Cellular α-synuclein pathology is associated with bioenergetic dysfunction in Parkinson's iPSC-derived dopamine neurons.

Parkinson's disease (PD) is the second most common neurodegenerative disorder and a central role for α-synuclein (αSyn; SNCA) in disease aetiology has been proposed based on genetics and neuropathology. To better understand the pathological mechanisms of αSyn, we generated induced pluripotent stem cells (iPSCs) from healthy individuals and PD patients carrying the A53T SNCA mutation or a triplication of the SNCA locus and differentiated them into dopaminergic neurons (DAns). iPSC-derived DAn from PD patients carrying either mutation showed increased intracellular αSyn accumulation, and DAns from patients carrying the SNCA triplication displayed oligomeric αSyn pathology and elevated αSyn extracellular release. Transcriptomic analysis of purified DAns revealed perturbations in expression of genes linked to mitochondrial function, consistent with observed reduction in mitochondrial respiration, impairment in mitochondrial membrane potential, aberrant mitochondrial morphology and decreased levels of phosphorylated DRP1Ser616. Parkinson's iPSC-derived DAns showed increased endoplasmic reticulum stress and impairments in cholesterol and lipid homeostasis. Together, these data show a correlation between αSyn cellular pathology and deficits in metabolic and cellular bioenergetics in the pathology of PD.

RNA sequencing reveals MMP2 and TGFB1 downregulation in LRRK2 G2019S Parkinson's iPSC-derived astrocytes.

Non-neuronal cell types such as astrocytes can contribute to Parkinson's disease (PD) pathology. The G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) is one of the most common known causes of familial PD. To characterize its effect on astrocytes, we developed a protocol to produce midbrain-patterned astrocytes from human induced pluripotent stem cells (iPSCs) derived from PD LRRK2 G2019S patients and healthy controls. RNA sequencing analysis revealed the downregulation of genes involved in the extracellular matrix in PD cases. In particular, transforming growth factor beta 1 (TGFB1), which has been shown to inhibit microglial inflammatory response in a rat model of PD, and matrix metallopeptidase 2 (MMP2), which has been shown to degrade α-synuclein aggregates, were found to be down-regulated in LRRK2 G2019S astrocytes. Our findings suggest that midbrain astrocytes carrying the LRRK2 G2019S mutation may have reduced neuroprotective capacity and may contribute to the development of PD pathology.

An integrated transcriptomics and proteomics analysis reveals functional endocytic dysregulation caused by mutations in LRRK2.

BACKGROUND: Mutations in LRRK2 are the most common cause of autosomal dominant Parkinson's disease, and the relevance of LRRK2 to the sporadic form of the disease is becoming ever more apparent. It is therefore essential that studies are conducted to improve our understanding of the cellular role of this protein. Here we use multiple models and techniques to identify the pathways through which LRRK2 mutations may lead to the development of Parkinson's disease. METHODS: A novel integrated transcriptomics and proteomics approach was used to identify pathways that were significantly altered in iPSC-derived dopaminergic neurons carrying the LRRK2-G2019S mutation. Western blotting, immunostaining and functional assays including FM1-43 analysis of synaptic vesicle endocytosis were performed to confirm these findings in iPSC-derived dopaminergic neuronal cultures carrying either the LRRK2-G2019S or the LRRK2-R1441C mutation, and LRRK2 BAC transgenic rats, and post-mortem human brain tissue from LRRK2-G2019S patients. RESULTS: Our integrated -omics analysis revealed highly significant dysregulation of the endocytic pathway in iPSC-derived dopaminergic neurons carrying the LRRK2-G2019S mutation. Western blot analysis confirmed that key endocytic proteins including endophilin I-III, dynamin-1, and various RAB proteins were downregulated in these cultures and in cultures carrying the LRRK2-R1441C mutation, compared with controls. We also found changes in expression of 25 RAB proteins. Changes in endocytic protein expression led to a functional impairment in clathrin-mediated synaptic vesicle endocytosis. Further to this, we found that the endocytic pathway was also perturbed in striatal tissue of aged LRRK2 BAC transgenic rats overexpressing either the LRRK2 wildtype, LRRK2-R1441C or LRRK2-G2019S transgenes. Finally, we found that clathrin heavy chain and endophilin I-III levels are increased in human post-mortem tissue from LRRK2-G2019S patients compared with controls. CONCLUSIONS: Our study demonstrates extensive alterations across the endocytic pathway associated with LRRK2 mutations in iPSC-derived dopaminergic neurons and BAC transgenic rats, as well as in post-mortem brain tissue from PD patients carrying a LRRK2 mutation. In particular, we find evidence of disrupted clathrin-mediated endocytosis and suggest that LRRK2-mediated PD pathogenesis may arise through dysregulation of this process.

