[Investigation into the Effect of Shitei Extract, a Traditional Chinese Medicine Formulation, on Hiccups].

Hiccups are occasionally experienced by most individuals, and although not life-threatening, they may leadto a decline in quality of life. Shitei extract(shitei, persimmon calyx)may be usedfor the treatment of hiccups. The effects of shitei extract were investigatedin patients admittedto the Japanese RedCross Musashino Hospital between October 2011 andM ay 2016. Numerous prescriptions for shitei extract were reportedin the Department of Respiratory Organs andNeurosurgery. The primary causes of hiccups were chemotherapy andbrain disease. Of 149 patients, 107(71.8%)sufferedfrom hiccups. None of the patients reported adverse events related to the administration of shitei extract. The incidence of hiccups was significantly higher in patients with brain disease(p=0.0075), treatedwith chemotherapy for cancer(p=0.0402), or requiring the insertion of a gastric tube(p=0.0065). Among those treatedwith chemotherapy for cancer, shitei extract was effective against hiccups in 82.0% patients(45 of 55). Furthermore, four patients receivedprophylaxis with shitei extract for the prevention of hiccups after chemotherapy. In conclusion, these results indicate that shitei extract is an effective and safe therapeutic drug for the treatment of hiccups. In particular, shitei extract was effective in the prevention of hiccups after chemotherapy.

Paraneoplastic Neuromyelitis Optica Spectrum Disorder Related to Glucose-regulated Protein 78 (GRP78) Autoantibodies in a Patient with Lynch Syndrome-associated Colorectal Cancer.

Neuromyelitis optica spectrum disorders have been previously reported in a paraneoplastic context, although there is no clear consensus on their pathogenesis. We herein report a case of aquaporin-4 antibody-positive neuromyelitis optica spectrum disorder in a 64-year-old woman with colorectal cancer. She underwent tumor resection, resulting in serum aquaporin-4 antibody titers subsequently becoming negative. Serum samples were also positive for glucose-regulated protein 78 antibody, which has recently been suggested to be a novel factor in the disruption of the blood-brain barrier. Serological and pathological investigations in this case highlight the role and involvement of aquaporin-4 and glucose-regulated protein 78 antibodies in paraneoplastic conditions.

Spinocerebellar ataxia type 31 is associated with "inserted" penta-nucleotide repeats containing (TGGAA)n.

Spinocerebellar ataxia type 31 (SCA31) is an adult-onset autosomal-dominant neurodegenerative disorder showing progressive cerebellar ataxia mainly affecting Purkinje cells. The SCA31 critical region was tracked down to a 900 kb interval in chromosome 16q22.1, where the disease shows a strong founder effect. By performing comprehensive Southern blot analysis and BAC- and fosmid-based sequencing, we isolated two genetic changes segregating with SCA31. One was a single-nucleotide change in an intron of the thymidine kinase 2 gene (TK2). However, this did not appear to affect splicing or expression patterns. The other was an insertion, from 2.5-3.8 kb long, consisting of complex penta-nucleotide repeats including a long (TGGAA)n stretch. In controls, shorter (1.5-2.0 kb) insertions lacking (TGGAA)n were found only rarely. The SCA31 repeat insertion's length inversely correlated with patient age of onset, and an expansion was documented in a single family showing anticipation. The repeat insertion was located in introns of TK2 and BEAN (brain expressed, associated with Nedd4) expressed in the brain and formed RNA foci in the nuclei of patients' Purkinje cells. An electrophoretic mobility-shift assay showed that essential splicing factors, serine/arginine-rich splicing factors SFRS1 and SFRS9, bind to (UGGAA)n in vitro. Because (TGGAA)n is a characteristic sequence of paracentromeric heterochromatin, we speculate that the insertion might have originated from heterochromatin. SCA31 is important because it exemplifies human diseases associated with "inserted" microsatellite repeats that can expand through transmission. Our finding suggests that the ectopic microsatellite repeat, when transcribed, might cause a disease involving the essential splicing factors.

Cardioembolic Stroke Due to Cardiac Sarcoidosis Diagnosed by Pathological Evaluation of the Retrieved Thrombus.

