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Abstract Hereditary transthyretin (ATTRv) amyloidosis is a rare and autosomal dominant disorder associated with mutations in the transthyretin gene. Patients present with diverse symptoms related to sensory, motor, and autonomic neuropathy, as well as gastrointestinal, ocular, cardiac, renal and orthopedic symptoms, resulting from the deposition of transthyretin amyloid fibrils in multiple organs. The progressive nature of ATTRv amyloidosis necessitates pre- and post-onset monitoring of the disease. This review article is primarily based on a collation of discussions from a medical advisory board meeting in August 2021. In this article, we summarize the best practices in amyloidosis centers in three major endemic countries for ATTRv amyloidosis (Japan, Brazil, and Portugal), where most patients carry the Val30Met mutation in the transthyretin gene and the patients’ genetic background was proven to be the same. The discussions highlighted the similarities and differences in the management of asymptomatic gene mutation carriers among the three countries in terms of the use of noninvasive tests and tissue biopsies and timing of starting the investigations. In addition, this article discusses a set of practical tests and examinations for monitoring disease progression applicable to neurologists working in diverse medical settings and generalizable in non-endemic countries and areas. This set of assessments consists of periodic (every 6 to 12 months) evaluations of patients’ nutritional status and autonomic, renal, cardiac, ophthalmologic, and neurological functions. Physical examinations and patient-reported outcome assessments should be also scheduled every 6 to 12 months. Programs for monitoring gene mutation carriers and robust referral networks can aid in appropriate patient management in pre- to post-onset stages. For pre- and post-symptom onset testing for ATTRv amyloidosis, various noninvasive techniques are available; however, their applicability differs depending on the medical setting in each country and region, and the optimal option should be selected in view of the clinical settings, medical environment, and available healthcare resources in each region.

Hereditary transthyretin (TTR) amyloidosis (ATTRv amyloidosis) is autosomal dominant and caused by mutation of TTR gene. Heterozygous ATTR Tyr114Cys (p.Tyr134Cys) amyloidosis is a lethal disease with a life expectancy of about 10 years after onset of the disease. However, the molecular pathogenesis of ATTR Tyr114Cys amyloidosis is still largely unknown. In this study, we took advantage of disease-specific induced pluripotent stem (iPS) cells and generated & characterized the heterozygous ATTR Tyr114Cys amyloidosis-specific iPS cells (Y114C iPS cells), to determine whether Y114C iPS cells could be useful for elucidating the pathogenesis of ATTR Tyr114Cys amyloidosis. We successfully differentiated heterozygous Y114C iPS cells into hepatocyte like cells (HLCs) mainly producing TTR protein. On day 27 after differentiation, the expression of hepatocyte maker albumin was detected, and TTR expression was significantly increased in HLCs differentiated from Y114C iPS cells. LC–MS/MS analysis showed that both WT TTR & ATTR Y114C protein were indeed expressed in the HLCs differentiated from Y114C iPS cells. Notably, the number of detected peptides derived from ATTR Y114C protein was lower than that of WT TTR protein, indeed indicating the clinical phenotype of ATTR Tyr114Cys amyloidosis. Taken together, we first reported the heterozygous Y114C iPS cells generated from patient with ATTR Tyr114Cys amyloidosis, and suggested that Y114C iPS cells could be a potential pathological tool, which may contribute to elucidating the molecular pathogenesis of heterozygous ATTR Tyr114Cys amyloidosis.

A mitochondrial tRNA modification enzyme, NSUN3, is found to be essential for embryonic development in mice, with whole-body knockout causing embryonic lethality and heart-specific Nsun3 knockout leading to heart aberration in older mice.

Abstract In patients with Parkinson’s disease (PD), α-synuclein pathology is thought to spread to the brain via the dorsal motor nucleus of the vagus nerve. The link between the gut microbiome and PD has been explored in various studies. The appendix might play an important role in immunity by maintaining the microbiota as a reservoir. In recent times, appendectomy has been linked to a lower risk of PD, possibly owing to the role of the appendix in altering the gut microbiome. We aimed to elucidate whether the gut microbiota affects PD development in the appendectomy cohort. We analyzed the fecal microbial composition in patients with PD and healthy controls with and without a history of appendectomy. The abundance of microbes from the family Enterobacteriaceae was higher in feces samples from patients with Parkinson’s disease compared to that in samples collected from healthy controls. Furthermore, there was a significant phylogenetic difference between patients with PD and healthy controls who had undergone appendectomy. There was a significant phylogenetic difference between patients with PD and HCs who had undergone APP. These results suggest the correlation between gut microbiota and PD in patients who have undergone APP.

Transthyretin (TTR), the precursor protein for amyloidogenic TTR (ATTR) amyloidosis, forms tetramers and escapes glomerular filtration by binding with thyroxine and retinol-binding protein. However, variant TTRs are unstable as tetramers, so monomeric TTR has become the precursor protein of amyloid deposits, via protein misfolding. The aim of the study was to evaluate the utility of urinary TTR in the diagnosis of ATTRv amyloidosis. Urinary samples from healthy volunteers, ATTRv V50M amyloidosis patients, and asymptomatic carriers of the ATTRv V50M gene were analysed using ELISA. To analyse the different forms of TTR secreted to the urine, we performed Western blotting and mass spectrometry. Urinary TTR concentrations were significantly higher in the ATTRv V50M amyloidosis patients than they were in the healthy volunteers and asymptomatic carriers of the gene. Although the TTR concentrations were negligible in the healthy volunteers, they were correlated with disease progression and urinary albumin concentrations in the ATTRv V50M amyloidosis patients. The Western blotting and mass spectrometry revealed the presence of monomeric wild-type and variant TTRs in the urine. Urinary TTR concentrations may become a more sensitive biomarker of ATTRv progression than albumin.