Transcriptomic profiling of purified patient-derived dopamine neurons identifies convergent perturbations and therapeutics for Parkinson's disease.

While induced pluripotent stem cell (iPSC) technologies enable the study of inaccessible patient cell types, cellular heterogeneity can confound the comparison of gene expression profiles between iPSC-derived cell lines. Here, we purified iPSC-derived human dopaminergic neurons (DaNs) using the intracellular marker, tyrosine hydroxylase. Once purified, the transcriptomic profiles of iPSC-derived DaNs appear remarkably similar to profiles obtained from mature post-mortem DaNs. Comparison of the profiles of purified iPSC-derived DaNs derived from Parkinson's disease (PD) patients carrying LRRK2 G2019S variants to controls identified significant functional convergence amongst differentially-expressed (DE) genes. The PD LRRK2-G2019S associated profile was positively matched with expression changes induced by the Parkinsonian neurotoxin rotenone and opposed by those induced by clioquinol, a compound with demonstrated therapeutic efficacy in multiple PD models. No functional convergence amongst DE genes was observed following a similar comparison using non-purified iPSC-derived DaN-containing populations, with cellular heterogeneity appearing a greater confound than genotypic background.

The Mechanism Underlying Change in the Sex Gap in Life Expectancy at Birth: An Extended Decomposition.

The relationship between differential mortality rates and differences in life expectancy is well understood, but how changing differential rates translate into changing differences in life expectancy has not been fully explained. To elucidate the mechanism involved, this study extends existing decomposition methods. The extended method decomposes change in the sex gap in life expectancy at birth into three components capturing the effects of the sex difference in mortality improvement (ρ-effect), life table deaths density by age (f-effect), and remaining life expectancy by age (e-effect). These three effects oppose and augment each other, depending on relative change in sex-differential mortality rates. The new method is applied to period data for 35 countries and cohort data for 25 countries. The results demonstrate how the mechanism, involving the three effects, operates to determine change in the sex difference in life expectancy. We observe the pivotal importance of the f-effect, which is predominantly negative because of lower female mortality, in favoring narrowing rather than widening of the sex gap, in shifting the overall effect to younger ages, and in exaggerating fluctuations due to crisis mortality. The new decomposition provides a more detailed basis for substantive analyses examining change in differences in life expectancy.

Projections of multi-morbidity in the older population in England to 2035: estimates from the Population Ageing and Care Simulation (PACSim) model.

Background: models projecting future disease burden have focussed on one or two diseases. Little is known on how risk factors of younger cohorts will play out in the future burden of multi-morbidity (two or more concurrent long-term conditions). Design: a dynamic microsimulation model, the Population Ageing and Care Simulation (PACSim) model, simulates the characteristics (sociodemographic factors, health behaviours, chronic diseases and geriatric conditions) of individuals over the period 2014-2040. Population: about 303,589 individuals aged 35 years and over (a 1% random sample of the 2014 England population) created from Understanding Society, the English Longitudinal Study of Ageing, and the Cognitive Function and Ageing Study II. Main outcome measures: the prevalence of, numbers with, and years lived with, chronic diseases, geriatric conditions and multi-morbidity. Results: between 2015 and 2035, multi-morbidity prevalence is estimated to increase, the proportion with 4+ diseases almost doubling (2015:9.8%; 2035:17.0%) and two-thirds of those with 4+ diseases will have mental ill-health (dementia, depression, cognitive impairment no dementia). Multi-morbidity prevalence in incoming cohorts aged 65-74 years will rise (2015:45.7%; 2035:52.8%). Life expectancy gains (men 3.6 years, women: 2.9 years) will be spent mostly with 4+ diseases (men: 2.4 years, 65.9%; women: 2.5 years, 85.2%), resulting from increased prevalence of rather than longer survival with multi-morbidity. Conclusions: our findings indicate that over the next 20 years there will be an expansion of morbidity, particularly complex multi-morbidity (4+ diseases). We advocate for a new focus on prevention of, and appropriate and efficient service provision for those with, complex multi-morbidity.

Structural and functional social network attributes moderate the association of self-rated health with mental health in midlife and older adults.