A 70-year-old woman undergoing glucocorticoid therapy for cardiac sarcoidosis was brought to our hospital with the sudden onset of right hemiplegia and aphasia. Brain magnetic resonance imaging showed a high diffusion-weighted imaging signal in the left frontotemporal lobe and disruption of blood flow in the M1 segment of the left middle cerebral artery. Hence, she underwent thrombolysis and mechanical thrombectomy, resulting in marked improvement in her neurological symptoms. A pathologic evaluation of the thrombus suggested its cardiogenicity, and the absence of any obvious abnormality other than a left ventricular aneurysm indicated stroke due to a cardioembolic etiology secondary to cardiac sarcoidosis.

The carboxy-terminal fragment of alpha(1A) calcium channel preferentially aggregates in the cytoplasm of human spinocerebellar ataxia type 6 Purkinje cells.

Spinocerebellar ataxia type 6 (SCA6) is an autosomal dominant neurodegenerative disease caused by a small polyglutamine (polyQ) expansion (control: 4-20Q; SCA6: 20-33Q) in the carboxyl(C)-terminal cytoplasmic domain of the alpha(1A) voltage-dependent calcium channel (Ca(v)2.1). Although a 75-85-kDa Ca(v)2.1 C-terminal fragment (CTF) is toxic in cultured cells, its existence in human brains and its role in SCA6 pathogenesis remains unknown. Here, we investigated whether the small polyQ expansion alters the expression pattern and intracellular distribution of Ca(v)2.1 in human SCA6 brains. New antibodies against the Ca(v)2.1 C-terminus were used in immunoblotting and immunohistochemistry. In the cerebella of six control individuals, the CTF was detected in sucrose- and SDS-soluble cytosolic fractions; in the cerebella of two SCA6 patients, it was additionally detected in SDS-insoluble cytosolic and sucrose-soluble nuclear fractions. In contrast, however, the CTF was not detected either in the nuclear fraction or in the SDS-insoluble cytosolic fraction of SCA6 extracerebellar tissues, indicating that the CTF being insoluble in the cytoplasm or mislocalized to the nucleus only in the SCA6 cerebellum. Immunohistochemistry revealed abundant aggregates in cell bodies and dendrites of SCA6 Purkinje cells (seven patients) but not in controls (n = 6). Recombinant CTF with a small polyQ expansion (rCTF-Q28) aggregated in cultured PC12 cells, but neither rCTF-Q13 (normal-length polyQ) nor full-length Ca(v)2.1 with Q28 did. We conclude that SCA6 pathogenesis may be associated with the CTF, normally found in the cytoplasm, being aggregated in the cytoplasm and additionally distributed in the nucleus.

[Molecular genetic approach to spinocerebellar ataxias].

Spinocerebellar ataxia (SCA) is a group of degenerative ataxias with autosomal dominant inheritance. The most common form of mutation that causes SCA is the expansion of trinucleotide (CAG) repeat encoding polyglutamine. These "polyglutamine disorders" are, SCA1, SCA2, Machado-Joseph disease, SCA6, SCA7, SCA17 and DRPLA. Another dynamic mutation, yet a non-coding one, has been identified as the cause of SCA8, SCA10 and SCA12. This mutation includes, trinucleotide (CAG/CTG) expansion causing SCA8 and SCA12, and pentanuclotide (ATTCT) expansion leading SCA10. In addition to these dynamic mutations, static mutations, such as missense mutations and deletions, have been identified to cause SCA5, SCA11, SCA13, SCA14, SCA15 and SCA27. Since 1992, authors have been involved in identifying the mutation (s) of autosomal dominant cerebellar ataxia with rather pure cerebellar syndrome (ADCAIII). About a half of our cohort with ADCAIII were SCA6, caused by a small CAG repeat expansion in the alpha1A-voltage-dependent calcium channel gene. Recent study in patients' brains suggested that a small polyglutamine expansion leads a portion of this channel protein to aggregate in the Purkinje cell. Another type of ADCAIII is the chromosome 16q22.1-linked ADCA. By a comprehensive positional cloning strategy, we have found a genetic change that segregate with the disease. Identifying the mutation of 16q-ADCA is imperative for understanding molecular basis of this disease.

Myocardial nerve fibers are preserved in MPTP-treated mice, despite cardiac sympathetic dysfunction.

