Polygenic Risk Scores Validated in Patient-Derived Cells Stratify for Mitochondrial Subtypes of Parkinson's Disease.

OBJECTIVE: The aim of our study is to better understand the genetic architecture and pathological mechanisms underlying neurodegeneration in idiopathic Parkinson's disease (iPD). We hypothesized that a fraction of iPD patients may harbor a combination of common variants in nuclear-encoded mitochondrial genes ultimately resulting in neurodegeneration. METHODS: We used mitochondria-specific polygenic risk scores (mitoPRSs) and created pathway-specific mitoPRSs using genotype data from different iPD case-control datasets worldwide, including the Luxembourg Parkinson's Study (412 iPD patients and 576 healthy controls) and COURAGE-PD cohorts (7,270 iPD cases and 6,819 healthy controls). Cellular models from individuals stratified according to the most significant mitoPRS were subsequently used to characterize different aspects of mitochondrial function. RESULTS: Common variants in genes regulating Oxidative Phosphorylation (OXPHOS-PRS) were significantly associated with a higher PD risk in independent cohorts (Luxembourg Parkinson's Study odds ratio, OR = 1.31[1.14-1.50], p-value = 5.4e-04; COURAGE-PD OR = 1.23[1.18-1.27], p-value = 1.5e-29). Functional analyses in fibroblasts and induced pluripotent stem cells-derived neuronal progenitors revealed significant differences in mitochondrial respiration between iPD patients with high or low OXPHOS-PRS (p-values < 0.05). Clinically, iPD patients with high OXPHOS-PRS have a significantly earlier age at disease onset compared to low-risk patients (false discovery rate [FDR]-adj p-value = 0.015), similar to prototypic monogenic forms of PD. Finally, iPD patients with high OXPHOS-PRS responded more effectively to treatment with mitochondrially active ursodeoxycholic acid. INTERPRETATION: OXPHOS-PRS may provide a precision medicine tool to stratify iPD patients into a pathogenic subgroup genetically defined by specific mitochondrial impairment, making these individuals eligible for future intelligent clinical trial designs. ANN NEUROL 2024;96:133-149.

Burnout and Physical Activity as Predictors of Job Satisfaction Among Peruvian Nurses: The Job Demands-Resources Theory.

BACKGROUND: The Job Demands-Resources (JD-R) theory suggests that an imbalance between job demands and available resources can lead to burnout, negatively affecting job satisfaction. Physical activity is recognized for its positive effects on psychological well-being and could play a crucial role in mitigating burnout and improving job satisfaction, especially in high-demand professions such as nursing. OBJECTIVE: This study investigates the relationship between burnout, physical activity, and job satisfaction in Peruvian nurses, using the JD-R theory as a theoretical framework. METHODS: A cross-sectional and explanatory analysis was conducted on a sample of 420 Peruvian nurses, using a Structural Equation Modeling (SEM) design to analyze the relationships between burnout, physical activity, and job satisfaction. The instruments included the Ultra-Short Burnout Measure (IUB), the General Job Satisfaction Scale NTP 394, and the International Physical Activity Questionnaire (IPAQ). RESULTS: The findings showed a significant negative correlation between burnout and physical activity (ß = -.40, P < .001) and between burnout and job satisfaction (ß = -.46, P < .001). Physical activity exhibited a significant positive correlation with job satisfaction (ß = .22, P < .001). Moreover, mediation analysis confirmed that physical activity mediates the relationship between burnout and job satisfaction (ß = -.106, P < .001). CONCLUSIONS: The findings emphasize the importance of physical activity as a mediator in the relationship between burnout and job satisfaction among Peruvian nurses, highlighting the need to promote physical activity as a strategy to improve workplace well-being. It is suggested that enhancing access to and promotion of physical activity could mitigate the effects of burnout and improve job satisfaction, which is essential for the quality of care and the well-being of nursing staff. These findings underscore the need for organizational and public health strategies that promote a healthy work environment and balance between the demands and resources available.

Alpha-synuclein pathology is associated with astrocyte senescence in a midbrain organoid model of familial Parkinson's disease.

