported a novel pathogenic gene of familial Parkinson's disease （PD） with autosomal recessive inheritance. The patients presented rigid-akinesia parkinsonism, young age at onset and cognitive dysfunction. We aim to investigate the prevalence and symptoms of the patients harbouring the variants of VPS13C. [Methods] We obtained DNA samples and clinical records of 445 PD patients including 229 familial and 216 sporadic, and 216 in-house controls, and screened entire exons in VPS13C. The variants were filtered as follows; coding region and splicing site ± 20 bp, estimated minor allele frequency less than 1% in controls, to compare the number of each rare variant-positive subjects. We established induced pluripotent stem cell
（iPSC） from two patients, each having compound heterozygote and homozygote variant, and induced dopaminergic neurons to analyze the mitochondrial abilities and protein function. [Results] Rare variants were identified in 27.8% of PD and in 20.3% of controls （OR 1.51, p = 0.0379）. Next, we surveyed allele frequency, using the public gene database. The detected variants were rare in the Japanese population and significantly relate to both familial and sporadic PD. The symptoms harboring the rare variants presented a typical parkinsonism. The maximal respiration was increased in patient's iPSC-derived dopaminergic neurons. [Conclusions] The variants in VPS13C are possibly a novel relative risk for PD.
VPS13C variants could induce overactive mitochondrial respiration and may lead to neurodegeneration.
Pe-06-8
MutationanalysisofBSNgeneinpatientswith sporadicprogressivenuclearpalsy○MasahiroWakita1,TakeshiIkeuchi2,IchiroYabe1
1 Department of Neurology, Hokkaido University, Japan, 2 Department of Molecular Genetics, Brain Research Institute, Niigata University
[Objective] Clinical diagnosis of progressive supranuclear palsy （PSP） may be difficult because of various phenotypes. We have reported some mutations in the bassoon （BSN） gene in not only familial but also sporadic cases with PSP-like syndrome. In this study, we explore the mutations of the BSN gene in other patients with sporadic PSP, corticobasal syndrome （CBS） and frontotemporal lobar degeneration （FTLD）. [Methods] We included the patients with sporadic PSP, CBS and FTLD. The variants of the BSN gene were detected using the next-generation sequencer. Non-synonymous variants were screened with HGVD, iJGVD, 1000G, ExAC and gnomAD to distinguish between rare and common variants. The potential pathogenicity of the non-synonymous variants was predicted in silico using the SIFT, PolyPhen-2, PROVEAN and MutationTaster. [Results] A total of 41 patients were included （PSP, n=28; CBS, n=7; FTLD, n=6）. Non-synonymous variants in the BSN gene were detected in 7 patients （PSP, n=4; CBS, n=2; FTLD, n=1）. One patient with PSP harbors the heterozygous missense mutation in exon 5 （c.7625C>T, p.T2542M） that is very rare and predicted in silico to result in a disease-causing change in the protein. [Conclusions] We found the missense mutation in the BSN gene with a potential pathogenicity in the patient with sporadic PSP.