Ultraviolet (UV) irradiation originating in the space environment has been increasing because of the destruction of the ozone layer. Although UV irradiation damages DNA, peptides, and cell structures, it also has the beneficial effects of suppressing excessive immune responses and cell proliferation. However, the effects of UV irradiation on amyloid fibril formation are unknown. Here, we analyzed its effects on amyloid fibril formation of two types of amyloidogenic proteins, human amyloid β42 (Aβ42) and human transthyretin (TTR). To study these effects, we incubated Aβ42 samples in neutral pH buffer and wild-type TTR (TTRwt) samples in acidic buffer, with and without UV irradiation. We analyzed amyloidogenicity via a thioflavin T (ThT) method and morphological changes by using electron microscopy. UV irradiation reduced the ThT fluorescence intensity of the human Aβ42 samples. For the electron microscopic analysis of Aβ42 samples, the UV irradiation induced thinning of the amyloid fiber tips. Although UV irradiation reduced the ThT fluorescence intensity of human TTRwt samples, the amounts of the aggregates did not differ with or without UV irradiation. Our experiments suggest that UV irradiation may become a useful tool for reducing the amyloid fibrils and amyloid like substances.

We aimed to establish a novel murine model of autoimmune autonomic ganglionopathy (AAG), which represents autoimmune dysautonomia, associated with MHC class II to understand its pathomechanism and the pathogenicity of nicotinic acetylcholine receptor (nAChR) antibodies. The amino acid sequence of the mouse nAChRα3 protein was analyzed using an epitope prediction tool to predict the possible MHC class II binding mouse nAChRα3 peptides. We focused on two nAChRα3 peptides in the extracellular region, and experimental AAG (EAAG) was induced by immunization of C57BL/6 mice with these two different peptides. EAAG mice were examined both physiologically and histologically. Mice with EAAG generated nAChRα3 antibodies and exhibited autonomic dysfunction, including reduced heart rate, excessive fluctuations in systolic blood pressure, and intestinal transit slowing. Additionally, we observed skin lesions, such as alopecia and skin ulcers, in immunized mice. Neuronal cell density in the sympathetic cervical ganglia in immunized mice was significantly lower than that in control mice at the light microscopic level. We interpreted that active immunization of mice with nAChRα3 peptides causes autonomic dysfunction similar to human AAG induced by an antibody-mediated mechanism. We suggested a mechanism by which different HLA class II molecules might preferentially affect the nAChR-specific immune response, thus controlling diversification of the autoantibody response. Our novel murine model mimics AAG in humans and provides a useful tool to investigate its pathomechanism.

ObjectivesPatients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) show various clinical symptoms, including migraine, recurrent stroke, and cognitive impairment. We investigated the associations between magnetic resonance imaging (MRI) markers of small vessel disease and neuropsychological tests and identified the MRI characteristics for predicting cognitive impairment in patients with CADASIL.MethodsSubjects included 60 CADASIL patients diagnosed with genetic tests and registered in the Japanese CADASIL REDCap database between June 2016 and December 2020. Patient information including clinical data, modified Rankin Scale (mRS); MRI findings of small vessel disease including periventricular and deep white matter lesions (WML), lacunar infarcts, and cerebral microbleeds (CMBs); and neuropsychological tests, including the Japanese version of the Mini-Mental State Examination (MMSE), the Japanese version of the Montreal Cognitive Assessment (MoCA-J), and the Frontal Assessment Battery (FAB), were evaluated.ResultsData from 44 CADASIL patients were eligible for this study, compared between patients with and without dementia. Regarding the neuroimaging findings, the Fazekas score of periventricular and deep WML was higher in patients with dementia (periventricular, p = 0.003; deep, p = 0.009). The number of lacunar infarcts was higher in patients with dementia (p = 0.001). The standardized partial regression coefficient (SPRC) in MoCA-J was 0.826 (95% CI, 0.723–0.942; p = 0.005) for the number of CMBs. The SPRC in MMSE was 0.826 (95% CI, 0.719–0.949; p = 0.007) for the number of CMBs. The SPRC for FAB decreased significantly to 0.728 (95% CI, 0.551–0.960; p = 0.024) for the number of lacunar infarcts. Receiver operating characteristic (ROC) curves for dementia showed that in the number of lacunar infarcts, a cut-off score of 5.5 showed 90.9% sensitivity and 61.1% specificity. For the number of CMBs, a cut-off score of 18.5 showed 45.5% sensitivity and 100% specificity.ConclusionThe characteristic MRI findings were that CADASIL patients with dementia had severe WML, both periventricular and deep, and a larger number of lacunar infarcts than those without dementia. The risk of dementia may be associated with ≥ 6 lacunar infarcts, ≥19 CMBs, or a Fazekas scale score of 3 in periventricular and deep WML.

Abstract Aims Hereditary transthyretin (ATTRv) amyloidosis is the most frequent and representative form of autosomal dominant hereditary systemic amyloidosis. Disease‐modifying treatments of the disease are more effective during the early stages, and we require biomarkers to detect early pathological changes for prompt diagnosis. This study aimed to investigate whether plasma growth differentiation factor 15 (GDF‐15) levels could aid detection of early pathological changes in ATTRv amyloidosis. Methods and results We retrospectively studied 32 patients with ATTRv amyloidosis, eight asymptomatic TTR mutation carriers, and eight healthy volunteers. We evaluated plasma GDF‐15 levels in these subjects as related to levels of brain natriuretic peptide and high‐sensitivity troponin T, echocardiographic features, 99mTc‐pyrophosphate (PYP) scans, and cardiac magnetic resonance imaging findings. Plasma GDF‐15 levels significantly increased even in asymptomatic TTR mutation carriers compared with healthy volunteers (P