Parkinson's disease (PD) is a complex, progressive neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta in the midbrain. Despite extensive research efforts, the molecular and cellular changes that precede neurodegeneration in PD are poorly understood. To address this, here we describe the use of patient specific human midbrain organoids harboring the SNCA triplication to investigate mechanisms underlying dopaminergic degeneration. Our midbrain organoid model recapitulates key pathological hallmarks of PD, including the aggregation of α-synuclein and the progressive loss of dopaminergic neurons. We found that these pathological hallmarks are associated with an increase in senescence associated cellular phenotypes in astrocytes including nuclear lamina defects, the presence of senescence associated heterochromatin foci, and the upregulation of cell cycle arrest genes. These results suggest a role of pathological α-synuclein in inducing astrosenescence which may, in turn, increase the vulnerability of dopaminergic neurons to degeneration.

Multiomics analysis identifies novel facilitators of human dopaminergic neuron differentiation.

Midbrain dopaminergic neurons (mDANs) control voluntary movement, cognition, and reward behavior under physiological conditions and are implicated in human diseases such as Parkinson's disease (PD). Many transcription factors (TFs) controlling human mDAN differentiation during development have been described, but much of the regulatory landscape remains undefined. Using a tyrosine hydroxylase (TH) human iPSC reporter line, we here generate time series transcriptomic and epigenomic profiles of purified mDANs during differentiation. Integrative analysis predicts novel regulators of mDAN differentiation and super-enhancers are used to identify key TFs. We find LBX1, NHLH1 and NR2F1/2 to promote mDAN differentiation and show that overexpression of either LBX1 or NHLH1 can also improve mDAN specification. A more detailed investigation of TF targets reveals that NHLH1 promotes the induction of neuronal miR-124, LBX1 regulates cholesterol biosynthesis, and NR2F1/2 controls neuronal activity.

Impaired neuron differentiation in GBA-associated Parkinson's disease is linked to cell cycle defects in organoids.

The mechanisms underlying Parkinson's disease (PD) etiology are only partially understood despite intensive research conducted in the field. Recent evidence suggests that early neurodevelopmental defects might play a role in cellular susceptibility to neurodegeneration. To study the early developmental contribution of GBA mutations in PD we used patient-derived iPSCs carrying a heterozygous N370S mutation in the GBA gene. Patient-specific midbrain organoids displayed GBA-PD relevant phenotypes such as reduction of GCase activity, autophagy impairment, and mitochondrial dysfunction. Genome-scale metabolic (GEM) modeling predicted changes in lipid metabolism which were validated with lipidomics analysis, showing significant differences in the lipidome of GBA-PD. In addition, patient-specific midbrain organoids exhibited a decrease in the number and complexity of dopaminergic neurons. This was accompanied by an increase in the neural progenitor population showing signs of oxidative stress-induced damage and premature cellular senescence. These results provide insights into how GBA mutations may lead to neurodevelopmental defects thereby predisposing to PD pathology.

Emicizumab prophylaxis for people with hemophilia A: Waste estimation and the Brazilian perspective.

Costs of hemophilia A treatment are increasing. Waste of clotting products should be avoided. To estimate the first-year waste of emicizumab prophylaxis for people with hemophilia A and inhibitors (PwHAi) who failed immune tolerance induction (ITI), in Brazil. We evaluated the manufacturer and the Brazilian Ministry of Health (MoH) protocol-recommended regimens in a budget impact model. The loading dose consisted of 3.0 mg/kg/Q1W for 4 weeks, for both recommendations. The manufacturer maintenance regimens comprised 1.5 mg/kg/Q1W, 3.0 mg/kg/Q2W, and 6.0 mg/kg/Q4W. The MoH protocol maintenance regimen encompassed a hybrid Q1W/Q2W administration, depending on the body weight. The Q4W regimen was not recommended by the MoH protocol. Analyses were performed to estimate waste given its expense based on the World Health Organization body weight range (percentiles [P] 15, 50, and 85). The first-year emicizumab waste was estimated individually and for the disclosed PwHAi who failed ITI (n = 114). The highest emicizumab waste was estimated for the lowest body weights and the Q1W regimen. The Q4W regimen resulted in the lowest emicizumab waste, followed by the MoH protocol regimen. The total reconstituted costs estimated for the PwHAi who failed ITI according to the hybrid MoH protocol ranged from US$32,858,777 (P15) to US$47,186,858 (P85), with emicizumab waste ranging from 7.9 % (US$2,594,515) to 3.7 % (US$1,738,750), respectively. Lost resources due to current protocols for emicizumab prophylaxis for PwHAi who failed ITI in Brazil are considerable. Waste was more pronounced due to lower body weight and shorter administration intervals.