Pe-06-9
ThepathologicalroleofGBA2inGBA1-related centralnervoussystemdisorders○EtsuroNakanishi1,NorihitoUemura1,HisakoAkiyama2, MasatoKinoshita3,HodakaYamakado1,ShunichiTakeda4, YoshioHirabayasi5,RyosukeTakahashi1
1 Department of Neurology, Kyoto University Graduate School of Medicine, Japan, 2 Laboratory for Neural Cell Dynamics, RIKEN Center for Brain Science, 3 Division of Applied Bioscience, Kyoto University Graduate School of Agriculture, 4 Department of Radiation Genetics, Kyoto University Graduate School of Medicine, 5 Cellular Informatics Laboratory, RIKEN [Objective] Recent genetic studies have identified that heterozygous mutations in the GBA1 gene is a strong risk factor for Parkinson's disease （PD）. We have reported that the GBA1 knockout
（KO） medaka survived long enough for pathological analysis of disease progression in contrast to the perinatal death of GBA1 KO mice. The non-lysosomal β-Glucosidase （GBA2） also cleaves glucosylceramide to glucose and ceramide. A recent study has reported that the deletion of GBA2 rescues the visceral manifestations in type1 Gaucher's disease （GD） mice model through reduction of sphingosine. This study was performed to determine the pathological role of GBA2 in the central nervous system （CNS） in GD and GBA1-related PD. [Methods] We created GBA2 KO medaka by CRISPR/ Cas9 system. Then, we crossed GBA2 KO medaka with GBA1 KO medaka to create GBA1/GBA2 double-knockout （DKO） medaka. [Results] GBA2 KO medaka did not show apparent phenotypes. GBA1/GBA2 DKO medaka showed a shorter lifespan than GBA1 KO medaka. However, there were no differences in the loss of dopaminergic cells between GBA1 KO and GBA1/GBA2 DKO medaka. The deletion of GBA2 did not reduce the amount of sphingosine and the amount of α-synuclein （asyn） in GBA1 KO medaka brains. The biochemical analysis revealed a trend towards an increase in some sphingolipids and an increase in the amount of asyn in GBA2 KO medaka brains without autophagic dysfunction. [Conclusions]
GBA2 may be a novel modifier of GBA1-related CNS disorders. Howevere, the deletion of GBA2 did not rescue the CSN phenotypes of GBA1 KO medaka.
Pe-07-2
Antibodyindexofanti-agalactosylimmunoglobulin Gantibodyasabiomarkerofrheumatoidmeningitis○KeikoHatano1,GenkiShimizu1,KazutoKatsuse1,NaokoSato1, TakeshiSuzuki2,HidejiHashida1
1 Department of Neurology, Japanese Red Cross Medical Center, Tokyo, Japan, 2 Department of Allergology and Rheumatology, Japanese Red Cross Medical Center, Tokyo
Objective: Rheumatoid meningitis （RM） is a rare, diagnostically-challenging, extra-articular manifestation in rheumatoid arthritis （RA）. From 2003 to 2019, 56 cases of RM were reported, but the detection rate of rheumatoid nodules in the meninges was 60%, and MRI findings of RM are not specific for RM. We searched for biomarkers of RM. Method: We used cerebrospinal fluid （CSF） and serum of a 53-year-old female RM patient with a history of RA. Her symptoms and MRI findings deteriorated before treatment, but after steroid was initiated on day 8, they began to improve on day 13, and she was discharged on day 38. We followed the levels of rheumatoid factor （RF） in CSF and serum and antibody indices of anti-agalactosyl immunoglobulin G antibody
（CARF） and anti-cyclic citrullinated peptide antibody （anti-CCP） before （on day 4 and 7） and after （on day 27 and 45） treatment. The antibody index is the ratio between the CSF/serum quotient for specific IgG antibodies and the CSF/serum quotient for total IgG, and its elevation more than 1.3 suggests intracranial production of specific IgG antibodies. Results: RF in CSF was detectable before treatment and was undetectable after treatment. The levels of RF in serum was elevated before treatment and was reduced after treatment.
Antibody indices of CARF were 8.7, 26.8, 7.7, and 0, and those of anti-CCP were 4.9, 7.6, 9.4, and 6.7, on day 4, 7, 27, and 45. Conclusion: To our knowledge, this is the first report that antibody index of CARF, as well as RF in CSF and serum, can serve as a diagnostic and sensitive activity marker for RM.
Pe-07-3
AbnormalalpharhythminEEGanddisconnected corticalconnectivitynetworksafterHPVvaccination○YoshiyukiKuroiwa1,2,ToshiakiHirai1,KimihiroFujino1, SakuraNishijima3,ShoTsukiyama3,ToshimasaYamazaki3, ShumpeiYokota4,5,IkuroNakamura5,KusukiNishioka5,6
1 Department of Neurology and Stroke Center, Mizonokuchi Hospital, Teikyo University School of Medicine, Kawasaki, Japan, 2 Medical Office, Ministry of Japan, Tokyo, Japan, 3 Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Japan, 4 Therapeutic Center of Pediatric Intractable Diseases, Fuji Toranomon Orthopedic Hospital, Gotemba, Japan,
5 Japan Medical Research Foundation, Tokyo, Japan, 6 National Graduate Institute for Policy Studies, Tokyo, Japan
[Objective] HPV vaccination associated neuro-immunopathic syndrome （HANS） show cognitive disorders including prosopagnosia, Kanji writing disorders, topographical disorientation and hemispatial neglect.