Abstract Background Transthyretin amyloidosis (also known as ATTR amyloidosis) is a systemic, life-threatening disease characterized by transthyretin (TTR) fibril deposition in organs and tissue. A definitive diagnosis of ATTR amyloidosis is often a challenge, in large part because of its heterogeneous presentation. Although ATTR amyloidosis was previously considered untreatable, disease-modifying therapies for the treatment of this disease have recently become available. This article aims to raise awareness of the initial symptoms of ATTR amyloidosis among general practitioners to facilitate identification of a patient with suspicious signs and symptoms. Methods These consensus recommendations for the suspicion and diagnosis of ATTR amyloidosis were developed through a series of development and review cycles by an international working group comprising key amyloidosis specialists. This working group met to discuss the barriers to early and accurate diagnosis of ATTR amyloidosis and develop a consensus recommendation through a thorough search of the literature performed using PubMed Central. Results The cardiac and peripheral nervous systems are most frequently involved in ATTR amyloidosis; however, many patients often also experience gastrointestinal and other systemic manifestations. Given the multisystemic nature of symptoms, ATTR amyloidosis is often misdiagnosed as a more common disorder, leading to significant delays in the initiation of treatment. Although histologic evaluation has been the gold standard to confirm ATTR amyloidosis, a range of tools are available that can facilitate early and accurate diagnosis. Of importance, genetic testing should be considered early in the evaluation of a patient with unexplained peripheral neuropathy. Conclusions A diagnostic algorithm based on initial red flag symptoms and manifestations of cardiac or neurologic involvement will facilitate identification by the general practitioner of a patient with clinically suspicious symptoms, enabling subsequent referral of the patient to a multidisciplinary specialized medical center.

Abstract To date, almost all case reports of insulin‐derived amyloidosis described the presence of a subcutaneous mass that was observable on physical examination. This report presents two cases of insulin‐derived amyloidosis without palpable masses at insulin injection sites. In both cases, blood glucose concentrations improved, and the insulin dose could be reduced by an average of 45% after changing the insulin injection sites. The insulin absorption at the site was reduced to at most 40% of that at a normal site in one case. Magnetic resonance imaging and ultrasonography were useful to screen and differentiate insulin‐derived amyloidosis without a palpable mass. This report showed that insulin‐derived amyloidosis without a palpable mass can be present at the insulin injection site, and has similar clinical effects to insulin‐derived amyloidosis with palpable masses.

The MATP/tau protein is hyperphosphorylated in Alzheimer’s patients. Therefore, research into the regulation of tau protein phosphorylation is important for understanding Alzheimer’s disease. HASPIN is a serine/threonine kinase that is expressed in various cells. To examine whether HASPIN is involved in the onset of Alzheimer’s disease through tau protein phosphorylation, we investigated the effects of a diet including soybean sprouts rich in the HASPIN inhibitor coumestrol in a mouse model of Alzheimer’s disease (5xFAD). The results showed that HASPIN was expressed in the hippocampus and phosphorylated tau protein, while the ingestion of soybean sprouts containing coumestrol suppressed the development of spatial cognitive dysfunction in 5xFAD. These results indicate that HASPIN may be one of the target molecules for the repression of tau phosphorylation in the treatment of Alzheimer’s disease.

Transthyretin (TTR) is an amyloidogenic protein associated with hereditary and nonhereditary transthyretin amyloidoses (ATTR). Dissociation of the tetramer of TTR to the monomer induces TTR misfolding, which leads to amyloid fibril formation and triggers the onset of ATTR amyloidosis. Stabilizers of tetrameric TTR have been accepted as an effective ATTR amyloidosis treatment while effect is limited and they are too expensive. The aim of our study was to find more effective and cheep natural compound to suppress TTR amyloid formation. Glabridin, a prenylated isoflavan isolated from Glycyrrhiza glabra L., stabilized the TTR tetramer in vitro. The effects of licorice-derived flavonoid oil—Glavonoid, a natural substance that includes glabridin and several polyphenols—on stabilizing the TTR tetramer must still be elucidated. To examine plasma TTR stabilization by Glavonoid in vitro, we investigated the feasibility of utilizing glabridin plus Glavonoid to prevent TTR amyloid fibril formation. Glavonoid mixed with human plasma samples at 24 h incubation in vitro increased the tetramer level (P < 0.05) and reduced the monomer level (P < 0.01) and the monomer/tetramer ratio (P < 0.05) of TTR compared to those without Glavonoid by immunoblot analysis, such effect could not observe in the presence of glabridin. Oral Glavonoid (300 mg for 12 weeks) in 7 healthy volunteers effectively increased the plasma glabridin concentration. Glavonoid increased the TTR tetramer level and reduced the monomer/tetramer ratio of TTR (P < 0.05) in plasma at 12 weeks in healthy volunteers compared to those of age matched control subjects without the supplement. Thus, oral Glavonoid may effectively prevent TTR amyloid fibril formation via TTR tetramer stabilization. Glavonoid may become a promising supplement before onset of ATTR amyloidosis.

Abstract Objective To characterize the CSF cytokine profile in chronic inflammatory demyelinating polyneuropathy (CIDP) patients with IgG4 anti‐neurofascin 155 (NF155) antibodies (NF155+ CIDP) or those lacking anti‐NF155 antibodies (NF155− CIDP). Methods Twenty‐eight CSF cytokines/chemokines/growth factors were measured by multiplexed fluorescent immunoassay in 35 patients with NF155+ CIDP, 36 with NF155− CIDP, and 28 with non‐inflammatory neurological disease (NIND). Results CSF CXCL8/IL‐8, IL‐13, TNF‐α, CCL11/eotaxin, CCL2/MCP‐1, and IFN‐γ were significantly higher, while IL‐1β, IL‐1ra, and G‐CSF were lower, in NF155+ CIDP than in NIND. Compared with NF155− CIDP, CXCL8/IL‐8 and IL‐13 were significantly higher, and IL‐1β, IL‐1ra, and IL‐6 were lower, in NF155+ CIDP. CXCL8/IL‐8, IL‐13, CCL11/eotaxin, CXCL10/IP‐10, CCL3/MIP‐1α, CCL4/MIP‐1β, and TNF‐α levels were positively correlated with markedly elevated CSF protein, while IL‐13, CCL11/eotaxin, and IL‐17 levels were positively correlated with increased CSF cell counts. IL‐13, CXCL8/IL‐8, CCL4/MIP‐1β, CCL3/MIP‐1α, and CCL5/RANTES were decreased by combined immunotherapies in nine NF155+ CIDP patients examined longitudinally. By contrast, NF155− CIDP had significantly increased IFN‐γ compared with NIND, and exhibited positive correlations of IFN‐γ, CXCL10/IP‐10, and CXCL8/IL‐8 with CSF protein. Canonical discriminant analysis of cytokines/chemokines revealed that NF155+ and NF155− CIDP were separable, and that IL‐4, IL‐10, and IL‐13 were the three most significant discriminators. Interpretation Intrathecal upregulation of type 2 helper T (Th2) cell cytokines is characteristic of IgG4 NF155+ CIDP, while type 1 helper T cell cytokines are increased in CIDP regardless of the presence or absence of anti‐NF155 antibodies, suggesting that overproduction of Th2 cell cytokines is unique to NF155+ CIDP.