Comparison of two protocols for the generation of iPSC-derived human astrocytes.

BACKGROUND: Astrocytes have recently gained attention as key contributors to the pathogenesis of neurodegenerative disorders including Parkinson's disease. To investigate human astrocytes in vitro, numerous differentiation protocols have been developed. However, the properties of the resulting glia are inconsistent, which complicates the selection of an appropriate method for a given research question. Thus, we compared two approaches for the generation of iPSC-derived astrocytes. We phenotyped glia that were obtained employing a widely used long, serum-free ("LSF") method against an in-house established short, serum-containing ("SSC") protocol which allows for the generation of astrocytes and midbrain neurons from the same precursor cells. RESULTS: We employed high-content confocal imaging and RNA sequencing to characterize the cultures. The astrocytes generated with the LSF or SSC protocols differed considerably in their properties: while the former cells were more labor-intense in their generation (5 vs 2 months), they were also more mature. This notion was strengthened by data resulting from cell type deconvolution analysis that was applied to bulk transcriptomes from the cultures to assess their similarity with human postmortem astrocytes. CONCLUSIONS: Overall, our analyses highlight the need to consider the advantages and disadvantages of a given differentiation protocol, when designing functional or drug discovery studies involving iPSC-derived astrocytes.

Parkin Deficiency Impairs Mitochondrial DNA Dynamics and Propagates Inflammation.

BACKGROUND: Mutations in the E3 ubiquitin ligase parkin cause autosomal recessive Parkinson's disease (PD). Together with PTEN-induced kinase 1 (PINK1), parkin regulates the clearance of dysfunctional mitochondria. New mitochondria are generated through an interplay of nuclear- and mitochondrial-encoded proteins, and recent studies suggest that parkin influences this process at both levels. In addition, parkin was shown to prevent mitochondrial membrane permeability, impeding mitochondrial DNA (mtDNA) escape and subsequent neuroinflammation. However, parkin's regulatory roles independent of mitophagy are not well described in patient-derived neurons. OBJECTIVES: We sought to investigate parkin's role in preventing neuronal mtDNA dyshomeostasis, release, and glial activation at the endogenous level. METHODS: We generated induced pluripotent stem cell (iPSC)-derived midbrain neurons from PD patients with parkin (PRKN) mutations and healthy controls. Live-cell imaging, proteomic, mtDNA integrity, and gene expression analyses were employed to investigate mitochondrial biogenesis and genome maintenance. To assess neuroinflammation, we performed single-nuclei RNA sequencing in postmortem tissue and quantified interleukin expression in mtDNA/lipopolysaccharides (LPS)-treated iPSC-derived neuron-microglia co-cultures. RESULTS: Neurons from patients with PRKN mutations revealed deficits in the mitochondrial biogenesis pathway, resulting in mtDNA dyshomeostasis. Moreover, the energy sensor sirtuin 1, which controls mitochondrial biogenesis and clearance, was downregulated in parkin-deficient cells. Linking mtDNA disintegration to neuroinflammation, in postmortem midbrain with PRKN mutations, we confirmed mtDNA dyshomeostasis and detected an upregulation of microglia overexpressing proinflammatory cytokines. Finally, parkin-deficient neuron-microglia co-cultures elicited an enhanced immune response when exposed to mtDNA/LPS. CONCLUSIONS: Our findings suggest that parkin coregulates mitophagy, mitochondrial biogenesis, and mtDNA maintenance pathways, thereby protecting midbrain neurons from neuroinflammation and degeneration. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Willingness to pay for a hypothetical malaria vaccine in Brazil: a cross-sectional study and the implications.

Aim: Malaria is an infection caused by protozoa of genus Plasmodium, considered the one associated with increasingly large outbreaks. Methods: A cross-sectional study was conducted with residents in the northern region of Brazil on the willingness to pay (WTP) for a hypothetical vaccine against malaria (effective protection of 80%). Results: Of 616 people interviewed, most interviewees were female (61%) and were employed (97%). The median individual maximum WTP for a hypothetical malaria vaccine was US$11.90 (BRL 50). Conclusion: The northern region of Brazil is one of the largest markets for a malaria vaccine due to its epidemiological relevance. Consequently, economic studies will be important to assist in the assessment of the potential price and value of new vaccines.