We studied brain functional connectivity networks （BFCN）, to find the reason why they show such cognitive symptoms of cortical origin. [Methods] We performed routine EEG study in 26 HANS patients.
We constructed BFCN for 7 patients using synchronization likelihood （SL） for all frequency bands. The SL reflects the strength of coupling between any of two electrodes. [Results] We found abnormal alpha rhythm, characterized by absent waxing-waning, slow alpha, and loss of occipital dominance. The BFCN showed trans-hemispherical disconnections （Fp1-Fp2, F3-F4, C3-C4, P3-P4, O1-O2） for beta and gamma bands, and anterior-posterior disconnections between anterior （Fp1, Fp2, F3, F4, C3, C4） and posterior
（T5, T6, O1, O2） electrodes for all frequency bands. We also noted short distance disconnections in the left temporal （F7-T3-T5） and right temporal （F8-T4-T6） regions for all frequency bands, and in the left frontocentral （Fp1-F3-C3） and right frontocentral （Fp2-F4-C4） regions for beta and gamma bands.
[Conclusions] We now report for the first time both long distance （anterior-posterior networks）, and short distance disconnections （trans-hemispherical, temporal, and frontocentral networks） in HANS.
These cortical disconnections could explain cognitive disorders in HANS, and are probably related to reduced blood flow at cingulate gyrus and thalamus shown by our previous SPECT study.
Pe-07-1
Anti-NMDARpositivityinacaseoflymphomatosis cerebri（LC）presentedwithataxia:acasereport○NarudolJinkarn,JirapornJitprapaikulsan Siriraj hospital, Mahidol university, Thailand
Objective: Lymphomatosis cerebri （LC） is a rare variant of primary central nervous system lymphoma characterized by diffuse brain infiltration without mass formation. Anti-N-methyl-D-aspartate receptor （NMDAR） antibody tested by cell-based assay has high specificity for anti-NMDAR encephalitis and never been reported in lymphoma. Methods: A 63-year-old female presented with subacute ataxia and binocular hemianopia for 6 weeks.
Physical examination revealed afebrile, no rash, normal cranial nerves, vertical nystagmus, and impaired finger to nose test. MRI brain demonstrated multiple hyperintense T2w lesions without enhancement. Cerebrospinal fluid （CSF）
showed 9 WBC （L 100%）, normal protein and sugar, and 8 oligoclonal bands.
Anti-myelin oligodendrocyte glycoprotein, anti-aquaporin-4, and CSF flow cytometry were negative, but Anti-NMDAR antibody was positive. Acute disseminated encephalomyelitis with anti-NMDAR encephalitis was diagnosed.
Methylprednisolone was given. Symptoms and MRI progressed. Plasmapheresis was performed without improvement. Brain biopsy showed LC. Results: LC is able to present with subacute ataxia. The plausible explanation of positivity of anti-NMDAR antibody would be immune or bystander mechanism. Conclusion:
LC should be concerned in subacute progressive focal neurological deficits.
Co-existing autoantibodies may occur and lead to missed diagnosis. Other diseases should be aware in case of the unusual presentations of autoimmune encephalitis.