Abstract Background Insulin-derived amyloidosis is a skin-related complication of insulin therapy that interferes with insulin therapy. Although toxicities of in vitro-formed insulin amyloid fibrils have been well studied, the toxicity of insulin-derived amyloidosis remains to be clarified. Case presentation A 58-year-old man with type 2 diabetes mellitus underwent a lower limb amputation due to diabetic gangrene. Several antibiotics including minocycline were administered for infection and sepsis. A hard mass at the insulin injection sites in the lower abdomen was discovered by chance four months later. Although no abnormal findings in the surface skin of the mass were observed, necrotic tissue was seen around the mass when a biopsy was performed. Histological and toxicity studies were performed for this patient and four other patients with abdominal masses at insulin injection sites. Histological and immunohistochemical studies showed that the masses had typical characteristics of amyloid deposits in all cases, whereas necrotic findings were seen adjacent to the amyloid deposit only in the case presented. Toxicity studies indicated that the amyloid tissue from the present case had significant cell toxicity compared to the control skin tissue or the amyloid tissues from the other four cases. Conclusions This report showed that toxic insulin-derived amyloidosis can occur. In addition, this report suggested that toxic insulin-derived amyloidosis may cause necrosis in the surrounding tissue. Although the toxic amyloid deposit of insulin-derived amyloidosis was found in only one patient, no structural differences between toxic and non-toxic deposits were seen on histological and immunohistochemical studies.

Abstract Background Hereditary transthyretin amyloidosis (ATTRv amyloidosis) is caused by a variant transthyretin (TTR), which is a serum protein secreted by the liver. Mass spectrometry (MS) is a useful tool that can detect variant TTRs in serum samples from patients with ATTRv amyloidosis. We previously reported several mass spectrometric methods to detect variant TTRs in serum samples. Those methods require cumbersome immunoprecipitation with anti-TTR antibodies and significant time to analyze the variant TTRs. In our study here, we developed a new simple and quick method to detect variant TTRs in serum samples by means of matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) MS without immunoprecipitation (direct MALDI). Methods By using direct MALDI, we analyzed 288 serum samples obtained from patients who were clinically suspected having amyloidosis to investigate the usefulness of this direct MALDI method to detect variant TTRs in serum samples. Results The method completed the process within 30 min. We successfully identified variant TTRs in serum samples from patients, except for a few patients with TTR Glu61Lys and Glu89Gln mutations because of the small mass shift of those variant TTRs from wild-type TTR. We also found that the mass shifts of variant TTRs measured by direct MALDI corresponded to theoretical mass changes. Conclusion Our results suggest that the direct MALDI method is useful for the screening of ATTRv amyloidosis.

Abstract Sporadic cerebral amyloid angiopathy (CAA) is characterized by cerebrovascular amyloid-β (Aβ) deposition, which leads to lobar hemorrhage and dementia. Biological molecules affecting the development of CAA have not been fully characterized. In this study, we performed proteome analysis of biopsied leptomeningeal and cortical vessels obtained from 6 CAA patients and 5 non-CAA patients who underwent surgery for large lobar hemorrhages. We found that 6 proteins, including Aβ, apolipoprotein E (apoE), clusterin (CLU), albumin, complement C4 and vitronectin were significantly upregulated in the vessels of CAA patients as compared to non-CAA patients. ApoE and CLU were found in all CAA patients. We next examined the effects of apoE and CLU on the early phase of Aβ aggregation, using a simple yet powerful in vitro model of CAA, which recapitulates the intramural periarterial drainage pathway model. We found that physiological concentrations of apoE and CLU delayed the initiation time of amyloid growth kinetics in a concentration-dependent manner. These data indicate that apoE and CLU may act as extracellular chaperones to inhibit Aβ amyloid deposition in CAA.

Alzheimer’s disease (AD) is an age-related neurodegenerative disease that is characterized by irreversible memory loss and cognitive decline. The deposition of amyloid-β (Aβ), especially aggregation-prone Aβ42, is considered to be an early event preceding neurodegeneration in AD. Sirtuins (SIRT1–7 in mammals) are nicotinamide adenine dinucleotide-dependent lysine deacetylases/deacylases, and several sirtuins play important roles in AD. However, the involvement of SIRT7 in AD pathogenesis is not known. Here, we demonstrate that SIRT7 mRNA expression is increased in the cortex, entorhinal cortex, and prefrontal cortex of AD patients. We also found that Aβ42 treatment rapidly increased NADPH oxidase 4 (NOX4) expression at the post-transcriptional level, and induced reactive oxygen species (ROS) production and apoptosis in neuronal SH-SY5Y cells. In contrast, SIRT7 knockdown inhibited Aβ42-induced ROS production and apoptosis by suppressing the upregulation of NOX4. Collectively, these findings suggest that the inhibition of SIRT7 may play a beneficial role in AD pathogenesis through the regulation of ROS production.