BACKGROUND: Social relationships are multifaceted, and different social network components can operate via different processes to influence well-being. This study examined associations of social network structure and relationship quality (positive and negative social exchanges) with mental health in midlife and older adults. The focus was on both direct associations of network structure and relationship quality with mental health, and whether these social network attributes moderated the association of self-rated health (SRH) with mental health. METHODS: Analyses were based on survey data provided by 2001 (Mean age = 65, SD = 8.07) midlife and older adults. We used Latent Class Analysis (LCA) to classify participants into network types based on network structure (partner status, network size, contact frequency, and activity engagement), and used continuous measures of positive and negative social exchanges to operationalize relationship quality. Regression analysis was used to test moderation. RESULTS: LCA revealed network types generally consistent with those reported in previous studies. Participants in more diverse networks reported better mental health than those categorized into a restricted network type after adjustment for age, sex, education, and employment status. Analysis of moderation indicated that those with poorer SRH were less likely to report poorer mental health if they were classified into more diverse networks. A similar moderation effect was also evident for positive exchanges. CONCLUSIONS: The findings suggest that both quantity and quality of social relationships can play a role in buffering against the negative implications of physical health decline for mental health.

Education-related inequalities in cause-specific mortality: first estimates for Australia using individual-level linked census and mortality data.

BACKGROUND: Socioeconomic inequalities in mortality are evident in all high-income countries, and ongoing monitoring is recommended using linked census-mortality data. Using such data, we provide the first estimates of education-related inequalities in cause-specific mortality in Australia, suitable for international comparisons. METHODS: We used Australian Census (2016) linked to 13 months of Death Registrations (2016-17). We estimated relative rates (RR) and rate differences (RD, per 100 000 person-years), comparing rates in low (no qualifications) and intermediate (secondary school) with high (tertiary) education for individual causes of death (among those aged 25-84 years) and grouped according to preventability (25-74 years), separately by sex and age group, adjusting for age, using negative binomial regression. RESULTS: Among 13.9 M people contributing 14 452 732 person-years, 84 743 deaths occurred. All-cause mortality rates among men and women aged 25-84 years with low education were 2.76 [95% confidence interval (CI): 2.61-2.91] and 2.13 (2.01-2.26) times the rates of those with high education, respectively. We observed inequalities in most causes of death in each age-sex group. Among men aged 25-44 years, relative and absolute inequalities were largest for injuries, e.g. transport accidents [RR = 10.1 (5.4-18.7), RD = 21.2 (14.5-27.9)]). Among those aged 45-64 years, inequalities were greatest for chronic diseases, e.g. lung cancer [men RR = 6.6 (4.9-8.9), RD = 57.7 (49.7-65.8)] and ischaemic heart disease [women RR = 5.8 (3.7-9.1), RD = 20.2 (15.8-24.6)], with similar patterns for people aged 65-84 years. When grouped according to preventability, inequalities were large for causes amenable to behaviour change and medical intervention for all ages and causes amenable to injury prevention among young men. CONCLUSIONS: Australian education-related inequalities in mortality are substantial, generally higher than international estimates, and related to preventability. Findings highlight opportunities to reduce them and the potential to improve the health of the population.

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension.

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo.

Who cares? Provision of Care and Assistance Among Older Persons in the Philippines.

OBJECTIVE: This study aims to examine the provision of assistance among older persons in the Philippines who experience difficulty with activities of daily living (ADLs) or instrumental activities of daily living (IADLs). METHOD: Data for this research are drawn from the 2007 Philippine Study on Aging (PSOA). Cross-tabulations and regression analyses are used to identify who provides assistance to older Filipinos. RESULTS: The study shows that the Filipino family, particularly spouse and daughters, plays an important role in providing assistance to older Filipinos who need help in carrying out functional activities. This provision of caregiving, however, follows a gendered pattern. Specifically, the spouse provides assistance to older men, whereas daughters provide assistance to older women. Grandchildren and noncoresident family members, to some extent, are also active in providing care to older Filipinos. DISCUSSION: The Filipino family remains faithful to its filial duty toward older persons, but who provides care and the way it is provided are likely to change due to the demographic changes and values shifts that have swept the country.

The Role of Astrocyte Dysfunction in Parkinson's Disease Pathogenesis.

Astrocytes are the most populous glial subtype and are critical for brain function. Despite this, historically there have been few studies into the role that they may have in neurodegenerative diseases, such as Parkinson's disease (PD). Recently, however, several studies have determined that genes known to have a causative role in the development of PD are expressed in astrocytes and have important roles in astrocyte function. Here, we review these recent developments and discuss their impact on our understanding of the pathophysiology of PD, and the implications that this might have for its treatment.

Directing Differentiation of Pluripotent Stem Cells Toward Retinal Pigment Epithelium Lineage.