一 般 演 題
ポ ス タ ー （ 英 語 ）
Pe-06-1
Increasedβ-tubulinglutamylationintheataxia andmalesterilitymousehindersneuralmaturation○YoshieIto1,SheikhAbdullah2,ShozoYano2,TabassumShatera2, KojiOmura3,AsukaAraki3,SatoshiInagaki1,SatoshiAbe1, ShingoMitaki1,AtsushiNagai1
1 Department of Neurology, Shimane University Faculty of Medicine, Japan,
2 Department of Laboratory Medicine, Shimane University Faculty of Medicine, 3 Department of Organ Pathology, Shimane University Faculty of Medicine
【Background】 Ataxia and male sterility （AMS） is a mutant mouse strain, which contains a missense mutation in the coding region of NNA1 gene. AMS demonstrates cerebellar Purkinje cell （PC） degeneration at very early age, and ataxic phenotype is expressed in an autosomal recessive manner. 【Objective】 The objective of this study is to understand the molecular mechanism that is affected by this mutation during PC degeneration. 【Methods】 We have generated neuronal stem cell （NSC） lines from wild type （NMW7）, NNA1 mutation heterozygous （NME） and NNA1 mutation homozygous
（NMO1） mice brains. We also analyzed time-dependent changes in AMS cerebellum at pathological and molecular levels. 【Results】 The growth rate of NMO1 was faster than other NSC lines. Although NMO1 could make neurospheres, some of them were attached to the plate after 3 days' culture. Immunostaining revealed that SOX2 and nestin levels were decreased in NMO1-derived neurospheres. Conversely, β-tubulin level was high in NMO1 and NME. Neuron differentiation potentials of NMO1 were less than NME or NMW7. Further, β-tubulin was high in NMO1 after neuron differentiation. Although total β-tubulin was not changed, glutamylated tubulin level was increased in AMS mice cerebellum both at 15 and 30 days. Similarly, glutamylated tubulin level was increased in NMO1-derived neurospheres without changing β-tubulin transcription. 【Conclusion】
Thus, our results suggest that glutamylated β-tubulin level is increased early in the cerebellum, which disturbs neuron maturation and induce PC death in AMS mice.
Pe-06-2
Establishmentofspinocerebellarataxiatype 36modelfly○TomoyaTaminato1,MorioUeyama1,ToruYamashita3, YoshioIkeda2,KojiAbe3,YoshitakaNagai1
1 Dept Neurotherapeutics, Osaka Univ Grad Sch of Med, Osaka, Japan, 2 Dept Neurology, Gunma Univ Grad Sch of Med, Gunma, Japan, 3 Dept Neurology, Okayama Univ Grad Sch of Med, Okayama, Japan
OBJECTIVE:Spinocerebellar Ataxia type 36 （SCA36） is one of the non-coding repeat expansion disorders （NRDs） which is caused by an GGCCTG repeat expansion （>25） in the first intron of the NOP56 gene. Remarkably, SCA36 patients develop not only ataxia but also motor neuron symptoms similar to ALS. A pathological hallmark of NRDs, such as C9orf72-linked ALS/FTD, is the accumulation of expanded repeat RNA as RNA foci. Furthermore, it is known that the expanded repeat RNA is translated into proteins by unconventional repeat associated non-ATG （RAN） translation. METHODS:To elucidate the molecular mechanisms of SCA36, we established novel Drosophila models expressing GGCCTG repeat RNA. Drosophila is a powerful genetic system for identification of genetic modifier or drug candidates because of their short lifespan, large numbers of embryos and accumulated knowledge about their genetics. In order to obtain a long repeat sequence with flanking region of NOP56 gene, we amplified subcloned （GGCCTG）43 sequences from the SCA36 patient by PCR and ligated these amplicons. Then transgenic flies were made using these constructs.RESULTS:When we expressed GGCCTG repeats in the compound eyes, we observed obvious eye degeneration in （GGCCTG）118 expressing flies, while
（GGCCTG）7 expressing flies showed almost normal eye phenotype. Consistent with SCA36 patients, intranuclear RNA foci were detected by fluorescent in situ hybridization. Furthermore, we found the expression of poly （Gly-Pro）, which is one of the RAN proteins, in eye imaginal discs by immunohistochemistry.