Abstract Background Transthyretin amyloidosis is a systemic disorder caused by extracellular deposition of insoluble amyloid fibrils in peripheral and autonomic nerves, heart, kidney, gastrointestinal tract, and other organs. Hereditary transthyretin amyloidosis is an autosomal dominant disease. More than 120 mutations have been reported in the transthyretin gene with considerable phenotypic heterogeneity and geographic diversity. Among them, a sporadic case of hereditary transthyretin amyloidosis with cardiac-predominant phenotype is very rare, progressive, and potentially fatal if left undiagnosed. However, a clinical diagnosis of cardiac amyloidosis still remains challenging due to non-specific symptoms, and less sensitivity and specificity of medical examinations. Case presentation A 60-year-old Japanese man with a history of embolic stroke and hypertrophic cardiomyopathy visited our department for heart failure. The present case exhibited only cardiomyopathy without any clinical signs of systemic amyloidosis manifested as carpal tunnel syndrome, polyneuropathy, or autonomic dysfunction. An echocardiogram revealed severe asymmetric left ventricular hypertrophy, biatrial dilatation, pericardial effusion, and preserved left ventricular ejection fraction of 50% with severe diastolic dysfunction. Technetium pyrophosphate scintigraphy indicated marked diffuse myocardial uptake of technetium pyrophosphate, strongly suggesting transthyretin cardiac amyloidosis, which was firmly confirmed by a left ventricular endomyocardial biopsy. Genetic analysis demonstrated a transthyretin C70T (Pro24Ser) heterozygous mutation. Tafamidis, a transthyretin stabilizer, was started. His cardiac symptoms remained unchanged for 12 months. Conclusions Here we report the case of a patient with hereditary cardiac amyloidosis associated with a Pro24Ser mutation in transthyretin, which is the first case reported in Japan. Technetium pyrophosphate scintigraphy was extremely useful for definitive diagnosis. Thus, we propose that the nuclear imaging technique should be taken into account even for an exploratory diagnosis of transthyretin cardiac amyloidosis.

In the future, humans may live in space because of global pollution and weather fluctuations. In microgravity, convection does not occur, which may change the amyloidogenicity of proteins. However, the effect of gravity on amyloid fibril formation is unclear and remains to be elucidated. Here, we analyzed the effect of microgravity on amyloid fibril formation of amyloidogenic proteins including insulin, amyloid β42 (Aβ42), and transthyretin (TTR). We produced microgravity (10−3 g) by using the gravity controller Gravite. Human insulin, Aβ42, and human wild-type TTR (TTRwt) were incubated at pH 3.0, 7.0, and 3.5 at 37 °C, respectively, in 1 g on the ground or in microgravity. We measured amyloidogenicity via the thioflavin T (ThT) method and cell-based 1-fluoro-2,5-bis[(E)-3-carboxy-4-hydroxystyryl]benzene (FSB) assay. ThT fluorescence intensity and cell-based FSB assay results for human insulin samples were decreased in microgravity compared with results in 1 g. Aβ42 samples did not differ in ThT fluorescence intensity in microgravity and in 1 g on the ground. However, in the cell-based FSB assay, the staining intensity was reduced in microgravity compared with that on 1 g. Human TTRwt tended to form fewer amyloid fibrils in ThT fluorescence intensity and cell-based FSB assays in microgravity than in 1 g. Human insulin and Aβ42 showed decreased amyloid fibril formation in microgravity compared with that in 1 g. Human TTRwt tended to form fewer amyloid fibrils in microgravity. Our experiments suggest that the earth's gravity may be an accelerating factor for amyloid fibril formation.

HASPIN is a serine/threonine kinase that regulates mitosis by phosphorylating histone H3 at threonine 3. The expression levels of HASPIN in various cancers are associated with tumor malignancy and poor survival, suggesting that HASPIN inhibition may suppress cancer growth. As HASPIN mRNA levels are elevated in human breast cancer tissues compared with adjacent normal tissues, we examined the growth-suppressive effects of CHR-6494, a potent HASPIN inhibitor, in breast cancer cell lines in vitro and in vivo. We found that HASPIN was expressed in breast cancer cells of all molecular subtypes, as well as in immortalized mammary epithelial cells. HASPIN expression levels appeared to be correlated with the cell growth rate but not the molecular subtype of breast cancer. CHR-6494 exhibited potent antiproliferative effects against breast cancer cell lines and immortalized mammary epithelial cells in vitro, but failed to inhibit the growth of MDA-MB-231 xenografted tumors under conditions that have significant effects in a colorectal cancer model. These results imply that CHR-6494 does have antiproliferative effects in some situations, and further drug screening efforts are anticipated to identify more potent and selective HASPIN inhibition for use as an anticancer agent in breast cancer patients.

Objectives: To better determine the usefulness of mechanical thrombectomy for stroke patients with middle cerebral artery (MCA) branch occlusion, we investigated predictive factors related to poor outcome in acute, MCA branch occlusion stroke patients who had not undergone mechanical thrombectomy. Patients and methods: We retrospectively evaluated 204 consecutive acute stroke patients with MCA branch (M2-M4) occlusion. Two groups, based on activity of daily living scores at discharge, were compared: a dependent outcome group (mRS 3–6; n = 109) and an independent outcome group (mRS 0–2; n = 95), and the factors related to dependent outcomes at discharge determined. Results: The dependent group was older, had higher National Institutes of Health Stroke Scale (NIHSS) scores on admission, higher serum D-dimer and brain natriuretic peptide levels, and lower Alberta Stroke Program Early CT scores (ASPECTS) and rates of intravenous thrombolysis than the independent group. In multivariate analysis, age (OR 1.06; 95%CI 1.03–1.10), admission NIHSS score (OR 1.12; 95%CI 1.05–1.19), ASPECTS (OR 0.56; 95%CI 0.40–0.79), and intravenous thrombolysis (OR 0.22; 95%CI 0.08–0.57) were related to dependent outcomes. Decision curve analysis demonstrated that admission NIHSS scores were better predictors of dependency for threshold probabilities from 0.25 to 0.46. The optimal cutoff scores for predicting dependency were ≥8 (sensitivity 0.76, specificity 0.62) for the admission NIHSS score. Conclusions: Admission NIHSS scores might be useful predictors of dependent outcomes at discharge in acute ischemic stroke patients with MCA branch occlusion.