Development of efficient and reproducible conditions for directed differentiation of pluripotent stem cells into specific cell types is important not only to understand early human development but also to enable more practical applications, such as in vitro disease modeling, drug discovery, and cell therapies. The differentiation of stem cells to retinal pigment epithelium (RPE) in particular holds promise as a source of cells for therapeutic replacement in age-related macular degeneration. Here we show development of an efficient method for deriving homogeneous RPE populations in a period of 45 days using an adherent, monolayer system and defined xeno-free media and matrices. The method utilizes sequential inhibition and activation of the Activin and bone morphogenetic protein signaling pathways and can be applied to both human embryonic stem cells and induced pluripotent stem cells as the starting population. In addition, we use whole genome transcript analysis to characterize cells at different stages of differentiation that provides further understanding of the developmental dynamics and fate specification of RPE. We show that with the described method, RPE develop through stages consistent with their formation during embryonic development. This characterization- together with the absence of steps involving embryoid bodies, three-dimensional culture, or manual dissections, which are common features of other protocols-makes this process very attractive for use in research as well as for clinical applications. Stem Cells Translational Medicine 2017;6:490-501.

MAPT Genetic Variation and Neuronal Maturity Alter Isoform Expression Affecting Axonal Transport in iPSC-Derived Dopamine Neurons.

The H1 haplotype of the microtubule-associated protein tau (MAPT) locus is genetically associated with neurodegenerative diseases, including Parkinson's disease (PD), and affects gene expression and splicing. However, the functional impact on neurons of such expression differences has yet to be fully elucidated. Here, we employ extended maturation phases during differentiation of induced pluripotent stem cells (iPSCs) into mature dopaminergic neuronal cultures to obtain cultures expressing all six adult tau protein isoforms. After 6 months of maturation, levels of exon 3+ and exon 10+ transcripts approach those of adult brain. Mature dopaminergic neuronal cultures display haplotype differences in expression, with H1 expressing 22% higher levels of MAPT transcripts than H2 and H2 expressing 2-fold greater exon 3+ transcripts than H1. Furthermore, knocking down adult tau protein variants alters axonal transport velocities in mature iPSC-derived dopaminergic neuronal cultures. This work links haplotype-specific MAPT expression with a biologically functional outcome relevant for PD.

Cardiovascular mortality sex differentials in selected East Asian and Western populations.

BACKGROUND: Explaining patterns in the sex ratio (male/female) of cardiovascular disease (CVD) mortality would improve understanding of mortality transitions under modernisation. Little research has examined secular trends in this ratio across populations, taking age and cohort into account. We examine cohort effects in the ratios of CVD mortality (including ischaemic heart disease and cerebrovascular disease) among 4 East Asian populations that vary in the timing of their modernisation, and assess the effect of smoking on these patterns in comparison with Western populations. METHODS: The sequential method for log-linear models is applied to analyse age, period and cohort effects for sex ratios. Age and cohort effects are fitted first, with population as offset; period effects are fitted in a second model using the fitted values from the first model as the offset. Lung cancer mortality serves as a proxy for smoking. RESULTS: Increases in sex ratios of CVD mortality began in earlier cohorts in Western than in East Asian populations. Once begun, increases were more rapid in East Asia. The cohort effect for the sex ratio of CVD mortality differs from that for lung cancer mortality. Trends in sex ratios of CVD mortality by cohort are similar before and after adjustment for lung cancer mortality in East Asia; the increasing trend across 1900-1945 cohorts is maintained in Western populations after adjustment. CONCLUSIONS: The sex ratio of CVD mortality has increased across successive cohorts living in increasingly modernised environments. There is scant evidence that this increase is attributable to changing sex-specific rates of smoking.

ER Stress and Autophagic Perturbations Lead to Elevated Extracellular α-Synuclein in GBA-N370S Parkinson's iPSC-Derived Dopamine Neurons.

Heterozygous mutations in the glucocerebrosidase gene (GBA) represent the strongest common genetic risk factor for Parkinson's disease (PD), the second most common neurodegenerative disorder. However, the molecular mechanisms underlying this association are still poorly understood. Here, we have analyzed ten independent induced pluripotent stem cell (iPSC) lines from three controls and three unrelated PD patients heterozygous for the GBA-N370S mutation, and identified relevant disease mechanisms. After differentiation into dopaminergic neurons, we observed misprocessing of mutant glucocerebrosidase protein in the ER, associated with activation of ER stress and abnormal cellular lipid profiles. Furthermore, we observed autophagic perturbations and an enlargement of the lysosomal compartment specifically in dopamine neurons. Finally, we found increased extracellular α-synuclein in patient-derived neuronal culture medium, which was not associated with exosomes. Overall, ER stress, autophagic/lysosomal perturbations, and elevated extracellular α-synuclein likely represent critical early cellular phenotypes of PD, which might offer multiple therapeutic targets.