Pe-06-3
Geneticscreeningforpotassiumchannelmutations inautosomaldominantspinocerebellarataxia○HiroyukiMorino1,YuiTada1,2,KodaiKume1,YukikoMatsuda1, TakashiKurashige3,YuheiKanaya1,HayatoTabu4,SatoshiKaneko5, ToshihikoSuenaga6,AkiraKakizuka2,HideshiKawakami1
1 Department of Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan, 2 Laboratory of Functional Biology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan,
3 Department of Neurology, National Hospital Organization Kure Medical Center and Chugoku Cancer Center, Kure, Japan, 4 Department of Neurology, Kitano Hospital, Osaka, Japan, 5 Department of Neurology, Kansai Medical University, Osaka, Japan, 6 Department of Neurology, Tenri Hospital, Tenri, Japan
Objective: Spinocerebellar ataxia （SCA） is a genetically heterogeneous disease characterized by cerebellar ataxia, and many causative genes have been identified so far. The most common etiology is the abnormal expansion of repeat sequences, and mutations of ion channel genes also play an important role in the development of SCA. However, there are still few reports on the mutations of the potassium channel genes.
Methods: We screened 192 Japanese individuals with dominant inherited SCA, who had no abnormal repeat expansions of causative genes, for potassium channel mutations （KCNC3 for SCA13 and KCND3 for SCA19/
SCA22） by target sequencing. Results: Two variants were identified from two patients: c.1973G>A,p.R658Q and c.1018G>A,p.V340M in KCNC3, and no pathogenic variant was identified in KCND3. The newly identified p.V340M exists in the extracellular domain, and p.R658Q exists in the intracellular domain on the C-terminal side, although most of the reported KCNC3 mutations are present at the transmembrane site. Discussion: Adult-onset and slowly progressive cerebellar ataxia are the main clinical features of SCA13 and SCA19 caused by potassium channel mutations, which was similar in our cases. SCA13 caused by KCNC3 mutations may present with deep sensory loss and cognitive impairment in addition to cerebellar ataxia. In this study, mild deep sensory loss was observed in one case. SCA caused by potassium channel gene mutations is extremely rare, and more cases should be accumulated in the future to elucidate its pathogenesis due to channel dysfunction.
Pe-06-4
EffectsoftheoverexpressionofTFEBincellular andmousemodelsofneurodegenerativediseases○HiroakiAdachi1,ZheHuang2,KazumasaOkada1,KeikoOhnari1, TomoyoHashimoto1,TomokoToyota1,YukioIwanaka1
1 Department of Neurology, University of Occupational and Environmental Health School of Medicine, Japan, 2 Department of Community Healthcare and Geriatrics, Nagoya University Graduate School of Medicine
[Background] Macroautophagy is a set of bulk degradation processesin which cells form double-membrane vesicles, called autophagosomes,around a portion of the cytoplasm. The transcription factor EB （TFEB） has been reported to regulate autophagy by upregulating genes that belong to the coordinated lysosomal expression and regulation （CLEAR） network, thereby controlling lysosomal biogenesis. Spinal and bulbar muscular atrophy （SBMA） is an inherited motor neuron disease caused by the expansion of a polyglutamine （polyQ） tract within the androgen receptor （AR）. We examined the effects of the overexpression of TFEB in cultured cell models of neurodegenerative diseases and in a transgenic mouse model that overexpressed the mutant AR （SBMA mice）. [Materials and Methods] Cells were transfected with plasmids encoding mutant AR, huntingtin, ataxin-1, ataxin-3 and TFEB. We generated transgenic mice overexpressing full-length human TFEB under the control of prion protein promoter, and crossed the SBMA mice with mice overexpressing human TFEB. [Results] The overexpression of TFEB decreased the expression of each causative protein in the neuronal cell models （p< 0.01）. The expression of the autophagic marker LC-3 II was significantly elevated in the cells expressing TFEB （p< 0.01）. TFEB overexpression improved SBMA mouse phenotypes by inducing autophagy.
[Conclusions] These findings demonstrated that the high expression of TFEB induced autophagy and enhanced the preferential degradation of the disease-causative proteins, and promoted therapeutic effects in an SBMA model mice.