ObjectivesClinical characteristics of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) include migraine, recurrent stroke, white matter lesions, and vascular dementia. CADASIL is one of the most common hereditary cerebral small vessel diseases. Clinical presentation of CADASIL varies and a racial gap may exist between the Asian and Caucasian populations. This is the first nationwide epidemiological survey which aimed to elucidate the clinical features of CADASIL in Japan. Moreover, the registration database of CADASIL was constructed.MethodsSubjects included CADASIL patients who visited the hospitals (totally 1,448 hospitals) certified by the Japanese Society of Neurology and/or Japan Stroke Society in 2016. This study consisted of a two-step survey; patients with CADASIL were identified genetically by the first questionnaire, and their clinical features were assessed by the second questionnaire. Selected 6 hospitals registered the data of all CADASIL patients using a Research Electronic Data Capture (REDCap) system for the second questionnaire.ResultsBased on the criteria, 88 patients (50 male and 38 female) with CADASIL were enrolled. The mean age of symptom onset was 49.5 years. Sixteen (18.2%) patients had an elderly onset (>60 years). Thirteen patients (13.6%) had history of migraine with aura and 33 patients (37.5%) had vascular risk factor(s). From among the 86 patients who were examined using magnetic resonance imaging, abnormal deep white matter lesions were detected in 85 patients (98.8%), WMLs extending to anterior temporal pole in 73 patients (84.9%), and cerebral microbleeds in 41 patients (47.7%). Anti-platelet therapy was received by 65 patients (73.9%). Thirty-eight patients (43.2%) underwent treatment with lomerizine hydrochloride. Thirty-four different mutations of NOTCH3 were found in exons 2, 3, 4, 5, 6, 8, 11, 14, and 19. Most of the mutations existed in exon 4 (n = 44, 60.3%). The prevalence rate of CADASIL was 1.20 to 3.58 per 100,000 adults in Japan.ConclusionThis questionnaire-based study revealed clinical features and treatment status in Japanese CADASIL patient, although it may not be an exhaustive search. We have constructed the REDCap database for these CADASIL patients.

SP7/Osterix is a transcription factor involved in osteoblast differentiation. Here, the authors show that Sirtuin 7 activates Osterix posttranslationally by regulating its lysine acylation, and that mice lacking Sirtuin 7 in osteoblasts show reduced bone formation.

Background and purpose: Oxidative stress has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Edaravone, a free radical scavenger, was approved as a therapeutic drug for ALS in 2015 in Japan. A phase 3 clinical trial demonstrated a smaller decline in ALS functional scale scores compared with placebo. However, the long-term effects of edaravone on ALS patients remain unclear. This study aimed to retrospectively investigate the long-term effects of edaravone on the survival of ALS patients. Methods: We retrospectively analyzed 27 consecutive patients with ALS who were treated with edaravone and 30 consecutive ALS patients who were not treated with edaravone between 2010 and 2016. Results: The differences of ALSFRS-R scores from baseline to 6 months was significantly reduced in the edaravone group, compared to the control group. The changes in serum creatinine, as a possible marker of ALS severity, from baseline to 6 and 12 months were significantly improved in the edaravone group, compared to the control group. The survival rate was significantly improved in the edaravone group compared with control patients. Conclusion: Our retrospective single-center analysis suggests slower progression and better prognosis of ALS patients with edaravone treatment. Further investigation, including prospective multicenter analysis, is warranted to confirm the usefulness of edaravone for a better prognosis of ALS. Keywords: Amyotrophic lateral sclerosis, Oxidative stress, Edaravone, Long-term effect, Survival

Abstract Hereditary ATTR (ATTRm) amyloidosis (also called transthyretin-type familial amyloid polyneuropathy [ATTR-FAP]) is an autosomal-dominant, adult-onset, rare systemic disorder predominantly characterized by irreversible, progressive, and persistent peripheral nerve damage. TTR gene mutations (e.g. replacement of valine with methionine at position 30 [Val30Met (p.Val50Met)]) lead to destabilization and dissociation of TTR tetramers into variant TTR monomers, which form amyloid fibrils that deposit in peripheral nerves and various organs, giving rise to peripheral and autonomic neuropathy and several non-disease specific symptoms. Phenotypic and genetic variability and non–disease-specific symptoms often delay diagnosis and lead to misdiagnosis. Red-flag symptom clusters simplify diagnosis globally. However, in Japan, types of TTR variants, age of onset, penetrance, and clinical symptoms of Val30Met are more varied than in other countries. Hence, development of a Japan-specific red-flag symptom cluster is warranted. Presence of progressive peripheral sensory-motor polyneuropathy and ≥1 red-flag sign/symptom (e.g. family history, autonomic dysfunction, cardiac involvement, carpal tunnel syndrome, gastrointestinal disturbances, unexplained weight loss, and immunotherapy resistance) suggests ATTR-FAP. Outside of Japan, pharmacotherapeutic options are first-line therapy. However, because of positive outcomes (better life expectancy and higher survival rates) with living donor transplant in Japan, liver transplantation remains first-line treatment, necessitating a Japan-specific treatment algorithm. Herein, we present a consolidated review of the ATTR-FAP Val30Met landscape in Japan and summarize findings from a medical advisory board meeting held in Tokyo on 18th August 2016, at which a Japan-specific ATTR-FAP red-flag symptom cluster and treatment algorithm was developed. Beside liver transplantation, a TTR-stabilizing agent (e.g. tafamidis) is a treatment option. Early diagnosis and timely treatment using the Japan-specific red-flag symptom cluster and treatment algorithm might help guide clinicians regarding apt and judicious use of available treatment modalities.