Association of proton-pump inhibitor use with adverse health outcomes: A systematic umbrella review of meta-analyses of cohort studies and randomised controlled trials.

AIMS: The aim was to perform an umbrella review to summarise the existing evidence on proton-pump inhibitor (PPI) use and adverse outcomes and to grade the certainty of evidence. METHODS: Electronic databases were searched up to July 2021 for meta-analyses of cohort studies and/or randomised controlled trials (RCTs). Summary effect sizes from a random-effects model, between-study heterogeneity, 95% prediction interval, small-study effect, excess significance and credibility ceilings were devised to classify the credibility of evidence from meta-analyses of cohort studies, whereas the GRADE approach was used for meta-analyses of RCTs. RESULTS: In meta-analyses of cohort studies, 52 of the 91 examined associations were statistically significant (P ≤ .05). Convincing evidence emerged from main analysis for the association between PPI use and risk of all-site fracture and chronic kidney disease in the elderly population. However, none of these associations remained supported by convincing evidence after sensitivity analyses. The use of PPI is also associated with an increased risk of mortality due to COVID-19 infection and other related adverse outcomes, but the quality of evidence was weak. In meta-analyses of RCTs, 38 of the 63 examined associations were statistically significant. However, no associations were supported by high or moderate-quality evidence. CONCLUSION: This study's findings imply that most putative adverse outcomes associated with PPI use may not be supported by high-quality evidence and are likely to have been affected by underlying confounding factors. Future research is needed to confirm the causal association between PPI use and risk of fracture and chronic kidney disease.

Parkinson's Disease Phenotypes in Patient Neuronal Cultures and Brain Organoids Improved by 2-Hydroxypropyl-ß-Cyclodextrin Treatment.

BACKGROUND: The etiology of Parkinson's disease (PD) is only partially understood despite the fact that environmental causes, risk factors, and specific gene mutations are contributors to the disease. Biallelic mutations in the phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) gene involved in mitochondrial homeostasis, vesicle trafficking, and autophagy are sufficient to cause PD. OBJECTIVES: We sought to evaluate the difference between controls' and PINK1 patients' derived neurons in their transition from neuroepithelial stem cells to neurons, allowing us to identify potential pathways to target with repurposed compounds. METHODS: Using two-dimensional and three-dimensional models of patients' derived neurons we recapitulated PD-related phenotypes. We introduced the usage of midbrain organoids for testing compounds. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), we corrected the point mutations of three patients' derived cells. We evaluated the effect of the selected compound in a mouse model. RESULTS: PD patient-derived cells presented differences in their energetic profile, imbalanced proliferation, apoptosis, mitophagy, and a reduced differentiation efficiency to tyrosine hydroxylase positive (TH+) neurons compared to controls' cells. Correction of a patient's point mutation ameliorated the metabolic properties and neuronal firing rates as well as reversing the differentiation phenotype, and reducing the increased astrocytic levels. Treatment with 2-hydroxypropyl-ß-cyclodextrin increased the autophagy and mitophagy capacity of neurons concomitant with an improved dopaminergic differentiation of patient-specific neurons in midbrain organoids and ameliorated neurotoxicity in a mouse model. CONCLUSION: We show that treatment with a repurposed compound is sufficient for restoring the impaired dopaminergic differentiation of PD patient-derived cells. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Single-cell sequencing of human midbrain reveals glial activation and a Parkinson-specific neuronal state.