Recently, we reported a profound depletion of cardiac sympathetic nerve fibers in Parkinson's disease (PD). This cardiac sympathetic denervation is a characteristic hallmark of PD. Cardiac sympathetic dysfunction was also observed in 1-methyl-4-phenyl-1,2,3,6-tetrahydroxypyridine (MPTP)-treated mice, a model of PD. Although binding assay showed a decreased density of norepinephrine transporter (NET) in the hearts of the mice, their histopathological alterations have not been demonstrated. In this study, we investigated hearts of MPTP-treated mice with immunohistochemical method and Western blot analyses. MPTP-treated mice showed significant decreases in the contents of cardiac noradrenaline and dopamine, suggesting the sympathetic dysfunction. Synaptophysin-, tyrosine hydroxylase- or NET-immunoreactive nerve fibers were abundant in the hearts of control mice and MPTP-treated mice, without apparent differences between the two groups. Western blot analyses also showed no difference in the amounts of these proteins. Myocardial nerve fibers were well preserved in MPTP-treated mice, despite apparent cardiac sympathetic dysfunction.

Analyses of copy number and mRNA expression level of the alpha-synuclein gene in multiple system atrophy.

Multiple system atrophy (MSA) is a sporadic neurodegenerative disease manifested clinically by progressive ataxia, parkinsonism, and autonomic dysfunction. Its cause is unknown, and there is no curative therapy. Alpha-synuclein is an important protein forming aggregations called glial cytoplasmic inclusions (GCIs) in oligodendroglia; these aggregations are considered important in MSA pathogenesis. Overexpression of the human alpha-synuclein gene in mice induces the formation of GCI-like aggregations in oligodendrocytes, leading mice to exhibit neurological signs similar to those in MSA patients. However, previous studies have excluded mutations within the coding region of the alpha-synuclein gene in MSA patients. To determine whether alteration in the expression level of the alpha-synuclein gene is associated with MSA pathogenesis, we used TaqMan quantitative PCR assay to analyze the alpha-synuclein gene copy number in patients' genomes. We also used quantitative RT-PCR and in situ hybridization to analyze alpha-synuclein mRNA expression in MSA patients' brain tissues. We found no alteration in the alpha-synuclein gene copy number in the patients' genomes (n = 50). Quantitative analysis for alpha-synuclein mRNA by the TaqMan method showed that alpha-synuclein mRNA levels were comparable between control (n = 3) and MSA (n = 3) cerebella. On in situ hybridization, the number of neurons with alpha-synuclein mRNA expression was no greater in the cerebella of MSA patients (n = 3) than in the controls (n = 3). However, GCIs were seen in these MSA specimens on immunohistochemistry for alpha-synuclein. These results suggest that alpha-synuclein gene expression is not the fundamental cause of MSA.

Profound cardiac sympathetic denervation occurs in Parkinson disease.

In the last few years, cardiac sympathetic dysfunction in Parkinson disease (PD) has been postulated on the basis of decreased cardiac uptake of sympathoneural imaging tracers. However, the pathological substrate for the dysfunction remains to be established. We examined the left ventricular anterior wall from postmortem specimens with immunohistochemical staining for tyrosine hydroxylase (TH), neurofilament (NF) and S-100 protein in PD patients and control subjects, and quantified the immunoreactive areas. As TH-immunoreactive axons nearly disappeared and NF-immunoreactive axons drastically decreased in number, the morphological degeneration of the cardiac sympathetic nerves in PD was confirmed. Quantitative analysis showed that sympathetic nerves were preferentially involved. Triple immunofluorolabeling for NF, TH, and myelin basic protein showed clearly the profound involvement of sympathetic axons in PD. The extent of involvement of the cardiac sympathetic nerves seems likely to be equivalent to that in the central nervous system, including the nigrostriatal dopaminergic system. PD affects the cardiac sympathetic nervous system profoundly as well as nigrostriatal dopaminergic system.

[A useful marker for differential diagnosis of Parkinson's disease--MIBG myocardial scintigraphy].

We performed [123I]-meta-iodobenzylguanidine (MIBG) myocardial scintigraphy in 180 patients with PD, 24 patients with DLB and 73 patients with the other related diseases. The heart-to-mediastinum (H/M) ratio in PD and DLB was significantly low compared to that in MSA, PSP, CBD and AD. The H/M ratio tended to decrease with the disease progression in PD. These findings suggest that MIBG myocardial scintigraphy could be a marker for differential diagnosis of PD and DLB. We immunohistochemically examined heart tissues from patients with pathologically confirmed PD, DLB and the other related diseases using monoclonal antibody against tyrosine hydroxylase (TH). The number of TH-immunoreactive nerve fibers in the epicardium was moderate or abundant in most patients with MSA, PSP, CBD and AD as well as in the control subjects. Contrarily, the number of TH-immunoreactive nerve fibers in the epicardium had almost completely disappeared in nearly all the patients with PD and DLB. These findings suggest that postganglionic cardiac sympathetic nerve denervation occurs in PD and DLB but not in MSA, PSP, CBD and AD, which accounts for the decreased cardiac uptake of MIBG in PD and DLB, but not in the other diseases.

