Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods

The concept that abnormal protein aggregates show prion-like propagation between cells has been considered to explain the onset and progression of many neurodegenerative diseases. Indeed, both synthetic amyloid-like fibrils and pathogenic proteins extracted from patients’ brains induce self-templated amplification and cell-to-cell transmission in vitro and in vivo. However, it is unclear whether exposure to exogenous prion-like proteins can potentially cause these diseases in humans. Here, we investigated in detail the prion-like seeding activities of several kinds of pathogenic α-synuclein (α-syn), including synthetic fibrils and detergent-insoluble fractions extracted from brains of patients with α-synucleinopathies. Exposure to synthetic α-syn fibrils at concentrations above 100 pg/mL caused seeded aggregation of α-syn in SH-SY5Y cells, and seeded aggregation was also observed in C57BL/6 J mice after intracerebral inoculation of at least 0.1 μg/animal. α-Syn aggregates extracted from brains of multiple system atrophy (MSA) patients showed higher seeding activity than those extracted from patients with dementia with Lewy bodies (DLB), and their potency was similar to that of synthetic α-syn fibrils. We also examined the effects of various methods that have been reported to inactivate abnormal prion proteins (PrPSc), including autoclaving at various temperatures, exposure to sodium dodecyl sulfate (SDS), and combined treatments. The combination of autoclaving and 1% SDS substantially reduced the seeding activities of synthetic α-syn fibrils and α-syn aggregates extracted from MSA brains. However, single treatment with 1% SDS or generally used sterilization conditions proved insufficient to prevent accumulation of pathological α-syn. In conclusion, α-syn aggregates derived from MSA patients showed a potent prion-like seeding activity, which could be efficiently reduced by combined use of SDS and autoclaving

Prevalence of childhood obstructive sleep apnea syndrome and its role in daytime sleepiness

ObjectivesTo investigate childhood obstructive sleep apnea syndrome (OSAS) and its role in daytime sleepiness among school-age children.MethodsA questionnaire survey was conducted with 25,211 children aged 6–15 (mean, 10.39) years attending 148 elementary and 71 middle schools in 10 prefectures across Japan and their parents. Questions concerned 4 sleep habit items (bedtime, sleep onset latency, wake time after sleep onset, wake-up time) and 4 sleep disorder items (loud snoring, snorts/gasps, breathing pauses, seems very sleepy in the daytime). Total sleep time (TST) was calculated with sleep habits. Severe possible OSAS (p-OSAS) was defined as having loud snoring, snorts and gasps, or breathing pauses “frequently” (≥ 5 times per week), and mild p-OSAS was rated as having any of these “sometimes” (2–4 times per week). Severe daytime sleepiness was defined as seeming very sleepy “frequently” and mild daytime sleepiness as seeming very sleepy “sometimes”.ResultsMean prevalence of mild to severe p-OSAS and severe p-OSAS in children across all grade levels was 9.5% and 1.6%, respectively. p-OSAS was particularly prevalent in children at lower elementary levels, decreasing with advancing grade levels. Prevalence of mild and severe daytime sleepiness was 6.1% and 0.9%, respectively, among all children (7.0%). Prevalence of daytime sleepiness increased with advancing grade levels, particularly in middle-school level. Average TST was 8.4 ± 2.2 h in both elementary and middle-school levels, and decreased as grades advanced, particularly in middle-school levels. Multivariate logistic regression analysis showed that middle-school level, TST < 8 h, and p-OSAS were independent factors for daytime sleepiness. Strong correlations were found between severe daytime sleepiness and severe p-OSAS or TST < 6 h, and between daytime sleepiness and loud snoring or breathing pauses.Conclusionp-OSAS may be an independent factor influencing daytime sleepiness in school-age children. Loud snoring and breathing pauses could be clinical markers for children with severe daytime sleepiness

Prevalence of childhood obstructive sleep apnea syndrome and its role in daytime sleepiness