Pe-06-5
Biochemicalanalysisofmiddle-age-onsetSCAR causedbyabiallelicmutationofHSD17B4○YukikoMatsuda1,HiroyukiMorino1,TakashiKurashige2, RyosukeMiyamoto3,HirofumiMaruyama4,HideshiKwakami1
1 Dept. Epidemiology, RIRBM, Hiroshima Univ., Japan, 2 Dept. Neurology, Natl. Hosp. Org. Kure Med. Ctr. and Chugoku Cancer Ctr., 3 Dept. Clinical Neuroscience., Inst. Biomed. Sci., Tokushima Univ., 4 Dept. Clinical Neuroscience and Therapeutics, Hiroshima Univ.
[Objective] We identified a homozygous mutation in the dehydrogenase domain of HSD17B4 gene as a cause for slowly progressive spinocerebellar ataxia, autosomal recessive （SCAR） using exome sequencing. In order to determine the biochemical underpinnings, we conducted the functional analysis of D-bifunctional protein （DBP） encoded by HSD17B4. [Methods] We assessed the expression level of DBP and the amount of dimerized DBP by immunoblotting and quantitative reverse transcription polymerase chain reaction using patient-derived fibroblasts and HEK293T cells overexpressing mutant DBP. We also performed morphological evaluation using immunocytochemistry. [Results]
mRNA expression levels of DBP were normal. However, protein levels were diminished in the patient-derived fibroblasts, and relative amount of DBP in peroxisome was reduced. A decrease in DBP expression was observed in HEK293T cells overexpressing mutant DBP. Moreover, the dimer which is a functional form of DBP was also decreased. [Conclusions] DBP mutations that cause severe functional impairment lead to severe infant-onset abnormalities.
The DBP mutation causing mild dysfunction analyzed in this study showed mild clinical symptoms and a phenotype of middle-age-onset slowly progressive SCAR. This study broadens the scope of DBP deficiency phenotypes and indicates that residual functional DBP may be used to estimate the severity of DBP deficiencies.
Pe-06-6
AssociationofFMR1intermediatealleleswith multiplesystematrophy○MAsemAlmansour1,HiroyukiIshiura1,JunMitsui2,ShojiTsuji2,3, TatsushiToda1
1 Department of Neurology, Graduate School of Medicine, The University of Tokyo, Japan, 2 Department of Molecular Neurology, Graduate School of Medicine, The University of Tokyo, 3 International University of Health and Welfare
Objective: To investigate the correlation between intermediate CGG expansion in the FMR1 gene and patients with multiple system atrophy（MSA） Method:
391 Japanese patients with MSA （195 males/ 167 females, 29 not identified）and 105healthy Japanese male subjects as controls were included. 225 patients were MSA-C（133males/92females）,and 137 patients were MSA-P （62males/75females）. All subjects were genetically assessed forFMR1 by repeat-primed PCR（RP-PCR） and the expansion alleles were confirmed by fragment analysis and/or Sanger sequencing. The sizes of CGG repeats of normal, intermediate, permutation, and full mutation alleles are defined as <45,45-60,60-200, and>200 CGG repeats, respectively. Results: Of the 195 MSA male patients, we identified three patients with FMR1 intermediate, giving an estimation of 1.6% among the male patients with MSA and4.9% of62 male patients with MSA-P. FMR1intermediate carriers harbor 45, 46, and 60 CGG repeats. No intermediate was detected in control group. Premutation and fullmutation were found in MSA or control cohorts. Our study showed FMR1intermediate is overrepresented in patients with MSA-P. Intermediate alleles were not observed in females with MSA in Japan. Conclusion: We revealed that FMR1intermediate alleles are observed at a considerably increased frequency in MSA-P（4.9% in MSA-P versus null in control）.
Previous studies have also suggested an increased risk in patients with Parkinson disease carrying FMR1 intermediate. Further studies of a larger group of patients with MSA-Parkinsonism and those with Parkinson disease will be needed.