Idiopathic Parkinson's disease is characterized by a progressive loss of dopaminergic neurons, but the exact disease aetiology remains largely unknown. To date, Parkinson's disease research has mainly focused on nigral dopaminergic neurons, although recent studies suggest disease-related changes also in non-neuronal cells and in midbrain regions beyond the substantia nigra. While there is some evidence for glial involvement in Parkinson's disease, the molecular mechanisms remain poorly understood. The aim of this study was to characterize the contribution of all cell types of the midbrain to Parkinson's disease pathology by single-nuclei RNA sequencing and to assess the cell type-specific risk for Parkinson's disease using the latest genome-wide association study. We profiled >41 000 single-nuclei transcriptomes of post-mortem midbrain from six idiopathic Parkinson's disease patients and five age-/sex-matched controls. To validate our findings in a spatial context, we utilized immunolabelling of the same tissues. Moreover, we analysed Parkinson's disease-associated risk enrichment in genes with cell type-specific expression patterns. We discovered a neuronal cell cluster characterized by CADPS2 overexpression and low TH levels, which was exclusively present in idiopathic Parkinson's disease midbrains. Validation analyses in laser-microdissected neurons suggest that this cluster represents dysfunctional dopaminergic neurons. With regard to glial cells, we observed an increase in nigral microglia in Parkinson's disease patients. Moreover, nigral idiopathic Parkinson's disease microglia were more amoeboid, indicating an activated state. We also discovered a reduction in idiopathic Parkinson's disease oligodendrocyte numbers with the remaining cells being characterized by a stress-induced upregulation of S100B. Parkinson's disease risk variants were associated with glia- and neuron-specific gene expression patterns in idiopathic Parkinson's disease cases. Furthermore, astrocytes and microglia presented idiopathic Parkinson's disease-specific cell proliferation and dysregulation of genes related to unfolded protein response and cytokine signalling. While reactive patient astrocytes showed CD44 overexpression, idiopathic Parkinson's disease microglia revealed a pro-inflammatory trajectory characterized by elevated levels of IL1B, GPNMB and HSP90AA1. Taken together, we generated the first single-nuclei RNA sequencing dataset from the idiopathic Parkinson's disease midbrain, which highlights a disease-specific neuronal cell cluster as well as 'pan-glial' activation as a central mechanism in the pathology of the movement disorder. This finding warrants further research into inflammatory signalling and immunomodulatory treatments in Parkinson's disease.

Comportamiento de la mortalidad por COVID-19 en profesionales de enfermería de América Latina / Behavior of COVID-19 mortality in nursing professionals in Latin America / Comportamento de mortalidade dos enfermeiros da COVID-19 na América Latina

Los enfermeros han estado al frente de la crisis causada por la COVID-19 lo que puso de manifiesto las oportunidades del sistema sanitario y la necesidad del cuidado de este gremio profesional. La finalidad del presente estudio fue analizar el comportamiento de la mortalidad por COVID-19 en profesionales de enfermería de América Latina. Se fundamentó en una revisión sistemática de la literatura, donde se hizo seguimiento de la declaración PRISMA en las bases de datos SCIELO, EMBASE, MEDLINE, PsycoINFO y SCOPUS. Los resultados de la búsqueda y aplicación de los criterios de inclusión y exclusión permitieron la selección de 25 trabajos que fueron sintetizados en una matriz que promulgó el análisis de contenido por año, país del recurso y aportes sustanciales de la revisión. Se percibió el efecto del comportamiento del profesional de enfermería en un marco de bienestar en emergencia y frágil a consecuencia del COVID-19.

Nurses have been at the forefront of the crisis caused by COVID-19, which highlighted the opportunities of the health system and the need for the care of this professional guild. The purpose of this study was to analyze the behavior of COVID-19 mortality in nursing professionals in Latin America. It was based on a systematic review of the literature, where the PRISMA statement was followed up in the SCIELO, EMBASE, MEDLINE, PsycoINFO and SCOPUS databases. The results of the search and application of the inclusion and exclusion criteria allowed the selection of 25 papers that were synthesized in a matrix that promulgated the content analysis by year, country of the resource and substantial contributions of the review. The effect of nursing professional behavior in an emergency and fragile wellness setting as a result of COVID-19 was perceived.

Os enfermeiros têm estado na vanguarda da crise da COVID-19, destacando as oportunidades para o sistema de saúde e a necessidade de cuidados de enfermagem. O objetivo deste estudo foi analisar o comportamento da mortalidade dos enfermeiros da COVID-19 na América Latina. Foi baseado em uma revisão sistemática da literatura, onde a declaração PRISMA foi rastreada nas bases de dados SCIELO, EMBASE, MEDLINE, PsycoINFO e SCOPUS. Os resultados da busca e aplicação dos critérios de inclusão e exclusão permitiram a seleção de 25 trabalhos que foram sintetizados em uma matriz que promulgou a análise de conteúdo por ano, país do recurso e contribuições substanciais da revisão. O efeito do comportamento profissional de enfermagem em um ambiente de emergência e bem-estar frágil como resultado da COVID-19 foi percebido.

Gene-corrected p.A30P SNCA patient-derived isogenic neurons rescue neuronal branching and function.