Objectives To investigate childhood obstructive sleep apnea syndrome (OSAS) and its role in daytime sleepiness among school-age children. Methods A questionnaire survey was conducted with 25,211 children aged 6-15 (mean, 10.39) years attending 148 elementary and 71 middle schools in 10 prefectures across Japan and their parents. Questions concerned 4 sleep habit items (bedtime, sleep onset latency, wake time after sleep onset, wake-up time) and 4 sleep disorder items (loud snoring, snorts/gasps, breathing pauses, seems very sleepy in the daytime). Total sleep time (TST) was calculated with sleep habits. Severe possible OSAS (p-OSAS) was defined as having loud snoring, snorts and gasps, or breathing pauses frequently (>= 5 times per week), and mild p-OSAS was rated as having any of these sometimes (2-4 times per week). Severe daytime sleepiness was defined as seeming very sleepy frequently and mild daytime sleepiness as seeming very sleepy sometimes. Results Mean prevalence of mild to severe p-OSAS and severe p-OSAS in children across all grade levels was 9.5% and 1.6%, respectively. p-OSAS was particularly prevalent in children at lower elementary levels, decreasing with advancing grade levels. Prevalence of mild and severe daytime sleepiness was 6.1% and 0.9%, respectively, among all children (7.0%). Prevalence of daytime sleepiness increased with advancing grade levels, particularly in middle-school level. Average TST was 8.4 +/- 2.2 h in both elementary and middle-school levels, and decreased as grades advanced, particularly in middle-school levels. Multivariate logistic regression analysis showed that middle-school level, TST < 8 h, and p-OSAS were independent factors for daytime sleepiness. Strong correlations were found between severe daytime sleepiness and severe p-OSAS or TST < 6 h, and between daytime sleepiness and loud snoring or breathing pauses. Conclusion p-OSAS may be an independent factor influencing daytime sleepiness in school-age children. Loud snoring and breathing pauses could be clinical markers for children with severe daytime sleepiness

Liquorice-induced hypokalaemia in patients treated with Yokukansan preparations: identification of the risk factors in a retrospective cohort study

Objective To evaluate serum potassium levels and rates of hypokalaemia in patients treated with liquorice-containing Japanese traditional Kampo-medicines Yokukansan (YK) and Yokukansan-ka-chinpihange (YKCH).Design Retrospective cohort study.Setting Patients receiving YK preparations for dementia and other psychiatric disorders in the University of Tsukuba Hospital in Japan.Participants 389 patients (male/female: 174/215, 68.6±16.1 years) were treated with YK preparations for 231 days (range 6–2788 days). Patients whose potassium levels were <3.6 mEq/L before administration of YK preparations, and drug non-compliant patients, were excluded.Main outcome measure The occurrence rate of hypokalaemia and assessment of the risk factors for YK preparation-induced hypokalaemia.Results Of the 389 patients treated with YK preparations, 94 (24.2%) developed hypokalaemia (potassium levels <3.6 mEq/L) 34 days (range 1–1600 days) after administration of the preparations. 36 (38.3%) patients had co-administration with lower potassium-inducing drugs (LPIDs; diuretics, glucocorticoids, mineralocorticoids and glycyrrhizin), which was more frequent in the patients without hypokalaemia (17.3%) (p<0.05). A Cox proportional hazard model identified four risk factors for hypokalaemia: YK administration (not YKCH) (HR 3.093, 95% CI 1.408 to 6.798), co-administration of LPIDs (HR 2.743, 95% CI 1.754 to 4.289), hypoalbuminaemia at baseline (HR 2.145, 95% 1.360 to 3.384), and full dosage administration (7.5 g/day) (HR 1.600, 95% CI 1.005 to 2.549).Conclusions Serum potassium monitoring should be done at least monthly in patients with the following risk factors: LPID co-administration, YK administration, hypoalbuminaemia, and full dosage administration

iPSC-Based Compound Screening and In Vitro Trials Identify a Synergistic Anti-amyloid β Combination for Alzheimer’s Disease

In the process of drug development, in vitro studies do not always adequately predict human-specific drug responsiveness in clinical trials. Here, we applied the advantage of human iPSC-derived neurons, which offer human-specific drug responsiveness, to screen and evaluate therapeutic candidates for Alzheimer’s disease (AD). Using AD patient neurons with nearly 100% purity from iPSCs, we established a robust and reproducible assay for amyloid β peptide (Aβ), a pathogenic molecule in AD, and screened a pharmaceutical compound library. We acquired 27 Aβ-lowering screen hits, prioritized hits by chemical structure-based clustering, and selected 6 leading compounds. Next, to maximize the anti-Aβ effect, we selected a synergistic combination of bromocriptine, cromolyn, and topiramate as an anti-Aβ cocktail. Finally, using neurons from familial and sporadic AD patients, we found that the cocktail showed a significant and potent anti-Aβ effect on patient cells. This human iPSC-based platform promises to be useful for AD drug development