Abstract Muscle satellite cells are essential for muscle regeneration. However, efficient regeneration does not occur without muscle-resident mesenchymal progenitor cells. We show here that bone marrow-derived mesenchymal stromal cells (Bm-MSCs) also facilitate muscle regeneration in Duchenne muscular dystrophy (DMD) model mice. Bm-MSCs transplanted into peritoneal cavities of DMD model mice with severe muscle degeneration strongly suppressed dystrophic pathology and improved death-related symptoms, which resulted in dramatic lifespan extension. Isolated single myofibers from Bm-MSC-transplanted mice manifested considerably less myofiber splitting compared with myofibers from non-transplanted mice, which indicated that transplantation significantly ameliorated abnormal regeneration. With regard to the number of satellite cells, several cells remained on myofibers from Bm-MSC-transplanted model mice, but satellite cells rarely occurred on myofibers from non-transplanted mice. Also, CXCL12 was crucial for muscle regeneration. CXCL12 facilitated muscle regeneration and paired box protein–7 (PAX7) expression after cardiotoxin-related muscle injury in vivo. The majority of primary muscle satellite cells sorted by integrin-α7 and CD34 expressed CXCR4, a receptor specific for CXCL12. CXCL12 strongly suppressed p-STAT3 expression in these sorted cells in vitro. CXCL12 may therefore influence muscle regeneration through STAT3 signaling in satellite cells. Targeting these proteins in or on muscle satellite cells may improve many degenerative muscle diseases.

Abstract The relationship between familial amyloid polyneuropathy (FAP), which is caused by mutated transthyretin (TTR), and inflammation has only recently been noted. To determine whether inflammation is present in FAP carriers and patients, serum interleukin (IL)−6 concentration in 57 healthy donors (HD), 21 FAP carriers, and 66 FAP patients was examined, with the relationship between IL-6 and TTR assessed in each group by multiple regression analysis and structural equation models (SEM). Compared with HD, IL-6 concentration was elevated in FAP carriers (p = 0.001, 95% CI 0.398–1.571) and patients (p = 0.002, 95% CI 0.362–1.521). Further, SEM indicated a positive relationship between IL-6 and TTR in FAP carriers (p = 0.010, 95% CI 0.019–0.140), but not in HD and FAP patients. In addition, we determined whether TTR induces production of pro-inflammatory cytokines ex vivo. HD-derived CD14 + monocytes and induced pluripotent stem cell-derived myeloid lineage cells from a HD and FAP patient dose-dependently produced IL-6 under mutated and aggregated TTR conditions, compared with wild-type TTR. In conclusion, FAP carriers and patients are in an inflammatory state, with the presence of mutated TTR being a trigger of inflammation, especially in FAP carriers.

Cerebral amyloid angiopathy (CAA) is characterized by accumulation of amyloid β (Aβ) in walls of leptomeningeal vessels and cortical capillaries in the brain. The loss of integrity of these vessels caused by cerebrovascular Aβ deposits results in fragile vessels and lobar intracerebral hemorrhages. CAA also manifests with progressive cognitive impairment or transient focal neurological symptoms. Although development of therapeutics for CAA is urgently needed, the pathogenesis of CAA remains to be fully elucidated. In this review, we summarize the epidemiology, pathology, clinical and radiological features, and perspectives for future research directions in CAA therapeutics. Recent advances in mass spectrometric methodology combined with vascular isolation techniques have aided understanding of the cerebrovascular proteome. In this paper, we describe several potential key CAA-associated molecules that have been identified by proteomic analyses (apolipoprotein E, clusterin, SRPX1 (sushi repeat-containing protein X-linked 1), TIMP3 (tissue inhibitor of metalloproteinases 3), and HTRA1 (HtrA serine peptidase 1)), and their pivotal roles in Aβ cytotoxicity, Aβ fibril formation, and vessel wall remodeling. Understanding the interactions between cerebrovascular Aβ deposits and molecules that accumulate with Aβ may lead to discovery of effective CAA therapeutics and to the identification of biomarkers for early diagnosis.

Autonomic neuropathy has been reported in autoimmune rheumatic diseases (ARD) including Sjögren’s syndrome, systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus. However, the pathophysiological mechanism underlying autonomic dysfunction remains unknown to researchers. On the other hand, autoimmune autonomic ganglionopathy (AAG) is an acquired immune-mediated disorder, which causes dysautonomia that is mediated by autoantibodies against ganglionic acetylcholine receptors (gAChRs). The purpose of this review was to describe the characteristics of autonomic disturbance through previous case reports and the functional tests used in these studies and address the importance of anti-gAChR antibodies. We have established luciferase immunoprecipitation systems to detect antibodies against gAChR in the past and determined the prevalence of gAChR antibodies in various autoimmune diseases including AAG and rheumatic diseases. Autonomic dysfunction, which affects lower parasympathetic and higher sympathetic activity, is usually observed in ARD. The anti-gAChR antibodies may play a crucial role in autonomic dysfunction observed in ARD. Further studies are necessary to determine whether anti-gAChR antibody levels are correlated with the severity of autonomic dysfunction in ARD.

The purpose of this study was to evaluate the therapeutic potential of human induced pluripotent stem (iPS) cell-derived macrophage-like cells for Alzheimer's disease (AD). In previous studies, we established the technology to generate macrophage-like myeloid lineage cells with proliferating capacity from human iPS cells, and we designated the cells iPS-ML. iPS-ML reduced the level of Aβ added into the culture medium, and the culture supernatant of iPS-ML alleviated the neurotoxicity of Aβ. We generated iPS-ML expressing the Fc-receptor-fused form of a single chain antibody specific to Aβ. In addition, we made iPS-ML expressing Neprilysin-2 (NEP2), which is a protease with Aβ-degrading activity. In vitro, expression of NEP2 but not anti-Aβ scFv enhanced the effect to reduce the level of soluble Aβ oligomer in the culture medium and to alleviate the neurotoxicity of Aβ. To analyze the effect of iPS-ML expressing NEP2 (iPS-ML/NEP2) in vivo, we intracerebrally administered the iPS-ML/NEP2 to 5XFAD mice, which is a mouse model of AD. We observed significant reduction in the level of Aβ in the brain interstitial fluid following administration of iPS-ML/NEP2. These results suggested that iPS-ML/NEP2 may be a potential therapeutic agent in the treatment of AD.