Parkinson's disease (PD) is characterised by the degeneration of A9 dopaminergic neurons and the pathological accumulation of alpha-synuclein. The p.A30P SNCA mutation generates the pathogenic form of the alpha-synuclein protein causing an autosomal-dominant form of PD. There are limited studies assessing pathogenic SNCA mutations in patient-derived isogenic cell models. Here we provide a functional assessment of dopaminergic neurons derived from a patient harbouring the p.A30P SNCA mutation. Using two clonal gene-corrected isogenic cell lines we identified image-based phenotypes showing impaired neuritic processes. The pathological neurons displayed impaired neuronal activity, reduced mitochondrial respiration, an energy deficit, vulnerability to rotenone, and transcriptional alterations in lipid metabolism. Our data describes for the first time the mutation-only effect of the p.A30P SNCA mutation on neuronal function, supporting the use of isogenic cell lines in identifying image-based pathological phenotypes that can serve as an entry point for future disease-modifying compound screenings and drug discovery strategies.

iPSC-Derived Microglia as a Model to Study Inflammation in Idiopathic Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disease with unknown cause in the majority of patients, who are therefore considered "idiopathic" (IPD). PD predominantly affects dopaminergic neurons in the substantia nigra pars compacta (SNpc), yet the pathology is not limited to this cell type. Advancing age is considered the main risk factor for the development of IPD and greatly influences the function of microglia, the immune cells of the brain. With increasing age, microglia become dysfunctional and release pro-inflammatory factors into the extracellular space, which promote neuronal cell death. Accordingly, neuroinflammation has also been described as a feature of PD. So far, studies exploring inflammatory pathways in IPD patient samples have primarily focused on blood-derived immune cells or brain sections, but rarely investigated patient microglia in vitro. Accordingly, we decided to explore the contribution of microglia to IPD in a comparative manner using, both, iPSC-derived cultures and postmortem tissue. Our meta-analysis of published RNAseq datasets indicated an upregulation of IL10 and IL1B in nigral tissue from IPD patients. We observed increased expression levels of these cytokines in microglia compared to neurons using our single-cell midbrain atlas. Moreover, IL10 and IL1B were upregulated in IPD compared to control microglia. Next, to validate these findings in vitro, we generated IPD patient microglia from iPSCs using an established differentiation protocol. IPD microglia were more readily primed as indicated by elevated IL1B and IL10 gene expression and higher mRNA and protein levels of NLRP3 after LPS treatment. In addition, IPD microglia had higher phagocytic capacity under basal conditions-a phenotype that was further exacerbated upon stimulation with LPS, suggesting an aberrant microglial function. Our results demonstrate the significance of microglia as the key player in the neuroinflammation process in IPD. While our study highlights the importance of microglia-mediated inflammatory signaling in IPD, further investigations will be needed to explore particular disease mechanisms in these cells.

The Parkinson's-disease-associated mutation LRRK2-G2019S alters dopaminergic differentiation dynamics via NR2F1.

Increasing evidence suggests that neurodevelopmental alterations might contribute to increase the susceptibility to develop neurodegenerative diseases. We investigate the occurrence of developmental abnormalities in dopaminergic neurons in a model of Parkinson's disease (PD). We monitor the differentiation of human patient-specific neuroepithelial stem cells (NESCs) into dopaminergic neurons. Using high-throughput image analyses and single-cell RNA sequencing, we observe that the PD-associated LRRK2-G2019S mutation alters the initial phase of neuronal differentiation by accelerating cell-cycle exit with a concomitant increase in cell death. We identify the NESC-specific core regulatory circuit and a molecular mechanism underlying the observed phenotypes. The expression of NR2F1, a key transcription factor involved in neurogenesis, decreases in LRRK2-G2019S NESCs, neurons, and midbrain organoids compared to controls. We also observe accelerated dopaminergic differentiation in vivo in NR2F1-deficient mouse embryos. This suggests a pathogenic mechanism involving the LRRK2-G2019S mutation, where the dynamics of dopaminergic differentiation are modified via NR2F1.

Consumer willingness to pay for a hypothetical Chagas disease vaccine in Brazil: a cross-sectional study and the implications.

Aim: Chagas disease is a serious public health problem, endemic in 21 countries in Latin America. A future vaccine can contribute to decreasing the number of cases and its complications. Methods: A cross-sectional study was conducted with residents of the northern region of Brazil, on the willingness to pay for a hypothetical vaccine against Chagas disease (effective protection of 80%). Results: We interviewed 619 individuals and seven were excluded from the analysis and the value of willingness to pay was US$23.77 (100.00 BRL). Conclusion: The Northern region of Brazil is one of the largest markets for this vaccine, due to its epidemiological relevance, so economic studies with this vaccine will be important to assist in the assessment of technologies.