Biochemical classification of tauopathies by immunoblot, protein sequence and mass spectrometric analyses of sarkosyl-insoluble and trypsin-resistant tau

Intracellular filamentous tau pathology is the defining feature of tauopathies, which form a subset of neurodegenerative diseases. We have analyzed pathological tau in Alzheimer’s disease, and in frontotemporal lobar degeneration associated with tauopathy to include cases with Pick bodies, corticobasal degeneration, progressive supranuclear palsy, and ones due to intronic mutations in MAPT. We found that the C-terminal band pattern of the pathological tau species is distinct for each disease. Immunoblot analysis of trypsin-resistant tau indicated that the different band patterns of the 7–18 kDa fragments in these diseases likely reflect different conformations of tau molecular species. Protein sequence and mass spectrometric analyses revealed the carboxyl-terminal region (residues 243–406) of tau comprises the protease-resistant core units of the tau aggregates, and the sequence lengths and precise regions involved are different among the diseases. These unique assembled tau cores may be used to classify and diagnose disease strains. Based on these results, we propose a new clinicopathological classification of tauopathies based on the biochemical properties of tau

Accumulation of multiple neurodegenerative disease-related proteins in familial frontotemporal lobar degeneration associated with granulin mutation

In 2006, mutations in the granulin gene were identified in patients with familial Frontotemporal Lobar Degeneration. Granulin transcript haploinsufficiency has been proposed as a disease mechanism that leads to the loss of functional progranulin protein. Granulin mutations were initially found in tau-negative patients, though recent findings indicate that these mutations are associated with other neurodegenerative disorders with tau pathology, including Alzheimer’s disease and corticobasal degeneration. Moreover, a reduction in progranulin in tau transgenic mice is associated with increasing tau accumulation. To investigate the influence of a decline in progranulin protein on other forms of neurodegenerative-related protein accumulation, human granulin mutation cases were investigated by histochemical and biochemical analyses. Results showed a neuronal and glial tau accumulation in granulin mutation cases. Tau staining revealed neuronal pretangle forms and glial tau in both astrocytes and oligodendrocytes. Furthermore, phosphorylated α-synuclein-positive structures were also found in oligodendrocytes and the neuropil. Immunoblot analysis of fresh frozen brain tissues revealed that tau was present in the sarkosyl-insoluble fraction, and composed of three- and four-repeat tau isoforms, resembling Alzheimer’s disease. Our data suggest that progranulin reduction might be the cause of multiple proteinopathies due to the accelerating accumulation of abnormal proteins including TDP-43 proteinopathy, tauopathy and α-synucleinopathy

A novel approach for assessing neuromodulation using phase-locked information measured with TMS-EEG

Neuromodulation therapies such as electroconvulsive therapy (ECT) are used to treat several neuropsychiatric disorders, including major depressive disorder (MDD). Recent work has highlighted the use of combined transcranial magnetic stimulation and electroencephalography (TMS-EEG) to evaluate the therapeutic effects of neuromodulation; particularly, the phase locking factor (PLF) and phase locking value (PLV) can reportedly assess neuromodulation-induced functional changes in cortical networks. To examine changes in TMS-induced PLV and PLF before and after ECT, and their relationship with depression severity in patients with MDD, TMS-EEG and the Montgomery–Åsberg Depression Rating Scale (MADRS; depression severity) were implemented before and after ECT in 10 patients with MDD. Single-pulse TMS was applied to the visual and motor areas to induce phase propagation in the visuo-motor network at rest. Functional changes were assessed using PLF and PLV data. Pre-ECT TMS-induced alpha band (9–12 Hz) PLV was negatively correlated with depression severity, and increments of post-ECT from pre-ECT TMS-induced alpha band PLV were positively correlated with the reduction in depression severity. Moreover, we found a negative correlation between pre-ECT TMS-induced PLF at TMS-destination and depression severity. Finally, differences in post-ECT TMS-induced PLF peak latencies between visual and motor areas were positively correlated with depression severity. TMS-EEG-based PLV and PLF may be used to assess the therapeutic effects of neuromodulation and depressive states, respectively. Furthermore, our results provide new insights about the neural mechanisms of ECT and depression

蓄積蛋白質の構造異常による認知症の分子病理診断法およびモデル動物の開発

科学研究費助成事業 研究成果報告書：基盤研究(C)2014-2016課題番号 : 2646173