Familial amyloidotic polyneuropathy (FAP) is a hereditary amyloidosis induced by amyloidogenic transthyretin (ATTR). Because most transthyretin (TTR) in serum is synthesized by the liver, liver transplantation (LT) is today the only treatment available to halt the progression of FAP, even though LT is associated with several problems. Despite the urgent need to develop alternatives to LT, the detailed pathogenesis of FAP is still unknown; also, no model fully represents the relevant processes in patients with FAP. The induction of induced pluripotent stem (iPS) cells has allowed development of pluripotent cells specific for patients and has led to useful models of human diseases. Because of the need for a tool to elucidate the molecular pathogenesis of FAP, in this study we sought to establish heterozygous ATTR mutant iPS cells, and were successful, by using a Sendai virus vector mixture containing four transcription factors (Oct3/4, Sox2, Klf4, and c-Myc) to reprogram dermal fibroblasts derived from FAP patients. Moreover, FAP-specific iPS cells had the potential to differentiate into hepatocyte-like cells and indeed expressed ATTR. FAP-specific iPS cells demonstrated the possibility of serving as a pathological tool that will contribute to understanding the pathogenesis of FAP and development of FAP treatments.

We hypothesized that tissue-resident macrophages in familial amyloid polyneuropathy (FAP) patients will exhibit qualitative or quantitative abnormalities, that may accelerate transthyretin (TTR)-derived amyloid deposition. To evaluate this, we examined the number and subset of tissue-resident macrophages in heart tissue from amyloid-deposited FAP and control patients. In both FAP and control patients, tissue-resident macrophages in heart tissue were all Iba+/CD163+/CD206+ macrophages. However, the number of macrophages was significantly decreased in FAP patients compared with control patients. Furthermore, the proportion of intracellular TTR in CD14+ monocytes was reduced in peripheral blood compared with healthy donors. Based on these results, we next examined degradation and endocytosis of TTR in human induced pluripotent stem (iPS) cell-derived myeloid lineage cells (MLs), which function like macrophages. iPS-MLs express CD163 and CD206, and belong to the inhibitory macrophage category. In addition, iPS-MLs degrade both native and aggregated TTR in a cell-dependent manner in vitro. Further, iPS-MLs endocytose aggregated, and especially polymerized, TTR. These results suggest that decreased tissue-localized macrophages disrupt clearance of TTR-derived amyloid deposits, leading to progression of a pathological condition in FAP patients. To improve this situation, clinical application of pluripotent stem cell-derived MLs may be useful as an approach for FAP therapy.

OBJECTIVE: Secondary glaucoma is a serious complication in patients with transthyretin (TTR)-related familial amyloidotic polyneuropathy (FAP). We assessed the long-term outcomes and complications of trabeculectomy with mitomycin C (MMC) for secondary glaucoma associated with FAP. METHODS: Medical case records of Kumamoto University Hospital were retrospectively reviewed. Twenty-one eyes of 13 patients (10 with FAP ATTR Val30Met; 3 with FAP ATTR Tyr114Cys) underwent trabeculectomy with MMC and follow-up of at least 2 years. The primary outcome measure was Kaplan-Meier survival, with failure of this treatment being defined as an intraocular pressure (IOP) of ≤5 mm Hg or ≥22 mm Hg on two consecutive visits or as additional operations needed to reduce IOP. Secondary outcome measures included complications, bleb characteristics, and additional postoperative interventions required. RESULTS: The mean postoperative follow-up period was 5.7 years (range, 2.2-12.7 years). Kaplan-Meier analysis indicated probabilities of success of 0.76, 0.67, and 0.53 at 1, 2, and 3 years after operation, respectively. Significant complications included ocular decompression retinopathy in 7 eyes (33%) and bleb encapsulation in 10 eyes (48%). Twelve eyes (57%) needed additional surgery, such as bleb revision or trabeculectomy with MMC, to reduce IOP. CONCLUSIONS: Trabeculectomy with MMC may not be optimal for patients with FAP-related glaucoma and may have several significant complications.

We herein demonstrate the immune-regulatory effect of embryonic stem cell-derived dendritic cells (ES-DCs) using two models of autoimmune disease, namely non-obese diabetic (NOD) mice and experimental autoimmune encephalomyelitis (EAE). Treatment of pre-diabetic NOD mice with ES-DCs exerted almost complete suppression of diabetes development during the observation period for more than 40 weeks. The prevention of diabetes by ES-DCs was accompanied with significant reduction of insulitis and decreased number of Th1 and Th17 cells in the spleen. Development of EAE was also inhibited by the treatment with ES-DCs, and the therapeutic effect was obtained even if ES-DCs were administrated after the onset of clinical symptoms. Treatment of EAE-induced mice with ES-DCs reduced the infiltration of inflammatory cells into the spinal cord and suppressed the T cell response to the myelin antigen. Importantly, the ES-DC treatment did not affect T cell response to an exogenous antigen. As the mechanisms underlying the reduction of the number of infiltrating Th1 cells, we observed the inhibition of differentiation and proliferation of Th1 cells by ES-DCs. Furthermore, the expression of VLA-4α on Th1 cells was significantly inhibited by ES-DCs. Considering the recent advances in human induced pluripotent stem cell-related technologies, these results suggest a clinical application for pluripotent stem cell-derived dendritic cells as a therapy for T cell-mediated autoimmune diseases.