PINK1 deficiency impairs adult neurogenesis of dopaminergic neurons.

Recent evidence suggests neurogenesis is on-going throughout life but the relevance of these findings for neurodegenerative disorders such as Parkinson's disease (PD) is poorly understood. Biallelic PINK1 mutations cause early onset, Mendelian inherited PD. We studied the effect of PINK1 deficiency on adult neurogenesis of dopaminergic (DA) neurons in two complementary model systems. Zebrafish are a widely-used model to study neurogenesis in development and through adulthood. Using EdU analyses and lineage-tracing studies, we first demonstrate that a subset of ascending DA neurons and adjacent local-projecting DA neurons are each generated into adulthood in wild type zebrafish at a rate that decreases with age. Pink1-deficiency impedes DA neurogenesis in these populations, most significantly in early adult life. Pink1 already exerts an early effect on Th1+ progenitor cells rather than on differentiated DA neurons only. In addition, we investigate the effect of PINK1 deficiency in a human isogenic organoid model. Global neuronal differentiation in PINK1-deficient organoids and isogenic controls is similar, but PINK1-deficient organoids display impeded DA neurogenesis. The observation of impaired adult dopaminergic neurogenesis in Pink1 deficiency in two complementing model systems may have significant consequences for future therapeutic approaches in human PD patients with biallelic PINK1 mutations.

Integrated, automated maintenance, expansion and differentiation of 2D and 3D patient-derived cellular models for high throughput drug screening.

Patient-derived cellular models become an increasingly powerful tool to model human diseases for precision medicine approaches. The identification of robust cellular disease phenotypes in these models paved the way towards high throughput screenings (HTS) including the implementation of laboratory advanced automation. However, maintenance and expansion of cells for HTS remains largely manual work. Here, we describe an integrated, complex automated platform for HTS in a translational research setting also designed for maintenance and expansion of different cell types. The comprehensive design allows automation of all cultivation steps and is flexible for development of methods for variable cell types. We demonstrate protocols for controlled cell seeding, splitting and expansion of human fibroblasts, induced pluripotent stem cells (iPSC), and neural progenitor cells (NPC) that allow for subsequent differentiation into different cell types and image-based multiparametric screening. Furthermore, we provide automated protocols for neuronal differentiation of NPC in 2D culture and 3D midbrain organoids for HTS. The flexibility of this multitask platform makes it an ideal solution for translational research settings involving experiments on different patient-derived cellular models for precision medicine.

Mitochondrial and Clearance Impairment in p.D620N VPS35 Patient-Derived Neurons.

BACKGROUND: VPS35 is part of the retromer complex and is responsible for the trafficking and recycling of proteins implicated in autophagy and lysosomal degradation, but also takes part in the degradation of mitochondrial proteins via mitochondria-derived vesicles. The p.D620N mutation of VPS35 causes an autosomal-dominant form of Parkinson's disease (PD), clinically representing typical PD. OBJECTIVE: Most of the studies on p.D620N VPS35 were performed on human tumor cell lines, rodent models overexpressing mutant VPS35, or in patient-derived fibroblasts. Here, based on identified target proteins, we investigated the implication of mutant VPS35 in autophagy, lysosomal degradation, and mitochondrial function in induced pluripotent stem cell-derived neurons from a patient harboring the p.D620N mutation. METHODS: We reprogrammed fibroblasts from a PD patient carrying the p.D620N mutation in the VPS35 gene and from two healthy donors in induced pluripotent stem cells. These were subsequently differentiated into neuronal precursor cells to finally generate midbrain dopaminergic neurons. RESULTS: We observed a decreased autophagic flux and lysosomal mass associated with an accumulation of α-synuclein in patient-derived neurons compared to controls. Moreover, patient-derived neurons presented a mitochondrial dysfunction with decreased membrane potential, impaired mitochondrial respiration, and increased production of reactive oxygen species associated with a defect in mitochondrial quality control via mitophagy. CONCLUSION: We describe for the first time the impact of the p.D620N VPS35 mutation on autophago-lysosome pathway and mitochondrial function in stem cell-derived neurons from an affected p.D620N carrier and define neuronal phenotypes for future pharmacological interventions. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.