Residents' Morning Report in Psychiatry Training: Description of a Model and a Survey of Resident Attitudes.

A residents' morning report, adapted from the traditional internal medicine model, was introduced into a psychiatry residency curriculum to supplement bedside teaching for junior residents during inpatient service rotations by focusing on case presentations, clinical reasoning, and evidence-based decision-making skills. This paper describes the design of the report and findings from surveys of residents who participated in the report over a 3-year period. The surveys demonstrate high levels of satisfaction with the exercise and offer insight into factors that may optimize or impede learning in the morning report setting in psychiatry.

L-dopa reverses behavioral deficits in the Pitx3 mouse fetus.

Studies of fetal rodents have provided evidence that early emerging behaviors, such as the suckling response, are dependent on the developing dopaminergic system. Although connections have been made between manipulations of dopamine and altered behavioral responses, the specific neural pathways involved have yet to be discovered. In this study, we examined the neurobehavioral output of the nigrostriatal pathway, using the Pitx3ak/2J mouse model (Pitx3). Used extensively in the study of Parkinson's disease, the Pitx3 mouse has very specific prenatal loss of dopaminergic neurons solely in the nigrostriatal pathway. Because of this specificity, we hypothesized that behavioral deficits specific to the nigrostriatal pathway would be reversed with administration of the dopamine precursor 3,4-dihydroxyphenylalanine (L-dopa). To test this hypothesis, homozygous mutant and heterozygous control fetal subjects were administered 1 of 4 doses (0, 25, 50, or 75 mg/kg) of L-dopa on the day before birth. Quantification of fetal behavior was scored from video recordings of behavioral observations. The behavioral measures used were (a) spontaneous movement activity; (b) state organization, from quantifications of high- and low-amplitude movements; (c) interlimb movement synchrony, a measure of limb coordination; and (d) oral grasp, similar to a newborn infant suckling response. Specific behavioral deficits observed in the Pitx3 mutants were reversed by L-dopa administration in a dose-dependent manner. However, different deficits required dissimilar doses for reversal, suggesting that some early emerging behaviors may be more sensitive to the administration of L-dopa. Taken together, this study provides valuable information about prenatal behaviors dependent on the nigrostriatal pathway.

Coherent mortality forecasting: the product-ratio method with functional time series models.

When independence is assumed, forecasts of mortality for subpopulations are almost always divergent in the long term. We propose a method for coherent forecasting of mortality rates for two or more subpopulations, based on functional principal components models of simple and interpretable functions of rates. The product-ratio functional forecasting method models and forecasts the geometric mean of subpopulation rates and the ratio of subpopulation rates to product rates. Coherence is imposed by constraining the forecast ratio function through stationary time series models. The method is applied to sex-specific data for Sweden and state-specific data for Australia. Based on out-of-sample forecasts, the coherent forecasts are at least as accurate in overall terms as comparable independent forecasts, and forecast accuracy is homogenized across subpopulations.

Indigenous Australians are under-represented in longitudinal ageing studies.

OBJECTIVE: Evidence-based policy depends on the availability of high-quality research that is relevant to the population. This study aimed to identify the available data on the health of older Indigenous Australians in population-based longitudinal studies of ageing. APPROACH: Evaluation of the Dynamic Analyses to Optimise Ageing Project (DYNOPTA) dataset that has pooled nine Australian longitudinal ageing studies, six of which were analysed here. MAIN OUTCOME MEASURES: Proportions of the DYNOPTA sample identified as Indigenous. RESULTS: Indigenous participants made up 0.7% of males and 0.5% of females in the weighted sample, compared with 0.8% of both sexes in the Australian population. Indigenous under-representation is greater at ages 45-54 than at older ages, despite overall greater participation in this age range. CONCLUSIONS AND IMPLICATIONS: Within the existing Australian longitudinal ageing studies, Indigenous Australians are under-represented. This means there is a significant gap in the evidence base relating to the health of older Indigenous Australians. Research approaches specifically designed to address the health and wellbeing of older Indigenous Australians are urgently required.

The evolution of epidemic suicide on Guam: context and contagion.

Thirty years of suicide rates for Guam were analyzed by age, sex, period, and cohort. Youth suicide increased rapidly in the 1990s; certain cohorts have higher rates. Four explanatory factors are discussed, including ecological factors and migration from the Federated States of Micronesia. Direct and indirect suicide contagion followed the death by suicide of a respected politician, strongly influencing period and cohort patterns. Suicide pacts inflated suicide among young people. These factors acted in combination to produce epidemic levels of suicide in the 1990s.