Serum amyloid A level is increased in neuromyelitis optica and atypical multiple sclerosis with smaller T2 lesion volume in brain MRI.

Serum amyloid A (SAA) is known to promote the development of T helper 17 cells (Th17) and can be a critical mediator of disease pathogenesis. We analyzed SAA levels in 40 patients with multiple sclerosis (MS) and related disorders, and 10 with non-inflammatory neurological disease (NIND) as controls. We found that SAA levels were significantly increased in neuromyelitis optica (NMO) patients and relapsing and remitting MS (RRMS) patients showing atypical phenotype with spinal cord lesions and smaller T2 lesion volume in brain MRI, resembling NMO. Therefore, SAA levels can be associated with clinical phenotypes in MS and NMO.

Autoimmune polyendocrine syndrome-3 in a patient with late-onset multiple sclerosis.

We present here the rare case of a 73-year-old patient with very late-onset multiple sclerosis who developed autoimmune polyendocrine syndrome (APS)-3. Despite only a few reports describing the association between multiple sclerosis and APS, both of these diseases have been shown to be associated with HLA-DR4. Intriguingly, the HLA genotype profile of this patient included HLA-DR4 which, fine mapped to the DRB1*0405-DQA1*0303-DQB1*0401 extended haplotype, reported to be a susceptibility haplotype for APS-3 in Japan. This unique genetic background might explain the clinical picture of this patient.

[Dissecting molecular mechanism of spinocerebellar ataxia type 31].

Spinocerebellar ataxia is a group of neurodegenerative disorders clinically presenting adult onset cerebellar ataxia. To date, 21 different genes (SCA1, 2, 3, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 17, 23, 27, 28, 31, 35, 36 and DRPLA) and additionally 10 different gene loci (SCA4, 18, 19, 20, 21, 25, 26, 29, 30 and 32) are identified. Among these, SCA6 and SCA31 are the two common diseases clinically presenting as a relatively predominant cerebellar syndrome, whereas Machado-Joseph disease/SCA3, DRPLA, SCA1 and SCA2 are SCAs often associated with extra-cerebellar manifestations. SCA31 is a late-onset purely cerebellar ataxia caused by a complex pentanucleotide repeat containing (TGGAA)(n) lying in an intronic region shared by two genes, BEAN (brain expressed, associated with NEDD4) and TK2 (thymidine kinase 2). In situ hybridization analysis in patients' Purkinje cells demonstrated that pentanucleotide repeats transcribed in BEAN direction form RNA aggregates ("RNA foci"), and essential splicing factors, SFRS1 and SFRS9, bind to (UGGAA)(n), the transcript of (TGGAA)(n)in vitro. Our preliminary data also demonstrated that (UGGAA)(n) is toxic when expressed in cultured cells. These findings may imply that RNA-mediated pathogenesis is involved in SCA31. Further studies are needed to explore precise mechanism of this disease.

[Spinocerebellar ataxia type 31].

Spinocerebellar ataxia type 31 (SCA31) is a relatively common degenerative ataxia in Japan. We recently discovered SCA31 mutation as a complex pentanucleotide repeat containing (TAAAA)(n), (TAGAA)(n), and (TGGAA)(n). The size of this repeat ranged from 2.8 to 3.5 kilo-base pairs (kb). Among these repeats, (TGGAA)(n) repeat appears crucial for SCA31 pathogenesis. The length of this complex repeat inversely correlated with ages of onset in patients. The mutation lies in an intron shared by two different genes, BEAN (brain expressed, associated with NEDD4) and TK2 (thymidine kinase 2), which are transcribed in opposite directions. Thus, the complex pentanucleotide sequence is predicted to be transcribed in both directions, but not necessarily translated into proteins. In situ hybridization analysis in patients' Purkinje cells demonstrated that pentanucleotide repeats transcribed in BEAN direction form RNA aggregates ("RNA foci"). We further found that splicing factors, SFRS1 and SFRS9, binds to (UGGAA)(n), the transcript of (TGGAA)(n) in vitro. These findings may imply that SCA31 conforms to pathogenic mechanisms underlying non-coding repeat disorders, such as myotonic dystrophies (DM1 & DM2), and that SFRS1 and SFRS9 are involved in SCA31 pathogenesis.